Teni Lab, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai, 410210, India.
Radiat Oncol. 2012 Aug 8;7:135. doi: 10.1186/1748-717X-7-135.
Oral cancer is a common cancer and a major health problem in the Indian subcontinent. At our laboratory Mcl-1, an anti-apoptotic member of the Bcl-2 family has been demonstrated to be overexpressed in oral cancers and to predict outcome in oral cancer patients treated with definitive radiotherapy. To study the role of Mcl-1 isoforms in radiation response of oral squamous carcinoma cells (OSCC), we investigated in the present study, the association of Mcl-1 isoform expression with radiosensitivity of OSCC, using siRNA strategy.
The time course expression of Mcl-1 splice variants (Mcl-1L, Mcl-1S & Mcl-1ES) was studied by RT-PCR, western blotting & immunofluorescence, post-irradiation in oral cell lines [immortalized FBM (radiosensitive) and tongue cancer AW8507 & AW13516 (radioresistant)]of relatively differing radiosensitivities. The effect of Mcl-1L knockdown alone or in combination with ionizing radiation (IR) on cell proliferation, apoptosis & clonogenic survival, was investigated in AW8507 & AW13516 cells. Further the expression of Mcl-1L protein was assessed in radioresistant sublines generated by fractionated ionizing radiation (FIR).
Three to six fold higher expression of anti-apoptotic Mcl-1L versus pro-apoptotic Mcl-1S was observed at mRNA & protein levels in all cell lines, post-irradiation. Sustained high levels of Mcl-1L, downregulation of pro-apoptotic Bax & Bak and a significant (P < 0.05) reduction in apoptosis was observed in the more radioresistant AW8507, AW13516 versus FBM cells, post-IR. The ratios of anti to pro-apoptotic proteins were high in AW8507 as compared to FBM. Treatment with Mcl-1L siRNA alone or in combination with IR significantly (P < 0.01) increased apoptosis viz. 17.3% (IR), 25.3% (siRNA) and 46.3% (IR plus siRNA) and upregulated pro-apoptotic Bax levels in AW8507 cells. Combination of siRNA & IR treatment significantly (P < 0.05) reduced cell proliferation and clonogenic survival of radioresistant AW8507 & AW13516 cells, suggesting a synergistic effect of the Mcl-1L siRNA with IR on radiosensitivity. Interestingly, during the development of radioresistant sublines using FIR, high expression of Mcl-1L was observed.
Our studies suggest that Mcl-1L isoform has an important role in the survival and radioresistance of OSCC and may be a promising therapeutic target in oral cancers.
口腔癌是印度次大陆常见的癌症和主要健康问题。在我们的实验室中，已经证明 McI-1（Bcl-2 家族的一种抗凋亡成员）在口腔癌中过度表达，并可预测接受确定性放射治疗的口腔癌患者的预后。为了研究 Mcl-1 异构体在口腔鳞状细胞癌（OSCC）放射反应中的作用，我们在本研究中使用 siRNA 策略研究了 Mcl-1 异构体表达与 OSCC 放射敏感性的关系。
通过 RT-PCR、Western blot 和免疫荧光技术研究了 McI-1 剪接变体（Mcl-1L、Mcl-1S 和 Mcl-1ES）在口腔细胞系[永生化 FBM（放射敏感）和舌癌 AW8507 和 AW13516（放射抵抗）]中辐射后的时间过程表达。研究了单独使用 Mcl-1L 敲低或与电离辐射（IR）联合使用对 AW8507 和 AW13516 细胞增殖、凋亡和集落存活的影响。进一步评估了在分次电离辐射（FIR）生成的放射抗性亚系中 Mcl-1L 蛋白的表达。
在所有细胞系中，辐射后在 mRNA 和蛋白质水平上观察到抗凋亡 Mcl-1L 的表达增加了 3 到 6 倍，而促凋亡 Mcl-1S 的表达增加了 3 到 6 倍。在更具放射抵抗性的 AW8507、AW13516 与 FBM 细胞中，持续高水平的 Mcl-1L、下调促凋亡 Bax 和 Bak 以及显著（P<0.05）减少凋亡，在 AW8507 中观察到凋亡。与 FBM 相比，AW8507 中的抗凋亡蛋白与促凋亡蛋白的比值较高。单独用 Mcl-1L siRNA 处理或与 IR 联合处理，显著（P<0.01）增加了凋亡，分别为 17.3%（IR）、25.3%（siRNA）和 46.3%（IR+siRNA），并上调了 AW8507 细胞中促凋亡 Bax 的水平。siRNA 和 IR 联合治疗显著（P<0.05）降低了放射抗性 AW8507 和 AW13516 细胞的增殖和集落存活，表明 Mcl-1L siRNA 与 IR 对放射敏感性具有协同作用。有趣的是，在使用 FIR 开发放射抗性亚系期间，观察到 Mcl-1L 的高表达。
我们的研究表明，Mcl-1L 异构体在 OSCC 的存活和放射抗性中起重要作用，可能是口腔癌有前途的治疗靶点。
