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Inhibition of caspase-9 by oridonin, a diterpenoid isolated from Rabdosia rubescens, augments apoptosis in human laryngeal cancer cells

Inhibition of caspase-9 by oridonin, a diterpenoid isolated from Rabdosia rubescens, augments... INTERNATIONAL JOURNAL OF ONCOLOGY 47: 2045-2056, 2015 Inhibition of caspase-9 by oridonin, a diterpenoid isolated from Rabdosia rubescens, augments apoptosis in human laryngeal cancer cells 1,3 4 4 3 5 NING KANG , SHI-JIE CAO , YAN ZHOU , HAO HE , SHIN-ICHI TASHIRO , 6 2 3 SATOSHI ONODERA , FENG QIU and TAKASHI IKEJImA 1 2 School of Integrative medicine, College of Traditional Chinese medicine, Tianjin University of Traditional Chinese medicine, Tianjin 300193; China-Japan Research Institute of medical and Pharmaceutical Sciences, Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China; Institute for Clinical and Biomedical Sciences, Kyoto 603-8072; Department of Clinical and Biomedical Science, Showa Pharmaceutical University, Tokyo 194-8543, Japan Received July 16, 2015; Accepted September 11, 2015 DOI: 10.3892/ijo.2015.3186 Abstract. Rabdosia rubescens, a commonly used traditional activation of both the caspase-9-independent mitochondrial Chinese medicine, has increasingly gained attention for its pathway and death receptor pathways, and the autophagy had use as an antitumor herb. Oridonin, a bioactive diterpenoid an anti-apoptotic function in oridonin-treated HEp-2 cells. isolated from Rabdosia rubescens, has been reported to These collective results suggest that oridonin targets caspase-9 induce apoptosis in human laryngeal cancer HEp-2 cells by to alter ROS production and autophagy situation to promote our group. Here, we made unexpected observations that the HEp-2 cell apoptosis. Therefore, oridonin has the potential to caspase-9 inhibitor (C9i) enhanced apoptosis in response to be developed as an anticancer agent, and the combination of selected stimuli, and HEp-2 cells which were made dec fi ient oridonin with those agents leading to reduction of caspase-9 in caspase-9 using siRNA exhibited no resistance to apop- expression in tumor cells could represent a novel approach to totic signals and actually demonstrated increased apoptotic human laryngeal cancer treatment. sensitivity to oridonin. The results were reversed by the trans- fection of an exogenous caspase-9 expression vector. Caspase-9 Introduction reduced sensitivity to apoptotic stimuli through reactive oxygen species (ROS)-suppressing and autophagy-promoting Apoptosis (also known as type I programmed cell death) is a methods. ROS triggered the progression of apoptosis through cellular suicide program critical for development and tissue homeostasis (1). Excess apoptosis is associated with degen- erative disorders (2), while a failure of apoptosis contributes to oncogenesis (3). Anticancer agents can induce apoptosis Correspondence to: Dr Takashi Ikejima, China-Japan Research in tumor cells that is mediated by the common cell death Institute of m edical and Pharmaceutical Sciences, Shenyang Pharma- machinery. Central to this is a family of intracellular proteases, ceutical University, 103 Wenhua Road, Shenyang 110016, P.R. China known as caspases. During apoptosis, they can act either as E-mail: [email protected] initiators (caspases-8 and -9) in response to apoptotic signals or as effectors (caspase-3, -6 and -7) that finally cleave a number Dr Feng Qiu, College of Traditional Chinese medicine, Tianjin University of Traditional Chinese medicine, 312 Anshanxi Road, of vital proteins and lead to the demise of the cells (1). ROS are Nankai District, Tianjin 300193, P.R. China generated from the mitochondria and other cellular sources, E-mail: [email protected] and can oxidize a wide range of cell constituents, including lipids, proteins and DNA, thus damaging cell structures and Abbreviations: ROS, reactive oxygen species; C9i, caspase-9 compromising function. When antioxidant mechanisms are inhibitor; NAC, N-acetylcysteine; 3-m A, 3-methyladenine; LSCC, overwhelmed by ROS and subsequent oxidative stress occurs, laryngeal squamous cell carcinoma; PI, propidium iodide; m DC, cell damage and cell death are induced (4). High levels of ROS monodansylcadaverine; siRNA, small interfering RNA; CAT, induce cell death, which often involves apoptosis through catalase; FADD, Fas-associated protein with death domain; CAD, caspase activation (5,6). caspase-activated DNase; ICAD, inhibitor of caspase-activated As a mode of type II programmed cell death, autophagy is DNase a series of biochemical steps through which eukaryotic cells Key words: oridonin, caspase-9, apoptosis, autophagy, reactive oxygen commit suicide by degrading their own cytoplasm and organ- species elles through a process in which these components are engulfed and then digested in double membrane-bound vacuoles called KANG et al: INHIBITION OF CASPASE-9 AUGm ENTS APOPTOSIS autophagosomes (7). However, autophagy has recently gained (20). Oridonin was isolated from Rabdosia rubescens and much attention for its paradoxical roles in cell survival and cell was identified by comparing its physical and spectroscopic 1 13 death, particularly in the pathogenesis as well as the treatment ( H Nm R, C Nm R) data with those reported in the literature of cancer (8,9). Whether autophagy enables cells to survive or (21). The purity was measured by HPLC (column: 4.6x150 mm, enhances their death is context-driven, depending on the type Inertsil ODS-SP, 5 µm; solvent phase: methanol/H O, 55:45) of stimuli, nutrient availability, and apoptotic status (10). and determined to be 99.6%. Oridonin was dissolved in Dm SO Laryngeal squamous cell carcinoma (LSCC) is the most to obtain a stock solution. The Dm SO concentration was kept common squamous cell carcinoma of the head and neck, and below 0.05% in all the cell cultures so that it had no detectable there is a steady annual increase in new cases and deaths (11). effect on cell growth or cell death. Epidemiological studies have revealed that the incidence rates vary from country to country and in different population Growth inhibition assay. HEp-2 cells were incubated in groups (12). LSCC accounts for 1-8.4% of all human cancers 96-well tissue culture plates. After a 24-h incubation, the cells in China. However, only a minority of patients are eligible for were treated with, or without, pan-caspase inhibitor (z-VAD- radical treatment aimed at a cure. The availability of new cyto- fmk), caspase-9, -8, -3, -1 inhibitors [Ac-LEHD-cmk (C9i), toxic drugs has led to steady improvements, but a paradigm z-IETD-fmk, z-DEVD-fmk or Ac-YVAD-cmk], 3-m A or shift is required to signic fi antly affect the poor prognosis of NAC (Sigma) at the given concentrations for 1 h and subse- most patients. quently treated with oridonin for 24 h. The cytotoxic effect With a long history of cancer treatment, traditional Chinese was measured by m TT assay as described elsewhere (20). medicine (TCm ) is recognized as a valuable source for seeking bioactive anticancer compounds (13). Rabdosia rubescens Observation of morphological changes. HEp-2 cells were (Hemsl.) Hara (Lamiaceae), also known as Dong Ling Cao seeded into 6-well culture plates. After a 24-h incubation, in TCm , is commonly used in Chinese folk medicine to treat the cells were treated with, or without, C9i or NAC at the stomach aches, pharyngitis, sore throats, coughs and wrestling given concentrations for 1 h and subsequently incubated with injuries. This herb has increasingly gained attention because oridonin for 24 h. The cellular morphology was observed using of its use as an antitumor herb and because of the antitumor a phase contrast microscope (Leica, Nussloch, Germany). activities of its discovered bioactive constituents. The herb contains a variety of active components, including diterpe- Transmission electron microscopy. HEp-2 cells were treated noids, flavonoids, phenolic acids, triterpenoids and volatile with 36 µm oridonin for the indicated time periods. The oils (14). Among these compounds, the diterpenoid compound collected cells were fixed with PBS containing 3% glutar - oridonin (Fig. 1) was reported to possess inhibitory effects aldehyde, and postfixed with PBS containing 1% OsO . The on a variety of cancer cell lines, such as leukemia, colorectal samples were dehydrated in graded alcohol solutions, then cancer, esophageal cancer, liver cancer, epidermoid carcinoma, embedded and sectioned. Ultrathin sections were stained lung cancer and uterine cervical cancer (15,16). However, no with uranyl acetate and lead citrate, and examined using a reports has yet been found on its anticancer effects in human JEm -1200 transmission electron microscope (Jeol, Tokyo, laryngeal cancer cells until our laboratory demonstrated that Japan) (22). oridonin could induce apoptosis and G2-m phase arrest in human laryngeal squamous carcinoma HEp-2 cells (17). Flow cytometric analysis of cell apoptosis. After chemical During the course of characterizing the role of the mito- treatment, 1x10 cells were harvested. The collected cells were chondrial pathway in oridonin-induced apoptosis in HEp-2 fixed in 70% ethanol, stained for DNA content with propidium cells, we made an unexpected observation that a selective iodide (PI), and measured by flow cytometry (Becton- inhibitor of caspase-9 (C9i) could enhance (rather than retard) Dickinson, Franklin Lakes, NJ, USA) as previously described apoptosis in response to selected stimuli. Interestingly, our (20). The sub-G1 DNA content was used as an indicator of group also found that caspases did not mediate apoptosis, but apoptosis (23). protected L929 cells from oridonin-induced cell death (18). m oreover, Shah et al also found that a caspase-9 inhibitor Measurement of intracellular ROS generation. After treat- could enhance stress-induced apoptosis in B-lineage cells (19). ment with 36 µm oridonin for the indicated time periods, the Driven by the above-mentioned interesting phenomena, we set cells were incubated with 10 mM DCF-DA (Sigma) at 37˚C for out to delineate the signaling pathways and specic fi ally the role 30 min. The intracellular ROS caused oxidation of DCF-DA to of caspase-9 in oridonin-induced apoptosis in greater detail. the u fl orescent compound DCF. Then, the cells were harvested Our data showed that caspase-9 played an anti-apoptotic role and the pellets were suspended in 1 ml PBS. Samples were in HEp-2 cells through inhibition of ROS generation. Further, analyzed at an excitation wavelength of 480 nm and an emis- we discovered that caspase-9 promoted oridonin-induced sion wavelength of 525 nm by FACScan flow cytometry (18). autophagy, and in this context, autophagy was a protective mechanism against apoptosis. Determination of mitochondrial membrane potential. The mitochondrial membrane potential was measured using the Materials and methods u fl orescent dye rhodamine-123 (Sigma) by o fl w cytometry as previously described (20). Cell culture and reagent treatment. Human laryngeal cancer HEp-2 cells were obtained from the American Type Cell Measurement of autophagy. After incubation with 36 µm Culture Collection and were cultured as previously described oridonin for the fixed times, cells were cultured with 0.05 mM INTERNATIONAL JOURNAL OF ONCOLOGY 47: 2045-2056, 2015 against Beclin 1, LC3, Bax, Bcl-2, AIF, cytochrome c, ICAD, FADD, caspase-9, -8, -3, Hsp90, β-actin (Santa Cruz). Representative blots from three independent experiments are shown. Statistical analysis of the data. All the presented data and results were confirmed in at least three independent experi - ments. The data are expressed as means ± SD. Statistical comparisons were made by Student's t-test and P<0.05 was Figure 1. Chemical structure of oridonin. considered statistically signic fi ant. Results monodansylcadaverine (MDC) at 37˚C for 60 min. The cellular u fl orescent changes were observed under a u fl ores- The apoptotic effects of oridonin in HEp-2 cells are enhanced cence microscope (Olympus). The u fl orescence intensity of by an irreversible inhibitor of caspase-9. Some of our prelimi- cells was analyzed by FACScan flow cytometry (24). nary studies have indicated that oridonin is able to inhibit the proliferation of human laryngeal squamous carcinoma HEp-2 Assay for caspase-9 activity. A caspase-9 u fl orimetric assay cells, and the cell death is shown to be typical of apoptosis kit from Chemicon International (Temecula, CA, USA) was (17,20). Since it is well known that caspase family members used according to the manufacturer's instructions. Briey fl , cell play a pivotal role in the cell apoptosis process, the effect of lysates were incubated with peptide substrate, LEHD-pNA caspase inhibitors on oridonin-induced HEp-2 cell death was (Ac-Leu-Glu-His-Asp-pNA), in assay buffer [100 mm NaCl, examined. Unexpectedly, the C9i enhanced cell death due 50 mm HEPES, 10 m m DTT, 1 m m EDTA, 10% glyc - to oridonin stimuli, while z-DEVD-fmk, z-IETD-fmk and erol, 0.1% CHAPS (pH 7.4)] for 2 h at 37˚C. The release of Z-VAD-fmk blocked the cytotoxity of the response to oridonin p-nitroaniline was monitored at 405 nm. Caspase-9 activity in (Fig. 2A). cell lysates was expressed as the x-fold increase from baseline Further, we investigated whether inhibition of caspase-9 controls. affected drug sensitivity. As shown in Fig. 2B, pre-treatment of HEp-2 cells with 5-20 µm C9i markedly enhanced the RNA interference. HEp-2 cells were transfected with caspase-9 inhibitory ratio in a dose-dependent manner after treatment small interfering RNA (siRNA) and control siRNA (Shanghai with oridonin. In addition, we examined cellular morpho- GenePharma, China) by Lipofectamine 2000 (Invitrogen) logical changes by phase contrast microscopy. At 24 h, some according to the manufacturer's instructions. The sequences of oridonin-treated HEp-2 cells became round, with shrunken of the sense strands of the RNAs used in this study were as nuclei and membrane blebbing, while untreated cells did follows. Control siRNA: UUC UCC GAA CGU GUC ACG. not show these apoptotic characteristics (Fig. 2C). Whereas, Caspase-9 siRNA-1: CGG UGA AAG GGA UUU AUA ATT. the C9i-pre-treated group showed more marked apoptotic Caspase-9 siRNA-2: CCA AAG UUG UCG AAG CCA ATT changes in a dose-dependent manner (Fig. 2C). Quantic fi ation CGU UGA. Caspase-9 siRNA-3: GUG ACA UCU UUG UGU of apoptotic cells by flow cytometric analysis also showed CCU ATT. that combined treatment with oridonin and C9i caused more significant apoptosis, and the apoptotic ratio was observed Transient transfection. HEp-2 cells at 70% confluence to be signic fi antly increased from 15.08% in oridonin alone- were transfected with control pcDNA3.1-3FLAG and treated cells to 23.59, 31.54 and 37.26% in 5, 10 and 20 µm C9i pcDNA3.1-hCASP9-3FLAG plasmid (Shanghai GeneChem, pre-treated cells, respectively (Fig. 2D). China). Transfection of cells was performed by using Lipofectamine 2000 reagent according to the manufacturer's Oridonin-induced increase in ROS generation is enhanced protocol. The effectiveness of transfection was detected by by inhibiting caspase-9. Dysregulation of cellular redox status western blot analysis. can be a potent mechanism of cell death (26). Therefore, we tested the possibility that oridonin, with or without C9i, Preparation of mitochondrial and cytosolic extracts. HEp-2 induces apoptosis through ROS accumulation. Compared with cells were collected and then washed twice with ice-cold PBS. the control group, ROS generation was signic fi antly increased The cell pellets were resuspended in ice-cold homogenizing after exposure to oridonin for 24 h. C9i effectively increased buffer (250 mm sucrose, 20 m m H EPES, 10 mm KCl, 1.5 m m oridonin-induced intracellular ROS generation (Fig. 3A and B). mgCl , 1 mm EDTA, 1 m m EGTA, 1 m m dithiothreitol, As expected, the use of NAC, a well known ROS scavenger 0.1 mM phenylmethanesulfonyl u fl oride, 10 µg/ml pepstatin (27), almost completely blocked ROS generation induced by and 10 µg/ml leupeptin). The cells were then homogenized and oridonin alone or in combination with C9i (Fig. 3A and B). centrifuged at 14,000 g at 4˚C for 60 min. The supernatant was Whereas, NAC not only completely prevented HEp-2 cell used as the cytosol fraction and the pellet was resuspended in apoptosis induced by oridonin alone, but also reversed the cell lysis buffer as the membrane fraction (25). apoptosis induced by combined treatment with oridonin and C9i (Fig. 2C and D). Collectively, these results demonstrated Western blot analysis. Western blot analysis was performed that caspase-9 inhibition amplie fi d oridonin-induced oxidative as previously described (20) using corresponding antibodies stress, which leads to enhanced apoptosis. KANG et al: INHIBITION OF CASPASE-9 AUGm ENTS APOPTOSIS Figure 2. The growth-inhibitory ratio and apoptotic ratio were enhanced by C9i but inhibited by NAC in oridonin-treated HEp-2 cells. (A) The cells were treated with oridonin for 24 h, in the presence or absence of caspase-9, -8, -3 and -1 inhibitors or pan-caspase inhibitor, and the inhibitory ratio was measured by m TT assay, n=3, mean ± SD. After the cells were incubated with oridonin in the absence or presence of C9i or NAC for 24 h, the inhibitory ratio was measured by MTT assay, n=3, mean ± SD (B), and the morphological changes were examined by phase contrast microscopy; scale bar, 10 µm (C). The flow ** ## cytometric quantic fi ation of apoptotic cells (sub-G1 fraction) is shown (D). P<0.01 vs oridonin alone-treated group; P<0.01 vs oridonin co-treated with 5 µm C9i group. Oridonin triggers a marked loss of ∆ψm and caspase-9 Fig. 3C and D, exposure of HEp-2 cells to oridonin for 24 h inhibition produces a progressive loss of ∆ψm. It has been caused a marked loss of ∆ψm compared with the control reported that ROS generation can lead to mitochondrial group and C9i pre-treatment produced a progressive loss of damage and membrane depolarization (28). Therefore, we ∆ψm. Notably, pre-treatment with NAC reversed the disrup- investigated whether ROS targeted the mitochondria and tion in ∆ψm induced by oridonin alone or in combination thereby decreased the ∆ψm in our model. As shown in with C9i (Fig. 3C and D). These results suggested that the INTERNATIONAL JOURNAL OF ONCOLOGY 47: 2045-2056, 2015 Figure 3. C9i amplie fi s the ROS production induced by oridonin, and the ROS subsequently triggers the progression of apoptosis in HEp-2 cells. The cells were cultured in the presence of 36 µΜ oridonin for 0 or 24 h or co-incubated with 5 µΜ C9i or 2.5 mM NAC for 24 h. Then, ROS generation (A) and the changes in ∆ψm (C) were measured by o fl w cytometry. The corresponding linear diagrams of the FACScan histograms are shown in (B) and (D) respectively. The values are mean ± standard errors (n= 3 of individual experiments). The cells were treated with oridonin in the absence or presence of 2.5 mm NAC or 500 U/ml CAT for the indicated time periods, then Bax, Bcl-2, cytochrome c, AIF (E) and FADD, caspase-8, -3, ICAD (F) protein expression was detected ** ## by western blot analysis. The results shown here are representative of at least three independent experiments. P<0.01 vs control group. P<0.01 vs oridonin alone-treated group. oridonin, with or without C9i, was capable of inducing Oridonin-induced Bax activation, Bcl-2 degradation, cyto- mitochondrial dysfunction and that the loss of ∆ψm might be chrome c and AIF elevation are markedly blocked by NAC affected directly by ROS generation. and CAT treatment. To determine the possible causes and KANG et al: INHIBITION OF CASPASE-9 AUGm ENTS APOPTOSIS Figure 4. Caspase-9 facilitates oridonin-induced autophagy, and inhibition of autophagy upregulates apoptosis. (A) The cells were incubated with oridonin for 0 or 24 h, or co-incubated with 3-m A for 24 h. Then, the cellular morphological changes were observed by transmission electron microscopy (arrow indicates autophagic vacuoles; scale bar, 1 µm) or by u fl orescence microscopy with MDC staining (scale bar, 10 µm). The cells were cultured in the presence of oridonin or co-incubated with 1 mM 3-MA (B) or 5 µM C9i (C) for 24 h. The corresponding column or o fl w cytometric histograms of autophagic level changes are represented. (D) The cells were treated with oridonin in the presence or absence of 3-m A or C9i for the indicated time periods, followed by western blot analysis for Beclin 1 and LC3 levels. The results shown here are representative of at least three independent experiments. (E) The cells were treated with oridonin for 24 h, in the presence or absence of 0.4-1.6 mm 3- m A, and the inhibitory ratio was measured by m TT assay, n=3, mean ± SD. (F) The cells were * ** treated with oridonin for 24 h in the presence or absence of 1 mM 3-MA. The sub-G1 fraction was analyzed by flow cytometry. P <0.05 or P<0.01 vs group treated with oridonin alone. consequences of the ∆ψm decrease, we studied the presence chondrial permeability and two other proteins, cytochrome of two proteins, pro-apoptotic protein Bax and anti-apoptotic c and AIF, which are usually released from mitochon- protein Bcl-2, which are involved in the regulation of mito- dria during apoptosis. We found that oridonin treatment INTERNATIONAL JOURNAL OF ONCOLOGY 47: 2045-2056, 2015 signic fi antly increased and decreased levels of Bax and Bcl-2 proteins, respectively, in a time-dependent manner (Fig. 3E). The expression of cytochrome c was markedly elevated. Simultaneously, a visible increase in AIF levels was also detectable after 12- and 24-h incubation (Fig. 3E). However, NAC and catalase (CAT, hydrogen peroxide decomposer) pre-treatment completely reversed the changes in Bax, Bcl-2, cytochrome c and AIF (Fig. 3E). Changes stimulated by oridonin in FADD, caspase-8, caspase-3 and ICAD expression are inhibited by NAC and CAT. To assess whether the Fas-mediated pathway was activated in oridonin-treated cells, the expressions of Fas-associated protein with death domain (FADD), caspase-8 and -3 were determined by western blot analysis. The expres- sion of FADD was markedly elevated and there was clear cleavage of procaspase-8 after oridonin administration. A time-dependent cleavage of procaspase-3 was also observed in oridonin-treated cells (Fig. 3F). ICAD, an inhibitor of caspase-activated DNase (CAD), is a substrate for caspases such as caspase-3. When caspase-3 is activated by apoptotic stimuli, ICAD is cleaved, resulting in the release of CAD which appears to cause DNA fragmentation in the nuclei (29). As shown in Fig. 3F, oridonin induced degradation of ICAD in HEp-2 cells in a time-dependent manner. Addition of NAC and CAT markedly inhibited the changes in FADD, caspase-8, -3 Figure 5. Time-course of activation of caspase-9 is not dependent on proteo- and ICAD (Fig. 3F). lytic cleavage. (A) The cells were treated with oridonin for the indicated time periods, followed by western blot analysis for detection of caspase-9, Hsp90 Caspase-9 facilitates oridonin-induced autophagy. We first and cytochrome c (both in the cytosol and the mitochondria) levels. The results shown here are representative of at least three independent experi- investigated the effect of oridonin on cell autophagy. The ments. (B) Equivalent cultures were prepared and the cells were collected ultrastructural details displayed that the oridonin-treated to assess the enzymic activity of caspase-9 as described in m aterials and ** cells possessed typical characteristics of autophagy. These methods, n=3, mean ± SD. P<0.01 vs group treated with oridonin for 24 h. characteristic changes included extensive cytoplasmic vacu- olization, and some autophagic vacuoles contained degraded organelles (Fig. 4A). The formation of autophagic vacuoles was further assessed by MDC, a u fl orescent dye specic fi ally tion in the number of MDC-labeled u fl orescent particles in the staining autophagosomes. As shown in Fig. 4A, control cells cells (Fig. 4A). Flow cytometric analysis also indicated that presented diffused staining, and oridonin treatment resulted pre-treatment with 3-m A markedly reduced the autophagic in an extensive punctuate m DC staining pattern. Next, the ratio compared with the group treated with oridonin alone levels of Beclin 1 and LC3, two important proteins involved (Fig. 4B). Simultaneously, oridonin-induced Beclin 1 acti- in autophagy (10,30), were examined by western blot analysis. vation as well as the modic fi ation of LC3-I to LC3-II were As shown in Fig. 4D, the level of Beclin 1 and conversion markedly blocked when the cells were pre-treated with from LC3-I to LC3-II both increased with time after oridonin 3-m A (Fig. 4D). However, pre-treatment of HEp-2 cells with administration. These findings apparently reveal that 0.4-1.6 mm of 3- m A dramatically increased the inhibitory autophagy is induced as a response of HEp-2 cells to oridonin rate of cell growth in a dose-dependent manner after oridonin treatment. treatment (Fig. 4E). Further, inhibition of autophagy increased To explore the involvement of caspase-9 in the modula- the oridonin-induced sub-G1 cell proportion (an index of tion of autophagy, the autophagic ratio was measured by o fl w apoptosis) in HEp-2 cells (Fig. 4F). Collectively, these results cytometry. Compared with the oridonin treatment group, the suggested that 3-m A inhibited oridonin-mediated autophagy autophagic ratio was signic fi antly reduced by the combined and it also sensitized the cells to the cytotoxic actions of use of oridonin and C9i (Fig. 4C). Accordingly, the treatment oridonin with induction of apoptosis. In short, oridonin- of C9i markedly suppressed Beclin 1 upregulation and the induced autophagy inhibits apoptosis in our model. conversion from LC3-I to LC3-II (Fig. 4D), suggesting the autophagy-promoting effects of caspase-9. Evaluation of the expression pattern of critical apoptosome complex-related proteins. In the cytosol, cytochrome c binds Inhibition of autophagy upregulates apoptosis induced by to Apaf-1, allowing recruitment of caspase-9 and formation of oridonin in HEp-2 cells. To investigate the role of autophagy apoptosome, resulting in caspase-9 activation and execution of in oridonin-induced apoptosis in HEp-2 cells, 3-MA, a specic fi cell death (31,32). Here, the expression of critical apoptosome inhibitor of autophagy, was introduced. Treatment with 3-m A complex-related proteins was examined by means of western prior to the addition of oridonin induced a signic fi ant reduc - blot analysis. As shown in Fig. 5A, the level of cytochrome c KANG et al: INHIBITION OF CASPASE-9 AUGm ENTS APOPTOSIS Figure 6. Apoptotic sensitivity to oridonin in HEp-2 cells made dec fi ient in caspase-9 by siRNA. (A) The cells were transfected with 25 nM caspase-9 siRNA-1, -2, -3 or control siRNA for 24 h (upper panel) or transfected with 25-75 nm caspase-9 siRNA-1 or control siRNA for 24 h (lower panel), and the caspase-9 level was examined by western blot analysis. The cells were transfected with 25 nm caspase-9 siRNA-1 or control siRNA for 24 h, followed by stimulation with oridonin for 24 h. Then, the inhibitory ratio was determined by m TT assay (n=3, mean ± SD) (B), and the proportion of sub-G1 cells (C) and ROS genera- tion (D) were measured by flow cytometry. The MDC fluorescent intensity of oridonin-treated cells was analyzed by flow cytometry (E), and the Beclin 1 and ** LC3 levels were examined by western blot analysis (F). The results shown here are representative of at least three independent experiments. P<0.01 vs control siRNA group treated with oridonin. in mitochondria began to decrease at 6 h, which was consistent Lower expression of caspase-9 augments apoptosis in with the increase in cytochrome c in the cytosol. However, oridonin-treated HEp-2 cells. To determine whether the no apparent change was observed in the protein level of results obtained with C9i could be corroborated by another pro-caspase-9, and cleaved-caspase-9 could not be detected method, we turned to RNA interference using siRNA. Here, (Fig. 5A). Recently, several investigators have described nega- the role of caspase-9 in oridonin-mediated apoptosis was tive regulation of the apoptosome complex by Hsp90 (33,34). studied by knock-down of caspase-9 using 3 different specific Therefore, the effect of oridonin on expression of Hsp90 was siRNAs. The expression of capase-9 was markedly suppressed examined. The result shows that oridonin treatment increased in HEp-2 cells when transfected with caspase-9 siRNA-1 levels of Hsp90 in a time-dependent manner in HEp-2 cells while only slight downregulation was found with the other (Fig. 5A). two siRNAs (Fig. 6A). Therefore, caspase-9 siRNA-1 was Next, we directly examined the effect of drug treat- selected as a valid candidate for the subsequent investiga- ments on caspase-9 activation using functional assays. The tion. As shown in Fig. 6A, caspase-9 siRNA-1 effectively result shows that caspase-9 activity was activated in a time- and specifically suppressed HEp-2 caspase-9 protein in a dependent manner, and the maximal activity was seen after a dose-dependent manner. In contrast, an identical amount of 24-h incubation (Fig. 5B). As expected, marked suppression control siRNA had no effect on caspase-9. We then tested of the high levels of caspase-9 was noted in cells subjected to whether caspase-9-deficient HEp-2 cells would undergo combination-treatment with C9i (Fig. 5B). a heightened response to apoptotic stimuli. As shown in INTERNATIONAL JOURNAL OF ONCOLOGY 47: 2045-2056, 2015 Figure 7. Caspase-9 overexpression attenuates apoptosis in oridonin-treated HEp-2 cells. (A) The cells were transfected with CASP9-expressing plasmid or control plasmid for 24, 48 and 72 h, and the caspase-9 level was examined by western blot analysis. The cells were transfected with CASP9-expressing plasmid or control plasmid for 48 h, followed by stimulation with oridonin for 24 h. Then, the inhibitory ratio was determined by m TT assay (n=3, mean ± SD) (B), and the proportion of sub-G1 cells (C) and ROS generation (D) were measured by o fl w cytometry. The MDC u fl orescent intensity of oridonin-treated cells was analyzed by o fl w cytometry (E), and the Beclin 1 and LC3 levels were examined by western blot analysis (F). The results shown here are representative of at ** least three independent experiments. P<0.01 vs control plasmid group treated with oridonin. Fig. 6B, caspase-9-deficient HEp-2 cells were significantly of transfection, the effectiveness of transfection was detected more sensitive to oridonin than control siRNA-treated cells. by western blotting. As shown in Fig. 7A, caspase-9 protein Quantic fi ation of apoptotic cells by o fl w cytometric analysis level was signic fi antly increased in a time-dependent manner showed that the incidence of apoptosis after oridonin treatment in the HEp-2 cells transiently transfected with the CAS9- was increased by >10% in caspase-9 siRNA-1-transfected expressing plasmid. In contrast, the control plasmid had cells when compared with control siRNA (Fig. 6C). Next, no effect on caspase-9 expression. We then tested whether the degree of ROS generation was also determined by flow increased caspase-9 expression would promote survival and cytometric analysis. The percentage of DCF-positive cells diminish apoptosis in oridonin-treated HEp-2 cells. As shown was highest in caspase-9 siRNA-1-transfected cells treated in Fig. 7B, the inhibitory ratio of control plasmid-transfected with oridonin (Fig. 6D). However, caspase-9-deci fi ent HEp-2 cells is 42.80%, while, the inhibitory ratio of CAS9-expressing cells showed a much reduced autophagic ratio after oridonin plasmid-transfected cells is 29.64%, thus demonstrating treatment when compared with those transfected with control caspase-9-mediated increase in survival. Moreover, quantifi - siRNA (Fig. 6E). Accordingly, oridonin treatment, with or cation of apoptotic cells by o fl w cytometric analysis showed without control siRNA, enhanced both the expression level that the incidence of apoptosis after oridonin treatment was of Beclin 1 and the conversion from LC3-I to LC3-II, while decreased by nearly 10% in CAS9-expressing plasmid-trans- these enhancements were markedly suppressed in HEp-2 cells fected cells when compared with control plasmid (Fig. 7C). transfected with caspase-9 siRNA-1 (Fig. 6F). Next, the degree of ROS generation was also determined by flow cytometric analysis. The percentage of DCF-positive Caspase-9 overexpression attenuates apoptosis in oridonin- cells was signic fi antly decreased in CAS9-expressing plasmid- treated HEp-2 cells. To test the ability of caspase-9 to inhibit transfected cells compared with control plasmid (Fig. 7D). oridonin-induced apoptosis, we transfected HEp-2 cells with However, CAS9-expressing plasmid-transfected HEp-2 cells a CAS9-expressing plasmid (pcDNA3.1-hCASP9-3FLAG) showed a much increased autophagic ratio after oridonin or control plasmid (pcDNA3.1-3FLAG). After 24, 48 or 72 h treatment when compared with those transfected with control KANG et al: INHIBITION OF CASPASE-9 AUGm ENTS APOPTOSIS Figure 8. m olecular mechanisms by which caspase-9 resists apoptosis in human laryngeal cancer HEp-2 cells. Caspase-9 reduces sensitivity to apoptosis via ROS-suppressing and autophagy-promoting methods. plasmid (Fig. 7E). Accordingly, oridonin treatment, with or caspase-dependent apoptotic pathway (31). However, in this without control plasmid, enhanced both the expression level study, it was interesting to note that cleaved caspase-9 was of Beclin 1 and the conversion from LC3-I to LC3-II, while not detected by western blot analysis in oridonin-treated cells, these enhancements were markedly augmented in HEp-2 cells although increased expression of cytosolic cytochrome c was transfected with CAS9-expressing plasmid (Fig. 7F). observed. m oreover, inhibition of caspase-9 in HEp-2 cells did not protect the cells from oridonin-induced apoptosis, Discussion indicating that the apoptosis occurred via a caspase-9-inde- pendent mitochondrial pathway. Pre-treatment with NAC Some plant-derived agents have been shown to inhibit and CAT not only reversed the expression of Bax, Bcl-2, cell growth and induce apoptosis in numerous cancer cell AIF and cytochrome c, but also resulted in the complete types (35). Oridonin, an active diterpenoid isolated from inhibition of oridonin-induced ∆ψm collapse, suggesting that R. Rubescens, has been found to exert apoptotic effects in ROS was capable of functioning as an initial mediator in the human laryngeal cancer HEp-2 cells by our group (17,20). caspase-9-independent mitochondrial apoptotic pathway. In this study, we made unexpected observations that the C9i Fas receptor-mediated apoptotic signaling is one of the enhanced apoptosis to oridonin stimuli. The C9i effect was most important extrinsic apoptotic pathways in cells (40). ROS dose-dependent and could be detected at concentrations as low generation has been reported to induce the death receptor as 5 µm . This study also provides evidence that sensitization pathway and increase the activity of caspase-8 (41). In the to oridonin-mediated apoptosis by C9i is dependent on the present study, the increasing expression of FADD, cleaved amplic fi ation of ROS production induced by oridonin. caspase-8 and cleaved caspase-3 in the oridonin-treated HEp-2 ROS, the products of cellular oxidative stress, have been cells suggested the involvement of the death receptor pathway. suggested to regulate the process involved in the initiation of The activated caspase-3 was further supported by downregula- apoptotic signaling (26). Several studies have shown that ROS tion of ICAD, resulting in the release of CAD which caused are responsible for the execution of the mitochondrial pathway DNA fragmentation in the nuclei (29). ROS scavengers NAC of apoptosis (36-38). In this study, we observed that oridonin and CAT significantly inhibited all the participants in the treatment resulted in a signic fi ant increase in Bax expression death receptor pathway in the cells. These results support the and a decrease in Bcl-2 expression. Our findings also showed notion that ROS plays a primary role in triggering apoptosis a collapse of ∆ψm and a substantial increase in AIF and through activation of the extrinsic pathway in HEp-2 cells. cytochrome c. AIF will translocate to the nucleus where it is Despite the many studies on the relationship between capable of inducing nuclear chromatin condensation and large autophagy and apoptosis, the functional relationship between scale DNA fragmentation to mediate a caspase-independent caspases and autophagy is not well understood (42). Recent mitochondrial apoptotic pathway (39). Cytochrome c normally studies show that caspase-6 and -8 cleavage of p62 can inhibit functions via its association with other molecules to form a autophagy (43). On the other hand, the cleavage of Beclin 1 caspase-9-activating complex which plays a key role in the has been shown to be a critical event whereby caspases inhibit INTERNATIONAL JOURNAL OF ONCOLOGY 47: 2045-2056, 2015 autophagy (44,45). m oreover, clinical therapies involving exhibited enhanced growth inhibitory as well as an apoptotic caspase inhibitors may arrest apoptosis but also have the unan- response to oridonin, consistent with the results obtained with ticipated effect of promoting autophagic cell death (46). Thus, C9i. Moreover, caspase-9-deci fi ent HEp-2 cells also exhibited caspase, the hallmark of apoptosis, may be involved in the a significantly enhanced level of ROS and a much reduced execution of autophagy, pointing to major cross-talk between autophagic ratio after oridonin treatment. Overall, our findings the two lethal subroutines. In the present study, the results indi- support the idea that the promotion of ROS, but the repres- cated that caspase-9 participated in the autophagy process and sion of autophagy might contribute to the failure to reduce acted as a promoting factor in oridonin-induced autophagy. apoptotic sensitivity in caspase-9-deci fi ent HEp-2 cells. By The reason why we were interested in the role of the regulators contrast, overexpression of caspase-9 enhanced autophagy of apoptosis, such as caspase-9, in oridonin-induced autophagy and suppressed ROS production, showing a protective role on was that, depending on the circumstances, autophagy can oridonin-induced apoptosis in HEp-2 cells. protect cells from apoptosis (47) or kill cells by promoting In conclusion, our data indicate that targeting caspase- apoptosis (48). It is known in this regard that autophagy may 9-dependent signals can cooperate in promoting cell death act as a regulator of oridonin-induced apoptosis in HEp-2 cells. induced by anticancer drugs. Thus, the combination of oridonin Here, inhibition of autophagy increased the apoptotic ratio and those leading to a reduction of caspase-9 in tumor cells in oridonin-induced HEp-2 cells, suggesting that autophagy with agents, such as C9i and/or reduction in caspase-9, could has an anti-apoptotic function. Recently, Jeong et al showed represent a novel approach to human laryngeal cancer treat- that treatment of breast cancer m CF-7 cells with the NSAID ment. FR122047 led to caspase-mediated apoptosis and simultane- ously stimulated cytoprotective autophagy (42). In the present Acknowledgements study we have also identified a novel caspase -9-dependent mechanism that suppresses oridonin-induced apoptosis of This study was supported by National Natural Science HEp-2 cells by promoting autophagy. Foundation of China (no. 81102855), China Postdoctoral Of note, the release of cytochrome c from mitochondria Science Foundation (no. 2013m 541192) and China Postdoctoral results in the formation of an Apaf-1-caspase-9 apoptosome Science Special Foundation (no. 2014T70224). and induces the apoptotic protease cascade (31,32). Here, it is interesting to note that the HEp-2 cells treated with References oridonin are dec fi ient in their ability to cleave procaspase-9 1. Danial NN and Korsmeyer SJ: Cell death: Critical control points. in the presence of cytochrome c. The regulation and activa- Cell 116: 205-219, 2004. tion of caspase-9 in the apoptosome complex is poorly 2. Yuan J and Yankner BA: Apoptosis in the nervous system. understood. 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Inhibition of caspase-9 by oridonin, a diterpenoid isolated from Rabdosia rubescens, augments apoptosis in human laryngeal cancer cells

KANG, NING; CAO, SHI-JIE; ZHOU, YAN; HE, HAO; TASHIRO, SHIN-ICHI; ONODERA, SATOSHI; QIU, FENG; IKEJIMA, TAKASHI
International Journal of Oncology , Volume 47 (6) – Sep 28, 2015
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Abstract

INTERNATIONAL JOURNAL OF ONCOLOGY 47: 2045-2056, 2015 Inhibition of caspase-9 by oridonin, a diterpenoid isolated from Rabdosia rubescens, augments apoptosis in human laryngeal cancer cells 1,3 4 4 3 5 NING KANG , SHI-JIE CAO , YAN ZHOU , HAO HE , SHIN-ICHI TASHIRO , 6 2 3 SATOSHI ONODERA , FENG QIU and TAKASHI IKEJImA 1 2 School of Integrative medicine, College of Traditional Chinese medicine, Tianjin University of Traditional Chinese medicine, Tianjin 300193; China-Japan Research Institute of medical and Pharmaceutical Sciences, Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang 110016, P.R. China; Institute for Clinical and Biomedical Sciences, Kyoto 603-8072; Department of Clinical and Biomedical Science, Showa Pharmaceutical University, Tokyo 194-8543, Japan Received July 16, 2015; Accepted September 11, 2015 DOI: 10.3892/ijo.2015.3186 Abstract. Rabdosia rubescens, a commonly used traditional activation of both the caspase-9-independent mitochondrial Chinese medicine, has increasingly gained attention for its pathway and death receptor pathways, and the autophagy had use as an antitumor herb. Oridonin, a bioactive diterpenoid an anti-apoptotic function in oridonin-treated HEp-2 cells. isolated from Rabdosia rubescens, has been reported to These collective results suggest that oridonin targets caspase-9 induce apoptosis in human laryngeal cancer HEp-2 cells by to alter ROS production and autophagy situation to promote our group. Here, we made unexpected observations that the HEp-2 cell apoptosis. Therefore, oridonin has the potential to caspase-9 inhibitor (C9i) enhanced apoptosis in response to be developed as an anticancer agent, and the combination of selected stimuli, and HEp-2 cells which were made dec fi ient oridonin with those agents leading to reduction of caspase-9 in caspase-9 using siRNA exhibited no resistance to apop- expression in tumor cells could represent a novel approach to totic signals and actually demonstrated increased apoptotic human laryngeal cancer treatment. sensitivity to oridonin. The results were reversed by the trans- fection of an exogenous caspase-9 expression vector. Caspase-9 Introduction reduced sensitivity to apoptotic stimuli through reactive oxygen species (ROS)-suppressing and autophagy-promoting Apoptosis (also known as type I programmed cell death) is a methods. ROS triggered the progression of apoptosis through cellular suicide program critical for development and tissue homeostasis (1). Excess apoptosis is associated with degen- erative disorders (2), while a failure of apoptosis contributes to oncogenesis (3). Anticancer agents can induce apoptosis Correspondence to: Dr Takashi Ikejima, China-Japan Research in tumor cells that is mediated by the common cell death Institute of m edical and Pharmaceutical Sciences, Shenyang Pharma- machinery. Central to this is a family of intracellular proteases, ceutical University, 103 Wenhua Road, Shenyang 110016, P.R. China known as caspases. During apoptosis, they can act either as E-mail: [email protected] initiators (caspases-8 and -9) in response to apoptotic signals or as effectors (caspase-3, -6 and -7) that finally cleave a number Dr Feng Qiu, College of Traditional Chinese medicine, Tianjin University of Traditional Chinese medicine, 312 Anshanxi Road, of vital proteins and lead to the demise of the cells (1). ROS are Nankai District, Tianjin 300193, P.R. China generated from the mitochondria and other cellular sources, E-mail: [email protected] and can oxidize a wide range of cell constituents, including lipids, proteins and DNA, thus damaging cell structures and Abbreviations: ROS, reactive oxygen species; C9i, caspase-9 compromising function. When antioxidant mechanisms are inhibitor; NAC, N-acetylcysteine; 3-m A, 3-methyladenine; LSCC, overwhelmed by ROS and subsequent oxidative stress occurs, laryngeal squamous cell carcinoma; PI, propidium iodide; m DC, cell damage and cell death are induced (4). High levels of ROS monodansylcadaverine; siRNA, small interfering RNA; CAT, induce cell death, which often involves apoptosis through catalase; FADD, Fas-associated protein with death domain; CAD, caspase activation (5,6). caspase-activated DNase; ICAD, inhibitor of caspase-activated As a mode of type II programmed cell death, autophagy is DNase a series of biochemical steps through which eukaryotic cells Key words: oridonin, caspase-9, apoptosis, autophagy, reactive oxygen commit suicide by degrading their own cytoplasm and organ- species elles through a process in which these components are engulfed and then digested in double membrane-bound vacuoles called KANG et al: INHIBITION OF CASPASE-9 AUGm ENTS APOPTOSIS autophagosomes (7). However, autophagy has recently gained (20). Oridonin was isolated from Rabdosia rubescens and much attention for its paradoxical roles in cell survival and cell was identified by comparing its physical and spectroscopic 1 13 death, particularly in the pathogenesis as well as the treatment ( H Nm R, C Nm R) data with those reported in the literature of cancer (8,9). Whether autophagy enables cells to survive or (21). The purity was measured by HPLC (column: 4.6x150 mm, enhances their death is context-driven, depending on the type Inertsil ODS-SP, 5 µm; solvent phase: methanol/H O, 55:45) of stimuli, nutrient availability, and apoptotic status (10). and determined to be 99.6%. Oridonin was dissolved in Dm SO Laryngeal squamous cell carcinoma (LSCC) is the most to obtain a stock solution. The Dm SO concentration was kept common squamous cell carcinoma of the head and neck, and below 0.05% in all the cell cultures so that it had no detectable there is a steady annual increase in new cases and deaths (11). effect on cell growth or cell death. Epidemiological studies have revealed that the incidence rates vary from country to country and in different population Growth inhibition assay. HEp-2 cells were incubated in groups (12). LSCC accounts for 1-8.4% of all human cancers 96-well tissue culture plates. After a 24-h incubation, the cells in China. However, only a minority of patients are eligible for were treated with, or without, pan-caspase inhibitor (z-VAD- radical treatment aimed at a cure. The availability of new cyto- fmk), caspase-9, -8, -3, -1 inhibitors [Ac-LEHD-cmk (C9i), toxic drugs has led to steady improvements, but a paradigm z-IETD-fmk, z-DEVD-fmk or Ac-YVAD-cmk], 3-m A or shift is required to signic fi antly affect the poor prognosis of NAC (Sigma) at the given concentrations for 1 h and subse- most patients. quently treated with oridonin for 24 h. The cytotoxic effect With a long history of cancer treatment, traditional Chinese was measured by m TT assay as described elsewhere (20). medicine (TCm ) is recognized as a valuable source for seeking bioactive anticancer compounds (13). Rabdosia rubescens Observation of morphological changes. HEp-2 cells were (Hemsl.) Hara (Lamiaceae), also known as Dong Ling Cao seeded into 6-well culture plates. After a 24-h incubation, in TCm , is commonly used in Chinese folk medicine to treat the cells were treated with, or without, C9i or NAC at the stomach aches, pharyngitis, sore throats, coughs and wrestling given concentrations for 1 h and subsequently incubated with injuries. This herb has increasingly gained attention because oridonin for 24 h. The cellular morphology was observed using of its use as an antitumor herb and because of the antitumor a phase contrast microscope (Leica, Nussloch, Germany). activities of its discovered bioactive constituents. The herb contains a variety of active components, including diterpe- Transmission electron microscopy. HEp-2 cells were treated noids, flavonoids, phenolic acids, triterpenoids and volatile with 36 µm oridonin for the indicated time periods. The oils (14). Among these compounds, the diterpenoid compound collected cells were fixed with PBS containing 3% glutar - oridonin (Fig. 1) was reported to possess inhibitory effects aldehyde, and postfixed with PBS containing 1% OsO . The on a variety of cancer cell lines, such as leukemia, colorectal samples were dehydrated in graded alcohol solutions, then cancer, esophageal cancer, liver cancer, epidermoid carcinoma, embedded and sectioned. Ultrathin sections were stained lung cancer and uterine cervical cancer (15,16). However, no with uranyl acetate and lead citrate, and examined using a reports has yet been found on its anticancer effects in human JEm -1200 transmission electron microscope (Jeol, Tokyo, laryngeal cancer cells until our laboratory demonstrated that Japan) (22). oridonin could induce apoptosis and G2-m phase arrest in human laryngeal squamous carcinoma HEp-2 cells (17). Flow cytometric analysis of cell apoptosis. After chemical During the course of characterizing the role of the mito- treatment, 1x10 cells were harvested. The collected cells were chondrial pathway in oridonin-induced apoptosis in HEp-2 fixed in 70% ethanol, stained for DNA content with propidium cells, we made an unexpected observation that a selective iodide (PI), and measured by flow cytometry (Becton- inhibitor of caspase-9 (C9i) could enhance (rather than retard) Dickinson, Franklin Lakes, NJ, USA) as previously described apoptosis in response to selected stimuli. Interestingly, our (20). The sub-G1 DNA content was used as an indicator of group also found that caspases did not mediate apoptosis, but apoptosis (23). protected L929 cells from oridonin-induced cell death (18). m oreover, Shah et al also found that a caspase-9 inhibitor Measurement of intracellular ROS generation. After treat- could enhance stress-induced apoptosis in B-lineage cells (19). ment with 36 µm oridonin for the indicated time periods, the Driven by the above-mentioned interesting phenomena, we set cells were incubated with 10 mM DCF-DA (Sigma) at 37˚C for out to delineate the signaling pathways and specic fi ally the role 30 min. The intracellular ROS caused oxidation of DCF-DA to of caspase-9 in oridonin-induced apoptosis in greater detail. the u fl orescent compound DCF. Then, the cells were harvested Our data showed that caspase-9 played an anti-apoptotic role and the pellets were suspended in 1 ml PBS. Samples were in HEp-2 cells through inhibition of ROS generation. Further, analyzed at an excitation wavelength of 480 nm and an emis- we discovered that caspase-9 promoted oridonin-induced sion wavelength of 525 nm by FACScan flow cytometry (18). autophagy, and in this context, autophagy was a protective mechanism against apoptosis. Determination of mitochondrial membrane potential. The mitochondrial membrane potential was measured using the Materials and methods u fl orescent dye rhodamine-123 (Sigma) by o fl w cytometry as previously described (20). Cell culture and reagent treatment. Human laryngeal cancer HEp-2 cells were obtained from the American Type Cell Measurement of autophagy. After incubation with 36 µm Culture Collection and were cultured as previously described oridonin for the fixed times, cells were cultured with 0.05 mM INTERNATIONAL JOURNAL OF ONCOLOGY 47: 2045-2056, 2015 against Beclin 1, LC3, Bax, Bcl-2, AIF, cytochrome c, ICAD, FADD, caspase-9, -8, -3, Hsp90, β-actin (Santa Cruz). Representative blots from three independent experiments are shown. Statistical analysis of the data. All the presented data and results were confirmed in at least three independent experi - ments. The data are expressed as means ± SD. Statistical comparisons were made by Student's t-test and P<0.05 was Figure 1. Chemical structure of oridonin. considered statistically signic fi ant. Results monodansylcadaverine (MDC) at 37˚C for 60 min. The cellular u fl orescent changes were observed under a u fl ores- The apoptotic effects of oridonin in HEp-2 cells are enhanced cence microscope (Olympus). The u fl orescence intensity of by an irreversible inhibitor of caspase-9. Some of our prelimi- cells was analyzed by FACScan flow cytometry (24). nary studies have indicated that oridonin is able to inhibit the proliferation of human laryngeal squamous carcinoma HEp-2 Assay for caspase-9 activity. A caspase-9 u fl orimetric assay cells, and the cell death is shown to be typical of apoptosis kit from Chemicon International (Temecula, CA, USA) was (17,20). Since it is well known that caspase family members used according to the manufacturer's instructions. Briey fl , cell play a pivotal role in the cell apoptosis process, the effect of lysates were incubated with peptide substrate, LEHD-pNA caspase inhibitors on oridonin-induced HEp-2 cell death was (Ac-Leu-Glu-His-Asp-pNA), in assay buffer [100 mm NaCl, examined. Unexpectedly, the C9i enhanced cell death due 50 mm HEPES, 10 m m DTT, 1 m m EDTA, 10% glyc - to oridonin stimuli, while z-DEVD-fmk, z-IETD-fmk and erol, 0.1% CHAPS (pH 7.4)] for 2 h at 37˚C. The release of Z-VAD-fmk blocked the cytotoxity of the response to oridonin p-nitroaniline was monitored at 405 nm. Caspase-9 activity in (Fig. 2A). cell lysates was expressed as the x-fold increase from baseline Further, we investigated whether inhibition of caspase-9 controls. affected drug sensitivity. As shown in Fig. 2B, pre-treatment of HEp-2 cells with 5-20 µm C9i markedly enhanced the RNA interference. HEp-2 cells were transfected with caspase-9 inhibitory ratio in a dose-dependent manner after treatment small interfering RNA (siRNA) and control siRNA (Shanghai with oridonin. In addition, we examined cellular morpho- GenePharma, China) by Lipofectamine 2000 (Invitrogen) logical changes by phase contrast microscopy. At 24 h, some according to the manufacturer's instructions. The sequences of oridonin-treated HEp-2 cells became round, with shrunken of the sense strands of the RNAs used in this study were as nuclei and membrane blebbing, while untreated cells did follows. Control siRNA: UUC UCC GAA CGU GUC ACG. not show these apoptotic characteristics (Fig. 2C). Whereas, Caspase-9 siRNA-1: CGG UGA AAG GGA UUU AUA ATT. the C9i-pre-treated group showed more marked apoptotic Caspase-9 siRNA-2: CCA AAG UUG UCG AAG CCA ATT changes in a dose-dependent manner (Fig. 2C). Quantic fi ation CGU UGA. Caspase-9 siRNA-3: GUG ACA UCU UUG UGU of apoptotic cells by flow cytometric analysis also showed CCU ATT. that combined treatment with oridonin and C9i caused more significant apoptosis, and the apoptotic ratio was observed Transient transfection. HEp-2 cells at 70% confluence to be signic fi antly increased from 15.08% in oridonin alone- were transfected with control pcDNA3.1-3FLAG and treated cells to 23.59, 31.54 and 37.26% in 5, 10 and 20 µm C9i pcDNA3.1-hCASP9-3FLAG plasmid (Shanghai GeneChem, pre-treated cells, respectively (Fig. 2D). China). Transfection of cells was performed by using Lipofectamine 2000 reagent according to the manufacturer's Oridonin-induced increase in ROS generation is enhanced protocol. The effectiveness of transfection was detected by by inhibiting caspase-9. Dysregulation of cellular redox status western blot analysis. can be a potent mechanism of cell death (26). Therefore, we tested the possibility that oridonin, with or without C9i, Preparation of mitochondrial and cytosolic extracts. HEp-2 induces apoptosis through ROS accumulation. Compared with cells were collected and then washed twice with ice-cold PBS. the control group, ROS generation was signic fi antly increased The cell pellets were resuspended in ice-cold homogenizing after exposure to oridonin for 24 h. C9i effectively increased buffer (250 mm sucrose, 20 m m H EPES, 10 mm KCl, 1.5 m m oridonin-induced intracellular ROS generation (Fig. 3A and B). mgCl , 1 mm EDTA, 1 m m EGTA, 1 m m dithiothreitol, As expected, the use of NAC, a well known ROS scavenger 0.1 mM phenylmethanesulfonyl u fl oride, 10 µg/ml pepstatin (27), almost completely blocked ROS generation induced by and 10 µg/ml leupeptin). The cells were then homogenized and oridonin alone or in combination with C9i (Fig. 3A and B). centrifuged at 14,000 g at 4˚C for 60 min. The supernatant was Whereas, NAC not only completely prevented HEp-2 cell used as the cytosol fraction and the pellet was resuspended in apoptosis induced by oridonin alone, but also reversed the cell lysis buffer as the membrane fraction (25). apoptosis induced by combined treatment with oridonin and C9i (Fig. 2C and D). Collectively, these results demonstrated Western blot analysis. Western blot analysis was performed that caspase-9 inhibition amplie fi d oridonin-induced oxidative as previously described (20) using corresponding antibodies stress, which leads to enhanced apoptosis. KANG et al: INHIBITION OF CASPASE-9 AUGm ENTS APOPTOSIS Figure 2. The growth-inhibitory ratio and apoptotic ratio were enhanced by C9i but inhibited by NAC in oridonin-treated HEp-2 cells. (A) The cells were treated with oridonin for 24 h, in the presence or absence of caspase-9, -8, -3 and -1 inhibitors or pan-caspase inhibitor, and the inhibitory ratio was measured by m TT assay, n=3, mean ± SD. After the cells were incubated with oridonin in the absence or presence of C9i or NAC for 24 h, the inhibitory ratio was measured by MTT assay, n=3, mean ± SD (B), and the morphological changes were examined by phase contrast microscopy; scale bar, 10 µm (C). The flow ** ## cytometric quantic fi ation of apoptotic cells (sub-G1 fraction) is shown (D). P<0.01 vs oridonin alone-treated group; P<0.01 vs oridonin co-treated with 5 µm C9i group. Oridonin triggers a marked loss of ∆ψm and caspase-9 Fig. 3C and D, exposure of HEp-2 cells to oridonin for 24 h inhibition produces a progressive loss of ∆ψm. It has been caused a marked loss of ∆ψm compared with the control reported that ROS generation can lead to mitochondrial group and C9i pre-treatment produced a progressive loss of damage and membrane depolarization (28). Therefore, we ∆ψm. Notably, pre-treatment with NAC reversed the disrup- investigated whether ROS targeted the mitochondria and tion in ∆ψm induced by oridonin alone or in combination thereby decreased the ∆ψm in our model. As shown in with C9i (Fig. 3C and D). These results suggested that the INTERNATIONAL JOURNAL OF ONCOLOGY 47: 2045-2056, 2015 Figure 3. C9i amplie fi s the ROS production induced by oridonin, and the ROS subsequently triggers the progression of apoptosis in HEp-2 cells. The cells were cultured in the presence of 36 µΜ oridonin for 0 or 24 h or co-incubated with 5 µΜ C9i or 2.5 mM NAC for 24 h. Then, ROS generation (A) and the changes in ∆ψm (C) were measured by o fl w cytometry. The corresponding linear diagrams of the FACScan histograms are shown in (B) and (D) respectively. The values are mean ± standard errors (n= 3 of individual experiments). The cells were treated with oridonin in the absence or presence of 2.5 mm NAC or 500 U/ml CAT for the indicated time periods, then Bax, Bcl-2, cytochrome c, AIF (E) and FADD, caspase-8, -3, ICAD (F) protein expression was detected ** ## by western blot analysis. The results shown here are representative of at least three independent experiments. P<0.01 vs control group. P<0.01 vs oridonin alone-treated group. oridonin, with or without C9i, was capable of inducing Oridonin-induced Bax activation, Bcl-2 degradation, cyto- mitochondrial dysfunction and that the loss of ∆ψm might be chrome c and AIF elevation are markedly blocked by NAC affected directly by ROS generation. and CAT treatment. To determine the possible causes and KANG et al: INHIBITION OF CASPASE-9 AUGm ENTS APOPTOSIS Figure 4. Caspase-9 facilitates oridonin-induced autophagy, and inhibition of autophagy upregulates apoptosis. (A) The cells were incubated with oridonin for 0 or 24 h, or co-incubated with 3-m A for 24 h. Then, the cellular morphological changes were observed by transmission electron microscopy (arrow indicates autophagic vacuoles; scale bar, 1 µm) or by u fl orescence microscopy with MDC staining (scale bar, 10 µm). The cells were cultured in the presence of oridonin or co-incubated with 1 mM 3-MA (B) or 5 µM C9i (C) for 24 h. The corresponding column or o fl w cytometric histograms of autophagic level changes are represented. (D) The cells were treated with oridonin in the presence or absence of 3-m A or C9i for the indicated time periods, followed by western blot analysis for Beclin 1 and LC3 levels. The results shown here are representative of at least three independent experiments. (E) The cells were treated with oridonin for 24 h, in the presence or absence of 0.4-1.6 mm 3- m A, and the inhibitory ratio was measured by m TT assay, n=3, mean ± SD. (F) The cells were * ** treated with oridonin for 24 h in the presence or absence of 1 mM 3-MA. The sub-G1 fraction was analyzed by flow cytometry. P <0.05 or P<0.01 vs group treated with oridonin alone. consequences of the ∆ψm decrease, we studied the presence chondrial permeability and two other proteins, cytochrome of two proteins, pro-apoptotic protein Bax and anti-apoptotic c and AIF, which are usually released from mitochon- protein Bcl-2, which are involved in the regulation of mito- dria during apoptosis. We found that oridonin treatment INTERNATIONAL JOURNAL OF ONCOLOGY 47: 2045-2056, 2015 signic fi antly increased and decreased levels of Bax and Bcl-2 proteins, respectively, in a time-dependent manner (Fig. 3E). The expression of cytochrome c was markedly elevated. Simultaneously, a visible increase in AIF levels was also detectable after 12- and 24-h incubation (Fig. 3E). However, NAC and catalase (CAT, hydrogen peroxide decomposer) pre-treatment completely reversed the changes in Bax, Bcl-2, cytochrome c and AIF (Fig. 3E). Changes stimulated by oridonin in FADD, caspase-8, caspase-3 and ICAD expression are inhibited by NAC and CAT. To assess whether the Fas-mediated pathway was activated in oridonin-treated cells, the expressions of Fas-associated protein with death domain (FADD), caspase-8 and -3 were determined by western blot analysis. The expres- sion of FADD was markedly elevated and there was clear cleavage of procaspase-8 after oridonin administration. A time-dependent cleavage of procaspase-3 was also observed in oridonin-treated cells (Fig. 3F). ICAD, an inhibitor of caspase-activated DNase (CAD), is a substrate for caspases such as caspase-3. When caspase-3 is activated by apoptotic stimuli, ICAD is cleaved, resulting in the release of CAD which appears to cause DNA fragmentation in the nuclei (29). As shown in Fig. 3F, oridonin induced degradation of ICAD in HEp-2 cells in a time-dependent manner. Addition of NAC and CAT markedly inhibited the changes in FADD, caspase-8, -3 Figure 5. Time-course of activation of caspase-9 is not dependent on proteo- and ICAD (Fig. 3F). lytic cleavage. (A) The cells were treated with oridonin for the indicated time periods, followed by western blot analysis for detection of caspase-9, Hsp90 Caspase-9 facilitates oridonin-induced autophagy. We first and cytochrome c (both in the cytosol and the mitochondria) levels. The results shown here are representative of at least three independent experi- investigated the effect of oridonin on cell autophagy. The ments. (B) Equivalent cultures were prepared and the cells were collected ultrastructural details displayed that the oridonin-treated to assess the enzymic activity of caspase-9 as described in m aterials and ** cells possessed typical characteristics of autophagy. These methods, n=3, mean ± SD. P<0.01 vs group treated with oridonin for 24 h. characteristic changes included extensive cytoplasmic vacu- olization, and some autophagic vacuoles contained degraded organelles (Fig. 4A). The formation of autophagic vacuoles was further assessed by MDC, a u fl orescent dye specic fi ally tion in the number of MDC-labeled u fl orescent particles in the staining autophagosomes. As shown in Fig. 4A, control cells cells (Fig. 4A). Flow cytometric analysis also indicated that presented diffused staining, and oridonin treatment resulted pre-treatment with 3-m A markedly reduced the autophagic in an extensive punctuate m DC staining pattern. Next, the ratio compared with the group treated with oridonin alone levels of Beclin 1 and LC3, two important proteins involved (Fig. 4B). Simultaneously, oridonin-induced Beclin 1 acti- in autophagy (10,30), were examined by western blot analysis. vation as well as the modic fi ation of LC3-I to LC3-II were As shown in Fig. 4D, the level of Beclin 1 and conversion markedly blocked when the cells were pre-treated with from LC3-I to LC3-II both increased with time after oridonin 3-m A (Fig. 4D). However, pre-treatment of HEp-2 cells with administration. These findings apparently reveal that 0.4-1.6 mm of 3- m A dramatically increased the inhibitory autophagy is induced as a response of HEp-2 cells to oridonin rate of cell growth in a dose-dependent manner after oridonin treatment. treatment (Fig. 4E). Further, inhibition of autophagy increased To explore the involvement of caspase-9 in the modula- the oridonin-induced sub-G1 cell proportion (an index of tion of autophagy, the autophagic ratio was measured by o fl w apoptosis) in HEp-2 cells (Fig. 4F). Collectively, these results cytometry. Compared with the oridonin treatment group, the suggested that 3-m A inhibited oridonin-mediated autophagy autophagic ratio was signic fi antly reduced by the combined and it also sensitized the cells to the cytotoxic actions of use of oridonin and C9i (Fig. 4C). Accordingly, the treatment oridonin with induction of apoptosis. In short, oridonin- of C9i markedly suppressed Beclin 1 upregulation and the induced autophagy inhibits apoptosis in our model. conversion from LC3-I to LC3-II (Fig. 4D), suggesting the autophagy-promoting effects of caspase-9. Evaluation of the expression pattern of critical apoptosome complex-related proteins. In the cytosol, cytochrome c binds Inhibition of autophagy upregulates apoptosis induced by to Apaf-1, allowing recruitment of caspase-9 and formation of oridonin in HEp-2 cells. To investigate the role of autophagy apoptosome, resulting in caspase-9 activation and execution of in oridonin-induced apoptosis in HEp-2 cells, 3-MA, a specic fi cell death (31,32). Here, the expression of critical apoptosome inhibitor of autophagy, was introduced. Treatment with 3-m A complex-related proteins was examined by means of western prior to the addition of oridonin induced a signic fi ant reduc - blot analysis. As shown in Fig. 5A, the level of cytochrome c KANG et al: INHIBITION OF CASPASE-9 AUGm ENTS APOPTOSIS Figure 6. Apoptotic sensitivity to oridonin in HEp-2 cells made dec fi ient in caspase-9 by siRNA. (A) The cells were transfected with 25 nM caspase-9 siRNA-1, -2, -3 or control siRNA for 24 h (upper panel) or transfected with 25-75 nm caspase-9 siRNA-1 or control siRNA for 24 h (lower panel), and the caspase-9 level was examined by western blot analysis. The cells were transfected with 25 nm caspase-9 siRNA-1 or control siRNA for 24 h, followed by stimulation with oridonin for 24 h. Then, the inhibitory ratio was determined by m TT assay (n=3, mean ± SD) (B), and the proportion of sub-G1 cells (C) and ROS genera- tion (D) were measured by flow cytometry. The MDC fluorescent intensity of oridonin-treated cells was analyzed by flow cytometry (E), and the Beclin 1 and ** LC3 levels were examined by western blot analysis (F). The results shown here are representative of at least three independent experiments. P<0.01 vs control siRNA group treated with oridonin. in mitochondria began to decrease at 6 h, which was consistent Lower expression of caspase-9 augments apoptosis in with the increase in cytochrome c in the cytosol. However, oridonin-treated HEp-2 cells. To determine whether the no apparent change was observed in the protein level of results obtained with C9i could be corroborated by another pro-caspase-9, and cleaved-caspase-9 could not be detected method, we turned to RNA interference using siRNA. Here, (Fig. 5A). Recently, several investigators have described nega- the role of caspase-9 in oridonin-mediated apoptosis was tive regulation of the apoptosome complex by Hsp90 (33,34). studied by knock-down of caspase-9 using 3 different specific Therefore, the effect of oridonin on expression of Hsp90 was siRNAs. The expression of capase-9 was markedly suppressed examined. The result shows that oridonin treatment increased in HEp-2 cells when transfected with caspase-9 siRNA-1 levels of Hsp90 in a time-dependent manner in HEp-2 cells while only slight downregulation was found with the other (Fig. 5A). two siRNAs (Fig. 6A). Therefore, caspase-9 siRNA-1 was Next, we directly examined the effect of drug treat- selected as a valid candidate for the subsequent investiga- ments on caspase-9 activation using functional assays. The tion. As shown in Fig. 6A, caspase-9 siRNA-1 effectively result shows that caspase-9 activity was activated in a time- and specifically suppressed HEp-2 caspase-9 protein in a dependent manner, and the maximal activity was seen after a dose-dependent manner. In contrast, an identical amount of 24-h incubation (Fig. 5B). As expected, marked suppression control siRNA had no effect on caspase-9. We then tested of the high levels of caspase-9 was noted in cells subjected to whether caspase-9-deficient HEp-2 cells would undergo combination-treatment with C9i (Fig. 5B). a heightened response to apoptotic stimuli. As shown in INTERNATIONAL JOURNAL OF ONCOLOGY 47: 2045-2056, 2015 Figure 7. Caspase-9 overexpression attenuates apoptosis in oridonin-treated HEp-2 cells. (A) The cells were transfected with CASP9-expressing plasmid or control plasmid for 24, 48 and 72 h, and the caspase-9 level was examined by western blot analysis. The cells were transfected with CASP9-expressing plasmid or control plasmid for 48 h, followed by stimulation with oridonin for 24 h. Then, the inhibitory ratio was determined by m TT assay (n=3, mean ± SD) (B), and the proportion of sub-G1 cells (C) and ROS generation (D) were measured by o fl w cytometry. The MDC u fl orescent intensity of oridonin-treated cells was analyzed by o fl w cytometry (E), and the Beclin 1 and LC3 levels were examined by western blot analysis (F). The results shown here are representative of at ** least three independent experiments. P<0.01 vs control plasmid group treated with oridonin. Fig. 6B, caspase-9-deficient HEp-2 cells were significantly of transfection, the effectiveness of transfection was detected more sensitive to oridonin than control siRNA-treated cells. by western blotting. As shown in Fig. 7A, caspase-9 protein Quantic fi ation of apoptotic cells by o fl w cytometric analysis level was signic fi antly increased in a time-dependent manner showed that the incidence of apoptosis after oridonin treatment in the HEp-2 cells transiently transfected with the CAS9- was increased by >10% in caspase-9 siRNA-1-transfected expressing plasmid. In contrast, the control plasmid had cells when compared with control siRNA (Fig. 6C). Next, no effect on caspase-9 expression. We then tested whether the degree of ROS generation was also determined by flow increased caspase-9 expression would promote survival and cytometric analysis. The percentage of DCF-positive cells diminish apoptosis in oridonin-treated HEp-2 cells. As shown was highest in caspase-9 siRNA-1-transfected cells treated in Fig. 7B, the inhibitory ratio of control plasmid-transfected with oridonin (Fig. 6D). However, caspase-9-deci fi ent HEp-2 cells is 42.80%, while, the inhibitory ratio of CAS9-expressing cells showed a much reduced autophagic ratio after oridonin plasmid-transfected cells is 29.64%, thus demonstrating treatment when compared with those transfected with control caspase-9-mediated increase in survival. Moreover, quantifi - siRNA (Fig. 6E). Accordingly, oridonin treatment, with or cation of apoptotic cells by o fl w cytometric analysis showed without control siRNA, enhanced both the expression level that the incidence of apoptosis after oridonin treatment was of Beclin 1 and the conversion from LC3-I to LC3-II, while decreased by nearly 10% in CAS9-expressing plasmid-trans- these enhancements were markedly suppressed in HEp-2 cells fected cells when compared with control plasmid (Fig. 7C). transfected with caspase-9 siRNA-1 (Fig. 6F). Next, the degree of ROS generation was also determined by flow cytometric analysis. The percentage of DCF-positive Caspase-9 overexpression attenuates apoptosis in oridonin- cells was signic fi antly decreased in CAS9-expressing plasmid- treated HEp-2 cells. To test the ability of caspase-9 to inhibit transfected cells compared with control plasmid (Fig. 7D). oridonin-induced apoptosis, we transfected HEp-2 cells with However, CAS9-expressing plasmid-transfected HEp-2 cells a CAS9-expressing plasmid (pcDNA3.1-hCASP9-3FLAG) showed a much increased autophagic ratio after oridonin or control plasmid (pcDNA3.1-3FLAG). After 24, 48 or 72 h treatment when compared with those transfected with control KANG et al: INHIBITION OF CASPASE-9 AUGm ENTS APOPTOSIS Figure 8. m olecular mechanisms by which caspase-9 resists apoptosis in human laryngeal cancer HEp-2 cells. Caspase-9 reduces sensitivity to apoptosis via ROS-suppressing and autophagy-promoting methods. plasmid (Fig. 7E). Accordingly, oridonin treatment, with or caspase-dependent apoptotic pathway (31). However, in this without control plasmid, enhanced both the expression level study, it was interesting to note that cleaved caspase-9 was of Beclin 1 and the conversion from LC3-I to LC3-II, while not detected by western blot analysis in oridonin-treated cells, these enhancements were markedly augmented in HEp-2 cells although increased expression of cytosolic cytochrome c was transfected with CAS9-expressing plasmid (Fig. 7F). observed. m oreover, inhibition of caspase-9 in HEp-2 cells did not protect the cells from oridonin-induced apoptosis, Discussion indicating that the apoptosis occurred via a caspase-9-inde- pendent mitochondrial pathway. Pre-treatment with NAC Some plant-derived agents have been shown to inhibit and CAT not only reversed the expression of Bax, Bcl-2, cell growth and induce apoptosis in numerous cancer cell AIF and cytochrome c, but also resulted in the complete types (35). Oridonin, an active diterpenoid isolated from inhibition of oridonin-induced ∆ψm collapse, suggesting that R. Rubescens, has been found to exert apoptotic effects in ROS was capable of functioning as an initial mediator in the human laryngeal cancer HEp-2 cells by our group (17,20). caspase-9-independent mitochondrial apoptotic pathway. In this study, we made unexpected observations that the C9i Fas receptor-mediated apoptotic signaling is one of the enhanced apoptosis to oridonin stimuli. The C9i effect was most important extrinsic apoptotic pathways in cells (40). ROS dose-dependent and could be detected at concentrations as low generation has been reported to induce the death receptor as 5 µm . This study also provides evidence that sensitization pathway and increase the activity of caspase-8 (41). In the to oridonin-mediated apoptosis by C9i is dependent on the present study, the increasing expression of FADD, cleaved amplic fi ation of ROS production induced by oridonin. caspase-8 and cleaved caspase-3 in the oridonin-treated HEp-2 ROS, the products of cellular oxidative stress, have been cells suggested the involvement of the death receptor pathway. suggested to regulate the process involved in the initiation of The activated caspase-3 was further supported by downregula- apoptotic signaling (26). Several studies have shown that ROS tion of ICAD, resulting in the release of CAD which caused are responsible for the execution of the mitochondrial pathway DNA fragmentation in the nuclei (29). ROS scavengers NAC of apoptosis (36-38). In this study, we observed that oridonin and CAT significantly inhibited all the participants in the treatment resulted in a signic fi ant increase in Bax expression death receptor pathway in the cells. These results support the and a decrease in Bcl-2 expression. Our findings also showed notion that ROS plays a primary role in triggering apoptosis a collapse of ∆ψm and a substantial increase in AIF and through activation of the extrinsic pathway in HEp-2 cells. cytochrome c. AIF will translocate to the nucleus where it is Despite the many studies on the relationship between capable of inducing nuclear chromatin condensation and large autophagy and apoptosis, the functional relationship between scale DNA fragmentation to mediate a caspase-independent caspases and autophagy is not well understood (42). Recent mitochondrial apoptotic pathway (39). Cytochrome c normally studies show that caspase-6 and -8 cleavage of p62 can inhibit functions via its association with other molecules to form a autophagy (43). On the other hand, the cleavage of Beclin 1 caspase-9-activating complex which plays a key role in the has been shown to be a critical event whereby caspases inhibit INTERNATIONAL JOURNAL OF ONCOLOGY 47: 2045-2056, 2015 autophagy (44,45). m oreover, clinical therapies involving exhibited enhanced growth inhibitory as well as an apoptotic caspase inhibitors may arrest apoptosis but also have the unan- response to oridonin, consistent with the results obtained with ticipated effect of promoting autophagic cell death (46). Thus, C9i. Moreover, caspase-9-deci fi ent HEp-2 cells also exhibited caspase, the hallmark of apoptosis, may be involved in the a significantly enhanced level of ROS and a much reduced execution of autophagy, pointing to major cross-talk between autophagic ratio after oridonin treatment. Overall, our findings the two lethal subroutines. In the present study, the results indi- support the idea that the promotion of ROS, but the repres- cated that caspase-9 participated in the autophagy process and sion of autophagy might contribute to the failure to reduce acted as a promoting factor in oridonin-induced autophagy. apoptotic sensitivity in caspase-9-deci fi ent HEp-2 cells. By The reason why we were interested in the role of the regulators contrast, overexpression of caspase-9 enhanced autophagy of apoptosis, such as caspase-9, in oridonin-induced autophagy and suppressed ROS production, showing a protective role on was that, depending on the circumstances, autophagy can oridonin-induced apoptosis in HEp-2 cells. protect cells from apoptosis (47) or kill cells by promoting In conclusion, our data indicate that targeting caspase- apoptosis (48). It is known in this regard that autophagy may 9-dependent signals can cooperate in promoting cell death act as a regulator of oridonin-induced apoptosis in HEp-2 cells. induced by anticancer drugs. Thus, the combination of oridonin Here, inhibition of autophagy increased the apoptotic ratio and those leading to a reduction of caspase-9 in tumor cells in oridonin-induced HEp-2 cells, suggesting that autophagy with agents, such as C9i and/or reduction in caspase-9, could has an anti-apoptotic function. Recently, Jeong et al showed represent a novel approach to human laryngeal cancer treat- that treatment of breast cancer m CF-7 cells with the NSAID ment. FR122047 led to caspase-mediated apoptosis and simultane- ously stimulated cytoprotective autophagy (42). In the present Acknowledgements study we have also identified a novel caspase -9-dependent mechanism that suppresses oridonin-induced apoptosis of This study was supported by National Natural Science HEp-2 cells by promoting autophagy. Foundation of China (no. 81102855), China Postdoctoral Of note, the release of cytochrome c from mitochondria Science Foundation (no. 2013m 541192) and China Postdoctoral results in the formation of an Apaf-1-caspase-9 apoptosome Science Special Foundation (no. 2014T70224). and induces the apoptotic protease cascade (31,32). Here, it is interesting to note that the HEp-2 cells treated with References oridonin are dec fi ient in their ability to cleave procaspase-9 1. Danial NN and Korsmeyer SJ: Cell death: Critical control points. in the presence of cytochrome c. The regulation and activa- Cell 116: 205-219, 2004. tion of caspase-9 in the apoptosome complex is poorly 2. Yuan J and Yankner BA: Apoptosis in the nervous system. understood. 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Journal

International Journal of Oncology – Pubmed Central

Published: Sep 28, 2015

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