Red Light from Light-Emitting Diodes as a Melanoma Therapy

Abstract

Overall, annual cancer rates have decreased due to improved treatment and prevention, but the incidence of melanoma is rising. Outcomes for metastatic melanoma are poor, with five year survival rates as low as 30%. As a result, melanoma can be very expensive to treat, with costs rising to 200,000US dollars per year per person undergoing metastatic melanoma therapy. We aimed to determine the safety and efficacy of red light (RL) phototherapy in preclinical models of melanoma. RL represents a potentially promising therapeutic approach, as RL is inexpensive, noninvasive, easily combined with existing melanoma pharmacologic treatments, and associated with low morbidity and no known mortality.

In vitro, melanoma cells (A375, B16F10, MNT-1) were irradiated with RL at fluences up to 1280 J/cm2 , and cell count, cell cycle progression, and apoptosis were measured. 640-1280 J/cm2 significantly decreased cell proliferation in a dose-dependent manner. At 1280 J/cm2, decreases in cell count were associated with increased cell death via apoptosis. In A375cells and MNT-1 cells, RL increased the percentage of cells in the G1/0-phaseand decreased the percentage of cells in S-phase.

As RL decreased proliferation and increased cell apoptosis, protein expression and phosphorylation of the p53 and p21 were confirmed inA375 and B16F10 cells using western blot. In A375 cells, 640 and 1280 J/cm2 RL increased p53 phosphorylation by 1.8 and 2.7-fold, respectively. p21 expression was increased by 1.5 and 2.4-fold following 640 and 1280 J/cm2 RL. However, siRNA knockdown p21 did not restore proliferation following 640 J/cm2 RL phototherapy. In B16F10 cells, 640 and 1280 J/cm2 RL increased p53 phosphorylation by 1.5 and2.7-fold. p21 expression was increased by 1.6 following 640 J/cm2 RL, but 1280J/cm2 decreased p21 expression to 0.52-fold.

Cytochrome c oxidase (COX), a heme derivative, absorbs RL phototherapy and is believed to generate reactive oxygen species by activating oxidative phosphorylation. Cancers, including melanoma, have dysregulated ROS homeostasis and may be particularly sensitive to oxidative stress. Therefore, we tested whether 640 and 1280 J/cm2 RL increased intracellular ROS production in A375, MNT-1, and B16F10 cell lines. At 0 hours post-treatment, RL induced a dose-dependent increase in ROS. Inhibition of ROS with the antioxidant n-acetylcysteine restored proliferation following 640 J/cm2 RL in A375 andB16F10 cells.

Before translating findings to human clinical studies, multiple RL treatment regimens were tested using mouse models to assess safety, measure efficacy, and establish protocols for future clinical trials. C57BL/6mice were intradermally injected with B16F10 cells and irradiated with1280-2560 J/cm2 RL for up to 15 days. Phototherapy safety, tumor growth, and immunocyte infiltration were assessed in RL-treated and control mice. After euthanization, excised tumors were measured in three dimensions to determine the exact tumor size. We investigated how RL alters melanoma growth and immune response (i.e., tumor-infiltrating lymphocytes, dendritic cells, and macrophages) in skin and lymph nodes using hematoxylin and eosin and immunohistochemistry. Daily treatments of 2560 J/cm2 RL significantly decreased tumor volumes by 38%compared to control and increased the expression of CD103+ and CD4+ cells. These immunocyte markers are associated with favorable immune niches in cancer.

therapeutics. Therefore, RL has the potential to improvepatient care as an adjunct treatment.

Further research is needed to clarify the mechanism of RL inmelanoma and determine the optimal treatment for adjunctive regimens using RL.Clinical studies have demonstrated that patients can safely use LED devices athome, and RL may empower patients to participate in their cancer treatment.