The Effect Of Irinotecan Metabolic Enzyme Gene Expression Regulation By DNA Methylation On Colorectal Cancer

Background: Irinotecan (CPT-11) is widely used in the adjuvant therapy for colorectal cancer recently. CPT-11 can interfere and stabilize a cleavable complex of topoisomerase I (Top I) and DNA double strands by transforming into 7-ethyl-10-hydroxycamptothecin (SN-38), which exhibits its cytotoxicity via suppressing DNA replication. CPT-11 is hydrolyzed into an active metabolite SN-38 by carboxylesterase(CES). Subsequently, SN-38 is inactivated into an unreactive combining form SN-38G by glucose hydroformylation in uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) enzyme. Meanwhile, beta-glucuronidase (GUSB) can reversely convert SN-38G into SN-38 via dehydroformylation. Recent studies suggested that the activation of CPT-11 in tumor tissue was more meaningful than in liver. Therefore, the regulation for CPT-11 metabolic enzymes is undoubtedly important to the level of SN-38, which affects the efficiency of chemotherapeutic drugs greatly.Studies have indicated that gene silencing of itself phenogenetics presented in many chemotherapeutic drugs metabolic enzymes. However, methylation may be responsible for regulating the expressions of these enzymes. If methylation is confirmed in the expressions of CPT-11 metabolic enzymes related genes, we can develop new reversal strategy from methylation intervention view, which will be favorable in enhancing the chemotherapy efficiency and improving the prognosis of colorectal cancer.Objective: To observe role of abnormal methylation in regulating CPT-11 metabolic enzymes related genes CES2, UGT1A1 and GUSB expressions and effect of the targeted genes methylation on susceptibility of colorectal cancer cell to CPT-11. To illuminate the phenogenetics mechanism for chemotherapeutic drugs resistance of colorectal cancer and provide an new approach and strategy for tumor resistant reversal and chemotherapeutic sensitization. Methods: Colorectal cancer tissue samples in clinic and cell lines such as LoVo, HT-29, LS174T, DLD-1, HCT-116, RQO, HCT-15 and SW-48 were selected in present study.PartⅠ.Immunohistochemistry was employed to determine the expressions of CES2, UGT1A1 and GUSB proteins in 99 colorectal cancer samples, 23 corresponding peficancerous tissues and 8 enteric adenoma samples. Methylation specific polymerase chain reaction (MSP) was used to assay methylation levels of CES2, UGT1A1 and GUSB in colorectal and peficancerous tissues. SPSS13.0 software was used to analyze the correlationships between CES2, UGT1A1, GUSB proteins levels and clinical pathological parameters and relationships between methylation and corresponding gene expressions. All data are expressed as means±standard deviation. The student's t test was used to examine the differences between groups. One-factor analysis of variance (ANOVA) was used to examine the differences among groups. A p value of less than 0.05 was considered significant, and one less than 0.01 was considered highly significant.PartⅡCultured 8 colorectal cancer cell lines LoVo, HT-29, LS174T, DLD-1, HCT-116, RQO, HCT-15 and SW-48 in vitro. MSP was used to determine promoters methylation in CES2, UGT1A1 and GUSB. Divided each cell line into two groups: DAC group(5μM DAC) and control group(equal PBS). Replaced culture medium every 24 h and added 5μM DAC and equal PBS in DAC and control group, respectively. Collected the cells when cells covered 80%-90% of culture flask surface. Respectively, fluorescent quantitative PCR and western blot was employed to assay the mRNA and proteins levels of CES2, UGT1A1 and GUSB in each colorectal cancer cell line.PartⅢMTT was used to assay the susceptible change of each cell line to CPT-11 treated or untreated by DAC. 50% inhibiting concentration (IC50) was obtained by inhibition ratio. Meanwhile, the correlationships between IC50 and the expressions of CES2, UGT1A1 and GUSB were analyzed.Ⅱ. Prepared UGT1A1siRNA using siRNA gene transfection kit. Observed transfection efficiency under flow cytometry(FCM). Cytotoxicity was assayed with trypan blue exclusion. The effective interfering sequence for UGT1A1 SiRNA was screened by reverse transcription polymerase chain reaction(RT-PCR). Methyl thiazolyl tetrazolium(MTT) assay was used to determine cytotoxic change of CPT-11 after HT-29 cell was transfected by UGT1A1 SiRNA.Ⅲ. Also, MTT assay was employed to evaluate cytotoxic change of CPT-11 by the transfection of chemosynthetic UGT1A1 SiRNA when the expression of UGT1A1 in abnormal methylated RKO cell was restored after demethylation treatment or not.Results:PartⅠThe positive expressions rates of CES2, UGT1A1 and GUSB in adjacent tumor tissue, colorectal adenoma and colorectal cancer tissue were 56.5%(13/23) vs. 34.7% (8/23) vs. 47.8%(11/23), 50.0% (4/8) vs. 25%(2/8) vs. 50.0% (4/8) and 35.3% (35/99) vs. 19.2% (19/99) vs. 38.3%(38/99), respectively. The expression of CES2 and UGT1A1 in colorectal cancer tissue was weaker than that in tumor adjacent tissue, respectively (P<0.05). There were differences in the expression of CES2 among different clinical stages (P<0.05). The promotors of CES2 and GUSB in colorectal cancer and tumor adjacent tissue displayed a consistent unmethylated or hemimethylated status. The positive methylation rate of UGT1A1 in colorectal cancer tissue and tumor adjacent tissue was 86.4% (51/59) and 65.2% (15/23), respectively. The methylation of UGT1A1 was negatively related to the expression of UGT1A1(P<0.05).PartⅡⅠ. The straps for methylated CES2, UGT1A1, GUSB and unmethylated positive control were amplified clearly in the gels. Promotor of CES2 was in a unmethylated status. The positive methylation of UGT1A1 promotor was seen in LS174T, DLD-1, HCT-116, RKO and HCT-15 cell lines. The negative methylation was in LoVo, HT-29 and SW-480. Promotor of GUSB was in a condition of hemimethylation.Ⅱ. There were differences in the mRNAs and proteins expressions of CES2, UGT1A1 and GUSB in LoVo, HT-29, LS174T, HCT-116, RKO and SW480. The cell lines expressing low levels of CES2 and GUSB were not increased significantly after the treatment of decitabine(DAC). UGT1A1 expressed more in LoVo, HT-29, SW480 cell lines and less in LS174T, HCT-116, RKO, HCT-15 and DLD-1. The expression of UGT1A1 in LS174T, HCT-116, RKO, HCT-15 and DLD-1 enhanced significantly after DAC treatment. The expression of UGT1A1 mRNA increased by 17 folds and 26 folds in HCT-116 and RKO, respectively.PartⅢThere were significant differences in chemotherapeutic susceptibility to CPT-11 among different colorectal cancer cell lines(P<0.05). The chemotherapeutic susceptibility of LoVo cell line to CPT-11 was lest and the susceptibility of RKO cell line was most. The difference of IC50 was nearly 5 folds. The expressions of CES2 and GUSB in colorectal cancer cell lines were negatively correlated to IC50, respectively (r= -0.58651, P<0.01; r= -0.62527, P<0.01), suggesting more CES2 or GUSB expressed, more susceptible to CPT-11 chemotherapy. The intracellular expression of UGT1A1 was not related to IC50 significantly. However, it suggested that more expression of UGT1A1 was more closely related to resistance of CPT-11 chemotherapy(r=0.790648, P<0.05) when SW480 cell line was excluded. Effective interfering sequences for appropriate SiRNA (50nM) were screened and interfering efficiency was 78%. The IC50 decreased nearly 50% after HT-29 cell line was transfected(P<0.01). IC50 of RKO cell with methylated UGT1A1 gene increased after demethylation treatment. However, the IC50 in demethylation treatment group was reduced compared with non- treatment group after transfection of UGT1A1 siRNA.Conclusions:Ⅰ. CES2, UGT1A1 and GUSB were less expressed in pathological tissues of colorectum. Their expressions were weaker corresponding adjacent tumor tissue and colorectal adenoma, respectively.Ⅱ. Abnormal methylation of UGT1A1 gene in colorectal cancer and adjacent tumor tissue was related to the less expression of UGT1A1. On the whole, CES2 and GUSB was respectively in a unmethylated or hemimethylat status, which was not significantly related to the corresponding proteins expressions.Ⅲ. UGT1A1 in part colorectal cancer cell lines was highly methylated. Furthermore, DAC could reverse the low expression of UGT1A1, suggesting the presentation of methylated regulation in UGT1A1.Ⅳ. CES2 promotor in colorectal cancer cell line was unmethylated. However, methylation was seen in the promoter of GUSB. Cell lines with low level of CES2 and GUSB could not be reversed by DAC, suggesting methylation did not involve in the regulation of CES2 and GUSB.Ⅴ. Cytotoxicity of CPT-11 was negatively related to the expression of UGT1A1 in tumor cell, it was positively related to CES2 and GUSB. siRNA for UGT1A1 gene could significantly enhance chemotherapeutic susceptibility of colorectal cancer cell lines to CPT-11.Ⅵ. UGT1A1 methylation is an important factor in therapeutic susceptibility to CPT-11. Demethylation treatment may reduce therapeutic susceptibility to CPT-11 in colorectal cancer with abnormal methylated UGT1A1 gene.