Studies On The Relationship Between HSMG-1 And Chemosensitivity Of Human Tumor Cells

Chemotherapy is one of the most important means in clinical cancer treatment, but some tumor cells have inherent abilities that resistant to anti-cancer drugs, which often result in the failure of chemotherapy. There are many mechanisms that involved in the drug resistance of tumor cells, among them, the important one is the strong ability of DNA damage repair in some tumor cells. hSMG-1 (the human of suppressor of morphogenesis in genitalia-1) is a recently identified member of the PIKK family, and plays an important role in cellular DNA damage repair. The depletion of hSMG-1 leads to spontaneous apoptosis in lung cancer A549 cells, and increases the sensitivity to ionizing radiation, but the relationship between hSMG-1 and chemosensitivity of human tumor cells remains unclear.In this study, we choosed some human lung cancer, cervical cancer, pancreatic cancer cell lines, investigated the effect of gemcitabine and X-ray on the expression level of hSMG-1 mRNA in these cell lines. Then, the effect of hSMG-1 deletion by RNAi on the chemosensitivity of human lung cancer A549 and H1299 cells were researched. The main contents are as follows:1. The human non-small cell lung cancer cell lines A549 and H1299, human cervical carcinoma cell line HeLa, human pancreatic cancer cell line PANC-1 and human embryonic lung diploid cell CCC-HPF-1 were cultured to the state of logarithmic phase. Total RNA were extracted and the expression levels of p53 and hSMG-1 were mearsured by RT-PCR. The results showed that hSMG-1 gene was commonly expressed in these cells.2. After the treatment of gemcitabine (10 mg·L-1、1.0 mg·L-1、0.1 mg·L-1、0.01 mg·L-1,4h or 2d) and X-ray (2Gy、4Gy、8Gy,4h) on human non-small cell lung cancer A549 and H1299 cells, human cervical carcinoma HeLa cells and human embryonic lung diploid CCC-HPF-1 cells, the total RNA were extracted. Then the expression level of hSMG-1 was measured by quantitative real-time RT-PCR, the results showed that both these stimulus reduced the expression of hSMG-1 in CCC-HPF-1, A549 and HeLa, while H1299 cells showed an increasing trend, indicating that both treatments can influence on the expression of hSMG-1, but not obvious, and the trend is corelated with cell types.3. Studies on lung cancer cells apoptosis induced by RNAi and chemotherapy drugs. hSMG-1 mRNA expression was inhibited by hSMG-1 siRNA transfection in lung cancer A549 and H1299 cells (inhibition ratio were 53.2% and 75.2%, respectively), then the cells were treated with 0.1 mg·L-1 gemcitabine and 1.0 mg·L-1 DDP for 48 h, respectively. Apoptosis rate was determined by flow cytometry and fluorescence microscope, the results showed that hSMG-1 inhibition significantly increased the percent of apoptotic and necrotic cells, the chemosensitive rate of A549 cell increased 3.4 times and 11.7 times, H1299 cell are 56.0% and 85.9%. Further studies showed that the expression level of caspase 3 increased significantly (76% and 83%) in H1299 cells, indicating that mitochondrial apoptosis pathway is correlated with the hSMG-1 deletion induced chemosensitivity enhancement in H1299 cells.