The Effect Of P66Shc On Proliferation And Apoptosis Of Colorectal Cancer Cells And The Mechanism Research

Background and ObjectiveColorectal cancer is a kind of high-incidence carcinoma, it is the third most common cancer in worldwide. Mortality rates of colorectal cancer is the second in the all of carcinoma. Colorectal cancer is the third most common cancer in men and the second in women in worldwide. In 2014, International Agency for Research on Cancer reported on the latest investigate datas about the colorectal cancer in 184 countries. In 2012, There were 96,830 new cancer cases,39,590 cancer deaths in USA. Obviously, the therapeutic effect of colorectal cancer in the developed region still is not satisfying. In our country, colorectal cancer has become the fifth most common cancer and its incidence has shown an significant increasing trend over the past decade. At present, early stage colorectal cancer patients can be treated successfully with surgical resection, but owing to the non-invision examination are low sensitivity on the hollow organ and most of peoples can’t accept the colonoscopy before symptom arise.So most of patients were diagnosed in the late stage. Now patients with terminal stage are reftactory. Therefore the effective therapeutic approaches for advanced colorectal cancer patients are still needed.SHC family was consist of four gene members,including Shc A,ShcB, Shc C, Shc D. In mammalian, ShcA adaptor protein family encode three isoforms, p66Shc, p46Shc, p52Shc.Three isforms were controlled by different cell signal transduction pathway, and play different physiological functions. All three isoforms of the Shc family consist of the N-terminal phosphotyrosine binding damain (PTB),the middle collagen homology domain(CH1),and the C-terminal Src-homology domain (SH2). p66Shc contains unique amino-terminal structure (CH2) which distinguish this isoform from the others. CH2 domain is rich in glycine and proline which contain the cytochrome C binding domain.CH2 domain contains a very crucial S36 residue which is a potential phosphorylation site. Some studies have shown that p66Shc, p46Shc, p52Shc plays the roles of mitogenic and carcinoma arise by regulate the tyrosine kinase receptor. Because of the distinguish p66Shc from the p46Shc and p52Shc, p66Shc has some particular functions. Some research indicated that p66Shc was mainly find in cytoplasm, small part located in mitochondria. Several lines of evidences show that p66Shc is high-level expression in a variety of carcinomas, such as prostate cancer, breast cancer,ovarian cancer. In the fields about cancer and signal transduction pathway, p66Shc adaptor protein involved in a various of signal transduction pathway. Such as it can activate the RAS mitogenic signal pathway and take part in the phosphoinositide 3-kinase/protein kinase B(PI3K/Akt) signal pathway.In the past decades, many of researches which including isoform,stuctures, signal pathway, molecular mechanisms of She family have been gained significant progress.But the mechanism are incompletely understood. It is well known that phosphatidylinositol 3-kinase (PI3K)-AKT pathway is a important transduction pathway which involve in cell proliferation, tumor differentiation, metastasis, apoptosis and so on. PI3K (IA type) and Akt are correlated with tumor arise in human. This pathway was associated with regulation of tumor proliferation and survival. If the pathway out of control which not only lead to cell malignant transformation, also influence the process of cell migration, adhesion, tumor angiogenesis, degradation of extracellular matrix. Therefore,PI3K/Akt signal pathway has been regarded as a potential target for cancer therapy. Activated PI3K can bring out second messenger (PIP3) on the cytomembrane, PIP3 can combined the Akt with phosphoinositi-dedependent kinase-1,PDK1 and phosphorylated the Ser 308,finally induced the process of Akt Activation. Activated Akt can invoke or inhibit the protein factor such as Bad,Caspase-9,GSK-3,FKHR, p21Cip1,p27Kipl which can regulate the tumor cell proliferation, differentiation, apoptosis, metastasis. In addition,Akt can phosphorylate and inhibit BAD. BAD is a pro-apoptosis protein,it belong to Bcl-2 family. Generally, BAD can combined with the Bcl-2,and antagonized anti-apoptosis of Bcl-2.Akt can make the BAD depolymerized with Bcl-2, and induced Bcl-2 recover its anti-apoptosis function. Recent studies suggest that Akt signal pathway involve in regulating cell cycle by Mdm-2-p53,p53 is a classic tumor suppressor gene. When the dissociative Mdm-2 enters the nucleus and combines with p53 to form a Mdm-2-p53 compound, thus inhibited the transcriptional activity of p53 and induce p53 degradation. So many studies demonstrated that Mdm-2 can inhibit the functions of p53 and influenced the cell cycle. However,Mdm-2 is the downstream molecule of Akt,so we speculated that PI3K-Akt-Mdm-2-p53 is a important pathway for cell cycle. A mass of prevous studies demonstrated that PI3K-Akt signaling was activated and excessive expressed in multi cancer tissue, such as gastroenteric tumor,breast cancer and pancreatic cancer. This pathway serves to inhibit many tumor suppressor proteins such as the BAD,FOXO transcription factors. Akt can activated the BAD by phosphorylation, then BAD depolymerized with Bcl-2, and induced Bcl-2 recover its anti-apoptosis function.Thus blocking this pathway could therefore simultaneously inhibit the proliferation of tumor cells and sensitize them toward apoptosis. Some researches on breast cancer and prostate cancer have been demonstrate that p66Shc involve in the oxygen stress in mitochondrial which can induce cell apoptosis. In this experiment, p66Shc can promotes cell proliferation and has anti-apoptosis roles. In pilot trial, we found that p66Shc is associated with Bcl-2 and caspase. So we hypothesized that p66Shc involve in the colorectal cancer cell proliferation and apoptosis by PI3K-Akt-Mdm-2-p53 pathway.In this experiment, firstly, we detected the expression level of p66Shc in colorectal cancer tissue and cell lines.Secondly, we also detected the effects of silenced p66Shc in HCT8 cells on the proliferation, apoptosis. Lastly, we determined the pro-apoptotic and anti-apoptotic expression and the cell cycle distribution. This research aim at illustrating the mechanism of p66Shc on proliferation and apoptosis of colorectal cancer cells, and providing the theory basis for the research on colorectal cancer.Methods1. The expression of p66Shc in colorectal cancer tissue and cell lines was detected using quantitative RT-PCR and Western blotQuantitative reverse transcription-PCR(qRT-PCR) was employed to detecte the expression of p66Shc in colorectal cancer tissue and adjacent tissue. Meanwhile detect the expression of p66Shc in colorectal cancer cell lines(NCM460、HCT8 HCT116、SW620、CaCO2). (NCM460 normal colonic epithelial cells, others are colorectal cancer cell lines)2. To interfere the expression of P66Shc in colorectal cancer cell lines(1) Disigned the RNAi sequence by bioinformatics database(Ambion, Genesil, Genecard).(2) RNAi was synthesized by biotechnology company, the interfere the expression of P66Shc in colorectal cancer cell lines by RNA interference.(3)To detect the outcome of RNA interference by RT-PCR and western blot.3. Effect of silence the P66Shc in HCT-8 on cell proliferation and apoptosis(1) To detect the effect of silence the P66Shc in HCT-8 on cell proliferation by CCK8 assay.(2) To detect the effect of silence the p66Shc in HCT-8 on cell apoptosis by AnnexinV-FITC.4.Research on p66Shc cell signal pathway on colorectal cancer cell(1) To detect the effect of silence the p66Shc on the expression of caspase-3,caspase-9,Bax, Bcl-2 in HCT8 by western blot.(2) To detect the effect of silence the p66Shc on the expression of PI3K、AKT、 Mdm-2、p53 in HCT8 by western blot.5. To detect the effect on the cell cycle distribution by infere p66Shc in HCT8 cells 6.Statistical analysisSPSS 13.0 software was used for statistical analysis. Quantitative values of all experiments are expressed as the mean±standard deviation(SD).Relative quantific-ation value of qRT-PCR in cells were analysed by One-way ANOVA.It also analysed through two-tailed independent-samples t-test.The data of CCK8 assay was analysed by Factorial design analysis of variance.Differences were considered significant if P<0.05.Results1. p66Shc overexpression in CRC tissue and cell lines correlates with proliferation and growth.(1) We explored the expression of p66Shc in colon cancer tisuue by RT-PCR. The rusults demonstrated that p66Shc was highly expressed in colon cancer tissue than adjacent tissue(P<0.05).(2) There was also a statistic difference in the expreesion of p66Shc between colon cancer cell line(HCT8、HCT116、SW620、CaCO2) and NCM460 cells(P<0.05).2. Effect of silence the p66Shc in HCT-8 on cell proliferation and apoptosis(1) After knockdown p66Shc in colon cancer cells, There was also a statistic difference on proliferative abilities in the control group, si-scramble group、 si-p66Shc group(P<0.01).HCT8 cells was significantly prohibited from day3 to day6 when compared to the control and scramble siRNA groups.(2) After silence p66Shc in colon cancer cells, the datas shown that amount of live cells were decreased compared with control group and si-scramble group (si-scramble group is 97.0%, si-p66Shc group is 84.8%,P<0.05). There was also a statistic difference on the apoptosis cells between the si-p66Shc group with si-scramble group(si-scramble group is 0.112%, si-p66Shc group is 0.159%,P<0.05). The apoptosis of HCT8 cells was dramatically increased in p66Shc siRNA treated group.3. Research on p66Shc cell signal pathway on colorectal cancer cell(1) Definite the effect on the cell cycle distribution by infere p66Shc in HCT8 cells with flow cytometry.In this part, the results refer to the control and scramble siRNA treated group,the proportion of cells in G0/G1 phase(P<0.01) was increased and the proportion of cells in G2/M phase (P<0.05)was declined in p66Shc siRNA treated group, which suggests the silence of p66Shc induces cell cycle arrest in G0/G1 phase.(2) The effect of silence the p66Shc on the expression of apoptosis proteins in HCT8 by western blot.The expression of caspase-3,caspase-9,Bax was significantly enhanced in si-p66Shc groups than the control and scramble siRNA treated group (P<0.01).On the contrary, the expression of Bcl-2 was declined in si-p66Shc groups by western blot(P<0.01).(3)The effect of silence the p66Shc on the expression of PI3K-AKT signal pathway proteins in HCT8 by western blot.In this part,after silence the p66Shc in the HCT8,we found that the phosphorylation of PI3K and AKT was significantly suppressed in si-p66Shc group when compared to the control and si-scramble group. Also the express of Mdm-2, a downstream of AKT,was obviously prohibited when compared to the other groups. In contrast, the expression of p53 was enhanced in p66Shc siRNA treated group than the control and scramble siRNA group. The gray scale value analysis of PI3K, AKT, Mdm-2, p53 expression further confirmed this result. The results revealed that the silence of p66Shc played an important role in the viability inhibition in HCT8 cells via PI3K/AKT/Mdm-2/p53 signaling pathway.Conclusion1. p66Shc protein highly expressed in colon cancer tissue and colon cancer cell line cells. So we infer that p66Shc is able to promote colon cancer cell proliferation.2. Silence the p66Shc can inhibited the colon cancer cell proliferation and induced cell apoptosis. These datas demonstrated that infere the p66Shc is important to regulate colon cancer cell proliferation.3. Silence the p66Shc can induces cell cycle arrest in G0/G1 phase.4. Silence the p66Shc can promoted the expression of caspase-3,caspase-9,Bax, reduced the phosphorylation of PI3K and AKT.Based on the above, we can draw a conclusion that the silence of p66Shc played an important role in the viability inhibition in HCT8 cells via PI3K/AKT /Mdm-2/p53 signaling pathway.