Combined Cisplatin Treatment and Photobiomodulation at High Fluence Induces Cytochrome c Release and Cytomorphologic Alterations in HEp-2 Cells

Abstract

BACKGROUND: Photochemotherapy is thought to be a novel therapeutic modality for cancer. The photobiomodulation (PBM), applied through high fluence low-level laser irradiation (HF-LLLI), can be combined with the chemotherapeutic drug cisplatin to gain the benefit of potentiating its cytotoxic effect at possibly lower doses. AIM: The study aimed at investigation of the apoptotic effect of PBM, through LLLI (at HF), alone and in combination with cisplatin on cultured laryngeal cancer (HEp-2) cells. MATERIALS AND METHODS: In the current experimental in vitro research, cultured laryngeal cancer cell line (HEp-2) was treated with the half maximal inhibitory concentration of cisplatin, with and without LLLI. The study design consisted of four groups: Control (untreated), cisplatin-alone-treated, PBM-alone-treated, and combination cisplatin + PBM treated groups. Cells were irradiated once with diode laser (wavelength 808 nm, energy output 350 mW, 3 min, fluence 190.91 J/cm2, and continuous wave mode). Cytotoxicity was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay and the potential apoptotic effect was evaluated by cytochrome c (CYC) release through enzyme-linked immunosorbent assay (ELISA), in conjunction with visualization of cytomorphologic alterations by light microscopic examination, followed by digital morphometric analysis of nuclear changes through estimation of nuclear area factor (NAF). Analysis of variance and post hoc multiple-comparison tests were used for statistical analysis of the data of cytotoxicity assay, ELISA, and nuclear morphometric analysis. RESULTS: PBM alone had a neutral effect on viability of HEp-2 cells, but it induced CYC release and lowered NAF mean value, significantly. When PBM was combined with cisplatin, more conspicuous deterioration in bioavailability of HEp-2 cells was observed, a higher amount of CYC was liberated and NAF value dropped in HEp-2 cells, compared to those which received separate treatments with cisplatin alone or PBM alone. CONCLUSION: Based on the current findings, low-level laser photochemotherapy might be a promising adjunctive anticancer treatment for laryngeal cancer, as PBM at HF was able to augment the apoptotic effect of cisplatin on HEp-2 cancer cells.