Seo Kang-Sik, Kim Jong-Seok, Park Ji-Hoon, Song Kyoung-Sub, Yun Eun-Jin, Park Jong-Il, Kweon Gi Ryang, Yoon Wan-Hee, Lim Kyu, Hwang Byung-Doo
Department of Biochemistry, College of Medicine, Chungnam National University, Daejeon, Korea.
BMC Cancer. 2014 Jan 22;14:36. doi: 10.1186/1471-2407-14-36.
Combination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells.
Cell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively.
We found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner.
These results suggest that the synergy between PMA and apicularen A is involved by PKCα activation and microtubule disruption, and that may inform the development of novel approaches to treat cancer.
联合治疗是提高癌症治疗疗效的关键。佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）是一种著名的蛋白激酶C（PKC）激活剂，可增强多种抗癌药物的细胞毒性。顶孢霉素A通过下调微管蛋白破坏微管网络，从而诱导肿瘤细胞的细胞毒性。在本研究中，我们检测了PMA是否会增强顶孢霉素A对人宫颈癌HeLa细胞的细胞毒性。
采用噻唑蓝四氮唑溴盐（MTT）法检测细胞活力。为研究顶孢霉素A的凋亡潜力，在提取基因组DNA后进行DNA片段化分析，并使用荧光底物通过荧光分析法进行半胱天冬酶-3活性分析。在用碘化丙啶（PI）染色后，通过流式细胞术检测PMA与顶孢霉素A联合诱导的细胞周期分布。使用特异性抗体通过蛋白质免疫印迹分析测定靶蛋白的表达水平，并通过逆转录聚合酶链反应（RT-PCR）评估α-微管蛋白mRNA水平。为检测PMA与顶孢霉素A联合对微管结构的影响，分别使用抗α-微管蛋白抗体和PI通过免疫荧光染色观察α-微管蛋白和细胞核。
我们发现顶孢霉素A在HeLa细胞中诱导了半胱天冬酶依赖性凋亡。PMA协同增强了顶孢霉素A诱导的细胞毒性和凋亡亚G1期细胞群。PKC抑制剂Ro31 - 8220和Go6983完全阻断了这些效应，而Z-VAD-fmk对半胱天冬酶的抑制并未阻止细胞毒性。使用针对PKCα而非PKCβ和PKCγ的小干扰RNA（siRNA）进行RNA干扰，抑制了PMA与顶孢霉素A联合诱导的细胞毒性。PMA通过以PKC依赖性方式进一步降低α-和β-微管蛋白水平，增强了顶孢霉素A诱导的微管网络破坏。
这些结果表明，PMA与顶孢霉素A之间的协同作用涉及PKCα激活和微管破坏，这可能为开发新的癌症治疗方法提供思路。
