β-sitosterol induces G1 arrest and causes depolarization of mitochondrial membrane potential in breast carcinoma MDA-MB-231 cells

BMC Complementary and Alternative Medicine

Rana P Singh²  Raosaheb K Kale²  Shanthi Sri Vundru¹ 

[1] School of Life Sciences, Central University of Gujarat, Gandhinagar, Gujarat, India;Cancer and Radiation Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India

关键词: Cell cycle and apoptosis;    Cell death;    Cell proliferation;    Breast cancer;    Skin cancer;    Lung cancer;   

Others  :  1220880
DOI  :  10.1186/1472-6882-13-280

 received in 2013-06-14, accepted in 2013-09-24,  发布年份 2013

【 摘 要 】

Backgrounds

It is suggested that dietary phytosterols, such as β-sitosterol (ST), have cancer chemopreventive effects; however, studies are limited to support such claims. Here, we evaluated the efficacy of ST on three different human cancer cell lines including skin epidermoid carcinoma A431 cells, lung epithelial carcinoma A549 cells and breast adenocarcinoma MDA-MB-231.

Methods

Cell growth assay, cell cycle analysis, FACS, JC-1 staining, annexin V staining and immunoblotting were used to study the efficacy of ST on cancer cells.

Results

ST (30–90 μM) treatments for 48 h and 72 h did not show any significant effect on cell growth and death in A431 cells. Whereas similar ST treatments moderately inhibited the growth of A549 cells by up to 13% (p ≤ 0.05) in 48 h and 14% (p ≤ 0.05-0.0001) in 72 h. In MDA-MB-231 cells, ST caused a significant dose-dependent cell growth inhibition by 31- 63% (p ≤ 0.0001) in 48 h and 40-50% (p ≤ 0.0001) in 72 h. While exploring the molecular changes associated with strong ST efficacy in breast cancer cells, we observed that ST induced cell cycle arrest as well as cell death. ST caused G0/G1 cell cycle arrest which was accompanied by a decrease in CDK4 and cyclin D1, and an increase in p21/Cip1and p27/Kip1 protein levels. Further, cell death effect of ST was associated with induction of apoptosis. ST also caused the depolarization of mitochondrial membrane potential and increased Bax/Bcl-2 protein ratio.

Conclusions

These results suggest prominent in vitro anti-proliferative and pro-apoptotic effects of ST in MDA-MB-231 cells. This study provides valuable insight into the chemopreventive efficacy and associated molecular alterations of ST in breast cancer cells whereas it had only moderate efficacy on lung cancer cells and did not show any considerable effect on skin cancer cells. These findings would form the basis for further studies to understand the mechanisms and assess the potential utility of ST as a cancer chemopreventive agent against breast cancer.

【 授权许可】

   
2013 Vundru et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

【 参考文献 】

• [1]Ferlay J, Shin H, Bray F, Forman D, Mathers C, Parkin D: Estimates of worldwide burden of cancer in 2008. Int J Cancer 2010, 127:2893-2917.
• [2]American Cancer Society: Cancer Facts and Figures: 2010. Atlanta: American Cancer Society; 2010:1-62.
• [3]Anne S, Tsao M, Edward S, Kim M, Waun Ki Hong M: Chemoprevention of cancer. Am Cancer J Clin 2004, 54:150-180.
• [4]Nishino H, Tokuda H, Satomi Y, Masuda M, Onozuka M, Yamaguchi S, Takayasu J: Cancer chemoprevention by phytochemicals and their related compounds. Asian Pacific J Cancer 2000, 1:49-55.
• [5]Dragsted L, Strube M, Larsen J: Cancer-protective factors in fruits and vegetables: biochemical and biological background. Pharmacol Toxicol 1993, 72:116-135.
• [6]Demirkol O, Adams C, Ercal N: Biologically important thiols in various vegetables and fruits. J Agric Food Chem 2004, 52:8151-8154.
• [7]Chandra S, Sah K, Bagewadi A, Keluskar V, Shetty A: Additive and synergistic effect of phytochemicals in prevention of oral cancer. Eur J Gen Dent 2012, 1:142-147.
• [8]Valsta L, Lemström A, Ovaskainen M, Lampi A, Toivo J, Korhonen T, Piironen V: Estimation of plant sterol and cholesterol intake in Finland: quality of new values and their effect on intake. Br J Nutr 2007, 92:671-678.
• [9]Trautwein E, Lin Y, Mel S, Molhuizen H, Ntanios F: Proposed mechanisms of cholesterol-lowering action of plant sterols. Eur J Lipid Sci Technol 2003, 105:171-185.
• [10]Bouic P, Lamprecht J: Plant Sterols and Sterolins: A review of their Immune-modulating properties sterols & sterolins. Altern Med Rev 1999, 4:170-177.
• [11]Awad A, Chinnam M, Fink C, Bradford P: Beta-Sitosterol activates Fas signaling in human breast cancer cells. Phytomedicine 2007, 14:747-754.
• [12]Awad A, Burr A, Fink C: Effect of resveratrol and beta-sitosterol in combination on reactive oxygen species and prostaglandin release by PC-3 cells. Prostaglandins Leukot Essent Fat Acids 2005, 72:219-226.
• [13]Von Holtz R, Fink C, Awad A: Beta-Sitosterol activates the sphingomyelin cycle and induces apoptosis in LNCaP human prostate cancer cells. Nutr Cancer 1998, 32:8-12.
• [14]Bhat TA, Nambiar D, Pal A, Agarwal R, Singh RP: Fisetin inhibits various attributes of angiogenesis in vitro and in vivo–implications for angioprevention. Carcinog 2012, 33:385-393.
• [15]Yim D, Singh RP, Agarwal C, Lee S, Chi H: A novel anticancer agent, decursin, induces G1 arrest and apoptosis in human prostate carcinoma cells. Cancer Res 2005, 65:1035-1044.
• [16]Luo KW, Sun JG, Chan JY, Yang L, Wu SH, et al.: Anticancer effects of imperatorin isolated from Angelica dahurica: induction of apoptosis in HepG2 cells through both death-receptor- and mitochondria-mediated pathways. Chemother 2011, 57:449-459.
• [17]Agarwal C, Singh RP, Dhanalakshmi S, Tyagi AK, Tecklenburg M, Sclafani RA, Agarwal R: Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells. Oncogene 2003, 22:8271-8282.
• [18]Varghese L, Agarwal C, Tyagi A, Singh RP, Agarwal R: Silibinin efficacy against human hepatocellular carcinoma. Clin Cancer Res 2005, 11:8441-8448.
• [19]Sheu MJ, Huang GJ, Wu CH, Chen JS, Chang HY, Chang SJ, Chang JG: Ethanol extract of Dunaliella salina induces cell cycle arrest and apoptosis in A549 human non-small cell lung cancer cells. In Vivo 2004, 212:369-378.
• [20]Singh T, Sharma SD, Katiyar SK: Grape proanthocyanidins induce apoptosis by loss of mitochondrial membrane potential of human non-small cell lung cancer cells in vitro and in vivo. PLoS One 2011, 6:e27444.
• [21]Kakizoe T: Chemoprevention of cancer – focusing on clinical trials. Japanese J Clin Oncol 2003, 33:421-442.
• [22]Deep G, Singh RP, Agarwal C, Kroll DJ, Agarwal R: Silymarin and silibinin cause G1 and G2-M cell cycle arrest via distinct circuitries in human prostate cancer PC3 cells: a comparison of flavanone silibinin with flavanolignan mixture silymarin. Oncogene 2006, 25:1053-1069.
• [23]Kastan MB, Canman CE, Leonard CJ: P53, cell cycle control and apoptosis: implications for cancer. Cancer Metastasis Rev 1995, 14:3-15.
• [24]Blagosklonny MV, Pardee AB: The restriction point of the cell cycle. Cell Cycle 2002, 1:103-110.
• [25]Bartkova J, Lukas J, Strauss M, Bartek J: Cyclin D1 oncoprotein aberrantly accumulates in malignancies of diverse histogenesis. Oncogene 1995, 10:775-778.
• [26]Dong Y, Sui L, Sugimoto K, Tai Y, Tokuda M: Cyclin D1-CDK4 complex, a possible critical factor for cell proliferation and prognosis in laryngeal squamous cell carcinomas. Int J Cancer 2001, 95:209-215.
• [27]Wu A, Wu B, Guo J, Luo W, Wu D, Yang H, Zhen Y, et al.: Elevated expression of CDK4 in lung cancer. J Transl Med 2011, 9:1-9. BioMed Central Full Text
• [28]Lantsov D, Meirmanov S, Nakashima M, Kondo H, Saenko V, Naruke Y, Namba H: Cyclin D1 overexpression in thyroid papillary microcarcinoma: its association with tumour size and aberrant beta-catenin expression. Histopathol 2005, 47:248-256.
• [29]Singh RP, Agarwal R: Flavonoid antioxidant silymarin and skin cancer. Antioxid Redox Signal 2002, 4:655-663.
• [30]Fang L, Igarashi M, Leung J, Sugrue MM, Lee SW, Aaronson SA: p21Waf1/Cip1/Sdi1 induces permanent growth arrest with markers of replicative senescence in human tumor cells lacking functional p53. Oncogene 1999, 18:2789-2797.
• [31]Polyak K, Lee MH, Erdjument BH, Koff A, Roberts JM, Tempst P, Massagué J: Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 1994, 78:59-66.
• [32]Lloyd RV, Erickson LA, Jin L, Kulig E, Qian X, Cheville JC, Scheithauer BW: p27/kip1: A multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers. Am J Pathol 1999, 154:313-323.
• [33]Osborne C, Wilson P, Tripathy D: Oncogenes and tumor suppressor genes in breast cancer: potential diagnostic and therapeutic applications. Oncologist 2004, 9:361-377.
• [34]Singh RP, Agarwal C, Agarwal R: Inositol hexaphosphate inhibits growth, and induces G1 arrest and apoptotic death of prostate carcinoma DU145 cells: modulation of CDKI-CDK-cyclin and pRb-related protein-E2F complexes. Carcinog 2003, 24:555-563.
• [35]Cory S, Huang DCS, Adams JM: The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 2003, 22:8590-8607.
• [36]Katiyar SK, Roy AM, Baliga MS: Silymarin induces apoptosis primarily through a p53-dependent pathway involving Bcl-2/Bax, cytochrome c release, and caspase activation. Mol Cancer Ther 2005, 4:207-216.