Pyrrolidine dithiocarbamate reverses Bcl-xL-mediated apoptotic resistance to doxorubicin by inducing paraptosis

Pyrrolidine dithiocarbamate reverses Bcl-xL-mediated apoptotic resistance to doxorubicin by... Abstract Elevated Bcl-xL expression in cancer cells contributes to doxorubicin (DOX) resistance, leading to failure in chemotherapy. In addition, the clinical use of high-dose doxorubicin (DOX) in cancer therapy has been limited by issues with cardiotoxicity and hepatotoxicity. Here, we show that co-treatment with pyrrolidine dithiocarbamate (PDTC) attenuates DOX-induced apoptosis in Chang-L liver cells and human hepatocytes, but overcomes DOX resistance in Bcl-xL-overexpressing Chang-L cells and several hepatocellular carcinoma (HCC) cell lines with high Bcl-xL expression. Additionally, combined treatment with DOX and PDTC markedly retarded tumor growth in a Huh-7 HCC cell xenograft tumor model, compared to either mono-treatment. These results suggest that DOX/PDTC co-treatment may provide a safe and effective therapeutic strategy against malignant hepatoma cells with Bcl-xL-mediated apoptotic defects. We also found that induction of paraptosis, a cell death mode that is accompanied by dilation of the endoplasmic reticulum and mitochondria, is involved in this anti-cancer effect of DOX/PDTC. The intracellular glutathione levels were reduced in Bcl-xL-overexpressing Chang-L cells treated with DOX/PDTC, and DOX/PDTC-induced paraptosis was effectively blocked by pretreatment with thiol-antioxidants, but not by non-thiol antioxidants. Collectively, our results suggest that disruption of thiol homeostasis may critically contribute to DOX/PDTC-induced paraptosis in Bcl-xL-overexpressing cells. Introduction Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the third leading cause of cancer-related deaths worldwide (1). Surgical resection has been considered the optimal treatment approach for HCC, but relatively few patients qualify for surgery and the recurrence rate is high (2). Transcatheter arterial chemoembolization has become the mainstay management strategy for patients with unresectable HCC (3) and DOX is the most widely used single chemotherapeutic agent in chemoembolization (4). However, the clinical usefulness of DOX is often limited by its cardiotoxicity, nephrotoxicity and hepatotoxicity (5–7). Bcl-xL is a significant prognostic factor for disease progression in human HCC (8) and overexpression of Bcl-xL in HCC cells contributes to their therapeutic resistance against anti-cancer drugs, including DOX (9). Thus, we need to identify a therapeutic strategy for HCC that can overcome Bcl-xL-mediated apoptotic resistance to DOX and minimize the toxic effects of this agent on normal cells. For treating apoptosis-resistant tumors, the induction of non-apoptotic cell death could be an option. Pyrrolidine dithiocarbamate (PDTC) is a low-molecular weight thiol compound (10) that has potent antioxidant and NF-κB inhibitor activities (11,12). It was found to show opposing effects in a cell-context-specific manner, protecting rats against DOX-induced myocardial apoptosis (13), but sensitizing multidrug resistant HepG-2 cells to DOX (14). These results suggested that the combination of DOX and PDTC may act against liver cancer, but it was unclear why or how PDTC could demonstrate pro-survival or pro-death effects in DOX-treated cells under different cellular contexts. In this study, we investigated whether PDTC could exhibit opposing effects on normal liver cells and hepatoma cells treated with DOX and, if so, whether this differential effect was related to the expression of Bcl-xL. Our results showed that co-treatment with PDTC attenuated DOX-mediated apoptosis in Chang-L normal liver cells due to the antioxidant activity of PDTC. While DOX-induced apoptosis in Chang-L cells was effectively blocked by Bcl-xL overexpression, PDTC co-treatment reverses this acquired Bcl-xL-mediated DOX resistance by inducing paraptosis. Paraptosis (para = next to or beside, and apoptosis) is a cell death mode, which is characterized by dilation of the endoplasmic reticulum (ER) and/or mitochondria and the lack of characteristic apoptotic features (e.g. apoptotic body formation, chromatin condensation, DNA fragmentation and caspase dependency) (15). Recent work has shown that paraptosis is associated with the generation of reactive oxygen species (ROS) (16,17) and perturbation of cellular proteostasis via proteasome inhibition (16,17) and disruption of sulfhydryl homeostasis (18,19). We show here that in the presence of Bcl-xL, depletion of glutathione (GSH), including mitochondrial GSH, critically contributes to the ability of DOX plus PDTC (DOX/PDTC) to induce paraptosis. Our results indicate that a combined regimen of DOX and PDTC may provide an attractive strategy for effectively treating hepatomas while sparing normal cells from DOX toxicity. Materials and methods Chemicals and antibodies All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless indicated otherwise. Cycloheximide (CHX) was obtained from Calbiochem (San Diego, CA). The following antibodies were used: caspase-3 and PDI (Stressgen, Ann Arbor, MI); Bcl-xL (Santa Cruz Biotechnology, Santa Cruz, CA); α-tubulin, phospho-p65 (S536), total p65, Alix (Cell signaling, Beverly, MA); SDHA and Bcl-2 (Invitrogen, Carsbad, CA); GCLC and GSR (Novus Biologicals, Littleton, CO). Horseradish peroxidase-conjugated anti-rabbit IgG, Horseradish peroxidase-conjugated anti-mouse IgG, Alexa 488-conjugated anti-mouse IgG and Alexa 594-conjugated anti-rabbit IgG (Molecular Probes, Carlsbad, CA). Cell culture Chang cells were obtained from American Tissue Culture Collections (ATCC, Manassas, VA) in 2010 and Chang-L cell clone, denoted as Chang-L (20,21), which has higher hepatic characteristics (albumin production and liver-specific carbamoyl phosphate synthase-1 expression) were isolated by single cell dilution and expansion, were used for this study. Chang-L clones were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum. Huh-7 cells were obtained from JCRB Cell Bank (Japanese Collection of Research Bioresources Cell Bank) in 2015. Chang-L human liver cells and Huh-7 human hepatoma cells were cultured in Dulbecco’s modified Eagle’s medium medium supplemented with 10% fetal bovine serum. Human hepatocytes were purchased from Cambrex (East Rutherford, NJ) in 2015 and cultured in human hepatocyte basal medium supplemented with human hepatocyte medium (HHM) SingleQuots. SNU-182, SNU-368 and SNU-449 human hepatoma cells were obtained from the Korean Cell Line Bank in 2015 and were grown in RPMI 1640 supplemented with 10% fetal bovine serum. All cell lines were authenticated and characterized by the supplier and routinely tested by PCR for mycoplasma contamination by using the following primers: Myco_F: 5′-ACA CCA TGG GAG CTG GTA AT-3′, Myco_R: 5′-CTT CAT CGA CTT TCA GAC CCA AGG CA-3′. Establishment of the stable cell lines overexpressing the Bcl-xL Chang-L cells were transfected with the Flag-tag-Bcl-xL and stably transfected cells were selected using the fresh media containing puromycin 4 μg/ml for 3 weeks. Chang-L sublines stably transfected with an empty vector were used as a control. The overexpression of Bcl-xL in selected cell lines was confirmed by Western blot analysis using anti-Flag antibody. Measurement of cellular viability Cells (5 × 104 cells) were cultured in 24-well plates and treated as indicated. For measurement of cellular viability, 2 μM calcein-acetoxymethyl ester (calcein-AM), a green fluorescent indicator of the intracellular esterase activity of cells and 4 μM ethidium homodimer-1 (EthD-1), a red fluorescent indicator of membrane damaged (dead) cells, we added to each well, and the plates were incubated for 5 min in 5% CO2 at 37°C. Cells were then observed under a fluorescence microscope (Axiovert 200M; Carl Zeiss) equipped with Zeiss filter sets #46 and #64HE. Viable cells, corresponding to those that exclusively exhibited green fluorescence, were counted in five fields per well at 200× magnification. Only exclusively green cells were counted as live because bicolored (green and red cells) cannot be unambiguosly to live or dead groups. The percentage of viable cells (Viable %) calculated as green cells/(green + red + bicolored cells), was normalized to that of untreated control cells (100%). Cell cycle analysis Trypsinized and floating cells were pooled, washed with phosphate-buffered saline (PBS)-EDTA and fixed in 70% (v/v) ethanol. DNA contents were assessed by staining cells with propidium iodide and monitoring by FACSAriaTM III (BD Biosciences, San Jose, CA). The data was analyzed using FACSDiva software (BD Biosciences). Measurement of reactive ROS Cells were treated with 10 μM PDTC and/or 1 µg/ml DOX for the indicated time points, stained with 5 µM H2DCF-DA for 20 min at 37°C and subjected to flow cytometry. TUNEL assay Chang-L and Bcl-xL-Chang-L cells cultured on coverslips were treated with 1 μg/ml DOX and 1 μg/ml DOX plus 10 μM PDTC for the indicated time points and fixed with 4% paraformaldehyde (pH 7.4) for 10 min. TUNEL assay of the fragmented DNAs was performed as recommended by the manufacturer (Molecular Probes). Transmission electron microscopy Cells were prefixed in Karnovsky’s solution [1% paraformaldehyde, 2% glutaraldehyde, 2 mM calcium chloride, 0.1 M cacodylate buffer (pH 7.4)] for 2 h and washed with cacodylate buffer. Postfixing was carried out in 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 h. After dehydration with 50–100% alcohol, the cells were embedded in Poly/Bed 812 resin (Pelco, Redding, CA) and polymerized and observed under electron microscope (EM 902A, Zeiss). Immunocytochemistry After treatments, cells were fixed with acetone/methanol (1:1) for 5 min at –20°C and blocking in 5% bovine serum albumin in PBS for 30 min. Fixed cells were incubated overnight at 4°C with primary antibody [anti-SDHA (1:500, mouse, Invitrogen] and anti-PDI [1:500, rabbit, Stressgen)] diluted in PBS and then washed three times in PBS and incubated for 1 h at room temperature with anti-mouse or anti-rabbit Alexa Fluor 488 or 594 (1:500, Molecular Probes). Slides were mounted with ProLong Gold antifade mounting reagent (Molecular probes) and cell staining was visualized with a fluorescence microscope using Zeiss filter sets #46 and #64HE (excitation band pass, 598/25 nm; emission band pass, 647/70 nm). Glutathione assay Bcl-xL-Chang-L cells were treated with 1 µg/ml DOX and/or 10 μM PDTC for the indicated time points. The intracellular level of reduced GSH was determined by using a kit from Cayman Chemical (Ann Arbor, MI). Equal amounts of the proteins (150 μg/reaction) were used for the determination of GSH according to the manufacturer’s instructions. Similar results were obtained in three independent experiments. Two-photon fluorescence microscopy Two-photon fluorescence microscopy (TPM) images of probe-labeled cells were obtained with multiphoton microscopes (Leica TCS SP8 MP, Wetzlar, Germany) using 40× oil objective and a numerical aperture (NA) setting of 1.30. The TPM images were obtained by exciting the probes with a mode-locked titanium-sapphire laser source (Mai Tai HP; Spectra Physics, 80 MHz, 100 fs) set at wavelength 740 nm and an output power of 2490 mW, which corresponded to an average power of approximately 4.14 × 108 mW cm-2 in the focal plane. Live-cell imaging was performed using live-cell incubator systems (Chamlide IC; Live Cell Instrument). We obtained images in the 400–450 nm (Fblue) and 500–600 nm (Fyellow) ranges, and ratiometric image processing and analyses were carried out using the MetaMorph software. Small interfering RNAs The small interfering RNA (siRNA) duplexes used in this study were purchased from OriGene Technologies (Rockville, MD) and have the following sequences: GCLC#1 (Cat. No. SR301815A), UGGAUGGAGAGUAGAA UUUCGACCC; GCLC#2 (Cat. No. SR301815B), CGACUUGACGAUAGAUAAA GAGATC; GSR#2 (Cat. No. SR301982B), CCAGAAUACCAACGUCAAAGGCATC; GSR#3 (Cat. No. SR301982C), GGAAGAUUCCAAAUUAGAUUAUAAC. Bcl-xL-Chang-L cells plated in 24-well plates were transfected with the respective siRNA oligonucleotides (50 nM) using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions. After 24 h, knockdown of GCLC and GSR in transfected cells was confirmed by Western blotting. In vivo tumor growth inhibition and immunohistochemistry Huh-7 cells were used to produce a xenograft tumor model in male BALB/c nude mice (nu/nu, 5 weeks old, Japan SLC, Hamamatsu, Japan). A suspension of 1 × 107 cells in a 50 μl volume (saline) was subcutaneously injected into the right hind limb of mice. Tumors were grown for 3 weeks until average tumor volume reached 70–100 mm3. Mice were randomized into six groups (n = 4 per group) and mice were received twice-weekly intraperitoneal (i.p.) injections of either DOX (2 mg/kg), PDTC (25, 50 mg/kg), a combination of each agent, or saline (control) for 2 weeks. PDTC was injected 1 h prior to DOX injection. DOX and PDTC were dissolved in saline and D.W, respectively. Tumor size and body weights were measured three times a week and tumor volume was calculated using the formula [V = (L × W2) × 0.5, where V = volume, L = length and W = width]. All experiments were performed following the guidelines and regulations approved by the Institutional Animal Care and Use Committee of the Asan Institute for Life Science. On the 20th day, mice were sacrificed and the tumors were isolated, fixed in 4% paraformaldehyde and then embedded into paraffin. Sections of 5 μm were stained with H&E, assayed for Ki67 using anti-Ki67 antibody (1:100; Abcam) and the ABC peroxidase labeling procedure (Vector Laboratories, Inc., Burlingame, CA) for immunologic detection. The image on the tissue sections was observed and photographed with microscopy set (Olympus, Tokyo, Japan). Statistical analysis All data are presented as mean ± SEM or ± SD. To perform statistical analysis, GraphPad Prism (GraphPad Software Inc, Sandiego, CA) was used. Each experiment was repeated at least three times. Normality of data was assessed by Kolmogorov–Smirnov testes and equal variance using Bartlett’s test. For normal distribution, statistical differences were determined using an analysis of variance (ANOVA) followed by followed by Bonferroni multiple comparison test. If the data were not normally distributed, Kruskal–Wallis test was performed followed by Dunn’s test. P < 0.05 was considered statistically significant. Results PDTC reverses the resistance of HCC cells to DOX both in vitro and in vivo To investigate whether PDTC could sensitize various HCC cells to DOX, we treated HCC cell lines (Huh-7, SNU-368, SNU-449 and SNU-182) with different doses of DOX and/or PDTC and analyzed cell viability using calcein-AM and EthD-1 to detect viable and dead cells, respectively. Although these HCC cell lines varied in their sensitivities to DOX, treatment with sub-lethal doses of PDTC commonly and dose-dependently reduced the viabilities of these cells when combined with DOX (Figure 1A). Interestingly, the cell death induced by DOX/PDTC was commonly accompanied by extensive vacuolation in these HCC cells (Figure 1B). Next, we evaluated the anti-cancer effect of DOX/PDTC in vivo using mouse Huh-7 cell xenograft models. Mice received twice-weekly intraperitoneal (i.p.) injections of DOX (2 mg/kg), PDTC (25 or 50 mg/kg), a combination of the two agents, or saline (control) for 2 weeks. Treatment with DOX or PDTC alone had no notable effect on the growth of Huh-7 xenograft tumors, whereas DOX/PDTC inhibited tumor growth in a PDTC-dose-dependent manner (Figure 1C) without triggering any loss of body weight (Supplementary Figure 1, available at Carcinogenesis Online). Tumor growth was decreased by 37.4% in mice treated with 2 mg/kg DOX plus 25 mg/kg PDTC and by 49.2% in mice treated with 2 mg/kg DOX plus 50 mg/kg PDTC, as compared to the control group. The cell proliferation marker, Ki67, was also significantly reduced in DOX/PDTC-treated tumor (Figure 1D). Hematoxylin and eosin (H&E) staining showed the presence of severe cellular vacuolation in Huh-7 xenograft sections obtained from mice treated with DOX/PDTC (Figure 1E), similar to our findings in DOX/PDTC-treated Huh-7 cells in vitro (Figure 1B). Collectively, these results indicate that PDTC co-treatment may effectively overcome DOX resistance in malignant HCC cells via a form of cell death that is accompanied by vacuolation. Figure 1. View largeDownload slide Co-treatment with PDTC overcomes the DOX resistance in HCC cells. (A) Viability of cells treated with DOX and/or PDTC for 72 h was assessed using calcein-AM and EthD-1 as described in Materials and Methods. The percentage of live cells was normalized to that of untreated control cells (100%). Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. **P < 0.01, *P < 0.001 versus DOX-treated cells. (B) Cellular morphologies following treatment with DOX and/or PDTC for 48 h in Huh-7 (20 μM PDTC and/or 5 μg/ml DOX); SNU-182 (10 μM PDTC and/or 0.25 μg/ml DOX); SNU-368 (50 μM PDTC and/or 5 μg/ml DOX); SNU-449 (40 μM PDTC and/or 10 μg/ml DOX) cells. Bars, 20 μm. (C) The relative tumor volume of Huh-7 xenograft tumor after administrated with the indicated amount of DOX and/or PDTC, as described in Material and Methods. Tumor size was measured three times a week for 20 days and plotted for growth curve (left). Data represent the means ± SD. Kruskal–Wallis test was performed followed by Dunn’s test. **P < 0.05, **P < 0.01 versus DOX-treated mice. On the 20th day, all tumors were isolated and photographed (right). (D, E) Tumor tissues sections were prepared from the mice harboring Huh-7 xenograft tumors following treatment with control, 2 mg//kg DOX, 50 mg/kg PDTC or 2 mg//kg DOX plus 50 mg//kg PDTC. (D) Representative pictures of the immunohistochemical staining of Ki67 (left). Bar, 50 μm. Intensities of Ki67 staining were quantified and denoted as a graph (right). Data represent the means ± S.D. Kruskal-Wallis test was performed followed by Dunn’s test. *P < 0.05* versus DOX-treated mice. (E) The results of H&E staining. Bar, 50 μm. Figure 1. View largeDownload slide Co-treatment with PDTC overcomes the DOX resistance in HCC cells. (A) Viability of cells treated with DOX and/or PDTC for 72 h was assessed using calcein-AM and EthD-1 as described in Materials and Methods. The percentage of live cells was normalized to that of untreated control cells (100%). Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. **P < 0.01, *P < 0.001 versus DOX-treated cells. (B) Cellular morphologies following treatment with DOX and/or PDTC for 48 h in Huh-7 (20 μM PDTC and/or 5 μg/ml DOX); SNU-182 (10 μM PDTC and/or 0.25 μg/ml DOX); SNU-368 (50 μM PDTC and/or 5 μg/ml DOX); SNU-449 (40 μM PDTC and/or 10 μg/ml DOX) cells. Bars, 20 μm. (C) The relative tumor volume of Huh-7 xenograft tumor after administrated with the indicated amount of DOX and/or PDTC, as described in Material and Methods. Tumor size was measured three times a week for 20 days and plotted for growth curve (left). Data represent the means ± SD. Kruskal–Wallis test was performed followed by Dunn’s test. **P < 0.05, **P < 0.01 versus DOX-treated mice. On the 20th day, all tumors were isolated and photographed (right). (D, E) Tumor tissues sections were prepared from the mice harboring Huh-7 xenograft tumors following treatment with control, 2 mg//kg DOX, 50 mg/kg PDTC or 2 mg//kg DOX plus 50 mg//kg PDTC. (D) Representative pictures of the immunohistochemical staining of Ki67 (left). Bar, 50 μm. Intensities of Ki67 staining were quantified and denoted as a graph (right). Data represent the means ± S.D. Kruskal-Wallis test was performed followed by Dunn’s test. *P < 0.05* versus DOX-treated mice. (E) The results of H&E staining. Bar, 50 μm. PDTC attenuates DOX-induced apoptosis in Chang-L liver cells Next, to investigate the effect of DOX and/or PDTC in normal liver cells, we employed Chang-L liver cells. This Chang cell clone was isolated by single cell dilution and expansion, and selected for the distinct hepatic characteristics of albumin production and liver-specific carbamoyl synthetase-1 expression (20,21). Compared to the tested HCC cells (Figure 1A), these cells were sensitive to the cytotoxic effect of DOX, and treatment with 1 μg/ml DOX alone for 16 h reduced cell viability by ~50% (Figure 2A). While treatment with PDTC alone up to 10 μM did not affect the viability of Chang-L cells, PDTC pretreatment dose-dependently attenuated the cytotoxicity of DOX (Figure 2A). We found that 1 μg/ml DOX treatment induced apoptosis, as evidenced by the apoptotic morphologies, such as cellular shrinkage, blebbing and apoptotic body formation (Figure 2B), increased sub-G1 hypoploid cell population (Figure 2C), DNA fragmentation (as assessed by TUNEL assays) (Figure 2D), and proteolytic processing of caspase-3 (Figure 2E). In contrast, co-treatment with 10 μM PDTC attenuated all these apoptotic features induced by DOX (Figure 2B–E). In addition, the death of human hepatocytes treated with 1 μg/ml DOX was also significantly reduced by 10 μM PDTC (Figure 2F), suggesting that PDTC may alleviate DOX-induced hepatotoxicity. Taken together, these results suggest that PDTC may chemosensitize resistant HCC cells to DOX while chemoprotecting hepatocytes, and thus the combined regimen of DOX and PDTC could offer a safe and effective therapeutic strategy against malignant HCCs. Figure 2. View largeDownload slide PDTC pretreatment attenuates DOX-induced apoptosis in Chang-L liver cells. (A) Viability of Chang-L cells untreated or pretreated with PDTC and further treated with DOX for 16 h was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. *P < 0.001 versus cells treated with DOX. (B) The representative pictures from Chang-L cells treated with the indicated concentrations of DOX and/or PDTC for 16 h were taken under a phase-contrast microscope. Bar, 20 μm. (C, D) Chang-L cells were treated with 1 μg/ml DOX and/or 10 μM PDTC for 24 h. FACS analysis to measure DNA content (C) and TUNEL assay (D) were performed. (E) Following treatment of Chang-L cells with 1 μg/ml DOX alone or 1 μg/ml DOX plus 10 μM PDTC, Western blotting of Caspase-3 was performed. Western blotting of α-tubulin was served as a loading control. (F) Viability of human hepatocytes treated with 1 μg/ml DOX and/or 10 μM PDTC for 24 h was measured using calcein-AM and EthD-1. Data represent the means ± SD. Kruskal–Wallis test was performed followed by Dunn’s test. #P < 0.01 versus untreated cells, *P < 0.01 versus DOX-treated cells. Figure 2. View largeDownload slide PDTC pretreatment attenuates DOX-induced apoptosis in Chang-L liver cells. (A) Viability of Chang-L cells untreated or pretreated with PDTC and further treated with DOX for 16 h was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. *P < 0.001 versus cells treated with DOX. (B) The representative pictures from Chang-L cells treated with the indicated concentrations of DOX and/or PDTC for 16 h were taken under a phase-contrast microscope. Bar, 20 μm. (C, D) Chang-L cells were treated with 1 μg/ml DOX and/or 10 μM PDTC for 24 h. FACS analysis to measure DNA content (C) and TUNEL assay (D) were performed. (E) Following treatment of Chang-L cells with 1 μg/ml DOX alone or 1 μg/ml DOX plus 10 μM PDTC, Western blotting of Caspase-3 was performed. Western blotting of α-tubulin was served as a loading control. (F) Viability of human hepatocytes treated with 1 μg/ml DOX and/or 10 μM PDTC for 24 h was measured using calcein-AM and EthD-1. Data represent the means ± SD. Kruskal–Wallis test was performed followed by Dunn’s test. #P < 0.01 versus untreated cells, *P < 0.01 versus DOX-treated cells. PDTC overcomes Bcl-xL-mediated apoptotic resistance to DOX via non-apoptotic cell death accompanied by vacuolation It is intriguing to consider how, when combined with DOX, PDTC could enhance the death of resistant HCC cells but attenuate that of normal liver cells. What is the determining factor in the ability of DOX or DOX/PDTC to induce differential fates in HCC cells versus hepatocytes? As a candidate protein, we selected Bcl-xL, because Bcl-xL is over-expressed in two-thirds of human HCC (8), and this upregulation enables HCC cells to resist chemotherapeutic agents, including DOX (9,22). Overexpression of Bcl-xL was detected also in Huh-7, SNU-182, SNU-368 and SNU-449 HCC cells (Figure 3A). To test whether the HCC cellular responses to DOX or DOX/PDTC are reproduced in Chang-L cells by overexpression of Bcl-xL, we established Chang-L sublines stably expressing Flag-tagged Bcl-xL (Bcl-xL-Chang-L cells). We found that the cell death induced by 1 μg/ml DOX was very effectively inhibited in three different subclones of Bcl-xL-Chang-L cells (Figure 3B). In addition, the DOX-induced apoptotic morphologies (Figure 3C), loss of mitochondrial membrane potential (MMP), which was assessed using Rhodamine-123 (Supplementary Figure 2A and B, available at Carcinogenesis Online) and release of mitochondrial Cytochrome c (Supplementary Figure 2C, available at Carcinogenesis Online) were also dramatically blocked in these Bcl-xL-overexpressing cells. The viability of Bcl-xL-Chang-L cells was not markedly affected by DOX alone, even under an increased dose (2 μg/ml) and incubation time (72 h); contrastingly, DOX/PDTC treatment dose-dependently and significantly enhanced cell death (Figure 3D). Furthermore, combination of DOX and PDTC for 72 h induced the cell death accompanied by extensive vacuolation (Figure 3E), as similar to its effect in the tested HCC cells (Figure 1). These results indicate that PDTC co-treatment can reverse Bcl-xL-mediated DOX resistance by inducing cell death associated with vacuolation. In addition, these finding suggest that Bcl-xL is a key factor in determining the survival or death of liver cells following treatment with DOX or DOX/PDTC. Figure 3. View largeDownload slide Bcl-xL-mediated resistance to DOX is overcome by PDTC co-treatment via induction of cell death accompanied by vacuolation. (A) Western blot to detect endogenous Bcl-xL protein levels in the indicated cells. Western blotting of α-tubulin was performed to confirm equal loading of protein samples. (B) In Chang-L sublines stably transfected with the expression vector encoding Flag-tagged Bcl-xL, the protein levels of Bcl-xL were examined by western blotting using anti-Flag antibody (upper). Control cells transfected with mock vector and three different Bcl-xL-Chang-L sublines were treated with 1 μg/ml DOX for 16 h and then cellular viabilities were analyzed using calcein-AM and EthD-1 (bottom). Data represent the means ± SD. Kruskal–Wallis test was performed followed by Dunn’s test. *P < 0.001 versus DOX-treated Chang-L cells. (C) Chang-L and Bcl-xL-Chang-L (#10) cells were treated with 1 μg/ml DOX for 16 h and the representative pictures were taken under a phase-contrast microscope. Bar, 20 μm. (D) Bcl-xL-Chang-L cells (#10) were treated with DOX and/or PDTC at the indicated concentrations for 72 h. Cellular viabilities were assessed using calcein-AM and EthD-1. Data represent the means ± S.E.M. One-way ANOVA and Bonferroni’s post hoc test. *P < 0.001 versus DOX-treated cells. (E) Bcl-xL-Chang-L cells treated with 1 μg/ml DOX and/or 10 μM PDTC for 72 h were observed by phase-contrast microscopy. Bar, 20 μm. Figure 3. View largeDownload slide Bcl-xL-mediated resistance to DOX is overcome by PDTC co-treatment via induction of cell death accompanied by vacuolation. (A) Western blot to detect endogenous Bcl-xL protein levels in the indicated cells. Western blotting of α-tubulin was performed to confirm equal loading of protein samples. (B) In Chang-L sublines stably transfected with the expression vector encoding Flag-tagged Bcl-xL, the protein levels of Bcl-xL were examined by western blotting using anti-Flag antibody (upper). Control cells transfected with mock vector and three different Bcl-xL-Chang-L sublines were treated with 1 μg/ml DOX for 16 h and then cellular viabilities were analyzed using calcein-AM and EthD-1 (bottom). Data represent the means ± SD. Kruskal–Wallis test was performed followed by Dunn’s test. *P < 0.001 versus DOX-treated Chang-L cells. (C) Chang-L and Bcl-xL-Chang-L (#10) cells were treated with 1 μg/ml DOX for 16 h and the representative pictures were taken under a phase-contrast microscope. Bar, 20 μm. (D) Bcl-xL-Chang-L cells (#10) were treated with DOX and/or PDTC at the indicated concentrations for 72 h. Cellular viabilities were assessed using calcein-AM and EthD-1. Data represent the means ± S.E.M. One-way ANOVA and Bonferroni’s post hoc test. *P < 0.001 versus DOX-treated cells. (E) Bcl-xL-Chang-L cells treated with 1 μg/ml DOX and/or 10 μM PDTC for 72 h were observed by phase-contrast microscopy. Bar, 20 μm. Combination of DOX and PDTC induces paraptosis in Bcl-xL-Chang-L cells We next examined whether PDTC promotes cell death in Bcl-xL-Chang-L cells that are resistant to DOX via apoptosis. However, compared to DOX alone, DOX/PDTC did not notably increase the sub-G1 cell population, TUNEL-positivity, caspsae-3 cleavage, and the release of mitochondrial cytochrome c (Supplementary Figure 3A—D, available at Carcinogenesis Online). Interestingly, cytochrome c was localized within the dilated mitochondria in spite of DOX/PDTC treatment (Supplementary Figure 3D, available at Carcinogenesis Online). Taken together, these results suggest that apoptosis is not responsible for the enhanced cytotoxicity of DOX/PDTC in Bcl-xL-Chang-L cells. Next, we performed electron microscopy to investigate the origins of vacuoles observed in Bcl-xL-overexpressing cells treated with DOX/PDTC. At 48 h post-treatment of Bcl-xL-Chang-L cells with DOX/PDTC, swelling of mitochondria with disruption of the mitochondrial cristae as well as the swelling and fusion of swollen ER structures were noted (Figure 4A). At 72 h, there was further ER fusion and the cellular space was fully occupied with ER-derived vacuoles. Immunocytochemistry using specific antibodies against subunit A of succinate dehydrogenase (SDHA, a mitochondrial protein), and protein disulfide-isomerase (PDI, an ER-resident protein) exhibited ring-shaped SDHA expression around the nuclei and polygonal PDI expression at the boundaries of the larger vacuoles not only in Bcl-xL-Chang-L but also in Huh-7 cells treated with DOX/PDTC for 48 h (Figure 4B). Dilation of the ER and/or mitochondria is a common feature of paraptosis and this mode of cell death is known to require protein synthesis (15,19,23). Therefore, we tested the effect of CHX in our system. Indeed, CHX pretreatment effectively inhibited the cellular vacuolation (Figure 4C) and cell death (Figure 4D) induced by DOX/PDTC in both Bcl-xL-Chang-L and Huh-7 cells. In addition, the protein levels of Alix, which is an inhibitor of paraptosis (23), were reduced by DOX/PDTC co-treatment, but not by either single treatment, in both Bcl-xL-Chang-L and Huh-7 cells (Figure 4E). Taken together, these results indicate that PDTC overcomes Bcl-xL-mediated resistance to DOX via the induction of paraptosis. Figure 4. View largeDownload slide Combination of DOX and PDTC induces paraptosis in Bcl-xL-Chang-L cells. (A) Electron microscopy was performed in Bcl-xL-Chang-L cells treated with 1 μg/ml DOX alone or 1 μg/ml DOX plus 10 μM PDTC for the indicated time points. Arrows indicate dilated mitochondria and arrowheads indicate dilated ER in cells treated with DOX/PDTC. Bar, 2 μm. (B) Bcl-xL-Chang-L cells or Huh-7 were treated with 1 μg/ml DOX plus 10 μM PDTC or 5 μg/ml DOX plus 10 μM PDTC for 48 h, respectively, and immunocytochemistry of SDHA and PDI was performed. Representative phase-contrast and fluorescence microscopic images of cells are shown. Bar, 10 μm. (C) Bcl-xL-Chang-L cells were pretreated with 2 μM CHX and further treated with 1 μg/ml DOX plus 10 μM PDTC for 48 h. Huh-7 cells were pretreated with 1 μM CHX and further treated with 5 μg/ml DOX plus 10 μM PDTC for 48 h. Cellular morphologies were observed by phase-contrast microscopy. Bar, 20 μm. (D) Cells were pretreated or not with CHX at the indicated concentrations and further treated with 1 μg/ml DOX plus 10 μM PDTC or 5 μg/ml DOX plus 10 μM PDTC for 72 h, respectively. Cellular viability was assessed using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus DOX-treated cells, **P < 0.05, **P < 0.001 versus DOX/PDTC-treated cells. (E) Bcl-xL-Chang-L cells were treated with 1 μg/ml DOX and/or 10 μM PDTC for 72 h. Huh-7 cells were treated with 5 μg/ml DOX and/or 10 μM PDTC for 48 h. Western blotting of Alix and α-tubulin was performed. Figure 4. View largeDownload slide Combination of DOX and PDTC induces paraptosis in Bcl-xL-Chang-L cells. (A) Electron microscopy was performed in Bcl-xL-Chang-L cells treated with 1 μg/ml DOX alone or 1 μg/ml DOX plus 10 μM PDTC for the indicated time points. Arrows indicate dilated mitochondria and arrowheads indicate dilated ER in cells treated with DOX/PDTC. Bar, 2 μm. (B) Bcl-xL-Chang-L cells or Huh-7 were treated with 1 μg/ml DOX plus 10 μM PDTC or 5 μg/ml DOX plus 10 μM PDTC for 48 h, respectively, and immunocytochemistry of SDHA and PDI was performed. Representative phase-contrast and fluorescence microscopic images of cells are shown. Bar, 10 μm. (C) Bcl-xL-Chang-L cells were pretreated with 2 μM CHX and further treated with 1 μg/ml DOX plus 10 μM PDTC for 48 h. Huh-7 cells were pretreated with 1 μM CHX and further treated with 5 μg/ml DOX plus 10 μM PDTC for 48 h. Cellular morphologies were observed by phase-contrast microscopy. Bar, 20 μm. (D) Cells were pretreated or not with CHX at the indicated concentrations and further treated with 1 μg/ml DOX plus 10 μM PDTC or 5 μg/ml DOX plus 10 μM PDTC for 72 h, respectively. Cellular viability was assessed using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus DOX-treated cells, **P < 0.05, **P < 0.001 versus DOX/PDTC-treated cells. (E) Bcl-xL-Chang-L cells were treated with 1 μg/ml DOX and/or 10 μM PDTC for 72 h. Huh-7 cells were treated with 5 μg/ml DOX and/or 10 μM PDTC for 48 h. Western blotting of Alix and α-tubulin was performed. PDTC may act as an antioxidant in rescuing DOX-induced apoptosis but as a prooxidant in reversing Bcl-xL-mediated DOX resistance Next, we investigated the underlying mechanism through which PDTC demonstrates pro-survival or pro-death effects in cells treated with DOX, depending on their Bcl-xL expression. ROS generation has been implicated in the apoptosis-inducing effect of DOX (24), and PDTC was previously shown to act as an antioxidant (25). Therefore, we first examined whether PDTC modulates DOX-induced ROS generation in Chang-L cells. FACS analysis using H2DCF-DA showed that following treatment with 1 μg/ml DOX, ROS levels exhibited a biphasic pattern: a marked but transient increase was seen at 6 h, and a second increase was observed at 24 h (Figure 5A). While treatment with PDTC alone did not generate ROS (data not shown), PDTC co-treatment markedly reduced DOX-induced ROS generation both in the duration and extent. While DOX-induced ROS generation was inhibited in Bcl-xL-Chang-L cells, ROS levels were markedly increased at a late phase of DOX/PDTC treatment in these cells. Together, these results indicate that PDTC co-treatment attenuates DOX-induced ROS generation in Chang-L cells, but recovers it in Bcl-xL-Chang-L cells. Pretreatment with various antioxidants, including NAC, GSH, GEE (the cell permeable ethyl ester form of GSH), Trolox, BHA or ascorbic acid commonly alleviated DOX-induced cell death in Chang-L cells (Figure 5B), similar to the effect of PDTC. These results suggest that PDTC may rescue DOX-induced apoptosis in Chang-L cells via its antioxidant activity. The increased ROS levels in Bcl-xL-Chang-L cells treated with DOX/PDTC suggest that PDTC may act as a prooxidant in reversing Bcl-xL-mediated DOX resistance. Thus, we next examined whether the cell death induced by DOX/PDTC in Bcl-xL-Chang-L cells could be inhibited by various antioxidants. Interestingly, the cell death (Figure 5C) and vacuolation (Figure 5D) induced by DOX/PDTC in these cells were significantly inhibited by pretreatment with thiol antioxidants, including GSH, GEE or NAC, but not by other non-thiol ROS scavengers, including Trolox, BHA or ascorbic acid. Immunocytochemical analysis of SDHA and PDI showed that GSH pretreatment effectively blocked the dilation of mitochondria and the ER induced by DOX/PDTC (Figure 5E). Similarly, the cell death as well as vacuolation induced by DOX/PDTC was also very effectively blocked by NAC pretreatment in Huh-7 cells (Supplementary Figure 4, available at Carcinogenesis Online). These results suggest the possibility that thiol-mediated regulation, rather than ROS generation, may be important for DOX/PDTC-induced paraptosis. Figure 5. View largeDownload slide PDTC co-treatment attenuates DOX-induced ROS generation in Chang-L cells, but recovers it in Bcl-xL-Chang-L cells. (A) Cells were treated with 1 μg/ml DOX or 10 μM PDTC plus 1 μg/ml DOX for the indicated time points, incubated with 5 μM H2DCF-DA for 20 min, and FACS analysis was performed. H2DCF-DA fluorescence intensities (FI) in cells treated with DOX or DOX/PDTC were compared with that of untreated cells and denoted in the graph. (B) Chang-L cells were pretreated with various antioxidants at the indicated concentrations for 30 min and further treated with 1 μg/ml DOX for 16 h. Cellular viability was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus untreated cells, *P < 0.001 versus cells treated with DOX. (C) Bcl-xL-Chang-L cells were pretreated with various antioxidants at the indicated concentrations and further treated with 1 μg/ml DOX plus 10 μM PDTC. Cellular viability was measured at 72 h post-treatment using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus untreated cells, *P < 0.001 versus DOX/PDTC-treated cells. Similar results were obtained from three independent experiments. (D) Bcl-xL-Chang-L cells were pretreated with 5 mM NAC, 5 mM GSH, 5 mM GEE, 100 μM Trolox, 50 μM BHA or 250 μM ascorbic acid and further treated with 10 μM PDTC plus 1 μg/ml DOX for 48 h. Representative phase-contrast images of cells are also shown. Bar, 20 μm. (E) Bcl-xL-Chang-L cells were pretreated with 5 mM GSH and further treated with 1 μg/ml DOX plus 10 μM PDTC for 48 h and immunocytochemistry of SDHA and PDI was performed. Representative phase-contrast and fluorescence microscopic images of cells are shown. Bar, 10 μm. Figure 5. View largeDownload slide PDTC co-treatment attenuates DOX-induced ROS generation in Chang-L cells, but recovers it in Bcl-xL-Chang-L cells. (A) Cells were treated with 1 μg/ml DOX or 10 μM PDTC plus 1 μg/ml DOX for the indicated time points, incubated with 5 μM H2DCF-DA for 20 min, and FACS analysis was performed. H2DCF-DA fluorescence intensities (FI) in cells treated with DOX or DOX/PDTC were compared with that of untreated cells and denoted in the graph. (B) Chang-L cells were pretreated with various antioxidants at the indicated concentrations for 30 min and further treated with 1 μg/ml DOX for 16 h. Cellular viability was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus untreated cells, *P < 0.001 versus cells treated with DOX. (C) Bcl-xL-Chang-L cells were pretreated with various antioxidants at the indicated concentrations and further treated with 1 μg/ml DOX plus 10 μM PDTC. Cellular viability was measured at 72 h post-treatment using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus untreated cells, *P < 0.001 versus DOX/PDTC-treated cells. Similar results were obtained from three independent experiments. (D) Bcl-xL-Chang-L cells were pretreated with 5 mM NAC, 5 mM GSH, 5 mM GEE, 100 μM Trolox, 50 μM BHA or 250 μM ascorbic acid and further treated with 10 μM PDTC plus 1 μg/ml DOX for 48 h. Representative phase-contrast images of cells are also shown. Bar, 20 μm. (E) Bcl-xL-Chang-L cells were pretreated with 5 mM GSH and further treated with 1 μg/ml DOX plus 10 μM PDTC for 48 h and immunocytochemistry of SDHA and PDI was performed. Representative phase-contrast and fluorescence microscopic images of cells are shown. Bar, 10 μm. Disruption of thiol homeostasis may critically contribute to DOX/PDTC-induced paraptosis Measurement of GSH levels revealed that PDTC alone had no effect, DOX alone slightly reduced these levels and DOX/PDTC progressively and markedly reduced GSH levels (Figure 6A). In contrast to the effects of NAC or GSH (Figure 5C and 5D), pretreatment with buthionine sulphoximine (BSO, which inhibits GSH synthesis), diethyl maleate (DEM, which depletes GSH) or carmustine (which inhibits glutathione reductase) dose-dependently enhanced the cell death (Figure 6B) and accelerated vacuolation leading to cell death (Figure 6C) induced by DOX/PDTC. Figure 6. View largeDownload slide GSH depletion may be critical for the paraptosis induced by DOX/PDTC. (A) Bcl-xL-Chang-L cells were treated with 1 μg/ml DOX and/or 10 μM PDTC for the indicated time points and GSH assay was performed as described in Materials and Methods. (B) Bcl-xL-Chang-L cells were pretreated with GSH, BSO, DEM or carmustine for 30 min at the indicated concentrations and further treated with 1 μg/ml DOX plus 10 μM PDTC for 72 h. Cellular viability was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus DOX-treated cells, *P < 0.001 versus cells treated with DOX/PDTC. Similar results were obtained from three independent experiments. (C) Bcl-xL-Chang-L cells were pretreated with 3 mM BSO, 80 μM DEM or 80 μg/ml carmustine for 30 min and further treated with 1 μg/ml DOX plus 10 μM PDTC for 24 or 48 h. Representative phase-contrast images of cells are also shown. Bar, 20 μm. (D–F) Bcl-xL-Chang-L cells were transfected with the scrambled negative control RNA, two different GCLC siRNAs, or GSR siRNAs, and incubated for 24 h. (D) Knockdown of GCLC and GSR was confirmed by Western blotting. (E) Transfected Bcl-xL-Chang-L cells were treated with 1 μg/ml DOX plus 10 μM PDTC for 72 h. Cellular viability was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus cells treated with DOX, *P < 0.001 versus cells treated with DOX/PDTC. Similar results were obtained from three independent experiments. (F) Transfected Bcl-xL-Chang-L cells were treated with1 μg/ml DOX plus 10 μM PDTC for 24 or 48 h. Representative phase-contrast images of cells are shown. Bar, 20 μm. (G) Bcl-xL-Chang-L cells were incubated with 1 μg/ml DOX and 10 μM PDTC for 48 h, or treated with DOX/PDTC together with 3 mM BSO, 5 mM GSH or 5 mM GEE for 48 h. Cells were stained with SSH-Mito, and images were acquired using 740 nm excitation and fluorescent emission windows of 425–475 nm (blue) and 525–575 nm (yellow). Bar, 25 μm. Representative images from replicate experiments (n = 5) and pseudocolored ratiometric TPM images (Fyellow/Fblue) are shown. (H) Hypothetical model for different cellular fates in response to DOX or DOX/PDTC depending on Bcl-xL expression. DOX-induced apoptosis is blocked by Bcl-xL overexpression. Co-treatment with PDTC attenuates DOX-induced apoptosis via antioxidant activity. In contrast, co-treatment with PDTC overcomes Bcl-xL-mediated DOX resistance via induction of paraptosis. In this process, disruption of thiol homeostasis by GSH depletion may be critically involved. Figure 6. View largeDownload slide GSH depletion may be critical for the paraptosis induced by DOX/PDTC. (A) Bcl-xL-Chang-L cells were treated with 1 μg/ml DOX and/or 10 μM PDTC for the indicated time points and GSH assay was performed as described in Materials and Methods. (B) Bcl-xL-Chang-L cells were pretreated with GSH, BSO, DEM or carmustine for 30 min at the indicated concentrations and further treated with 1 μg/ml DOX plus 10 μM PDTC for 72 h. Cellular viability was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus DOX-treated cells, *P < 0.001 versus cells treated with DOX/PDTC. Similar results were obtained from three independent experiments. (C) Bcl-xL-Chang-L cells were pretreated with 3 mM BSO, 80 μM DEM or 80 μg/ml carmustine for 30 min and further treated with 1 μg/ml DOX plus 10 μM PDTC for 24 or 48 h. Representative phase-contrast images of cells are also shown. Bar, 20 μm. (D–F) Bcl-xL-Chang-L cells were transfected with the scrambled negative control RNA, two different GCLC siRNAs, or GSR siRNAs, and incubated for 24 h. (D) Knockdown of GCLC and GSR was confirmed by Western blotting. (E) Transfected Bcl-xL-Chang-L cells were treated with 1 μg/ml DOX plus 10 μM PDTC for 72 h. Cellular viability was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus cells treated with DOX, *P < 0.001 versus cells treated with DOX/PDTC. Similar results were obtained from three independent experiments. (F) Transfected Bcl-xL-Chang-L cells were treated with1 μg/ml DOX plus 10 μM PDTC for 24 or 48 h. Representative phase-contrast images of cells are shown. Bar, 20 μm. (G) Bcl-xL-Chang-L cells were incubated with 1 μg/ml DOX and 10 μM PDTC for 48 h, or treated with DOX/PDTC together with 3 mM BSO, 5 mM GSH or 5 mM GEE for 48 h. Cells were stained with SSH-Mito, and images were acquired using 740 nm excitation and fluorescent emission windows of 425–475 nm (blue) and 525–575 nm (yellow). Bar, 25 μm. Representative images from replicate experiments (n = 5) and pseudocolored ratiometric TPM images (Fyellow/Fblue) are shown. (H) Hypothetical model for different cellular fates in response to DOX or DOX/PDTC depending on Bcl-xL expression. DOX-induced apoptosis is blocked by Bcl-xL overexpression. Co-treatment with PDTC attenuates DOX-induced apoptosis via antioxidant activity. In contrast, co-treatment with PDTC overcomes Bcl-xL-mediated DOX resistance via induction of paraptosis. In this process, disruption of thiol homeostasis by GSH depletion may be critically involved. To confirm how chemical depletors of GSH affect the cellular responses to DOX/PDTC, we knocked down genes related to glutathione metabolism, including GCLC (Glutamate-cysteine ligase catalytic subunit) and GSR (glutathione-disulfide reductase). GCLC is a target of BSO and is an essential subunit of γGCS (γ-glutamate-cysteine ligase) (26). The γGCS-mediated synthesis of the γ-glutamylcysteine from L-glutamate and cysteine is a rate-limiting step in glutathione synthesis (27). GSR is a target of carmustine and is responsible for reducing oxidized glutathione (GSSG) to reduced glutathione (GSH) (28). Transfection of Bcl-xL-Chang cells with two siRNAs each against GCLC or GSR potentiated DOX/PDTC-induced cell death and accelerated vacuolation (Figure 6D–F). These results suggest that GSH depletion may be critical for the paraptosis induced by DOX/PDTC treatment in Bcl-xL-overexpressing cells. We further investigated whether DOX/PDTC affects the mitochondrial thiol content employing a ratiometric two-photon probe (SSH-Mito) (29), which shows a marked blue-to-yellow emission color change in response to mitochondrial thiols (RSH), including GSH and cysteine, when visualized under two-photon microscopy (TPM). Upon TP excitation at 740 nm, the ratio image of SSH-Mito-labeled Bcl-xL-Chang-L cells constructed from two collection windows gave an average emission ratio of 1.12 (Figure 6G and Supplementary Figure 5, available at Carcinogenesis Online). The Fyellow/Fblue ratio decreased to 0.64 in Bcl-xL-Chang-L cells treated with DOX/PDTC for 48 h, and it was further decreased to 0.48 by addition of BSO. In contrast, pretreatment with GSH or GEE effectively recovered the value to the original level seen in untreated cells. These results suggest DOX/PDTC treatment effectively reduces the mitochondrial thiol content. Collectively, our results suggest that the disruption of thiol homeostasis, including mitochondrial thiol homeostasis, may be critical for the paraptosis induced by DOX/PDTC in Bcl-xL-overexpressing cells. In summary, our results show that DOX mono-treatment and DOX/PDTC co-treatment induce different cellular fates depending on the expression of Bcl-xL (Figure 6H). Bcl-xL overexpression confers resistance to DOX-induced apoptosis, while PDTC attenuates DOX-induced apoptosis in cells that do not highly express Bcl-xL, but induces paraptosis in Bcl-xL-overexpressing cells treated with DOX. Therefore, PDTC co-treatment may overcome DOX resistance in Bcl-xL-expressing cells (and thus possibly in resistant hepatoma cells), whereas it attenuates the cytotoxicity of DOX in the absence of Bcl-xL expression (such as found in normal hepatocytes). Discussion Acquired resistance is a major obstacle for successful cancer treatment, and we urgently need novel drugs that exhibit improved efficacy against tumor cells with less toxicity toward normal cells. The anthracycline, DOX, is one of the most effective drugs for treating solid tumors (30), including unresectable HCC (31), however, high plasma concentrations of DOX tend to cause severe toxicity to normal tissues, including cardiotoxicity (6,31), nephrotoxicity (7) and hepatotoxicity (5), limiting the administration of high doses of this agent. DOX is thought to act through DNA intercalation/binding, inhibition of topoisomerase II, free radical generation, and/or cell membrane damage (32,33). DOX induces apoptosis by disrupting mitochondrial membrane potential and activating caspases (34) via a pathway that is believed to be critically controlled by ROS (33) and GSH (35). Bcl-xL is a well-known anti-apoptotic protein that acts by preventing the cytotoxic-stimulus-induced release of mitochondrial cytochrome c into cytosol (36) and reducing proapoptotic-stimulus-induced mitochondrial dysfunction and ROS hyperproduction (37). In HCC, Bcl-xL is overexpressed in cancerous specimens from two-thirds of patients, and Bcl-xL expression is significantly correlated with overall and disease-free survival (8). Since Bcl-xL overexpression is known to contribute to cancer development and therapeutic resistance in many types of malignant tumors (38,39), we need to identify sensitizers that effectively overcome the Bcl-xL-mediated resistance of cancer cells to anti-cancer drugs. In tumors that are resistant to proapoptotic anti-cancer agents, strategies to induce an alternative cell death mode could prove helpful. PDTC is a pyrrolidine derivative of dithiocarbamate (11). It is a functionally versatile molecule that can confer numerous effects (e.g., NF-κB inhibition (11), metal chelation (40), antioxidant activity (12,25) and prooxidant activity (41)) depending on the cellular model and microenvironment. PDTC was previously shown to attenuate DOX-induced cardiotoxicity (13,42,43) and nephrotoxicity (7). The effect of PDTC on DOX-treated hepatocytes has not been studied, but micellar nanoparticle-mediated co-delivery of DOX and PDTC was shown to effectively overcome DOX resistance in HCC cells (14). The clinical use of PDTC has not yet been investigated in a published report, but the existing literature suggests that this agent could be a clinically important adjuvant drug for cancer therapy. In the present study, we investigated whether administration of PDTC could improve DOX treatment by protecting hepatocytes while increasing the therapeutic efficacy in resistant HCC cells. We employed Chang-L cells and Chang-L sublines stably overexpressing Bcl-xL (Bcl-xL-Chang-L cells) as a model system to mimic human hepatocytes (which are sensitive to DOX) and malignant HCC cells (which are resistant to DOX), respectively. We found that co-treatment with PDTC attenuates DOX-induced apoptosis in Chang-L liver cells and human hepatocytes (Figure 2), but overcomes DOX resistance in Bcl-xL-overexpressing Chang-L cells and HCC cells by inducing paraptosis (a cell death mode that is accompanied by dilation of the ER and/or mitochondria) (Figures 3 and 4). Moreover, the anti-cancer effect and vacuolation by DOX/PDTC in vivo was also confirmed in mice bearing Huh-7-derived xenograft tumors (Figure 1C–E). Therefore, our results suggest that PDTC may protect normal non-Bcl-xL-expressing cells (i.e. hepatocytes) against DOX, while sensitizing Bcl-xL-overexpressing cancer cells (i.e. resistant HCC cells) to this agent. It is very intriguing to consider how PDTC demonstrates opposing activities towards normal and cancer cells exposed to DOX. Previous studies showed that PDTC attenuated DOX-induced myocardial apoptosis (13,24,42,43) and acute myocardial injury in rats by inhibiting NF-κB (43). When we assessed NF-κB activity by p65 phosphorylation at serine 536 (44), DOX increased NF-κB activity in Chang-L cells and this DOX-induced NF-κB activation was considerably attenuated by PDTC and by Bcl-xL overexpression (Supplementary Figure 6A, available at Carcinogenesis Online). However, DOX-induced cell death was unaffected by other NF-κB inhibitors (Supplementary Figure 6B, available at Carcinogenesis Online). In contrast, DOX-induced ROS generation and subsequent apoptosis in Chang-L cells were effectively attenuated by various antioxidants (Figure 5A and B) and by Bcl-xL overexpression (Figures 3B, 3C and 5A). These results suggest that the antioxidant activity of PDTC, rather than its NF-κB-inhibiting activity, may be more important for the attenuation of DOX-induced cytotoxicity in these cells. However, in Bcl-xL-overexpressing Chang-L cells, perturbation of thiol homeostasis, rather than ROS generation, appears to critically contribute to the paraptosis induced by DOX/PDTC, as supported by the following findings: (a) The intracellular content of GSH levels were reduced and ROS generation was followed in DOX/PDTC-induced paraptosis (Figures 5A and 6A). (b) Thiol-containing antioxidants with reducing activity (e.g. GSH, GEE and NAC) abrogated the vacuolation and cell death induced by DOX/PDTC in Bcl-xL-Chang-L cells, whereas non-thiol antioxidants (e.g., BHA, Trolox and ascorbic acid) did not (Figure 5C–E). (c) Depletion of GSH employing either pharmacological (using BSO, DEM or carmustine) or genetic (knockdown of GCLC or GSR) tools accelerated the vacuolation and subsequent cell death induced by DOX/PDTC (Figure 6B–F). GSH not only acts as a major antioxidant by protecting cells against the damaging effects of free radicals and ROS; it also functions in reducing the disulfide linkages of proteins (45,46). NAC, an acetylated variant of L-cysteine, possesses both direct (i.e., via oxidizable sulfhydryl groups) and indirect (i.e. as a substrate for the biosynthesis of GSH) antioxidant activities (47). Similar to our results in Bcl-xL-Chang-L cells treated with DOX/PDTC, Kar et al. (18). found that the cytoplasmic vacuolation and cell death induced by 15d-PGJ2 was effectively blocked by various thiol-antioxidants, but not by non-thiol antioxidants. The authors argued that the effects of 15d-PGJ2 may be mediated not through ROS generation, but through the ability of this agent to covalently modify free sulfhydryl groups on proteins. Therefore, we speculate that the perturbation of thiol homeostasis in cells co-treated with PDTC may also critically contribute to enhancement of cell death in Bcl-xL-expressing cells treated with DOX. Although additional work is needed to clarify how PDTC switches the cellular fates of DOX-treated cells depending on their expression of Bcl-xL, this ability may rely on the versatility of PDTC in exhibiting both anti- and prooxidant activities (48). PDTC induces differential effects on the redox equilibrium according to: (a) its ability to decrease single-electron radical species, such as superoxide anion (O2-·) (25), and hydroxyl radical (HO·) (49) via scavenging (an antioxidant effect); and (b) its capacity to oxidize GSH and related thiol compounds (a prooxidant effect) (49), and thus modulate glutathione recycling. PDTC may undergo oxidation by ROS, which are derived from DOX treatment, generating dithiocarbamate thiyl radical and further dimerization of the radicals to thiuram disulfide (41). In Bcl-xL-overexpressing Chang-L cells, prolonged incubation with DOX/PDTC may enable thiuram disulfides to oxidize glutathione, leading to the formation of GSSG and eventual cell death. In this case, PDTC would act as a prooxidant and thiol group modulator, as the up-regulated GSSG may promote the formation of disulfide bonds in cellular polypeptides through the oxidation of cysteinyl thiols (50,51), resulting in accumulation of misfolded polypeptides. Future work is needed to clarify whether disruption of thiol homeostasis directly triggers the accumulation of misfolded proteins, leading to cellular vacuolation and paraptotic cell death in Bcl-xL-overexpressing cells treated with DOX/PDTC. In our study, Bcl-xL overexpression in Chang-L cells very effectively blocked DOX-induced apoptosis and markedly inhibited ROS generation. These results suggest that Bcl-xL may act, at least indirectly, as an antioxidant. The anti-apoptotic roles of Bcl-2 and Bcl-xL have been widely linked to the GSH content. The anti-apoptotic effect of Bcl-xL has been attributed to its ability to regulate GSH homeostasis by preventing GSH loss (52). Bcl-2 has been shown to regulate the GSH content in different cellular compartments (53), and Bcl-2 overexpression is known to increase GSH levels and inhibit the mitochondria-induced cell death elicited by GSH-depleting reagents (54). Consistent with this, depletion of intracellular GSH has been reported to overcome the Bcl-2-mediated resistance to apoptosis (55). When we assessed the mitochondrial GSH levels using the SSH-Mito probe in Chang-L and Bcl-xL-Chang-L cells, we found that the mitochondrial GSH levels were significantly higher (1.6- fold) in the presence of Bcl-xL (Supplementary Figure 7, available at Carcinogenesis Online). Our results therefore suggest that Bcl-xL-induced increase in mitochondrial GSH may help enhance the ability of cells to scavenge DOX-induced ROS and block apoptosis. In contrast, prolonged exposure of Bcl-xL-Chang-L cells to DOX/PDTC progressively reduced GSH levels, ultimately leading to paraptosis. We also found that Bcl-xL shares with Bcl-2 in the ability to regulate apoptosis and paraptosis in response to DOX or DOX/PDTC. DOX-induced apoptosis was effectively blocked by Bcl-2 overexpression, but not by Mcl-1 overexpression (Supplementary Figure 8, available at Carcinogenesis Online). In addition, PDTC co-treatment overcame Bcl-2-mediated resistance to DOX via a cell death accompanied by severe vacuolation, whereas it slightly protected Mcl-1-overexpressing cells from DOX-induced cytotoxicity (Supplementary Figure 9, available at Carcinogenesis Online). Furthermore, we found that PDTC co-treatment attenuated daunorubicin- or etoposide-induced apoptosis (Supplementary Figure 10A and B, available at Carcinogenesis Online). While Bcl-xL overexpression effectively blocked daunorubicin- or etoposide-induced cell death, PDTC co-treatment overcame this Bcl-xL-mediated resistance (Supplementary Figure 10C and D, available at Carcinogenesis Online). These results suggest that PDTC may play a cytoprotective role in the absence of Bcl-xL and a chemo-sensitizing role in the presence of Bcl-xL when combined with anti-cancer drugs that share the action mechanism of DOX (e.g. ROS generation and DNA damage). In summary, we herein show that PDTC co-treatment attenuates DOX-mediated toxicity in normal (non-Bcl-xL-expressing) Chang liver cells by inhibiting ROS-mediated apoptosis, but overcomes Bcl-xL-mediated DOX resistance in Bcl-xL-overexpressing (cancer-like) cells by inducing paraptosis, possibly through the disruption of thiol homeostasis. These novel findings suggest that DOX/PDTC co-treatment may provide a safe and effective therapeutic strategy against Bcl-xL-overexpressing malignant HCC cells, which are resistant many chemotherapeutic agents. Funding This work was supported by the National Research Foundation of Korea (NRF) grants (Mid-Career Researcher Program No. 2015R1A2A2A1006966 and 2011-0030043(SRC)) funded by the Korean government and a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare (HI14C2230). Conflict of Interest Statement None declared. Supplementary material Supplementary data are available at Carcinogenesis online. Acknowledgements We thank Dr. Eun Hee Kim (UNIST, Korea) for the helpful discussion. References 1. El-Serag, H.B. ( 2012) Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology , 142, 1264– 1273.e1. Google Scholar CrossRef Search ADS PubMed  2. Hung, H. ( 2005) Treatment modalities for hepatocellular carcinoma. Curr. Cancer Drug Targets , 5, 131– 138. Google Scholar CrossRef Search ADS PubMed  3. Takayasu, K.et al.  ; Liver Cancer Study Group of Japan. 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FASEB J ., 16, 1263– 1265. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Carcinogenesis Oxford University Press

Pyrrolidine dithiocarbamate reverses Bcl-xL-mediated apoptotic resistance to doxorubicin by inducing paraptosis

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10.1093/carcin/bgy003
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Abstract

Abstract Elevated Bcl-xL expression in cancer cells contributes to doxorubicin (DOX) resistance, leading to failure in chemotherapy. In addition, the clinical use of high-dose doxorubicin (DOX) in cancer therapy has been limited by issues with cardiotoxicity and hepatotoxicity. Here, we show that co-treatment with pyrrolidine dithiocarbamate (PDTC) attenuates DOX-induced apoptosis in Chang-L liver cells and human hepatocytes, but overcomes DOX resistance in Bcl-xL-overexpressing Chang-L cells and several hepatocellular carcinoma (HCC) cell lines with high Bcl-xL expression. Additionally, combined treatment with DOX and PDTC markedly retarded tumor growth in a Huh-7 HCC cell xenograft tumor model, compared to either mono-treatment. These results suggest that DOX/PDTC co-treatment may provide a safe and effective therapeutic strategy against malignant hepatoma cells with Bcl-xL-mediated apoptotic defects. We also found that induction of paraptosis, a cell death mode that is accompanied by dilation of the endoplasmic reticulum and mitochondria, is involved in this anti-cancer effect of DOX/PDTC. The intracellular glutathione levels were reduced in Bcl-xL-overexpressing Chang-L cells treated with DOX/PDTC, and DOX/PDTC-induced paraptosis was effectively blocked by pretreatment with thiol-antioxidants, but not by non-thiol antioxidants. Collectively, our results suggest that disruption of thiol homeostasis may critically contribute to DOX/PDTC-induced paraptosis in Bcl-xL-overexpressing cells. Introduction Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the third leading cause of cancer-related deaths worldwide (1). Surgical resection has been considered the optimal treatment approach for HCC, but relatively few patients qualify for surgery and the recurrence rate is high (2). Transcatheter arterial chemoembolization has become the mainstay management strategy for patients with unresectable HCC (3) and DOX is the most widely used single chemotherapeutic agent in chemoembolization (4). However, the clinical usefulness of DOX is often limited by its cardiotoxicity, nephrotoxicity and hepatotoxicity (5–7). Bcl-xL is a significant prognostic factor for disease progression in human HCC (8) and overexpression of Bcl-xL in HCC cells contributes to their therapeutic resistance against anti-cancer drugs, including DOX (9). Thus, we need to identify a therapeutic strategy for HCC that can overcome Bcl-xL-mediated apoptotic resistance to DOX and minimize the toxic effects of this agent on normal cells. For treating apoptosis-resistant tumors, the induction of non-apoptotic cell death could be an option. Pyrrolidine dithiocarbamate (PDTC) is a low-molecular weight thiol compound (10) that has potent antioxidant and NF-κB inhibitor activities (11,12). It was found to show opposing effects in a cell-context-specific manner, protecting rats against DOX-induced myocardial apoptosis (13), but sensitizing multidrug resistant HepG-2 cells to DOX (14). These results suggested that the combination of DOX and PDTC may act against liver cancer, but it was unclear why or how PDTC could demonstrate pro-survival or pro-death effects in DOX-treated cells under different cellular contexts. In this study, we investigated whether PDTC could exhibit opposing effects on normal liver cells and hepatoma cells treated with DOX and, if so, whether this differential effect was related to the expression of Bcl-xL. Our results showed that co-treatment with PDTC attenuated DOX-mediated apoptosis in Chang-L normal liver cells due to the antioxidant activity of PDTC. While DOX-induced apoptosis in Chang-L cells was effectively blocked by Bcl-xL overexpression, PDTC co-treatment reverses this acquired Bcl-xL-mediated DOX resistance by inducing paraptosis. Paraptosis (para = next to or beside, and apoptosis) is a cell death mode, which is characterized by dilation of the endoplasmic reticulum (ER) and/or mitochondria and the lack of characteristic apoptotic features (e.g. apoptotic body formation, chromatin condensation, DNA fragmentation and caspase dependency) (15). Recent work has shown that paraptosis is associated with the generation of reactive oxygen species (ROS) (16,17) and perturbation of cellular proteostasis via proteasome inhibition (16,17) and disruption of sulfhydryl homeostasis (18,19). We show here that in the presence of Bcl-xL, depletion of glutathione (GSH), including mitochondrial GSH, critically contributes to the ability of DOX plus PDTC (DOX/PDTC) to induce paraptosis. Our results indicate that a combined regimen of DOX and PDTC may provide an attractive strategy for effectively treating hepatomas while sparing normal cells from DOX toxicity. Materials and methods Chemicals and antibodies All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless indicated otherwise. Cycloheximide (CHX) was obtained from Calbiochem (San Diego, CA). The following antibodies were used: caspase-3 and PDI (Stressgen, Ann Arbor, MI); Bcl-xL (Santa Cruz Biotechnology, Santa Cruz, CA); α-tubulin, phospho-p65 (S536), total p65, Alix (Cell signaling, Beverly, MA); SDHA and Bcl-2 (Invitrogen, Carsbad, CA); GCLC and GSR (Novus Biologicals, Littleton, CO). Horseradish peroxidase-conjugated anti-rabbit IgG, Horseradish peroxidase-conjugated anti-mouse IgG, Alexa 488-conjugated anti-mouse IgG and Alexa 594-conjugated anti-rabbit IgG (Molecular Probes, Carlsbad, CA). Cell culture Chang cells were obtained from American Tissue Culture Collections (ATCC, Manassas, VA) in 2010 and Chang-L cell clone, denoted as Chang-L (20,21), which has higher hepatic characteristics (albumin production and liver-specific carbamoyl phosphate synthase-1 expression) were isolated by single cell dilution and expansion, were used for this study. Chang-L clones were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum. Huh-7 cells were obtained from JCRB Cell Bank (Japanese Collection of Research Bioresources Cell Bank) in 2015. Chang-L human liver cells and Huh-7 human hepatoma cells were cultured in Dulbecco’s modified Eagle’s medium medium supplemented with 10% fetal bovine serum. Human hepatocytes were purchased from Cambrex (East Rutherford, NJ) in 2015 and cultured in human hepatocyte basal medium supplemented with human hepatocyte medium (HHM) SingleQuots. SNU-182, SNU-368 and SNU-449 human hepatoma cells were obtained from the Korean Cell Line Bank in 2015 and were grown in RPMI 1640 supplemented with 10% fetal bovine serum. All cell lines were authenticated and characterized by the supplier and routinely tested by PCR for mycoplasma contamination by using the following primers: Myco_F: 5′-ACA CCA TGG GAG CTG GTA AT-3′, Myco_R: 5′-CTT CAT CGA CTT TCA GAC CCA AGG CA-3′. Establishment of the stable cell lines overexpressing the Bcl-xL Chang-L cells were transfected with the Flag-tag-Bcl-xL and stably transfected cells were selected using the fresh media containing puromycin 4 μg/ml for 3 weeks. Chang-L sublines stably transfected with an empty vector were used as a control. The overexpression of Bcl-xL in selected cell lines was confirmed by Western blot analysis using anti-Flag antibody. Measurement of cellular viability Cells (5 × 104 cells) were cultured in 24-well plates and treated as indicated. For measurement of cellular viability, 2 μM calcein-acetoxymethyl ester (calcein-AM), a green fluorescent indicator of the intracellular esterase activity of cells and 4 μM ethidium homodimer-1 (EthD-1), a red fluorescent indicator of membrane damaged (dead) cells, we added to each well, and the plates were incubated for 5 min in 5% CO2 at 37°C. Cells were then observed under a fluorescence microscope (Axiovert 200M; Carl Zeiss) equipped with Zeiss filter sets #46 and #64HE. Viable cells, corresponding to those that exclusively exhibited green fluorescence, were counted in five fields per well at 200× magnification. Only exclusively green cells were counted as live because bicolored (green and red cells) cannot be unambiguosly to live or dead groups. The percentage of viable cells (Viable %) calculated as green cells/(green + red + bicolored cells), was normalized to that of untreated control cells (100%). Cell cycle analysis Trypsinized and floating cells were pooled, washed with phosphate-buffered saline (PBS)-EDTA and fixed in 70% (v/v) ethanol. DNA contents were assessed by staining cells with propidium iodide and monitoring by FACSAriaTM III (BD Biosciences, San Jose, CA). The data was analyzed using FACSDiva software (BD Biosciences). Measurement of reactive ROS Cells were treated with 10 μM PDTC and/or 1 µg/ml DOX for the indicated time points, stained with 5 µM H2DCF-DA for 20 min at 37°C and subjected to flow cytometry. TUNEL assay Chang-L and Bcl-xL-Chang-L cells cultured on coverslips were treated with 1 μg/ml DOX and 1 μg/ml DOX plus 10 μM PDTC for the indicated time points and fixed with 4% paraformaldehyde (pH 7.4) for 10 min. TUNEL assay of the fragmented DNAs was performed as recommended by the manufacturer (Molecular Probes). Transmission electron microscopy Cells were prefixed in Karnovsky’s solution [1% paraformaldehyde, 2% glutaraldehyde, 2 mM calcium chloride, 0.1 M cacodylate buffer (pH 7.4)] for 2 h and washed with cacodylate buffer. Postfixing was carried out in 1% osmium tetroxide and 1.5% potassium ferrocyanide for 1 h. After dehydration with 50–100% alcohol, the cells were embedded in Poly/Bed 812 resin (Pelco, Redding, CA) and polymerized and observed under electron microscope (EM 902A, Zeiss). Immunocytochemistry After treatments, cells were fixed with acetone/methanol (1:1) for 5 min at –20°C and blocking in 5% bovine serum albumin in PBS for 30 min. Fixed cells were incubated overnight at 4°C with primary antibody [anti-SDHA (1:500, mouse, Invitrogen] and anti-PDI [1:500, rabbit, Stressgen)] diluted in PBS and then washed three times in PBS and incubated for 1 h at room temperature with anti-mouse or anti-rabbit Alexa Fluor 488 or 594 (1:500, Molecular Probes). Slides were mounted with ProLong Gold antifade mounting reagent (Molecular probes) and cell staining was visualized with a fluorescence microscope using Zeiss filter sets #46 and #64HE (excitation band pass, 598/25 nm; emission band pass, 647/70 nm). Glutathione assay Bcl-xL-Chang-L cells were treated with 1 µg/ml DOX and/or 10 μM PDTC for the indicated time points. The intracellular level of reduced GSH was determined by using a kit from Cayman Chemical (Ann Arbor, MI). Equal amounts of the proteins (150 μg/reaction) were used for the determination of GSH according to the manufacturer’s instructions. Similar results were obtained in three independent experiments. Two-photon fluorescence microscopy Two-photon fluorescence microscopy (TPM) images of probe-labeled cells were obtained with multiphoton microscopes (Leica TCS SP8 MP, Wetzlar, Germany) using 40× oil objective and a numerical aperture (NA) setting of 1.30. The TPM images were obtained by exciting the probes with a mode-locked titanium-sapphire laser source (Mai Tai HP; Spectra Physics, 80 MHz, 100 fs) set at wavelength 740 nm and an output power of 2490 mW, which corresponded to an average power of approximately 4.14 × 108 mW cm-2 in the focal plane. Live-cell imaging was performed using live-cell incubator systems (Chamlide IC; Live Cell Instrument). We obtained images in the 400–450 nm (Fblue) and 500–600 nm (Fyellow) ranges, and ratiometric image processing and analyses were carried out using the MetaMorph software. Small interfering RNAs The small interfering RNA (siRNA) duplexes used in this study were purchased from OriGene Technologies (Rockville, MD) and have the following sequences: GCLC#1 (Cat. No. SR301815A), UGGAUGGAGAGUAGAA UUUCGACCC; GCLC#2 (Cat. No. SR301815B), CGACUUGACGAUAGAUAAA GAGATC; GSR#2 (Cat. No. SR301982B), CCAGAAUACCAACGUCAAAGGCATC; GSR#3 (Cat. No. SR301982C), GGAAGAUUCCAAAUUAGAUUAUAAC. Bcl-xL-Chang-L cells plated in 24-well plates were transfected with the respective siRNA oligonucleotides (50 nM) using Lipofectamine RNAiMAX reagent (Invitrogen) according to the manufacturer’s instructions. After 24 h, knockdown of GCLC and GSR in transfected cells was confirmed by Western blotting. In vivo tumor growth inhibition and immunohistochemistry Huh-7 cells were used to produce a xenograft tumor model in male BALB/c nude mice (nu/nu, 5 weeks old, Japan SLC, Hamamatsu, Japan). A suspension of 1 × 107 cells in a 50 μl volume (saline) was subcutaneously injected into the right hind limb of mice. Tumors were grown for 3 weeks until average tumor volume reached 70–100 mm3. Mice were randomized into six groups (n = 4 per group) and mice were received twice-weekly intraperitoneal (i.p.) injections of either DOX (2 mg/kg), PDTC (25, 50 mg/kg), a combination of each agent, or saline (control) for 2 weeks. PDTC was injected 1 h prior to DOX injection. DOX and PDTC were dissolved in saline and D.W, respectively. Tumor size and body weights were measured three times a week and tumor volume was calculated using the formula [V = (L × W2) × 0.5, where V = volume, L = length and W = width]. All experiments were performed following the guidelines and regulations approved by the Institutional Animal Care and Use Committee of the Asan Institute for Life Science. On the 20th day, mice were sacrificed and the tumors were isolated, fixed in 4% paraformaldehyde and then embedded into paraffin. Sections of 5 μm were stained with H&E, assayed for Ki67 using anti-Ki67 antibody (1:100; Abcam) and the ABC peroxidase labeling procedure (Vector Laboratories, Inc., Burlingame, CA) for immunologic detection. The image on the tissue sections was observed and photographed with microscopy set (Olympus, Tokyo, Japan). Statistical analysis All data are presented as mean ± SEM or ± SD. To perform statistical analysis, GraphPad Prism (GraphPad Software Inc, Sandiego, CA) was used. Each experiment was repeated at least three times. Normality of data was assessed by Kolmogorov–Smirnov testes and equal variance using Bartlett’s test. For normal distribution, statistical differences were determined using an analysis of variance (ANOVA) followed by followed by Bonferroni multiple comparison test. If the data were not normally distributed, Kruskal–Wallis test was performed followed by Dunn’s test. P < 0.05 was considered statistically significant. Results PDTC reverses the resistance of HCC cells to DOX both in vitro and in vivo To investigate whether PDTC could sensitize various HCC cells to DOX, we treated HCC cell lines (Huh-7, SNU-368, SNU-449 and SNU-182) with different doses of DOX and/or PDTC and analyzed cell viability using calcein-AM and EthD-1 to detect viable and dead cells, respectively. Although these HCC cell lines varied in their sensitivities to DOX, treatment with sub-lethal doses of PDTC commonly and dose-dependently reduced the viabilities of these cells when combined with DOX (Figure 1A). Interestingly, the cell death induced by DOX/PDTC was commonly accompanied by extensive vacuolation in these HCC cells (Figure 1B). Next, we evaluated the anti-cancer effect of DOX/PDTC in vivo using mouse Huh-7 cell xenograft models. Mice received twice-weekly intraperitoneal (i.p.) injections of DOX (2 mg/kg), PDTC (25 or 50 mg/kg), a combination of the two agents, or saline (control) for 2 weeks. Treatment with DOX or PDTC alone had no notable effect on the growth of Huh-7 xenograft tumors, whereas DOX/PDTC inhibited tumor growth in a PDTC-dose-dependent manner (Figure 1C) without triggering any loss of body weight (Supplementary Figure 1, available at Carcinogenesis Online). Tumor growth was decreased by 37.4% in mice treated with 2 mg/kg DOX plus 25 mg/kg PDTC and by 49.2% in mice treated with 2 mg/kg DOX plus 50 mg/kg PDTC, as compared to the control group. The cell proliferation marker, Ki67, was also significantly reduced in DOX/PDTC-treated tumor (Figure 1D). Hematoxylin and eosin (H&E) staining showed the presence of severe cellular vacuolation in Huh-7 xenograft sections obtained from mice treated with DOX/PDTC (Figure 1E), similar to our findings in DOX/PDTC-treated Huh-7 cells in vitro (Figure 1B). Collectively, these results indicate that PDTC co-treatment may effectively overcome DOX resistance in malignant HCC cells via a form of cell death that is accompanied by vacuolation. Figure 1. View largeDownload slide Co-treatment with PDTC overcomes the DOX resistance in HCC cells. (A) Viability of cells treated with DOX and/or PDTC for 72 h was assessed using calcein-AM and EthD-1 as described in Materials and Methods. The percentage of live cells was normalized to that of untreated control cells (100%). Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. **P < 0.01, *P < 0.001 versus DOX-treated cells. (B) Cellular morphologies following treatment with DOX and/or PDTC for 48 h in Huh-7 (20 μM PDTC and/or 5 μg/ml DOX); SNU-182 (10 μM PDTC and/or 0.25 μg/ml DOX); SNU-368 (50 μM PDTC and/or 5 μg/ml DOX); SNU-449 (40 μM PDTC and/or 10 μg/ml DOX) cells. Bars, 20 μm. (C) The relative tumor volume of Huh-7 xenograft tumor after administrated with the indicated amount of DOX and/or PDTC, as described in Material and Methods. Tumor size was measured three times a week for 20 days and plotted for growth curve (left). Data represent the means ± SD. Kruskal–Wallis test was performed followed by Dunn’s test. **P < 0.05, **P < 0.01 versus DOX-treated mice. On the 20th day, all tumors were isolated and photographed (right). (D, E) Tumor tissues sections were prepared from the mice harboring Huh-7 xenograft tumors following treatment with control, 2 mg//kg DOX, 50 mg/kg PDTC or 2 mg//kg DOX plus 50 mg//kg PDTC. (D) Representative pictures of the immunohistochemical staining of Ki67 (left). Bar, 50 μm. Intensities of Ki67 staining were quantified and denoted as a graph (right). Data represent the means ± S.D. Kruskal-Wallis test was performed followed by Dunn’s test. *P < 0.05* versus DOX-treated mice. (E) The results of H&E staining. Bar, 50 μm. Figure 1. View largeDownload slide Co-treatment with PDTC overcomes the DOX resistance in HCC cells. (A) Viability of cells treated with DOX and/or PDTC for 72 h was assessed using calcein-AM and EthD-1 as described in Materials and Methods. The percentage of live cells was normalized to that of untreated control cells (100%). Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. **P < 0.01, *P < 0.001 versus DOX-treated cells. (B) Cellular morphologies following treatment with DOX and/or PDTC for 48 h in Huh-7 (20 μM PDTC and/or 5 μg/ml DOX); SNU-182 (10 μM PDTC and/or 0.25 μg/ml DOX); SNU-368 (50 μM PDTC and/or 5 μg/ml DOX); SNU-449 (40 μM PDTC and/or 10 μg/ml DOX) cells. Bars, 20 μm. (C) The relative tumor volume of Huh-7 xenograft tumor after administrated with the indicated amount of DOX and/or PDTC, as described in Material and Methods. Tumor size was measured three times a week for 20 days and plotted for growth curve (left). Data represent the means ± SD. Kruskal–Wallis test was performed followed by Dunn’s test. **P < 0.05, **P < 0.01 versus DOX-treated mice. On the 20th day, all tumors were isolated and photographed (right). (D, E) Tumor tissues sections were prepared from the mice harboring Huh-7 xenograft tumors following treatment with control, 2 mg//kg DOX, 50 mg/kg PDTC or 2 mg//kg DOX plus 50 mg//kg PDTC. (D) Representative pictures of the immunohistochemical staining of Ki67 (left). Bar, 50 μm. Intensities of Ki67 staining were quantified and denoted as a graph (right). Data represent the means ± S.D. Kruskal-Wallis test was performed followed by Dunn’s test. *P < 0.05* versus DOX-treated mice. (E) The results of H&E staining. Bar, 50 μm. PDTC attenuates DOX-induced apoptosis in Chang-L liver cells Next, to investigate the effect of DOX and/or PDTC in normal liver cells, we employed Chang-L liver cells. This Chang cell clone was isolated by single cell dilution and expansion, and selected for the distinct hepatic characteristics of albumin production and liver-specific carbamoyl synthetase-1 expression (20,21). Compared to the tested HCC cells (Figure 1A), these cells were sensitive to the cytotoxic effect of DOX, and treatment with 1 μg/ml DOX alone for 16 h reduced cell viability by ~50% (Figure 2A). While treatment with PDTC alone up to 10 μM did not affect the viability of Chang-L cells, PDTC pretreatment dose-dependently attenuated the cytotoxicity of DOX (Figure 2A). We found that 1 μg/ml DOX treatment induced apoptosis, as evidenced by the apoptotic morphologies, such as cellular shrinkage, blebbing and apoptotic body formation (Figure 2B), increased sub-G1 hypoploid cell population (Figure 2C), DNA fragmentation (as assessed by TUNEL assays) (Figure 2D), and proteolytic processing of caspase-3 (Figure 2E). In contrast, co-treatment with 10 μM PDTC attenuated all these apoptotic features induced by DOX (Figure 2B–E). In addition, the death of human hepatocytes treated with 1 μg/ml DOX was also significantly reduced by 10 μM PDTC (Figure 2F), suggesting that PDTC may alleviate DOX-induced hepatotoxicity. Taken together, these results suggest that PDTC may chemosensitize resistant HCC cells to DOX while chemoprotecting hepatocytes, and thus the combined regimen of DOX and PDTC could offer a safe and effective therapeutic strategy against malignant HCCs. Figure 2. View largeDownload slide PDTC pretreatment attenuates DOX-induced apoptosis in Chang-L liver cells. (A) Viability of Chang-L cells untreated or pretreated with PDTC and further treated with DOX for 16 h was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. *P < 0.001 versus cells treated with DOX. (B) The representative pictures from Chang-L cells treated with the indicated concentrations of DOX and/or PDTC for 16 h were taken under a phase-contrast microscope. Bar, 20 μm. (C, D) Chang-L cells were treated with 1 μg/ml DOX and/or 10 μM PDTC for 24 h. FACS analysis to measure DNA content (C) and TUNEL assay (D) were performed. (E) Following treatment of Chang-L cells with 1 μg/ml DOX alone or 1 μg/ml DOX plus 10 μM PDTC, Western blotting of Caspase-3 was performed. Western blotting of α-tubulin was served as a loading control. (F) Viability of human hepatocytes treated with 1 μg/ml DOX and/or 10 μM PDTC for 24 h was measured using calcein-AM and EthD-1. Data represent the means ± SD. Kruskal–Wallis test was performed followed by Dunn’s test. #P < 0.01 versus untreated cells, *P < 0.01 versus DOX-treated cells. Figure 2. View largeDownload slide PDTC pretreatment attenuates DOX-induced apoptosis in Chang-L liver cells. (A) Viability of Chang-L cells untreated or pretreated with PDTC and further treated with DOX for 16 h was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. *P < 0.001 versus cells treated with DOX. (B) The representative pictures from Chang-L cells treated with the indicated concentrations of DOX and/or PDTC for 16 h were taken under a phase-contrast microscope. Bar, 20 μm. (C, D) Chang-L cells were treated with 1 μg/ml DOX and/or 10 μM PDTC for 24 h. FACS analysis to measure DNA content (C) and TUNEL assay (D) were performed. (E) Following treatment of Chang-L cells with 1 μg/ml DOX alone or 1 μg/ml DOX plus 10 μM PDTC, Western blotting of Caspase-3 was performed. Western blotting of α-tubulin was served as a loading control. (F) Viability of human hepatocytes treated with 1 μg/ml DOX and/or 10 μM PDTC for 24 h was measured using calcein-AM and EthD-1. Data represent the means ± SD. Kruskal–Wallis test was performed followed by Dunn’s test. #P < 0.01 versus untreated cells, *P < 0.01 versus DOX-treated cells. PDTC overcomes Bcl-xL-mediated apoptotic resistance to DOX via non-apoptotic cell death accompanied by vacuolation It is intriguing to consider how, when combined with DOX, PDTC could enhance the death of resistant HCC cells but attenuate that of normal liver cells. What is the determining factor in the ability of DOX or DOX/PDTC to induce differential fates in HCC cells versus hepatocytes? As a candidate protein, we selected Bcl-xL, because Bcl-xL is over-expressed in two-thirds of human HCC (8), and this upregulation enables HCC cells to resist chemotherapeutic agents, including DOX (9,22). Overexpression of Bcl-xL was detected also in Huh-7, SNU-182, SNU-368 and SNU-449 HCC cells (Figure 3A). To test whether the HCC cellular responses to DOX or DOX/PDTC are reproduced in Chang-L cells by overexpression of Bcl-xL, we established Chang-L sublines stably expressing Flag-tagged Bcl-xL (Bcl-xL-Chang-L cells). We found that the cell death induced by 1 μg/ml DOX was very effectively inhibited in three different subclones of Bcl-xL-Chang-L cells (Figure 3B). In addition, the DOX-induced apoptotic morphologies (Figure 3C), loss of mitochondrial membrane potential (MMP), which was assessed using Rhodamine-123 (Supplementary Figure 2A and B, available at Carcinogenesis Online) and release of mitochondrial Cytochrome c (Supplementary Figure 2C, available at Carcinogenesis Online) were also dramatically blocked in these Bcl-xL-overexpressing cells. The viability of Bcl-xL-Chang-L cells was not markedly affected by DOX alone, even under an increased dose (2 μg/ml) and incubation time (72 h); contrastingly, DOX/PDTC treatment dose-dependently and significantly enhanced cell death (Figure 3D). Furthermore, combination of DOX and PDTC for 72 h induced the cell death accompanied by extensive vacuolation (Figure 3E), as similar to its effect in the tested HCC cells (Figure 1). These results indicate that PDTC co-treatment can reverse Bcl-xL-mediated DOX resistance by inducing cell death associated with vacuolation. In addition, these finding suggest that Bcl-xL is a key factor in determining the survival or death of liver cells following treatment with DOX or DOX/PDTC. Figure 3. View largeDownload slide Bcl-xL-mediated resistance to DOX is overcome by PDTC co-treatment via induction of cell death accompanied by vacuolation. (A) Western blot to detect endogenous Bcl-xL protein levels in the indicated cells. Western blotting of α-tubulin was performed to confirm equal loading of protein samples. (B) In Chang-L sublines stably transfected with the expression vector encoding Flag-tagged Bcl-xL, the protein levels of Bcl-xL were examined by western blotting using anti-Flag antibody (upper). Control cells transfected with mock vector and three different Bcl-xL-Chang-L sublines were treated with 1 μg/ml DOX for 16 h and then cellular viabilities were analyzed using calcein-AM and EthD-1 (bottom). Data represent the means ± SD. Kruskal–Wallis test was performed followed by Dunn’s test. *P < 0.001 versus DOX-treated Chang-L cells. (C) Chang-L and Bcl-xL-Chang-L (#10) cells were treated with 1 μg/ml DOX for 16 h and the representative pictures were taken under a phase-contrast microscope. Bar, 20 μm. (D) Bcl-xL-Chang-L cells (#10) were treated with DOX and/or PDTC at the indicated concentrations for 72 h. Cellular viabilities were assessed using calcein-AM and EthD-1. Data represent the means ± S.E.M. One-way ANOVA and Bonferroni’s post hoc test. *P < 0.001 versus DOX-treated cells. (E) Bcl-xL-Chang-L cells treated with 1 μg/ml DOX and/or 10 μM PDTC for 72 h were observed by phase-contrast microscopy. Bar, 20 μm. Figure 3. View largeDownload slide Bcl-xL-mediated resistance to DOX is overcome by PDTC co-treatment via induction of cell death accompanied by vacuolation. (A) Western blot to detect endogenous Bcl-xL protein levels in the indicated cells. Western blotting of α-tubulin was performed to confirm equal loading of protein samples. (B) In Chang-L sublines stably transfected with the expression vector encoding Flag-tagged Bcl-xL, the protein levels of Bcl-xL were examined by western blotting using anti-Flag antibody (upper). Control cells transfected with mock vector and three different Bcl-xL-Chang-L sublines were treated with 1 μg/ml DOX for 16 h and then cellular viabilities were analyzed using calcein-AM and EthD-1 (bottom). Data represent the means ± SD. Kruskal–Wallis test was performed followed by Dunn’s test. *P < 0.001 versus DOX-treated Chang-L cells. (C) Chang-L and Bcl-xL-Chang-L (#10) cells were treated with 1 μg/ml DOX for 16 h and the representative pictures were taken under a phase-contrast microscope. Bar, 20 μm. (D) Bcl-xL-Chang-L cells (#10) were treated with DOX and/or PDTC at the indicated concentrations for 72 h. Cellular viabilities were assessed using calcein-AM and EthD-1. Data represent the means ± S.E.M. One-way ANOVA and Bonferroni’s post hoc test. *P < 0.001 versus DOX-treated cells. (E) Bcl-xL-Chang-L cells treated with 1 μg/ml DOX and/or 10 μM PDTC for 72 h were observed by phase-contrast microscopy. Bar, 20 μm. Combination of DOX and PDTC induces paraptosis in Bcl-xL-Chang-L cells We next examined whether PDTC promotes cell death in Bcl-xL-Chang-L cells that are resistant to DOX via apoptosis. However, compared to DOX alone, DOX/PDTC did not notably increase the sub-G1 cell population, TUNEL-positivity, caspsae-3 cleavage, and the release of mitochondrial cytochrome c (Supplementary Figure 3A—D, available at Carcinogenesis Online). Interestingly, cytochrome c was localized within the dilated mitochondria in spite of DOX/PDTC treatment (Supplementary Figure 3D, available at Carcinogenesis Online). Taken together, these results suggest that apoptosis is not responsible for the enhanced cytotoxicity of DOX/PDTC in Bcl-xL-Chang-L cells. Next, we performed electron microscopy to investigate the origins of vacuoles observed in Bcl-xL-overexpressing cells treated with DOX/PDTC. At 48 h post-treatment of Bcl-xL-Chang-L cells with DOX/PDTC, swelling of mitochondria with disruption of the mitochondrial cristae as well as the swelling and fusion of swollen ER structures were noted (Figure 4A). At 72 h, there was further ER fusion and the cellular space was fully occupied with ER-derived vacuoles. Immunocytochemistry using specific antibodies against subunit A of succinate dehydrogenase (SDHA, a mitochondrial protein), and protein disulfide-isomerase (PDI, an ER-resident protein) exhibited ring-shaped SDHA expression around the nuclei and polygonal PDI expression at the boundaries of the larger vacuoles not only in Bcl-xL-Chang-L but also in Huh-7 cells treated with DOX/PDTC for 48 h (Figure 4B). Dilation of the ER and/or mitochondria is a common feature of paraptosis and this mode of cell death is known to require protein synthesis (15,19,23). Therefore, we tested the effect of CHX in our system. Indeed, CHX pretreatment effectively inhibited the cellular vacuolation (Figure 4C) and cell death (Figure 4D) induced by DOX/PDTC in both Bcl-xL-Chang-L and Huh-7 cells. In addition, the protein levels of Alix, which is an inhibitor of paraptosis (23), were reduced by DOX/PDTC co-treatment, but not by either single treatment, in both Bcl-xL-Chang-L and Huh-7 cells (Figure 4E). Taken together, these results indicate that PDTC overcomes Bcl-xL-mediated resistance to DOX via the induction of paraptosis. Figure 4. View largeDownload slide Combination of DOX and PDTC induces paraptosis in Bcl-xL-Chang-L cells. (A) Electron microscopy was performed in Bcl-xL-Chang-L cells treated with 1 μg/ml DOX alone or 1 μg/ml DOX plus 10 μM PDTC for the indicated time points. Arrows indicate dilated mitochondria and arrowheads indicate dilated ER in cells treated with DOX/PDTC. Bar, 2 μm. (B) Bcl-xL-Chang-L cells or Huh-7 were treated with 1 μg/ml DOX plus 10 μM PDTC or 5 μg/ml DOX plus 10 μM PDTC for 48 h, respectively, and immunocytochemistry of SDHA and PDI was performed. Representative phase-contrast and fluorescence microscopic images of cells are shown. Bar, 10 μm. (C) Bcl-xL-Chang-L cells were pretreated with 2 μM CHX and further treated with 1 μg/ml DOX plus 10 μM PDTC for 48 h. Huh-7 cells were pretreated with 1 μM CHX and further treated with 5 μg/ml DOX plus 10 μM PDTC for 48 h. Cellular morphologies were observed by phase-contrast microscopy. Bar, 20 μm. (D) Cells were pretreated or not with CHX at the indicated concentrations and further treated with 1 μg/ml DOX plus 10 μM PDTC or 5 μg/ml DOX plus 10 μM PDTC for 72 h, respectively. Cellular viability was assessed using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus DOX-treated cells, **P < 0.05, **P < 0.001 versus DOX/PDTC-treated cells. (E) Bcl-xL-Chang-L cells were treated with 1 μg/ml DOX and/or 10 μM PDTC for 72 h. Huh-7 cells were treated with 5 μg/ml DOX and/or 10 μM PDTC for 48 h. Western blotting of Alix and α-tubulin was performed. Figure 4. View largeDownload slide Combination of DOX and PDTC induces paraptosis in Bcl-xL-Chang-L cells. (A) Electron microscopy was performed in Bcl-xL-Chang-L cells treated with 1 μg/ml DOX alone or 1 μg/ml DOX plus 10 μM PDTC for the indicated time points. Arrows indicate dilated mitochondria and arrowheads indicate dilated ER in cells treated with DOX/PDTC. Bar, 2 μm. (B) Bcl-xL-Chang-L cells or Huh-7 were treated with 1 μg/ml DOX plus 10 μM PDTC or 5 μg/ml DOX plus 10 μM PDTC for 48 h, respectively, and immunocytochemistry of SDHA and PDI was performed. Representative phase-contrast and fluorescence microscopic images of cells are shown. Bar, 10 μm. (C) Bcl-xL-Chang-L cells were pretreated with 2 μM CHX and further treated with 1 μg/ml DOX plus 10 μM PDTC for 48 h. Huh-7 cells were pretreated with 1 μM CHX and further treated with 5 μg/ml DOX plus 10 μM PDTC for 48 h. Cellular morphologies were observed by phase-contrast microscopy. Bar, 20 μm. (D) Cells were pretreated or not with CHX at the indicated concentrations and further treated with 1 μg/ml DOX plus 10 μM PDTC or 5 μg/ml DOX plus 10 μM PDTC for 72 h, respectively. Cellular viability was assessed using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus DOX-treated cells, **P < 0.05, **P < 0.001 versus DOX/PDTC-treated cells. (E) Bcl-xL-Chang-L cells were treated with 1 μg/ml DOX and/or 10 μM PDTC for 72 h. Huh-7 cells were treated with 5 μg/ml DOX and/or 10 μM PDTC for 48 h. Western blotting of Alix and α-tubulin was performed. PDTC may act as an antioxidant in rescuing DOX-induced apoptosis but as a prooxidant in reversing Bcl-xL-mediated DOX resistance Next, we investigated the underlying mechanism through which PDTC demonstrates pro-survival or pro-death effects in cells treated with DOX, depending on their Bcl-xL expression. ROS generation has been implicated in the apoptosis-inducing effect of DOX (24), and PDTC was previously shown to act as an antioxidant (25). Therefore, we first examined whether PDTC modulates DOX-induced ROS generation in Chang-L cells. FACS analysis using H2DCF-DA showed that following treatment with 1 μg/ml DOX, ROS levels exhibited a biphasic pattern: a marked but transient increase was seen at 6 h, and a second increase was observed at 24 h (Figure 5A). While treatment with PDTC alone did not generate ROS (data not shown), PDTC co-treatment markedly reduced DOX-induced ROS generation both in the duration and extent. While DOX-induced ROS generation was inhibited in Bcl-xL-Chang-L cells, ROS levels were markedly increased at a late phase of DOX/PDTC treatment in these cells. Together, these results indicate that PDTC co-treatment attenuates DOX-induced ROS generation in Chang-L cells, but recovers it in Bcl-xL-Chang-L cells. Pretreatment with various antioxidants, including NAC, GSH, GEE (the cell permeable ethyl ester form of GSH), Trolox, BHA or ascorbic acid commonly alleviated DOX-induced cell death in Chang-L cells (Figure 5B), similar to the effect of PDTC. These results suggest that PDTC may rescue DOX-induced apoptosis in Chang-L cells via its antioxidant activity. The increased ROS levels in Bcl-xL-Chang-L cells treated with DOX/PDTC suggest that PDTC may act as a prooxidant in reversing Bcl-xL-mediated DOX resistance. Thus, we next examined whether the cell death induced by DOX/PDTC in Bcl-xL-Chang-L cells could be inhibited by various antioxidants. Interestingly, the cell death (Figure 5C) and vacuolation (Figure 5D) induced by DOX/PDTC in these cells were significantly inhibited by pretreatment with thiol antioxidants, including GSH, GEE or NAC, but not by other non-thiol ROS scavengers, including Trolox, BHA or ascorbic acid. Immunocytochemical analysis of SDHA and PDI showed that GSH pretreatment effectively blocked the dilation of mitochondria and the ER induced by DOX/PDTC (Figure 5E). Similarly, the cell death as well as vacuolation induced by DOX/PDTC was also very effectively blocked by NAC pretreatment in Huh-7 cells (Supplementary Figure 4, available at Carcinogenesis Online). These results suggest the possibility that thiol-mediated regulation, rather than ROS generation, may be important for DOX/PDTC-induced paraptosis. Figure 5. View largeDownload slide PDTC co-treatment attenuates DOX-induced ROS generation in Chang-L cells, but recovers it in Bcl-xL-Chang-L cells. (A) Cells were treated with 1 μg/ml DOX or 10 μM PDTC plus 1 μg/ml DOX for the indicated time points, incubated with 5 μM H2DCF-DA for 20 min, and FACS analysis was performed. H2DCF-DA fluorescence intensities (FI) in cells treated with DOX or DOX/PDTC were compared with that of untreated cells and denoted in the graph. (B) Chang-L cells were pretreated with various antioxidants at the indicated concentrations for 30 min and further treated with 1 μg/ml DOX for 16 h. Cellular viability was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus untreated cells, *P < 0.001 versus cells treated with DOX. (C) Bcl-xL-Chang-L cells were pretreated with various antioxidants at the indicated concentrations and further treated with 1 μg/ml DOX plus 10 μM PDTC. Cellular viability was measured at 72 h post-treatment using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus untreated cells, *P < 0.001 versus DOX/PDTC-treated cells. Similar results were obtained from three independent experiments. (D) Bcl-xL-Chang-L cells were pretreated with 5 mM NAC, 5 mM GSH, 5 mM GEE, 100 μM Trolox, 50 μM BHA or 250 μM ascorbic acid and further treated with 10 μM PDTC plus 1 μg/ml DOX for 48 h. Representative phase-contrast images of cells are also shown. Bar, 20 μm. (E) Bcl-xL-Chang-L cells were pretreated with 5 mM GSH and further treated with 1 μg/ml DOX plus 10 μM PDTC for 48 h and immunocytochemistry of SDHA and PDI was performed. Representative phase-contrast and fluorescence microscopic images of cells are shown. Bar, 10 μm. Figure 5. View largeDownload slide PDTC co-treatment attenuates DOX-induced ROS generation in Chang-L cells, but recovers it in Bcl-xL-Chang-L cells. (A) Cells were treated with 1 μg/ml DOX or 10 μM PDTC plus 1 μg/ml DOX for the indicated time points, incubated with 5 μM H2DCF-DA for 20 min, and FACS analysis was performed. H2DCF-DA fluorescence intensities (FI) in cells treated with DOX or DOX/PDTC were compared with that of untreated cells and denoted in the graph. (B) Chang-L cells were pretreated with various antioxidants at the indicated concentrations for 30 min and further treated with 1 μg/ml DOX for 16 h. Cellular viability was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus untreated cells, *P < 0.001 versus cells treated with DOX. (C) Bcl-xL-Chang-L cells were pretreated with various antioxidants at the indicated concentrations and further treated with 1 μg/ml DOX plus 10 μM PDTC. Cellular viability was measured at 72 h post-treatment using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus untreated cells, *P < 0.001 versus DOX/PDTC-treated cells. Similar results were obtained from three independent experiments. (D) Bcl-xL-Chang-L cells were pretreated with 5 mM NAC, 5 mM GSH, 5 mM GEE, 100 μM Trolox, 50 μM BHA or 250 μM ascorbic acid and further treated with 10 μM PDTC plus 1 μg/ml DOX for 48 h. Representative phase-contrast images of cells are also shown. Bar, 20 μm. (E) Bcl-xL-Chang-L cells were pretreated with 5 mM GSH and further treated with 1 μg/ml DOX plus 10 μM PDTC for 48 h and immunocytochemistry of SDHA and PDI was performed. Representative phase-contrast and fluorescence microscopic images of cells are shown. Bar, 10 μm. Disruption of thiol homeostasis may critically contribute to DOX/PDTC-induced paraptosis Measurement of GSH levels revealed that PDTC alone had no effect, DOX alone slightly reduced these levels and DOX/PDTC progressively and markedly reduced GSH levels (Figure 6A). In contrast to the effects of NAC or GSH (Figure 5C and 5D), pretreatment with buthionine sulphoximine (BSO, which inhibits GSH synthesis), diethyl maleate (DEM, which depletes GSH) or carmustine (which inhibits glutathione reductase) dose-dependently enhanced the cell death (Figure 6B) and accelerated vacuolation leading to cell death (Figure 6C) induced by DOX/PDTC. Figure 6. View largeDownload slide GSH depletion may be critical for the paraptosis induced by DOX/PDTC. (A) Bcl-xL-Chang-L cells were treated with 1 μg/ml DOX and/or 10 μM PDTC for the indicated time points and GSH assay was performed as described in Materials and Methods. (B) Bcl-xL-Chang-L cells were pretreated with GSH, BSO, DEM or carmustine for 30 min at the indicated concentrations and further treated with 1 μg/ml DOX plus 10 μM PDTC for 72 h. Cellular viability was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus DOX-treated cells, *P < 0.001 versus cells treated with DOX/PDTC. Similar results were obtained from three independent experiments. (C) Bcl-xL-Chang-L cells were pretreated with 3 mM BSO, 80 μM DEM or 80 μg/ml carmustine for 30 min and further treated with 1 μg/ml DOX plus 10 μM PDTC for 24 or 48 h. Representative phase-contrast images of cells are also shown. Bar, 20 μm. (D–F) Bcl-xL-Chang-L cells were transfected with the scrambled negative control RNA, two different GCLC siRNAs, or GSR siRNAs, and incubated for 24 h. (D) Knockdown of GCLC and GSR was confirmed by Western blotting. (E) Transfected Bcl-xL-Chang-L cells were treated with 1 μg/ml DOX plus 10 μM PDTC for 72 h. Cellular viability was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus cells treated with DOX, *P < 0.001 versus cells treated with DOX/PDTC. Similar results were obtained from three independent experiments. (F) Transfected Bcl-xL-Chang-L cells were treated with1 μg/ml DOX plus 10 μM PDTC for 24 or 48 h. Representative phase-contrast images of cells are shown. Bar, 20 μm. (G) Bcl-xL-Chang-L cells were incubated with 1 μg/ml DOX and 10 μM PDTC for 48 h, or treated with DOX/PDTC together with 3 mM BSO, 5 mM GSH or 5 mM GEE for 48 h. Cells were stained with SSH-Mito, and images were acquired using 740 nm excitation and fluorescent emission windows of 425–475 nm (blue) and 525–575 nm (yellow). Bar, 25 μm. Representative images from replicate experiments (n = 5) and pseudocolored ratiometric TPM images (Fyellow/Fblue) are shown. (H) Hypothetical model for different cellular fates in response to DOX or DOX/PDTC depending on Bcl-xL expression. DOX-induced apoptosis is blocked by Bcl-xL overexpression. Co-treatment with PDTC attenuates DOX-induced apoptosis via antioxidant activity. In contrast, co-treatment with PDTC overcomes Bcl-xL-mediated DOX resistance via induction of paraptosis. In this process, disruption of thiol homeostasis by GSH depletion may be critically involved. Figure 6. View largeDownload slide GSH depletion may be critical for the paraptosis induced by DOX/PDTC. (A) Bcl-xL-Chang-L cells were treated with 1 μg/ml DOX and/or 10 μM PDTC for the indicated time points and GSH assay was performed as described in Materials and Methods. (B) Bcl-xL-Chang-L cells were pretreated with GSH, BSO, DEM or carmustine for 30 min at the indicated concentrations and further treated with 1 μg/ml DOX plus 10 μM PDTC for 72 h. Cellular viability was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus DOX-treated cells, *P < 0.001 versus cells treated with DOX/PDTC. Similar results were obtained from three independent experiments. (C) Bcl-xL-Chang-L cells were pretreated with 3 mM BSO, 80 μM DEM or 80 μg/ml carmustine for 30 min and further treated with 1 μg/ml DOX plus 10 μM PDTC for 24 or 48 h. Representative phase-contrast images of cells are also shown. Bar, 20 μm. (D–F) Bcl-xL-Chang-L cells were transfected with the scrambled negative control RNA, two different GCLC siRNAs, or GSR siRNAs, and incubated for 24 h. (D) Knockdown of GCLC and GSR was confirmed by Western blotting. (E) Transfected Bcl-xL-Chang-L cells were treated with 1 μg/ml DOX plus 10 μM PDTC for 72 h. Cellular viability was measured using calcein-AM and EthD-1. Data represent the means ± SEM. One-way ANOVA and Bonferroni’s post hoc test. #P < 0.001 versus cells treated with DOX, *P < 0.001 versus cells treated with DOX/PDTC. Similar results were obtained from three independent experiments. (F) Transfected Bcl-xL-Chang-L cells were treated with1 μg/ml DOX plus 10 μM PDTC for 24 or 48 h. Representative phase-contrast images of cells are shown. Bar, 20 μm. (G) Bcl-xL-Chang-L cells were incubated with 1 μg/ml DOX and 10 μM PDTC for 48 h, or treated with DOX/PDTC together with 3 mM BSO, 5 mM GSH or 5 mM GEE for 48 h. Cells were stained with SSH-Mito, and images were acquired using 740 nm excitation and fluorescent emission windows of 425–475 nm (blue) and 525–575 nm (yellow). Bar, 25 μm. Representative images from replicate experiments (n = 5) and pseudocolored ratiometric TPM images (Fyellow/Fblue) are shown. (H) Hypothetical model for different cellular fates in response to DOX or DOX/PDTC depending on Bcl-xL expression. DOX-induced apoptosis is blocked by Bcl-xL overexpression. Co-treatment with PDTC attenuates DOX-induced apoptosis via antioxidant activity. In contrast, co-treatment with PDTC overcomes Bcl-xL-mediated DOX resistance via induction of paraptosis. In this process, disruption of thiol homeostasis by GSH depletion may be critically involved. To confirm how chemical depletors of GSH affect the cellular responses to DOX/PDTC, we knocked down genes related to glutathione metabolism, including GCLC (Glutamate-cysteine ligase catalytic subunit) and GSR (glutathione-disulfide reductase). GCLC is a target of BSO and is an essential subunit of γGCS (γ-glutamate-cysteine ligase) (26). The γGCS-mediated synthesis of the γ-glutamylcysteine from L-glutamate and cysteine is a rate-limiting step in glutathione synthesis (27). GSR is a target of carmustine and is responsible for reducing oxidized glutathione (GSSG) to reduced glutathione (GSH) (28). Transfection of Bcl-xL-Chang cells with two siRNAs each against GCLC or GSR potentiated DOX/PDTC-induced cell death and accelerated vacuolation (Figure 6D–F). These results suggest that GSH depletion may be critical for the paraptosis induced by DOX/PDTC treatment in Bcl-xL-overexpressing cells. We further investigated whether DOX/PDTC affects the mitochondrial thiol content employing a ratiometric two-photon probe (SSH-Mito) (29), which shows a marked blue-to-yellow emission color change in response to mitochondrial thiols (RSH), including GSH and cysteine, when visualized under two-photon microscopy (TPM). Upon TP excitation at 740 nm, the ratio image of SSH-Mito-labeled Bcl-xL-Chang-L cells constructed from two collection windows gave an average emission ratio of 1.12 (Figure 6G and Supplementary Figure 5, available at Carcinogenesis Online). The Fyellow/Fblue ratio decreased to 0.64 in Bcl-xL-Chang-L cells treated with DOX/PDTC for 48 h, and it was further decreased to 0.48 by addition of BSO. In contrast, pretreatment with GSH or GEE effectively recovered the value to the original level seen in untreated cells. These results suggest DOX/PDTC treatment effectively reduces the mitochondrial thiol content. Collectively, our results suggest that the disruption of thiol homeostasis, including mitochondrial thiol homeostasis, may be critical for the paraptosis induced by DOX/PDTC in Bcl-xL-overexpressing cells. In summary, our results show that DOX mono-treatment and DOX/PDTC co-treatment induce different cellular fates depending on the expression of Bcl-xL (Figure 6H). Bcl-xL overexpression confers resistance to DOX-induced apoptosis, while PDTC attenuates DOX-induced apoptosis in cells that do not highly express Bcl-xL, but induces paraptosis in Bcl-xL-overexpressing cells treated with DOX. Therefore, PDTC co-treatment may overcome DOX resistance in Bcl-xL-expressing cells (and thus possibly in resistant hepatoma cells), whereas it attenuates the cytotoxicity of DOX in the absence of Bcl-xL expression (such as found in normal hepatocytes). Discussion Acquired resistance is a major obstacle for successful cancer treatment, and we urgently need novel drugs that exhibit improved efficacy against tumor cells with less toxicity toward normal cells. The anthracycline, DOX, is one of the most effective drugs for treating solid tumors (30), including unresectable HCC (31), however, high plasma concentrations of DOX tend to cause severe toxicity to normal tissues, including cardiotoxicity (6,31), nephrotoxicity (7) and hepatotoxicity (5), limiting the administration of high doses of this agent. DOX is thought to act through DNA intercalation/binding, inhibition of topoisomerase II, free radical generation, and/or cell membrane damage (32,33). DOX induces apoptosis by disrupting mitochondrial membrane potential and activating caspases (34) via a pathway that is believed to be critically controlled by ROS (33) and GSH (35). Bcl-xL is a well-known anti-apoptotic protein that acts by preventing the cytotoxic-stimulus-induced release of mitochondrial cytochrome c into cytosol (36) and reducing proapoptotic-stimulus-induced mitochondrial dysfunction and ROS hyperproduction (37). In HCC, Bcl-xL is overexpressed in cancerous specimens from two-thirds of patients, and Bcl-xL expression is significantly correlated with overall and disease-free survival (8). Since Bcl-xL overexpression is known to contribute to cancer development and therapeutic resistance in many types of malignant tumors (38,39), we need to identify sensitizers that effectively overcome the Bcl-xL-mediated resistance of cancer cells to anti-cancer drugs. In tumors that are resistant to proapoptotic anti-cancer agents, strategies to induce an alternative cell death mode could prove helpful. PDTC is a pyrrolidine derivative of dithiocarbamate (11). It is a functionally versatile molecule that can confer numerous effects (e.g., NF-κB inhibition (11), metal chelation (40), antioxidant activity (12,25) and prooxidant activity (41)) depending on the cellular model and microenvironment. PDTC was previously shown to attenuate DOX-induced cardiotoxicity (13,42,43) and nephrotoxicity (7). The effect of PDTC on DOX-treated hepatocytes has not been studied, but micellar nanoparticle-mediated co-delivery of DOX and PDTC was shown to effectively overcome DOX resistance in HCC cells (14). The clinical use of PDTC has not yet been investigated in a published report, but the existing literature suggests that this agent could be a clinically important adjuvant drug for cancer therapy. In the present study, we investigated whether administration of PDTC could improve DOX treatment by protecting hepatocytes while increasing the therapeutic efficacy in resistant HCC cells. We employed Chang-L cells and Chang-L sublines stably overexpressing Bcl-xL (Bcl-xL-Chang-L cells) as a model system to mimic human hepatocytes (which are sensitive to DOX) and malignant HCC cells (which are resistant to DOX), respectively. We found that co-treatment with PDTC attenuates DOX-induced apoptosis in Chang-L liver cells and human hepatocytes (Figure 2), but overcomes DOX resistance in Bcl-xL-overexpressing Chang-L cells and HCC cells by inducing paraptosis (a cell death mode that is accompanied by dilation of the ER and/or mitochondria) (Figures 3 and 4). Moreover, the anti-cancer effect and vacuolation by DOX/PDTC in vivo was also confirmed in mice bearing Huh-7-derived xenograft tumors (Figure 1C–E). Therefore, our results suggest that PDTC may protect normal non-Bcl-xL-expressing cells (i.e. hepatocytes) against DOX, while sensitizing Bcl-xL-overexpressing cancer cells (i.e. resistant HCC cells) to this agent. It is very intriguing to consider how PDTC demonstrates opposing activities towards normal and cancer cells exposed to DOX. Previous studies showed that PDTC attenuated DOX-induced myocardial apoptosis (13,24,42,43) and acute myocardial injury in rats by inhibiting NF-κB (43). When we assessed NF-κB activity by p65 phosphorylation at serine 536 (44), DOX increased NF-κB activity in Chang-L cells and this DOX-induced NF-κB activation was considerably attenuated by PDTC and by Bcl-xL overexpression (Supplementary Figure 6A, available at Carcinogenesis Online). However, DOX-induced cell death was unaffected by other NF-κB inhibitors (Supplementary Figure 6B, available at Carcinogenesis Online). In contrast, DOX-induced ROS generation and subsequent apoptosis in Chang-L cells were effectively attenuated by various antioxidants (Figure 5A and B) and by Bcl-xL overexpression (Figures 3B, 3C and 5A). These results suggest that the antioxidant activity of PDTC, rather than its NF-κB-inhibiting activity, may be more important for the attenuation of DOX-induced cytotoxicity in these cells. However, in Bcl-xL-overexpressing Chang-L cells, perturbation of thiol homeostasis, rather than ROS generation, appears to critically contribute to the paraptosis induced by DOX/PDTC, as supported by the following findings: (a) The intracellular content of GSH levels were reduced and ROS generation was followed in DOX/PDTC-induced paraptosis (Figures 5A and 6A). (b) Thiol-containing antioxidants with reducing activity (e.g. GSH, GEE and NAC) abrogated the vacuolation and cell death induced by DOX/PDTC in Bcl-xL-Chang-L cells, whereas non-thiol antioxidants (e.g., BHA, Trolox and ascorbic acid) did not (Figure 5C–E). (c) Depletion of GSH employing either pharmacological (using BSO, DEM or carmustine) or genetic (knockdown of GCLC or GSR) tools accelerated the vacuolation and subsequent cell death induced by DOX/PDTC (Figure 6B–F). GSH not only acts as a major antioxidant by protecting cells against the damaging effects of free radicals and ROS; it also functions in reducing the disulfide linkages of proteins (45,46). NAC, an acetylated variant of L-cysteine, possesses both direct (i.e., via oxidizable sulfhydryl groups) and indirect (i.e. as a substrate for the biosynthesis of GSH) antioxidant activities (47). Similar to our results in Bcl-xL-Chang-L cells treated with DOX/PDTC, Kar et al. (18). found that the cytoplasmic vacuolation and cell death induced by 15d-PGJ2 was effectively blocked by various thiol-antioxidants, but not by non-thiol antioxidants. The authors argued that the effects of 15d-PGJ2 may be mediated not through ROS generation, but through the ability of this agent to covalently modify free sulfhydryl groups on proteins. Therefore, we speculate that the perturbation of thiol homeostasis in cells co-treated with PDTC may also critically contribute to enhancement of cell death in Bcl-xL-expressing cells treated with DOX. Although additional work is needed to clarify how PDTC switches the cellular fates of DOX-treated cells depending on their expression of Bcl-xL, this ability may rely on the versatility of PDTC in exhibiting both anti- and prooxidant activities (48). PDTC induces differential effects on the redox equilibrium according to: (a) its ability to decrease single-electron radical species, such as superoxide anion (O2-·) (25), and hydroxyl radical (HO·) (49) via scavenging (an antioxidant effect); and (b) its capacity to oxidize GSH and related thiol compounds (a prooxidant effect) (49), and thus modulate glutathione recycling. PDTC may undergo oxidation by ROS, which are derived from DOX treatment, generating dithiocarbamate thiyl radical and further dimerization of the radicals to thiuram disulfide (41). In Bcl-xL-overexpressing Chang-L cells, prolonged incubation with DOX/PDTC may enable thiuram disulfides to oxidize glutathione, leading to the formation of GSSG and eventual cell death. In this case, PDTC would act as a prooxidant and thiol group modulator, as the up-regulated GSSG may promote the formation of disulfide bonds in cellular polypeptides through the oxidation of cysteinyl thiols (50,51), resulting in accumulation of misfolded polypeptides. Future work is needed to clarify whether disruption of thiol homeostasis directly triggers the accumulation of misfolded proteins, leading to cellular vacuolation and paraptotic cell death in Bcl-xL-overexpressing cells treated with DOX/PDTC. In our study, Bcl-xL overexpression in Chang-L cells very effectively blocked DOX-induced apoptosis and markedly inhibited ROS generation. These results suggest that Bcl-xL may act, at least indirectly, as an antioxidant. The anti-apoptotic roles of Bcl-2 and Bcl-xL have been widely linked to the GSH content. The anti-apoptotic effect of Bcl-xL has been attributed to its ability to regulate GSH homeostasis by preventing GSH loss (52). Bcl-2 has been shown to regulate the GSH content in different cellular compartments (53), and Bcl-2 overexpression is known to increase GSH levels and inhibit the mitochondria-induced cell death elicited by GSH-depleting reagents (54). Consistent with this, depletion of intracellular GSH has been reported to overcome the Bcl-2-mediated resistance to apoptosis (55). When we assessed the mitochondrial GSH levels using the SSH-Mito probe in Chang-L and Bcl-xL-Chang-L cells, we found that the mitochondrial GSH levels were significantly higher (1.6- fold) in the presence of Bcl-xL (Supplementary Figure 7, available at Carcinogenesis Online). Our results therefore suggest that Bcl-xL-induced increase in mitochondrial GSH may help enhance the ability of cells to scavenge DOX-induced ROS and block apoptosis. In contrast, prolonged exposure of Bcl-xL-Chang-L cells to DOX/PDTC progressively reduced GSH levels, ultimately leading to paraptosis. We also found that Bcl-xL shares with Bcl-2 in the ability to regulate apoptosis and paraptosis in response to DOX or DOX/PDTC. DOX-induced apoptosis was effectively blocked by Bcl-2 overexpression, but not by Mcl-1 overexpression (Supplementary Figure 8, available at Carcinogenesis Online). In addition, PDTC co-treatment overcame Bcl-2-mediated resistance to DOX via a cell death accompanied by severe vacuolation, whereas it slightly protected Mcl-1-overexpressing cells from DOX-induced cytotoxicity (Supplementary Figure 9, available at Carcinogenesis Online). Furthermore, we found that PDTC co-treatment attenuated daunorubicin- or etoposide-induced apoptosis (Supplementary Figure 10A and B, available at Carcinogenesis Online). While Bcl-xL overexpression effectively blocked daunorubicin- or etoposide-induced cell death, PDTC co-treatment overcame this Bcl-xL-mediated resistance (Supplementary Figure 10C and D, available at Carcinogenesis Online). These results suggest that PDTC may play a cytoprotective role in the absence of Bcl-xL and a chemo-sensitizing role in the presence of Bcl-xL when combined with anti-cancer drugs that share the action mechanism of DOX (e.g. ROS generation and DNA damage). In summary, we herein show that PDTC co-treatment attenuates DOX-mediated toxicity in normal (non-Bcl-xL-expressing) Chang liver cells by inhibiting ROS-mediated apoptosis, but overcomes Bcl-xL-mediated DOX resistance in Bcl-xL-overexpressing (cancer-like) cells by inducing paraptosis, possibly through the disruption of thiol homeostasis. These novel findings suggest that DOX/PDTC co-treatment may provide a safe and effective therapeutic strategy against Bcl-xL-overexpressing malignant HCC cells, which are resistant many chemotherapeutic agents. 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CarcinogenesisOxford University Press

Published: Mar 1, 2018

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