Bcl-2 family proteins and cytoskeleton changes involved in DM-1 cytotoxic effect on melanoma cells
Bcl-2 family proteins and cytoskeleton changes involved in DM-1 cytotoxic effect on melanoma cells
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DOI：
10.1007/s13277-013-0666-6
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发表时间：
2013-04-01
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影响因子：
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通讯作者：
Maria, Durvanei Augusto
中科院分区：
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作者：
Faiao-Flores, Fernanda;Quincoces Suarez, Jose Agustin;Maria, Durvanei Augusto
黑色素瘤是最具侵袭性的皮肤癌之一，其发病率仍在上升。所有现有的治疗方法都是最低限度的。因此，应该开发新的治疗黑色素瘤的药物。DM-1化合物是姜黄素类似物，具有姜黄素的几种特性，如抗增殖、抗肿瘤和抗转移特性。本研究的目的是评估DM-1对黑色素瘤细胞的细胞毒性作用中涉及的不同信号通路。免疫印迹法和免疫荧光法分别对黑素瘤细胞的凋亡过程和细胞骨架变化进行了评价。DM-1治疗后，SK-MEL-5黑色素瘤细胞显示肌动蛋白丝组织紊乱，整个细胞骨架形成针状结构，局灶性粘连显著减少，并且它们仅存在于细胞末端，导致细胞与底物之间连接不良。除此之外，还有明显的丝足缩回和典型细胞骨架支架的丢失。这些修饰导致细胞脱离，随后细胞死亡。此外，dm -1诱导的细胞凋亡是由多种Bcl-2蛋白触发的，这些Bcl-2蛋白参与了外源性和内源性凋亡途径。SK-MEL-5细胞的死亡机制主要是Bcl-2/Bax比值降低，而A375细胞则是通过下调Mcl-1和Bcl-xL诱导凋亡。在SK-MEL-5和A375黑色素瘤细胞中，caspase 9的活性形式显著增加，而效应caspase 3的非活性形式在两种细胞系中都有所减少。DM-1处理后，这些黑色素瘤细胞系中cleaved poly ADP核糖聚合酶的表达增加，表明发生了凋亡过程。总之，这些数据阐明了抗肿瘤药物DM-1在黑色素瘤细胞中诱导细胞毒性的细胞和分子机制。
Melanoma is one of the most aggressive types of skin cancer and its incidence rate is still increasing. All existing treatments are minimally effective. Consequently, new therapeutic agents for melanoma treatment should be developed. The DM-1 compound is a curcumin analog that possesses several curcumin characteristics, such as antiproliferative, antitumor, and anti-metastatic properties. The aim of this study was to evaluate the different signaling pathways involved in the cytotoxic effect of DM-1 on melanoma cells. The apoptotic process and cytoskeletal changes were evaluated by immunoblotting and immunofluorescence, respectively, in melanoma cells. After DM-1 treatment, SK-MEL-5 melanoma cells showed actin filament disorganization with spicule formation throughout the cytoskeleton and significant reduction of focal adhesion as well as they were present only at cell extremities, conferring a poor connection between the cell and the substrate. Besides this, there was significant filopodium retraction and loss of typical cytoskeleton scaffold. These modifications contributed to cell detachment followed by cell death. Furthermore, DM-1-induced apoptosis was triggered by multiple Bcl-2 proteins involved in both the extrinsic and the intrinsic apoptotic pathways. SK-MEL-5 cells showed a death mechanism mainly by Bcl-2/Bax ratio decrease, whereas A375 cells presented apoptosis induction by Mcl-1 and Bcl-xL downregulation. In SK-MEL-5 and A375 melanoma cells, there was a significant increase in the active form of caspase 9, and the inactive form of the effector caspase 3 was decreased in both cell lines. Expression of cleaved poly ADP ribose polymerase was increased after DM-1 treatment in these melanoma cell lines, demonstrating that the apoptotic process occurred. Altogether, these data elucidate the cellular and molecular mechanisms involved in the cytotoxicity induced by the antitumor agent DM-1 in melanoma cells.