Yuan Falu, Liu Junyong, Qiao Tingting, Li Ting, Shen Qi, Peng Fang
College of Pharmacy and Chemistry, Dali University, Dali 671000, Yunnan, China.
College of Pharmacy, Shanghai Jiaotong University, Shanghai 200240, China.
Molecules. 2016 Jun 28;21(7):852. doi: 10.3390/molecules21070852.
The present study reports the reversing effects of extracts from P. americana on multidrug resistance of BEL-7402/5-FU cells, as well as a preliminary investigation on their mechanism of action. A methylthiazolyl tetrazolium (MTT) method was applied to determine the multidrug resistance of BEL-7402/5-FU, while an intracellular drug accumulation assay was used to evaluate the effects of a column chromatography extract (PACC) and defatted extract (PADF) from P. americana on reversing multi-drug resistance. BEL-7402/5-FU reflected high resistance to 5-FU; PACC and PADF could promote drug accumulation in BEL-7402/5-FU cells, among which PADF was more effective than PACC. Moreover, results from the immunocytochemical method showed that PACC and PADF could downregulate the expression of drug resistance-associated proteins (P-gp, MRP, LRP); PACC and PADF had no effects on the expression of multidrug resistance-associated enzymes (GST-π), but PACC could increase the expression of multidrug resistance-associated enzymes (PKC). Results of real-time fluorescence quantitative PCR revealed that PACC and PADF were able to markedly inhibit the expression of multidrug resistance-associated genes (MDR1, LRP and MRP1); PACC presented a significant impact on the gene expression of multidrug resistance-associated enzymes, which increased the gene expression of GST-π and PKC. However, PADF had little impact on the expression of multidrug resistance-associated enzymes. These results demonstrated that PACC and PADF extracted from P. americana could effectively reverse MDR in BEL-7402/5-FU cells, whose mechanism was to inhibit the expression of P-gp, MRP, and LRP, and that PADF was more effective in the reversal of MDR than did PACC. In addition, some of extracts from P. americana altered (sometimes increasing) the expression of multidrug resistance-associated enzymes.
本研究报道了美洲商陆提取物对BEL-7402/5-FU细胞多药耐药性的逆转作用及其作用机制的初步研究。采用甲基噻唑基四氮唑蓝（MTT）法测定BEL-7402/5-FU的多药耐药性，同时采用细胞内药物蓄积试验评价美洲商陆柱色谱提取物（PACC）和脱脂提取物（PADF）对多药耐药逆转的影响。BEL-7402/5-FU对5-FU表现出高耐药性；PACC和PADF可促进药物在BEL-7402/5-FU细胞中的蓄积，其中PADF比PACC更有效。此外，免疫细胞化学方法结果显示，PACC和PADF可下调耐药相关蛋白（P-gp、MRP、LRP）的表达；PACC和PADF对多药耐药相关酶（GST-π）的表达无影响，但PACC可增加多药耐药相关酶（PKC）的表达。实时荧光定量PCR结果显示，PACC和PADF能够显著抑制多药耐药相关基因（MDR1、LRP和MRP1）的表达；PACC对多药耐药相关酶的基因表达有显著影响，增加了GST-π和PKC的基因表达。然而，PADF对多药耐药相关酶的表达影响较小。这些结果表明，美洲商陆提取的PACC和PADF可有效逆转BEL-7402/5-FU细胞的多药耐药性，其机制是抑制P-gp、MRP和LRP的表达，且PADF在逆转多药耐药方面比PACC更有效。此外，美洲商陆的一些提取物改变（有时增加）了多药耐药相关酶的表达。
