Effects of Combined Blue light and 5-ALA on cell death in B16F1 melanoma and HaCaT cells

Effects of Combined Blue light and 5-ALA on cell death in B16F1 melanoma and HaCaT cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Effects of Combined Blue light and 5-ALA on cell death in B16F1 melanoma and HaCaT cells Kazuomi Sato, Taiki Sato, Riku Hirotani, Munetsugu Bam This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3894182/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Investigating the effect of blue light and 5-aminolevulinic acid (5-ALA) co-treatment, this study examined its effects on B16F1 melanoma cells and HaCaT keratinocytes. Our focus was on cellular responses including mitochondrial function, DNA integrity, and gene expression. Co-treatment significantly damaged mitochondria, alters their morphology, induced mitochondrial membrane depolarization, increased intracellular reactive oxygen species (ROS), and led to cardiolipin peroxidation in both cell types. This approach also increased DNA fragmentation and apoptosis. Notably, it triggers complex, time-dependent changes in gene expression, particularly upregulating MMP-1 and p21 in HaCaT cells. Our findings reveal that blue light and 5-ALA co-treatment causes substantial cellular stress and damage, suggesting its therapeutic potential against melanoma, while also highlighting the need for caution and precision in its application to avoid harming normal cells. This underscores the necessity for further research to refine therapeutic approaches. Biological sciences/Molecular biology/Dna damage and repair/Dna damage response Biological sciences/Molecular biology/Dna damage and repair/Double strand dna breaks Biological sciences/Molecular biology/Dna damage and repair/Single strand dna breaks Biological sciences/Molecular biology/Cell division/Cell cycle exit Biological sciences/Cell biology/Cell death/Apoptosis Biological sciences/Cell biology/Cell division/Cell growth Biological sciences/Cancer/Skin cancer/Melanoma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Ultraviolet (UV) light, a component of solar radiation, exerts profound effects on various forms of life, particularly animals. Chronic exposure to solar UV light, particularly in humans, has been linked to photoaging, which is characterized by UV-induced oxidative damage and induction of matrix metalloproteinase (MMP) expression [ 1 ]. Such exposure can significantly alter skin properties, with oxidative stress playing a central role in the damage process. In mice, detrimental effects of UV radiation are evident in the skin, where UV exposure can underlie protein oxidation, a hallmark of photoaging [ 1 ]. At the cellular level, UV radiation can cause DNA damage, leading to mutations that result in cancer growth. UV radiation also induces the formation of cyclobutane pyrimidine dimers (CPDs) between neighboring pyrimidine bases [ 2 ]. Blue light, a component of visible light, has been extensively studied for its effects on various biological processes in animals and cells. Increasing use of blue light in various applications gives impetus to studies aimed at understanding its effects on cellular and metabolic processes. In animal husbandry, Xia et al. found that irradiating ducks with blue light (460 nm) decreased their body weight and enhanced their anti-inflammatory and antioxidant capacities. DNA damage and somatic mutations have been observed in mammalian cells irradiated with a nail polish dryer [ 3 ]. Phototoxicity, especially in live-cell fluorescence microscopy, is a concern associated with blue light exposure. Alghamidi et al. showed that even low levels of blue light can affect the motility of PC3 human prostate cancer cells [ 4 ]. In human SH-SY5Y neuroblastoma cells, intriguing interactions were observed between blue light and a 50 Hz magnetic field (MF) [ 5 ]. Combined MF and blue light exposure have been shown to modulate superoxide levels. Additionally, we have reported that blue light induces the collapse of mitochondrial membrane potential and subsequent cell death [ 6 , 7 ]. 5-aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid that serves as a precursor in heme biosynthesis [ 8 , 9 ]. In plants, 5-ALA improves tomato coloration by regulating carotenoid metabolism and promoting fruit maturation [ 10 ]. In contrast, 5-ALA exerts cytotoxic effects in malignant glioblastoma cells by increasing apoptosis, altering the expression of apoptosis-related genes, and enhancing reactive oxygen species (ROS) generation [ 11 ]. These findings highlight the multifaceted impacts of 5-ALA in cells ranging from human cancer cells to plant cells. In photodynamic therapy (PDT), 5-ALA is converted to protoporphyrin IX, which acts as a photosensitizer by producing ROS in response to light exposure [ 12 ]. Halander et al. [ 13 ] demonstrated that the efficacy of PDT was several times higher with blue light than with red light in several cancer cell lines. We have previously demonstrated that co-treatment with blue light and 5-ALA induces severe DNA damage, including double- and single-strand breaks (SSB), in both melanoma and normal keratinocyte cell lines [ 14 ]. Although numerous studies have described ALA-PDT, the effect of 5-ALA on intracellular metabolism remains unknown. This study aimed to elucidate the effects of 5-ALA on the intracellular metabolism of murine B16F1 melanoma and human HaCaT keratinocytes treated with blue light. We aimed to investigate the specific metabolic pathways influenced by 5-ALA and blue light exposure, focusing on cell viability, intracellular ROS, mitochondrial membrane potential and morphology, and expression of apoptosis-related genes. Materials and Methods Cell culture B16F1 melanoma cells (RIKEN BioResource Research Center, Tsukuba, Japan) and HaCaT cells (Cosmo Bio, Tokyo, Japan) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM; Sigma, St Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin, and 100 µg/mL streptomycin. Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO 2 . For blue light treatments, we used blue LED lamps with peak emission at 465 nm. Detailed information on the irradiance of LED lamps is available in our previous study [ 6 ]. Cell viability and proliferation To assess cell proliferation and viability, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assays were performed. For experiments involving varying concentrations of 5-ALA, HaCaT and B16F1 melanoma cells were seeded into 96-well plates at a density of 2.0 × 10 3 cells/well. After 24 h incubation, cells were treated with different concentrations of 1 mM 5-ALA (Sigma-Aldrich, St. Louis, MO, USA) for specified durations. After treatment, cells were washed with phosphate-buffered saline (PBS), and culture medium was replaced. Following a 72-h incubation, 5 mg/mL MTT was added and cells were incubated for 3.5 h at 37°C. Resultant formazan crystals were dissolved in dimethyl sulfoxide and absorbance at 590 nm was measured using a microplate reader. For experiments involving co-treatment with blue light and 5-ALA in the presence of N-acetyl cysteine (NAC), B16F1 and HaCaT cells were seeded in 35 mm dishes at a density of 2 × 10 4 cells/dish. Cells were pre-treated with 5 mM NAC (Fujifilm Wako, Osaka, Japan) and 1 mM 5-ALA for 1 h followed by exposure to blue light for an additional hour. After exposure, cells were incubated for another hour with NAC. Subsequently, cells were cultured for 72 h in NAC- and 5-ALA-free medium. MTT assays were then performed as described above. Intracellular superoxide anion production and mitochondria membrane potential (ΔΨm) Intracellular ROS levels were measured as previously described [ 6 ]. B16F1 melanoma and HaCaT cells were seeded into 35 mm dishes at a density of 1 × 10 5 cells/dish. After incubation for 24 h, cells were pre-treated with 1 mM 5-ALA for 1 h, followed by irradiation with blue light for 30 or 60 min in the presence of 5-ALA. After treatment, cells were incubated in DMEM containing 5 µM hydroethidine (HE, Thermo Fisher Scientific, Waltham, MA, USA) for superoxide anion detection, or 50 nM 3,3’-dihexyloxacarbocyanine iodide (DiOC6; Invitrogen) to assess ΔΨm. After incubation for 20 min at 37°C, cells were collected by trypsinization, washed twice with PBS, and analyzed using flow cytometry (FACSCalibur, BD, Franklin Lakes, NJ, USA). Cardiolipin peroxidation To assess cardiolipin status, we used nonyl acridine orange (NAO; Sigma), a fluorescent dye that binds with high affinity to non-oxidized cardiolipin. B16F1 melanoma and HaCaT cells were seeded onto 35 mm dishes at a density of 1 × 10 5 cells/dish. After 24 h of incubation, the cells were pre-treated with 1 mM 5-ALA for 1 h, then irradiated with blue light for 30 or 60 min in the presence of 5-ALA. After irradiation, cells were incubated in DMEM containing 50 µM NAO for 20 min at 37°C. Cells were collected by trypsinization, washed twice with PBS, and analyzed by flow cytometry. Mitochondrial fluorescence staining To evaluate the impact of blue light and 5-ALA treatment on mitochondrial morphology, we performed MitoTracker Green staining, a widely used technique for visualizing mitochondria in live cells. B16F1 and HaCaT cells were seeded onto glass-bottomed 35-mm dishes at 1 × 10 5 cells/dish. After 24 h of incubation, the cells were treated with vehicle or several concentrations of 5-ALA for 1 h, and subsequently exposed to blue light. Following treatment, the culture medium was replaced with pre-warmed serum-free fresh medium containing 200 nM MitoTracker Green FM (Thermo Fisher Scientific, Waltham, MA, USA), and cells were incubated for 30 min at 37°C. After incubation, cells were washed three times to remove excess probe. For nuclear staining, cells were incubated with 1 µM Hoechst 33342 (Thermo Fisher Scientific) in DMEM for 10 min at 37°C. Finally, cells were visualized using an LSM 700 confocal microscope (Carl Zeiss AG, Oberkochen, Germany). Transmission electron microscopy (TEM) To elucidate ultrastructural changes in mitochondria following treatment, we used the TEM. After blue light and 5-ALA treatment as described above, cells were fixed using 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h at room temperature in the dark. Subsequently, fixed cells were rinsed with phosphate buffer. Secondary fixation was carried out using 1% osmium tetroxide (OsO 4 ) in 0.1 M phosphate buffer (pH 7.2). After fixation, cells were washed and embedded in 1% agar. Agarose-embedded samples were trimmed into 1 mm cubes and dehydrated using an ethanol series. Samples were subsequently substituted with propylene oxide and embedded in an epoxy–resin mixture. These samples were ultrathin sectioned to a thickness of 70 nm and stained with an EM Stainer (Nisshin EM Co., Ltd., Tokyo, Japan) and Reynolds lead citrate. Analyses were conducted using a TEM JEM-2100F (JEOL Co., Ltd., Tokyo, Japan) at an acceleration voltage of 120 kV. Mitochondrial ROS (mtROS) detection We used MitoSOX Red as a probe (Thermo Fisher Scientific) to detect mtROS levels. After blue light and 5-ALA treatment, as described above, cells were incubated with DMEM containing 5 µM MitoSOX Red for 10 min at 37°C. After incubation, cells were washed and stained with Hoechst 33342 following the protocol outlined in the MitoTracker staining section. Cells were visualized using confocal microscopy. Cell cycle analysis and subG1 cell detection To analyze cell cycle progression and detect apoptotic cells in subG1 phase, B16F1 melanoma and HaCaT cells were subjected to identical intracellular ROS treatment. After treatment, both attached and detached cells were harvested, fixed with ice-cold 70% ethanol, and stored at least 24 h at -20 ℃. After fixation, cells were washed, resuspended in PBS containing RNase A (0.1 mg/mL, Sigma), and incubated at 37°C for 30 min to digest RNA. Aqueous propidium iodide (50 µg/mL, Sigma) was added to stain DNA. The DNA content of the cells was determined by flow cytometry. To accurately assess cell-cycle disruption and identify the subG1 population, we employed a method described by Nunez et al. [ 15 ], which effectively discriminates between single cells and doublets or aggregates. In our cell cycle analysis, we reported proportions of cells in G1, S, and G2/M phases, explicitly excluding the subG1 population from these calculations. Evaluation of DNA damage using γ-H2AX immunofluorescence To assess DNA damage, cells were seeded into 35 mm glass-bottom dishes at a density of 1 × 10 5 cells/well and incubated overnight. Cells were treated with or without 5-ALA and exposed to blue light. Following treatment, cells were briefly washed with PBS and fixed with 2% paraformaldehyde (Fujifilm Wako) for 10 min at room temperature. Cells were then rinsed with PBS and permeabilized in 0.2% PBS-Tween for 5 min at room temperature. After three washes with PBS, the cells were blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. For immunofluorescence staining, cells were incubated with primary anti-γH2AX antibodies (dilution 1:200; Merck Millipore, Burlington, MA, USA) overnight at 4°C. Cells were then incubated with a secondary anti-mouse IgG (Alexa Fluor 488) antibody (dilution 1:200; Abcam, Cambridge, UK) for 2 h at room temperature. Finally, cells were mounted in VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories, Inc., Newark, CA, USA) and observed using an EVOS FL imaging system (Thermo Fisher Scientific) or a confocal microscope. Detection of CPD, 6 − 4 PP, and Dewar PP via enzyme-linked immunosorbent assay (ELISA) To evaluate DNA base damage induced by blue light and 5-ALA, we first employed an ELISA-based approach. After treatment, cells were lysed, and genomic DNA was extracted using a QIAamp DNA Blood Mini kit (Qiagen, Venlo, Netherlands) following the manufacturer’s protocol. DNA concentration was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). The extracted DNA was denatured by heating at 100 ºC for 10 min, then rapidly cooled on ice. Subsequently, 50 µL aliquots of DNA at specific concentrations (0.2 µg/mL for CPD, 4.0 µg/mL for 6 − 4 PP, and 10 µg/mL for Dewar PP detection, respectively) were prepared and applied to protamine sulfate-coated 96-well plates. Plates were dried overnight at 37°C in the dark. After drying, wells were washed with PBS containing 0.05% Tween 20 (PBS-T). Blocking was performed using 2% FBS for 30 min at 37°C. After five washes with PBS-T, wells were incubated with antibodies (1:1000 anti-CPDs antibody, 1:1500 anti-6-4 PPs antibody, and 1:5000 anti-Dewar PPs antibody; Cosmo Bio) for 30 min at 37°C. After primary antibody incubation, wells were washed and incubated with biotinylated secondary anti-mouse antibody for 30 min at 37°C, followed by 1:10000 peroxidase-streptavidin for 30 min at 37°C. After washing, wells were treated with PBS-T and once with citrate-phosphate buffer (containing 25 mM citric acid monohydrate and 50 mM Na 2 HPO 4 , pH 5.0), then incubated with 100 µL/well of substrate buffer (4 mM o-phenylenediamine, 0.007% H 2 O 2 in citrate-phosphate buffer) for 30 min at 37°C. The enzymatic reaction was stopped using 50 µL of 2 M sulfuric acid (H 2 SO 4 ). The absorbance of each well was measured at 492 nm using a microplate reader. Immunofluorescent CPD staining Cells were seeded into 35 mm glass-bottom dishes at a density of 1 × 10 5 cells and incubated overnight. Cells were treated with or without 5-ALA and exposed to blue light. After treatment, cells were fixed with 4% paraformaldehyde for 10 min at room temperature, rinsed with PBS, and permeabilized with PBS-Tween (0.2%) for 5 min at room temperature. Cells were subsequently blocked with 1% BSA in PBS for 1 h at room temperature. For CPD detection, cells were treated with 2 M HCl for 30 min to denature cellular DNA. After blocking, cells were incubated with an anti-CPD antibody (1:200; Cosmo Bio). Cells were then incubated with secondary anti-mouse IgG (Alexa Fluor 594) antibodies (1:200; Abcam) for 2 h at room temperature. Finally, cells were mounted in VECTASHIELD Vibrance Antifade Mounting Medium supplemented with DAPI and observed under a confocal microscope. Quantitative RT-PCR Total RNA was extracted from treated cells using ISOGEN 2 (Nippon Gene, Tokyo, Japan), following the manufacturer’s instructions. The extracted RNA was reverse transcribed into cDNA using AMV Reverse Transcriptase (TaKaRa, Shiga, Japan). The primer sequences are listed in Supplementary Table 1. PCR amplification was performed as previously described [ 7 ] using a 7500 Fast Real-Time PCR system (Thermo Fisher Scientific). Relative target gene expression levels were normalized to that of GAPDH and calculated using the 2 −ΔΔCt method. All experiments were performed in triplicate. Statistical analysis Data were analyzed using GraphPad Prism 9 software (GraphPad Software, Inc., La Jolla, CA, USA). All numerical data are presented as means ± standard error of the mean (SEM) of experiments performed at least in triplicate. Results were considered statistically significant at P < 0.05. Results Effect of 5-ALA on cell viability and proliferation We previously reported that co-treatment with blue light and 5-ALA induced significant cell death [ 14 ]. We thus aimed to further understand the effects of 5-ALA on cell proliferation. B16F1 and HaCaT cells were treated with various concentrations of 5-ALA (0.1 to 10 mM) and their proliferation was assessed. As shown in Supplementary Fig. 1A, even at the lowest concentration (0.1 mM), 5-ALA significantly inhibited cell proliferation in B16F1 cells. Similarly, HaCaT cells exhibited significant proliferation arrest in 0.1 mM 5-ALA (Supplementary Fig. 1B). To explore the temporal effect of 5-ALA, we treated cells with the same concentrations of 5-ALA for a shorter duration of 2 h, followed by an additional 72 h incubation in 5-ALA-free medium. Notably, a slight but significant inhibition of B16F1 cell proliferation was observed starting at 0.2 mM (Supplementary Fig. 1C). In contrast, HaCaT cells did not show significant inhibition of cell proliferation at any concentration tested (Supplementary Fig. 1D). In addition to our previous experiments, we investigated the protective role of NAC, an antioxidant, against the cytotoxic effects of blue light and 5-ALA co-treatment. NAC at 5 mM was included in the medium from one h before to one h after blue light exposure. Following this treatment, cells were incubated for 72 h in medium lacking 5-ALA or NAC. As shown in Supplementary Fig. 1E, B16F1 melanoma cells exhibited significantly reduced viability after exposure to blue light alone or in combination with 5-ALA, consistent with our previous findings. NAC did not ameliorate the decreased cell viability induced by blue light exposure. Similar results were observed in HaCaT cells (Supplementary Fig. 1F). Co-treatment with blue light and 5-ALA increased intracellular ROS levels, induced depolarization of mitochondrial membrane potential (ΔΨm), and peroxidation of cardiolipin. To elucidate the impact of blue light exposure and 5-ALA treatment on intracellular ROS levels, we performed flow cytometry using HE, which detects intracellular superoxide anions. In B16F1 melanoma cells, exposure to blue light for 60 min did not significantly increase ROS levels compared to those in the control (Fig. 1 A). Similarly, 5-ALA treatment alone did not significantly change ROS levels. However, when cells were co-treated with blue light and 5-ALA, a significant increase in the number of HE-positive cells was observed after 60 min of exposure (four-fold increase after 60 min of blue light exposure). We also assessed intracellular ROS levels in HaCaT cells, and exposure to blue light alone for 60 min decreased ROS levels (Fig. 1 B). Treatment with 5-ALA alone, similar to results with B16F1 melanoma cells, did not result in significant differences. Given that the data from HaCaT cells indicated that blue light treatment reduced the proportion of HE-positive cells, we conducted a time-course experiment with only blue light exposure. Blue light treatment resulted in a consistent decrease in ROS levels at all examined time points (Supplementary Fig. 2C). Following our assessment of intracellular ROS, we investigated ΔΨm as an indicator of early apoptotic events. Flow cytometric analysis was conducted using the cell-permeable probe DiOC6, which selectively accumulates in mitochondria with an intact membrane potential. In B16F1 melanoma cells, neither blue light exposure nor 5-ALA treatment alone significantly changed ΔΨm (Fig. 1 C). However, depolarization of the ΔΨm was observed in cells co-treated with blue light and 5-ALA. A similar pattern was observed in HaCaT cells, where co-treatment for 60 min resulted in a significant collapse of ΔΨm, indicating the potential for initiating apoptosis (Fig. 1 D). Additionally, we also assessed cardiolipin peroxidation, which is a marker of mitochondrial oxidative stress and apoptosis. NAO preferentially binds to non-oxidized cardiolipin, thus a decrease in NAO fluorescence is indicative of cardiolipin peroxidation [ 16 , 17 ]. As shown in Fig. 1 E, F, co-treatment with blue light and 5-ALA significantly reduced NAO fluorescence intensity in both cell lines. We also conducted these three experiments with cells co-treated for 30 min, which showed results similar to those of the 60 min co-treatment (Supplementary Fig. 2, 3, and 3, respectively). Mitochondrial morphology alterations induced by combined blue light and 5-ALA Given our finding that co-treatment with blue light and 5-ALA induced ΔΨm collapse in B16F1 and HaCaT cells, we hypothesized that these treatments might also affect mitochondrial morphology. To investigate, we visualized mitochondria in live cells subjected to blue light and 5-ALA treatment using MitoTracker Green. As shown in Fig. 2 A, B16F1 cells exposed only to blue light exhibited mitochondria with tubular and branched morphologies, similar to those of the untreated controls. In cells co-treated with blue light and 5-ALA (at 0.2 and 1 mM concentrations), mitochondrial fluorescence appeared diffuse, and clear shapes were not discernible, suggesting potential mitochondrial disruption. In HaCaT cells (Fig. 2 B), the mitochondria maintained their typical morphology under blue light exposure alone. In contrast, cells treated with blue light and 0.2 mM 5-ALA showed numerous fragmented and rounded mitochondria that exhibited tremulous movements (data not shown). Mitochondria tended to cluster around the nucleus. More severe mitochondrial damage was evident in HaCaT cells co-treated with 1 mM 5-ALA, in which the mitochondrial structure was significantly disrupted. We also investigated the effects of co-exposure of B16F1 and HaCaT cells to blue light and 5-ALA using TEM. Cells were pre-treated with 0.2 or 0.02 mM 5-ALA for 1 h, followed by exposure to blue light for 1 h. As shown in Fig. 2 C, exposure to blue light alone resulted in noticeable mitochondrial membrane damage in B16F1 cells. Co-treatment with 0.02 mM 5-ALA resulted in similar levels of mitochondrial damage to those observed with blue light alone. However, co-treatment with high concentrations of 5-ALA (0.2 mM) led to severe mitochondrial damage in B16F1 cells. HaCaT cells also exhibited no significant mitochondrial damage when exposed to blue light alone or in combination with 0.02 mM 5-ALA, but co-treatment of HaCaT with 0.2 mM 5-ALA resulted in evident mitochondrial damage. This experiment revealed not only membrane disruption but also the disappearance of cristae. Furthermore, extreme cellular damage was observed in both cell lines co-treated with 1 mM 5-ALA (data not shown). mtROS levels in response to blue light and 5-ALA To assess the effect of blue light and 5-ALA co-treatment on mtROS levels, we used MitoSOX Red with confocal microscopic analysis. In B16F1 melanoma cells, exposure to blue light alone or co-treatment with 0.02 mM 5-ALA significantly increased mtROS levels (Fig. 3 A, B). However, cells co-treated with higher concentrations of 5-ALA (0.2 mM) displayed reduced mtROS levels compared with those exposed only to blue light. Exposure to blue light alone did not increase mtROS levels in HaCaT cells (Fig. 3 C, D). Co-treatment with 0.2 mM 5-ALA in HaCaT cells slightly increased mtROS levels, in contrast to results observed in B16F1 cells. Cell cycle analyses and SubG1 population cells To assess the extent of DNA fragmentation, which is an indicator of apoptosis, we quantified the subG1 cell population in cells treated with blue light and 5-ALA. As shown in Fig. 4 A, B, co-treatment with blue light and 5-ALA significantly increased the subG1 population in both B16F1 melanoma and HaCaT cells. This increase was particularly pronounced after 60 min of exposure (a 60% increase in B16F1 cells). Further cell cycle analysis revealed that B16F1 cells exhibited altered cell cycle dynamics after treatment. Specifically, there was a significant decrease in the G1 phase coupled with an increase in the G2/M phase after 60 min of blue light exposure, with or without 5-ALA (Fig. 4 C). These results suggest that blue light disrupts cell-cycle progression, potentially leading to cell-cycle arrest in the G2/M phase. HaCaT cells did not show significant alterations in the proportion of cells in different cell cycle phases after 30 min of blue light exposure, regardless of 5-ALA treatment (Supplementary Fig. 5C). However, after 60 min of 5-ALA exposure, cells in G1 were significantly increased, and cells in S and G2/M were decreased (Fig. 4 D). These results indicate that combined treatment with blue light and 5-ALA affects cell cycle progression in response to checkpoint activation or DNA damage. Co-treatment with blue light and 5-ALA increases γH2AX expression To further investigate DNA damage, we assessed γH2AX, a well-established marker of DNA double-strand breaks (DSBs) [ 18 ]. Previously, we performed a two-tailed comet assay to detect DSB and SSB in B16F1 and HaCaT cells, finding that co-treatment with blue light and 5-ALA induced DSB and SSB formation [ 14 ]. We thus attempted to quantify γH2AX expression by immunofluorescence. As shown in Fig. 5 A, B, UVC treatment, performed as a positive control, significantly enhanced γH2AX fluorescence intensity in both cell lines. Notably, co-treatment with blue light and 5-ALA also increased γH2AX expression in both B16F1 and HaCaT cells, suggesting induction of DNA damage. In B16F1 cells exposed to blue light, γH2AX expression was enhanced, whereas HaCaT cells exposed to blue light exhibited only a slight decrease in γH2AX fluorescence intensity compared to the control group. We employed confocal microscopy to observe γH2AX foci formation in treated cells. As shown in Fig. 5 C, a significant increase in γH2AX foci was observed in all treatment groups, excluding the control, in B16F1 melanoma cells, indicating DNA damage response following exposure to blue light, 5-ALA co-treatment, and UV irradiation. In contrast, HaCaT cells exhibited a different response (Fig. 5 D). While UV and 5-ALA co-treatment with blue light caused a similar response as in B16F1 cells, exposure to blue light alone markedly reduced numbers of cells with γH2AX foci. Co-treatment with blue light and 5-ALA did not enhance formation of CPDs, 6-4PPs, or Dewar PPs In our previous study, we established that visible light, including blue and red light, did not enhance CPD or 6-4PP formation [ 7 ]. We here investigated the potential formation of CPD, 6-4PP, and Dewar PP in cells co-treated with blue light and 5-ALA. As shown in Fig. 6 A–D, co-treatment with blue light and 5-ALA did not enhance CPD or 6-4PP formation in either cell line. In contrast, UVC treatment significantly increased CPD and 6-4PP production. Notably, the presence of 5-ALA did not increase UVC-induced formation of these photoproducts. Furthermore, Dewar PP levels did not change in any treatment group (Fig. 6 E, F). We performed confocal laser scanning microscopy to visualize CPD formation in B16F1 and HaCaT cells (Supplementary Fig. 6). These microscopy results were consistent with our ELISA data, demonstrating that blue light and 5-ALA did not significantly induce CPD formation. Influence of blue light and 5-ALA on gene expression We previously reported blue-light induced alterations in the expression of apoptotic and melanogenic genes [ 7 ]. In this study, we investigated the effects of blue light and 5-ALA co-treatment on transcription of a broad spectrum of genes in B16F1 and HaCaT cells. B16F1 melanoma cells were exposed to blue light for 30 min in the presence of 0.2 mM 5-ALA (Fig. 7 A). Time-point analyses of mRNA levels were conducted at 2, 4, 8, 16, and 24 h post-treatment. We performed similar analyses after 1 h of blue light exposure, but cells were severely damaged under these conditions, indicating that the data were unreliable (data not shown). An increase in p21 mRNA, a well-known cyclin-dependent kinase inhibitor involved in cell cycle regulation [ 23 ], peaking at 4 h, was observed after blue light exposure. Expression of survivin , a gene associated with cell survival and inhibition of apoptosis, was also enhanced following irradiation. Transcription levels of Bax and Bcl-2 , key genes in the apoptotic pathway, were also assessed. While Bax mRNA levels exhibited variability and lacked consistency post-treatment, Bcl-2 mRNA levels were consistently suppressed immediately after treatment for up to 24 h. The Bcl-2 gene, which is typically involved in inhibiting apoptosis, may indicate a shift towards a pro-apoptotic state [ 21 ]. Caspase-3 mRNA levels also increased 8 h post-irradiation. HaCaT cells were exposed to blue light for 1 h in the presence of 0.2 mM 5-ALA (Fig. 7 B). After treatment, we conducted a time-course analysis of mRNA levels at 2, 4, 8, 16, and 24 h. Matrix Metalloproteinase-1 ( MMP-1 ), a gene associated with extracellular matrix remodeling and skin aging [ 22 ], showed a gradual increase in mRNA levels post-irradiation. p21 exhibited an early response to treatment. Hyaluronan synthase ( HAS ) -2 mRNA levels also significantly increased. COL1a , a key component of type I collagen, exhibited a slight increase in mRNA levels 4 h post-irradiation. Sirtuin 1 ( SIRT-1 ) is an NAD + -dependent class III protein deacetylase recognized for its role in promoting cell survival in response to DNA damage and associated with enhanced cellular longevity and stress resistance [ 24 , 25 ]. Our findings demonstrate a modest elevation in SIRT-1 levels beginning at 8 h post-irradiation. This observation is consistent with the role of SIRT -1 in cellular defense mechanisms against oxidative damage. Nuclear factor erythroid 2-related factor 2 ( Nrf-2 ), a critical early regulator of the antioxidant response, initially increased 2 h post-irradiation and subsequently decreased. HaCaT cells were treated with various durations of blue light exposure (15, 30, 60, and 120 min) in the presence or absence of 1 mM 5-ALA. Following irradiation, cells were incubated for an additional 3 h before quantification by qRT-PCR. Figure 7 C shows the responses of specific genes to blue light exposure with and without 5-ALA treatment. MMP-1 mRNA levels increased significantly following blue light exposure alone, but in the presence of 5-ALA, MMP-1 levels initially increased at 15 min and subsequently decreased. p21 mRNA levels consistently increased in response to irradiation, regardless of 5-ALA presence, displayed time-dependent upregulation. Exposure to blue light alone increased HAS-2 mRNA levels at 15 and 30 min, but a slight decrease was observed at 120 min. When combined with 5-ALA, HAS-2 levels increased only at 15 min, followed by a decrease after longer exposure. COL1a mRNA levels modestly increased after blue light exposure. However, in the presence of 5-ALA, a decrease was observed after 30 min of exposure. SIRT-1 and Nrf-2 mRNA levels slightly increased upon blue light exposure alone, but decreased when cells were co-treated with 5-ALA. Discussion In this study, we investigated the effects of blue light exposure alone and in combination with 5-ALA on B16F1 melanoma and HaCaT cells. In our previous study, we demonstrated that blue and green light elevate ROS levels in B16F1 cells, leading to cell death [ 6 ]. Additionally, we found that co-treatment with 5-ALA severely damaged DNA in both B16F1 and HaCaT cells [ 14 ]. This study aimed to elucidate relevant cellular mechanisms by which these effects are exerted, including impacts on mitochondrial morphology, DNA integrity, and gene transcription. We first assessed the effects of 5-ALA on B16F1 and HaCaT cell proliferation. In B16F1 cells, we observed suppression of cell proliferation during short-term treatment. No significant inhibition of proliferation was observed in HaCaT cells. Notably, prolonged treatment (72 h) with 5-ALA significantly suppressed proliferation in both cell lines. Kumar et al. [ 26 ] reported similar findings, in which the proliferation of three types of hepatocarcinoma cell lines was significantly suppressed in the presence of 0.6 mM 5-ALA. In addition, Sparsa et al. [ 27 ] found that 5-ALA suppressed the viability of B16F1 melanoma cells. Grigalavicius et al. suggested that 5-ALA might act as a lactate dehydrogenase inhibitor to induce cancer cell death [ 28 ]. These results suggest that 5-ALA or its intermediates can affect the cell cycle and cell proliferation. We also investigated the ameliorating effects of NAC on the inhibition of cell proliferation induced by co-treatment with blue light and 5-ALA. Notably, NAC was unable to rescue the inhibition of proliferation. These results suggest that while NAC can mitigate additional oxidative stress, it does not significantly counteract the reduction in cell viability caused by blue light and 5-ALA co-treatment in both B16F1 melanoma and HaCaT cells. We next examined the effects of blue light and 5-ALA co-treatment on intracellular ROS, ΔΨm, and cardiolipin peroxidation in B16F1 and HaCaT cells. B16F1 cells did not exhibit increased ROS levels upon exposure to blue light alone; however, a significant increase was observed upon 5-ALA co-treatment. ROS levels were also enhanced in HaCaT cells after co-treatment. Notably, blue light alone decreased ROS levels in HaCaT cells, suggesting a potential cell-protective effect of blue light in certain cell types. Sutterby et al. demonstrated that monochromatic visible light (yellow, orange, and red light) induces proliferation and wound healing in HaCaT cells [ 29 ]. The effect of blue light was not assessed, thus this question requires further investigation. Neither blue light nor 5-ALA alone affected ΔΨm in either cell line. However, co-treatment resulted in depolarization of ΔΨm in both cell lines. As ΔΨm depolarization is an early indicator of apoptosis [ 30 ], this result suggests that co-treatment promotes cell death. We also assessed cardiolipin peroxidation, a process that weakens cytochrome c retention in the inner mitochondrial membrane, triggering subsequent apoptosis [ 31 ]. Our findings showed that neither blue light nor 5-ALA alone induced cardiolipin peroxidation in either cell line. However, co-treatment led to significant cardiolipin peroxidation. These observations suggest that co-treatment with blue light and 5-ALA induce significant cell death in both B16F1 and HaCaT cells, primarily through elevating ROS levels and promoting mitochondrial dysfunction. Notably, a previous report indicated that the combination of ultrasound and 5-ALA increased ROS levels, promoting subsequent cell death in a human leukemia cell line [ 32 ]. Given that co-treatment induced ΔΨm depolarization and cardiolipin peroxidation, we investigated the effect of blue light and 5-ALA co-treatment on mitochondrial morphology using imaging techniques. Using MitoTracker imaging, we observed that blue light alone did not significantly change mitochondrial morphology in either B16F1 or HaCaT cells. However, co-treatment with 0.2 mM 5-ALA in B16F1 cells induced a noticeable loss of mitochondrial structure, suggesting severe damage. In HaCaT cells, the same co-treatment caused elongated mitochondria to become increasingly smaller and more granular. Such mitochondrial fragmentation, often linked to ΔΨm depolarization [ 33 ], may aid in removing damaged mitochondria and help cells recover from stress [ 34 ]. This effect was even more evident at 1 mM 5-ALA, which apparently dispersed the mitochondria. Both cell lines showed mitochondrial clustering near the nucleus (nuclear periphery) after co-treatment, a response possibly related to increased intracellular ROS levels and cellular stress. This morphological change was more pronounced at 1 mM 5-ALA, where the mitochondria appeared to be dispersed. Some reports have indicated that perinuclear clustering of mitochondria occurs in response to intracellular ROS accumulation and several other types of cell stress [ 35 , 36 ]. These observations suggest that co-treatment with blue light and 5-ALA can induce significant mitochondrial stress and morphological changes. TEM also provided intriguing insights into mitochondrial damage in response to various treatment conditions. In B16F1 cells, exposure to blue light alone was sufficient to induce significant mitochondrial membrane damage, suggesting that blue light exerts a detrimental effect on mitochondrial functionality. Moreover, increased mitochondrial damage was observed in B16F1 cells co-treated with a higher concentration of 5-ALA (0.2 mM). This exacerbated damage was attributed to the potential synergistic effects of blue light and 5-ALA on mitochondrial integrity. In contrast, HaCaT cells exhibited resistance to mitochondrial damage upon blue light exposure and low-concentration 5-ALA co-treatment. However, similar to B16F1 cells, HaCaT cells exhibit clear mitochondrial damage when treated with 0.2 mM 5-ALA. TEM analysis revealed a notable disappearance of mitochondrial cristae after co-treatment. Fu et al. reported that mitochondrial DNA DSBs result in loss of membrane potential and abnormal or missing cristae [ 37 ]. The alterations in mitochondrial morphology observed in our study could be a consequence of similar DNA damage, potentially affecting both mitochondrial and nuclear DNA. We also assessed mtROS levels in B16F1 and HaCaT cells following blue light exposure and 5-ALA co-treatment. In B16F1 cells, blue light alone increased mtROS. This increase was not observed upon co-treatment with 0.2 mM 5-ALA, which may be attributable to severe mitochondrial damage. In contrast, HaCaT cells did not show a significant increase in mtROS levels with either blue light alone or with low 5-ALA concentration. Notably, an increase in mtROS was detected with 0.2 mM 5-ALA co-treatment. These results suggest that the differential responses to treatment may be due to variations in cellular sensitivity. We also explored the effects of blue light and 5-ALA co-treatment on the subG1 cell population and on cell cycle progression. Co-treatment markedly increased the number of subG1 events in both cell lines, indicating the induction of DNA fragmentation, which is a hallmark of apoptosis. Co-treatment of B16F1 cells led to cell cycle arrest at the G2/M checkpoint, a response associated with DNA damage [ 38 ]. In contrast, HaCaT cells exposed to blue light for 60 min showed an increase in cells in G1 and a decrease in cells in G2/M. These findings suggest that co-treatment may induce cell cycle arrest in response to DNA damage. Consequently, we assessed multiple types of DNA damage following co-treatment. To investigate DNA damage, we first assessed γH2AX, a marker of DNA DSBs. In both B16F1 and HaCaT cells, γH2AX levels increased following UVC irradiation. Notably, in B16F1 cells, exposure to blue light alone also elevated γH2AX levels. Co-treatment with blue light and 5-ALA increased γH2AX levels in both cell lines. These findings reinforce those of our previous study, which indicated the induction of DSBs and SSBs under similar experimental conditions [ 14 ]. In HaCaT cells, blue light alone decreased γH2AX fluorescence intensity compared with control levels. This observation suggests a potential reduction in cellular stress. Regarding CPD and 6-4PP, key markers of DNA base damage, neither blue light exposure nor co-treatment induced an increase in any of the cell lines. It is important to note that shorter wavelengths of visible light may cause the formation of CPD and other photoproducts [ 39 ]. In contrast, UVC irradiation significantly elevated CPD and 6-4PP levels in both cell lines. However, co-treatment with 5-ALA and UVC did not affect the production of these photoproducts. Regarding Dewar PP, which are formed through photoisomerization of 6-4PP under UVA [ 40 , 41 ], no significant changes were observed under any treatment conditions. Our study revealed significant changes in gene expression in B16F1 and HaCaT cells following exposure to blue light and co-treatment with 5-ALA. In B16F1 cells, we observed upregulation of p21 and caspase-3 , alongside downregulation of Bcl-2 . These changes in gene expression suggest the initiation of apoptosis. Survivin also exhibited variable responses, in contrast to our previous findings, where long-term blue light exposure significantly inhibited survivin transcription in B16F1 cells [ 7 ]. This discrepancy may be attributed to differences in experimental conditions, particularly in the duration of blue light exposure. Notable increases in MMP-1 , p21 , and HAS-2 levels, with slight alterations in COL1a and SIRT-1 levels were observed. UV radiation can enhance MMP-1 levels, leading to collagen fiber degradation, contributing to skin aging and wrinkling [ 42 , 43 ]. MMPs play a role in cancer invasion [ 44 – 46 ]. Our results suggest that blue light and co-treatment with 5-ALA also elevates MMP-1 expression, potentially leading to similar detrimental effects. In our study, we observed a notable upregulation of p21 , a cyclin-dependent kinase inhibitor, in both B16F1 and HaCaT cells following co-treatment with blue light and 5-ALA. p21, a critical regulator of cell cycle progression, is often induced in response to DNA damage, and serves as a major target of the tumor suppressor p53 [ 47 , 48 ]. p21 is also implicated in cellular responses to stress and can confer protection against apoptosis under certain condition [ 49 ]. The enhanced p21 expression observed in our experiments suggests that co-treatment with blue light and 5-ALA induces cell cycle arrest. Combined blue light and 5-ALA exposure induced complex, time-dependent alterations in gene expression, rendering discussion difficult. Further research is needed to fully understand these complex changes. However, our findings provide foundational data for future studies in this area. In conclusion, this study elucidated the effects of co-treatment with blue light and 5-ALA on B16F1 melanoma and HaCaT cells. Our findings reveal that this co-treatment induced significant cellular stress, as evidenced by the elevation in intracellular ROS levels, ΔΨm collapse, and DNA damage. Additionally, alterations in mitochondrial morphology and induction of apoptosis-related gene expression were observed following blue light exposure with 5-ALA. Although these results advance our understanding of the cellular responses to blue light and 5-ALA, they also raise concerns regarding potential adverse effects of 5-ALA in normal cells. This study highlights the need for cautious application of 5-ALA in clinical settings, considering its potential to induce damage in target cancer cells and normal cells. Declarations Data availability The datasets use/or analyzed during the current study available from the corresponding author on reasonable request. Author Contribution K.S. designed the experiments. K.S., T.S., R.H., and M.B. performed the experiments and analyzed the data. K.S. and M.B. wrote the manuscript. All authors have approved the final manuscript. The authors have read and approved the final manuscript. References Vayalil, P.K., Mittal, A., Hara, Y., Elmets, C.A. & Katiyar, S.K. Green tea polyphenols prevent ultraviolet light-induced oxidative damage and matrix metalloproteinase expression in mouse skin. J. Invest Dermatol. 2004, 122, 1480–1487. Zhivagui, M. et al. DNA damage and somatic mutations in mammalian cells after irradiation with a nail polish dryer. Nat. Commun. 2021, 14, 276. Xia, D. et al. Combined Analysis of the Effects of Exposure to Blue Light in Ducks Reveals a Reduction in Cholesterol Accumulation Through Changes in Methionine Metabolism and the Intestinal Microbiota. Front. Nutr. 2021, 8, 737059. Alghamdi, R.A. et al. Assessing Phototoxicity in a Mammalian Cell Line: How Low Levels of Blue Light Affect Motility in PC3 Cells. Front. Cell Dev. Biol. 2021, 9, 738786. Höytö, A. et al. Cellular detection of 50 Hz magnetic fields and weak blue light: effects on superoxide levels and genotoxicity. Int. J. Radiat. Biol. 2017, 93, 646–652. Sato, K., Minai, Y. & Watanabe, H. Effect of monochromatic visible light on intracellular superoxide anion production and mitochondrial membrane potential of B16F1 and B16F10 murine melanoma cells. Cell Biol. Int. 2013, 37, 633–637. Nishio, T., Kishi, R. & Sato, K. Blue light exposure enhances oxidative stress, causes DNA damage, and induces apoptosis signaling in B16F1 melanoma cells. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 2022, 883–884, 503562. Kang, Z. et al. Recent advances in production of 5-aminolevulinic acid using biological strategies. World J. Microbiol. Biotechnol. 2017, 33, 200. Kiening, M. & Lange, N. A Recap of Heme Metabolism towards Understanding Protoporphyrin IX Selectivity in Cancer Cells. Int. J. Mol. Sci. 2022, 23, 7974. Wang, J. et al. Exogenous Application of 5-Aminolevulinic Acid Promotes Coloration and Improves the Quality of Tomato Fruit by Regulating Carotenoid Metabolism. Front. Plant Sci. 2021, 12, 683868. Jalili-Nik, M. et al. Antitumor Effects of 5-Aminolevulinic Acid on Human Malignant Glioblastoma Cells. Int. J. Mol. Sci. 2021, 22, 5596. Ryabova, A. et al. Detection of Changes in Macrophage Polarization as a Result of 5-Aminolevulinic Acid Photodynamic Therapy Using Fluorescence-Lifetime Imaging Microscopy. Photonics. 2022, 9, 961. Helander, L. et al. Red versus blue light illumination in hexyl 5-aminolevulinate photodynamic therapy: The influence of light color and irradiance on the treatment outcome in vitro. J. Biomed. Opt. 2018, 19, 088002. Sato, T. & Sato, K. Adverse Effect of Blue Light on DNA Integrity Is Accelerated by 5-Aminolevulinic Acid in HaCaT Human Keratinocyte Cells and B16F1 Murine Melanoma Cells. Biology. 2022, 11, 1743. Nunez, R. DNA Measurement and Cell Cycle Analysis by Flow Cytometry. Curr. Issues Mol. Biol. 2001, 3, 67–70. etrosillo, G., Ruggiero, F.M. & Paradies, G. Role of reactive oxygen species and cardiolipin in the release of cytochrome c from mitochondria. FASEB J. 2003, 17, 2202–2208. Zeno, S. et al. CoCl(2) induces apoptosis via the 18 kDa translocator protein in U118MG human glioblastoma cells. Biochemistry. 2009, 48, 4652–4661. Redon, C.E. et al. γ-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin. Adv. Space Res. 2009, 43, 1171–1178. Nishimura, K. et al. Seasonal Differences in the UVA/UVB Ratio of Natural Sunlight Influence the Efficiency of the Photoisomerization of (6 – 4) Photoproducts into their Dewar Valence Isomers. Photochem. Photobiol. 2021, 97, 582–588. D ouki, T. Relative Contributions of UVB and UVA to the Photoconversion of (6 – 4) Photoproducts into their Dewar Valence Isomers. Photochem. Photobiol. 2016, 92, 587–594. Qian, S. et al. The role of BCL-2 family proteins in regulating apoptosis and cancer therapy. Front. Oncol. 2022, 12, 985363. Karadeniz, F., Oh, J.H., Kim, H.R., Ko, J. and Kong, C.-S. Camellioside A, isolated from Camellia japonica flowers, attenuates UVA-induced production of MMP-1 in HaCaT keratinocytes via suppression of MAPK activation. Exp. Ther. Med. 2021, 21, 16. Bitar, S.A. & Gali-Muhtasib, H. The Role of the Cyclin Dependent Kinase Inhibitor p21cip1/waf1 in Targeting Cancer: Molecular Mechanisms and Novel Therapeutics. Cancers. 2019, 11, 1475. Kume, S. et al. Silent information regulator 2 (SIRT1) attenuates oxidative stress-induced mesangial cell apoptosis via p53 deacetylation. Free Radic. Biol. Med. 2006, 40, 2175–2182. Luo, J. et al. Negative control of p53 by Sir2alpha promotes cell survival under stress. Cell. 2001, 107, 137–148. Kumar, A. et al. An Efficient 5-Aminolevulinic Acid Photodynamic Therapy Treatment for Human Hepatocellular Carcinoma. Int. J. Mol. Sci. 2023, 24, 10426. Sparsa, A. et al. Photodynamic treatment induces cell death by apoptosis or autophagy depending on the melanin content in two B16 melanoma cell lines. Oncol. Rep. 2013, 29, 1196–1200. Grigalavicius, M. et al. 5-ALA Is a Potent Lactate Dehydrogenase Inhibitor but Not a Substrate: Implications for Cell Glycolysis and New Avenues in 5-ALA-Mediated Anticancer Action. Cancers. 2022, 14, 4003. Sutterby, E. et al. Investigating the effects of low intensity visible light on human keratinocytes using a customized LED exposure system. Sci. Rep. 2022, 12, 18907. Nabekura, T., Ishikawa, S., Tanase, M., Okumura, T., Kawasaki, T. Antidepressants induce toxicity in human placental BeWo cells. Curr. Res. Toxicol. 2022, 3, 100073. Zhang, G.-X. et al. Inhibition of cytochrome c release by 10-N-nonyl acridine orange, a cardiolipin-specific dye, during myocardial ischemia-reperfusion in the rat. Am. J. Physiol. Heart Circ. Physiol. 2010, 298, H433-H439. Sun, X. et al. Potential involvement of the 18 kDa translocator protein and reactive oxygen species in apoptosis of THP-1 macrophages induced by sonodynamic therapy. PLOS One. 2018, 13, e0196541. Machiela, E. et al. Disruption of mitochondrial dynamics increases stress resistance through activation of multiple stress response pathways. FASEB J. 2020, 34, 8475–8492. Fernandes, T. et al. Structural and Functional Alterations in Mitochondria-Associated Membranes (MAMs) and in Mitochondria Activate Stress Response Mechanisms in an In Vitro Model of Alzheimer's Disease. Biomedicines. 2021, 9, 881. Bolfer, L. et al. Functional Consequences of PDK4 Deficiency in Doberman Pinscher Fibroblasts. Sci. Rep. 2020, 10, 3930. Al-Mehdi, A.-B. et al. Perinuclear mitochondrial clustering creates an oxidant-rich nuclear domain required for hypoxia-induced transcription. Sci. Signal. 2012, 5, ra47. Fu, Y. et al. Mitochondrial DNA breaks activate an integrated stress response to reestablish homeostasis. Mol. Cell. 2023, 83, 3740–3753. Lim, Y., Dorstyn, L. & Kumar, S. The p53-caspase-2 axis in the cell cycle and DNA damage response. Exp. Mol. Med. 2021, 53, 517–527. Lawrence, K.P. et al. The UV/Visible Radiation Boundary Region (385–405 nm) Damages Skin Cells and Induces "dark" Cyclobutane Pyrimidine Dimers in Human Skin in vivo. Sci. Rep. 2018, 8, 12722. Douki, T. Pyrimidine (6 – 4) Pyrimidone Photoproducts in UVA-Irradiated DNA: Photosensitization or Photoisomerization? ChemPhotoChem. 2020, 4, 294–299. Douki, T. Relative Contributions of UVB and UVA to the Photoconversion of (6 – 4) Photoproducts into their Dewar Valence Isomers. Photochem. Photobiol. 2016, 92, 587–594. Hwang, E., Lee, T.H., Park, S.-Y. & Kim, S.Y. Enzyme-modified Panax ginseng inhibits UVB-induced skin aging through the regulation of procollagen type I and MMP-1 expression. Food Funct. 2014, 5, 265–274. Park, E.K. et al. The Anti-Wrinkle Mechanism of Melatonin in UVB Treated HaCaT Keratinocytes and Hairless Mice via Inhibition of ROS and Sonic Hedgehog Mediated Inflammatory Proteins. Int. J. Mol. Sci. 2018, 19, 1995. Akter, H. et al. Activation of matrix metalloproteinase-9 (MMP-9) by neurotensin promotes cell invasion and migration through ERK pathway in gastric cancer. Tumour Biol. 2015, 436, 6053–6062. Soto-Guzman, A., Navarro-Tito, N., Castro-Sanchez, L., Martinez-Orozco, R., Salazar, E.P. Oleic acid promotes MMP-9 secretion and invasion in breast cancer cells. Clin. Exp. Metastasis. 2010, 27, 505–515. Rattanasinchai, C., Llewellyn, B.J., Conrad, S.E. & Gallo, K.A. MLK3 regulates FRA-1 and MMPs to drive invasion and transendothelial migration in triple-negative breast cancer cells. Oncogenesis. 2017, 6, e345. Liu, G. et al. RNA-binding protein CELF6 is cell cycle regulated and controls cancer cell proliferation by stabilizing p21. Cell Death Dis. 2019, 10, 688. Engeland, K. Cell cycle regulation: p53-p21-RB signaling. Cell Death Differ. 2022, 29, 946–960. Karimian, A., Ahmadi, Y. & Yousefi, B. Multiple functions of p21 in cell cycle, apoptosis and transcriptional regulation after DNA damage. DNA Repair 2016, 42, 63–71. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3894182","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":271524994,"identity":"d52ec959-a841-4cb5-81af-69af1f899272","order_by":0,"name":"Kazuomi Sato","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIiWNgGAWjYBACCQhlgynDjFfLAYY0EMXYQIqWw5hacALJ/uMPP3+oOC8vH32A/cHPHDu7/hnJDxh+1DCwm+PQIs1wxljiwJnbhhvPJTA29m5LTp5xI82AsecYA7MlDkvlGHsYJA623Wbc2AN0GO825mSG2wkGDLwNDMwGB3BoYWZ//ONg2zl7kJbGv9vqk+Vvp39g/ItHizQbgxnQlgOJ83kYGJt5tx22M7idY8CMzxbJHh4zizNnkpM38DA2zpbddjzB8P6bgsMyxyRw+kXi/PHHNyoq7Gzn9zAf+Ph2W7W93JnjGx++qbFJxhVicGBwABItiSAS6CSJZANCWuShzrCHCdgR1DIKRsEoGAUjBQAALjJeHuFvayAAAAAASUVORK5CYII=","orcid":"","institution":"Tamagawa University","correspondingAuthor":true,"prefix":"","firstName":"Kazuomi","middleName":"","lastName":"Sato","suffix":""},{"id":271524995,"identity":"0137d301-a640-4b47-98b4-e3b2e10b3d26","order_by":1,"name":"Taiki Sato","email":"","orcid":"","institution":"Tamagawa University","correspondingAuthor":false,"prefix":"","firstName":"Taiki","middleName":"","lastName":"Sato","suffix":""},{"id":271524996,"identity":"289d8fd1-e348-4bf9-9008-a96d078aa1b1","order_by":2,"name":"Riku Hirotani","email":"","orcid":"","institution":"Tamagawa University","correspondingAuthor":false,"prefix":"","firstName":"Riku","middleName":"","lastName":"Hirotani","suffix":""},{"id":271524997,"identity":"fd5157aa-7787-404d-b215-4746ca0ffccd","order_by":3,"name":"Munetsugu Bam","email":"","orcid":"","institution":"University of Yamanashi","correspondingAuthor":false,"prefix":"","firstName":"Munetsugu","middleName":"","lastName":"Bam","suffix":""}],"badges":[],"createdAt":"2024-01-24 13:24:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3894182/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3894182/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":50833193,"identity":"341fc8e0-d422-4992-9f08-4037fa451e7d","added_by":"auto","created_at":"2024-02-08 04:43:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":83093,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of blue light and 5-ALA on intracellular ROS levels, mitochondrial membrane potential, and cardiolipin peroxidation.\u003c/p\u003e\n\u003cp\u003eEffect of 5-ALA (1 mM) and 50 W/m2 blue light exposure for 60 min on B16F1 and HaCaT cells. Intracellular ROS levels in B16F1 (A) and HaCaT cells (B). Mitochondrial membrane potential (ΔΨm) depolarized cells were detected in B16F1 (C) and HaCaT cells (D). The proportion of cells exhibiting low nonyl acridine orange fluorescence, indicative of cardiolipin peroxidation, was determined in B16F1 (E) and HaCaT cells (F). All measurements were conducted using flow cytometry. Data represent means ± standard error of the mean obtained from at least three independent experiments. \u003csup\u003e＊\u003c/sup\u003ep \u0026lt;0.05 and \u003csup\u003e＊＊\u003c/sup\u003ep \u0026lt;0.01 versus control group. \u003csup\u003e#\u003c/sup\u003ep \u0026lt;0.05, and\u003csup\u003e ##\u003c/sup\u003ep \u0026lt;0.01 was considered significant.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3894182/v1/f2b204f97e47712f21128458.png"},{"id":50833196,"identity":"3361ac09-744b-419b-851d-ec172d5e9609","added_by":"auto","created_at":"2024-02-08 04:43:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5849830,"visible":true,"origin":"","legend":"\u003cp\u003eMitochondrial morphology of B16F1 and HaCaT cells\u003c/p\u003e\n\u003cp\u003eMitochondrial morphology in B16F1 (A) and HaCaT cells (B) was assessed using MitoTracker Green. Cells were pre-treated with or without 0.2 or 1 mM 5-ALA for 1 h, followed by exposure to blue light (50 W/m\u003csup\u003e2\u003c/sup\u003e) for 1 h. After treatment, cells were stained with MitoTracker, then imaged using a confocal microscope. (C) Transmission electron microscopy (TEM) images of B16F1 and HaCaT cells. Cells were pre-treated with 0.02 or 0.2 mM 5-ALA for 1 h, followed by exposure to blue light (50 W/m\u003csup\u003e2\u003c/sup\u003e) for another 1 h. After treatment, cells were fixed and processed for TEM to visualize ultrastructural changes. Arrows indicate damaged mitochondria.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3894182/v1/5e20eace4324174d1be3d703.png"},{"id":50833195,"identity":"51c13494-8a9c-49ed-9e93-74b85d209aeb","added_by":"auto","created_at":"2024-02-08 04:43:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1422654,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of blue light and 5-ALA on mitochondrial ROS levels in B16F1 and HaCaT cells\u003c/p\u003e\n\u003cp\u003eMitochondrial ROS levels were assessed in B16F1 (A, B) and HaCaT (B, D) cells after treatment with blue light ± 5-ALA. Cells were pre-treated with or without 5-ALA (0.02, 0.2 mM) and exposed to blue light (50 W/m\u003csup\u003e2\u003c/sup\u003e) for 1 h. After treatment, cells were stained with MitoSOX Red and imaged using a confocal microscope. Quantitative analysis of MitoSOX fluorescence intensity was performed using Zeiss Zen software. \u003csup\u003e＊\u003c/sup\u003ep \u0026lt;0.05 and \u003csup\u003e＊＊\u003c/sup\u003ep \u0026lt;0.01 versus control group.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3894182/v1/f6e66f0dcc67f2afd490db2b.png"},{"id":50833194,"identity":"61d03d09-b1d3-45f2-8a52-d90bd7363c97","added_by":"auto","created_at":"2024-02-08 04:43:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":62559,"visible":true,"origin":"","legend":"\u003cp\u003esubG1 population and cell cycle in B16F1 and HaCaT cells\u003c/p\u003e\n\u003cp\u003eEffect of blue light and 5-ALA treatment on the subG1 cell population and cell cycle distribution in B16F1 (A, B, E, F) and HaCaT cells (C, D, G, H).\u003c/p\u003e\n\u003cp\u003eFor subG1 population analysis, B16F1 (A, B) and HaCaT cells (C, D) were pre-treated ± 1 mM 5-ALA for 1 h, followed by exposure to blue light (50 W/m\u003csup\u003e2\u003c/sup\u003e) for either 30 min (A, C) or 60 min (B, D). The subG1 population, indicative of apoptotic cells, was quantified using flow cytometry.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;For cell cycle analysis, B16F1 (E, F) and HaCaT (G, H) cells were treated as described previously. Blue light exposure was performed for 30 (E, G) or 60 min (F, H). After treatment, the proportion of cells in each phase of the cell cycle (G1, S, and G2/M) was determined using flow cytometry.\u003c/p\u003e\n\u003cp\u003eData represent means ± standard error of the mean obtained from at least three independent experiments. \u003csup\u003e＊\u003c/sup\u003ep \u0026lt;0.05 and \u003csup\u003e＊＊\u003c/sup\u003ep \u0026lt;0.01 versus control group. \u003csup\u003e#\u003c/sup\u003ep \u0026lt;0.05, and\u003csup\u003e ##\u003c/sup\u003ep \u0026lt;0.01 was considered significant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3894182/v1/c415fc404c6b12c971e67db1.png"},{"id":50833201,"identity":"408407fb-2654-407c-865c-2639a5039e60","added_by":"auto","created_at":"2024-02-08 04:43:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":616836,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of γH2AX formation in response to blue light and 5-ALA treatment in B16F1 and HaCaT cells\u003c/p\u003e\n\u003cp\u003e(A, B) Respective quantitative analyses of γH2AX fluorescence intensity in B16F1 and HaCaT cells. (C, D) Confocal microscopic images of B16F1 and HaCaT cells post-treatment, highlighting γH2AX localization and intensity.\u003c/p\u003e\n\u003cp\u003eCells were pre-treated with or without 5-ALA and exposed to blue light for 1 h. After irradiation, cells were stained with anti-γH2AX antibodies to detect DNA damage response and visualized using a confocal microscope (C, D). The methodology for these procedures is described in detail in Materials and Methods. Data represent means ± standard error of the mean obtained from at least three independent experiments.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3894182/v1/1ca89c46826f3b8884652f09.png"},{"id":50833198,"identity":"2c2a7e98-0271-4c15-8ccd-fba69251838b","added_by":"auto","created_at":"2024-02-08 04:43:54","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":62058,"visible":true,"origin":"","legend":"\u003cp\u003eAssessment of photoproduct formation in B16F1 and HaCaT cells following blue light and 5-ALA treatment\u003c/p\u003e\n\u003cp\u003e(A, B) Formation of CPD in B16F1 and HaCaT cells, respectively. (C, D) Formation of 6-4 PP in B16F1 and HaCaT cells, respectively. (E, F) Formation of Dewar PP in B16F1 and HaCaT cells, respectively. For these assays, cells were pre-treated with or without 5-ALA, then exposed to blue light for 1 h. After irradiation, DNA was extracted from the cells, and enzyme-linked immunosorbent assay was performed to quantify formation of these photoproducts. Detailed methodologies are described in Materials and Methods. The data represent means ± SEM obtained from at least three independent experiments. \u003csup\u003e＊\u003c/sup\u003ep\u0026lt;0.05 and \u003csup\u003e＊＊\u003c/sup\u003ep\u0026lt;0.01 versus control group.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3894182/v1/efc2cc4dd42a2e3f83ac0d75.png"},{"id":50833202,"identity":"a5b57219-8dbe-4854-8054-30d6ed05de1c","added_by":"auto","created_at":"2024-02-08 04:43:54","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":151194,"visible":true,"origin":"","legend":"\u003cp\u003eReal-time qRT-PCR analysis\u003c/p\u003e\n\u003cp\u003e(A) Effect of combined blue light and 5-ALA on gene transcription in B16F1 melanoma cells. Cells were treated with blue light for 30 min in the presence of 0.2 mM 5-ALA. Subsequently, a time-course analysis of mRNA levels was conducted at intervals of 2, 4, 8, 16, and 24 h post-treatment. (B) Effect of combined blue light and 5-ALA on gene transcription in HaCaT cells. Cells were exposed to blue light for 1 h in the presence of 0.2 mM 5-ALA. A time-course analysis of mRNA levels was then performed at intervals of 2, 4, 8, 16, and 24 h post-treatment. (C) Effect of blue light and co-treatment with 5-ALA on gene transcription in HaCaT cells. Cells were exposed to blue light for durations ranging from 15–120 min ± 1 mM 5-ALA. After an additional 3 h of incubation, mRNA levels were assessed using qRT-PCR. Data represent means ± standard error of the mean obtained from at least three independent experiments. \u003csup\u003e＊\u003c/sup\u003ep \u0026lt;0.05 and \u003csup\u003e＊＊\u003c/sup\u003ep \u0026lt;0.01 versus control group. \u003csup\u003e#\u003c/sup\u003ep \u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003ep \u0026lt;0.01 versus the 5-ALA-alone-treated group.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3894182/v1/95d5e7e5993d4ba3a29836ae.png"},{"id":53403448,"identity":"3b5f1658-aebc-4399-aaa3-c5014efcde6d","added_by":"auto","created_at":"2024-03-25 15:08:41","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1219183,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3894182/v1/503997b5-3ae8-445e-a42c-38ea14d62c6e.pdf"},{"id":50833701,"identity":"806c150e-c965-43f6-bf69-8d0cfbed8f47","added_by":"auto","created_at":"2024-02-08 04:51:54","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":945297,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3894182/v1/88b23f19b5bc2b03fee66114.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Combined Blue light and 5-ALA on cell death in B16F1 melanoma and HaCaT cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUltraviolet (UV) light, a component of solar radiation, exerts profound effects on various forms of life, particularly animals. Chronic exposure to solar UV light, particularly in humans, has been linked to photoaging, which is characterized by UV-induced oxidative damage and induction of matrix metalloproteinase (MMP) expression [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Such exposure can significantly alter skin properties, with oxidative stress playing a central role in the damage process. In mice, detrimental effects of UV radiation are evident in the skin, where UV exposure can underlie protein oxidation, a hallmark of photoaging [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. At the cellular level, UV radiation can cause DNA damage, leading to mutations that result in cancer growth. UV radiation also induces the formation of cyclobutane pyrimidine dimers (CPDs) between neighboring pyrimidine bases [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBlue light, a component of visible light, has been extensively studied for its effects on various biological processes in animals and cells. Increasing use of blue light in various applications gives impetus to studies aimed at understanding its effects on cellular and metabolic processes. In animal husbandry, Xia et al. found that irradiating ducks with blue light (460 nm) decreased their body weight and enhanced their anti-inflammatory and antioxidant capacities. DNA damage and somatic mutations have been observed in mammalian cells irradiated with a nail polish dryer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Phototoxicity, especially in live-cell fluorescence microscopy, is a concern associated with blue light exposure. Alghamidi et al. showed that even low levels of blue light can affect the motility of PC3 human prostate cancer cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In human SH-SY5Y neuroblastoma cells, intriguing interactions were observed between blue light and a 50 Hz magnetic field (MF) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Combined MF and blue light exposure have been shown to modulate superoxide levels. Additionally, we have reported that blue light induces the collapse of mitochondrial membrane potential and subsequent cell death [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e5-aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid that serves as a precursor in heme biosynthesis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In plants, 5-ALA improves tomato coloration by regulating carotenoid metabolism and promoting fruit maturation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In contrast, 5-ALA exerts cytotoxic effects in malignant glioblastoma cells by increasing apoptosis, altering the expression of apoptosis-related genes, and enhancing reactive oxygen species (ROS) generation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These findings highlight the multifaceted impacts of 5-ALA in cells ranging from human cancer cells to plant cells.\u003c/p\u003e \u003cp\u003eIn photodynamic therapy (PDT), 5-ALA is converted to protoporphyrin IX, which acts as a photosensitizer by producing ROS in response to light exposure [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Halander et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] demonstrated that the efficacy of PDT was several times higher with blue light than with red light in several cancer cell lines. We have previously demonstrated that co-treatment with blue light and 5-ALA induces severe DNA damage, including double- and single-strand breaks (SSB), in both melanoma and normal keratinocyte cell lines [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Although numerous studies have described ALA-PDT, the effect of 5-ALA on intracellular metabolism remains unknown.\u003c/p\u003e \u003cp\u003eThis study aimed to elucidate the effects of 5-ALA on the intracellular metabolism of murine B16F1 melanoma and human HaCaT keratinocytes treated with blue light. We aimed to investigate the specific metabolic pathways influenced by 5-ALA and blue light exposure, focusing on cell viability, intracellular ROS, mitochondrial membrane potential and morphology, and expression of apoptosis-related genes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eB16F1 melanoma cells (RIKEN BioResource Research Center, Tsukuba, Japan) and HaCaT cells (Cosmo Bio, Tokyo, Japan) were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s medium (DMEM; Sigma, St Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), 50 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin. Cultures were maintained at 37\u0026deg;C in a humidified atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e. For blue light treatments, we used blue LED lamps with peak emission at 465 nm. Detailed information on the irradiance of LED lamps is available in our previous study [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell viability and proliferation\u003c/h2\u003e \u003cp\u003eTo assess cell proliferation and viability, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assays were performed. For experiments involving varying concentrations of 5-ALA, HaCaT and B16F1 melanoma cells were seeded into 96-well plates at a density of 2.0 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well. After 24 h incubation, cells were treated with different concentrations of 1 mM 5-ALA (Sigma-Aldrich, St. Louis, MO, USA) for specified durations. After treatment, cells were washed with phosphate-buffered saline (PBS), and culture medium was replaced. Following a 72-h incubation, 5 mg/mL MTT was added and cells were incubated for 3.5 h at 37\u0026deg;C. Resultant formazan crystals were dissolved in dimethyl sulfoxide and absorbance at 590 nm was measured using a microplate reader. For experiments involving co-treatment with blue light and 5-ALA in the presence of N-acetyl cysteine (NAC), B16F1 and HaCaT cells were seeded in 35 mm dishes at a density of 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/dish. Cells were pre-treated with 5 mM NAC (Fujifilm Wako, Osaka, Japan) and 1 mM 5-ALA for 1 h followed by exposure to blue light for an additional hour. After exposure, cells were incubated for another hour with NAC. Subsequently, cells were cultured for 72 h in NAC- and 5-ALA-free medium. MTT assays were then performed as described above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular superoxide anion production and mitochondria membrane potential (ΔΨm)\u003c/h2\u003e \u003cp\u003eIntracellular ROS levels were measured as previously described [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. B16F1 melanoma and HaCaT cells were seeded into 35 mm dishes at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/dish. After incubation for 24 h, cells were pre-treated with 1 mM 5-ALA for 1 h, followed by irradiation with blue light for 30 or 60 min in the presence of 5-ALA. After treatment, cells were incubated in DMEM containing 5 \u0026micro;M hydroethidine (HE, Thermo Fisher Scientific, Waltham, MA, USA) for superoxide anion detection, or 50 nM 3,3\u0026rsquo;-dihexyloxacarbocyanine iodide (DiOC6; Invitrogen) to assess ΔΨm. After incubation for 20 min at 37\u0026deg;C, cells were collected by trypsinization, washed twice with PBS, and analyzed using flow cytometry (FACSCalibur, BD, Franklin Lakes, NJ, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCardiolipin peroxidation\u003c/h2\u003e \u003cp\u003eTo assess cardiolipin status, we used nonyl acridine orange (NAO; Sigma), a fluorescent dye that binds with high affinity to non-oxidized cardiolipin. B16F1 melanoma and HaCaT cells were seeded onto 35 mm dishes at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/dish. After 24 h of incubation, the cells were pre-treated with 1 mM 5-ALA for 1 h, then irradiated with blue light for 30 or 60 min in the presence of 5-ALA. After irradiation, cells were incubated in DMEM containing 50 \u0026micro;M NAO for 20 min at 37\u0026deg;C. Cells were collected by trypsinization, washed twice with PBS, and analyzed by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial fluorescence staining\u003c/h2\u003e \u003cp\u003eTo evaluate the impact of blue light and 5-ALA treatment on mitochondrial morphology, we performed MitoTracker Green staining, a widely used technique for visualizing mitochondria in live cells. B16F1 and HaCaT cells were seeded onto glass-bottomed 35-mm dishes at 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/dish. After 24 h of incubation, the cells were treated with vehicle or several concentrations of 5-ALA for 1 h, and subsequently exposed to blue light. Following treatment, the culture medium was replaced with pre-warmed serum-free fresh medium containing 200 nM MitoTracker Green FM (Thermo Fisher Scientific, Waltham, MA, USA), and cells were incubated for 30 min at 37\u0026deg;C. After incubation, cells were washed three times to remove excess probe. For nuclear staining, cells were incubated with 1 \u0026micro;M Hoechst 33342 (Thermo Fisher Scientific) in DMEM for 10 min at 37\u0026deg;C. Finally, cells were visualized using an LSM 700 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTransmission electron microscopy (TEM)\u003c/h2\u003e \u003cp\u003eTo elucidate ultrastructural changes in mitochondria following treatment, we used the TEM. After blue light and 5-ALA treatment as described above, cells were fixed using 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h at room temperature in the dark. Subsequently, fixed cells were rinsed with phosphate buffer. Secondary fixation was carried out using 1% osmium tetroxide (OsO\u003csub\u003e4\u003c/sub\u003e) in 0.1 M phosphate buffer (pH 7.2). After fixation, cells were washed and embedded in 1% agar. Agarose-embedded samples were trimmed into 1 mm cubes and dehydrated using an ethanol series. Samples were subsequently substituted with propylene oxide and embedded in an epoxy\u0026ndash;resin mixture. These samples were ultrathin sectioned to a thickness of 70 nm and stained with an EM Stainer (Nisshin EM Co., Ltd., Tokyo, Japan) and Reynolds lead citrate. Analyses were conducted using a TEM JEM-2100F (JEOL Co., Ltd., Tokyo, Japan) at an acceleration voltage of 120 kV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial ROS (mtROS) detection\u003c/h2\u003e \u003cp\u003eWe used MitoSOX Red as a probe (Thermo Fisher Scientific) to detect mtROS levels. After blue light and 5-ALA treatment, as described above, cells were incubated with DMEM containing 5 \u0026micro;M MitoSOX Red for 10 min at 37\u0026deg;C. After incubation, cells were washed and stained with Hoechst 33342 following the protocol outlined in the MitoTracker staining section. Cells were visualized using confocal microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analysis and subG1 cell detection\u003c/h2\u003e \u003cp\u003eTo analyze cell cycle progression and detect apoptotic cells in subG1 phase, B16F1 melanoma and HaCaT cells were subjected to identical intracellular ROS treatment. After treatment, both attached and detached cells were harvested, fixed with ice-cold 70% ethanol, and stored at least 24 h at -20 ℃. After fixation, cells were washed, resuspended in PBS containing RNase A (0.1 mg/mL, Sigma), and incubated at 37\u0026deg;C for 30 min to digest RNA. Aqueous propidium iodide (50 \u0026micro;g/mL, Sigma) was added to stain DNA. The DNA content of the cells was determined by flow cytometry. To accurately assess cell-cycle disruption and identify the subG1 population, we employed a method described by Nunez et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], which effectively discriminates between single cells and doublets or aggregates. In our cell cycle analysis, we reported proportions of cells in G1, S, and G2/M phases, explicitly excluding the subG1 population from these calculations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of DNA damage using γ-H2AX immunofluorescence\u003c/h2\u003e \u003cp\u003eTo assess DNA damage, cells were seeded into 35 mm glass-bottom dishes at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well and incubated overnight. Cells were treated with or without 5-ALA and exposed to blue light. Following treatment, cells were briefly washed with PBS and fixed with 2% paraformaldehyde (Fujifilm Wako) for 10 min at room temperature. Cells were then rinsed with PBS and permeabilized in 0.2% PBS-Tween for 5 min at room temperature. After three washes with PBS, the cells were blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at room temperature. For immunofluorescence staining, cells were incubated with primary anti-γH2AX antibodies (dilution 1:200; Merck Millipore, Burlington, MA, USA) overnight at 4\u0026deg;C. Cells were then incubated with a secondary anti-mouse IgG (Alexa Fluor 488) antibody (dilution 1:200; Abcam, Cambridge, UK) for 2 h at room temperature. Finally, cells were mounted in VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories, Inc., Newark, CA, USA) and observed using an EVOS FL imaging system (Thermo Fisher Scientific) or a confocal microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetection of CPD, 6\u0026thinsp;\u0026minus;\u0026thinsp;4 PP, and Dewar PP via enzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eTo evaluate DNA base damage induced by blue light and 5-ALA, we first employed an ELISA-based approach. After treatment, cells were lysed, and genomic DNA was extracted using a QIAamp DNA Blood Mini kit (Qiagen, Venlo, Netherlands) following the manufacturer\u0026rsquo;s protocol. DNA concentration was quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). The extracted DNA was denatured by heating at 100 \u0026ordm;C for 10 min, then rapidly cooled on ice. Subsequently, 50 \u0026micro;L aliquots of DNA at specific concentrations (0.2 \u0026micro;g/mL for CPD, 4.0 \u0026micro;g/mL for 6\u0026thinsp;\u0026minus;\u0026thinsp;4 PP, and 10 \u0026micro;g/mL for Dewar PP detection, respectively) were prepared and applied to protamine sulfate-coated 96-well plates. Plates were dried overnight at 37\u0026deg;C in the dark. After drying, wells were washed with PBS containing 0.05% Tween 20 (PBS-T). Blocking was performed using 2% FBS for 30 min at 37\u0026deg;C. After five washes with PBS-T, wells were incubated with antibodies (1:1000 anti-CPDs antibody, 1:1500 anti-6-4 PPs antibody, and 1:5000 anti-Dewar PPs antibody; Cosmo Bio) for 30 min at 37\u0026deg;C. After primary antibody incubation, wells were washed and incubated with biotinylated secondary anti-mouse antibody for 30 min at 37\u0026deg;C, followed by 1:10000 peroxidase-streptavidin for 30 min at 37\u0026deg;C. After washing, wells were treated with PBS-T and once with citrate-phosphate buffer (containing 25 mM citric acid monohydrate and 50 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, pH 5.0), then incubated with 100 \u0026micro;L/well of substrate buffer (4 mM o-phenylenediamine, 0.007% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in citrate-phosphate buffer) for 30 min at 37\u0026deg;C. The enzymatic reaction was stopped using 50 \u0026micro;L of 2 M sulfuric acid (H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e). The absorbance of each well was measured at 492 nm using a microplate reader.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescent CPD staining\u003c/h2\u003e \u003cp\u003eCells were seeded into 35 mm glass-bottom dishes at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells and incubated overnight. Cells were treated with or without 5-ALA and exposed to blue light. After treatment, cells were fixed with 4% paraformaldehyde for 10 min at room temperature, rinsed with PBS, and permeabilized with PBS-Tween (0.2%) for 5 min at room temperature. Cells were subsequently blocked with 1% BSA in PBS for 1 h at room temperature. For CPD detection, cells were treated with 2 M HCl for 30 min to denature cellular DNA. After blocking, cells were incubated with an anti-CPD antibody (1:200; Cosmo Bio). Cells were then incubated with secondary anti-mouse IgG (Alexa Fluor 594) antibodies (1:200; Abcam) for 2 h at room temperature. Finally, cells were mounted in VECTASHIELD Vibrance Antifade Mounting Medium supplemented with DAPI and observed under a confocal microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative RT-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from treated cells using ISOGEN 2 (Nippon Gene, Tokyo, Japan), following the manufacturer\u0026rsquo;s instructions. The extracted RNA was reverse transcribed into cDNA using AMV Reverse Transcriptase (TaKaRa, Shiga, Japan). The primer sequences are listed in Supplementary Table\u0026nbsp;1. PCR amplification was performed as previously described [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] using a 7500 Fast Real-Time PCR system (Thermo Fisher Scientific). Relative target gene expression levels were normalized to that of GAPDH and calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. All experiments were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were analyzed using GraphPad Prism 9 software (GraphPad Software, Inc., La Jolla, CA, USA). All numerical data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) of experiments performed at least in triplicate. Results were considered statistically significant at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffect of 5-ALA on cell viability and proliferation\u003c/h2\u003e \u003cp\u003eWe previously reported that co-treatment with blue light and 5-ALA induced significant cell death [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. We thus aimed to further understand the effects of 5-ALA on cell proliferation. B16F1 and HaCaT cells were treated with various concentrations of 5-ALA (0.1 to 10 mM) and their proliferation was assessed. As shown in Supplementary Fig.\u0026nbsp;1A, even at the lowest concentration (0.1 mM), 5-ALA significantly inhibited cell proliferation in B16F1 cells. Similarly, HaCaT cells exhibited significant proliferation arrest in 0.1 mM 5-ALA (Supplementary Fig.\u0026nbsp;1B). To explore the temporal effect of 5-ALA, we treated cells with the same concentrations of 5-ALA for a shorter duration of 2 h, followed by an additional 72 h incubation in 5-ALA-free medium. Notably, a slight but significant inhibition of B16F1 cell proliferation was observed starting at 0.2 mM (Supplementary Fig.\u0026nbsp;1C). In contrast, HaCaT cells did not show significant inhibition of cell proliferation at any concentration tested (Supplementary Fig.\u0026nbsp;1D).\u003c/p\u003e \u003cp\u003eIn addition to our previous experiments, we investigated the protective role of NAC, an antioxidant, against the cytotoxic effects of blue light and 5-ALA co-treatment. NAC at 5 mM was included in the medium from one h before to one h after blue light exposure. Following this treatment, cells were incubated for 72 h in medium lacking 5-ALA or NAC. As shown in Supplementary Fig.\u0026nbsp;1E, B16F1 melanoma cells exhibited significantly reduced viability after exposure to blue light alone or in combination with 5-ALA, consistent with our previous findings. NAC did not ameliorate the decreased cell viability induced by blue light exposure. Similar results were observed in HaCaT cells (Supplementary Fig.\u0026nbsp;1F).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCo-treatment with blue light and 5-ALA increased intracellular ROS levels, induced depolarization of mitochondrial membrane potential (ΔΨm), and peroxidation of cardiolipin.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo elucidate the impact of blue light exposure and 5-ALA treatment on intracellular ROS levels, we performed flow cytometry using HE, which detects intracellular superoxide anions. In B16F1 melanoma cells, exposure to blue light for 60 min did not significantly increase ROS levels compared to those in the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Similarly, 5-ALA treatment alone did not significantly change ROS levels. However, when cells were co-treated with blue light and 5-ALA, a significant increase in the number of HE-positive cells was observed after 60 min of exposure (four-fold increase after 60 min of blue light exposure).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also assessed intracellular ROS levels in HaCaT cells, and exposure to blue light alone for 60 min decreased ROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Treatment with 5-ALA alone, similar to results with B16F1 melanoma cells, did not result in significant differences.\u003c/p\u003e \u003cp\u003eGiven that the data from HaCaT cells indicated that blue light treatment reduced the proportion of HE-positive cells, we conducted a time-course experiment with only blue light exposure. Blue light treatment resulted in a consistent decrease in ROS levels at all examined time points (Supplementary Fig.\u0026nbsp;2C).\u003c/p\u003e \u003cp\u003eFollowing our assessment of intracellular ROS, we investigated ΔΨm as an indicator of early apoptotic events. Flow cytometric analysis was conducted using the cell-permeable probe DiOC6, which selectively accumulates in mitochondria with an intact membrane potential. In B16F1 melanoma cells, neither blue light exposure nor 5-ALA treatment alone significantly changed ΔΨm (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). However, depolarization of the ΔΨm was observed in cells co-treated with blue light and 5-ALA. A similar pattern was observed in HaCaT cells, where co-treatment for 60 min resulted in a significant collapse of ΔΨm, indicating the potential for initiating apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003eAdditionally, we also assessed cardiolipin peroxidation, which is a marker of mitochondrial oxidative stress and apoptosis. NAO preferentially binds to non-oxidized cardiolipin, thus a decrease in NAO fluorescence is indicative of cardiolipin peroxidation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F, co-treatment with blue light and 5-ALA significantly reduced NAO fluorescence intensity in both cell lines.\u003c/p\u003e \u003cp\u003eWe also conducted these three experiments with cells co-treated for 30 min, which showed results similar to those of the 60 min co-treatment (Supplementary Fig.\u0026nbsp;2, 3, and 3, respectively).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMitochondrial morphology alterations induced by combined blue light and 5-ALA\u003c/h2\u003e \u003cp\u003eGiven our finding that co-treatment with blue light and 5-ALA induced ΔΨm collapse in B16F1 and HaCaT cells, we hypothesized that these treatments might also affect mitochondrial morphology. To investigate, we visualized mitochondria in live cells subjected to blue light and 5-ALA treatment using MitoTracker Green. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B16F1 cells exposed only to blue light exhibited mitochondria with tubular and branched morphologies, similar to those of the untreated controls. In cells co-treated with blue light and 5-ALA (at 0.2 and 1 mM concentrations), mitochondrial fluorescence appeared diffuse, and clear shapes were not discernible, suggesting potential mitochondrial disruption. In HaCaT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), the mitochondria maintained their typical morphology under blue light exposure alone. In contrast, cells treated with blue light and 0.2 mM 5-ALA showed numerous fragmented and rounded mitochondria that exhibited tremulous movements (data not shown). Mitochondria tended to cluster around the nucleus. More severe mitochondrial damage was evident in HaCaT cells co-treated with 1 mM 5-ALA, in which the mitochondrial structure was significantly disrupted.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also investigated the effects of co-exposure of B16F1 and HaCaT cells to blue light and 5-ALA using TEM. Cells were pre-treated with 0.2 or 0.02 mM 5-ALA for 1 h, followed by exposure to blue light for 1 h. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, exposure to blue light alone resulted in noticeable mitochondrial membrane damage in B16F1 cells. Co-treatment with 0.02 mM 5-ALA resulted in similar levels of mitochondrial damage to those observed with blue light alone. However, co-treatment with high concentrations of 5-ALA (0.2 mM) led to severe mitochondrial damage in B16F1 cells. HaCaT cells also exhibited no significant mitochondrial damage when exposed to blue light alone or in combination with 0.02 mM 5-ALA, but co-treatment of HaCaT with 0.2 mM 5-ALA resulted in evident mitochondrial damage. This experiment revealed not only membrane disruption but also the disappearance of cristae. Furthermore, extreme cellular damage was observed in both cell lines co-treated with 1 mM 5-ALA (data not shown).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003emtROS levels in response to blue light and 5-ALA\u003c/h2\u003e \u003cp\u003eTo assess the effect of blue light and 5-ALA co-treatment on mtROS levels, we used MitoSOX Red with confocal microscopic analysis. In B16F1 melanoma cells, exposure to blue light alone or co-treatment with 0.02 mM 5-ALA significantly increased mtROS levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). However, cells co-treated with higher concentrations of 5-ALA (0.2 mM) displayed reduced mtROS levels compared with those exposed only to blue light. Exposure to blue light alone did not increase mtROS levels in HaCaT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). Co-treatment with 0.2 mM 5-ALA in HaCaT cells slightly increased mtROS levels, in contrast to results observed in B16F1 cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCell cycle analyses and SubG1 population cells\u003c/h2\u003e \u003cp\u003eTo assess the extent of DNA fragmentation, which is an indicator of apoptosis, we quantified the subG1 cell population in cells treated with blue light and 5-ALA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B, co-treatment with blue light and 5-ALA significantly increased the subG1 population in both B16F1 melanoma and HaCaT cells. This increase was particularly pronounced after 60 min of exposure (a 60% increase in B16F1 cells). Further cell cycle analysis revealed that B16F1 cells exhibited altered cell cycle dynamics after treatment. Specifically, there was a significant decrease in the G1 phase coupled with an increase in the G2/M phase after 60 min of blue light exposure, with or without 5-ALA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These results suggest that blue light disrupts cell-cycle progression, potentially leading to cell-cycle arrest in the G2/M phase. HaCaT cells did not show significant alterations in the proportion of cells in different cell cycle phases after 30 min of blue light exposure, regardless of 5-ALA treatment (Supplementary Fig.\u0026nbsp;5C). However, after 60 min of 5-ALA exposure, cells in G1 were significantly increased, and cells in S and G2/M were decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results indicate that combined treatment with blue light and 5-ALA affects cell cycle progression in response to checkpoint activation or DNA damage.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eCo-treatment with blue light and 5-ALA increases γH2AX expression\u003c/h2\u003e \u003cp\u003eTo further investigate DNA damage, we assessed γH2AX, a well-established marker of DNA double-strand breaks (DSBs) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Previously, we performed a two-tailed comet assay to detect DSB and SSB in B16F1 and HaCaT cells, finding that co-treatment with blue light and 5-ALA induced DSB and SSB formation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. We thus attempted to quantify γH2AX expression by immunofluorescence. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B, UVC treatment, performed as a positive control, significantly enhanced γH2AX fluorescence intensity in both cell lines. Notably, co-treatment with blue light and 5-ALA also increased γH2AX expression in both B16F1 and HaCaT cells, suggesting induction of DNA damage. In B16F1 cells exposed to blue light, γH2AX expression was enhanced, whereas HaCaT cells exposed to blue light exhibited only a slight decrease in γH2AX fluorescence intensity compared to the control group. We employed confocal microscopy to observe γH2AX foci formation in treated cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, a significant increase in γH2AX foci was observed in all treatment groups, excluding the control, in B16F1 melanoma cells, indicating DNA damage response following exposure to blue light, 5-ALA co-treatment, and UV irradiation. In contrast, HaCaT cells exhibited a different response (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). While UV and 5-ALA co-treatment with blue light caused a similar response as in B16F1 cells, exposure to blue light alone markedly reduced numbers of cells with γH2AX foci.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eCo-treatment with blue light and 5-ALA did not enhance formation of CPDs, 6-4PPs, or Dewar PPs\u003c/em\u003e \u003c/p\u003e \u003cp\u003eIn our previous study, we established that visible light, including blue and red light, did not enhance CPD or 6-4PP formation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. We here investigated the potential formation of CPD, 6-4PP, and Dewar PP in cells co-treated with blue light and 5-ALA. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;D, co-treatment with blue light and 5-ALA did not enhance CPD or 6-4PP formation in either cell line. In contrast, UVC treatment significantly increased CPD and 6-4PP production. Notably, the presence of 5-ALA did not increase UVC-induced formation of these photoproducts. Furthermore, Dewar PP levels did not change in any treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, F). We performed confocal laser scanning microscopy to visualize CPD formation in B16F1 and HaCaT cells (Supplementary Fig.\u0026nbsp;6). These microscopy results were consistent with our ELISA data, demonstrating that blue light and 5-ALA did not significantly induce CPD formation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eInfluence of blue light and 5-ALA on gene expression\u003c/h2\u003e \u003cp\u003eWe previously reported blue-light induced alterations in the expression of apoptotic and melanogenic genes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In this study, we investigated the effects of blue light and 5-ALA co-treatment on transcription of a broad spectrum of genes in B16F1 and HaCaT cells.\u003c/p\u003e \u003cp\u003eB16F1 melanoma cells were exposed to blue light for 30 min in the presence of 0.2 mM 5-ALA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Time-point analyses of mRNA levels were conducted at 2, 4, 8, 16, and 24 h post-treatment. We performed similar analyses after 1 h of blue light exposure, but cells were severely damaged under these conditions, indicating that the data were unreliable (data not shown). An increase in \u003cem\u003ep21\u003c/em\u003e mRNA, a well-known cyclin-dependent kinase inhibitor involved in cell cycle regulation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], peaking at 4 h, was observed after blue light exposure. Expression of \u003cem\u003esurvivin\u003c/em\u003e, a gene associated with cell survival and inhibition of apoptosis, was also enhanced following irradiation. Transcription levels of \u003cem\u003eBax\u003c/em\u003e and \u003cem\u003eBcl-2\u003c/em\u003e, key genes in the apoptotic pathway, were also assessed. While \u003cem\u003eBax\u003c/em\u003e mRNA levels exhibited variability and lacked consistency post-treatment, \u003cem\u003eBcl-2\u003c/em\u003e mRNA levels were consistently suppressed immediately after treatment for up to 24 h. The \u003cem\u003eBcl-2\u003c/em\u003e gene, which is typically involved in inhibiting apoptosis, may indicate a shift towards a pro-apoptotic state [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Caspase-3 mRNA levels also increased 8 h post-irradiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHaCaT cells were exposed to blue light for 1 h in the presence of 0.2 mM 5-ALA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). After treatment, we conducted a time-course analysis of mRNA levels at 2, 4, 8, 16, and 24 h. \u003cem\u003eMatrix Metalloproteinase-1\u003c/em\u003e (\u003cem\u003eMMP-1\u003c/em\u003e), a gene associated with extracellular matrix remodeling and skin aging [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], showed a gradual increase in mRNA levels post-irradiation. \u003cem\u003ep21\u003c/em\u003e exhibited an early response to treatment. \u003cem\u003eHyaluronan synthase\u003c/em\u003e (\u003cem\u003eHAS\u003c/em\u003e)\u003cem\u003e-2\u003c/em\u003e mRNA levels also significantly increased. \u003cem\u003eCOL1a\u003c/em\u003e, a key component of type I collagen, exhibited a slight increase in mRNA levels 4 h post-irradiation. \u003cem\u003eSirtuin 1\u003c/em\u003e (\u003cem\u003eSIRT-1\u003c/em\u003e) is an NAD\u003csup\u003e+\u003c/sup\u003e-dependent class III protein deacetylase recognized for its role in promoting cell survival in response to DNA damage and associated with enhanced cellular longevity and stress resistance [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Our findings demonstrate a modest elevation in \u003cem\u003eSIRT-1\u003c/em\u003e levels beginning at 8 h post-irradiation. This observation is consistent with the role of \u003cem\u003eSIRT\u003c/em\u003e-1 in cellular defense mechanisms against oxidative damage. \u003cem\u003eNuclear factor erythroid 2-related factor 2\u003c/em\u003e (\u003cem\u003eNrf-2\u003c/em\u003e), a critical early regulator of the antioxidant response, initially increased 2 h post-irradiation and subsequently decreased.\u003c/p\u003e \u003cp\u003eHaCaT cells were treated with various durations of blue light exposure (15, 30, 60, and 120 min) in the presence or absence of 1 mM 5-ALA. Following irradiation, cells were incubated for an additional 3 h before quantification by qRT-PCR. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC shows the responses of specific genes to blue light exposure with and without 5-ALA treatment. \u003cem\u003eMMP-1\u003c/em\u003e mRNA levels increased significantly following blue light exposure alone, but in the presence of 5-ALA, \u003cem\u003eMMP-1\u003c/em\u003e levels initially increased at 15 min and subsequently decreased. \u003cem\u003ep21\u003c/em\u003e mRNA levels consistently increased in response to irradiation, regardless of 5-ALA presence, displayed time-dependent upregulation. Exposure to blue light alone increased \u003cem\u003eHAS-2\u003c/em\u003e mRNA levels at 15 and 30 min, but a slight decrease was observed at 120 min. When combined with 5-ALA, \u003cem\u003eHAS-2\u003c/em\u003e levels increased only at 15 min, followed by a decrease after longer exposure. \u003cem\u003eCOL1a\u003c/em\u003e mRNA levels modestly increased after blue light exposure. However, in the presence of 5-ALA, a decrease was observed after 30 min of exposure. \u003cem\u003eSIRT-1\u003c/em\u003e and \u003cem\u003eNrf-2\u003c/em\u003e mRNA levels slightly increased upon blue light exposure alone, but decreased when cells were co-treated with 5-ALA.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the effects of blue light exposure alone and in combination with 5-ALA on B16F1 melanoma and HaCaT cells. In our previous study, we demonstrated that blue and green light elevate ROS levels in B16F1 cells, leading to cell death [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Additionally, we found that co-treatment with 5-ALA severely damaged DNA in both B16F1 and HaCaT cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. This study aimed to elucidate relevant cellular mechanisms by which these effects are exerted, including impacts on mitochondrial morphology, DNA integrity, and gene transcription.\u003c/p\u003e \u003cp\u003eWe first assessed the effects of 5-ALA on B16F1 and HaCaT cell proliferation. In B16F1 cells, we observed suppression of cell proliferation during short-term treatment. No significant inhibition of proliferation was observed in HaCaT cells. Notably, prolonged treatment (72 h) with 5-ALA significantly suppressed proliferation in both cell lines. Kumar et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] reported similar findings, in which the proliferation of three types of hepatocarcinoma cell lines was significantly suppressed in the presence of 0.6 mM 5-ALA. In addition, Sparsa et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] found that 5-ALA suppressed the viability of B16F1 melanoma cells. Grigalavicius et al. suggested that 5-ALA might act as a lactate dehydrogenase inhibitor to induce cancer cell death [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These results suggest that 5-ALA or its intermediates can affect the cell cycle and cell proliferation. We also investigated the ameliorating effects of NAC on the inhibition of cell proliferation induced by co-treatment with blue light and 5-ALA. Notably, NAC was unable to rescue the inhibition of proliferation. These results suggest that while NAC can mitigate additional oxidative stress, it does not significantly counteract the reduction in cell viability caused by blue light and 5-ALA co-treatment in both B16F1 melanoma and HaCaT cells.\u003c/p\u003e \u003cp\u003eWe next examined the effects of blue light and 5-ALA co-treatment on intracellular ROS, ΔΨm, and cardiolipin peroxidation in B16F1 and HaCaT cells. B16F1 cells did not exhibit increased ROS levels upon exposure to blue light alone; however, a significant increase was observed upon 5-ALA co-treatment. ROS levels were also enhanced in HaCaT cells after co-treatment. Notably, blue light alone decreased ROS levels in HaCaT cells, suggesting a potential cell-protective effect of blue light in certain cell types. Sutterby et al. demonstrated that monochromatic visible light (yellow, orange, and red light) induces proliferation and wound healing in HaCaT cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The effect of blue light was not assessed, thus this question requires further investigation. Neither blue light nor 5-ALA alone affected ΔΨm in either cell line. However, co-treatment resulted in depolarization of ΔΨm in both cell lines. As ΔΨm depolarization is an early indicator of apoptosis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], this result suggests that co-treatment promotes cell death. We also assessed cardiolipin peroxidation, a process that weakens cytochrome c retention in the inner mitochondrial membrane, triggering subsequent apoptosis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Our findings showed that neither blue light nor 5-ALA alone induced cardiolipin peroxidation in either cell line. However, co-treatment led to significant cardiolipin peroxidation. These observations suggest that co-treatment with blue light and 5-ALA induce significant cell death in both B16F1 and HaCaT cells, primarily through elevating ROS levels and promoting mitochondrial dysfunction. Notably, a previous report indicated that the combination of ultrasound and 5-ALA increased ROS levels, promoting subsequent cell death in a human leukemia cell line [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGiven that co-treatment induced ΔΨm depolarization and cardiolipin peroxidation, we investigated the effect of blue light and 5-ALA co-treatment on mitochondrial morphology using imaging techniques. Using MitoTracker imaging, we observed that blue light alone did not significantly change mitochondrial morphology in either B16F1 or HaCaT cells. However, co-treatment with 0.2 mM 5-ALA in B16F1 cells induced a noticeable loss of mitochondrial structure, suggesting severe damage. In HaCaT cells, the same co-treatment caused elongated mitochondria to become increasingly smaller and more granular. Such mitochondrial fragmentation, often linked to ΔΨm depolarization [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], may aid in removing damaged mitochondria and help cells recover from stress [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This effect was even more evident at 1 mM 5-ALA, which apparently dispersed the mitochondria. Both cell lines showed mitochondrial clustering near the nucleus (nuclear periphery) after co-treatment, a response possibly related to increased intracellular ROS levels and cellular stress. This morphological change was more pronounced at 1 mM 5-ALA, where the mitochondria appeared to be dispersed. Some reports have indicated that perinuclear clustering of mitochondria occurs in response to intracellular ROS accumulation and several other types of cell stress [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These observations suggest that co-treatment with blue light and 5-ALA can induce significant mitochondrial stress and morphological changes.\u003c/p\u003e \u003cp\u003eTEM also provided intriguing insights into mitochondrial damage in response to various treatment conditions. In B16F1 cells, exposure to blue light alone was sufficient to induce significant mitochondrial membrane damage, suggesting that blue light exerts a detrimental effect on mitochondrial functionality. Moreover, increased mitochondrial damage was observed in B16F1 cells co-treated with a higher concentration of 5-ALA (0.2 mM). This exacerbated damage was attributed to the potential synergistic effects of blue light and 5-ALA on mitochondrial integrity. In contrast, HaCaT cells exhibited resistance to mitochondrial damage upon blue light exposure and low-concentration 5-ALA co-treatment. However, similar to B16F1 cells, HaCaT cells exhibit clear mitochondrial damage when treated with 0.2 mM 5-ALA. TEM analysis revealed a notable disappearance of mitochondrial cristae after co-treatment. Fu et al. reported that mitochondrial DNA DSBs result in loss of membrane potential and abnormal or missing cristae [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The alterations in mitochondrial morphology observed in our study could be a consequence of similar DNA damage, potentially affecting both mitochondrial and nuclear DNA.\u003c/p\u003e \u003cp\u003eWe also assessed mtROS levels in B16F1 and HaCaT cells following blue light exposure and 5-ALA co-treatment. In B16F1 cells, blue light alone increased mtROS. This increase was not observed upon co-treatment with 0.2 mM 5-ALA, which may be attributable to severe mitochondrial damage. In contrast, HaCaT cells did not show a significant increase in mtROS levels with either blue light alone or with low 5-ALA concentration. Notably, an increase in mtROS was detected with 0.2 mM 5-ALA co-treatment. These results suggest that the differential responses to treatment may be due to variations in cellular sensitivity.\u003c/p\u003e \u003cp\u003eWe also explored the effects of blue light and 5-ALA co-treatment on the subG1 cell population and on cell cycle progression. Co-treatment markedly increased the number of subG1 events in both cell lines, indicating the induction of DNA fragmentation, which is a hallmark of apoptosis. Co-treatment of B16F1 cells led to cell cycle arrest at the G2/M checkpoint, a response associated with DNA damage [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In contrast, HaCaT cells exposed to blue light for 60 min showed an increase in cells in G1 and a decrease in cells in G2/M. These findings suggest that co-treatment may induce cell cycle arrest in response to DNA damage. Consequently, we assessed multiple types of DNA damage following co-treatment.\u003c/p\u003e \u003cp\u003eTo investigate DNA damage, we first assessed γH2AX, a marker of DNA DSBs. In both B16F1 and HaCaT cells, γH2AX levels increased following UVC irradiation. Notably, in B16F1 cells, exposure to blue light alone also elevated γH2AX levels. Co-treatment with blue light and 5-ALA increased γH2AX levels in both cell lines. These findings reinforce those of our previous study, which indicated the induction of DSBs and SSBs under similar experimental conditions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In HaCaT cells, blue light alone decreased γH2AX fluorescence intensity compared with control levels. This observation suggests a potential reduction in cellular stress. Regarding CPD and 6-4PP, key markers of DNA base damage, neither blue light exposure nor co-treatment induced an increase in any of the cell lines. It is important to note that shorter wavelengths of visible light may cause the formation of CPD and other photoproducts [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In contrast, UVC irradiation significantly elevated CPD and 6-4PP levels in both cell lines. However, co-treatment with 5-ALA and UVC did not affect the production of these photoproducts. Regarding Dewar PP, which are formed through photoisomerization of 6-4PP under UVA [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], no significant changes were observed under any treatment conditions.\u003c/p\u003e \u003cp\u003eOur study revealed significant changes in gene expression in B16F1 and HaCaT cells following exposure to blue light and co-treatment with 5-ALA. In B16F1 cells, we observed upregulation of \u003cem\u003ep21\u003c/em\u003e and \u003cem\u003ecaspase-3\u003c/em\u003e, alongside downregulation of \u003cem\u003eBcl-2\u003c/em\u003e. These changes in gene expression suggest the initiation of apoptosis. \u003cem\u003eSurvivin\u003c/em\u003e also exhibited variable responses, in contrast to our previous findings, where long-term blue light exposure significantly inhibited \u003cem\u003esurvivin\u003c/em\u003e transcription in B16F1 cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This discrepancy may be attributed to differences in experimental conditions, particularly in the duration of blue light exposure. Notable increases in \u003cem\u003eMMP-1\u003c/em\u003e, \u003cem\u003ep21\u003c/em\u003e, and \u003cem\u003eHAS-2\u003c/em\u003e levels, with slight alterations in \u003cem\u003eCOL1a\u003c/em\u003e and \u003cem\u003eSIRT-1\u003c/em\u003e levels were observed. UV radiation can enhance \u003cem\u003eMMP-1\u003c/em\u003e levels, leading to collagen fiber degradation, contributing to skin aging and wrinkling [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. MMPs play a role in cancer invasion [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Our results suggest that blue light and co-treatment with 5-ALA also elevates \u003cem\u003eMMP-1\u003c/em\u003e expression, potentially leading to similar detrimental effects. In our study, we observed a notable upregulation of \u003cem\u003ep21\u003c/em\u003e, a cyclin-dependent kinase inhibitor, in both B16F1 and HaCaT cells following co-treatment with blue light and 5-ALA. p21, a critical regulator of cell cycle progression, is often induced in response to DNA damage, and serves as a major target of the tumor suppressor p53 [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. p21 is also implicated in cellular responses to stress and can confer protection against apoptosis under certain condition [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The enhanced \u003cem\u003ep21\u003c/em\u003e expression observed in our experiments suggests that co-treatment with blue light and 5-ALA induces cell cycle arrest. Combined blue light and 5-ALA exposure induced complex, time-dependent alterations in gene expression, rendering discussion difficult. Further research is needed to fully understand these complex changes. However, our findings provide foundational data for future studies in this area.\u003c/p\u003e \u003cp\u003eIn conclusion, this study elucidated the effects of co-treatment with blue light and 5-ALA on B16F1 melanoma and HaCaT cells. Our findings reveal that this co-treatment induced significant cellular stress, as evidenced by the elevation in intracellular ROS levels, ΔΨm collapse, and DNA damage. Additionally, alterations in mitochondrial morphology and induction of apoptosis-related gene expression were observed following blue light exposure with 5-ALA. Although these results advance our understanding of the cellular responses to blue light and 5-ALA, they also raise concerns regarding potential adverse effects of 5-ALA in normal cells. This study highlights the need for cautious application of 5-ALA in clinical settings, considering its potential to induce damage in target cancer cells and normal cells.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eThe datasets use/or analyzed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e \u003c/div\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eK.S. designed the experiments. K.S., T.S., R.H., and M.B. performed the experiments and analyzed the data. K.S. and M.B. wrote the manuscript. All authors have approved the final manuscript. The authors have read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVayalil, P.K., Mittal, A., Hara, Y., Elmets, C.A. \u0026amp; Katiyar, S.K. Green tea polyphenols prevent ultraviolet light-induced oxidative damage and matrix metalloproteinase expression in mouse skin. J. Invest Dermatol. 2004, 122, 1480\u0026ndash;1487.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhivagui, M. et al. DNA damage and somatic mutations in mammalian cells after irradiation with a nail polish dryer. Nat. Commun. 2021, 14, 276.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXia, D. et al. Combined Analysis of the Effects of Exposure to Blue Light in Ducks Reveals a Reduction in Cholesterol Accumulation Through Changes in Methionine Metabolism and the Intestinal Microbiota. Front. Nutr. 2021, 8, 737059.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlghamdi, R.A. et al. Assessing Phototoxicity in a Mammalian Cell Line: How Low Levels of Blue Light Affect Motility in PC3 Cells. Front. Cell Dev. Biol. 2021, 9, 738786.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eH\u0026ouml;yt\u0026ouml;, A. et al. Cellular detection of 50 Hz magnetic fields and weak blue light: effects on superoxide levels and genotoxicity. Int. J. Radiat. Biol. 2017, 93, 646\u0026ndash;652.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato, K., Minai, Y. \u0026amp; Watanabe, H. Effect of monochromatic visible light on intracellular superoxide anion production and mitochondrial membrane potential of B16F1 and B16F10 murine melanoma cells. Cell Biol. Int. 2013, 37, 633\u0026ndash;637.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishio, T., Kishi, R. \u0026amp; Sato, K. Blue light exposure enhances oxidative stress, causes DNA damage, and induces apoptosis signaling in B16F1 melanoma cells. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 2022, 883\u0026ndash;884, 503562.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang, Z. et al. Recent advances in production of 5-aminolevulinic acid using biological strategies. World J. Microbiol. Biotechnol. 2017, 33, 200.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKiening, M. \u0026amp; Lange, N. A Recap of Heme Metabolism towards Understanding Protoporphyrin IX Selectivity in Cancer Cells. Int. J. Mol. Sci. 2022, 23, 7974.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, J. et al. Exogenous Application of 5-Aminolevulinic Acid Promotes Coloration and Improves the Quality of Tomato Fruit by Regulating Carotenoid Metabolism. Front. Plant Sci. 2021, 12, 683868.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJalili-Nik, M. et al. Antitumor Effects of 5-Aminolevulinic Acid on Human Malignant Glioblastoma Cells. Int. J. Mol. Sci. 2021, 22, 5596.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRyabova, A. et al. Detection of Changes in Macrophage Polarization as a Result of 5-Aminolevulinic Acid Photodynamic Therapy Using Fluorescence-Lifetime Imaging Microscopy. Photonics. 2022, 9, 961.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHelander, L. et al. Red versus blue light illumination in hexyl 5-aminolevulinate photodynamic therapy: The influence of light color and irradiance on the treatment outcome in vitro. J. Biomed. Opt. 2018, 19, 088002.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato, T. \u0026amp; Sato, K. Adverse Effect of Blue Light on DNA Integrity Is Accelerated by 5-Aminolevulinic Acid in HaCaT Human Keratinocyte Cells and B16F1 Murine Melanoma Cells. Biology. 2022, 11, 1743.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNunez, R. DNA Measurement and Cell Cycle Analysis by Flow Cytometry. Curr. Issues Mol. Biol. 2001, 3, 67\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eetrosillo, G., Ruggiero, F.M. \u0026amp; Paradies, G. Role of reactive oxygen species and cardiolipin in the release of cytochrome c from mitochondria. FASEB J. 2003, 17, 2202\u0026ndash;2208.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeno, S. et al. CoCl(2) induces apoptosis via the 18 kDa translocator protein in U118MG human glioblastoma cells. Biochemistry. 2009, 48, 4652\u0026ndash;4661.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRedon, C.E. et al. γ-H2AX as a biomarker of DNA damage induced by ionizing radiation in human peripheral blood lymphocytes and artificial skin. Adv. Space Res. 2009, 43, 1171\u0026ndash;1178.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNishimura, K. et al. Seasonal Differences in the UVA/UVB Ratio of Natural Sunlight Influence the Efficiency of the Photoisomerization of (6\u0026thinsp;\u0026ndash;\u0026thinsp;4) Photoproducts into their Dewar Valence Isomers. Photochem. Photobiol. 2021, 97, 582\u0026ndash;588.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD ouki, T. Relative Contributions of UVB and UVA to the Photoconversion of (6\u0026thinsp;\u0026ndash;\u0026thinsp;4) Photoproducts into their Dewar Valence Isomers. Photochem. Photobiol. 2016, 92, 587\u0026ndash;594.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQian, S. et al. The role of BCL-2 family proteins in regulating apoptosis and cancer therapy. Front. Oncol. 2022, 12, 985363.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaradeniz, F., Oh, J.H., Kim, H.R., Ko, J. and Kong, C.-S. Camellioside A, isolated from Camellia japonica flowers, attenuates UVA-induced production of MMP-1 in HaCaT keratinocytes via suppression of MAPK activation. Exp. Ther. Med. 2021, 21, 16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBitar, S.A. \u0026amp; Gali-Muhtasib, H. The Role of the Cyclin Dependent Kinase Inhibitor p21cip1/waf1 in Targeting Cancer: Molecular Mechanisms and Novel Therapeutics. Cancers. 2019, 11, 1475.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKume, S. et al. Silent information regulator 2 (SIRT1) attenuates oxidative stress-induced mesangial cell apoptosis via p53 deacetylation. Free Radic. Biol. Med. 2006, 40, 2175\u0026ndash;2182.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo, J. et al. Negative control of p53 by Sir2alpha promotes cell survival under stress. Cell. 2001, 107, 137\u0026ndash;148.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar, A. et al. An Efficient 5-Aminolevulinic Acid Photodynamic Therapy Treatment for Human Hepatocellular Carcinoma. Int. J. Mol. Sci. 2023, 24, 10426.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSparsa, A. et al. Photodynamic treatment induces cell death by apoptosis or autophagy depending on the melanin content in two B16 melanoma cell lines. Oncol. Rep. 2013, 29, 1196\u0026ndash;1200.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrigalavicius, M. et al. 5-ALA Is a Potent Lactate Dehydrogenase Inhibitor but Not a Substrate: Implications for Cell Glycolysis and New Avenues in 5-ALA-Mediated Anticancer Action. Cancers. 2022, 14, 4003.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSutterby, E. et al. Investigating the effects of low intensity visible light on human keratinocytes using a customized LED exposure system. Sci. Rep. 2022, 12, 18907.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNabekura, T., Ishikawa, S., Tanase, M., Okumura, T., Kawasaki, T. Antidepressants induce toxicity in human placental BeWo cells. Curr. Res. Toxicol. 2022, 3, 100073.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, G.-X. et al. Inhibition of cytochrome c release by 10-N-nonyl acridine orange, a cardiolipin-specific dye, during myocardial ischemia-reperfusion in the rat. Am. J. Physiol. Heart Circ. Physiol. 2010, 298, H433-H439.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun, X. et al. Potential involvement of the 18 kDa translocator protein and reactive oxygen species in apoptosis of THP-1 macrophages induced by sonodynamic therapy. PLOS One. 2018, 13, e0196541.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachiela, E. et al. Disruption of mitochondrial dynamics increases stress resistance through activation of multiple stress response pathways. FASEB J. 2020, 34, 8475\u0026ndash;8492.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFernandes, T. et al. Structural and Functional Alterations in Mitochondria-Associated Membranes (MAMs) and in Mitochondria Activate Stress Response Mechanisms in an In Vitro Model of Alzheimer's Disease. Biomedicines. 2021, 9, 881.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolfer, L. et al. Functional Consequences of PDK4 Deficiency in Doberman Pinscher Fibroblasts. Sci. Rep. 2020, 10, 3930.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Mehdi, A.-B. et al. Perinuclear mitochondrial clustering creates an oxidant-rich nuclear domain required for hypoxia-induced transcription. Sci. Signal. 2012, 5, ra47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu, Y. et al. Mitochondrial DNA breaks activate an integrated stress response to reestablish homeostasis. Mol. Cell. 2023, 83, 3740\u0026ndash;3753.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLim, Y., Dorstyn, L. \u0026amp; Kumar, S. The p53-caspase-2 axis in the cell cycle and DNA damage response. Exp. Mol. Med. 2021, 53, 517\u0026ndash;527.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawrence, K.P. et al. The UV/Visible Radiation Boundary Region (385\u0026ndash;405 nm) Damages Skin Cells and Induces \"dark\" Cyclobutane Pyrimidine Dimers in Human Skin in vivo. Sci. Rep. 2018, 8, 12722.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDouki, T. Pyrimidine (6\u0026thinsp;\u0026ndash;\u0026thinsp;4) Pyrimidone Photoproducts in UVA-Irradiated DNA: Photosensitization or Photoisomerization? ChemPhotoChem. 2020, 4, 294\u0026ndash;299.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDouki, T. Relative Contributions of UVB and UVA to the Photoconversion of (6\u0026thinsp;\u0026ndash;\u0026thinsp;4) Photoproducts into their Dewar Valence Isomers. Photochem. Photobiol. 2016, 92, 587\u0026ndash;594.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHwang, E., Lee, T.H., Park, S.-Y. \u0026amp; Kim, S.Y. Enzyme-modified Panax ginseng inhibits UVB-induced skin aging through the regulation of procollagen type I and MMP-1 expression. Food Funct. 2014, 5, 265\u0026ndash;274.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark, E.K. et al. The Anti-Wrinkle Mechanism of Melatonin in UVB Treated HaCaT Keratinocytes and Hairless Mice via Inhibition of ROS and Sonic Hedgehog Mediated Inflammatory Proteins. Int. J. Mol. Sci. 2018, 19, 1995.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAkter, H. et al. Activation of matrix metalloproteinase-9 (MMP-9) by neurotensin promotes cell invasion and migration through ERK pathway in gastric cancer. Tumour Biol. 2015, 436, 6053\u0026ndash;6062.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoto-Guzman, A., Navarro-Tito, N., Castro-Sanchez, L., Martinez-Orozco, R., Salazar, E.P. Oleic acid promotes MMP-9 secretion and invasion in breast cancer cells. Clin. Exp. Metastasis. 2010, 27, 505\u0026ndash;515.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRattanasinchai, C., Llewellyn, B.J., Conrad, S.E. \u0026amp; Gallo, K.A. MLK3 regulates FRA-1 and MMPs to drive invasion and transendothelial migration in triple-negative breast cancer cells. Oncogenesis. 2017, 6, e345.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, G. et al. RNA-binding protein CELF6 is cell cycle regulated and controls cancer cell proliferation by stabilizing p21. Cell Death Dis. 2019, 10, 688.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEngeland, K. Cell cycle regulation: p53-p21-RB signaling. Cell Death Differ. 2022, 29, 946\u0026ndash;960.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarimian, A., Ahmadi, Y. \u0026amp; Yousefi, B. Multiple functions of p21 in cell cycle, apoptosis and transcriptional regulation after DNA damage. DNA Repair 2016, 42, 63\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3894182/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3894182/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eInvestigating the effect of blue light and 5-aminolevulinic acid (5-ALA) co-treatment, this study examined its effects on B16F1 melanoma cells and HaCaT keratinocytes. Our focus was on cellular responses including mitochondrial function, DNA integrity, and gene expression. Co-treatment significantly damaged mitochondria, alters their morphology, induced mitochondrial membrane depolarization, increased intracellular reactive oxygen species (ROS), and led to cardiolipin peroxidation in both cell types. This approach also increased DNA fragmentation and apoptosis. Notably, it triggers complex, time-dependent changes in gene expression, particularly upregulating MMP-1 and p21 in HaCaT cells. Our findings reveal that blue light and 5-ALA co-treatment causes substantial cellular stress and damage, suggesting its therapeutic potential against melanoma, while also highlighting the need for caution and precision in its application to avoid harming normal cells. This underscores the necessity for further research to refine therapeutic approaches.\u003c/p\u003e","manuscriptTitle":"Effects of Combined Blue light and 5-ALA on cell death in B16F1 melanoma and HaCaT cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-08 04:43:49","doi":"10.21203/rs.3.rs-3894182/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"25e70f41-dd98-4740-95f2-a9fca0def0ae","owner":[],"postedDate":"February 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28625236,"name":"Biological sciences/Molecular biology/Dna damage and repair/Dna damage response"},{"id":28625237,"name":"Biological sciences/Molecular biology/Dna damage and repair/Double strand dna breaks"},{"id":28625238,"name":"Biological sciences/Molecular biology/Dna damage and repair/Single strand dna breaks"},{"id":28625239,"name":"Biological sciences/Molecular biology/Cell division/Cell cycle exit"},{"id":28625240,"name":"Biological sciences/Cell biology/Cell death/Apoptosis"},{"id":28625241,"name":"Biological sciences/Cell biology/Cell division/Cell growth"},{"id":28625242,"name":"Biological sciences/Cancer/Skin cancer/Melanoma"}],"tags":[],"updatedAt":"2024-03-25T15:00:30+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-08 04:43:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3894182","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3894182","identity":"rs-3894182","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}