木犀草素逆转宫颈癌细胞对阿霉素耐药性的机制研究

Luteolin reverses doxorubicin resistance in cervical cancer by regulating PI3K/Akt signaling pathway

目的探讨木犀草素(Lut)逆转宫颈癌对阿霉素(Dox)耐药性的效应及其机制。方法建立Dox耐药的宫颈癌HeLa/Dox细胞株,利用Dox、Lut单独或联合作用于细胞,分为control组、Dox组、Lut组、Dox+Lut组,检测各组细胞存活率及凋亡率。构建小鼠移植瘤模型,经腹腔注射3 mg·kg^(-1)Dox和/或5 mg·kg^(-1)Lut,检测小鼠肿瘤生长情况及肿瘤中相关蛋白的表达。结果(1)CCK-8检测结果显示,Dox+Lut组细胞的生长活性明显受到抑制,存活率显著低于Dox组和Lut组(P<0.01)。细胞克隆形成实验显示出与CCK-8实验相似的细胞生长趋势。流式细胞术检测结果显示,Dox单药对HeLa/Dox细胞的凋亡无明显促进作用,而Lut单药可显著诱导HeLa/Dox细胞凋亡。(2)Dox单药对HeLa/Dox细胞迁移的影响较小(P<0.05),而Lut单药处理的HeLa/Dox细胞迁移数目明显减少,Dox与Lut联合应用对HeLa/Dox细胞迁移能力的抑制作用最强(P<0.01)。(3)与对照组相比,Dox组、Lut组、Dox+Lut组HeLa/Dox细胞中PI3K、Cleaved caspase-3蛋白表达均显著上调,p-Akt、p-mTOR、p70S6K蛋白表达显著下调(P<0.05),且Dox+Lut组蛋白表达水平变化最为显著。(4)对照组、Dox组、Lut组、Dox+Lut组小鼠肿瘤体积分别为(1265.8±123.3)mm^(3)、(456.4±68.7)mm^(3)、(479.9±57.2)mm^(3)、(108.1±14.0)mm^(3),差异具有统计学意义(P<0.05)。联合应用Dox和Lut后,肿瘤组织中PI3K和Cleaved caspase-3的表达显著上调(P<0.01),而p70S6K和Ki-67的表达显著下调(P<0.01)。结论Lut可通过调节PI3K/Akt信号通路的活性逆转宫颈癌细胞对阿霉素的耐药性,从而抑制肿瘤细胞的增殖和转移,并促进其凋亡。

Objective To investigate the effects and mechanism of luteolin(Lut)in reversing doxorubicin(Dox)resistance of cervical cancer.Methods Dox-resistant HeLa/Dox cell line was established,and the HeLa/Dox cells were divided into control group,Dox group,Lut group,Dox+Lut group after treated with Dox or/and Lut to detect the survival rate and apoptosis rate.The model of transplanted tumor in mice was established,and they were treated with 3 mg·kg^(-1) Dox or/and 5 mg·kg^(-1) Lut.Detect the growth of the tumor in mice and the changes of related proteins in tumor.Results(1)The results of CCK-8 assay showed that the cell growth of Dox+Lut group was significantly inhibited and its survival rate was lower than that of the Lut group and Dox group(P<0.01).Cell clone formation experiments showed a similar trend of cell growth as CCK-8 assay.Flow cytometry showed that Dox alone did not promote the apoptosis of HeLa/Dox cells,but Lut alone could significantly cause the apoptosis of HeLa/Dox cells.(2)Dox alone showed few effects on the migration of HeLa/Dox cells(P<0.05),while Lut alone significantly inhibited the migration of HeLa/Dox cells.Dox combined with Lut showed the strongest inhibitory effects on HeLa/Dox cell migration(P<0.01).(3)Compared with the control group,the expressions of PI3K and cleaved caspase-3 proteins in Dox group,Lut group and Dox+Lut group were up-regulated.The expression levels of PI3K and cleaved caspase-3 proteins in Dox+Lut group were the highest.The protein expressions of p-Akt,p-mTOR and p70S6K proteins was significantly decreased in Dox+Lut group(P<0.05).(4)The tumor volume of control group,Dox group,Lut group and Dox+Lut group were respectively(1265.8+123.3)mm^(3),(456.4+68.7)mm^(3),(479.9+57.2)mm^(3) and(108.1+14.0)mm^(3),with statistical differences(P<0.05).Immunohistochemistry showed that the expression of PI3K and cleaved caspase-3 increased significantly in Dox+Lut group(P<0.01),while the expression of P70S6K and Ki-67 decreased significantly(P<0.01).Conclusion Lut can reverse the drug resistance of cervical cancer cells to doxorubicin by regulating the activity of PI3K/Akt signaling pathway,and inhibit the proliferation and metastasis and promote the apoptosis of tumor cells.