The Research Of Human Pancreatic Cancer Cell PANC-1 Proliferation Inhibited By NCTD In Vitro

Background:The incidence and mortality of pancreatic cancer lines in the top 10 among all the malignant tumors, it's incidence rate increase gradually. As the pancreas particular location, physical characteristics and non-specific symptoms of pancreatic cancer, although in recent years, laboratory tests, imaging techniques and endoscopic technology has made tremendous progress, the early diagnosis of pancreatic cancer is still very difficult. Diagnosed patients with pancreatic cancer, only about 20% of patients have surgery opportunities, over 80% of patients already diagnosed in the late time, lost surgery opportunities, even if the surgical resection, the 5-year survival rate of only 18%, the proportion of local recurrence and distant metastasis remains high. For patients with unresectable tumor, or postoperative recurrence and metastasis, it must find out non-surgical treatment to prolong patient's survival and improve patient's quality of life. Combined with chemotherapy in comprehensive treatment was the major treatment for pancreatic cancer for the locally advanced, metastatic and post-operative recurrence pancreatic cancer. In recent years with the research and development of anti-tumor medicines, cantharidin shows a unique effect for digestive system cancers and a number of complex diseases, especially has a certain effect in the treatment of advanced cancer. Studies have shown that NCTD can inhibit the synthesis of protein in tumor cells, thus affecting the synthesis of RNA and DNA and affecting cell cycle progression, promoting tumor cell apoptosis and inhibiting tumor cell proliferation. The cantharidin can stimulate bone marrow to increase the peripheral blood white blood cell, thus Improving the patient's resistance. But the studies was less for the NCTD inhibit the pancreatic cancer, so far little studies was made that if NCTD has effect on pancreatic cancer. Objective:This study want to discuss the mechanism how the NCTD inhibit pancreatic cancer cells PANC-1,through the study of NCTD inhibiting human pancreatic cancer cell cells PANC-1 growth,and inhibiting human pancreatic cancer cell cells PANC-1 producing the VEGF and Akt In vitro. Provide a theoretical basis for the NCTD as a Chemotherapy on the pancreatic cancer patients. Methods:Put the DMEM medium with different concentration of NCTD and human pancreatic cancer cell PANC-1 together to culture, collect cells doing the MTT experiments at 12 h,24 h,36 h,48 h,60 h separately, Calculate Cell growth rate. Culture the cells with 1,16μg/ml concentrations of drug for 24h,then detecte VEGF, Akt expression by immunohistochemistry. Use SPSS 17.0 statistical analysis software for statistical analysis.Significant deviation was confirmed if P<0.05. Results:The inhibition result for the NCTD on human pancreatic cancer cell PANC-1 shows that:PANC-1 cells were inhibited by different concentrations of NCTD, Absorbance decreased with the concentrations of NCTD increased, Mean pairwise multiple comparison results compared with different concentration groups were statistically significant (P<0.05); the inhibition result for the different concentrations of NCTD on human pancreatic cancer cell PANC-1 in different time shows that:in the concentration range of 1～4μg/ml, the inhibition of PANC-1 cells was lower than 12% at the time of 12h, 1μg/ml and 2μg/ml group does not show significantly difference; the inhibition rate was significantly higher with the concentrations of NCTD increased,and with time goes by; the inhibition rate will reach more than 30% when 4μg/ml NCTD act on PANC-1 cells for 24h, the inhibition rate will reach to 60% at the time of 60h; the inhibition rate will reache to 73.86% when 16μg/ml NCTD act on PANC-1 cells for 60h; Half inhibition concentration (IC50) decreased as the time goes by, the half inhibitory concentration was 17.62μg/ml at 12h, the half inhibitory concentration dropped to 6.43μg/ml at 60h. the Akt and VEGF protein in the PANC-1 cells immunocytochemistry results:According to MTT results, we chose thel and 16μg/ml concentration of the NCTD to represent the low-dose and high-dose stimulation, immunocytochemistry technology were used to identify the Akt and VEGF protein expression in PANC-1 cells after cultured 24h.Akt, VEGF in the PANC-1 cells showed a brown positive reaction. Brown cytoplasmic granules in the lug/ml concentration significantly reducted compared with the control group, the 16ug/ml concentration of group changes more obviously than the gorup of lug/ml. The expression of VEGF and Akt protein in PANC-1 cells significantly reduced under the action of NCTD;The higher the concentration of NCTD, the lower expression of VEGF and Akt protein in PANC-1 cells. The Akt expression difference was significant between the experimental and control groups (F=7.410, P<0.01),expression difference was significant between the different experimental groups(F=2.538, P<0.01); The VEGF expression difference was significant between the experimental and control groups (F=6.991,P<0.01), expression difference was significant between the different experimental groups(F= 0.849, P<0.01). Conclusion:1 NCTD can inhibit the grouth of human pancreatic cancer cells PANC-1, the inhibition became more obvious with the concentration of NCTD goes higher, the inhibition became more obvious with the time of action goes, the time-dosage dependent relationship existed in the inhibition of PANC-1cells.2 The NCTD inhibit the VEGF expression in the PANC-1 cells,the inhibition became more obvious with the concentration of NCTD goes higher.3 The NCTD inhibit the Akt expression in the PANC-1 cells,the inhibition became more obvious with the concentration of NCTD goes higher.