Selective apoptotic cell death effects of oral cancer cells treated with destruxin B
- Rosa Huang Liu†1,
- Shih-Pin Chen†2, 3, 4,
- Tsong-Ming Lu3, 5,
- Wei-Yu Tsai6,
- Chung-Hung Tsai2, 3, 7,
- Chi-Chiang Yang2, 6, 8Email author and
- Yew-Min Tzeng9Email author
© Huang Liu et al.; licensee BioMed Central Ltd. 2014
Received: 31 March 2014
Accepted: 24 June 2014
Published: 28 June 2014
Recent studies have revealed that destruxins (Dtx) have potent cytotoxic activities on individual cancer cells, however, data on oral cancer cells especial human are absent.
Destruxin B (DB) was isolated and used to evaluate the selective cytotoxicity with human oral cancer cell lines, GNM (Neck metastasis of gingival carcinoma) and TSCCa (Tongue squamous cell carcinoma) cells, and normal gingival fibroblasts (GF) were also included as controls. Cells were tested with different concentrations of DB for 24, 48, and 72 h by MTT assay. Moreover, the mechanism of cytotoxicity was investigated using caspase-3 Immunofluorescence, annexin V/PI staining, and the expression of caspase-3, Bax, and Bcl-2 by western blotting after treated with different concentrations of DB for 72 h as parameters for apoptosis analyses.
The results show that DB exhibited significant (p < 0.01) and selective time- and dose-dependent inhibitory effects on GNM and TSCCa cells viability but not on GF cells. The data suggested that DB is capable to induce tumor specific growth inhibition in oral GNM and TSCCa cancer cells via Bax/Bcl-2-mediated intrinsic mitochondrial apoptotic pathway in time- and dose-dependent manners.
This is the first report on the anti-proliferation effect of DB in oral cancer cells. The results reported here may offer further evidences to the development of DB as a potential complementary chemotherapeutic target for oral cancer complications.
KeywordsDestruxin Cytotoxicity Oral cancer Apoptosis
Destruxins (Dtx) are the most abundant secondary metabolites of the entomopathogenic fungus Metarhizium anisopliae and usually secreted into the culture medium during growth . Destruxins, especially Destruxin A, B and E (DA, DB, and DE), are a class of insecticidal cyclic depsipeptides . Previous studies have also shown destruxins exhibited strong biological effects; for example, destruxins disturbs macromolecular syntheses (DNA, RNA and protein synthesis) , produces anti-hepatitis B effects [4–6] and modifies the DNA content of murine leukemia cells [7–9]in vitro. Recently, Dtx came into focus of interest as an anticancer therapeutics [10–13]. Our previous data revealed a significant inhibition of colorectal cancer cell (HT-29) viability was observed with DB treatment in time- and dose-dependent manners . Moreover, DB administered subcutaneously daily at 0.6-15 mg/kg was safe and effective in inhibiting the growth of colorectal cancer cells in a murine xenograft animal model. Expression of Bax, cleaved poly (ADP-ribose) polymerase and active caspase-3 were observed with DB treatment and the increase in tumor volumes of treated groups were significantly (P < 0.05) less than those of the mock-treated group. Therefore DB was suggested to have the potential as a new therapeutic agent against human colorectal cancer.
Oral cancer, a subtype of head and neck cancer, may arise as a primary lesion originating in the oral tissues that line the mouth, lips, gingiva (gums) and tongue. Around 90% are squamous cell carcinomas. In the battle against cancer, one of the main technologies used for treatment is chemotherapy. Chemotherapy is the use of chemicals. Chemotherapy works by interfering with the cancer cell’s ability to grow or destroying the cancer cells. Biological agent, such as cetuximab has recently been shown to be effective in the treatment of squamous cell head and neck cancers, and is likely to has an increasing role in the future management of this condition when used in conjunction with other established treatment modalities . However, both mortality and morbidity of patients with oral cancer have not significantly much improved in the past decades . New chemicals against oral cancer are desperate in needs.
Previously, we have demonstrated some chemicals including caffeic acid phenethyl ester analogues , Methylantcinate A , and derivatives from Antrodia camphorate exhibit significant anti-oral cancer activities. Our previous data also revealed a significant inhibition of colorectal cancer cell viability was observed with DB treatment both in vitro and in vivo. Although recent studies have revealed that Dtx have potent cytotoxic activities on individual cancer cells [7–13], data on oral cancer cells are absent. Consequently, this is the first study aimed to investigate the impact of DB on human oral cancer cells in vitro growth and survival, as well as with special focus on the apoptotic cell death pathway. In this study, DB was isolated and used to evaluate the selective cytotoxicity with human oral cancer cell lines, GNM (Neck metastasis of gingival carcinoma) and TSCCa (Tongue squamous cell carcinoma) cells, and normal gingival fibroblasts (GF) were also included as controls. Hopefully, together with previous findings, we could evaluate different aspects of different cancer cells and molecular biological characteristics and assess potential novel cancer treatment regimens of DB.
Production of destruxins
A culture of M. anisopliae F061 var. anisopliae kindly provided by Dr. Suey-Sheng Kao, Taiwan Agricultural Chemicals and Toxic Research Institute (Wufeng, Taiwan), was used in this study. The culture method was used as described previously . Briefly, the spore suspension culture from -80°C was thawed at room temperature and inoculated into a 500-ml Erlenmeyer flask with a baffle containing 200 ml of 3% (w/v) Czapek-Dox (CD) broth (BD, Spark, MD, USA) and 0.75% bacto-peptone (BD, Spark, MD, USA) as seed culture. The flask was cultivated in an incubator (LM-575R, Yih-Der Co., Taipei, Taiwan) at 200 rpm, 28°C for 4 days. For the stirred-tank cultivation, the inoculum (10% of the working volume) was transferred from the flask of the 4 day old seed culture to the reactor, which contained 3 L of the desired medium. Cultivations were conducted in a 5 L stirred tank reactor (BTF-A5L, Bio-Top Inc, Taichung, Taiwan) at 28°Cwith the aeration rate regulated at 0.3 vvm (volume air/volume liquid/min). The culture medium (pH 9.0) was maintained by automatic addition of 2 N NaOH or 1 N HCl at a agitation rate of 150 rpm. After 14 days, the fermentation broth was harvested and purified as the following procedures.
Purification of destruxins
The destruxins were isolated and purified according to the method of Chen et al. . The culture medium was harvested after incubation for 14 days and centrifuged at 9000 rpm for 20 min. The supernatant was adjusted to pH 4.0 by 1 N HCl then extracted with ethyl acetate (sample: EA = 5:2, v/v), and the organic phase was evaporated with a rotary vacuum evaporator (model N-1, Eyela, Tokyo, Japan) at 45°C. The concentrate was diluted with 2 times volume of acetonitrile and filtered through a 0.22 μm chromatodisc unit before HPLC analysis. The sample (800 μ L) was injected into a preparative column (Cosmosil 15 C18-AR-II column, 28 × 250 mm, 15 μm). The eluent from the column was monitored at 215 nm with a L-7100 pump and a L-7400 UV detector (Hitachi, Tokyo, Japan). The mobile phase was: 80% Methanol/H2O. The eluting solvent was set at 10 mL/min. Fractionated samples were characterized by analytic HPLC, ESI-MASS and 1H NMR spectroscopes.
The GNM, TSCCa, and GF cells used in this study have been reported previously [16–18, 21]. Briefly, GNM cells were in RPMI 1640 with 10% supplemented with 10% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA, USA). TSCCa and GF cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Life Technologies, Carlsbad, CA, USA). Both medium were added with penicillin (100 IU/ml) and streptomycin (100 μg/ml). Briefly, the cells were maintained in the appropriate growth medium at 37°C in a humidified atmosphere of 5% CO2 and 95% air and used over a restricted culture period of 10 passages.
The colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, tetrazolium) assay will be used to observe the survival ratio of cells which has been used in our lab before [16, 21–24]. Cells will be seeded in 96-wells plates in triplicate in medium containing 2% (V/V) serum. After 24 h, 20 μl MTT solution (5 mg/ml) were added to each well of cells group and then incubated 4 h in a 5% CO2 incubator 37°C. After the reaction was finished, the supernatant was discarded and 200 μl DMSO (dimethyl sulfoxide) was added to each well. A blank control which contained medium and MTT, but no cells, was also included. Cell growth and growth inhibition will be measured at 570 nm with a spectrophotometer (Model 550, BIO-RAD, Palo Alto, California, USA).
This is manipulated as described before [21, 23]. Briefly, cells were incubated and grown as a monolayer in chamber slides at 37°C in humidified 5% CO2/95% air atmosphere. After treatment, cells were rinsed twice in phosphate-buffered saline (PBS, pH 7.4), immediately fixed with ice-cold acetone at -20°C for 20 min. After 3 washes with PBS, cells were incubated with 10% normal goat serum (NGS) in PBS for 1 h and then incubated with 1% NGS in PBS for 10 min to block nonspecific binding sites. Two-hundred microliters of rabbit polyclonal anti-caspase-3 primary IgG antibody (Clone H-277, Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) diluted 1:100 in PBS-10% FCS was added to each well for 45 min at 37°C. After washing three times in PBS, 200 μl of secondary goat anti-rabbit IgG-FITC (fluorescein isothiocyanate) conjugated antibody (sc-2012, Santa Cruz Biotechnology, Inc., Dallas, Texas, USA) was diluted 1:200 in PBS-10% FBS and then added to each well for 45 min at 37°C. After washing three times in PBS, cells were visualized using a fluorescent microscope.
Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining
Annexin V binding was assayed by fluorescence-activated cell sorter (FACS) analysis according to the ApoAlert Annexin V protocol (BD Biosciences Clontech Palo Alto, CA, USA) as previously described [17, 21, 23, 24]. Briefly, the tested cells were washed with PBS, detached from the culture plates with 0.25% trypsin and gently washed once with the growth medium. Then 5 × 105 cells were washed once and resuspended in 200 μL of the 1X binding buffer, mixed with 5 μL of annexin V-FITC and 10 μL of PI, and incubated at room temperature for 5 min in the dark. Then the reaction volume was adjusted with the binding buffer to 500 μL. The cells were subjected to FACS analysis in a flow cytometer (Becton Dickinson, San Jose, CA) using a single laser emitting excitation light at 488 nm.
Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and western blotting
SDS-PAGE [25–27] and western blotting were performed as previously described [17, 24]. Total proteins were solubilized and extracted from tissue with 300 μl lysis buffer. The lysates were used to estimate the protein content with Bradford protein assay. Equal amounts of protein (50 μg) from each sample were then subjected to electrophoresis on a 12% (v/v) SDS-polyacrylamide gel. After electrophoresis, proteins were electroblotted to a PolyScreen® PVDF hybridization transfer membrane (PerkinElmer, Boston, MA, USA). The membrane was blocked at room temperature with blast blocking buffer [1% blast blocking reagent (PerkinElmer, Boston, MA, USA) in Tris buffered saline (TBS) containing 0.05% (v/v) Tween (TBS-T)]. Then the membrane was washed three times with TBS-T and incubated overnight at 4°C with the primary antibody, mouse monoclonal anti-caspases-3 (1:1000, v/v), or rabbit monoclonal antibodies to poly (ADP-ribose) polymerase (PARP) (1:4000, v/v), Bcl-2-associated X protein (Bax) (1:8000, v/v) and β-actin (1:2000, v/v), followed by 1 h incubation with a 1:10000 (v/v) dilution of the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody. Primary antibodies (β-actin, PARP, Bax) were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA), whereas secondary antibodies (anti-rabbit IgG and anti-mouse IgG HRP-labeled) were purchased from PerkinElmer (Boston, MA, USA). After incubation, the membrane was washed with TBS-T three times, and the antigen-antibody complexes were visualized by the enhanced chemiluminescence system (PerkinElmer). The relative protein expression with the control defined as 100% was measured by Image J software (National Institutes of Health, USA).
The Kruskal-Wallis test of analysis in SPSS (version 10.0) software will be used to study the significant change between different cells in the experiments used in this study. The p value < 0.05 will be taken to be regarded as significant.
Selective cytotoxicity of DB for oral squamous cell carcinoma cell lines
The IC 50 concentration of DB for GNM and TSCCa cells after 24, 48, and 72 h treatment
The mechanism of DB-induced cell death
Oral squamous cell carcinoma, which arises from the mucosa lining, accounts for over 90% of oral malignancies . In addition, squamous cell carcinoma of the oral cavity is the 11th most common global cancer, accounting for 3% of all newly diagnosed cancer cases [32, 33]; it is also the eighth most predominant cancer in males [33, 34]. In those countries in which chewing betel quid and/or tobacco is highly popular including Taiwan [35–37], squamous cell carcinoma of the buccal mucosa is the most common oral cancer . Despite the large amount of research into tumor cells and molecular biology, as well as recent advances in oncology and surgery, the rates of both mortality and morbidity in oral squamous cell carcinoma patients have remained unimproved .
In the search of anti-cancer chemicals, previous studies in our lab have shown the potential of several chemicals including a series of caffeic acid phenethyl ester-like compounds, methylantcinate A and triterpenoids derived from Antrodia camphorata to be promising anti-oral cancer drugs [16–18]. The activity of DB against cancer cells have been suggested or studied on murine leukemia, epidermal carcinoma, non-Hodgkin lymphoma, and non-small cell lung cancer cells [7–9, 11, 12]. We also report that DB has potential as a new therapeutic agent against human colorectal cancer and non-small cell lung cancer [10, 13]. This study provided the first evidence that DB attenuates the growth of human oral cancer GNM and TSCCA cells, and might exert pro-apoptotic effects through a Bax-mediated mitochondrial apoptotic pathway and caspase cascade in time- and dose-dependent manners. Comparing with the aforementioned other chemicals, DB has the advantages on the accessibility and cost in addition to the prominent cytotoxicity against cancer cells. DB, one of the most abundant secondary metabolites of M. anisopliae, is secreted into the culture medium during growth. It is relatively cheap and easy to produce and purify DB from the medium rather than to synthesis it chemically. Moreover, as the safety of using DB in vivo has been demonstrated through the use of drug-related death rate, body weight loss, splenomegaly and pathological examination as parameters for evaluation in our previous report , results reported here may offer further impulse to the development of DB as a potential chemotherapeutic drug for oral and other cancers.
Apoptosis is a physiological cell death process to prevent individuals from tumor genesis or damage cancer cells. A recent study by Dornetshuber-Fleiss et al.  tested Dtx antiproliferative potential against a panel of human cancer cell lines and suggested that DB, in accordance with our previous study , being most effective in apoptosis induction in cancer cells of colorectal origin. However, the non-small cell lung cancer cell line A549 was remarkably resistant to it. In this study, the selective apoptotic cell death effects of oral cancer GNM and TSCCa cells nevertheless have been demonstrated with DB treatment. In addition, it was also suggested that 48 h treatment is necessary for DB to exert profound cell death, at least in clonogenicity assays. This is in accordance with our data, in our hands, the IC50s of DB treatment for 24 h are far greater than that of 48 and 72 h (Table 1).
In summary, this is the first report on the anti-proliferation effect of DB in oral cancer cells. These data suggested that DB is capable to induce tumor specific growth inhibition in oral GNM and TSCCa cancer cells via Bax/Bcl-2-mediated intrinsic mitochondrial apoptotic pathway in time- and dose-dependent manners. Many lines of evidence demonstrate that Bax/Bcl-2-related proteins play an important role in either inhibition or promotion of apoptosis . The Bcl-2 acts as an anti-apoptotic protein to prevent permeabilization of the outer mitochondrial membrane by inhibiting the action of the pro-apoptotic protein Bax . Although the detailed molecular mechanism by which apoptosis is induced by this natural compound remains to investigated, and of course further studies including in vivo animal model are needed, the results reported here may offer further evidences to the development of DB as a potential complementary chemotherapeutic target for oral cancer complications.
Yang CC is one of the editors of the following journals, including Cellular and Molecular Immunology (2010-present), Virus Genes (2010-present), The Open Infectious Disease Journal (SJR) (2008-present), Oriental Pharmacy and Experimental Medicine (2006-present), World Journal of AIDS (2011-present), World Journal of Virology (2011-present), and International Journal of Genuine Traditional Medicine, TANG (2011-present). Moreover, he is the executive editor of Chung Shan Medical Journal since 2012. Tzeng YM is the president of Biochemical Engineering Society of Taiwan (2010 ~ 2013). He is currently on the executive board of Asian Federation of Biotechnology (AFOB) and on the editorial board of Evidence-Based Complementary and Alternative Medicine, Biotechnology and Applied Biochemistry, and Journal of Bioscience and Bioengineering.
Fluorescence-activated cell sorter
Fetal bovine serum
Neck metastasis of gingival carcinoma
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, tetrazolium
Normal goat serum
Polyacrylamide gel electrophoresis
Poly (ADP-ribose) polymerase
Sodium dodecyl sulfate
Tris buffered saline
Tongue squamous cell carcinoma.
Flow cytometry was performed in the Instrument Center of Chung Shan Medical University, which is supported by National Science Council, Ministry of Education and Chung Shan Medical University. This study was funded by grants from the National Science Council (NSC 99-2320-B-040-004-MY3 to Yang CC; NSC 100-2113-M-324-001-MY3 to Tzeng YM).
- Dong C, Zhang J, Huang H, Chen W, Hu Y: Pathogenicity of a new China variety of Metarhizium anisopliae (M. Anisopliae var. Dcjhyium) to subterranean termite Odontotermes formosanus. Microbiol Res. 2009, 164 (1): 27-35.View ArticlePubMedGoogle Scholar
- Cavelier F, Verducci J, Andre F, Haraux F, Sigalat C, Traris M, Vey A: Natural cyclopeptides as leads for novel pesticides: tentoxin and destruxins. Pestic Sci. 1998, 52 (1): 81-89.View ArticleGoogle Scholar
- Quiot JM, Vey A, Vago C: Effects of mycotoxins on invertebrate cells in vitro. Adv Cell Cult. 1985, 4: 199-212.Google Scholar
- Sun CM, Chen HC, Yeh SF: Suppressive effects of metabolites from Alternaria brassicaea on the hepatitis B surface antigen. Planta Med. 1994, 60 (1): 87-88.View ArticlePubMedGoogle Scholar
- Yeh SF, Pan W, Ong GT, Chiou AJ, Chuang CC, Chiou SH, Wu SH: Study of structure-activity correlation in destruxins, a class of cyclodepsipeptides possessing suppressive effect on the generation of hepatitis B virus surface antigen in human hepatoma cells. Biochem Biophys Res Commun. 1996, 4;229 (1): 65-72.View ArticleGoogle Scholar
- Chen HC, Chou CK, Sun CM, Yeh SF: Suppressive effects of destruxin B on hepatitis B virus surface antigen gene expression in human hepatoma cells. Antiviral Res. 1997, 34 (3): 137-144.View ArticlePubMedGoogle Scholar
- Morel E, Pais M, Turpin M, Guyot M: Cytotoxicity of cyclodepsipeptides on murine lymphocytes and on L 1210 leukemia cells. Biomed Pharmacother. 1983, 37 (4): 184-185.PubMedGoogle Scholar
- Odier F, Vago P, Quiot JM, Devauchelle G, Bureau JP: Cytometric study of the effects of destruxin E on leukemic cells in mice. CR Acad Sci III. 1987, 305 (14): 575-578.Google Scholar
- Odier F, Vey A, Bureau JP: In vitro effect fungal cyclodepsipeptides on leukemic cells: study of destruxins A, B and E. Biol Cell. 1992, 74: 267-271.View ArticlePubMedGoogle Scholar
- Lee YP, Wang CW, Liao WC, Yang CR, Yeh CT, Tsai CH, Yang CC, Tzeng YM: In vitro and in vivo xenograft mice inhibitory effects of Destruxin B on human colorectal cancer. Anticancer Res. 2012, 32 (7): 2735-2745.PubMedGoogle Scholar
- Dornetshuber-Fleiss R, Heffeter P, Mohr T, Hazemi P, Kryeziu K, Seger C, Berger W, Lemmens-Gruber R: Destruxins: fungal-derived cyclohexadepsipeptides with multifaceted anticancer and antiangiogenic activities. Biochem Pharmacol. 2013, 1;86 (3): 361-377.View ArticleGoogle Scholar
- Chao PZ, Chin YP, Hsu IU, Liu CM, Yu YC, Leung TK, Lee YJ, Chen CH, Lin YF: Apoptotic toxicity of destruxin B in human non-Hodgkin lymphoma cells. Toxicol In Vitro. 2013, 27: 1870-1876.View ArticlePubMedGoogle Scholar
- Wu CC, Chen TH, Liu BL, Wu LC, Chen YC, Tzeng YM, Hsu SL: Destruxin B isolated from entomopathogenic fungus Metarhizium anisopliae induces apoptosis via a Bcl-2 family-dependent mitochondrial pathway in human nonsmall cell lung cancer cells. Evid Based Complement Alternat Med. 2013, 2013: 548929-PubMedPubMed CentralGoogle Scholar
- Ye AY, Hay JH, Laskin JJ, Wu JS, Ho CC: Toxicity and outcomes in combined modality treatment of head and neck squamous cell carcinoma: cisplatin versus cetuximab. J Cancer Res Ther. 2013, 9 (4): 607-612.View ArticlePubMedGoogle Scholar
- Sankaranarayanan R, Black RJ, Parkin DM: Cancer Survival in Developing Countries. 1998, Lyon, France: International Agency for Research on Cancer, IARC Scientific Publication No. 145Google Scholar
- Lee YJ, Liao PH, Chen WK, Yang CC: Preferential cytotoxicity of caffeic acid phenethyl ester analogues on oral cancer cells. Cancer Lett. 2000, 153: 51-56.View ArticlePubMedGoogle Scholar
- Tsai WC, Rao YK, Lin SS, Chou MY, Shen YT, Wu CH, Geethangili M, Yang CC, Tzeng YM: Methylantcinate A induces tumor specific growth inhibition in oral cancer cells via Bax-mediated mitochondrial apoptotic pathway. Bioorg Med Chem Lett. 2010, 20: 6145-6148.View ArticlePubMedGoogle Scholar
- Lee YP, Tsai WC, Ko CJ, Rao YK, Yang CR, Chen DR, Yang MH, Yang CC, Tzeng YM: Anticancer effects study of eleven triterpenoids derived from Antrodia camphorata. Anticancer Res. 2012, 32 (7): 2727-2734.PubMedGoogle Scholar
- Liu BL, Rou TM, Rao YK, Tzeng YM: Effect of pH and aeration rate on the production of destruxins A and B from Metarhizium anisopliae. Int J Appl Sci Eng. 2007, 5 (1): 17-26.Google Scholar
- Chen JW, Liu BL, Tzeng YM: Purification and quantification of destruxins A and B from Metarhizium anisopliae. J Chromatogr A. 1999, 830 (1): 115-125.View ArticleGoogle Scholar
- Huang CR, Lin SS, Chou MY, Ho CC, Wang L, Lee YL, Chen CS, Yang CC: Demonstrating different mode of cell death upon HSV-1 infection in different oral cell populations. Acta Virol. 2005, 49: 7-15.PubMedGoogle Scholar
- Huang TH, Yang CC, Ding SJ, Yen M, Chou MY, Kao CT: Biocompatibility of human osteosarcoma cells to root end filling materials. J Biomed Mater Res. 2005, 72B (1): 140-145.View ArticleGoogle Scholar
- Xu FL, Lee YL, Tsai WY, Lin SJ, Yang ZQ, Yang CC, Liu HY, Cheng L, Xiao H, Wen L: Effect of cordycepin on hantaan virus 76–118 infection of primary human embryonic pulmonary fibroblasts- characterization of apoptotic effects. Acta Virol. 2005, 49: 183-193.PubMedGoogle Scholar
- Cheng MH, Cheng HT, Lin SS, Young SC, Pai CJ, Liao PH, Chen SC, Chou MY, Yang JJ, Yang CC: Apoptotic characteristics of MMC-treated HeLa cells and cellular localization and characteristics of induced P-glycoprotein 170. Drug Chem Toxicol. 2009, 32: 158-168.View ArticlePubMedGoogle Scholar
- Lin HW, Hsieh YS, Yang YY, Hu CC, Chiou SC, Yang CC: A novel modified silver staining for detecting protein, DNA and RNA in polyacrylamide gels. J Biomed Lab Sci. 1999, 11 (4): 122-127.Google Scholar
- Yang CC, Lin SJ, Lin KL: A newly identified true late protein of HSV-1 possibly encoded by UL49.5. J Biomed Lab Sci. 1999, 11 (4): 111-117.Google Scholar
- Yang CC, Yang YY, Lin KL, Lin SJ: The different forms of HSV-1 VP22a within purified virion and infected cells. J Microbiol Immunol Infect. 2000, 33: 63-68.Google Scholar
- Pradelli LA, Beneteau M, Ricci JE: Mitochondrial control of caspase-dependent and -independent cell death. Cell Mol Life Sci. 2010, 67: 1589-1597.View ArticlePubMedGoogle Scholar
- Kroemer G, Galluzzi L, Brenner C: Mitochondrial membrane permeabilization in cell death. Physiol Rev. 2007, 87: 99-163.View ArticlePubMedGoogle Scholar
- Ashkenazi A: Directing cancer cells to self-destruct with pro-apoptotic receptor agonists. Nat Rev Drug Disc. 2008, 7: 1001-1012.View ArticleGoogle Scholar
- Silverman S: Demographics and occurrence of oral and oropharyngeal cancers. The outcomes, the trends, the challenge. J Am Dent Assoc. 2001, 132 (1): 7S-11S.View ArticlePubMedGoogle Scholar
- Parkin DM, Bray F, Ferlay J, Pisani P: Global cancer statistics. CA Cancer J Clin. 2005, 55 (2): 74-108.View ArticlePubMedGoogle Scholar
- Parkin DM, Pisani P, Ferlay J: Estimates of the worldwide incidence of 25 major cancers in 1990. Int J Cancer. 1999, 80 (6): 827-841.View ArticlePubMedGoogle Scholar
- Warnakulasuriya S: Global epidemiology of oral and oropharyngeal cancer. Oral Oncol. 2009, 45 (4–5): 309-316.View ArticlePubMedGoogle Scholar
- Chen YK, Huang HC, Lin LM, Lin CC: Primary oral squamous cell carcinomas: an analysis of 703 cases in southern Taiwan. Oral Oncol. 1999, 35 (2): 173-179.View ArticlePubMedGoogle Scholar
- Reichart PA, Nguyen XH: Betel quid chewing, oral cancer and other oral mucosal diseases in Vietnam: a review. J Oral Pathol Med. 2008, 37 (9): 511-514.View ArticlePubMedGoogle Scholar
- Zhang X, Li C, Liao Q, Reichart PA: Areca chewing in Xiangtan, Hunan province, China: interviews with chewers. J Oral Pathol Med. 2008, 37 (7): 423-429.View ArticlePubMedGoogle Scholar
- Diaz EM, Holsinger C, Zuniga ER, Roberts DB, Sorensen D: Squamous cell carcinoma of the buccal mucosa: one institution’s experience with 119 previously untreated patients. Head Neck. 2003, 25 (4): 267-273.View ArticlePubMedGoogle Scholar
- Richardson A, Kaye SB: Pharmacological inhibition of the bcl-2 family of apoptosis regulators as cancer therapy. Curr Mol Pharmacol. 2008, 1: 244-254.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/14/207/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.