| Objective:Ovarian cancer is one of the three malignant tumors of femalereproductive system, which is threatening the female health severely.Currently, in addition to the surgical treatment, chemotherapy is the mainmeans of adjuvant therapeutic ways. Narcotine is a nontoxic benzylisoquinoline alkaloid, has been used as an antitussive. In recent years, researchhas confirmed that it can act on the microtubules, inhibit the dynamicinstability, interfere with tumor cell mitosis process, and promote tumor cellapoptosis. In the experimental study of tumor cells and animal tumor models,it's showed good anti-tumor activity in viv and no significant side effects. Sonarcotine is expected to become the new tubulin inhibitors for the treatment ofovarian cancer. Through this study, to invest the effects and mechanisms ofhow Ovarian Cancer Cell Line SKOV3cells proliferate and apoptosis afterusing narcotine and combined with cisplatin.Methods:Ovarian cancer cell line SKOV-3were planted on four96-wellplate in the same density and maintained in RPMI-1640vehicle containing10％FCS(fetal calf serum)in37℃,5％C02for24hour.then changed RPMI-1640vehicle containing different concentration narcotine ranging from5,10,20,40,80,160μmol/L,incubated for24h,48h,72h respectively.Proliferation was determined spectophotometrically by the incorporation oftetrazolium dye；optical density at490nm was then determined using anenzyme-linked immunosorbent reader.20,40μmol/L narcotine,10μg/mlcisplatin, and nosapine combined with cisplatin to SKOV-3cells, Incubated at24,48and72hours. Morphologic changes of apoptotic cells were observedwith optical microscope. Cell apoptosis and cell cycle Was detected by cellflow cytometry(FCM).Flow cytometry analysis narcotine, cisplatin, andnarcotine combined with cisplatin of aurora-A and FGF-2protein expression. Statistical treatment using SPSSl7.0, representation of measurement datausing Mean±SD. Multiple-group analysis was performed by one-way ANOVA,inter-group analysis was performed by LSD-t test. P<0.05the deference havestatistic significance.Results:1The effects of narcotine on proliferation inhibition:Narcotine(5,10,20,40,80μmol／L) exerted inhibitory effects on proliferation of SKOV3cells, after24,48and72hours later,the survival rate of SKOV-3cells were88.27±11.71％,82.89±14.13％,67.97±15.3％,39.41±12.28％,38.45±7.86％and84.56±9.36％,79.03±13.37％,62.18±9.90％,23.49±9.34％,11.90±2.64％and82.40±8.21％,73.54±12.05％,59.38±4.22％,11.17±3.63％,07.78±1.34％.its effect presented a time and dose-dependentway. There was a significant difference between the treatment group and thecontrol group(P<0.05).2Morphological observation of SKOV3cells:Control group:SKOV-3cells arranged close adherent growth, cell diamond-shaped or spindle-shaped,grew well, the cells round and bright and full, close connections between thecells.Treatment group: narcotine alone (40μmol/L), and combined withcisplatin (10μg/ml),48hours later, Cell shrinkage, cracken, shed insuspended like, connections between cells reduced. We could observe thatthese morphological changes of combination group are more obvious.3Cell cycle rate detect using Flow cytometry:The percentage of G0／G1,G2／M stages of SKOV-3cells of control grou were64.77±4.97％and16.87±2.65％.The percentage of G0／G1,G2／M stages of SKOV-3cellswith narcotine (20,40μmol／L) were50.73±2.66％,31.77±9.38％and22.53±2.53％,32.00±1.80％,but percentage of G0／G1,G2／M stages ofSK-OV-3cells with narcotine (20,40μmol／L)and cisplatin (10μg/ml)were27.47±5.77％,6.55±3.59％and37.17±6.66％,55.10±6.36％,(p<0.001).It can be detected in G2/M arrest and apoptosis using narcotine.4Flow cytometry analysis detected of aurora-A protein:In comparisionwith control group,20,40μ mol／L narcotine for48h,aurora-A protein expression were1.047±0.030,1.089±0.039,narcotine (20,40μmol／L)combine with cisplatin (10μg/ml) were1.120±0.036,1.160±0.054.Compared with narcotine alone way, aurora-A protein expression ofcombination group was elevated, the difference was statistically significant (P<0.05).Conclusions:1Narcotine exerted inhibitory effects on proliferation of SKOV3cellsand its effect presented a time and dose-dependent way.2Morphological observation of SKOV3cells: the morphological changesof narcotine combined with cisplatin group is significantly higher thannarcotine alone group.3Narcotine can block ovarian cancer SKOV-3in G2/M phase andinduce its apoptosis.4Aurora-A protein levels rise after narcotine acted on SKOV3cells,which provide experimental basis for narcotine by blocking the G2/M phaseto induce apoptosis for further. |
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