Background: Tetrandrine, a bisbenzylisoquinoline alkaloid that was isolated from the medicinal plant Stephania tetrandrine S. Moore, was recently identified as a novel chemotherapy drug. Tetrandrine exhibited a potential antitumor effect on multiple types of cancer. Notably, an enhanced therapeutic efficacy was identified when tetrandrine was combined with a molecularly targeted agent. H89 is a potent inhibitor of protein kinase A and is an isoquinoline sulfonamide. Methods: The effects of H89 combined with tetrandrine were investigated in vitro with respect to cell viability, apoptosis and autophagy, and synergy was assessed by calculation of the combination index. The mechanism was examined by western blot, flow cytometry and fluorescence microscopy. This combination was also evaluated in a mouse xenograft model; tumor growth and tumor lysates were analyzed, and a TUNEL assay was performed. Results: Combined treatment with H89 and tetrandrine exerts a mostly synergistic anti-tumor effect on human cancer cells in vitro and in vivo while sparing normal cells. Mechanistically, the combined therapy significantly induced cancer cell apoptosis and autophagy, which were mediated by ROS regulated PKA and ERK signaling. Moreover, Mcl-1 and c-Myc were shown to play a critical role in H89/tetrandrine combined treatment. Mcl-1 ectopic expression significantly diminished H89/tetrandrine sensitivity and amplified c-Myc sensitized cancer cells in the combined treatment. Conclusion: Our findings demonstrate that the combination of tetrandrine and H89 exhibits an enhanced therapeutic effect and may become a promising therapeutic strategy for cancer patients. They also indicate a significant clinical application of tetrandrine in the treatment of human cancer. Moreover, the combination of H89/tetrandrine provides new selectively targeted therapeutic strategies for patients with c-Myc amplification. Keywords: Combination therapy, H89, Tetrandrine, Apoptosis, Autophagy, C-Myc Background from the actual form [3, 4]. Thus, natural products have Cancer is a multigenic disease caused by the abnormal garnered increased attention in the chemotherapy drug proliferation and differentiation of cells governed by discovery field because they are biologically friendly tumorigenic factors . Chemotherapy is one of the and have high therapeutic effects [5, 6]. major cancer treatment strategies, and it functions by Tetrandrine (Tet), a bisbenzylisoquinoline alkaloid targeting the biological capabilities of cancer cells, including isolated from the medicinal plant Stephania tetrandrine sustained proliferation, the evasion of programmed cell S. Moore, has been widely used as an effective agent to death and tissue migration . Remarkably, among the FDA treat patients with hypertension, arrhythmia, arthritis, approved anticancer drugs, more than 75% originate from inflammation, and silicosis in traditional Chinese medicine natural sources (e.g., Taxol, doxorubicin, or vincristine) and . Of note, tetrandrine has recently been identified as a are used in their actual form or with simple modifications potential leading compound among anticancer agents with various pharmacological effects, including the regula- tion of cell viability, migration, invasion, angiogenesis and * Correspondence: email@example.com multidrug resistance of tumors [8, 9]. Our previous studies Hubei Key Laboratory of Cell Homeostasis, College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Yu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:114 Page 2 of 16 have indicated that tetrandrine induced apoptosis at a These various cell lines were cultured in DMEM. The high concentration and induced autophagy at low human renal carcinoma cell lines (769-P, ACHN, and concentrations [10–12]. Moreover, tetrandrine showed 786-O) and the human lung cancer cell line (A549) potential anti-tumor activity in leukemia and hepatocellular were purchased from ATCC and maintained in 1640 carcinoma [13, 14]. RPMI. The human colon cancer cell lines (LOVO and However, tetrandrine, as a promising chemotherapeutic HCT116) were purchased from ATCC and cultured in candidate, was in the preclinical phase [9, 12]. At times, it McCoy’s 5A. All cells were cultured in media supple- has been observed that certain phytochemicals are active mented with 10% fetal bovine serum (FBS, HyClone), − 1 − 1 only when they are in combination with other metabolites penicillin 100 U·mL and 100 μg·ml streptomycin and of the source material . In addition, as a result of the were incubated at 37 °C in a humidified atmosphere with complexity of cancer with the involvement of multiple 5% CO . signaling pathways, it is difficult for a single compound to combat cancer [16, 17]. Nevertheless, if a compound Reagents exhibits a potent anticancer effect, there is a chance for The reagents used in this study are listed as follows: the development of resistance against the compound by H89 2HCl (10 mM, dissolved in DMSO, S1582, Selleck, tumor cells, thereby making the drug ineffective . Houston, TX), forskolin (FSK) (10 mM, dissolved in Thus, combination therapy may be an available strategy to DMSO, S2449, Selleck, Houston, TX), PKI (14–22) improve the treatment efficacy [19, 20]. Increasing studies amide myristoylated (PKI) (0.5 mg, dissolved in DMSO, have shown that tetrandrine may induce synergistic Sc471154, Santa Cruz, CA), Tetrandrine (10 mM, dissolved activity to enhance cytotoxicity when combined with in DMSO, CAS:518–34-3, Shanghai Ronghe Medical, molecularly targeted drugs, such as sorafenib , Shanghai, China), Caspase inhibitor z-VAD-fmk (10 mM, methylprednisolone  and glucocorticoids . dissolved in DMSO, S7023, Selleck, Houston, TX), H89, a potent protein kinase A (PKA) inhibitor, has the Bafilomycin A1 (dissolved in DMSO, S1413, Selleck, ability to readily cross the cell membrane, with preclinical Houston, TX), Chloroquine (CQ) (C6628, Sigma-Aldrich, activity demonstrated in vitro and in vivo [24–26]. H89 USA), PD98059 (Beverly, MA, USA), N-acetyl-L-cysteine attenuates airway inflammation in mouse models of (NAC) (dissolved in ddH O, A0150000, Sigma-Aldrich, asthma . Of note, recent efforts have focused on its USA), 3-Methyladenine (3-MA) (dissolved in ddH O, pharmacological activities against cancer. Numerous M9281-100MG, Sigma-Aldrich, USA), 2′,7′-dichlorodihy- studies have demonstrated that H89 showed chemotherapy drofluorescein diacetate (DCFH-DA) (Invitrogen Carlsbad, sensitization activity. Reports have documented that H89 CA), and an FITC Annexin V Apoptosis Detection Kit enhanced HA22 (Moxetumomab pasudotox) treatment (556,547, BD Pharmingen). All reagents were formulated as of CD22-positive ALL and mesothelin-expressing solid recommended by their suppliers. tumors . H89 has also been shown to dramatically synergize with oncolytic virus M1 to improve tumor Cell viability assay regression and trigger apoptosis in aggressive cancer cells To measure viability following H89 and/or tetrandrine when combined with glyceryl trinitrate (GTN) [29, 30]. treatment, cells were seeded on 96-well plates at a density In this work, we discovered that H89 and tetrandrine of 4 × 10 cells per well. Cells were allowed to attach over- showed synergistic anti-tumor effects on various cancer night in complete media and were subsequently treated cells in vitro and in vivo, and we investigated the under- with the indicated concentrations of tetrandrine and/or lying mechanisms of their anti-tumor activities. In addition, H89 for an additional 72 h. Control cells received DMSO we determined that c-Myc amplified cells are more sensi- (< 0.1%) that contained medium. The cell viability was tive to H89/tetrandrine combined treatment, which may determined using the trypan blue dye exclusion assay represent a novel, selective therapeutic strategy for cancer according to established protocols. patients. Methods Drug combination analysis Cell lines and cell culture A drug combination analysis was performed using the The human breast cancer cell lines (MDA-MB-231, method described by Chou and Talalay . Briefly, cells MDA-MB-468, and MCF-7) were purchased from ATCC were plated in 96-well plates and treated with 1–5 μM (Manassas, VA, USA). The human hepatoma cell lines tetrandrine and 1–10 μM H89 alone or in combination (Hep3B and Huh7) and the normal cell lines (L02, for 72 h; the cell viability was subsequently assessed. HBL-100, and HEK293T) were purchased from CCTCC Multiple drug dose-effect calculations and the combination (Wuhan, China). The cell line HCCLM9 was purchased index plots were generated using Calcusyn 2.1 software from the Liver Cancer Institute (Fudan University, China). (Biosoft, Cambridge, UK). CI values < 1, = 1 and > 1 indicate Yu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:114 Page 3 of 16 synergism, additive and antagonism between two drugs, (# 9197), which were obtained from Cell Signaling respectively. Technology. Antibodies for LC3B (# L7543) and anti- β-actin (# A2228) were obtained from Sigma-Aldrich. Apoptosis assay Antibody for c-Myc (9E10) (sc-40) was obtained from Apoptosis was determined using an Annexin V-FITC/PI Santa Cruz Biotechnology. apoptosis detection kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Determination of intracellular ROS Briefly, the untreated and treated cells were washed with The intracellular ROS levels were detected by a flow PBS and gently suspended in Annexin V binding buffer, cytometer utilizing Dichloro-dihydro-fluorescein diacetate followed by incubation with Annexin V-FITC and PI at (DCFH-DA). Briefly, 7 × 10 cells were plated on 12-well room temperature for 15 min. Finally, the fluorescent plates, allowed to attach overnight and then treated with intensities were determined by a flow cytometer (Beckman the indicated concentrations of tetrandrine and/or H89 for Coulter, Indianapolis, CA, USA), and the data were the indicated times. NAC pretreatment, where indicated, analyzed using FlowJo software (Tree Star Inc., San Carlos, was conducted for 1 h. Cells were stained with DCFH-DA CA, USA). (1 μM) in serum-free media at 37 °C for 30 min in the dark. DCF fluorescence (produced in the presence of ROS) Colony formation assay was analyzed using flow cytometry. For the detection of Cell were seeded at 2500 cells per well in 6-well plates the mitochondrial membrane potential, the cell pre- and treated with H89, tetrandrine or the combination. treatments were performed as described for the ROS − 1 Cells were washed with fresh medium after 24 h, allowed detection protocol. A 5 μLsolution of 0.1 mg·mL to grow for 8 days under drug-free conditions, and stained Rho123 (Sigma-Aldrich, USA) was added, and the cells with crystal violet (Sigma-Aldrich, USA). Colonies with were incubated for 30 min at 37 °C. The fluorescence more than 50 cells were counted. was measured via flow cytometry. Western blot and antibodies Tandem mRFP-GFP-LC3 reporter assay After various treatments, both floating and adherent Cells were transfected with the mRFP-GFP-LC3 plasmid cells were harvested and subsequently washed with cold (Addgene, # 21074) and were subsequently treated with PBS. The cells were then lysed with 1% SDS on ice. The DMSO or H89/tetrandrine. Autophagy flux was assessed by + + cell lysates were subsequently heated at 95 °C for 20 min counting cells that were mRFP GFP LC3 (yellow puncta), + − and centrifuged at 12,000×g for 10 min, and the super- which represents autophagosomes, and mRFP GFP LC3 natant was collected. For the tumor tissue, the samples (red puncta), which represents autolysosomes. were homogenized and sonicated in RIPA buffer (Beyotime, Nantong, China) in the presence of protease inhibitor cock- Lentiviral transduction tail on ice. The tissue lysates were subsequently centrifuged pLKO.1 plasmids that contained shRNA sequences target- at 12,000×g for 15 min at 4 °C, and the supernatant was ing c-Myc (shc-Myc#1: target sequence: CCTGAGACA collected for Western blotting analysis. Protein was quanti- GATCAGCAACAA; shc-Myc#2: target sequence CAGT fied using a BCA Protein Assay Kit (Thermo Scientific, TGAAACACAAACTTGAA) were established. The empty MA, USA). The proteins were separated by 8–12% vector wasusedasa negative control. PHAGE-puro-c-- SDS-PAGE and transferred to PVDF membranes (Millipore, Myc and the control plasmids were kindly provided by Dr. Billerica, MA, USA). The blots were blocked for 2 h at room Youjun Li (Wuhan University). A PHAGE-puro-Mcl-1 temperature with freshly prepared 5% nonfat milk (Bio-Rad, plasmid was constructed. To generate virus, 293 T cells USA) in TBST and were subsequently incubated with spe- were seeded in each 10 cm dish. After 24 h, the control cific primary antibodies overnight at 4 °C. The membranes and shRNA constructs that targeted c-Myc (12 μgeach), were then washed with TBST and incubated with HRP con- packaging plasmid (psPAX2; 6 μg) and envelope plasmid jugated secondary antibodies for 1 h at room temperature. (pMD2.G; 6 μg) were diluted with 0.5 mL Opti-MEM After washing with TBST, the immunoblots were visualized medium (Gibco, Invitrogen, Carlsbad, CA, USA) and by Immobilon™ Western HRP Substrate peroxide (Millipore, mixed with transfection reagent FuGENE™ HD (Roche, Billerica, MA, USA). The following antibodies were USA). Twelve hours after transfection, the culture medium employed: anti-PARP (# 9542), anti-Caspase-3 (# 9662), was changed to fresh culture medium; after 48 h, the anti-Caspase-9 (# 9502), anti-Bcl-2 (# 2872), anti-Bcl-xL virus-containing supernatants were collected and used − 1 (# 2762), anti-Mcl-1 (# 4572), anti-Bim (# 2819), anti-Bid to infect cells in the presence of 8 μg·mL polybrene (Sig- (# 2002), anti-cytochrome c (# 4272), anti-p-ERK1/2 ma-Aldrich, USA). The cells were subsequently selected in − 1 (Thr202/Tyr204, # 9101), anti-T-ERK (# 9102), p-MEK thepresenceofpuromycin(5 μg·mL ;Sigma-Aldrich, (# 9121S), anti-p-CREB1-s133 (# 9198), and anti-CREB1 USA) to establish stable clones. Yu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:114 Page 4 of 16 RT-PCR the experiments, the mice were euthanized after being RNA was isolated from cultured cells using an OMEGA- anesthetized with an i.p. injection with pentobarbital − 1 RNA Miniprep kit, and RNA was reverse-transcribed (50 mg·kg ); their tumors were isolated by dissection, into cDNA molecules using a cDNA synthesis kit (Roche weighed and used for in vitro experiments. Samples Applied Science, USA). The numbers of Mcl-1 and were prepared for histology and protein assays. All β-actin molecules were monitored in real time on a studies involving animals are reported in accordance 7500 Fast Real-Time PCR System (Applied Biosystems) with the ARRIVE guidelines for reporting experiments by measuring the fluorescence increases of SYBR Green involving animals . (Roche Applied Science, USA). The primer sequences for PCR were as follows: Mcl-1 forward 5’-CCAAGAAA Malondialdehyde (MDA) assay GCTGCATCGAACCAT-3′ and Mcl-1 reverse 5’-CAGC Tumor samples from mice were homogenized and soni- ACATTCCTGATGCCACCT-3′; β-actin forward 5’-GGCA cated. Tissue lysates were subsequently centrifuged at TGGGTCAGAAGGATT-3′ and β-actin reverse 5’-AGGA 12000×g for 10 min at 4 °C to collect the supernatant. TGCCTCTCTTGCTCTG-3′. To determine the relative The total protein content was determined using the abundance of Mcl-1 in relation to β-actin, the Δ-ΔCT (cycle Bradford assay. The MDA levels were measured by the threshold) method was utilized. Lipid Peroxidation MDA assay kit (Beyotime Institute of Biotechnology). Tumor xenograft Animals were handled according to the Guidelines of Statistical analysis the China Animal Welfare Legislation, as provided by the All experiments were randomized and repeated at least Committee on Ethics in the Care and Use of Laboratory three times. Data analysis was performed using Microsoft Animals of Wuhan University. The experimental protocols Excel and GraphPad Prism Software version 5.0 (GraphPad were approved by the Experimental Animal Centre of Software, La Jolla, CA, USA). All data are expressed as the Wuhan University. Female nude nu/nu BALB/c mice mean ± SD. Student’s two-tailed t-tests were performed to (14–16 g; 4–5 weeks of age) were purchased from calculate P values unless otherwise specified. P <0.05 was Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). considered statistically significant. Animals were housed at a constant room temperature with a 12/12 h light/dark cycle and were fed a standard rodent Results diet. Combination treatment with H89 and tetrandrine yields MDA-MB-231 cells (5 × 10 ), MDA-MB-231 control synergistic anti-tumor effects in multiple human cancer or Mcl-1 cells (5 × 10 ), or MDA-MB-231 shCtrl or cells shc-Myc cells (5 × 10 ) were subcutaneously implanted To examine whether a cooperative effect exists between into the right flank of each mouse in 0.2 mL PBS. Once the H89 and tetrandrine in tumor chemotherapy, we treated tumor volume of MDA-MB-231 cells reached 50–100 mm , cancer cells with a series of concentrations of H89 and the tumor-bearing mice were randomly separated into four tetrandrine alone or in combination. Dose-response studies − 1 groups (n = 6) and treated via gavage of 25 mg·kg - indicated that 0–5 μM tetrandrine or 0–10 μMH89 were tetrandrine with 0.5% sodium carboxyl methylcellulose, only minimally toxic by themselves (Fig. 1, a and b). − 1 i.p. injection of 10 mg·kg H89 (in a solution of PBS However, 4 μM tetrandrine substantially increased the with 1% DMSO+ 30% polyethylene glycol+ 1%Tween 80) cell death of H89 at 6–10 μM(Fig. 1c). Analogously, − 1 or a combination of H89 and tetrandrine (10 mg·kg H89 the lethality of the marginally toxic tetrandrine concentra- − 1 and 25 mg·kg tetrandrine) every other day for 28 days. tions (3–5 μM) was significantly increased by co-exposure The control group received the same vehicle. For the to 6 μM H89 (Fig. 1d). In addition, we determined that MDA-MB-231 control and Mcl-1 cells, tumor-bearing 4 μM tetrandrine plus 6 μM H89 had a clear effect on mice were randomized into two test groups (n =6) and most human cancer cells (Fig. 1e). In contrast, normal were administered vehicle (0.5% carboxymethylcellulose cells, such as HBL-100, L02 and HEK-293 T, were sub- − 1 − 1 sodium) or H89 (10 mg·kg ) and tetrandrine (25 mg·kg ) stantially less sensitive to H89/tetrandrine, which suggests every other day for 36 days. For the MDA-MB-231 that the H89/tetrandrine combined treatment is relatively shCtrl and shc-Myc cells, tumor-bearing mice were selective towards cancer cells (Fig. 1f). The use of the randomized into two test groups (n =6) and were Chou-Talalay method to calculate the H89/tetrandrine administered vehicle (0.5% carboxymethylcellulose sodium) interaction showed combination indexes < 1, which indi- − 1 − 1 or H89 (10 mg·kg )and tetrandrine(25 mg·kg )every cated their synergistic effects under a number of the treat- other day for 30 days. The tumor volumes were determined ment conditions (Fig. 1g). Furthermore, the analysis of by measuring the length (l) and width (w) and calculating long-term cell survival via colony formation assay indi- the volume (V = 0.5 × l × w ) every other day. At the end of cated that the combination of H89 and tetrandrine Yu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:114 Page 5 of 16 a b cd ef gh Fig. 1 Combination treatment with H89 and tetrandrine yields a synergistic antitumor effect across different types of cancer cells. a Cell viabilities were determined by a trypan blue dye exclusion assay in cancer cells treated with increasing doses of tetrandrine (0–5 μM) or (b) H89 (0–20 μM) alone for 72 h. c Cancer cells were exposed to the designated concentrations of H89 in combination with tetrandrine (4 μM) for 72 h. Cell viabilities were determined as previously described. d Cells were exposed to the designated concentrations of tetrandrine in combination with H89 (6 μM) for 72 h, following which the cell viabilities were determined. e Cell viabilities were determined in cells incubated with H89 (6 μM) and tetrandrine (4 μM) alone or in combination for 72 h. f HBL-100, L02 and HEK293T cells were treated with H89 (6 μM) and tetrandrine (4 μM) alone or in combination for 72 h. Cells viabilities were determined. g The combination index (CI) values for LOVO, MDA-MB-231, AGS, and Hep3B cells were constructed by CalcuSyn 2.1 software. h Cells were treated with H89 and tetrandrine alone or in combination for 24 h. The attached cells were stained with crystal violet after 8 days. The number of colonies was quantified. All representative images are from three independent experiments. Data are reported as the mean ± SD and were analyzed by Student’s t-test; all data represent at least n = 3 independent experiments, *P < 0.05 compared with DMSO control suppressed colony formation significantly more than tetran- H89 in combination with tetrandrine synergistically drine or H89 alone in cancer cells, whereas the effect on induced concomitant apoptosis and autophagy normal cells was not significant (Fig. 1h). These data suggest To determine whether the reduction in cell survival in- that a combination treatment of H89 and tetrandrine has a duced by H89/tetrandrine was associated with cell apop- significant synergistic anti-tumor effect on cancerous cells. tosis, cancer cells were treated with H89 and tetrandrine Yu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:114 Page 6 of 16 alone or in combination for 48 h and were subsequently tetrandrine combination treatment increased apoptotic analyzed via flow cytometry with Annexin V/PI. As cell death compared with each agent alone. Consistently, shown in Fig. 2a and Additional file 1: Figure S1A, H89/ apoptosis was also evident from Western blots, which ab e f Fig. 2 H89 in combination with tetrandrine synergistically induced concomitant apoptosis and autophagy. a FACS analysis of apoptosis following treatment with H89 (6 μM) and tetrandrine (4 μM) alone or combination on Hep3B, AGS, MDA-MB-231, and LOVO for 48 h. b Western blot analysis of PARP, caspase 9, caspase 3 and cytochrome c in the cells after treatment with H89 and/or tetrandrine for 48 h. c The cells were pretreated with z-VAD-fmk (50 μM) for 1 h, followed by treatment with H89 and/or tetrandrine for 72 h; cell viabilities were subsequently evaluated. Z-VAD, z-VAD-fmk. d Western blot analysis of the level of the autophagy-related protein LC3 in the cells after treatment with H89 and/ or tetrandrine for 24 h. e Cancer cells were transiently transfected with GFP-LC3 plasmid and subsequently treated as in panel (d); the percentage of cells with GFP-LC3 puncta was used to quantify the percentage of autophagic cells, and 150–160 cells per condition were counted. Representative images are shown to indicate the cellular localization patterns of the GFP-LC3 fusion protein (× 40 magnification). f Fluorescence microscopy analysis of AGS cells expressing tandem mRFP-GFP-LC3 reporter treated with H89/tetrandrine, rapamycin (500 nM) and chloroquine (10 μM) for 36 h; the ratio of mRFP vs GFP puncta was used to quantify the autophagy flux. Scale bars, 5 μm. g Cell viability was determined in cells pretreated with 3-MA (2 mM) or bafilomycin A1 (100 nM) for 1 h following H89/tetrandrine combination treatment for 48 h. Baf, Bafilomycin A1. Data are reported as the mean ± SD and were analyzed by Student’s t-test; all data represent at least n = 3 independent experiments; *P <0.05. All images are representative of three independent experiments Yu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:114 Page 7 of 16 indicated that the cleavages of PARP, caspase-3, caspase-9 is related to synergistic antitumor activity, we initially and cytochrome c were substantially increased in the detected the levels of phosphorylated cAMP-dependent combination treatment (Fig. 2b). To ascertain whether response element (CREB ser133). As shown in Fig. 4a, H89/tetrandrine induced cell apoptosis was dependent on H89 alone or combined with tetrandrine significantly caspase activation, we pre-treated cells with z-VAD-fmk decreased the phosphorylation of the transcription factor for 1 h. The results showed that z-VAD-fmk partially CREB in cancer cells. In contrast, forskolin (FSK), an rescued H89/tetrandrine induced cell death (Fig. 2c). Our established activator of adenylyl cyclase, significantly previous studies have shown that tetrandrine is a potent reduced the proportion of cells undergoing apoptosis autophagy agonist. We subsequently examined whether and autophagy (Fig. 4b and c, and Additional file 1: the H89/tetrandrine combination synergistically induced Figure S2, A and B). Our previous studies have shown autophagy. Western blot and GFP-LC3 fluorescence that tetrandrine activates intracellular ERK . We assays showed that the H89/tetrandrine combination treat- subsequently examined the role of MEK/ERK signaling ment synergistically increased autophagy (Fig. 2d and e, in H89/tetrandrine combination treatment. As shown and Additional file 1:FigureS1B). In addition,weused in Fig. 4d, H89/tetrandrine transiently activated MEK/ an mRFP-EGFP-LC3 tandem-tagged fluorescent protein ERK1/2 signaling, and these effects mainly originated (ptf-LC3) to determine the dynamic process of autophagic from tetrandrine (Additional file 1: Figure S2C). Inhibition flux [33, 34]. The results suggested that the combination of ERK1/2 with PD98059 could partially rescue H89/ of H89/tetrandrine promotes functional autophagy in tetrandrine induced cell apoptosis (Fig. 4e and f), cancer cells (Fig. 2f). Next, to investigate the role of whereas it had no effect on autophagy (Fig. 4g). However, autophagy in cell death, we pre-treated cells with 3-MA or concomitant pretreatment of cells with FSK and PD98059 bafilomycinA1for 1h.As shown in Fig. 2g, the autophagy almost completely restored cell viability, which implies inhibitor also partially inhibited H89/tetrandrine induced that PKA and ERK signaling are simultaneously involved cell death. Collectively, these data indicate that apoptosis in H89/tetrandrine induced cell death (Fig. 4h). Moreover, and autophagy act simultaneously in H89/tetrandrine NAC could mitigate H89/tetrandrine regulated PKA and induced cancer cell death. ERK activities, whereas FSK and PD98059 had no effect on H89/tetrandrine increased ROS (Fig. 4i and j), which H89/tetrandrine induced cell death is associated with the indicates that PKA and ERK signaling are involved in generation of ROS H89/tetrandrine induced cell death and are initiated by Increasing evidence suggests that chemotherapeutic agents ROS. Therefore, we demonstrated that the inhibition of activate intracellular reactive oxygen species (ROS) accu- PKA and activation of ERK activity play a role in H89/ mulation and subsequently induce cell death [35, 36]. We tetrandrine induced cell death. assessed the intracellular ROS levels via flow cytometry with DCFH-DA, which specifically detects peroxides. As Mcl-1 plays a critical role in H89/tetrandrine induced cell showninFig. 3a,the H89/tetrandrine combination treat- death ment substantially increased intracellular ROS compared Bcl-2-family proteins play a role in chemotherapy induced with either agent alone in cancer cells. Pretreatment of cells cell death, including apoptosis and autophagy . We with the ROS scavenger NAC not only strikingly abrogated first detected the Bcl-2, Bcl-xl and Mcl-1 expression levels H89/tetrandrine-induced ROS production (Fig. 3b)butalso by Western blotting after AGS and MDA-MB-231 cells significantly rescued them from cell death induced by underwent treatment with H89 and tetrandrine alone or in combination treatment (Fig. 3c). Moreover, NAC rescued combinationfor 48 h. As showninFig. 5a, the expression cells from apoptosis (Fig. 3d) and blocked both PARP of Mcl-1 was significantly decreased after H89/tetrandrine cleavage and caspase 3/caspase 9 activation in AGS and treatment in both AGS and MDA-MB-231 cells. Con- MDA-MB-231 cells (Fig. 3e). In addition, pretreatment with sistent with its protein level, the Mcl-1 mRNA expres- NAC also significantly decreased the intracellular LC3-II sion was downregulated by H89/tetrandrine (Fig. 5b). expression and GFP-LC3 fluorescent punctate dots We subsequently assessed the role of Mcl-1 in H89/tet- induced by H89/tetrandrine (Fig. 3f and g). Therefore, randrine mediated cell death. AGS and MDA-MB-231 these results clearly demonstrated that the H89/tetran- were established stably expressing a control or Mcl-1 drine combination treatment-induced apoptosis and and were subsequently treated with H89/tetrandrine. In autophagy were initiated by ROS production. the presence of H89/tetrandrine, overexpression of Mcl-1 diminished cell sensitivity to the combined treatment PKA and ERK signaling are involved in H89/tetrandrine (Fig. 5c) and attenuated cell apoptosis (Fig. 5d and e); induced cell death however, it had no effect on autophagy (Fig. 5g). As an H89 is a strong inhibitor of cyclic AMP-dependent protein anti-apoptotic Bcl-2 family member, Mcl-1 appeared to kinase A (PKA) . To determine whether PKA activity inhibit apoptosis by preventing mitochondrial dysfunction Yu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:114 Page 8 of 16 ab cd Fig. 3 H89/tetrandrine induced cell death is associated with ROS generation. a The intracellular ROS were detected by flow cytometry after the cells had been treated with 6 μM H89 and 4 μM tetrandrine alone or in combination for 24 h. b The cells were pretreated with 15 mM NAC for 1 h, followed by treatment with DMSO or H89/tetrandrine. The intracellular ROS were measured by flow cytometry after 24 h of treatment. c Cell viability was determined after 72 h of treatment. d Apoptosis was measured by flow cytometry, and (e) PARP, caspase 3, and caspase 9 levels were analyzed by Western blot after 48 h of treatment. f After 24 h of treatment, LC3 expression was measured by Western blot and (g) GFP-LC3 was observed using a fluorescence microscope. Scale bars, 5 μm. Data are reported as the mean ± SD and were analyzed by Student’s t-test; all data represent at least n = 3 independent experiments; *P < 0.05 compared with DMSO control. All images are representative of three independent experiments . We determined that overexpression of Mcl-1 signifi- apoptotic protein Mcl-1 considerably contributes to H89/ cantly rescued the H89/tetrandrine-induced mitochondrial tetrandrine induced cell death. transmembrane potential drop (Fig. 5f). In addition, NAC could partially impede the H89/tetrandrine triggered C-Myc sensitizes cancer cells to H89/tetrandrine Mcl-1 decrease (Fig. 5h); however, Mcl-1 overexpression combination treatment had no significant effect on the ROS increases (Fig. 5i), Deregulated and enhanced expression of c-Myc contributes which demonstrates that the induction of intracellular to the genesis of a substantial fraction of human tumors ROS occurs upstream of the inhibition of Mcl-1 activity. . Paradoxically, high expression levels of c-Myc not only Therefore, we concluded that the activity of the anti- promote transformation but also sensitize cells against a Yu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:114 Page 9 of 16 ab Fig. 4 PKA and ERK signaling are involved in H89/tetrandrine induced cell death. a Western blot of p-CREB1-s133 and CREB1 in AGS and MDA-MB-231 cells exposed to H89 and/or tetrandrine for 24 h. b The cells were pre-treated with FSK (10 μM) for 1 h, followed by treatment with DMSO or H89/tetrandrine, and apoptosis was measured by flow cytometry after 36 h of treatment. c GFP-LC3 punctate was determined using a fluorescence microscope after 24 h of treatment. Scale bars, 5 μm. d Western blot of p-ERK1/2, T-ERK and p-MEK in cells incubated with H89/tetrandrine for 0, 2, 4, 8, 12 & 24 h. e MDA-MB-231 was pre-treated with 20 μM PD98059 for 1 h prior to exposure to H89/tetrandrine. Apoptosis was detected by flow cytometry after 36 h, and (f)p-ERKandPARP levels were analyzed by Western blot at 8 h and 36 h, respectively. g After 24 h of treatment, LC3 expression was measured by Western blot. h Cells were pre-treated with PD98059 and FSK alone or in combination for 1 h prior to exposure to H89/tetrandrine for 72 h. Cell viability was determined by trypan blue staining. PD, PD98059. i Analysis of p-CREB1-s133 and p-ERK levels in the cells pretreated with NAC for 1 h prior to exposure to H89/tetrandrine for 8 h. j The cells were pre-treated with FSK or PD98059 for 1 h followed by H89/tetrandrine combination treatment for 24 h; intracellular ROS were subsequently detected by flow cytometry. Data are reported as the mean ± SD and were analyzed by Student’s t-test; all data represent at least n = 3 independent experiments; *P < 0.05 compared with DMSO control. All images are representative of three independent experiments broad range of pro-apoptotic stimuli, such as growth factor (Fig. 6a,Fig. 1e, and Additional file 1:FigureS3A). To deprivation, hypoxia, ionizing radiation, or exposure to further verify this observation, we ectopically expressed chemotherapy [42–44]. Consistently, in this study, we c-Myc in HCT116 and A549 cells, which express low determined that H89/tetrandrine sensitive cells showed levels of c-Myc and are resistant to H89/tetrandrine higher levels of c-Myc expression than resistant cells treatment. Interestingly, the overexpression of c-Myc Yu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:114 Page 10 of 16 Fig. 5 Mcl-1 plays a critical role in H89/tetrandrine induced cell death. a Western blot analysis of Bcl-2 family members after treatment for 48 h. b AGS and MDA-MB-231 cells were treated with DMSO or H89/tetrandrine for 24 h; the relative Mcl-1 mRNA expression was determined by qPCR, and β-actin was used as a reference. c The cells stably transfected with Mcl-1 were treated with DMSO or H89/tetrandrine for 72 h and subjected to viability assays. d MDA-MB-231 Mcl-1 overexpression and control cells were treated with DMSO or H89/tetrandrine for 48 h. Apoptosis was evaluated by flow cytometry. e Western blot analysis of PARP, caspase 9 and Mcl-1 in the cells after treatment with DMSO or H89/ tetrandrine for 48 h. f The mitochondrial membrane potential was measured by flow cytometry with Rho123 in the cells after treatment with DMSO or H89/tetrandrine for 48 h. g LC3 was determined in the cells after treatment with DMSO or H89/tetrandrine for 24 h. h AGS and MDA-MB-231 cells were pre-treated with NAC for 1 h followed by H89/tetrandrine for 36 h; Mcl-1 levels were subsequently determined by Western blot. i Cells with control or Mcl-1 overexpression were treated with DMSO or H89/tetrandrine for 24 h; intracellular ROS levels were subsequently measured. Data are reported as the mean ± SD and were analyzed by Student’s t-test; all data represent at least n = 3 independent experiments; *P <0.05, **P < 0.01 compared with DMSO control. All images are representative of three independent experiments enhanced the effects of combination treatment induced determined that c-Myc significantly regulated Mcl-1 cell death and apoptosis (Fig. 6b and c). In contrast, the expression (Fig. 6g). Furthermore, we determined that knockdown of c-Myc in AGS and MDA-MB-231, which knockdown of c-Myc in AGS and MDA-MB-231 cells, express high levels of c-Myc, decreased the sensitivity cell autophagy, and intracellular ROS generation trig- of cells to H89/tetrandrine (Fig. 6d-f). Importantly, we gered by H89/tetrandrine combination treatment were Yu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:114 Page 11 of 16 ab Fig. 6 c-Myc sensitizes cancer cells to H89/tetrandrine combination treatment. a Western blot of c-Myc in H89/tetrandrine sensitive cells (MDA-MB-231, AGS, LOVO and Hep3B) and resistant cells (Huh7, A549, HCCLM9 and HCT116). b HCT116 and A549 cells engineered to overexpress c-Myc and exposed to H89/tetrandrine combined treatment. Cell viabilities and PARP activation were determined at 72 h and 48 h, respectively. c Apoptosis was detected by flow cytometry in HCT116 c-Myc overexpressing cells after H89/tetrandrine combination treatment for 48 h. d AGS and MDA-MB-231 cells were stably transduced with lentiviral vectors that expressed c-Myc shRNAs (#1 or #2) and negative control vector PLKO.1 (shCtrl); c-Myc protein levels were subsequently detected to confirm the knockdown efficiency. e shRNA mediates c-Myc knockdown in AGS and MDA-MB-231 cells undergoing H89/ tetrandrine combination treatment. Cell viabilities and PARP were determined as previously described. f MDA-MB-231 knockdown of c-Myc following treatment with H89/tetrandrine for 48 h. Apoptosis was detected by Annexin V/PI staining. g c-Myc and Mcl-1 levels were determined by Western blot in AGS and MDA-MB-231 cells after knockdown of c-Myc and HCT116, A549 cells overexpressing c-Myc. h Western blot of LC3 in AGS and MDA-MB-231 c-Myc knockdown cells following H89/tetrandrine combination treatment for 24 h. i AGS and MDA-MB-231 cells were transduced with c-Myc shRNA#1 or shCtrl. Intracellular ROS were determined by flow cytometry after H89/tetrandrine treatment for 24 h. Data are reported as the mean ± SD and were analyzed by Student’s t-test; all data represent at least n = 3 independent experiments; *P < 0.05, **P < 0.01. All images are representative of at least three independent experiments suppressed (Fig. 6h and i). In contrast, ectopically Together, these data indicate that c-Myc amplification expressed c-Myc in HCT116 and A549 cells significantly downregulates Mcl-1 expression and increases intracellular elevated intracellular ROS (Additional file 1: Figure S3B). ROS, which contributes to H89/tetrandrine sensitivity. Yu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:114 Page 12 of 16 Combination of H89 and tetrandrine causes tumor Bim and Bid, and downregulated the expression of the regression in xenograft models anti-apoptotic protein Mcl-1. Surprisingly, H89/tetrandri- To assess the therapeutic efficacy of H89 and tetrandrine ne-induced cell death could not be completely reversed by combination therapy in vivo, we established MDA-MB-231 the apoptosis inhibitor z-VAD-fmk, which implies that subcutaneous tumor xenograft models with female athymic apoptosis was not the only contributor. Autophagy is nude mice. After six days, tumor-bearing mice were involved in type-II programmed cell death, particularly randomly assigned to four groups and were administered in apoptosis-deficient cells, and may be exploited to − 1 − 1 vehicle, H89 (10 mg·kg ), tetrandrine (25 mg·kg )or suppress tumor growth [47, 48]. Our results showed the combination treatment every other day for 28 days. At that H89/tetrandrine-induced cell death was moderately 18 days, treatment with H89 or tetrandrine inhibited the diminished by the autophagy inhibitor, which demonstrates growth of tumor xenografts; however, there was an the contribution of autophagy to cell death in response to enhanced effect in the combination group (Fig. 7a). treatment. Therefore, a combination of agents that induce Consistent with the tumor volumes, the mean tumor both apoptotic and autophagic cell death may have greater weights substantially decreased in the H89/tetrandrine advantages during the treatment of cancer. group compared with the other groups (Fig. 7b). Notably, H89 is a strong PKA inhibitor. Moreover, our study the body weight measurement indicated that the dose of suggests that PKA and ERK signaling are involved in the H89/tetrandrine combination was tolerable to the animals response to the PKA kinase inhibitor H89 and tetran- (Additional file 1: Figure S4A). Furthermore, TUNEL drine synergistic anti-tumor activity. H89/tetrandrine assays showed significant apoptosis in the tumor tissue resulted in almost complete abrogation of the expression from the animals treated with H89/tetrandrine (Fig. 7c). of phosphorylated CREB1. Moreover, pretreatment with The level of lipid peroxidation product MDA, which indi- the adenylyl cyclase activator FSK partially rescued cells cates the level of oxidative stress in tissues, was increased from death, which suggests that combined treatment in the H89/tetrandrine group (Fig. 7d). Consistent with exerted anti-tumor effects in a cAMP/PKA-dependent the observations in vitro, H89/tetrandrine increased the manner. To mimic the H89-mediated inhibition of PKA, levels of cleaved PARP and LC3-II and decreased the PKI (14–22) amide, another PKA special inhibitor, was levels of Mcl-1 in vivo (Fig. 7e). Next, to examine the role used in combination with tetrandrine to treat AGS, 769-P of Mcl-1 in H89/tetrandrine combination treatment in and 786-O cells (Additional file 1: Figure S2D). PKI and vivo, we performed xenograft assays in MDA-MB-231 ec- tetrandrine also acted synergistically on cancer cells, topically expressing Mcl-1. The tumor regression in which implies that the suppression of PKA activity plays a response to H89/tetrandrine was statistically significant in role in the anti-tumor activity of H89 plus tetrandrine. the control tumors, but not in the Mcl-1 overexpressing However, the relationship between PKA and ERK tumors (Fig. 7f and g). Tumor lysates showed the Mcl-1 signaling was not investigated in this study. Previous overexpression group exhibited decreased cleavage of reports have demonstrated that cAMP increases inhibitory PARP in response to H89/tetrandrine treatment (Fig. 7h). Raf-1 phosphorylation at Ser-259 and reduces activating Again, the combination therapy was well tolerated by Raf-1 phosphorylation at Ser-338 in a PKA-dependent evidence of weight sustainability (Additional file 1: manner, thereby inducing ERK deactivation [49, 50], which Figure S4B). To be consistent with the previous experi- is consistent with our finding that H89 induced PKA ment, we continued to experiment with MDA-MB-231 inhibition and tetrandrine induced ERK activation are and established xenograft models using c-Myc knockdown concomitantly involved in H89/tetrandrine combination MDA-MB-231 cells. The in vivo results showed that treatment induced cell death. c-Myc depletion exhibited resistance to H89/tetrandrine Mcl-1 is a pro-survival member of the Bcl-2 family treatment (Fig. 7i and j). Consistent with the in vitro find- and is highly expressed in various types of malignancy. ings, c-Myc knockdown increased the Mcl-1 expression in Thus, Mcl-1 has emerged as a promising target for cancer vivo (Fig. 7k). The combination-treated mice did not ex- treatment . In our study, we determined that H89/tet- hibit a reduction in weight gain during the treatment randrine treatment synergistically inhibited Mcl-1 in period (Additional file 1: Figure S4C). cancer cells at both the transcription and protein expres- sion levels. Furthermore, we identified that Mcl-1 plays an Discussion important role in H89/tetrandrine anti-tumor activity in Apoptosis, defined as type-I programmed cell death (PDC), vitro and in vivo. Mechanistically, Mcl-1 appears to inhibit is considered to be a major route by which chemotherapeu- apoptosis by preventing mitochondrial dysfunction, with a tic agents eradicate cancer cells [45, 46]. In this study, we limited effect on autophagy. However, it is not clear why showed that H89/tetrandrine activated caspase-dependent the expression of the anti-apoptotic protein Mcl-1 was de- apoptosis through mitochondrial-mediated pathways, creased in response to H89/tetrandrine treatment. This upregulated the expression of the pro-apoptotic proteins finding must be investigated in our future studies. Yu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:114 Page 13 of 16 ab c de f gh i jk Fig. 7 Combination treatment with H89 and tetrandrine leads to a regression of xenograft tumors. a MDA-MB-231 cells were inoculated into BALB/c mice (via s.c. injection) to establish a tumor model as indicated in the Materials and Methods section. Mice bearing tumors were randomly assigned to groups (six mice per group) and treated with vehicle, H89 (10 mg/kg), tetrandrine (25 mg/kg) alone or a combination of H89 and tetrandrine (H89 10 mg/kg and tetrandrine 25 mg/kg) every other day. The tumor volume was measured (the bars represent the means ± SD). b Scatter plots display the quantitative tumor weights at the end of the experiment. c MDA-MB-231 transplanted tumors were dissected and subjected to a TUNEL assay. Scale bars, 100 μm. d The level of the oxidative stress marker MDA was measured in the tumor tissues. e Cleaved-PARP, Mcl-1 and LC3 were analyzed by Western blot from tumor tissue lysates. f BALB/c mice (n = 6) were transplanted with MDA-MB-231 Ctrl or Mcl-1 overexpressing cells and treated with DMSO or H89/tetrandrine (H89 10 mg/kg and tetrandrine 25 mg/kg) every other day; tumor volumes were measured during the study. g Scatter plots display the quantitative tumor weights. h Western blot of tumor lysates from MDA-MB-231 Ctrl or Mcl-1- bearing mice with the indicated treatments. i BALB/c mice (n = 6) were transplanted with MDA-MB-231 shCtrl or shc-Myc cells and treated as previously described, and the tumor volume and j tumor weight are shown. k Western blot of tumor lysates from MDA-MB-231 shCtrl or shc-Myc-bearing mice with the indicated treatments. Data are reported as the mean ± SD and were analyzed by Student’st-test; n =6 mice per group; *P <0.05, **P <0.01 and NS = not significant. All images are representative of six mice per group c-Myc, a commonly activated oncogene, also increases in mouse xenograft models. We showed that the knock- cellular susceptibility to apoptosis . In this study, we down of c-Myc significantly increased the Mcl-1 expres- interestingly determined that c-Myc-overexpressing sion, and the overexpression of c-Myc decreased the Mcl-1 cancer cells are more sensitive to H89/tetrandrine combin- levels, which indicates that c-Myc regulating sensitivity to ation therapy. Consistently, the knockdown of c-Myc H89/tetrandrine may be associated with downregulating attenuated the sensitivity to H89/tetrandrine in vitro and Mcl-1. Other researchers have previously reported that Yu et al. Journal of Experimental & Clinical Cancer Research (2018) 37:114 Page 14 of 16 oncogenes such that c-Myc activation induced DNA dam- Additional file age in human normal fibroblasts, which was correlated Additional file 1: Supplementary figures and figure legends. (DOCX 1246 kb) with the induction of ROS without induction of apoptosis . Having uncovered that c-Myc regulates ROS gen- eration in cancer cells and affects chemotherapeutic Abbreviations 3-MA: 3-methyladenine; DCFH-DA: 2′,7′-dichlorodihydrofluorescein diacetate; sensitivity, in this study, we determined that c-Myc FSK: Forskolin; NAC: N-acetyl-L-cysteine; PKA: Protein kinase A; PKI: PKI (14–22) knockdown or ectopic expression significantly diminished amide (myristoylated); ROS: Reactive oxygen species; Tet: Tetrandrine or increased ROS generation, respectively. These findings Funding may explain why c-Myc amplified cells are more sensitive This project was supported by The National Basic Research Program of China to H89/tetrandrine treatment. Although intracellular ROS (2014CB910600), The National Natural Science Foundation of China were increased in AGS and MDA-MB-231 shc-Myc cells (81273540 and 81472684) and Fundamental Research Funds for the Central Universities (2042017KF0242). when treated with H89/tetrandrine, the levels remained lower than in shCtrl cells, which implies that an appropriate Availability of data and materials ROS threshold is necessary for H89/tetrandrine induced All data analyzed during this study are included in this manuscript. cell death. Authors’ contributions MY conducted the experiments, created the figures and wrote the manuscript; TL and YC performed the research and analyzed and interpreted Conclusions the data; YL performed the data analyses and reviewed the manuscript; WL Our data indicate that H89/tetrandrine showed a synergistic supervised and designed the research, analyzed and interpreted the data and co-wrote the manuscript. All authors reviewed the results and approved anti-tumor activity by inducing concomitant cell apoptosis the final version of the manuscript. and autophagy in vitro and in vivo. The potential molecular mechanisms involved ROS regulated PKA and ERK Ethics approval and consent to participate signaling and the anti-apoptotic protein Mcl-1 (Fig. 8). For the animal study, all animal care and experiments were approved by the Experimental Animal Center of Wuhan University. Furthermore, c-Mycamplifiedcells aremoresensitive to H89/tetrandrine combined treatment. 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Published: Jun 4, 2018