自噬对苯丙素苷逆转耐药作用的影响

Effect of autophagy on reversing multidrug resistance of phenylpropanoid glycosides

目的探讨自噬对苯丙素苷(PPG)逆转大肠癌耐药株LoVo/Adr细胞耐药作用的机制。方法 MTT法检测PPG对LoVo/Adr细胞增殖及耐药性的影响;Western-blotting检测P-糖蛋白(P-gp)的表达;流式细胞术测定阿霉素(ADR)在LoVo/Adr细胞中的摄入;单丹磺酰尸胺(MDC)染色后在荧光显微镜下观察细胞自噬体及通过流式细胞仪定量分析自噬率;ATP检测试剂盒测定细胞内的ATP水平。结果 80、160 mg/L的PPG可抑制LoVo/Adr细胞增殖,20、40 mg/L的PPG无明显细胞毒作用;20、40 mg/L的PPG对ADR的逆转倍数分别为5.31和7.51倍,加入自噬特异抑制剂3-甲基腺嘌呤(3-MA)后,2组的逆转倍数分别增至11.84和19.25倍;20、40 mg/L的PPG及5 mmol/L的3-MA均不影响P-gp的表达;20、40 mg/L的PPG作用于LoVo/Adr细胞后,细胞内ADR荧光强度显著增加(P<0.01),联合3-MA后,细胞内ADR的荧光强度进一步增加;在荧光显微镜下,LoVo/Adr细胞很少或几乎没有点状绿色荧光,自噬率为(3.17±0.52)%,20、40 mg/L的PPG作用后,均可见大量点状绿色荧光即自噬体分布于胞浆及核周,自噬率分别升至(45.82±5.47)%、(76.23±8.28)%(P<0.01);当3-MA与PPG合用时,自噬泡的数量较单用PPG明显减少,自噬率分别降至(24.87±2.43)%、(39.56±4.64)%(P<0.05);对照组LoVo/Adr细胞中ATP的浓度为(1.92±0.23)nmol/mg,20、40 mg/L的PPG作用后,细胞内ATP的水平分别下降至(0.97±0.14)、(0.63±0.13)nmol/mg;联合3-MA后,细胞内ATP水平分别进一步降至(0.58±0.12)、(0.33±0.10)nmol/mg。结论 PPG具有诱导大肠癌耐药LoVo/Adr细胞自噬和逆转耐药的特性,这种特性与PPG降低细胞ATP水平,激活自噬、抑制ATP依赖性P-gp的外泵功能有关;同时抑制自噬可导致细胞内ATP水平进一步降低,加重抑制P-gp功能,提高PPG的逆转作用。

Objective To explore the mechanism of autophagy on the reversing multidrug resistance （MDR） of phenylpropanoid glycosides （PPG） for cocorectal carcinoma resistant strains LoVo/Adr. Methods The effects of PPG on cell proliferation and MDR in LoVo/Adr were measured by MTT assay and the expression of P-glycoprotein （P-gp） was determined by Western-blotting. The uptake of adriamycin （ADR） in LoVo/Adr cells was detected by flow cytometry （FCM）; Autophagosomes were tested by MDC staining and fluorescence microscope. Autophagic rate was measured by FCM and the ATP level of cells was measured by ATP assay kit. Results PPG at high concentration （80 and 160 mg/L） inhibited the proliferation of cells while PPG at low concentration （20 and 40 mg/L） had no cytotoxicity to cells. Reverse fold of PPG on ADR was 5.31 and 7.51 respectively when PPG was 20 and 40 mg/L, and significantly increased to 11.84 and 19.25 respectively after adding the autophagic inhibitor 3-MA. The expression of P-gp was not affected by PPG （20 and 40 mg/L） or 3-MA （5 mmol/L）. The fluorescence intensity of ADR was increased （P 〈 0.01） after treatment with PPG （20 and 40 mg/L） on LoVo/Adr respectively, and after PPG combined with 3-MA, the intensity was further increased. Under fluorescence microscopy green fluorescence dots were rarely observed in LoVo/ADR cells, and the autophagic rate was （3.17 ± 0.52） %. The green fluorescence dots （autophagosomes） which distributed in cytoplasm and around the nucleus appeared abundantly in the LoVo/ADR cells after the treatment with PPG （20 and 40 nag/L） and the autophagic rate increased to （45.82± 5.47）% and （76.23± 8.28）% （P 〈 0.01）, respectively. The quantity of autophagosomes was more significantly decreased after the treatment with PPG combined with 3-MA than with PPG alone. The autophagic rates decreased to （24.87±2.43）% and （39.56 ± 4.64）% （P 〈 0.05）, respectively. The ATP level was （1.92 ± 0.23） nmol/mg in LoVo/Adr cells and declined to （0.97 ± 0.14） and （0.63 ± 0.13） nmol/mg, respectively when PPG was 20 and 40 mg/L. The ATP levels were further declined to （0.58 ± 0.12） and （0.33 ± 0.10） nmol/mg, respectively after the treatment with PPG combined with 3-MA. Conclusion PPG could induce autophagy and reversing MDR of the LoVo/ADR cells and this character might be related with decreasing the level of ATP in cells, activating autophagy, and inhibiting the ATP-dependent effiux function of P-gp by PPG Autophagic inhibition promotes the reverse efficiency of PPG by decreasing the level of ATP and inhibiting the function of P-gp further.