| Objective: Gastric cancer is the most common enteron malignant tumor. The mortality of gastric cancer is in the first range of all kinds of malignant tumors in our country. Chemotherapy, as one of the major methods to anticancer, has got influential effect in the prevention and cure recurrence and metastasis of malignant tumor. Chemotherapy is constant consummated, however, the curative effect of chemotherapy to parts of gastric cancer patients is not ideal because of the multi-drug resistance (MDR). With the development of the modern pharmaceutical technology, P-glycoprotein inhibitors on tumor tolerance reversal by the extensive attention from scholars at home and abroad, showing good prospects. Tetrandrine(Tet),a bisbenzy lisoquinoline albaloid isolated from the Chinese herb"Hanfangji",could effectively reverse P-glycoprotein-mediated multidrug resistance. Studies have shown that tetrandrine on the drug-resistant cells have a strong reversal effect in vivo, it could be used for the clinical treatment of advanced gastric cancer .L-OHP, the third generation drug followed by C-DDP and carboplatin, has been used in advanced gastric cancer widely and shows great prospect.Until now, few experiment that Tet reverse MDR tumor cells in vitro has been launched.In our study, immunocytochemistry, flow cytometry and MTT will be utilized to observe the anticancer effect of Tet combined with L-OHP on SGC7901/VCR in vitro in order to provide relative experiment foundation for clinical treatment.Methods:1.This experiment is based on human gastric cancer cell line SGC7901 and SGC7901/VCR, the methods as follows1.1 By inverted microscope observation of morphological changes in different cell groups.1.2 L-OHP that in different times and different concentrations was adopted to different cell groups by MTT assay .1.4 Flow cytometry was adopted to detect the P-gp, Survivin, GST-πprotein in diffirent groups.1.3 Immunohistochemical was adopted to detect the P-gp, Survivin, GST-πprotein in diffirent groups.2. Statistical methodAll statistical analyses were carried out with SPSS 13.0 and P<0.05 was considered significant.Results:1. The morphology changes of SGC7901 and SGC7901/VCR cells when Tetrandrine and L-OHP on them1.1 The morphological characteristics of SGC7901 and SGC7901/VCR cellsUnder inverted microscope ,the SGC7901/VCR cells compared to the SGC7901 cells are irregular shape, larger, more particles and floating cells can be seen in the fluid.1.2 The morphology changes of SGC7901/VCR cells when Tetrandrine on themObserved under the inverted microscope, we can see a slight loose in cells, smaller in size, more cell debris and floating cells,when the SGC7901/VCR cells were cultured by tetrandrine after 48h.1.3 The morphology changes of SGC7901/VCR cells when Tetrandrine combined L-OHP on themObserved under the inverted microscope, we can see a slight loose in cells, smaller in size, the nucleus and cytoplasm difficult to separate, when the SGC7901/VCR cells were cultured by tetrandrine and after L-OHP 48h later. At the same time, we can also see a large number of cell debris and more floating cells.2. The killer activity that Tet and L-OHP on SGC7901/VCR and SGC7901 cells2.1 The killer activity that L-OHP against SGC7901/VCR and SGC7901 cellsAt 24 hours , The IC50 that L-OHP on SGC7901 was 11.4μg/ml. The IC50 that L-OHP on SGC7901/VCR was 55.98μg/ml. SGC7901/VCR cells'tolerance to L-OHP cells were 4.91 times more than sensitive strain SGC7901 cells.At 48 hours , The IC50 that L-OHP on SGC7901 was 6.58μg/ml. The IC50 that L-OHP on SGC7901/VCR was 42.03μg/ml. SGC7901/VCR cells'tolerance to L-OHP cells were 6.39 times more than sensitive strain SGC7901 cells.At 72 hours , The IC50 that L-OHP on SGC7901 was 3.86μg/ml. The IC50 that L-OHP on SGC7901/VCR was 19.5μg/ml. SGC7901/VCR cells'tolerance to L-OHP cells were 5.05 times more than sensitive strain SGC7901 cells.The inhibition in vitro of L-OHP on SGC7901/VCR or SGC7901 cells cells was the most powerful at 72 hours. The inhibition grew followed the density of L-OHP. But the IC50 that L-OHP on SGC7901/VCR to SGC7901 cells was the hightest at 48 hours.2.2 The killer activity that Tet on SGC7901/VCRAs the resistence of SGC7901/VCR to L-OHP was the hightest at 48 hours,we take the 10% inhibition(IC10) concentrion of Tet to SGC7901/VCR cells, which is 1.28μg/ ml. In this experimen we take 1.0μg / ml as the dose of Tet (IC10) at 48 hours.2.3 The killer activity that Tet combined L-OHP on SGC7901/VCR cellsThe killer activity of Tet combined with L-OHP on SGC7901/VCR was obviously higher than L-OHP worked alone(P<0.05), and the killer activity reinforced with the Tet or density of L-OHP growing. Combined with Tet, the IC50 of L-OHP on SGC7901/VCR cells can obviously reduced. Tet combined with L-OHP had better killer effect to SGC7901/VCR cells.3. The expression of P-gp, Survivin and GST-πin SGC7901/VCR by flow cytometry and immunohistochemical The expression of P-gp and Survivin in the SGC7901/VCR cell are higher than which in the SGC7901 cells, it shows that the exprssion can reduced by tetrandrine. (P <0.05). The expression of GST-πis similary both in SGC7901 and GC7901/VCR cells,and the the expression of GST-πcan not reduced by tetrandrine. (P >0.05).4 The apoptosis rate of SGC7901/VCR cell is Measured by flow cytometryAt 48 hours , the apoptosis rate of SGC7901/VCR cells that Tet (1.0μg/ml) on them was 6.55%. Compared with natural apoptosis rate(4.80%), Tet was not significantly induce apoptosis (P> 0.05).Conclusion:1. Compared to SGC7901 cells, SGC7901/VCR has strongly multi-drug resistance.2. At 48 hours , the apoptosis rate of SGC7901/VCR cells is similary with natural apoptosis rate when Tet (IC10:1.0μg/ml) on them. So Tet was not significantly induce apoptosis of SGC7901/VCR cells.3. Compared to SGC7901 cells, the anti-tumor activity of L-OHP on SGC7901/VCR was lower. The inhibition grew follow the concentration of L-OHP. And it was the highest at 72 hours. The resistance folds of SGC7901/VCR were 6.39 times when L-OHP on them. But the IC50 that L-OHP on SGC7901/VCR to SGC7901 cells was the hightest at 48 hours.4. The expression of P-gp and Survivin in the SGC7901/VCR cell are higher than which in the SGC7901 cells, it shows that the exprssion can reduced by tetrandrine. But the expression of GST-πis similary both in SGC7901 and GC7901/VCR cells,and the the expression of GST-πcan not reduced by tetrandrine.5. Tetrandrine combined L-OHP on SGC7901/VCR can significantly reduce the dose of L-OHP ,increase the effect of chemotherapy. The mechanism of tetrandrine on gastric cancer cells is related to multidrug resistance.6.Tetrandrine can reverse the MDR in vitro, whichhas no significant cytotoxicity. Tetrandrine can enhance the the killer activity of L-OHP...
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