Meschini Stefania, Condello Maria, Calcabrini Annarica, Marra Manuela, Formisano Giuseppe, Lista Pasquale, De Milito Angelo, Federici Elena, Arancia Giuseppe
Department of Technology and Health, Istituto Superiore di Sanità, Rome, Italy.
Autophagy. 2008 Nov;4(8):1020-33. doi: 10.4161/auto.6952. Epub 2008 Nov 9.
In our previous studies, the bisindolic alkaloid voacamine (VOA), isolated from the plant Peschiera fuchsiaefolia, proved to exert a chemosensitizing effect on cultured multidrug resistant (MDR) osteosarcoma cells exposed to doxorubicin (DOX). In particular, VOA was capable of inhibiting P-glycoprotein action in a competitive way, thus explaining the enhancement of the cytotoxic effect induced by DOX on MDR cells. Afterwards, preliminary observations suggested that such an enhancement did not involve the apoptotic process but was due instead to the induction of autophagic cell death. The results of the present investigation demonstrate that the plant alkaloid VOA is an autophagy inducer able to exert apoptosis-independent cytotoxic effect on both wild-type and MDR tumor cells. In fact, under treatment condition causing about 50 percent of cell death, no evidence of apoptosis could be revealed by microscopical observations, Annexin V-FITC labeling and analysis of PARP cleavage, whereas the same cells underwent apoptosis when treated with apoptosis inducers, such as doxorubicin and staurosporine. Conversely, VOA-induced autophagy was clearly evidentiated by electron microscopy observations, monodansylcadaverine staining, LC3 expression, and conversion. These results were confirmed by the analysis of the modulating effects of the pretreatment with autophagy inhibitors prior to VOA administration. In addition, transfection of osteosarcoma cells with siRNA against ATG genes reduced VOA cytotoxicity. In conclusion, considering the very debated dual role of autophagy in cancer cells (protective or lethal, pro- or anti- apoptotic) our findings seem to demonstrate, at least in vitro, that a natural product able to induce autophagy can be effective against drug resistant tumors, either used alone or in association with conventional chemotherapeutics.
在我们之前的研究中，从植物佩氏倒挂金钟中分离得到的双吲哚生物碱沃卡明（VOA），被证明对暴露于阿霉素（DOX）的培养多药耐药（MDR）骨肉瘤细胞具有化学增敏作用。特别是，VOA能够以竞争性方式抑制P-糖蛋白的作用，从而解释了DOX对MDR细胞诱导的细胞毒性作用增强的原因。此后，初步观察表明，这种增强并不涉及凋亡过程，而是由于自噬性细胞死亡的诱导。本研究结果表明，植物生物碱VOA是一种自噬诱导剂，能够对野生型和MDR肿瘤细胞发挥不依赖于凋亡的细胞毒性作用。事实上，在导致约50%细胞死亡的处理条件下，通过显微镜观察、膜联蛋白V-FITC标记和PARP裂解分析，未发现凋亡证据，而当用阿霉素和星形孢菌素等凋亡诱导剂处理时，相同的细胞发生凋亡。相反，通过电子显微镜观察、单丹磺酰尸胺染色、LC3表达和转化，VOA诱导的自噬明显可见。在用自噬抑制剂预处理VOA给药前的调节作用分析中，这些结果得到了证实。此外，用针对ATG基因的siRNA转染骨肉瘤细胞可降低VOA的细胞毒性。总之，考虑到自噬在癌细胞中极具争议的双重作用（保护或致死、促凋亡或抗凋亡），我们的研究结果似乎至少在体外表明，一种能够诱导自噬的天然产物，无论是单独使用还是与传统化疗药物联合使用，都可以有效对抗耐药肿瘤。
