Ovarian Cancer, Paclitaxel Resistance And Protein P-cofilin Correlation Study

Background and objectiveOvarian cancer is the third leading cause of death and has the highest mortality rate among the gynecologic malignancies.Although the combination chemotherapy including taxol and cisplatin has improved the prognosis of initial treatment of ovarian cancer,five-year survival rate of advanced stage rambled between 15%to 20%,one of the reasons is primary or acquired drug resistance after chemotherapy.The mechanisms by chemoresistance is thoroughly recognized,but all of them could not predict the prognosis of cancers very well and inconsistent expression between mRNA and relative protein possibly is main reason.Proteomics approach provides a new tool for further investigation on chemoresistance of ovarian cancers.The study was supported by key foundation of Peking Union Medical College Hospital in 2003.We had performed a proteomics analysis on seeking chemoresistant marker of taxol,on the basis of SKOV3 taxol resistant ovarian cancer cell line (TJ2500) induced by different way in vitro and A2780 taxol sensitive and taxol resistant cell lines(TA2780) purchased,adopted 2-DE technique,protein expression map of taxol-sensitive and resistant ovarian cell lines were successful prepared,and cofilinl was selected as the marker of taxol drug resistance.The different expression of phosphorylation of cofilin(p-cofilin) between sensitive and resistant cell line of ovarian cancer was found by western-blot technique,but the same expression was not exist on cofilin1.Expression of different forms of cofilin1 was detected in human ovarian cancer specimes including 24 clinical chemosensitive cases and 22 clinical chemoresistant cases by immunohistochemistry.The expression of cofilin 1 had no significant difference between the groups of chemosensitivity and chemoresistance. However,the expression of p-cofilin displayed much higher in chemoresistant group than in sensitive one,which further confirmed that p-cofilin is possibly related with chemoresistance of taxol on ovarian cancer.Cofilin is a binding protein of actin,which control the polymerization and disaggregation of actin.Phos-cofilin is inactive and dephos-cofilin is active,dynamic switch of cofilin is the premise for cofilin action.Both actin and tubulin(target of taxol) are the major components of cytoskeleton.The function of cytoskeleton involves the maintance of cell morphology,material transportation,cell cleavage,cell mobility and signal transduction.The purpose of this research is to further explore the relationship between phosphorylation procedure of cofilin 1 and chemoresistance of taxol,and through cell cycle synchronization,more resistant cells are concentrated in S phase and enter to M phase at equal pace,then the taxol's antitumous effects are investigated.Materials and methods1.MTT(for drug resistant index),cell staining,growing curve and cell cycle were used on comparison the change of cell appearance and bionomics on the ovarian cancer taxol-sensitive and resistant cell lines(SKOV3/TJ2500 and A2780/TA2780), which had been induced and cultured for three years.2.In order to removed the influence of exogenous factors on p-cofilin,four cell lines(SKOV3/TJ2500 and A2780/TA2780) were cultured to 70%confluens,then replaced with serum-free medium,48h later different concentrations of taxol were added and cells were collected in different time point.Western-blot technique was used to detect the expression of p-cofilin and analysize the effect of taxol on endogenous p-cofilin.3.Investigate the function of p-cofilin as taxol resistance relative protein.Plasmids containing S3D and cofilin were transfected into sensitive cell lines A2780 respectively.Western-blot and MTT technique were used to detect the expression of pcofilin and total cofilin and resistant index respectively.4.The study investiagted the effects of cell cycle synchronization of taxol sensitive and resistant cell lines to lethal effect of taxol.The change of cell cycle was analysed with flow cytometry in different time after adding distinct concentration taxol in sensitive cell lines.Thymidine could center cells in S phase through cell cycle synchronization.medium without thymidine was changed after most cells had entered S phase in same step,8-10 hours later taxol was added into medium.The apoptosis rate of sensitive and resistant cells was analysed with flow cytometry in different time after adding taxol 12 hours,and the cells cultured without thymidine was as control.Results1.The results of MTT showed that if we setted the resistant index(RI) of SKOV3, A2780 as 1,the RI of TJ2500 and TA2780 were 62.35±11.3,25±6.5 respectively. The results of cell stain showed that aberrant nucleus in TJ2500 were more than in SKOV3 and appearance of TA2780 most turned into fusiform from round shape of A2780.The rate of resistant cells in G0-G1 phase increased and in S phase reduced, the difference was significantly.Population doubling time of two kinds of taxol resistant cells became longer.2.After the starvated cells were stimulated with different concentrations and time of taxol,western blot results showed that expression of endogenous p-cofilin were all high in sensitive and resistant cell lines.Down-regulation of p-cofilin in sensitive cells were displayed in definitive time point after stimulated with different concentrations of taxol,and the time prolonged with the increase of taxol concentrations,while the levels of p-cofilin in resistant cells had no significant changes after stimulated with different concentration of taxol.3.After transfected with cofilin plasmid,A2780 displayed more resistant to taxol and RI improved from 1 to 11.5-11.8 compared with parent cells.Expressions of cofilin and p-cofilin on western blot image all up-regulated.After transfected with S3D plasmid,RI of A2780 decreased from 1 to 0.1 to 0.16 compared with parent cells and expressions of p-cofilin slightly down-regulated.4.The effect of low concentration taxol-10nmol/L and high concentration -100nmol/l on G2-M phase blockage had significant difference.the difference between 100 and 1000nmol/L was not striking.With the action time of taxol extension,the effectiveness of taxol blockage on G2-M phase is increasing and become most significant 24 hours later.Apoptosis peak usually occurred 48 hours later after using taxol.with the technique of Annexin-v FITC,apoptotic rate in taxol sensitive and resistant cells were all higher than control,especially 48 hours later after the cells dealed with thymidine and withdraw taxol in short time. Conclusions1.After resuscitated and cultured in vitro for three years,ovarian cancer taxol-sensitive and resistant cell line-A2780/TA2780,SKOV3/TJ2500 displayed little change in bionomics compared with the cells induced on beginning.Although the RI decreased,the characteristics of taxol-resistant cell lines still exist.2.Taxol-sensitive cells(SKOV3,A2780) and resistant cells(TJ2500,TA2780) were cultured with serum-free medium for 48 hours.Western blot image displayed high level expressions of endogenous p-cofilin exist in four cell lines.With increasing concentrations of taxol,expression of p-cofilin in sensitive cell lines down-regulated, but in resistant cell lines did not change significantly.Dynamic switch of cofilin is the premise for its action.Phosphorylation of cofilin is inactive type and dephosphorylation is active type.Down-regulation of endogenous p-ofilin in sensitive cell lines after taxol action means more cofilin converted into active form-dephosphorylation,which could improve the function of actin,facilitate proliferation of cells and profit cytotoxic action of taxol.High level expressions of endogenous p-cofilin in resistant cell lines still sustained after taxol action,which indirectly demonstrated the relationship between p-cofilin and drug resistance of taxol.3.After transfected with cofilin plasmid,taxol-sensitive cells A2780 displayed up-regulation of cofilin,p-cofilin and RI.After transfected with S3D plasmid,A2780 showed slightly down-regulation of p-cofilin and RI,which further confirmed the relationship between high expression of p-cofilin and taxol resistance on ovarian cancer cells.4.The effectiveness of taxol will be increasing with the elevation of concentration from 10 to 100 nmol/L,but this phenomena will be disappear when the concentration of taxol rise to some degree,which could illustrate that the action of taxol exist saturation.The blockage in G2-M phase of taxol will be strengthen by the prolongation of time and 24 hours is the best.long time medication will cause serious side-effect,then hinder the later therapy.The means of cell cycle in same step will be better way to shorter the drug action time,strengthen the effectiveness and reverse the drug resistance of taxol.