Irradiance plays a significant role in photobiomodulation of B16F10 melanoma cells by increasing reactive oxygen species and inhibiting mitochondrial function

Abstract

Melanoma is a type of aggressive cancer. Recent studies have indicated that blue light has an inhibition effect on melanoma cells, but the effect of photobiomodulation (PBM) parameters on the treatment of melanoma remains unknown. Thus, this study was aimed to investigate B16F10 melanoma cells responses to PBM with varying irradiance and doses, and further explored the molecular mechanism of PBM. Our results suggested that the responses of B16F10 melanoma cells to PBM with varying irradiance and dose were different and the inhibition of blue light on cells under high irradiance was better than low irradiance at a constant total dose (0.04, 0.07, 0.15, 0.22, 0.30, 0.37, 0.45, 0.56 or 1.12 J/cm²), presumably due to that high irradiance can produce more ROS, thus disrupting mitochondrial function.

Fig. 1.

Light humidified incubator and spectrum of the respective LED light source. (A) Light humidified incubator. (B) The spectra of blue light (418 nm,457 nm) and red light (630 nm) used in this study. The LEDs light sources are on top of the incubator and can be replaced.

Fig. 2.

B16F10 melanoma cells were treated with light at 418 nm and 457 nm of blue, and 630 nm of red. The 630 nm had no effect on cells, 418 nm and 457 nm inhibited cells. At 900 s, 457 nm inhibited cells were better than 418 nm(p < 0.05).

Fig. 3.

Cell migration results after irradiation for 450 s at 2.48 mW/cm² (418 nm, 457 nm, and 630 nm). (A) Photograph of cell migration, the area between the two yellow lines was the wound area (B) Statistical analysis of migration ratio,*** p < 0.001

Fig. 4.

In vitro cell culture temperature

Fig. 5.

Treatment of melanoma cells with various irradiance (0.31-19.84 mW/cm²) for 450 seconds, the inhibition ratio of the control group was 0.

Fig. 6.

The inhibition ratio of cells treated with three different irradiance (0.31,0.62,0.93 mW/cm²) at the same doses (0, 0.04, 0.07, 0.15, 0.22, 0.30, 0.37, 0.45, 0.56 or 1.12 J/cm²), the inhibition ratio of the control group was 0.

Fig. 7.

The inhibition ratio of cells treated with three different irradiance (0.31,0.62,0.93 mW/cm²) at the same doses (0, 0.04, 0.07, 0.15, 0.22, 0.30, 0.37, 0.45, 0.56 or 1.12 J/cm²), the inhibition ratio of the control group was 0. * p < 0.05, ** p < 0.01, *** p < 0.001

Fig. 8.

Effect of 457 nm irradiation on the production of ROS in B16F10 melanoma cells. (A) Fluorescence of cellular reactive oxygen species (B) Statistical analysis of the average fluorescence intensity of reactive oxygen species. * p < 0.05, *** p < 0.001

Fig. 9.

Effect of 457 nm irradiation on MMP in B16F10 melanoma cells. The higher the irradiance, the more obvious the loss of MMP (mt.ΔΨ) (A) Fluorescence of MMP (B) Statistical analysis of the fluorescence rate of MMP * p < 0.05, ** p < 0.01