Leite Daniela F, Kassuya Cândida A, Mazzuco Tânia L, Silvestre Aline, de Melo Lucilia V, Rehder Vera L, Rumjanek Vivian M, Calixto João B
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, UFRJ, Rio de Janeiro, RJ, Brazil.
Planta Med. 2006 Dec;72(15):1353-8. doi: 10.1055/s-2006-951708. Epub 2006 Oct 20.
Multidrug resistance (MDR) constitutes the major obstacle to the successful treatment of cancer. In several cancer cells, MDR is thought to be mediated by the super-expression of P-glycoprotein (Pgp). Pgp extrudes drugs from the cells, therefore reducing their cytotoxicity, and its activity inhibition may reverse the MDR phenotype. The present study evaluated the possible cytotoxic effect and MDR reversing properties of the extract and compounds isolated from Phyllanthus amarus. To this purpose, two human leukaemia cell lines were employed: K-562 and its vincristine-resistant counterpart Lucena-1, a Pgp-overexpressing subline. We report here that Lucena-1 was significantly more resistant to the cytotoxicity of P. amarus derivatives: the hexane extract (HE, 100 microg/mL), the lignans-rich fraction (LRF, 100 microg/mL) and the lignans nirtetralin (NIRT, 43.2 microg/mL), niranthin (NIRA, 43 microg/mL) or phyllanthin (PHYLLA, 43 microg/mL) exerted cytotoxic effects on K-562 cells with 40.3, 66.0, 62.0, 61.0 or 24.1% of cell death, respectively. The cellular toxicity observed on Lucena-1 was 16.3, 40.4, 29.4, 30.2, or 24.8%, respectively. However, cell treatment with the lignan phyltetralin (PHYLT) up to 41.6 microg/mL had no cytotoxic action on either of the cell lines. P. amarus derivatives were also found to be effective in inhibiting Pgp activity as assessed by rhodamine accumulation in Lucena-1 cells, as were the classical Pgp inhibitors, cyclosporine A (160 nM), PSC-833 (2 microM) and verapamil (5 microM). The lignan NIRT produced the most potent inhibition (EC (50) = 29.4 microg/mL) followed by NIRA (44.3 microg/mL), LRF (49.1 microg/mL), PHYLT (99.4 microg/mL), PHYLLA and HE (> 100 microg/mL). Lucena-1 cells were more resistant to daunorubicin-induced cell death (LC (50) = 50 microM) than K562 cells (LC (50) = 4.95 microM). Of note, the P. amarus derivatives significantly potentiated 5 microM daunorubicin-induced cell death in Lucena-1 cells (P < 0.01) but not in K562 cells. After treatment only with P. amarus derivatives (100 microg/mL HE, 30 microg/mL LRF, 12.9 microg/mL NIRA, 43.2 microg/mL NIRT, 43 microg/mL PHYLLA or 41.6 microg/mL PHYLT), the Lucena-1 cellular viability was 83.7, 85.3, 101, 69.7, 75.6 or 88.7%, respectively, whereas the in the presence of daunorubincin, which was not cytotoxic PER SE, the cell viability decreased to 42.9, 42.2, 64.2, 35.4, 30.4 or 52.6%, respectively. Together, these results suggest a potential action of P. amarus derivatives as MDR reversing agents, mainly due to their ability to synergize with the action of conventional chemotherapeutics.
多药耐药性（MDR）是癌症成功治疗的主要障碍。在几种癌细胞中，MDR被认为是由P-糖蛋白（Pgp）的超表达介导的。Pgp将药物从细胞中排出，从而降低其细胞毒性，抑制其活性可能会逆转MDR表型。本研究评估了苦味叶下珠提取物及其分离化合物可能的细胞毒性作用和MDR逆转特性。为此，使用了两种人类白血病细胞系：K-562及其长春新碱耐药对应物Lucena-1，后者是Pgp过表达亚系。我们在此报告，Lucena-1对苦味叶下珠衍生物的细胞毒性明显更具抗性：己烷提取物（HE，100μg/mL）、富含木脂素的部分（LRF，100μg/mL）以及木脂素尼替他林（NIRT，43.2μg/mL）、尼兰辛（NIRA，43μg/mL）或叶下珠素（PHYLLA，43μg/mL）对K-562细胞分别产生40.3%、66.0%、62.0%、61.0%或24.1%的细胞死亡的细胞毒性作用。在Lucena-1上观察到的细胞毒性分别为细胞死亡的16.3%、40.4%、29.4%、30.2%或24.8%。然而，用高达41.6μg/mL的木脂素叶下珠脂素（PHYLT）处理细胞对两种细胞系均无细胞毒性作用。通过Lucena-1细胞中罗丹明积累评估发现，苦味叶下珠衍生物在抑制Pgp活性方面也很有效，经典的Pgp抑制剂环孢素A（160 nM）、PSC-833（2μM）和维拉帕米（5μM）也是如此。木脂素NIRT产生的抑制作用最强（半数有效浓度（EC50）=29.4μg/mL），其次是NIRA（44.3μg/mL）、LRF（49.1μg/mL）、PHYLT（99.4μg/mL）、PHYLLA和HE（>100μg/mL）。Lucena-1细胞比K562细胞对柔红霉素诱导细胞死亡更具抗性（半数致死浓度（LC50）=50μM对4.95μM）。值得注意的是，苦味叶下珠衍生物显著增强了5μM柔红霉素在Lucena-1细胞中诱导的细胞死亡（P<0.01），但在K562细胞中未观察到这种现象。仅用苦味叶下珠衍生物（100μg/mL HE、30μg/mL LRF、12.9μg/mL NIRA、43.2μg/mL NIRT、43μg/mL PHYLLA或41.6μg/mL PHYLT）处理后，Lucena-1细胞的活力分别为83.7%、85.3%、101%、69.7%、75.6%或88.7%，而在存在本身无细胞毒性的柔红霉素的情况下，细胞活力分别降至42.9%、42.2%、64.2%、35.4%、30.4%或52.6%。总之，这些结果表明苦味叶下珠衍生物作为MDR逆转剂具有潜在作用，主要是由于它们与传统化疗药物协同作用的能力。
