Zhu Kunshou, Fang Weimin, Chen Yuanmei, Lin Shaofeng, Chen Xiaohui
Department of Thoracic Surgery, Fujian Provincial Cancer Hospital, Fuzhou, Fujian 350014, P.R. China.
Oncol Rep. 2014 Sep;32(3):1234-42. doi: 10.3892/or.2014.3324. Epub 2014 Jul 11.
Non-small cell lung cancer (NSCLC) is relatively insensitive to chemotherapy. NP [vinorelbine (NVB) + cisplatin] is the standard chemotherapy regimen in clinical treatment; however, its side-effects are intolerable for most patients. In some reports, the TNF-related apoptosis-inducing ligand (TRAIL) can enhance the sensitivity of chemotherapy drugs by inducing apoptosis without toxicity to normal cells. In the present study, we evaluated the antitumor effects of the two drugs (TRAIL and NVB alone or in combination) by inducing apoptosis in vitro and in vivo. Using the human NSCLC cell line (A549) and a BALB/c nude mice model, we observed the cell viability (MTT assay), cell apoptosis [Hoechst staining, Annexin V/propidium iodide (PI) staining assay, immunohistochemical staining, RT-PCR and western blotting] and cell proliferation (soft agar colony formation and cell cycle assay). The results showed that TRAIL and NVB alone inhibited tumor growth both in vivo and in vitro. However, the combination of the two drugs produced a more potent antitumor effect (P<0.05) and caused more severe apoptosis in tumor tissue (P<0.05). The key molecular protein level of the mitochondrial apoptotic pathway (Bax and caspase-3) was further upregulated by the combination of the two drugs (P<0.01) and either drug alone (P<0.05). The mRNA level of Bcl-2 and Bax in the combination group was downregulated and upregulated respectively, when compared with the control group (P<0.01). The combination group of A549 cells also showed lower viability compared with the one drug alone group (P<0.05). Moreover, the Hoechst staining assay and Annexin V/PI staining assay showed that the combination of the two drugs induced a more potent apoptosis than either drug alone (P<0.05, early apoptosis P<0.01, respectively). In addition, the cell colony numbers were deduced after treatment with TRAIL or NVB alone (P<0.05) and the two drug combination (P<0.01). Cell cycle analysis showed that TRAIL and NVB alone markedly increased the percentage of cells in G1 phase (P<0.05) and the combination of the two drugs decreased the percentage of cells in G2 and S phase (P<0.05). Thus, the combination of TRAIL and NVB can inhibit lung cancer cell growth by affecting the level of key signaling protein expression of the apoptosis pathway.
非小细胞肺癌（NSCLC）对化疗相对不敏感。NP方案[长春瑞滨（NVB）+顺铂]是临床治疗中的标准化疗方案；然而，其副作用对大多数患者来说难以耐受。在一些报道中，肿瘤坏死因子相关凋亡诱导配体（TRAIL）可通过诱导凋亡增强化疗药物的敏感性，且对正常细胞无毒性。在本研究中，我们通过体内外诱导凋亡评估了两种药物（单独使用TRAIL和NVB或两者联合）的抗肿瘤作用。使用人NSCLC细胞系（A549）和BALB/c裸鼠模型，我们观察了细胞活力（MTT法）、细胞凋亡[Hoechst染色、膜联蛋白V/碘化丙啶（PI）染色法、免疫组织化学染色、RT-PCR和蛋白质印迹法]以及细胞增殖（软琼脂集落形成和细胞周期检测）。结果显示，单独使用TRAIL和NVB在体内外均能抑制肿瘤生长。然而，两种药物联合产生了更强的抗肿瘤作用（P<0.05），并导致肿瘤组织中更严重的凋亡（P<0.05）。两种药物联合（P<0.01）及单独使用任一药物（P<0.05）均进一步上调了线粒体凋亡途径的关键分子蛋白水平（Bax和caspase-3）。与对照组相比，联合组中Bcl-2和Bax的mRNA水平分别下调和上调（P<0.01）。A549细胞联合组的活力也低于单药组（P<0.05）。此外，Hoechst染色法和膜联蛋白V/PI染色法显示，两种药物联合诱导的凋亡比单独使用任一药物更强（P<0.05，早期凋亡分别为P<0.01）。另外，单独使用TRAIL或NVB（P<0.05）以及两种药物联合（P<0.01）处理后细胞集落数均减少。细胞周期分析显示，单独使用TRAIL和NVB显著增加了G1期细胞百分比（P<0.05），两种药物联合降低了G2期和S期细胞百分比（P<0.05）。因此，TRAIL和NVB联合可通过影响凋亡途径关键信号蛋白表达水平来抑制肺癌细胞生长。
