Alphitolic acid, an anti-inflammatory triterpene, induces apoptosis
and autophagy in oral squamous cell carcinoma cells, in part,
through a p53-dependent pathway
Li-Yuan Baia,b, Chang-Fang Chiub,c, Shih-Jiuan Chiud, Yu-Wen Chene, Jing-Lan Hue, and Jing-Ru Wenge,*
aCollege of Medicine, China Medical University, Taichung, 404, Taiwan, bDivision of Hematology and Oncology, Department of Internal Medicine, China Medical
University Hospital, Taichung 404, Taiwan, cCancer Center; China Medical University Hospital, Taichung 404, Taiwan, dSchool of Pharmacy, Taipei Medical University, Taipei 250, Taiwan, eDepartment of Biological Science and Technology, China Medical University, Taichung 404, Taiwan
Short title:
Alphitolic acid induces apoptosis and autophagy *To whom correspondence should be address. Jing-Ru Weng, Ph.D.
Tel: (886)-4-22053366 ext.2511; Fax: (886)-4-22071507 91 Hsueh-Shih Road, Taichung 404, Taiwan
Abstract
In this study, we interrogated the antitumor mechanism of alphitolic acid (2-hydroxybetulinic acid, ALA), an anti-inflammatory triterpene from medicinal plants, in oral squamous cell carcinoma (OSCC) cells. ALA suppressed the proliferation of SCC4 and SCC2095 OSCC cells with IC50 of 12 and 15 µM, respectively, through apoptotic death. Mechanistically, the effect of ALA on apoptosis was, in part, associated with its ability to block the Akt-NF-B signaling axis. Moreover, ALA induced autophagy, as evidenced by increased expression of the autophagy markers Beclin 1, Atg7, and LC3B-II, and autophagosome formation. This autophagy induction played a protective role as co-treatment with the autophagy inhibitors chloroquine and bafilomycin A1 increased the sensitivity of OSCC cells to ALA-induced apoptosis. Pursuant to our finding that p53-null HSC-3 cells were resistant to ALA, we obtained evidence that p53 played a role in ALA-mediated cytotoxicity. ALA increased p53 phosphorylation/expression, accompanied by parallel decreases in the expression of the E3 ligase MDM2, and equally important, shRNA-mediated knockdown of p53 partially rescued ALA-mediated cytotoxicity. Together, these findings suggest the translational value of ALA as a potential therapeutic agent for OSCC.
1. Introduction
Oral squamous cell carcinoma (OSCC) is the fifth most common cause of cancer death in Taiwan. It has been reported that tobacco use, betel quid chewing, and alcohol drinking represent major risk factors of OSCC (Chen et al., 2008). In addition, certain environmental contaminants, including arsenic, chromium and nickel, have also been linked to OSCC (Chiang et al., 2011; Su et al., 2010; Yuan et al., 2011). Mechanistically, dysregulated oncogenes and/or tumor suppressor genes are associated with oral carcinogenesis. For example, p53 mutation is found in approximately 70% in human OSCC (Jurel, Gupta, Singh, Singh, & Srivastava, 2014). The 5-year survival rate for OSCC patients is below 50%, even with the treatment of surgery, radiation, and chemotherapy (Chin et al., 2006). Current chemotherapeutic agents for OSCC treatment, including 5-fluorouracil, platinum, taxane, ifosfamide, and methotrexate, have side effects limiting their use, and patients succumb eventually to their disease once chemoresistance is developed. Therefore, there is an urgent need to develop new chemopreventive strategies for this debilitating disease. Pharmacological exploitation of natural products to develop new chemopreventive agents for OSCC has been the focus of many investigation in the past three decades (Newman & Cragg, 2012), as exemplified by the reported activities of garlic, flavonoids, and resveratrol in suppressing oral carcinogenesis (Bhavana &
Lakshmi, 2014; Campos, Campos, Aarestrup, & Aarestrup, 2014). From a translational perspective, understanding the mechanism underlying the antitumor effects of bioactive phytochemicals provide a viable approach to identify new chemopreventive agents.
Syzygium kusukusense (Myrtaceae) is an indigenous plant distributed in Southern Taiwan (Chang, 1982). The Syzygium genus is well known for producing polyphenol, flavonoids, alkaloids, terpenoids, and saponin (Chaieb et al., 2007; Helmstadter, 2008; Shad et al., 2014). In our previous study of secondary metabolites from S.
kusukusense, we isolated one of the constituents, alphitolic acid (ALA) (Bai, Lin, Chiu, & Weng, 2014), a naturally occurring triterpene that displays a spectrum of activities, including those of anti-inflammation (Aguirre et al., 2006), anti-parasitites (Suksamrarn et al., 2006), and anti-cancer (Kim, Choi, & Lee, 2012). In the context of antitumor activity, ALA was reported to inhibit the transcriptional activity of Hedgehog/Gli1 in HaCaT cells (Arai et al., 2008). In this study, we interrgoated the mechanism underlying the antitumor effect of ALA (chemical structure, Fig. 1A) in OSCC cells.
2. Materials and methods 2.1. Sample preparation
(collected in PinTung County, Taiwan) was described in the previous report (Bai et al., 2014). Structure identification and purity of the compound were verified by published spectroscopic data (Djoukeng et al., 2005). For in vitro experiments, ALA was dissolved in dimethyl sulphoxide (DMSO), and was added at the indicated concentrations to culture medium with a final DMSO concentration of less than 0.1%. Rabbit polyclonal antibodies against various biomarkers were obtained from the following sources: p-473Ser-Akt, IB, p-32Ser-IB, Atg7, beclin 1, p-389 Thr-p70S6K, S6, p-235Ser-S6, LC3B, p-15Ser-p-53, and NF-B, Cell Signaling Technologies (Beverly, MA, USA); Akt, p-166Ser-MDM2, MDM2, Bcl-2, and p70S6K, Santa Cruz Biotechnology (Santa Cruz, CA, USA); -actin, Sigma-Aldrich (St. Louis, MO, USA). The enhanced chemiluminescence (ECL) system for the detection of immunoblotted proteins was from GE Healthcare Bioscience (Piscataway, NJ, USA). The GFP-LC3 plasmid was kindly provided by Professor Ching-Shih Chen (The Ohio State University), and p53 shRNA was purchased from the National RNAi Core Facility (Taiwan). Other chemical and biochemical reagents were obtained from Sigma-Aldrich unless otherwise mentioned.
2.2. Cell culture
Susan R. Mallery (The Ohio State University). HSC-3 human oral cancer cells were purchased from Japanese Collection of Research Bioresources (Tokyo, Japan). All cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA), 5 mg/ml of penicillin and 5 mg/ml of streptomycin. All cells were cultured at 37 oC in a humidified incubator containing 5% CO2.
2.3. Cell Viability Analysis
The effect of ALA on cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Weng et al., 2013) in 6 replicates. Cells (5 × 103) were seeded and incubated in 96-well, flat-bottomed plates in 10% FBS-supplemented DMEM/F12 for 24 h, and were exposed to test agents at the indicated concentrations in 5% DMEM/F12 medium for different time intervals. Medium was removed and replaced by 200 L of 0.5 mg/ml MTT in 5% FBS-DMEM/F12. After 2 h incubation, the reduced MTT dye was solubilized in 200 L/well of DMSO. Absorbance was determined with a Synergy HT (Bio-Tek) at 570 nm.
2.4. Assessments of apoptosis
Drug-treated cells (2 × 105) were grown to subconfluency on 18-mm coverslips for 72 h, washed, and fixed with 3% formaldehyde in PBS for 15 min at 4 oC. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature and incubated with 20 ng/mL of DAPI (Invitrogen, Carlsbad, CA) for 30 min. The condensed chromatin were visualized and captured at 200× magnification by a fluorescence microscope.
2.4.2. Flow cytometric analysis of Annexin V-propidium iodide (PI) staining OSCC cells (5 × 104) were plated and treated with ALA at indicated concentrations in 5 % FBS-supplemented DMEM/F12 medium for 72 h. Cells were harvested, washed twice in ice-cold phosphate-buffered saline (PBS), fixed in 70% cold ethanol at 4 oC for 4 h, followed by spinning at 1200 rpm for 5 min and re-suspending in ice-cold PBS containing 2% PBS. Cells were stained with Annexin V-FITC and PI according to the vendor’s protocols (BD Pharmingen, San Diego, USA) and analyzed by using BD FACSAria flow cytometer (Becton, Dickinson and Co., Franklin Lakes, NY, USA).
2.5. Western Blotting
Drug-treated cells were collected, washed with ice-cold PBS, and resuspended in lysis buffer [20 mM Tris-HCl (pH 8), 137 mM NaCl, 1 mM CaCl2, 10% glycerol, 1%
Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 100 M 4-(2-aminoethyl)-benzenesulfonyl fluoride, leupeptin at 10 g/mL, and aprotinin at 10 g/mL]. Soluble cell lysates were collected after centrifugation at 1500g for 5 min. Equivalent amounts of protein (60-100 g) from each lysate were resolved in 10% SDS-polyacrylamide gels. Bands were transferred to nitrocellulose membranes and blocked with 5% nonfat milk in PBS containing 0.1% Tween 20 (PBST) and incubated overnight with the corresponding primary antibodies at 4oC. After washing with PBST three times, the membrane was incubated at room temperature for 1 h with the secondary antibody with PBST and visualized by the ECL.
2.6. Transient transfection and confocal microscopy
The GFP-LC3 plasmid was transiently transfected into SCC4 cells by using the Fugene HD reagent (Roche, Mannheim, Germany) according to the manufacture’s protocol. Cells (2 105/3 mL) were seeded in each well of a six-well plate. After 24-h incubation, cells were treated with ALA at indicated concentrations or rapamycin (20 µM) as positive control for 48 h, fixed in 2% paraformaldehyde (Merck) for 30 min at room temperature, permeabilized with 0.1% Triton X-100 for 20 min, and washed with PBS. GFP fluorescence was visualized on a Leica TCS SP2 confocal microscope (Leica Biosystems Nussloch GmbH, Heidelberg, Germany).
For transient gene knockdown, SCC4 cells (2 105/3 mL) were seeded in each well of a six-well plate. After 24-h incubation, cells were transfected with p53 shRNA using Fugene HD reagent (Roche) for 24 h. Then the cells were treated with ALA for 24 h and collected the protein for Western blotting.
2.7. Transmission electron microscopy
Samples were prepared according to an established procedure (Weng et al., 2013). Briefly, SCC4 cells were fixed in a solution containing 2% paraformaldehyde, 2.5% glutaraldehyde, 0.2M sodium cacodylate for 1 h. The fixed cells were suspended in a buffered solution containing 1% osmic acid for 1 h, followed by dehydration in a graded ethanol series, wash with acetone, and embedding into EPON epoxy resin. Ultrathin sections (60-80 nm) were prepared on an ultramicrotome and double-stained with uranyl acetate and lead citrate. All sections were examined and photographed with a Hitach H-600 transmission electron microscope.
2.8. Statistical Analysis
All data are presented as mean S.D. obtained from three independent experiments. Statistical differences were calculated using Student’s t-test, with the following symbols of significance level: *P < 0.05, **P< 0.005.
3. Results
3.1. Anti-proliferative effect of ALA in oral cancer cells
The antiproliferative efficacy of ALA was assessed in 3 human OSCC cell lines, HSC-3, SCC2095, and SCC4, by using MTT assays (Fig. 1B). ALA exhibited a dose-dependent suppressive effect on the viability of SCC4 and SCC2095 with IC50 values of 12 M and 15 µM, respectively. However, p53-null HSC-3 cells were resistant to ALA (Fig. 1B), suggesting that ALA-mediated cytotoxicity might be p53-dependent.
3.2. ALA induces apoptosis
To shed light onto the mode of antitumor action of ALA, we conducted flow cytometric analysis of Annexin V-PI staining in ALA-treated SCC4 cells. As shown, ALA induced a concentration-dependent increase in the population of apoptotic cells (defined as Annexin V-positive cells) after 72 h of treatment (Fig. 2A and 2B; etoposide as a positive control). The ability of ALA to induce apoptosis in SCC4 cells was further confirmed by DAPI assays, which showed increased chromatin condensation in ALA-treated cells, as evidenced by intense bluish-white fluorescence in pyknotic nuclei (Fig. 2C; etoposide as a positive control).
Consistent with the functional role of Akt in regulating the proliferation of OSCC cells (Nakayama, Ikebe, Beppu, & Shirasuna, 2001), ALA was effective in blocking Akt signaling, as manifested by decreased phosphorylation of Akt and its downstream substrates, including p70S6K, S6, and IB(Fig. 3). Equally important, the expression of NF-B and its downstream target gene product Bcl-2 were also down-regulated by ALA treatment (Fig. 3).
3.4. ALA induces autophagy
We obtained several lines of evidence that ALA shared the ability of many plant natural compounds to induce autophagy in cancer cells (X. Zhang, Chen, Ouyang, Cheng, & Liu, 2012), a cellular catabolic response to starvation or stress (Mathew, Karantza-Wadsworth, & White, 2007). Transmission electron microscopy demonstrated the formation of autophagosomes (arrow) after exposing SCC4 cells to ALA (20 µM) for 24 h (Fig. 4A), which was further confirmed by the concentration-and time-dependent cellular accumulations of LC3B-II, an autophagosome marker for monitoring autophagy (Fig. 4B and 4C). Our data show that this ALA-induced autophagy was associated with elevated expression levels of two important autophagy-regulatory proteins: autophagy-related protein (Atg)7 (Cui, Gong, & Shen, 2013; Fu, Cheng, & Liu, 2013) and beclin 1 (Fig. 4B). In addition, SCC4 cells were
transiently transfected with GFP-tagged LC3 (GFP-LC3), and exposed to ALA (20 or 25 µM) or the positive control rapamycin (20 µM). Confocal fluorescence imaging indicates the accumulation of LC3-positive puncta in the cytoplasm of SCC4 cells in a manner similar to that of rapamycin (Fig. 4D).
3.5. Pharmacological inhibition of autophagy enhances the cellular sensitivity to ALA-induced apoptosis
Autophagy has been reported to mediate either a protective or enhancing effect on drug-induced cell death (Ajabnoor, Crook, & Coley, 2012; Bincoletto et al., 2013). To study the role of autophagy in ALA-induced cytotoxicity, cells were co-treated with chloroquine (CQ; a late stage autophagy inhibitor) or bafilomycin A1 (a vacuolar-type H+-ATPase inhibitor that blocks autophagosome-lysosome fusion) for 72 h. As shown in Fig. 5A and 5B, co-treatment of cells with CQ or bafilomycin A1 increased ALA-mediated apoptotic death. Consistent with the flow cytometric analysis, Western blotting showed that co-treatment with CQ or bafilomycin A1 led to a greater extent of PARP cleavage as compared to ALA alone (Fig. 5C). Together, these data suggested that ALA induced protective autophagy rather than autophagic cell death in OSCC.
Pursuant to our finding that p53-functional SCC4 (Lai et al., 2014) and p53-null HSC-3 cells (Kaneuchi et al., 1999) exhibited differential sensitivity to ALA (Fig. 1B), we hypothesized that ALA-induced cytotoxicity was mediated through a p53-dependent pathway. This premise was supported by the ability to upregulate the p53 phosphorylation/expression, accompanied by the concomitant downregulation of the phosphorylation/expression of MDM2 (Fig. 6A), an E3 ubiquitin ligase which targets p53 for proteasomal degradation (Chao, 2014; Manfredi, 2010).
To investigate the role of p53 in ALA-mediated cytotoxicity, SCC4 cells were transiently transfected with p53 shRNA to knockdown the expression of p53 (Fig. 6B). Compared with cells transfected with vector control, p53 knockdown partially protected SCC4 cells from ALA-mediated cytotoxicity (Fig. 6C). These results suggested that the antitumor activity of ALA was, at least, partially mediated through p53 activation.
4. Discussion
Substantial evidence has demonstrated the promising chemopreventive and/or therapeutic activities of many phytochemicals isolated from medicinal plants, including terpenoids, flavanoids, isothiocynates, and polyphenolics (Darvesh & Bishayee, 2013; Gonzalez-Vallinas, Gonzalez-Castejon, Rodriguez-Casado, &
Ramirez de Molina, 2013; Singh & Singh, 2012; Spagnuolo et al., 2012; Wang et al., 2012), in different types of cancer, including oral cancer (Bhavana & Lakshmi, 2014), in light of their pleiotropic modes of action. In this study, we report the anti-tumor activity of the triterpene ALA in OSCC cells, in part, through the concurrent downregulation of the Akt-mTOR/NF-B signaling axis and activation of p53.
The unique ability of ALA to target Akt and its downstream targets, mTOR and NF-B is noteworthy because of the involvement of these signaling effectors in the development of invasive [i.e., Akt and mTOR (Giudice, Dal Vechio, Abrahao, Sperandio, & Pinto-Junior Ddos, 2011; Hsu et al., 2010)] and chemo-resistant [NF-B (Lee et al., 2007; Tamatani et al., 2004)] phenotypes of OSCC cells.
However, because of its ability to blocking mTOR/p70S6K signaling, ALA also caused autophagy, which is a process by which cells undergo partial autodigestion that prolong survival under stress conditions (Tsujimoto & Shimizu, 2005). Environmental and metabolic stresses, including starvation, growth factor withdrawal, high temperature, hypoxia, or accumulation of misfolded proteins, can induce autophagy (Kundu & Thompson, 2008). Many phytochemicals, including triterpenoid, epigallocatechin-3-gallate, curcumin, genistein, flavones, and resveratrol have been shown to induce autophagy through distinct mechanisms (Garcia-Zepeda, Garcia-Villa, Diaz-Chavez, Hernandez-Pando, & Gariglio, 2013; Nakamura et al.,
2009; Weng et al., 2013; Ye, Li, Yin, & Zhang, 2012; Zhou et al., 2014). In cancer cells, autophagy is a double-edge sword in the context of cancer therapy (F. Zhang & Cheong, 2013), having tumor-promoting or tumor suppressing properties under different cellular contexts (Janku, McConkey, Hong, & Kurzrock, 2011). Many of the aforementioned phytochemicals, autophagy was reported to underlie their antitumor effects. In contrast, autophagy represents a protective mechanism for ALA-induced cell death in OSCC cells as CQ and bafilomycin A1 enhanced the cytotoxic effect of ALA in SCC4 cells. From a mechanistic perspective, this discrepancy might be attributable to differences in the mechanisms by which these plant natural compounds induce autophagy (mTOR/p70S6K inhibition versus ROS production).
In response to stress signals, tumor suppressor p53 prevents the development of cancer by inducing DNA repair, apoptosis and cell cycle arrest (Jurel et al., 2014; Partridge, Costea, & Huang, 2007). In light of the intimate link between p53 function and tumorigenesis, regulation of p53 activity and stability becomes an therapeutic target (Senderowicz, 2004). In this present study, we found that ALA facilitated p53 activation by downregulating the phosphorylation/expression of its regulator MDM2 (Fig. 6A). Equally important, this p53 activation is integral to the antitumor activity of ALA as knockdown of p53 partially protected OSCC cells from ALA-mediated cytotoxicity.
5. Conclusion
In summary, the triterepene ALA exerts anti-proliferative effects by inducing apoptosis through the inhibition of the Akt/mTOR-NF-B signaling axis and p53 activation in OSCC cells. These findings provide new insight into the mode of antitumor mechanism of ALA. Further investigations on the in vivo efficacy of ALA in prevent oral carcinogenesis in pertinent animal models are warranted for the preclinical development of this chemopreventive agent.
Conflict of interest statement
The authors declare no competing financial interests.
Acknowledgements
This work was supported by grant from Ministry of Science and Technology grant (MOST 103-2320-B-039-023-MY3).
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Figures legends
Fig. 1. The antiproliferative effects of ALA in three oral cancer cell lines (SCC4, SCC2095, and HSC-3). (A) The chemical structure of alphotolic acid (ALA) (B) Effect of ALA at the indicated concentrations on the viability of oral cancer cells. Cells were treated with ALA in 5% FBS-supplemented DMEM/F12 medium in 96-well plates at 72 h, and cell viability was assessed by MTT assays. Points, mean; bars, S.D. (n = 6). **P< 0.005 compared to the control group.
Fig. 2. Evidence of apoptosis for ALA-induced cell death. (A) Annexin V-FITC/ propidium iodide staining. SCC4 cells were treated with DMSO vehicle or ALA at the indicated concentrations in 5% FBS-supplemented DMEM/F12 medium for 72 h. (B) Cells were analyzed by flow cytometry after staining with fluorescein-conjugated Annexin V and PI. Percentages in the graphs represent the percent of cells in the respective quadrants. Columns, means; bar, S.D. *P< 0.05 compared to the control group. (C) DAPI staining of SCC4 cells showing pyknotic nuclei (arrows) as an indicator of apoptosis after 72 h of ALA treatment.
Fig. 3. Dose-dependent effects of ALA on the phosphorylation/expression of Akt, p70S6K, S6, NF-B, IB, and Bcl-2 in SCC4 cells. Cells were treated with ALA in
5% FBS-supplemented DMEM/F12 medium for 72h, and cell lysates were immunoblotted as described in Material and Methods.
Fig. 4. ALA induced autophagy. (A) Electron microscopic analysis of autophagosome formation in vehicle- or drug-treated SCC4 cells as described in materials and methods. Arrow: autophagosomes (B) Dose-dependent effects of ALA on the expression of LC3B, Beclin-1, and Atg7. (C) Time-dependent effects of ALA on the conversion of LC3B-I to LC3B-II. (D) Fluorescent confocal microscopic analysis of drug-induced autophagosome formation in SCC4 cells ectopically expressing GFP-LC3. SCC4 cells transiently transfected with GFP-LC3 plasmids were treated with DMSO, 20 or 25 M ALA, or 20 M rapamycin for 48 h and then fixed by 3.7% paraldehyde and examined by confocal microscopy.
Fig. 5. Effects of autophagic inhibitors on ALA-induced autophagy and apoptosis. (A) SCC4 cells were treated with 20 M ALA alone or in combination with 20 M chloroquine (CQ) or 0.25 M bafilomycin A1 (Bafilo.) for 72 h, and then Annexin V-FITC/PI double-staining analysis was performed. Cells exposed to etoposide at 1 µM were used as a positive control. (C) Western blot analyses of PARP cleavage in SCC4 cells co-treated with 20 M ALA with CQ (20 M) or bafilomycin A1 (0.25 M).
Fig. 6. Restoration of apoptotic activity of ALA by activating p53. (A) Dose-dependent effects of ALA on the phosphorylation/expression of p53 and MDM2 in SCC4 cells. Cells were treated with ALA in 5% FBS-supplemented DMEM/F12 medium for 72 h, and cell lysates were immunoblotted as described in Material and Methods. (B) Western blotting of SCC4 cells transfected with control or p53 shRNA 24 h post-transfection. The expression and phosphorylation of p53 were analysed. (C) The cytotoxicity of ALA at 24 h after the knockdown by 24h-p53 shRNA transfection as described in materials and Methods.
