Blue light‐emitting diodes induce autophagy in colon cancer cells by Opsin 3

Abstract Background Light emitting‐diodes (LED) have various effects on living organisms and recent studies have shown the efficacy of visible light irradiation from LED for anticancer therapies. However, the mechanism of LED's effects on cancer cells remains unclear. The aim of the present study was to investigate the effects of LED on colon cancer cell lines and the role of photoreceptor Opsin 3 (Opn3) on LED irradiation in vitro. Methods Human colon cancer cells (HT‐29 or HCT‐116) were seeded onto laboratory dishes and irradiated with 465‐nm LED at 30 mW/cm2 for 30 minutes. Cell Counting Kit‐8 was used to measure cell viability, and apoptosis and caspase 3/8 expression were evaluated by AnnexinV/PI and reverse transcription‐polymerase chain reaction (RT‐PCR), respectively. Autophagy and expression of LC‐3 and beclin‐1 were also evaluated by autophagy assays, RT‐PCR and Western blotting. We further tested Opn3 knockdown by Opn3 siRNA and the Gi/o G‐protein inhibitor NF023 in these assays. Results Viability of HT‐29 and HCT‐116 cells was lower in 465‐nm LED‐irradiated cultures than in control cultures. LC‐3 and beclin‐1 expressions were significantly higher in LED‐irradiated cultures, and autophagosomes were detected in irradiated cells. The reductive effect of cancer cell viability following blue LED irradiation was reversed by Opn3 knockdown or NF023 treatment. Furthermore, increased LC‐3 and beclin‐1 expression that resulted from blue LED irradiation was suppressed by Opn3 knockdown or NF023 treatment. Conclusion Blue LED irradiation suppressed the growth of colon cancer cells and Opn3 may play an important role as a photoreceptor.


| INTRODUCTION
Light-emitting diodes (LED) are the preferred light source for phototherapy and have been developed to replace traditional light bulbs because of their lower cost and high variability of wavelengths. 1 LED have been used to treat dermatitis 2 and muscle analgesia, 3 as well as to remove bacteria in vitro. 4 Recent studies have reported that blue light irradiation inhibits cell growth in various cancers in vitro and in vivo. 1,[5][6][7] Phototoxic and antiproliferative mechanisms of blue light LED include inducing apoptosis, 6 autophagy 1 and cell cycle arrest. 7 We also reported that blue LED irradiation at 465 nm inhibited proliferation of HT-29 and HCT-116 cells by extrinsic apoptosis and mitogen-activated protein kinase (MAPK) pathways. 8 However, the mechanism of light reception in these cells, especially "non-visual" tumor cells, is unclear.
In animals, opsin-based photopigments act as photoreceptors for vision and other non-visual functions, such as circadian photoentrainment and pupil response. 9 Most animal opsin-based pigments are typical G protein-coupled receptors (GPCR). 10 Based on their amino acid sequences, opsins are currently classified into seven distinct groups, and more than 1000 opsin subtypes have been found in vertebrates. 11 Within those subtypes, Opsin 3 (Opn3), originally called encephalopsin, panopsin or teleost multiple tissue opsin, was first identified in the deep brain and internal organs of humans and mice. 10 Opn3 exhibits an absorption maxima at 460-470 nm and activation efficiencies of the G i and G o subtypes of G protein, which inhibits adenylate cyclase and decreases cyclic adenosine monophosphate (cAMP). 12,13 Interestingly, Opn3 and its homologs are expressed in various non-visual tissues, such as the brain, liver, kidney and heart; furthermore, they work as photoreceptors and regulate GPCR signaling. 12 A previous report showed that opsins relate to phototherapy as photoreceptors in malignant melanocytes; 14 however, the role of Opn3 in colon cancer is unclear.
The aim of the present study was to investigate the effects of blue LED irradiation on colon cancer cells and to further dissect whether Opn3 functions as a photoreceptor in this process.

| Light-emitting diodes irradiation
A 465-nm blue light LED (NCSB119) was used for irradiation experiments (NICHIA Corporation, Tokushima, Japan). A photo-radiometer (MCPD-370A; Otsuka Electronics, Tokyo, Japan) was used to measure light intensity. An LED irradiation device (Department of Electrical and Electronic Engineering, Faculty of Engineering, The University of Tokushima, Tokushima, Japan) was used as a platform for the light source. We used the continuous mode and a distance of 15 cm from the light source to the cells for each irradiation; irradiance was set at 30 mW/cm 2 (1.0 A). For all experiments, 5 9 10 4 cells/well were seeded with medium onto 35-mm plates (BD Biosciences, Franklin Lakes, NJ, USA). Cells in the LED treatment group were exposed to 465-nm wavelength light for 30 minutes in a dark room. For Opn3 knockdown studies, Opn3 siRNAtransfected cells were exposed to 465 nm wavelength light for 30 minutes 24 hours after transfection. For G i/o G-protein inhibitor studies, NF023 was used at 50 lmol L À1 , 24 hours after seeding, and NF023-administered cells were exposed to 465 nm wavelength light for 30 minutes after 24 hours NF023 treatment. Control cells were treated in the same way except for light exposure. No treatments increased culture medium temperature.

| Cell proliferation assay
Cell Counting Kit-8 (CCK8; Dojindo, Kumamoto, Japan) was used to measure cell proliferation. Briefly, 48 hours after LED irradiation, 200 lL CCK8 was added to each plate. After 2 hours incubation, optical density was biochromatically measured with an enzyme calibrator at 450 and 630 nm.

| Opsin 3 expression analysis by immunohistochemistry
This study was carried out in accordance with the Helsinki Declaration of the World Medical Association. Retrospectively, tumor samples of colon cancer were collected from formalin-fixed paraffin-embedded specimens that were resected at surgical operation in our institute.

| Statistical analysis
All statistical analyses were done using Stat View version 5.0 (SAS Institute, Cary, NC, USA). Mann-Whitney U-test and the Wilcoxon signed-rank test were used for statistical comparisons. Differences were considered statistically significant at P < .05.

| Effects of blue LED irradiation on cell proliferation
Light-emitting diodes emitting 465 nm light were used to assess the effects of blue LED irradiation on HT-29 and HCT-116 cell proliferation. Cells were irradiated once for 30 minutes, and then CCK-8 assays were carried out 48 hours after irradiation to measure cell viability. Viability of HT-29 and HCT-116 cells exposed to blue LED was significantly reduced (Figure 1).  To examine protein expressions of these autophagy-related proteins, Western blotting was carried out. In HT-29 and HCT-116 cells, expressions of LC-3 and beclin-1 were increased by blue LED irradiation, compared to control cells. In contrast, these upregulations of LC-3 and beclin-1 were suppressed in Opn3 knockdown cells and in cells treated with NF023 ( Figure 5E).

| Opn3 protein expression in human colon cancer tissue
To investigate protein expression of Opn3 in human colon cancer, immunohistochemistry staining was carried out in tissue resected by surgical operation in our institute. In our case, Opn3 expression was observed in human colon cancer cells and normal colon tissue was weakly positive for Opn3 ( Figure 6).

| DISCUSSION
This study showed that 30 minutes irradiation with 465-nm blue LED inhibited the growth of human colon cancer cell lines, and blue LED-induced autophagy was shown to be the mechanism of cell growth inhibition. Additionally, the effects of LED were inhibited by Blue LED have been reported to have phototoxic and antiproliferative effects through the generation of intracellular reactive oxygen species (ROS); 15,16 blue LED irradiation has also been reported to induce apoptosis by a mitochondrially mediated signaling pathway that reduces early-stage melanoma growth. 6 Recently, we reported that 10 min/d 9 5 d of blue LED irradiation induced apoptosis in colon cancer cells. 8 In this study, the irradiation method was changed to 30 minutes once, and cell growth was inhibited to the same degree; however, there was not a significant induction of apoptosis.
Interestingly, we found that autophagy markers were upregulated and autophagosomes were detected in irradiated cells. This exciting result suggested that the inhibitory effect on cell growth in cancer cells differed according to the blue LED irradiation protocol.
Autophagy is one of the catabolic cellular responses that sustains cellular metabolism against starvation or stress, whereby cellular proteins and organelles are engulfed, digested and recycled. 17,18 Significant evidence supports a role for autophagy in sustaining cell survival, but, paradoxically, progressive cellular consumption by unrestrained autophagy results in cell death. 19,20 In cancer cells,  6 In regard to autophagy, longer irradiation time with blue LED increased LC-3 expression and induced the formation of autophagosomes. 1 Our previous report showed induction of apoptosis by short daily blue LED irradiation times; however, this study showed that autophagy was induced after a single irradiation of longer duration. The relationship between apoptosis and autophagy is rather complicated, 8 as autophagy can either delay or promote apoptosis. 23,24 This study showed that blue LED irradiation has time-dependent effects on colon cancer cells.
Several reports have shown the cytotoxic effect of blue LED, but the cellular mechanism of light reception remained unclear. A wide variety of opsins exist in vertebrates as photoreceptors, and each has an optimal wavelength. The first report of the relationship between opsins and tumor suppression was reported by Yang et al, 25 who showed that opsin gene transfers with blue LED irradiation inhibited the growth of malignant glioma. In the opsin family, Opn3 is known as a blue light receptor and is widely expressed in non-visual cells. In this study, human colon cancer cell lines also expressed Opn3 and, from the fact that blue LED's effect was inhibited by Opn3 knockdown or inhibiting the G i/o G-protein a-subunit, Opn3 may play an important role as a G protein-coupled photoreceptor by cAMP signaling in inducing autophagy.
Based on these results, we conclude that blue LED irradiation induces autophagy and suppresses the growth of colon cancer cells by the Opn3 photoreceptor pathway, which regulates the G i/o G-protein a-subunit. Although further investigation is required to fully elucidate the molecular mechanism of blue LED irradiation, including phosphorylation, G-protein function, cAMP signaling and their relationship with apoptosis, this study showed a novel mechanism of blue LED irradiation by Opn3 and we consider that Opn3 could be an important target of colon cancer in clinical settings. In the future, there is the possibility of the development of phototherapy for colon cancer by irradiation from the tip of an endoscope for example.