Effects Of TRAIL And CPT-11 Combination On Apoptosis Of Colon Carcinoma Cell Line HT-29

Objective: The present study was to observe the effects of TRAIL, CPT-11 alone and combination on cell viability and apoptosis of colon carcinoma cell line HT-29, to investigate the synergistic effect of TRAIL and CPT-11 combination and to explore potential mechanisms of inducing apoptosis.Methods: Cell viability was measured by Sulforhodamine B (SRB) method. Synergistic effects of TRAIL and CPT-11 combination were analyzed by Weeb coefficient. Apoptosis was determined by DNA ladder and flow cytometry. The expression of DR4 and DR5 mRNA were determined by RT-PCR. The protein expression of Bax and Caspase-9 was measured by Western blot.Results:(1) Colon carcinoma cell line HT-29 was treated by TRAIL (0～10μg/ml), there was significant negative correlation between cell viability and TRAIL concentration (r=-0.779, P＞0.05) . IC₅₀＞10μg/ml, which suggesting that colon carcinoma cell line HT-29 was resistant to TRAIL. Cell viability was obviously decreased in 10μg/ml TRAIL compared with other groups (P＜0.05) (2) Colon carcinoma cell line HT-29 was treated by CPT-11(0～10μg/ml), there was distinctly negative correlation between cell viability and CPT-11 concentration (r=-0.863, P＜0.05) . IC₅₀＜10μg/ml, which suggesting that colon carcinoma cell line HT-29 was sensitive to CPT-11. Cell viability was obviously reduced in 10μg/ml CPT-11 than other groups (P＜0.05) . Cell viability was obviously reduced in 1μg/ml, 5μg/ml and 10μg/ml than those in negative control, 0.1μg/ml and 0.5μg/ml CPT-11 groups (P＜0.05) .(3) Compared with TRAIL and CPT-11 alone, respectively, cell viability was apparently receded than those in groups of TRAIL and CPT-11 combination (P＜0.05), except of the groups of 0.1μg/ml TRAIL+1μg/ml CPT-11, 0.1μg/ml TRAIL+10μg/ml CPT- 11 and 1μg/ml TRAIL+10μg/ml CPT-11 combination.(4) Cell viability in group of 0.1μg/ml TRAIL and 5μg/ml CPT-11 combination was less than 70% estimated effect, which indicated that synergistic effect occurred in the group of 0.1μg/ml TRAIL and 5μg/ml CPT- 11 combination.(5) The results of DNA ladder and flow cytometry showed that CPT-11 alone could not induce apoptosis of colon carcinoma cell line HT-29, but TRAIL alone and combination of TRAIL and CPT-11 could significantly attenuate apoptosis, and more in the group of TRAIL and CPT-11 combination.(6) Although the expression of DR4 mRNA showed no significant difference among different groups, the expression of DR5 mRNA was augmented in the group of TRAIL and CPT-11 combination than other groups (P＜ 0.05).(7) Protein expression levels of Bax and Caspase-9 were enhanced in the group of TRAIL and CPT-11 combination compared with other groups (P＜0.05).Conclusions:(1) Colon carcinoma cell line HT-29 was resistant to TRAIL, but sensitive to CPT-11.(2) Cell proliferation was obviously inhibited in some concentration of combination of TRAIL and CPT-11.(3) Combination of 0.1μg/ml TRAIL and 5μg/ml CPT-11 showed synergistic effect on colon carcinoma cell line HT-29.(4) Apoptosis of colon carcinoma cell line HT-29 was more obviously induced by TRAIL and CPT-11 combination compared with TRAIL and CPT-11 alone.(5) Combination of TRAIL and CPT-11 induced apoptosis of colon carcinoma cell line HT-29, which may attribute to up-regulating the expression of DR5 mRNA and increasing the protein expression of Bax and Caspase-9.