Association of anti-apoptotic Mcl-1L isoform expression with radioresistance of oral squamous carcinoma cells

Abstract

Background: 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.

Methods: 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).

Results: 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.

Conclusion: 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.

Figure 1

Radiosensitivity assessment and expression of Mcl-1 isoforms in oral cell lines. (a) Clonogenic cell survival assay of FBM, AW8507 & AW13516 oral cell lines. Data given as percentage survival of untreated cell cultures and represent the means (±SD) of three independent colony formation experiments. (b) Expression of Mcl-1 isoforms in oral cell lines. RT-PCR and western blot analysis of Mcl-1 isoforms in the three oral cell lines (FBM, AW13516 & AW8507). A representative blot was shown for three independent experiments. Histogram indicates quantitative expression of Mcl-1 isoforms (Mcl-1L & Mcl-1S) at both mRNA & protein level in oral cell lines.

Figure 2

Time course profile of Mcl-1 splice variants & apoptosis related proteins post-IR. (a) Expression of Mcl-1L, Mcl-1S transcripts and proteins at different time points post-IR in FBM & AW8507. Post-IR cells were harvested at different time points, used for RT-PCR and Western blotting. (b) Western blot illustrates expression of Bax, Bak, Bcl-xl & Bcl-2 proteins at different time points post-IR in oral cell lines, using β-actin as loading control. A representative blot for three independent experiments is shown.

Figure 3

Ratios of anti to pro-apoptotic proteins; Mcl-1L/Mcl-1S (a-b), Mcl-1L/Bax (c-d) and Bcl-xl/Bax (e-f) in AW8507 & FBM cell lines. The relative ratios of expression of proteins were obtained by densitometry of blots using ImageJ software (NIH, USA).

Figure 4

Apoptosis induction and localization of Mcl-1 post-IR. (a) Percentage of apoptosis induction at different time points post-IR by Annexin-V & PI staining analyzed by FACS. The flow cytometry data shown is representative of three independent experiments (*P < 0.05 & **P < 0.01) (b) Immunofluorescence staining of Mcl-1 protein counterstained with DAPI (blue) shows peri-nuclear accumulation (inset) and additional nuclear localization 4 hrs post-IR in AW8507 cells.

Figure 5

Western blot analysis of Mcl-1L knockdown. (a) Mcl-1L downregulation using different concentrations of siRNA and unaltered expression of Mcl-1S in AW8507 cells. The effect of Mcl-1L siRNA was analyzed upto 96 hrs post transfection. (b) Expression of Mcl-1L, Bak, Bax & Bcl-xl proteins 24 hrs after transfection of AW8507 cells: [Experimental control without siRNA (EC); universal control siRNA (UC); Mcl-1L siRNA (siRNA); irradiation (IR)]. A representative blot of three independently performed experiments is shown.

Figure 6

Microscopic analyses of cell proliferation and apoptosis after different treatment combinations. (a & b) After different treatment combinations as described, cell growth of AW8507 & AW13516 cells were determined by counting cell numbers using Trypan blue dye exclusion assay. Cell numbers are given in percent of untreated controls. Data are means ± SD of three independent experiments. (c & d) Apoptosis was determined by DAPI staining, cells either untreated (Control) or treated with UC siRNA, Mcl-1L siRNA alone or in combination with IR. Apoptotic cells were counted in different fields and shown as percent of apoptosis relative to control. Data are means ± SD of three independent experiments. *indicates P < 0.05, ** P < 0.01.

Figure 7

Clonogenic survival analysis after Mcl-1L knockdown and/or irradiation. (a & b) AW8507 & AW13516 cells were either untreated, or treated with siRNA. After 24 hrs, cell culture dishes were treated with increasing doses of ionizing irradiation (0, 2, 4, 6 8 and 10 Gy) as indicated. Survival was assessed by performing clonogenic assay as described in Methods. Data are given as percentage survival of untreated cells and represent the means (±SD) of three independent colony formation experiments.

Figure 8

Expression of Mcl-1L in radio resistant sublines. (a & b) AW8507 & AW13516 cells were treated with fractionated irradiation and the radio resistant sublines were assayed for expression of Mcl-1L. β-actin blot from the same lysates serve as loading control.