叶酸受体单抗Farletuzumab对卵巢癌细胞多药耐药逆转的机制

Mechanism of Folate Receptor Monoclonal Antibody Farletuzumab Reversing Multidrug Resistance in Ovarian Cancer Cells

目的探讨叶酸受体-α(folate receptor-α,FR-α)的单克隆抗体Farletuzumab对卵巢癌细胞多药耐药特性的逆转机制。方法激光共聚焦及流式细胞术检测卵巢癌细胞SKOV3及耐药细胞株SKOV3-ts中FR-α的表达。Western blot法检测卵巢癌细胞在Farletuzumab作用前后P-糖蛋白(P-glycoprotein,P-gp)的表达。MTT法检测耐药细胞在Farletuzumab作用前后针对化疗药紫杉醇(paclitaxel,PTX)及阿霉素(doxorubicin,DOX)的半数抑制浓度(inhibitory concentration 50,IC_(50))改变。激光共聚焦显微镜下观察并测定细胞在Farletuzumab作用前后DOX的细胞内红色荧光储备改变。结果激光共聚焦及流式细胞术检测显示,耐药细胞株SKOV3-ts细胞膜层有FR-α强表达,而亲本细胞SKOV3则表达较弱。Western blot显示,耐药细胞株SKOV3-ts有P-gp强表达,但亲本细胞SKOV3表达较弱,Farletuzumab作用后,2种细胞P-gp表达无明显改变。MTT法显示,SKOV3-ts细胞针对PTX及DOX的IC_(50)明显高于SKOV3细胞(均P<0.05)。SKOV3-ts细胞经Farletuzumab作用后,IC_(50)明显降低,作用前后比较,差异有统计学意义(均P<0.05)。激光共聚焦显示,经Farletuzumab作用后,SKOV3-ts细胞内DOX红色荧光蓄积较作用前明显增加。结论随着卵巢癌细胞多药耐药特性的产生,FR-α及P-gp呈强表达。Farletuzumab可逆转卵巢癌细胞SKOV3-ts多药耐药,这一作用可能通过拮抗FR-α,从而削弱P-gp将化疗药外泵至细胞外的作用实现。

Objective To investigate the mechanism by which Farletuzumab,a monoclonal antibody of folate receptor-α(FR-α)reverses the multidrug resistance of ovarian cancer cells.Methods The confocal laser scanning microscopy and flow cytometry were used to detect the expression of FR-αin ovarian cancer cells SKOV3 and SKOV3-ts(a drug-resistant cell line),Western blotting to detect P-glycoprotein(P-gp)expression in SKOV3 and SKOV3-ts cells before and after Farletuzumab treatment,and MTT assay to detect the 50%inhibiting concentration(IC 50)of drug-resistant cells SKOV3-ts to paclitaxel(PTX)and doxorubicin(DOX)before and after Farletuzumab treatment.The confocal laser scanning microscopy was used to determine the change of intracellular red fluorescence of DOX in cells before and after farletuzumab treatment.Results Confocal laser scanning microscopy and flow cytometry showed that FR-αwas strongly expressed on the SKOV3-ts cell membrane,whereas it was weakly expressed in the parental cell line SKOV3.Western blotting showed that the SKOV3-ts cell line had strong expression of P-gp,but the expression of P-gp was weak in SKOV3 cells.After Farletuzumab treatment,the expression of P-gp was not significantly changed in both cells.MTT assay showed that the IC 50 of SKOV3-ts cells against PTX and DOX was significantly higher than that of SKOV3 cells(all P<0.05).The IC 50 of SKOV3-ts cells treated with Farletuzumab was significantly decreased,and the difference was statistically significant before and after the treatment(all P<0.05).Laser confocal microscopy showed that the accumulation of DOX red fluorescence in SKOV3-ts cells was significantly increased after farletuzumab treatment.Conclusion FR-αand P-gp are strongly expressed in ovarian cancer cells with multidrug resistance.Farletuzumab can reverse the multidrug resistance of ovarian cancer cells SKOV3-ts,which may be achieved by antagonizing FR-α,thereby impairing the effect of P-gp pumping extracellular chemotherapeutic drugs out of the cell.