Regulation Of ASPP1 And ASPP2 In Resveratrol Induced Apoptosis In Human Breast Cancer Cells

Identification and functional analysis of safe and effective anti-cancer active ingredient from plants is one of the ways to find new anti-cancer drugs. Resveratrol (3,5,4'-trihydroxystilbene) is a natural polyphenolic compound that is commonly found in grape skin, red wine, peanuts and some Chinese herbs Previous studies have demonstrated that resveratrol has a nμMber of biological activities that might be beneficial to human health, including anti-oxidan, anti-inflammatory, cardioprotective and phytoestrogen properties. Importantly, resveratrol may also act as an anti-cancer agent by inhibition of cell proliferation/transformation and induction of apoptosis. Although resveratrol associated growth inhibition and apoptosis has been associated with a number of molecules and pathways, the precise mechanisms of resveratrol mediated activities remain unclear. This is exemplified by resveratrol mediated regulation of the p53 pathway.p53 is a tumor suppressor mutated in about 50% of human cancers. Activation of p53 pathway plays a critical role in homeostatic maintenance and cellular responses to anti-cancer therapeutics. Regulation of the p53 pathway in resveratrol induced apoptosis appears to be complicated. It was demonstrated that resveratrol induced apoptosis in cells expressing wild-type p53 (p53+/+), but not in p53-deficient (p53-/-) cells. Reports from different studies, however, indicated that some cancer cells with mutant p53 remain sensitive to resveratrol. Since the p53 pathway can also be regulated by other p53 family members and p53 interacting proteins, such as p73, p63 and the ASPP family members, it is important to understand whether and how these p53 interacting molecules are regulated in resveratrol induced apoptosis.ASPP (apoptosis stimulation protein of p53) is a new family of proteins including ASPP1, ASPP2 and iASPP.This family is characterized by sharing a highly conserved carboxyl terminus, which contains a proline-rich region, four ankyrin repeats and an SH3 domain, and p53 binding capacity. Mechanistic analysis indicates ASPP1 and ASPP2 proteins enhance the apoptotic function of p53 by stimulating the selection of proapoptotic promoters, whereas iASPP-p53 interaction inactivates p53 mediated apoptosis. As putative tumor suppressors, ASPP1 and ASPP2 are frequently downregulated in human breast tumors and leukemia, which may involve hypermethylation of its promoter. Importantly, ASPP1 and ASPP2 may also interact and stimulate p73 and p63 mediated apoptosis in a manner similar to p53, which underscores the role of ASPP1 and ASPP2 in tμMor cells with different p53 status. Nevertheless, the role of ASPP proteins in resveratrol induced apoptosis has not been documented.In this report, we studied the regulation of ASPP 1 and ASPP2 in resveratrol treated breast cancer cells. Our results indicate that resveratrol upregulates the expression of ASPP1 and ASPP2 though E2F-1 mediated transcription. Resveratrol induced ASPP1 and ASPP2 expression plays a critical role in resveratrol induced apoptosis.PART I Expression of ASPP1 and ASPP2 in MCF-7 and MDA-MB-231 cells treated with resveratrolObjectiveExamine gene expression of ASPP1 and ASPP2 in resveratrol-induced apoptosis in breast cancer cells.Methods1. MTT assay was used to evaluate the growth inhibition of MCF-7 and MDA-MB-231 cells in responses to various doses of resveratrol.2. RT-PCR, Western blot were applied to detect the expression of ASPP1 and ASPP2 mRNA and protein levels in MCF-7 and MDA-MB-231 cells treated with resveratrol.Results1. Proliferation of MCF-7 and MDA-MB-231 cells was inhibited by resveratrol in a dose dependent manner.2. The expression of ASPP1 and ASPP2 mRNA and protein in MCF-7 and MDA-MB-231 cells treated with resveratrol were increased in a dose-dependentmanner as determined by RT-PCR, Western blot analysis.ConclusionResveratrol induced apoptosis correlates with induction of ASPP1 and ASPP2 expression. PARTⅡOver-expression of ASPP1 and ASPP2 Enhances Resveratrol-induced apoptosis in MCF-7 breast cancer cellsObjectiveTo test whether over-expression of ASPP1 and ASPP2 gene sensitizes to MCF-7 and MBA-MD-231 cells to resveratrol induce apoptosis.Methods1. pcDNA3/ASPP1 and pcDNA3/ASPP2 were transfected into MCF-7 cells by Fugen-6, respectively. Positive clone cells were obtained by persistent G418 selection. Overexpression of ASPP1 and ASPP2 expression in the transfected cells was assessed by Western blot.2. MCF-7, MCF-7/ASPP1 and MCF-7/ASPP2 cells were treated with resveratrol at indicated concentrations for 5 days following by MTT assay. Apoptosis Elisa Assay was used to evaluate the percentage of apoptosis cells.After treated with resveratrol for 24 hours to MCF-7,MCF-7/ASPP1 and MCF-7/ASPP2 cells, RT-PCR was applied to detect the expression of apoptosis associated protein such as Bax,p21.3. pcDNA3/ASPP1 and pcDNA3/ASPP2 were transiently transfeced to MDA-MB-231 cells with or without resveratrol treatment. Then MDA-MB-231 cells were treated with resveratrol at indicated concentrations for 5 days following by MTT assay.4. MTT assay was preformed to evaluate the percentage of survival cells; Apoptosis Elisa Assay was used to evaluate the percentage of apoptostic activties in resveratrol treated MCF-7 and MCF-7/Caspase 3.5. pcDNA3/ASPP1 and pcDNA3/ASPP2 were transiently transfeced to MCF-7/Caspase 3 cells with or without t resveratrol treatment, followed by MTT assay to evaluate the percentage of survival cells. Results1. Stable cell lines that over-expression ASPP1or ASPP2 obtained after G418 selection for 4 weeks. ASPP1 or ASPP2 overexpression was confirmed by Western-bolt.2. MTT and Apoptosis Elisa Assays showed that ASPP1 or ASPP2 overexpression sensitized resveratrol mediated apoptosis in MCF-7/ASPP1, MCF-7/ASPP2 cells, which was accompanied by higher expression of bax, p21, as compared to control.3. MTT showed that the percentage of survival cells were decreased after transient transfection of ASPP1 or ASPP2 plasmid in MDA-MB231 cells, as compared to control. Combination of ASPP1 or ASPP2 transfection and resveratrol treatment results in significant inhibtion of MDA-MB231 cells.4. MTT and Apoptosis Elisa Assay showed that MCF-7/Caspase 3 was more sensitive to resveratrol treatment compared with MCF-7 cells.5. Much more apoptotic cells were observed in the group treated by the combination compared with those transfected by ASPP1 and ASPP2 plamid and resveratrol.ConclusionOver expression of ASPP1 and ASPP2 rendered MCF-7 cells more sensitive to resveratrol.PARTⅢObjectiveInvestigate the role of E2F-1 in resveratrol-induced upregulation of ASPP1 and ASPP2. Methods1. RT-PCR, Western-blot were applied to detect the expression of E2F-1 mRNA and protein in MCF-7 and MDA-MB-231 cells treated with resveratrol.2. MCF-7 and MB-231 cells were infected with adenoviral E2F-1 (Ad-E2F-1) and control Ad., and then treated with resveratrol (50μM) 2 hours after infection. Western-blot was used to detect the expression of E2F-1, ASPP1 and ASPP2 protein in MCF-7 and MDA-MB-231 cells treated with resveratrol.3. Small interfering RNA Transfection siRNA-mediated silencing of E2F-1 in MCF-7 and MDA-MB-231 cells, followed by resveratrol treatment. RT-PCR was applied to evaluate the mRNA levels of E2F-1,ASPP1 and ASPP2.Results1. RT-PCR and Western Blot showed that both E2F-1 mRNA and protein levels were increased in resveratrol-treated MCF-7 and MDA-MB-231 cells.2. Western Blot analysis showed that the levels of ASPP1 and ASPP2 were induced by Ad-E2F-1 infection.3. RT-PCR analysis indicated that resveratrol induced ASPP1 and ASPP2 expression was inhibited by E2F-1siRNA transfection.ConclusionResveratrol induced expression of ASPP1 and ASPP2 was mediated by the activation of the E2F-1 pathway.