The Antitumor Effects Produced By Manumycin Combined With Cisplatin On The Ovarian Transplantation Tumors

Objective: To investigate the cytotoxic effects of manumy- cin combined with cisplatin (DDP) on human ovarian cancer 3AO cells and the transplantation tumors of nude mice. To analyze the possible mechanisms produced by the combination of manumycin and cisplatin to ovarian transplantation tumors.Methods:1 Human ovarian cancer 3AO cells were cultured in vitro.The cytotoxic effects of manumycin on the proliferation of 3AO cells was measured by MTT colorimetric method, and the morphological changes of 3AO cells were observed.2 The cytotoxic effects of manumycin combined with cispltin on the proliferation of 3AO cells was measured by MTT colorimetric method. Jin's formula was used to analyze the interaction effects. If the interaction index is more than 0.85, synergy is thought to be produced. Otherwise, antagonism is produced.3 3AO cells were injected into the nude mice. After the xenografted tumors grow for two weeks, the mice were randomly divided into 4 groups (control group, manumy- cin group, DDP group and DDP+ manumycin group). The tumor volumes of the mice were measured and the responses of the nude mice were also observed during the whole observation time. The curves of tumor growth were draw. Moreover, the tumor growth inhibiting rate was calculated.4 The expression of Survivin, NF-κB, VEGF, Caspase-3 proteins were checked by immuno-histochemical method.5 Cell cycle analysis and apoptosis were performed by flow cytometry.Results:1 The proliferation inhibition of 3AO cells can be produced by manumycin at different concentrations in a time- and dose-denpendent way. After the cells were dealed with different concentrations manumycin for 24~72 hours, compared with the control group, the OD values of the treatment group decreased significantly(p<0.05). Moreover, cells morphology was observ- ed under light microscope, showing that cells became shrinked and were broked down into irregular strips, much floating cells could be seen in the supernatants.2 10, 20, 40, 60μmol/L manumycin can strengthen the cytotoxic effects of cisplatin to 3AO cells, and the synergic effects which further be testified by interaction index from Jin's formula could enhanced with the increasing manumycin.3 The ovarian transplantation tumors grew in subcutaneou- ly of nude mice successfully. The tumor size of the treated group was significantly smaller than that of the control group (p<0.05). Furthermore, compared with DDP group and manumycin group the tumor size was also smaller than that of the combination group (p<0.05). The inhibition ratio was 0.00% for the control group, 29.85% for the manumycin group, 37.36% for the DDP group, 64.60% for the combination group, respectively.4 The expression of Survivin, NF-κB, VEGF, Caspase-3 were determinated by immuno-histochemical method. For each of these indicators, there was a great difference between the treatment groups and the control group by IHS score (p<0.05), Furthermore, compared with DDP group and manumycin group the expression of Survivin, NF-κB, VEGF protein were significantly down-regulated in the combination group (p<0.05). However, the expression of Caspase-3 protein were significantly up-regualetd in the combination group (p<0.05).5 The apoptotic percentage raised up gradually, and the apoptotic percentage was 5.24±0.94% for the control group, 12.16±1.08% for the manumycin group, 17.41±2.26% for the DDP group, 22.93±3.22% for the combination group, respectively. The number of cells in G0/G1 phase decreased gradually, while the number of cells in G2/M phase increased gradually, S phase did not show changes.Conclusions:1 Manumycin can inhibit the proliferation of 3AO cells in a time- and dose-dependent way. Manumycin can enhance the cytotoxic effects of cisplatin to 3AO cells.2 Manumycin could inhibit the growth of the xenograft tumors in nude mice. The cytotoxic effects of DDP could be enhanced by its combination with manumycin. 3 Manumycin caused G2/M phase arrest as well as apoptosis in 3AO cells, and the apoptosis of 3AO cells treated with the combination of manumycin and DDP became more significant.4 The expression of Survivin, NF-κB, VEGF can be reduced by Manumycin and the expression of Caspase-3 can be increased by Manumycin, which may be the possible machenism of enhancing the cell-killing effects of DDP to 3AO cells.