Relationship Between Multidrug Resistance (MDR1) Gene Polymorphism And Radiosensitivity Of Nasopharyngeal Carcinoma

1.Background and objectiveNasopharyngeal carcinoma (NPC) is one of the most common head and neck malignancies in Southern China and southeast Asia, with incidence rates of between 15 to 50 per 100000, for which radiation therapy is always selected as the major tehrapeutic modality. Local control of radical radiotherapy, i.e. radiation response, which indicates the radiosensitivy of nasopharyngeal carcinoma, is essential for the outcome and prognosis of NPC patients.The multidrug resistance (MDR1) gene product P-glycoprotein is a membrane protein that functions as an ATP-dependent effiux pump, transporting exogenous and endogenous substrates from the inside of cells to the outside, which plays an important role in mediating mulitdrug resistance to chemotherapeutic agents. Over last decades human P-gp has been demonstrated to regulate the metabolism, proliferation, differentiation and apoptosis of the cell, which not only causes multidrng resistance, but also confers radiation resistance. It has been elucidated that polymorphisms ofMDR1 gene may have an impact on the expression and function of P-gp, and might also correlate with the radiosensitivity of NPC primary mass, and thereby influence the outcome of local control. This study is to investigate the relationship between the polymorphisms of human MDR1 gene and radiosensitivity of NPC.2.MethodsCase-control study was pratised in this study .Three main steps were performed as followed: (1) primers were designed for detecting the 28 exons, iatron-exon boundaries, and core promoter regions of MDR1 with the web-based software Primer 3.0. in order to verify the known SNPs and find the new SNPs for further study. PCR was performed to amplify the target fragments from a small sample of 5 radioresistant cases and 5 radiosensitive cases of Cantonese-speaking population in Guangdong Province. After sequencing the PCR products by ABI 3730 DNA sequencer, the results were analyzed by Chromas software and a few SNPs were chosen for further study. (2) 18 patients with radioresistant NPC and 30 patients with radiosensitive NPC are included in the case-control study. Genomic DNA was extracted from peripheral blood lymphocytes by proteinase K digestion and phenol/chloroform extraction. PCR-DNA sequencing was performed for genotyping of the selected SNPs above. The comparison of the distributions of the genotypes at selected SNP loci among the radioresistant group and radiosensitive group were made using a chi-square test. The hardy-Weinberg equilibrium of alleles at selected individual loci were tested with a goodness-of-fit x~2 test with one degree of freedom to compare the observed genotype frequencies with the expected genotype frequencies among the subjects. Haplotypes and their frequencies were estimated based on the Bayesian algorithm using the PHASE program, and comparisons of the distributions of the haplotypes between two groups were made using a chi-square test. All analyses were performed using the SPSS, version 13.0. (3) Six cell lines of NPC, i.e. CNE1, CNE2,HNE1, HONE1, 5-8F, 6-10B, were genotyped by the PCR-DNA sequencing method described above. We identified that the cell lines of CNE1, HNE1 and 5-8F are different in the seclected SNPs which were significantly related to the radiosensitivity of NPC tumors, and these cell lines are different in the radiosensitivity according to their radiobiological characteristics. So the realtime RT-PCR is performed on them to compare the differences of MDR1 mRNA expression.3.Results3.1 The distributions of both the genotypes and the alleles of G2677T of the radioresistant group were significantly different from those of the radiosensitive group(P=0.009, 0.004 respectively). The Odds Ratio(OR) value of G2677T alleles was 3.538(95% CI:1.488～8.416), which indicates the higher radioresistant risk of NPC with the 2677T allele compared with the 2677G allele.3.2 There were statistically significant differences of the distribution of both the genotypes and alleles of C3435T(P=0.012, 0.017 respctively). The OR value of C3435T allele were 2.800(95% CI:1.194～6.569), which indicated that the risk for NPC haboring 3435T allele to develop radioresistant was 2.800 times that of 3435C allele.3.3 The 1236 TT genotype or 1236 T allele was associated with a worse radiation response compared with the combined 1236 CC and CT genotypes or 1236 C allele, although it was not statistically significant(P≥0.100).3.4 There were not statistically significant differences of the genotype or allele distribution of both T-129C and C1149T(P≥0.136), although the heterozygosity only appeared in the radioresistant group. The 1400 GA genotype was also associated with a worse radiation response, although it was not statistically significant(P=0.142).3.5 Consistent with the result of genotyping analyses, patients harboring the 1236T-2677T or 2677T-3435T haplotype had a significantly worse radiation response compared with the other haplotypes conbined (P= 0.033, 0.013 respectively).3.6 Six NPC cell lines of CNE1, CNE2, HNE1, HONE1, 5-8F, 6-10B were genotyped by the PCR-DNA sequencing methods as described above.CNE1,HNE1 and 5-8F were identified to be different in the genotypes which were found significantly related to radiosensitivity of NPC according to the results above: CNE1 haboring 1236 CT, 2677 GT, 3435 CT; HNE1 haboring 1236 CT, 2677 GT, 3435 CC; 5-8F haboring 1236 CT, 2677 GG,3435 CC. According to their radiobiological characteristics, CNE1 is a more radioresistant one compared with HNE1 and 5-8F. So they were chosen for the quantitiation of the mRNA expression of MDR1 via realtime RT-PCR. The expression of MDR1 mRNA of the CNE1 is 2.34 times that of HNE1, and 2.42 times that of 5-8F.4. Conclusions:The MDR1 polymorphisms could be used for predicting the response to radiotherapy as genetic markers in NPC patients. The G2677T and C3435T polymorphisms were siginificantly correlated with a worse radiation response of NPC. Patients harboring the 2677TT, 3435TT genotypes, or 1236T-3435T,2677T-3435T haplotype had a statistically significant higher risk to develop resistance to radiotherapy of NPC. The realtime RT-PCR results also indicated that the radiosensitivity of NPC cell lines might correlate with the MDR1 mRNA expression, and the latter might be associated with MDR1 gene polymorphisms of NPC cell lines. Further studies with a larger sample size are required to comfirme our results.