Cytotoxic Effects Of Manumycin On The Proliferation Of Human Ovarian Cancer Cell Line 3AO And Enhanced By The Combination With Cisplatin

Objective: To investigate the effects of manumycin on the proliferation of human ovarian cancer 3AO cells. Moreover, to check the effects of manumycin combined with an anticancer agent cisplatin (DDP) to human ovarian cancer 3AO cells, and to analyze the mechanisms of inhibiting the proliferation and inducing apoptosis of 3AO cells by manumycin.Methods: 1 Human ovarian cancer 3AO cells were incubat -ed with different concentration of manumycin in vitro. The cytotoxic effect of manumycin on the proliferation of 3AO cells was measured by MTT colorimetric method. 2 Cell cycle analysis was performed by flow cytometry. 3 Morphological changes of 3AO cells were observed by light microscopy and transmission electron microscopy, and morphological changes of 3AO cells of Giemsa staining were further observed by light microscope. 4 Extracted DNA of 3AO cells before and after treated with manumycin were run by agarose gel electrophoresis. 5 The expression of survivin and NF-κB(P65) protein of 3AO cells treated with manumycin were analyzed by flow cytometric indirect immunofluorescent technique. 6 MTT colorimetric method was performed to evaluate the effect of manumycin combined with DDP to 3AO cells. Jin's formula was performed to determine whether the combination of manumycin with DDP could result in a synergistic effect or not. More than 0.85 of the interaction index was thought to be as a synergic effect, while less than 0.85 was thought to be as antagonism.Results: 1The proliferation of 3AO cells could be inhibited by the different concentrations of manumycin (5, 10, 20, 40, 80μmol/L). With the increasing concentration of manumycin, the cytotoxic effects were enhanced, furthermore, with the prolonged treatment time, the cytotoxic effects were also enhanced, and there was a significant difference between the treatment group and the control group (P<0.05), which means manumycin could inhibit the proliferation of 3AO cells significantly and this inhibition was dose- and time-dependent.2 After treated with 0, 5, 10, 20, 40, 80μmol/L manumycin for 48h, the number of cells in G0/G1 phase decreased gradually, while the number of cells in G2/M phase increased gradually, which was in dose-dependent manner, but S phase did not show changes obviously. Furthermore, after treated with 0, 5, 10, 20, 40, 80μmol/L manumycin for 48h, there were obvious apoptosis of 3AO cells, the apoptotic percentage raised up gradually with the increasing concentration of manumycin, and the apoptotic percentage was 1.73%, 2.39%, 3.76%, 14.37%, 28.86%, 36.32% respectively.3 When treated with manumycin, 3AO cells changed signif -icantly in morphology observed by light microscopy. 3AO cells untreated with manumycin were fusiform, diamond, and they were satiation; while the cells treated with manumycin were shri -nked and broken, became irregular in shape, spreading in a thin and long pseudopodium in both end points, vacuolus could also be seen in some cells kytoplasm, floating and cast-off cells increased. Morphological changes of 3AO cells of Giemsa staining were further observed by light microscopy, 3AO cells untreated with manumycin were fusiform, diamond, and they were satiation, furthermore, the cell nucleus staining were uniform and clear, no floating cells. But 3AO cells treated with manumycin became rounding, the cell kytoplasm was concentrated, the staining cell nucleus was uneven, there were also caryocinesis phenomenon, showing the typical apoptotic morphological changes. The ultramicrostructure of 3AO cells changed obviously, which were observed by transmission electron microscope, for example, the nucelus overshoot in acute angle outwards way, chromatin concentrated highly, electron density raised and they were enriched as crescent below the nuclear membrane, karyopycnosis could also be seen.4 Extracted DNA of manumycin-treated group cells were analyzed by agarose gel electrophoresis, there were typical ladder-form electrophoresis strips, which means the DNA were degrdned to regular size fragments, while control group cells showed no degrdned fragments.5 After 3AO cells were treated with 0, 10, 20, 40μmol/L manumycin for 48 hours,the expression of survivin and NF-κB (P65) protein decreased gradually with the increasing concentration of manumycin. There was significantly difference of survivin and NF-κB(P65) protein among each treated group and control group (P<0.05).6 Manumycin combined with DDP could inhibit the proliferation of 3AO cells. The results showed that 10, 20, 40μmol/L manumycin combined with DDP induced synergic cytotoxic effects to 3AO cells, and the synergic effects which further be testified by interaction index from Jin's formula could enhanced with the increasing manumycin.Conclusions: 1 Manumycin could induce cytotoxic effects on proliferation of human ovarian cancer 3AO cells and apoptosis, which provided some theoretical foundation for the clinical use of manumycin. 2 We suppose the mechanisms of manumycin inducing the cytotoxic effects and apoptosis to 3AO cells as follows: firstly, down-regulated the expression of survivin and arrested 3AO cell cycle in G2/M phase, secondly, down-regulated the expression of NF-κB(P65), which could enhance the cytotoxic effects of 3AO cells to manumycin. 3 Manumycin combined with DDP could inhibit the proliferation of 3AO cells and produce synergic cytotoxic effects to 3AO cells.