Interferon-α enhances etoposide-induced apoptosis and cell cycle arrest

Abstract

Objective To study effect of interferon-α (IFN-α) on etoposide-induced cell cycle arrest and apoptosis in human osteosarcoma cells with its mechanisms. Methods Osteosacoma U2OS cells were treated with IFN-α and etoposide, alone or in combination, for 72 h. Growth inhibition was determined with trypan blue exclude test. Cell cycle arrest was evaluated with FACS. Apoptosis was detected through Hoechst33258 staining. The expression of p53 was determined with reverse transcriptase-polymerase chain reaction (RT-PCR) in mRNA level. small interfering RNA (siRNA) interference was used to silence p53. Western blotting was used to evaluate the expression of p53 and poly adenosine diphosphate-ribose polymerase (PARP). Results After treatment for 72 h, the growth inhibition was (2.29±1.03)%, (23.85±3.09)% and (50.51±3.77)% in IFN-α, Etoposide and combination group. IFN-α enhanced etoposide-induced growth inhibiton. There were statistic differences (P=0.008). There were apoptotic morphological changes in etoposide and combination groups. Cells in S phase were (19.68±3.62)% and (11.33±2.94)% in Etoposide and combination group, while G2/M cells were (65.02±2.55)% and (76.09±2.87)%. There were statistic differences (P=0.037). IFN-α did not further increase the expression of p53 in mRNA level. The inhibition rate was (55.72±3.88)%, (51.63±4.36)% and (36.40±3.95)% in blank, control-siRNA and p53-siRNA group after p53 silence. There were statistic differences (P=0.023). p53 silence largely decreased PARP cleavage in response to the combination. After pan cysteinyl aspartate-specific protease (Caspase) inhibitor Z-VAD-FMK pre-treatment, the growth inhibition rate was (3.52±1.98)%, (27.11±4.06)%, (56.44±4.78)%, (21.37±3.55)% and (23.81±4.22)% in Z-VAD-FMK, Etoposide, IFN-α+ Etoposide, etoposide+ Z-VAD-FMK and IFN-α+ Etoposide+ Z-VAD-FMK group. Z-VAD-FMK decreased growth inhibition caused by combination. There were statistic defferences (P=0.015). PARP cleavage caused by combination was also inhibited by Z-VAD-FMK. Conclusion IFN-α enhances etoposide-mediated growth inhibition, cell cycle arrest and apoptosis in human osteosarcoma U2OS cells. The growth inhibiton and apoptosis are p53-dependent and Caspase-dependent. Key words: Interferon-α; Etoposide; Osteosarcoma; Cell cycle arrest; Apoptosis