Experimental Research Of Antiproliferative Effect And Apoptosis-related Gene Expression On Human Pancreatic Carcinoma Cell Line BxPC-3 Induced By 5-FU In Combination With Sulfasalazine

To study the Anti-proliferative effect and influence of cell cycle apoptosis-related protein cell morphous changes induced by 5-FU in combination with Sulfasalazine on human pancreatic carcinoma cell line BxPC-3.MethodsMTT was used to evaluate the Anti-proliferative effect of 5-FU combined with Sulfasalazine on human pancreatic carcinoma cell line BxPC-3. Cell cycle a-nalysis apoptotic rate apoptosis-related protein expression were measured by flow cytometry ; Typically apoptotic changes in cell morphosis were observed by phase-contrast microscope and transmission electron microscopy.Results1. 100ug/ml 5-FU combining with 0.5mmol/L Sulfasalazine significantly inhibited the growth of BxPC-3 cell with a dose-,time-dependent manner,which was the more obvious than 5-FU treating group did.2 . After different time treatment with 100 ug / ml 5 - FU in combination with 0. 5mmol/L Sulfasalazine ,it appeared to find the hypodyploid peak and showed that apoptotic rate significantly increased from 62. 90% to 92. 64% and cell cycle was arrested in G0/G1 phase. S phase cell gradually decreased, while G2/M phase cell did not obviously change.3. The level of Bcl-2 expression gradually decreased while that of Bax ex-pression gradually increased; meanwhile the ratio of Bax to Bcl-2 significantly increased with time increasing in the treating group 5-FU combining Sulfasalazine at different time, there was significant difference between the treating group and the control, P < 0. 01. After 24 hour treatment of 5-FU, Sulfasalazine,5-FU with Sulfasalazine, respectively in the expression of ratio of Bax to Bcl-2 the above treatment groups comparing with the control had significant difference , P < 0. 01; there was significant difference between the combining group and the Sulfasalazine treating group or the 5-FU treating group, P < 0. 05. After 24 hour treatment, the level of Bax expression in the treatment groups was significantly the more different than in the control, P <0. 05, while the level of Bcl-2 expression decreased in the combining treatment group than that in 5-FU treatment. The ratio of Bax to Bcl-2 was significantly higer in the combining treatment than that in 5-FU or Sulfasalazine group alone.4. Typically apoptotic changes were observed in all treament groups at different times by phase-contrast microscopy. Cell started in the pyknosis fom in 12 hour, cell body splitting and unregular margin in 24 hour, the latter change was obviously seen in 36 48 hour. After 12 24 hour treatment of 5-FU, cell had not any change,cell body swelling and the edge of cell broken in 36 hour, swelling and breaking was more obvious in 48 hour.5. Transmission electron microscopy in the different treated group for 24 hours observing cell changes: Bubble apophysis was presented on the cell surface in the combination of 5-FU with Sulfasalazine, meanwhile lots of apoptotic body was formed in the cytoplasm, chromatin located in the margin of nuclear membrane. Microvillus abscission was evident with cytoplasm vacuolation forming and nuclear chromatin representing pyknosis and splitting in group treated by Sulfasalazine. Exocytosis corpuscle was bulky on the cell membrane, microvillus abscission not being obvious with nuclear membrane incisuring and fragmentation of intranulear chromatin by the treating group of 5-FU.Conclusion1. The study proved that 5-FU in combination with Sulfasalazine made a co-. operative inhibition effect on BxPC-3 cell.2. Sulfasalazine intensified cell apoptotic effect induced by 5-FU. During the process of cell apoptosis induced by Sulfasalazine with 5-FU, we found that up-regulating Bax expression, down- regulating Bcl-2 and NF-kB (P65) may be one of apoptotic mechanisms and was correlated with cell cycle arrested in G0/G1 phase, increase of apoptotic rate and cooperative anti-tumour effect.3. Characteristic changes of a lot of apoptotic body appeared in the BxPC-3 cell line treated by 5-FU combining Sulfasalazine and was obviously better than those by 5-FU or Sulfasalazi...