鞣酸对LLC/cMOAT细胞多药耐药性的逆转机制

目的本研究探讨鞣酸(Tannic acid,TA)对LLC/cMOAT细胞多药耐药性的逆转作用及其可能机制。方法采用MTT法检测LLC/CMV和LLC/cMOAT细胞对各种化疗药物的敏感性以及在不同浓度的TA处理下细胞的存活状态;确定TA的非细胞毒性浓度以及在该浓度下各梯度浓度处理时细胞的存活率;采用流式细胞仪技术检测使用非细胞毒性浓度的TA处理前后LLC/cMOAT细胞内柔红霉素(DNR)浓度变化、细胞周期阻滞及对凋亡的影响。结果LLC/cMOAT细胞对羟基喜树碱(CPT-11),阿霉素(ADM),顺铂(DDP)和长春新碱(VCR)均有不同程度的耐药,其耐药倍数分别是2.08、3.02、4.01和9.31,对依托泊苷(VP-16)、紫衫醇(TAX)无明显耐药,10.0μmol/L浓度以下的TA对LLC/CMV和LLC/cMOAT细胞无明显细胞毒性,2.5、5.0、10.0μmol/L浓度的TA能显著降低LLC/cMOAT细胞的IC₅₀(P<0.05),其逆转倍数分别为5.09、6.33、7.76倍;细胞内DNR荧光强度随着TA浓度的增高而明显增强;TA作用细胞12h可使G₁期细胞数百分比由(46.35±3.74) %增至(66. 43 ±5. 87) % , 阻滞细胞于G₁期; TA 与VCR 联合处理细胞24 h 和48 h 时凋亡率分别为12. 1 %和19. 0 % ,与处理前(1. 65 %) 相比提高明显;使用2. 5μmol/ L 、5.0μmol/ L 和10. 0μmol/ L 浓度的TA 与VCR 共同处理细胞24 h 后,凋亡率由处理前的4. 96 %分别提高到11. 2 %、17. 9 %和25. 1 %。结论　TA能部分逆转LLC/ cMOAT 细胞的多药耐药,显著提高耐药细胞内DNR 荧光强度并呈浓度依赖性,阻滞细胞周期于G₁ 期,并可诱导细胞凋亡,呈时间、剂量依赖性,其机制可能与抑制化疗药物外排、增加细胞内药物浓度以及诱导细胞凋亡有关。

Reversal Mechanism of Tannic Acid on Multidrug Resistant of LLC/cMOAT Cells

Objective To investigate the reversal effects of tannic acid(TA) on the multidrug resistance of LLC/cMOAT cells and the possible mechanism.Methods MTT assay was used to determine the sensitivity of LLC/CMV and LLC/cMOAT cells to the various kinds of chemotherapeutics,and the activity of LLC/CMV and LLC/cMOAT cells treated with different concentrations of TA. The non-cytotoxic concentrations of TA were determined for the cells and the reversal effects of the chemotherapeutics were observed. The intracellular concent ration of DNR , the cell cycle dist ribution and the apoptosis rate of thecells t reated with different concent rations of TA were determined by flow cytomet ry ( FCM) . Results 　LLC/ cMOAT cells were resistant to CPT-11 ,ADM ,DDP ,VCR to a certain extent , and not resistant toVP-16 、TAX. At 10. 0μmol/ L and below , TA was not significantly cytotoxic to LLC/ CMV and LLC/cMOAT cells. TA at the concent ration of 2. 5 ,5. 0 ,10. 0μmol/ L may remarkably decrease IC50 of LLC/cMOAT cells ( P < 0. 05) . The int racellular DNR fluorescence intensity in LLC/ cMOAT cells was significantly enhanced with the increase of TA concent ration. The cells at G₁ stage increased f rom (46. 35 ±3. 74) % to (66. 43 ±5. 87) % when t reated with TA at 5. 0μmol/ L for 12 hours. The cell apoptosis wasenhanced in a time-and concent ration-dependent manner by t reating with TA. Conclusion 　TA is able toreverse the multidrug resistance of LLC/ cMOAT cells and may remarkably raise drug concent rations inconcent ration-dependent manner ,TA is able to block cell cycle at G₁ stage ,and it s function of inducingapoptosis act s in time-and dose-depended manner. The mechanism may involve the decrease of chemotherapeutics excretion ,the increase of int racellular drug concent ration , growth arrest at G₁ and apoptosis.