| Objective: Ovarian cancer is a serious threat to women’s health, commonmalignancy, accounting for a mortality rate in the female genital malignancies.The treatment of ovarian cancer with surgery and chemotherapy because of itsoccult onset, early screening difficult, about2/3of the patients at diagnosishad occurred widely planted within the abdominal cavity. In recent years,along with the cytoreductive surgery continues to improve, as well as thewidely used platinum-based combination chemotherapy,80%of untreatedpatients obtain clinical remission. However, the ensuing early relapse,resistance to chemotherapy orders over the years the efficacy of ovariancancer has not significantly improved. Ovarian cancer resistant toplatinum-based chemotherapy drugs affect the effect of chemotherapy, causingimportant cause of tumor recurrence, metastasis, and mortality is high.Therefore, the search for new, effective therapies, and to elucidate itsmechanism of action, becomes increasingly important and urgent in thetreatment of ovarian cancer, gynecological oncology community at home andabroad to pay close attention to the research focus. Noscapine, also known asnocatine, one from the benzyl isoquinoline alkaloids in opium poppy. Over thepast decades it has been used as antitussives, the research in recent yearsfound noscapine can inhibit the proliferation of various tumor cells. Noscapineand its derivatives antitumor activity, inconspicuous toxicity, recurrence anddrug resistance of tumor cells was inhibited.The experimental researchnoscapine on human ovarian cancer cisplatin-resistant strains SKOV3/DDP(ovarian serous cystadenocarcinoma the cisplatin resistant strains) role, and bymeasuring the expression of apoptosis-related proteins Bcl2and Baxprotein,further study the mechanism of action of noscapine-induced apoptosis.Methods:Cultured in vitro of human ovarian cancer the cisplatin resistant strains SKOV3/DDP, planting the same concentration in96-well plates, thenoscapine divided into seven concentration (0.625,1.25,2.5,5,10,20,40,80μmol/L) acting on SKOV3/DDP cells and sub-24h,48h,72h three timeperiods, using the MTT assay to detect cell proliferation, and using SPSS13.0software package for data analysis, log fitting calculated IC50and IC10. Withdifferent concentrations of cisplatin (1,2,4,8,16μg/mL) and the United2.5μmol/L noscapine role SKOV3/DDP cells48h after using the MTT assayto detect cell proliferation, SPSS13.0software package for data analysis, usingthe log fitting calculated IC50. Cisplatin (4μg/ml) and noscapine (2.5μmol/L), alone or combined effects of the two drugs acting on SKOV3/DDP cellsafter48h using an inverted phase contrast microscope to observe changes incell morphology, and then the cells were collected made single cell suspension,fixed with70%alcohol, apoptosis, Bcl2and Bax protein expression levels wasdetected by flow cytometry.Statistical treatment using SPSSl3.0, representation of measurement datausing Mean±SD. Multiple-group analysis was performed by one-way ANOVA,inter-group analysis was performed by LSD-t test. With P=0.05as thestandard, P<0.05the deference have statistic significance.Results:1The proliferation inhibition of noscapine to the SKOV3/DDP cell:(0.625,1.25,2.5,5,10,20,40,80μmol/L)8concentration noscapine acting onthe SKOV3/DDP cells24h the inhibition rates were1.33±0.42%,2.53±0.98%,5.21±1.25%,15.26±0.85%,22.21±2.73%,29.14±1.62%,37.33±3.48%,63.23±2.27%, as in SKOV3/DDP cells48h inhibition rates were1.21±0.38%,3.71±0.65%,7.89±1.23%,18.32±1.13%,25.97±1.18%,36.52±3.51%,63.47±3.55%,72.12±2.85%, acting on, respectively, theSKOV3/DDP cells72h cells inhibition rate to2.17±0.43%,4.85±1.21%,9.78±1.55%,22.16±2.46%,31.54±3.17%,41.63±4.92%,69.53±2.12%,78.72±2.96%, the comparison between groups more than2.5μmol／L, thedifference was statistically significant (P <0.05). With the increase of theconcentration of the drug and drug action time extension, SKOV3/DDP cell inhibition rate showed an upward trend.2Cisplatin and its combined with noscapine on SKOV3/DDP cellproliferation inhibition:1,2,4,8,16, μg/mL concentrations of cisplatin, acton the SKOV3/DDP cells for48h, inhibition rate were10.45±1.21%,17.89±1.01%,25.77±1.56%,34.46±1.45%,52.23±1.33%, various concentrationsof cisplatin combined with2.5μmol/L noscapine for48h with SKOV3/DDPcells the inhibition rate was13.79±1.38%,25.76±1.17%,32.24±1.40%,45.16±1.34%,64.36±1.96%. The inhibition rate after cisplatin combinedwith noscapine was significantly higher than cisplatin alone with group, andthe difference was statistically significant (P <0.05).3SKOV3/DDP cell morphological observation: noscapine (2.5μmol/L) and cisplatin (4μg/ml) alone and combination role in SKOV3/DDP cellsafter48h observed under an inverted phase contrast microscope, the cells ofthe control group rowcloth tightly adherent growth, cells were spindle orpolygonal transparent cytoplasm, refraction, nucleus looming growth in goodcondition. noscapine role in cell morphology and growth of no significantchange. Cisplatin after visible contact to reduce some of the cells, a small partof the cells became round, losing adherent growth morphology, sheddingsuspended in the medium. the morphological changes of noscapine combinedwith cisplatin was obviously, compared with the single cisplatin group, visiblereduce contact between cells, some cells became round, shrinkage, cracking,shedding in suspension.4Flow cytometry detection SKOV3/DDP cell apoptosis rate: controlgroup cells48h apoptosis rate:1.21±0.23%.4μg/ml of cisplatin role in thecells after48h apoptosis rate:18.49±2.11%.2.5μmol/L noscapine role in theapoptosis rate of cells after48h:2.34±0.98%.2.5μmol/L noscapine theUnited4μg/ml cisplatin role in the apoptotic rate of cells after48h:27.67±3.25%. The experiments show that, apoptosis rate of the alone noscapinegroup showed no significant difference to the control group, while the groupof cisplatin and cisplatin combined noscapine,the difference was statisticallysignificant role in the apoptosis rate with the control group in the SKOV3/DDP cells, which the noscapine associated cisplatin group apoptosiswas significantly higher than the other groups, the difference was statisticallysignificant (P <0.05).5Flow cytometry to detect Bcl2and Bax protein expression:⑴4μg/mLcisplatin role in SKOV3/DDP cells after48h, Bcl2and Bax fluorescence index(FI) is0.934±0.037,1.213±0.028, compared with the control group1.000±0.009,1.000±0.023, the difference was statistically significant (P<0.05).⑵2.5μmol/L noscapine role in SKOV3/DDP cells after48h, Bcl2and Bax fluorescence index (FI) is0.995±0.024,1.039±0.016, the differencewas no statistically significant (P>0.05).⑶4μg/mL of cisplatin combined2.5μmol/L noscapine role SKOV3/DDPcells after48h, Bcl2and Baxfluorescence index (FI) was0.842±0.019,1.334±0.041, the difference wasstatistically significant (P <0.05). The results showed that the cisplatin groupand noscapine plus cisplatin group the Bcl2protein fluorescence indexdecreased, and Bax protein fluorescence index rises, the more obviouschanges in the combination group.Conclusions:1Alone noscapine able to inhibit the proliferation of ovarian cancer cellSKOV3/DDP in a certain concentration range, and presents a time and dosedependent effects.2Cisplatin combined with non-cytotoxic drug solubility noscapine canincrease of cisplatin sensitivity to the SKOV3/DDP cells, it can serve as a kindof ideal cisplatin sensitization agent.3The noscapine synergy with cisplatin may by lower Bcl2expressionand increased expression of Bax, down Bcl2/Bax ratio to induced apoptosis,and enhance the sensitivity of cisplatin to the cisplatin-resistant ovarian cancercells SKOV3/DDP, noscapine as a new kind of chemotherapy drugs tochemoresistant ovarian cancer, its mechanism remains to be further research. |
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