The Effect And Regulation Mechanism Of BNIP3 On Chemosensitivity In Colorectal Cancer

Colorectal cancer is one of the most common malignant tumors and chemotherapy plays an important role in its treatment. However, drug resistance leads to chemotherapy failure in most colorectal cancer patients. Impaired apoptosis is is one of the major reasons rendering the tumor cells resistant to cytotoxic chemotherapy . Bcl-2 family proteins play a pivotal role in the regulation of the intrinsic apoptotic pathway induced by cytotoxic stress. Whether a cell undergoes apoptosis in response to cellular stress, including chemotherapy, is determined largely by the balance between pro- and antiapoptotic members of the Bcl-2 protein family.Bcl-2/ adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is a important pro- poptosis member of the Bcl-2 protein family. Induction of BNIP3 leads to apoptosis by two different pathways:(1) heterodimerization with the antiapoptotic protein Bcl-2 or Bcl-Xl , and (2) opening the mitochondrial permeability transition pores by direct contact with its outer membrane. Overexpression of BNIP3 can also induce necrosis-like cell death and autophagic cell death. The BNIP3 gene has been found to be decreased or silenced in pancreatic cancer, colorectal cancer and some hematological tumors and downregulation of BNIP3 is associated with a chemoresistant cell phenotype in pancreatic cancer. This imply that silence of BNIP3 may be a impotant factor that rendering tumor cells resistant to cytotoxic chemotherapy by escaping chemotherapy-induced cell death. However, the precise therapeutic significance of BNIP3 in colorectal cancer is unknown.BNIP3 expression was found to be downregulated by hypermethylation of the gene promoter. DNA methylation is catalyzed by DNA methyltransferase and in mammalian cells DNA methylation is mainly carried out by three DNA methyltransferases: DNMT1, DNMT3a, and DNMT3b. DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-aza-dC) has been extensively used as a demethylating agent which covalently bind with the DNMTs during the methylation reaction. Treatment with 5-aza-dC in pancreatic and colorectal cancer cancer cells could make BNIP3 gene promoter demethylated and restore the BNIP3 expression. Therefore,we assume that it is possible to increase tumor chemosensitivity through increasing the BNIP3 expression and drug-induced apoptosis by means of BNIP3 promoter demethylation. However, clinical application of DNA methyltransferase inhibitor is limited because of its cytotoxicity and lacking of specificity.RNA interference is a powerful technique that allows highly specific suppression of individual gene expression . Previous findings suggest that RNA interference either single targetting DNMT1 or dual targetting DNMT1 and DNMT3b had effect on DNA demethylation and reactivation of many tumor suppressor genes in cancer cells. However, the effect of RNA intefence targetting DNMTs on BNIP3 gene DNA demethylation and colorectal cancer chemosensitivity remains unknown .The aim of present study is to investigate the effect of BNIP3 on chemosensitivity and BNIP3 expression regulation mechanism in colorectal cancer. Thus, we detect the BNIP3 BNIP3 protein expression in colorectal cancer tissue and analyse its association with therapeutic effect of chemotherapy . We also observed the effect of BNIP3 inhibition on cell proliferation, apoptosis and chemosensitivity in colon cancer cells. Furthermore, the methylation status of BNIP3 promotor and the expression of DNMTs in colon cancer cell lines were detected and the influence of inhibition DNMTs by RNAi on BNIP3 expression, cell proliferation , apoptosis and chemosensitivity were also analyzed in colon cancer cells.Methods1. Immunohistochemical detection of BNIP3 proteins was done in tumor and normal samples from 81 advanced colorectal cancer patients who received"oxaliplatin plus 5-Fu and CF"regimens.The association between BNIP3 expression in tumor samples and the clinicopathologic variables, objective response rates (RR) to chenmotherapy, progression-free survival (PFS) and overall survival (OS) of these patients were analysed.2. The mRNA and protein expression of BNIP3 in eight human colon cancer cell lines were detected by RT-PCR and Western blot analysis respectively. The methylation statue of promoters of BNIP3 genes was measured by methylation specific PCR in these cells.3. The plasmid containing short hairpin RNA (shRNA) targeting at BNIP3 gene was constructed and was stably transfected into colon cancer LoVo cell line.The effect of BNIP3 silence were detected by RT-PCR and Western blotting. Cell growth was studied by cell counting using a hemocytometer. MTT assay were employed to detect the chemosensitivity of LoVo cells to 5-FU. Flow cytometry were analyseded to detect 5-FU-induced apoptosis rate.4. The mRNA and protein expression DNMT1 and DNMT3B in eight human colon cancer cell lines were detected by RT-PCR and Western blot analysis respectively.5. HT-29 cells are selected and interfered by DNMT1 siRNA ,or DNMT3B siRNA, or dual DNMT1 and DNMT3B siRNA, mock only and 5-aza-dC treatment as negative and positive control respectively. The expression of DNMT1, DNMT3B and BNIP3 genes are detected and compared by RT-PCR and Western Blot Analysis. The methylation statue of promoters of BNIP3 genes was measured by methylation specific PCR. Cell cycle, apoptotic rate ,cell growth and chemosensitivity to 5-FU were also assessed.Results1. BNIP3 expression was significantly decreased in the tumour sample compared to the normal (1.8±0.2 vs. 3.7±0.5, P<0.05). Patients with positive BNIP3 expression had significantly better objective response rate (53.8% v 28.6%; P= 0.021) and longer progression-free survival (median, 9.25 v 6.5 months; P=0.011) when compared with the BNIP3 negative expression patients. The improvement in overall survival did not reach significance (median, 17.25 v15.0 months; P=0 .143).2. Clear expression of BNIP3 were observed in three colon cancer cell lines (HCT-116, LoVo and LS-174T) and hypoxia enhanced BNIP3 expression in these cells. BNIP3 expression in SW620 was obscure in RT-PCR and Western Blot Analysis.BNIP3 was not expressed in the rest four cell lines (SW480,HT29,HCT-15 and RKO) and even hypoxia could not induce BNIP3 expression.BNIP3 promoter methylation were detected in all the five type of cells with decreased or silenced BNIP3 exprssion.3. The expression of BNIP3 gene of LoVo cells were efficiently blocked by RNA interference (RNAi), which inhibition rates were 73.1% and 75.4% at mRNA and protein levels respectively.4. Silencing BNIP3 gene increased cell proliferation of LoVo cells. Compared with those in the control group, the 5-FU-induced apoptotic rate of LoVo cells transfected with pGCsi3.0-BNIP3 plasmids was significantly decreased (8.35±0.46 vs 19.75±2.37, P< 0.05). The IC50 of 5-FU to LoVo cells transfected with pGCsi3.0-BNIP3 plasmids were 108.4±10.69μg/ml, significantly higher than those to the negative control group (50.08±0.85μg/ml ,P<0.05).5. High mRNA and protein expression of DNMT1 were observed in eight colon cancer cells and DNMT3B expression was different and apparent in these cell lines.6. The decrease in DNMT1 or DNMT3b after siRNA transfection was verified by immunoblotting analysis for HT-29 cells. When cells were treated with 40 nM siRNAs, we observed 76.0% decrease for DNMT1 and 71.1% decrease for DNMT3b compared to transfection control.7. In both DNMT1 and DNMT3b siRNA transfected and Single DNMT1 siRNA transfected cells the BNIP3 promoter were demethylted and BNIP3 expression were restored. The re-expression BNIP3 level after treatment with both DNMT siRNAs was much high than with DNMT1 siRNAs alone. The re-expression BNIP3 level after treatment with both DNMT siRNAs was comparable to 1μM of 5-aza-dC treatment positive control group. Single transfection of DNMT3b siRNA did not restore the BNIP3 expression and the BNIP3 promoter methylation was not changed.8. Co-transfection with DNMTl siRNA and DNMT3b siRNA and single transfection with DNMTl siRNA suppress the colon formation of HT-29 significantly. Compared with the negative control group, the ratio of LoVo cells co-transfected with DNMTl and DNMT3b siRNA and single transfected with DNMTl siRNA in G0/G1 phase was respectively increased by 21.72% and 28.33%,that in S phase was decreased by 30.86% and 44.56%, while apoptotic rate was increased by 6.1 and 13.34 times (both P<0.05). The IC50 of 5-FU to LoVo cells co-transfection with DNMTl and DNMT3b siRNA and single transfection with DNMTl siRNA were 45.54±12.88μg/ml and 171.5±15.20μg/ml respectively, significantly lower than that to the negative control group (376.5±33.69μg/ml, both P<0.05). The effects of co-transfection on colon formation , apoptotic rate and chemosensitivity to 5-Fu of HT-29 were significantly stronger than those of single transfection with DNMTl siRNA (all P<0.05).Meanwhile, single transfection with DNMT3b siRNA had not obvious effects on the biological characteristics of HT-29.Conclusion1. BNIP3 expression is decreared or silenced in most colorectal cancer tissues and colon cancer cells and gene promoter methylation is a important downregulation mechanism of BNIP3 expression.2. The BNIP3 expression is associated with clinical outcome of FOLFOX regimens in advanced colorectal cancer patients. Inteference of BNIP3 could increased the chemo- resistance of LoVo cells to 5-Fu through decreasing the 5-Fu induced apoptosis and promoting cell growth and prolifermion.These imply that BNIP3 may be a potential therapy target for CRC.3. SiRNA is a powerful tool to interfere DNMT1 and DNMT3B genes. Dual inteference of DNMT1 and DNMT3B and single inteference of DNMT1 could rendered BNIP3 promoter demethylated and restore the BNIP3 expression, and both inteference could suppress the colon formation, increase the apoptosis rate and enhance the chemosensitivity to 5-Fu.These suggests that the specific inhibition of DNMT1 and DNMT3B expression by RNAi could be a effective therapeutic approach for colorectal cancer.4. The biological effect of dual DNMT1 and DNMT3B inteference is significantly stronger than single inteference of DNMT1.DNMTl gene plays a key role in DNA methylation and DNMT3b gene may act as an accessory to support its function.Combination DNMTl and DNMT3b has a synergistic effect on DNA methylation.