Abnormal Methylation Of Mismatch Repair Genes MLH1 And MSH2 In Human SCLC Cell Line H446 Under Hypoxic Condition

Hypoxia is a common microenvironmental characteristic of human solid tumors including lung cancer. The selective pressure and induced mutations of hypoxia may be the strong driving force for genetic heterogeneity and instability, which contribute to a poor prognosis due to tumor progression towards a more malignant phenotype, with increased metastatic potential, and an increased resistance to treatment.Some evidence has indicated that hypoxia is related to inactive MMR, which plays an important role in maintaining genetic stability, but the mechanism is still unclear. It was previously thought that gene changes mainly meant mutation or deletion, namely at genetics level, but with recent progress of epigenetics, more and more people realized that the regulation of gene expression also was closely related to changes at epigenetics level such as DNA methylation, even more critical. Therefore, it is worth further exploring the relationship between DNA methylation and mechnism of hypoxia on MMR genes regulation.Furthermore, abnormal methylation patterns is closely related to cancer and some forces in cancer cells involved in. The relationship between hypoxia and methylation deserved attention.In conclusion, methylation is a reversible regulation program, which may become a new strategy of gene therapy for tumor. The treatment of demethylation drugs, such as 5-Aza-CdR, can restore the tumor suppressor gene and DNA repair gene expression and shows a certain antitumor activity in several tumors, but not mentioned in SCLC.Objective1. To observe the effect of hypoxia on biological characteristics of human SCLC cell line H446.2. To investigate the expression and the role of promoter methylation of DNA mismatch repair genes MLH1 and MSH2 in H446 cells under hypoxic condition.3. To study the growth inhibition effect of 5-Aza-CdR on H446 cells and the relationship with hypoxia.Materials and methods1. Light microscope and TEM were used to observe cell morphological changes. MTT was used to determine the proliferation and the 50% inhibitory concentration(IC50) for different drugs. FACs was emplored to detect the efficacy of drug exclusion and cell cycle. The caspase-3/7 activity and generation of ROS was assessed by luminometer, flow cytometry and fluorescence microscope, respectively.2. RT-PCR and Western blot were applied to detect MLH1 and MSH2 expression in human SCLC cell line H446 at the mRNA and the protein level, respectively, under the hypoxic condition and after 5-Aza-CdR treatment. Meanwhile, methylation-specific PCR(MSP) was used to determine promoter methylation of MLH1 and MSH2. MS-AP-PCR was emplored to detect the genomic methylation level of H446 cells under hypoxic condition.3. The proliferation, IC50, cell cycle caspase-3/7 activity, Rh123 exclusion efficacy and genomic methylation level of H446 cells with 5-Aza-CdR treatment were analyzed as above.Result1. Under hypoxic condition, the proliferation of H446 cell was decreased significantly. Mitochondria swelling,organelle bubbing and myelinogenesis were observed in H446 cells, even microadencavity emerged in some cells. Meanwhile, H446 cells were more resisted to VP-16 and doxorubicin (P<0.05) under hypoxic condition. Rh123 exclusion efficacy of H446 cells significantly increased (P<0.05). Hypoxia induced the arrest of H446 cells in S phase in the time manner. Moreover, the G2 phase cells decreased, finally disappeared after 48h, but the caspase-3/7 activity did not increase until 48h. FACs showed that ROS increased at first, but significantly decreased after 12h.2. The expression of MLH1 and MSH2 in H446 cells significantly decreased both at the mRNA and the protein level under the hypoxic condition. Meanwhile, the promoter methylation status changed with time of hypoxia: MSH2 gene promoter directly changed from unmethlation to complete methylation and all the CpG sites occurred methlation. Nevertheless, MLH1 gene promoter gradually transformed unmethylation into partial methylation, finally methylation completely and only part of CpG sites occurred methlation. 5-Aza-CdR treatment led to the restoration of MLH1 and MSH2 expression, while, both MLH1 and MSH2 were down-regulated again after removing 5-Aza-CdR. Some genes high-methylation and some genes low-methylation both appeared in H446 cells under hypoxic condition, which was different from H446 cells under normoxic condition.3. Under normoxic condition, 5-Aza-CdR could significantly decrease the proliferation of H446 cells in dose-indepentent manner, induce G1 phase arrest and apoptosis in time-independent manner. Simultaneously, Rh123 exclusion efficacy was increased, genomic methylation level was decreased, while the sensitivities to drugs were not affected. Hypoxia has a synergistic effect on growth inhibition, G1 phase arrest and genomic methylation level reduction.Conclusion1. Hypoxia could inhibit growth, induce S phase arrest, increase the efficacy of drug exclusion and decrease apoptotic potential cells, meanwhile, decrease the sensitivities of H446 cells to chemotherapeitic drugs. This shows that hypoxia may regulate the biological characteristics of tumor cells and its process through a variety of mechanisms.2. The promoter methylation of MLH1 and MSH2 may play an important role in its defective expression in H446 cells under the hypoxic conditon.3. Hypoxia might be one of forces inducing abnormal methylation of tumor cells.4. 5-Aza-CdR could inhibit cell growth, restore mismatch repair genes expression, induce G1 phase arrest and apoptosis, which might be important in antitumor effect of 5-Aza-CdR on H446 cell. Hypoxia has a certain synergy.