Research On Correlation Between The Expression Of Excision Repair Cross-complementing 1,Metallothionein1H And Acquired Platinum Resistance In Advanced Gastric Cancer

Gastric Cancer is one of the most common malignant tumors worldwide. The incidence and mortality rates are high and the rate of early diagnosis is low. Early gastric cancer is lack of specific performance. 5-year survival rate of the patients is usually no more than 20%. Platinum and fluorouracil are the most common drugs of neo-adjuvant chemotherapy and postoperative adjuvant chemotherapy for gastric cancer, palliative treatment for advanced gastric cancer patients. Drug resistance, especially Multidrug resistance (MDR), has seriously affected the effects of chemotherapy and prognosis. How to improve the sensitivity of chemotherapy in gastric cancer and the clinical response of chemotherapy is the focus of attention.Drug resistance of tumor cells is associated with the differences of DNA damage repair capacity in tumor cells and over-expression of thiol molecules in cell detoxification system. DNA damage is repaired primarily through the way of nucleotide excision repair (NER), and excision repair cross-complement gene (ERCC1) plays a key role in the NER. ERCC1 low expression can increase the incidence of cancer, and ERCCl over-expression may be against the anti-tumor effect of platinum. Metallothioneins (MTs) are a group of ubiquitous low molecular mass, cysteine-rich intracellular metal-binding proteins. In human, MT genes may involve at least 10 functional genes and 7 unfunctional genes. MT1H is one of the 10 functional genes. There are close relations between MT and the occurrence and development of tumor. MTs are involved in tumor cell proliferation, apoptosis, differentiation, drug resistance, prognosis. However, current studies are limited to histopathological observation, only a few was on hematology. For this, first we studied the correlation between the expression of ERCC1,MT1H and acquired cisplatin resistance in human gastric adenocarcinoma cell SGC-7901. Second,we collected the patients with circulating tumor cells, then the expression of ERCC1 and MT1H in peripheral bloods of advanced gastric cancer patients before and after platinum-based chemotherapy was investigated to explore the relationship with acquired platinum resistance.PartⅠSignificance of expression of ERCC1 and MT1H before and after the effect of cisplatin on human gastric adenocarcinoma cell SGC-7901Objective: We investigated the effect of cisplatin on human gastric adenocarcinoma cell SGC-7901 to confirm the correlation between the expression of ERCC1,MT1H and acquired cisplatin resistance .Methods: The way of methyl thiazolyl tetrazolium (MTT) was used to measure the anticancer activity. We measured the expression of ERCC1 and MT1H by semi-quantitative RT-PCR in human gastric adenocarcinoma cell SGC-7901 which was treated with cisplatin for different concentration and different hours. All dates were analyzed by using student's t-test by SPSS10.0 statistical package.Results:1. With the increase of the concentration of cisplatin, the inhibition to human gastric adenocarcinoma cell SGC-7901 showed a gradual increase. When treating with cisplatin for 24 or 48 hours, the IC50 was 14μmol/L and 11μmol/L, respectively.2. After treating with 2.5μmo/L cisplatin for 24 hours, the expression of ERCC1mRNA and MT1HmRNA increased. With the increase of the concentration of cisplatin, the expression increased gradually, and reached the top when treating with 40μmol/L cisplatin. The difference was statistically significant between the top group and control group (P=0.001,P=0.005).3. After treating with 20μmol/L cisplatin for 12 hours, the expression of ERCC1mRNA and MT1HmRNA increased in SGC-7901 cell .With the extension of the time, the expression also increased. When treated with 20μmol/L cisplatin for 72 hours, the expression reached the top. The difference was statistically significant between the top group and control group (P=0.005,P=0.001).Conclusions: The effect of cisplatin on human gastric adenocarcinoma cell SGC-7901 was of concentration-dependentment. With the increase of the concentration of DDP and the extension of time, the expression of ERCC1mRNA and MT1HmRNA increased gradually. ERCC1 and MT1H may be involved in acquired platinum resistanc in gastric carcinomor.PartⅡSignificance of expression of excision repair cross-complementing 1 and metallothionein1H in peripheral blood of advanced gastric cancer patients before and after platinum-based chemotherapyObjective: We studied the expression of ERCC1 and MT1H in peripheral blood of advanced gastric cancer patients with circulating tumor cells before and after platinum-based chemotherapy to investigate the relationship between the expression of ERCC1,MT1H and acquired platinum resistance.Methods: 162 advanced gastric cancer patients confirmed with pathology or cytology were included. The 15ml peripheral blood of every case before chemotherapy was collected. The sample (7.5ml) was filtrated with the polycarbonate membrane containing cylindrical pores with 8 um in diameter. The way was used to separate the tumor cells, then the cells were dyed with Cytokeratin 8 (CK8) antibody, Leukocyte common antigen (LCA) CD45 antibody and 4,6-diamidino-2-phenylindole (DAPI), and finally scanned and analyzed by laser scanning cytometry (SLC). The cells of DAPI+CK8+CD45- were selected and confirmed visually according to shape and size of the tumor cell nuclei. We collected the patients with circulating tumor cells. Then we measured the expression of ERCC1 and MT1H in their peripheral blood (7.5ml) before or after platinum-based chemotherapy by semi-quantitative RT-PCR, and all patients who received 2 cycles could be evaluated. All dates were analyzed by using student's t-test andχ2-test by SPSS10.0 statistical package.Results:1. The cells were identified as circulating tumor cells if they were DAPI+CK8+CD45- cells, and the cell nucleus was large, deep-dye, showed a high nuclear cytoplasm ratio, and cytoplasmic CK8 antibody staining showed a typical ring or cap-shaped. 108 samples (66.67﹪) in 162 patients were detected with the CTCs. However, CTCs were not detected in 35 healthy samples by control. The detection rate of CTCs in advanced gastric cancer patients was higher than in 35 healthy volunteers (P=0.000).2. 40 of 108 patients were responders to chemotherapy and 68 were non-responders. There was no case with complete response in 108 patients. 40 cases got partial response, 38 cases got stable disease and 30 cases got progressive disease. The responsive rate (RR) was 37.04% (40/108).3. The expression of ERCC1 before chemotherapy was correlated with histological grade (P=0.002). 76 samples (70.37﹪) in 108 patients expressed ERCC1. The level after chemotherapy was higher than before significantly (P=0.000). The difference of ERCC1 expression between the responders and non-responders after chemotherapy was significant (P=0.000). The difference of ERCC1 expression in the non-responders before and after chemotherapy was significant (P=0.000).4. The expression of MT1H before chemotherapy was correlated with histological grade (P=0.014). 94 samples (87.04﹪) in 108 patients expressed MT1H. The level after chemotherapy was higher than before significantly (P=0.037). The difference of MT1H expression in peripheral blood between the responders and non-responders before chemotherapy was not significant (P=0.199), and also after chemotherapy (P=0.118). The difference of MT1H expression in peripheral blood before and after chemotherapy in both responders and non-responders was not significant (P=0.109,P=0.082).Conclusions:1. The way to detect circulating tumor cells in advanced gastric cancer patients with cell filtration combined with laser scanning cytometry was high specificity. It could effectively detect circulating tumor cells. This provided a simple and effective method for early diagnosis of cancer.2. The expression of ERCC1 and MT1H before chemotherapy was correlated with histological grade. This suggested ERCC1 and MT1H may be involved in the tumorigenesis and development of gastric cancer.3. The expression of ERCC1 in peripheral blood was associated with acquired platinum resistance in advanced gastric cancer.4. There was no significant difference between the expression of MT1H in peripheral blood of advanced gastric cancer patients and platinum resistance.