| JD is from the structural modification of oridonin (active ingredient of Rubescens). The preliminary anti-tumor activity evaluation showed that JD has good activity, but the mechanism of anti-tumor is still unknown. Paclitaxel is effective for the treatment of gastric cancer. However, long-term use of normal doses of paclitaxel always result in adverse reactions and side effects, and the adverse reactions and drug resistance will be more obvious with the increasing dose of Paclitaxel, thus limiting the clinical application of paclitaxel. Combination therapy is a clinically important drug regimen. Therefore, to seek one strategy in combination with Paclitaxel with high efficacy and low toxicity is still significant for the treatment of gastric cancer. Main research work was described as follows:1. MTT assay was used to determine the cell viability of JD and paclitaxel monotheray on MKN45 cells. The correlation between dosage and viability was illustrated and indicated that both JD and paclitaxel decreased the viability of MKN45 in a stime-and concentration-dependent manner.2. The combination index (CI) values of JD plus paclitaxel were evaluated by the median-effect method. All CI values of JD plus paclitaxel in the tested combination groups were less than 1, which indicated all tested combination groups showed a synergistic effect. And paclitaxel (5 nM) in combination with JD (20μM) was the best.3. The proliferation of single MKN45 cell was detected by colony formation. Inhibition rate of colony formation was markedly increased after combined treatment, demonstrating that combined treatment with JD and low-dose paclitaxel plays a synergistic effect on inhibiting the colony formation (p<0.01).4. Flow cytometry analysis was performed to detect the apoptosis. The corresponding apoptosis rate of JD (20μM), paclitaxel (5 nM) and their combination treatment were (22.65±2.07)%, (13.12±1.75)% and (54.73±3.10)%. Cell apoptosis induced by paclitaxel was markedly increased when combined with JD (p< 0.01).5. Western blotting was performed to detect the expression levels of pro-and anti-apoptotic proteins. The results demonstrated that combined therapy with JD and paclitaxel activated pro-apoptotic proteins, such as Bax, Puma, Cleaved-caspase 3 and Cleaved-caspase 9 to a greater degree (p<0.05) and decreased anti-apoptotic proteins, such as Bcl-2, Mcl-1, Bcl-xL, PBKp110α, p-AKT, p-p70S6K and Raptor to a lower degree than either JD or paclitaxel treatment alone (p<0.05).6. Bax was knocked down by siRNA transfection to futher detect its role in the apoptotic-related pathways. Caspase 9 inhibitor (Z-LEHD-FMK) partially rescued JD plus paclitaxel-induced apoptosis (p<0.05). Consistent with this finding, the apoptosis induced by JD plus paclitaxel was partially reversed when Bax was knocked down by Bax-specific siRNA (p<0.05).7. Xenograft model was established to assess the inhibitory effect on tumor growth by JD plus low-dose paclitaxel. The combination of JD and low-dose paclitaxel showed a synergistic inhibitory effect similar with nomal-dose paclitaxel on tumor volume compared to either agent treatment alone (p<0.05), and the body weight of mice remained stable (p>0.05).8. Hematoxylin and eosin (HE) staining assay was performed to observe the changes of different treatment. HE staining exhibited increased necrosis in the group treated with JD and low-dose paclitaxel compared to that in the control group.9. TUNEL assay was performed to detect cell apoptosis in tumor tissues. TUNEL staining exhibited there were greater apoptosis in the combination group than in either agent monotherapy group.10. Western blotting was performed to detect the expression levels of pro-and anti-apoptotic proteins in tumor tissues. Western blotting results of tumor issues proteins demonstrated that combined therapy with JD and paclitaxel activated pro-apoptotic proteins, such as Bax, Puma, Cleaved-caspase 3 and Cleaved-caspase 9 to a greater degree (p<0.05) and decreased expression of anti-apoptotic proteins, such as Bcl-2, Mcl-1, Bcl-xL, PI3Kp110α, p-AKT, Raptor and p-p70S6K to a lower degree (p<0.05) than either JD or paclitaxel treatment alone.In summary, the combination of JD and low-dose paclitaxel showed a synergistic anti-tumor effects. The anti-tumor effects of low-dose paclitaxel was markedly increased when combined with JD. JD enhancing the anti-tumor effects of paclitaxel for the treatment of MKN45 gastric cancer was caused by the induction of apoptosis, and the mechanism may be related to the activited mitochondrial apoptosis pathway and the inhibited PI3K/AKT/mTOR pathway. |
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