| Objective:Pancreatic cancer which is an important solid tumor of internal organs, and so far, is one of the malignant tumors which are most difficult in the early diagnosis and treatment. Clinical practice shows that, for the treatment of pancreatic cancer, only surgical treatment is limited, while the effect of radiotherapy and chemotherapy and targeted treatment for pancreatic cancer is poor. Therefore, the morbidity and mortality of pancreatic cancer is basically same.So, many scholars believe that pancreatic cancer is "the king of the cancer" in the21st century. In order to improve the survival rate of patients with pancreatic cancers, the researching and developmenting of new internal medicines to treat pancreatic cancer is a very urgent task.As a Chinese medicine mixture, Jinlong Capsule is mainly composed of fresh Bungarus, fresh Agkistrodon and fresh Gecko. The active ingredients are extracted by the process of modern cryogenic and biochemical separation when the animal drugs are fresh. The previous experimental data had proved that Jinlong Capsule is multi-targeted in anti-tumors, but the basic research of its role of anti-tumors about pancreatic cancer has not been reported. Studying the impact of proliferation and apoptosis of Jinlong Capsule on human pancreatic cancer BXPC-3cells, in order to exploring the possible mechanism of Jinlong Capsule, will provides some theoretical support for its treatment of pancreatic cancer,and will pave the way for the next in-depth study.Methods:1The proliferation inhibition rate of different concentrations of Jinlong Capsule on human pancreatic cancer BXPC-3cells for24h,48h,72h,96h,120h were tested by MTT assay. And exploring the appropriate role time and dose to be used in the subsequent experiments.2The changes of the morphological of human pancreatic cancer BXPC-3cells stimulated by Jinlong Capsule for48h were stained by AV-FITC/PI and observed by the confocal laser scanning microscopy.3The apoptosis of human pancreatic cancer BXPC-3cells stimulated by different concentrations of Jinlong Capsule for48h were stained by AV-FITC/PI and observed by the flow cytometry. 4The cell cycle of pancreatic cancer BXPC-3cells stimulated by different concentrations of Jinlong Capsule for48h were stained by PI and observed by the flow cytometry.5The expression of the Calcium-binding protein S100A4in the human pancreatic cancer BXPC-3cells matrix, when the cells were stimulated by different concentrations of Jinlong Capsule for48h, was analyzed by ELISA.6The level of Proteins BNIP3, Bcl-2and Caspase-3in the human pancreatic cancer BXPC-3cells stimulated by different concentrations of Jinlong Capsule for48h was tested by Westen Blotting.Results:1MTT assay results showed that the Jinlong capsule has the role of inhibiting the proliferation of human pancreatic cancer cells BXPC-3. When cells were stimulated by Jinlong Capsule for48h,the difference between the0.4,0.6,0.8.1.0mg/ml of Jinlong Capsule group and the negative control group was significant; The difference between the0.8mg/ml of Jinlong Capsule group and positive group (GEM) was not significant; The inhibition of proliferation of the1.0mg/ml Jinlong Capsule group was better than the positive group. The cell metabolites was relatively less,and the interference to the results was smaller after human pancreatic cancer cells BXPC-3were stimulated by Jinlong Capsule for48h. Based on the above two factors, the follow-up experiment choosed the Jinlong Capsule in the concentration of0.4,0.6,0.8mg/ml and the reaction time of48h.2The morphological changes of the human pancreatic cancer cells BXPC-3observed by the confocal laser scanning microscopy showed that, the AV-FITC green fluorescence was enhanced in early apoptotic cells, and the AV-FITC green fluorescence and the PI red fluorescence were both enhanced in the late apoptotic cells, and the necrotic cells only had enhanced PI red fluorescence. The cell nucleus and the cell morphology of apoptosis and death cells were irregular and unequal.3The results of the apoptosis of human pancreatic cancer cells BXPC-3stained by AV-FITC/PI and observed by the flow cytometry showed that, the Jinlong capsule could significantly promote apoptosis of human pancreatic cancer cells BXPC-3, and had a dose-dependent. With the rise of the concentration of Jinlong capsule, late apoptotic cells increased more apparently.4The G2/M and S phase increased significantly of the human pancreatic cancer cells BXPC-3stimulated by Jinlong Capsule for48h in, while the cells in G1/G0phase reduced. This phenomenon was most obvious when the Jinlong capsule concentration was0.6mg/ml.5When the human pancreatic cancer cells BXPC-3were stimulated for48h by JinLong Capsule in the concentrations of0.4,0.6and0.8mg/ml, the ELISA test showed that the concentrations of the Calcium-binding protein S100A4in the cell matrix separately were17.982,17.907and16.782pg/ml, which is different from the concentration of normal group which was19.789pg/ml.6Western Blotting test showed that the expression of Caspase-3protein in different groups was not obviously different, and the expression of BNIP3, Bcl-2protein in different groups was obviously different. Comparing with normal group, the expression of BNIP3protein in JinLong Capsule group increased, and Bcl-2protein reduced.Conclusions:JinLong Capsule can significantly suppress the proliferation of human pancreatic cancer cells BXPC-3, and promote BXPC-3apoptosis. The inhibition of cell proliferation may be achieved through promoting cell apoptosis,and blocking cell cycle in S, G2/M phases which affecting the normal transforming of cell cycle, and reducing the calcium binding protein S100A4. The mechanism of promoting apoptosis may be according with raising the BNIP3proteins which promoting apoptosis, and reducing the expression of anti-apoptotic protein Bcl-2. |
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