Growth arrest and apoptosis via caspase activation of dioscoreanone in human non-small-cell lung cancer A549 cells

BMC Complementary and Alternative Medicine

Arunporn Itharat³  Sermkiat Tanuchit²  Kalaya Aree¹  Pintusorn Hansakul⁴ 

[1] Division of Microbiology, Department of Preclinical Science, Faculty of Medicine, Thammasat University, Rangsit Campus, Khlong Luang, Pathum Thani 12120, Thailand;Research Administration Office, Faculty of Medicine, Thammasat University, Rangsit Campus, Khlong Luang, Pathum Thani 12120, Thailand;Department of Applied Thai Traditional Medicine Center, Faculty of Medicine, Thammasat University, Rangsit Campus, Khlong Luang, Pathum Thani 12120, Thailand;Division of Biochemistry, Department of Preclinical Science, Faculty of Medicine, Thammasat University, Rangsit Campus, Khlong Luang, Pathum Thani 12120, Thailand

关键词: Caspase activation;    Apoptosis;    Antiproliferative;    Dioscorea membranacea;    Dioscoreanone;   

Others  :  1085832
DOI  :  10.1186/1472-6882-14-413

 received in 2014-01-06, accepted in 2014-10-16,  发布年份 2014

【 摘 要 】

Background

Dioscoreanone (DN) isolated from Dioscorea membranacea Pierre has been reported to exert potent cytotoxic effects against particular types of cancer. The present study was carried out to investigate the cytotoxicity of DN against a panel of different human lung cancer cell lines. The study further examined the underlying mechanisms of its anticancer activity in the human lung adenocarcinoma cell line A549.

Methods

Antiproliferative effects of DN were determined by SRB and CFSE assays. The effect of DN on cell cycle distribution was assessed by flow cytometric analysis. Apoptotic effects of DN were determined by sub-G₁ quantitation and Annexin V-FITC/PI flow cytometric analyses, as well as by changes in caspase-3 activity and relative levels of Bax and Bcl-2 mRNA.

Results

DN exerted antiproliferative and cytotoxic effects on all three subtypes of non-small cell lung cancer (NSCLC) cells, but not on small cell lung cancer (SCLC) cells and normal lung fibroblasts. DN slowed down the cell division and arrested the cell cycle at the G2/M phase in treated A549 cells, leading to a dose- and time- dependent increase of the sub-G1 population (apoptotic cells). Consistently, early apoptotic cells (AnnexinV ⁺/PI^-) were detected in those cells that were treated for 24 h and increased progressively over time. Moreover, the highest activity of caspase-3 in DN-treated A549 cells was detected within the first 24 h, and pretreatment with the general caspase inhibitor z-VAD-fmk completely abolished such activity and also DN-induced apoptosis in a dose-dependent manner. Additionally, DN increased the Bax/Bcl-2 ratio in treated A549 cells with time, indicating its induction of apoptosis via the mitochondrial pathway.

Conclusions

This study reveals for the first time that the anticancer activity of DN was induced through regulation of the Bcl-2 family protein-mediated mitochondrial pathway and the subsequent caspase-3 activation in A549 cancer cells, thus supporting its potential role as a natural apoptosis-inducing agent for NSCLC.

【 授权许可】

   
2014 Hansakul et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ: Cancer statistics, 2007. CA Cancer J Clin 2007, 57:43-66.
• [2]Wang L, Xiong Y, Sun Y, Fang Z, Li L, Ji H, Shi T: HLungDB: an integrated database of human lung cancer research. Nucleic Acids Res 2010, 38:D665-D669.
• [3]Fruh M: The search for improved systemic therapy of non-small cell lung cancer–what are today’s options? Lung Cancer 2011, 72:265-270.
• [4]Feng Y, Wang N, Zhu M, Li H, Tsao S: Recent progress on anticancer candidates in patents of herbal medicinal products. Recent Pat Food Nutr Agric 2011, 3:30-48.
• [5]Elmore S: Apoptosis: a review of programmed cell death. Toxicol Pathol 2007, 35:495-516.
• [6]Fesik SW: Promoting apoptosis as a strategy for cancer drug discovery. Nat Rev Cancer 2005, 5:876-885.
• [7]Ocker M, Hopfner M: Apoptosis-modulating drugs for improved cancer therapy. Eur Surg Res 2012, 48:111-120.
• [8]Wang SR, Fang WS: Pentacyclic triterpenoids and their saponins with apoptosis-inducing activity. Curr Top Med Chem 2009, 9:1581-1596.
• [9]Itharat A, Plubrukarn A, Kongsaeree P, Bui T, Keawpradub N, Houghton PJ: Dioscorealides and dioscoreanone, novel cytotoxic naphthofuranoxepins, and 1,4-phenanthraquinone from Dioscorea membranacea Pierre. Org Lett 2003, 5:2879-2882.
• [10]Tewtrakul S, Itharat A: Nitric oxide inhibitory substances from the rhizomes of Dioscorea membranacea. J Ethnopharmacol 2007, 109:412-416.
• [11]Itharat A, Houghton PJ, Eno-Amooquaye E, Burke PJ, Sampson JH, Raman A: In vitro cytotoxic activity of Thai medicinal plants used traditionally to treat cancer. J Ethnopharmacol 2004, 90:33-38.
• [12]Earnshaw WC, Martins LM, Kaufmann SH: Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 1999, 68:383-424.
• [13]Slee EA, Adrain C, Martin SJ: Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem 2001, 276:7320-7326.
• [14]Suen DF, Norris KL, Youle RJ: Mitochondrial dynamics and apoptosis. Genes Dev 2008, 22:1577-1590.
• [15]Lapenna S, Giordano A: Cell cycle kinases as therapeutic targets for cancer. Nat Rev Drug Discov 2009, 8:547-566.
• [16]Vermeulen K, Van Bockstaele DR, Berneman ZN: The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif 2003, 36:131-149.
• [17]Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C: A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995, 184:39-51.
• [18]Grutter MG: Caspases: key players in programmed cell death. Curr Opin Struct Biol 2000, 10:649-655.
• [19]Porter AG, Janicke RU: Emerging roles of caspase-3 in apoptosis. Cell Death Differ 1999, 6:99-104.
• [20]Susin SA, Daugas E, Ravagnan L, Samejima K, Zamzami N, Loeffler M, Costantini P, Ferri KF, Irinopoulou T, Prevost MC: Two distinct pathways leading to nuclear apoptosis. J Exp Med 2000, 192:571-580.
• [21]Li LY, Luo X, Wang X: Endonuclease G is an apoptotic DNase when released from mitochondria. Nature 2001, 412:95-99.
• [22]Martinou JC, Green DR: Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol 2001, 2:63-67.
• [23]Arnoult D, Gaume B, Karbowski M, Sharpe JC, Cecconi F, Youle RJ: Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization. EMBO J 2003, 22:4385-4399.