The Effect Of Silencing PI3K P85α Expression By RNA Interference On The Apoptosis Of Colorectal Cancer Cells Induced By 5-FU

Background and objectivesColorectal cancer (CRC) is one of the major malignancies in the world. The morbidity is higher in North America and Oceania than in Europe and Asia. In China, the morbidity is higher in South than in North,only after esophageal and gastric cancer. In the past twenty years,the morbidity of colorectal cancer has increased in most countries over the world,the morbidity of CRCs was increasing at a rate of 2% per year,while in our country,with many changes having taken place in the economy as well as people's diet and lifestyle which is rich of fat,protein,calorise and lack of fiber, the number of new cases in china arising each year is still increasing.According to the survey conducted by Shanghai, The morbidity of CRCs in china is increasing at an annual rate of 4% which is higher than the average incidence rate of the world. Despite the rate of improvements in surgery, radiotherapy and chemotherapy, unfortunately, the prognosis of CRCs has not been gained progress over the past decades.The local treatment of surgery and radiotherapy are powerless to control tumor recurrence and metastasis,while the conventional chemotherapy regiments for the treatment of colorectal cancer have limited efficacy and are associated with significant toxicity. Although FOLFOX regiments are recoganized at home and abroad,the resistance of 5-fluorouracil(5-FU) was not uncommon.Therefore, novels diagnose and treatments need to be developing in order to enrich the therapeutic armamentarium. With the rapid development of molecular biology recently, signaling pathways that involved in tumor progression have been payed more and more attention. Truly, an increasing knowledge about the genetic control of cellular proliferation and the modulation of the signaling pathways that are aberrant in colorectal cancer has the potential to provide an effective and better approach for its diagnosis,prevention and management.The data obtained have enriched our understanding of why 5-FU drug resistance taken place and how to increase the sensitivity of CRCs to 5-FU treatment.Phosphatidylinositol-3-kinase (PI3K) is a lipid kinase and is responsible for the phosphorylation of 3 position of the inositol ring of PI(4,5)P2, to generate PI(3,4,5) P3, a potent second-messenger required for fundamental cellular functions such as transcription, translation, proliferation, growth, metabolism, motility and survival. There are three members in PI3K family. Signaling pathway composed of Class-IA PI3K and serine/threonine kinase Akt/PKB has close relationship with tumor progression. This pathway regulates proliferation and survival of cancer cells. The disturbed activation of PI3K signaling leads to not only neoplastic transformation of normal cells, but also correlation with tumor cell migration, adhesion, tumor angiogenesis, as well as the degradation of extracellular matrix. Class-IA PI3K are heterodimers composed of a catalytic subunit (p110α/β/δ/γ) and an adapter/regulatory subunit (p85α/β/γ). P85 regulatory subunit is essential for stabilization and collection of P110 catalytic subunit and for activation of Class-IA PI3K. p85αis the most abundant regulatory subunit of PI3K family. Evidences have showed that PI3K p85αis a kind of oncogene.It has been reporetd that mutation of PI3K p85αcould activate PI3K/Akt signaling pathway and the mutation was higher in CRC. Our laboratory have proofed that PI3K p85αexpression is negative in normal colorectal mucosa and polypi, begins to increase in adenoma and over-expressed in primary colorectal adenocarcinoma, and there was a significant difference in the positive rates of the PI3K p85αexpression duiring different Dukes' stage. FoxO transcription factors belong to the Forkhead family of proteins, a family characterized by a conserved DNA binding domain termed the Forkhead box (Fox). The family is primarily regulated by PI3K pathway. Direct phosphorylation by PKB/Akt inhibits transcriptional activation by FoxO factors, causing their displacement from the nucleus into the cytoplasm. Deregulation of cell cycle and cell apoptosis are colosely related with tumor progression. The FoxO is inhibited when it is phosphorylated by active AKT, furthermore,the target genes of FoxO are inhibited either, they are apoptotic genes such as Bim, Bcl-6, Bax.Our laboratory has constructed stable transfected CRC cells LoVo and SW480 cells(L324 and SW1124),which were tranfected with shRNA vector constructed ourselves. We have proved that depletion of PI3K p85a could obviously induce cell cycle arrest, the cell cycle progression was arrested in GO/Gl phase.In this study, we are on the way to investigating the expression and significance of PI3K p85a in the progression of colorectal cancer, including normal colorectal mucosa, colorectal proliferational ployp, colorectal adenoma and primary colorectal carcinoma.Then we evaluated the 50% inhibitory concentration (IC50) values of 5-FU by MTT reduction assay after the cells treated with different concentration of 5-FU for 72 hours. Mitochondrial membrane potential was demonstrated by JC-1 fluorescence. Hoechest33258 staining was used to observate the changes of nulei morphology.Western blot was used to analyze the expression of apoptotic protein Bcl-6,Bim and Bax. We also investigate the effect of silencing PI3K p85a expression by RNA interference on the apoptosis of colorectal cancer cells induced by 5-Fluorouracil.Materials and methodsThe expression and significance of PI3K p85a in progression of colorectal cancerWestern blot was used to detect the expression and significance of PI3K p85a in the progression of colorectal cancer, including normal colorectal mucosa, colorectal proliferational ployp, colorectal adenoma and primary colorectal carcinoma.The effect of silencing PI3K p85a expression by RNA interference on the apoptosis of colorectal cancer cell induced by 5-Fluorouracil 1. The 50% inhibitory concentration (IC50) values of 5-FU were evaluated by MTT reduction assay after the cells treated with 5-FU for 72 hours.2. After the colorectal cancer cells being treated with 5-FU for 48 hours.cells were harvested and washed with PBS, fixed with 4% paraformaldehyde for 15 min at 25℃,washed three times with cold PBS, and exposed to lOmg/L Hoechst33258 in the dark at room temperature for 10 min. Images were acquired using a fluorescence microscope.The cells with condensed chromatin and shrunken nulei were counted as apoptotic cells.3. Mitochondrial membrane potential was demonstrated by JC-1 fluorescence after the colorectal cells being treated with 5-FU 48 hours.4. Western blot was used to analyze the expression of apoptotic protein Bcl-6,Bim and Bax in control group cells LoVo, SW480 and transfected group cells L324, SW1124 after they being treated with 5-FU for 48 hours.Statistical AnalysisAll experiments results were from at least three separate experiments. Dates are expressed as the mean±SD. one-way analysis of variance (ANOVA) and Student's t test were used in group comparison. A value of P<0.05 was considered statistically significant.ResultsThe expression and significance of PI3K p85a in progression of colorectal cancerWestern blot was performed to examine PI3K p85a expression levels in fresh tissue from colorectal mucosa, benign polyp and adenomas to primary colorectal cancers. The expression of PI3K p85a was increasing from colorecta mucosa. The diffierence for the expression of PI3K p85a protein in normal colorectal tissue, colorectal proliferational ployp, colorectal adenoma and primary colorectal carcinoma was significant, P=0.000.The effect of silencing PI3K p85a expression by RNA interference on the apoptosis of colorectal cancer cell induced by 5-Fluorouracil1. compared with LoVo(8.07±0.30μmol/L), SW480(33.61±12.19μmol/L) the IC50 of 5-FU were obviously decreased in the cells L324 (4.57±0.16μmol/L)and SW1124(19.04±5.22μmol/L), P=0.000.2. Under a fluorescence microscope, typical apoptotic morphological changes, such as condensed chromatin,shrunken nulei,and loss of cell volume,were more frequently observed in the transfected cells L324 and SW1124 than in the control group cells LoVo and SW480.3. The mitochondrial membrane potential of L324 and SW1124 cells were much lower than that of LoVo and SW480 cells,this suggested that silencing PI3K p85αexpression could increase the sensitivity of colorectal cancer cells to 5-FU treatment.4. We examined the expression levels of apoptotic protein Bcl-6,Bim,Bax in the L324 and SW1124 cells were much higher than the expression in the LoVo and SW480 cells, P=0.000.Conclusions1. Western blot results show that the expression levels of PI3K p85a gradually increased from normal colorectal mucosa, colorectal proliferational ployp,adenoma to primary colorectal adenocarcinoma, indicating that PI3K p85a plays an important role in the progression of colorectal cancer.2. MTT assay,Hoechst staining and JC-1 fluorescence were proved that down-regulate PI3K p85a could significantly enhance the sensitivity of colorectal cancer cells to 5-FU.3. Down-regulating PI3K p85a expression could active the activity of FoxO transcription factors, which, in turn, activated apoptotic genes such as Bcl-6,Bim,Bax, involved in cell apoptosis. Thus, PI3K p85a depletion could significantly enhance the apoptosis of colorectal cancer cells induced by 5-FU through increasing the expression of apoptotic protein such as Bax,Bim,Bcl-6.