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. 2024 Sep 11;76(6):795–816. doi: 10.1007/s10616-024-00654-x

Effect of combined blue light and 5-ALA on mitochondrial functions and cellular responses in B16F1 melanoma and HaCaT cells

Kazuomi Sato 1,2,✉, Taiki Sato 1, Riku Hirotani 1, Munetsugu Bam 3
PMCID: PMC11490642  PMID: 39435424

Abstract

In this study, we investigated the effects of blue light and 5-aminolevulinic acid (5-ALA) co-treatment on B16F1 melanoma cells and HaCaT keratinocytes. We focused on cellular responses, including mitochondrial function, DNA integrity, and gene expression. Co-treatment significantly damaged the mitochondria, altered their morphology, induced mitochondrial membrane depolarization, increased intracellular reactive oxygen species, and led to cardiolipin peroxidation in both cell types. This approach promoted DNA fragmentation and apoptosis. However, blue light and co-treatment with 5-ALA did not enhance the formation of cyclobutane pyrimidine dimers, 6–4 photoproducts, or Dewar photoproducts. Moreover, it triggered complex, time-dependent changes in gene expression, particularly the upregulation of MMP-1 and p21 in HaCaT cells. Our findings revealed that blue light and 5-ALA co-treatment caused substantial cellular stress and damage, suggesting their therapeutic potential against melanoma and highlighting the need for caution and precision in their application to avoid harming normal cells. This underscores the necessity for further research to refine therapeutic approaches.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10616-024-00654-x.

Keywords: Blue light, 5-Aminolevulinic acid, Cell death, Mitochondria, DNA damage

Introduction

Ultraviolet (UV) light, a component of solar radiation, has profound effects on various life forms, particularly animals. Chronic exposure to solar UV light, particularly in humans, has been linked to photoaging, characterized by UV-induced oxidative damage and the induction of matrix metalloproteinase (MMP) expression (Vayalil et al. 2004). Such exposure can significantly alter the skin’s properties, with oxidative stress playing a central role in the damage. In mice, the detrimental effects of UV radiation are evident in the skin, where UV exposure can cause protein oxidation, a hallmark of photoaging (Vayalil et al. 2004). At the cellular level, UV radiation causes DNA damage, leading to mutations that result in cancer initiation and growth. UV radiation induces the formation of cyclobutane pyrimidine dimers (CPDs) between neighboring pyrimidine bases (Zhivagui et al. 2021).

The effects of blue light, a component of visible light, on various biological processes in animals and cells have been extensively studied. The increasing use of blue light in various applications provides an impetus for studies aimed at understanding its effects on cellular and metabolic processes. Xia et al. (2021) reported that irradiating ducks with blue light (460 nm) decreased their body weight and enhanced their anti-inflammatory and antioxidant responses. DNA damage and somatic mutations have been observed in mammalian cells irradiated with a nail polish dryer (Xia et al. 2021). Phototoxicity, particularly in live-cell fluorescence microscopy, is a concern associated with blue light exposure. Alghamidi et al. (2021) showed that even low levels of blue light could affect the motility of PC3 human prostate cancer cells. In human SH-SY5Y neuroblastoma cells, notable interactions were observed between blue light and a 50 Hz magnetic field (MF) (Höytö et al. 2017). Combined exposure to MF and blue light modulates superoxide levels. Additionally, we reported that blue light induces the collapse of mitochondrial membrane potential and subsequent cell death (Sato et al. 2013; Nishio et al. 2022).

5-aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid that serves as a precursor for heme biosynthesis (Kang et al. 2017; Kiening and Lange 2022). In plants, 5-ALA improves tomato coloration by regulating carotenoid metabolism and promoting fruit maturation (Wang et al. 2021). In contrast, 5-ALA exerts cytotoxic effects on malignant glioblastoma cells by increasing apoptosis, altering the expression of apoptosis-related genes, and enhancing reactive oxygen species (ROS) generation (Jalili-Nik et al. 2021). These findings highlight the multifaceted effects of 5-ALA in cells, ranging from human cancer cells to plant cells.

In photodynamic therapy (PDT), 5-ALA is converted to protoporphyrin IX (PPIX), which acts as a photosensitizer by producing ROS in response to light exposure (Ryabova et al. 2022). Helander et al. (2018) demonstrated that the efficacy of PDT is several times higher with blue light than with red light in several cancer cell lines. We previously demonstrated that co-treatment with blue light and 5-ALA induces severe DNA damage, including double-strand breaks (DSBs) and single-strand breaks (SSBs), in both melanoma and normal keratinocyte cell lines (Sato and Sato 2022). Although numerous studies have described ALA-photodynamic therapy (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 cells 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, DNA damage, and the 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-Aldrich, 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% CO2. For the blue light treatments, we used blue light-emitting diode (LED) lamps with peak emission at 465 nm. Detailed information on the irradiance of the LED lamps is available in our previous study (Sato et al. 2022).

Cell viability and proliferation

To assess cell proliferation and viability, the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed. For experiments involving varying concentrations of 5-ALA, HaCaT and B16F1 cells were seeded into 96-well plates at a density of 2.0 × 103 cells/well. After 24 h of incubation, cells were treated with different concentrations of 5-ALA (Sigma) for specified durations. After treatment, the cells were washed with phosphate-buffered saline (PBS), and the 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. The resultant formazan crystals were dissolved in dimethyl sulfoxide, and the absorbance at 590 nm was measured using a microplate reader. For experiments involving co-treatment with 50 W/m2 blue light and 5-ALA in the presence of N-acetyl cysteine (NAC; Fujifilm Wako, Osaka, Japan), B16F1 and HaCaT cells were seeded into 35 mm dishes at a density of 2 × 104 cells/dish. The cells were pre-treated with 5 mM NAC 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 and the MTT assay was performed as described above.

Intracellular superoxide anion production and mitochondrial membrane potential (ΔΨm)

Intracellular ROS levels were measured as previously described (Sato et al. 2022). B16F1 and HaCaT cells were seeded in 35-mm dishes at a density of 1 × 105 cells/dish. After incubation for 24 h, cells were pretreated with 1 mM 5-ALA for 1 h, followed by irradiation with 50 W/m2 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 the level of cardiolipin peroxidation, we used nonyl acridine orange (NAO; Sigma), a fluorescent dye that binds with high affinity to non-oxidized cardiolipin. B16F1 and HaCaT cells were seeded into 35-mm dishes at a density of 1 × 105 cells/dish. After 24 h of incubation, the cells were pretreated with 1 mM 5-ALA for 1 h and then irradiated with 50 W/m2 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. The 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 into glass-bottomed 35-mm dishes at a density of 1 × 105 cells/dish. After 24 h of incubation, the cells were treated with the vehicle or various concentrations of 5-ALA for 1 h, and subsequently exposed to 50 W/m2 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, the cells were washed three times to remove the excess probe. For nuclear staining, cells were incubated with 1 µM Hoechst 33,342 (Thermo Fisher Scientific) in DMEM for 10 min at 37 °C. Finally, the cells were visualized using an LSM 700 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).

Transmission electron microscopy (TEM)

To elucidate ultrastructural changes in the mitochondria following treatment, we used TEM. After 50 W/m2 blue light and 5-ALA treatment, 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 (OsO4) in the phosphate buffer. After fixation, cells were washed and embedded in 1% agarose. Agarose-embedded samples were trimmed into 1 mm cubes and dehydrated using an ethanol series. Subsequently, the samples were substituted with propylene oxide and embedded in an epresin mixture. These samples were cut into ultrathin sections of 70 nm thickness and stained with EM Stainer (Nisshin EM Co., Ltd., Tokyo, Japan) and Reynolds lead citrate. The analyses were done using a TEM JEM-2100F (JEOL Co., Ltd., Tokyo, Japan) at an acceleration voltage of 120 kV.

Mitochondrial ROS (mtROS) detection

MitoSOX Red was used as a probe (Thermo Fisher Scientific) to detect mtROS levels. After 50 W/m2 blue light and 5-ALA treatment, cells were incubated with DMEM containing 5 µM MitoSOX Red for 10 min at 37 °C. After incubation, the cells were washed and stained with 1 µM Hoechst 33,342 in DMEM for 10 min at 37 °C. The cells were visualized by confocal microscopy.

Cell cycle analysis and subG1 cell detection

To analyze cell cycle progression and detect apoptotic cells in the subG1 phase, B16F1 and HaCaT cells were subjected to identical intracellular ROS treatments. After treatment, both attached (live) and detached (dead) cells were harvested, fixed with ice-cold 70% ethanol, and incubated for 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 the 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 (2001), which effectively discriminates between single cells and doublets or aggregates. For the cell cycle analysis, the proportions of cells in the G1, S, and G2/M phases was calculated by explicitly gating out the subG1 population.

Evaluation of DNA damage using γ-H2AX immunofluorescence

To assess DNA damage, cells were seeded into glass-bottomed 35-mm dishes at a density of 1 × 105 cells/dish and incubated overnight. The cells were treated with or without 5-ALA and exposed to 50 W/m2 blue light. Following treatment, the cells were briefly washed with PBS and fixed with 2% paraformaldehyde (Fujifilm Wako) for 10 min at room temperature. The cells were then rinsed with PBS and permeabilized in 0.2% PBS containing 0.05% Tween 20 (PBS-T) 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-γ-H2AX antibodies (dilution 1:200; Merck Millipore, Burlington, MA, USA) overnight at 4 °C. The 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, the cells were mounted in the VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories, Inc., Newark, CA, USA) and observed under a confocal microscope.

Detection of CPD, 6–4 photoproducts (PPs), and Dewar PPs using an enzyme-linked immunosorbent assay (ELISA)

To evaluate DNA base damage induced by 50 W/m2 blue light and 5-ALA, we employed and ELISA-based approach. After treatment, the cells were lysed, and genomic DNA was extracted using the QIAamp DNA Blood Mini kit (Qiagen, Venlo, Netherlands) following the manufacturer’s protocol. The extracted DNA was denatured by heating at 100 °C 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 6-4PPs, and 10 µg/mL for Dewar PPs detection, respectively) were prepared and added to protamine sulfate-coated 96-well plates. Plates were dried overnight at 37 °C in the dark. After drying, the wells were washed with PBS-T. Blocking was done using 2% FBS for 30 min at 37 °C. After five washes with PBS-T, the wells were incubated with primary antibodies (1:1000 anti-CPDs, 1:1500 anti-6–4 PPs, and 1:5000 anti-Dewar PPs; Cosmo Bio) for 30 min at 37 °C. The wells were washed and incubated with a biotinylated secondary anti-mouse antibody for 30 min at 37 °C, followed by 1:10,000 streptavidin-conjugated peroxidase for 30 min at 37 °C. The wells were washed with PBS-T, once with citrate–phosphate buffer (25 mM citric acid monohydrate and 50 mM Na2HPO4; pH 5.0), and then incubated with 100 µL/well of substrate buffer (4 mM o-phenylenediamine, 0.007% H2O2 in citrate–phosphate buffer). The absorbance of each well was measured at 492 nm using a microplate reader.

Immunofluorescent CPD staining

Cells were seeded into glass-bottomed 35-mm dishes at a density of 1 × 105 cells/dish and incubated overnight. The cells were treated with or without 5-ALA and exposed to 50 W/m2 blue light. After treatment, the 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 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). The cells were then incubated with a secondary anti-mouse IgG (Alexa Fluor 594) antibody (1:200; Abcam) for 2 h at room temperature. Finally, the 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 the 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 (Nishio et al. 2022) using a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). Target gene expression levels were normalized to those of GAPDH and calculated using the 2-ΔΔCt method. All experiments were performed in triplicate.

Statistical analysis

Data were analyzed using the GraphPad Prism 9 software (GraphPad Software, Inc., La Jolla, CA, USA). All numerical data are expressed as means ± standard error of the mean (SEM) of experiments performed at least in triplicate. Multiple comparisons were done using Dunnett’s test or Tukey’s test. Results were considered statistically significant at P < 0.05.

Results

Effect of 5-ALA, blue light, and NAC on cell viability and proliferation

We previously reported that co-treatment with blue light and 5-ALA induces significant cell death (Sato et al. 2022). Therefore, we aimed to further understand the effects of 5-ALA on cell proliferation. B16F1 and HaCaT cells were treated with various concentrations of 5-ALA (0.1–10 mM), and their proliferation was assessed. As shown in Fig. 1a, even at the lowest concentration (0.1 mM), 5-ALA significantly inhibited the cell proliferation of B16F1 cells. Similarly, HaCaT cells exhibited significant proliferation arrest with 0.1 mM 5-ALA (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 the 5-ALA-free medium. A slight but significant inhibition of B16F1 cell proliferation was observed starting at 0.2 mM (Fig. 1c). In contrast, HaCaT cells did not show significant inhibition of proliferation at any of the concentrations tested (Fig. 1d).

Fig. 1.

Fig. 1

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Effect of 5-ALA on cell viability. B16F1 (a) and HaCaT (b) cells were treated with various concentrations of 5-ALA for 72 h. Cell viability was evaluated using the MTT assay. In B16F1 (c) and HaCaT (d) cells, short-duration treatment (2 h) with 5-ALA was followed by 72 h of incubation in 5-ALA-free media. Data are represented as the means ± SEM (n = 8). Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. *p < 0.05 and **p < 0.01 versus the control group

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 (5 mM) was added to the medium for 1 h before to 1 h after blue light exposure. Following this treatment, the cells were incubated for 72 h in a medium lacking 5-ALA or NAC. As shown in Fig. 2a, B16F1 cells exhibited significantly reduced viability after exposure to blue light alone or in combination with 5-ALA, which is consistent with our previous findings. NAC did not ameliorate the blue light-induced decrease in cell viability. Similar results were observed in HaCaT cells (Fig. 2b).

Fig. 2.

Fig. 2

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Effect of co-treatment with blue light, 5-ALA, and NAC on cell viability. B16F1 (a) and HaCaT (b) cells were pre-treated with 5 mM NAC and 1 mM 5-ALA for 1 h, followed by exposure to blue light. After exposure, the cells were incubated for an additional hour with NAC and cultured for 72 h in NAC- and 5-ALA- free media. Cell viability was assessed using the MTT assay. Data are represented as the means ± SEM (n = 8). Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05 and **p < 0.01 versus the control group, ns = non-significant

Co-treatment with blue light and 5-ALA increased intracellular ROS levels, induced de-polarization of mitochondrial membrane potential (ΔΨm), and peroxidation of cardiolipin

To elucidate the effects 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 cells, exposure to blue light for 60 min did not significantly increase ROS levels compared to the control cells (Fig. 3a). Similarly, 5-ALA treatment alone did not significantly change ROS levels. However, when the cells were co-treated with blue light and 5-ALA, a four-fold increase in the number of HE-positive cells was observed after 60 min of exposure.

Fig. 3.

Fig. 3

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Effect of blue light and 5-ALA on intracellular ROS levels in B16F1 and HaCaT cells. Effect of 5-ALA (1 mM) and 50 W/m2 blue light for 60 min on B16F1 and HaCaT cells. Intracellular ROS levels in B16F1 (a) and HaCaT cells (b). c HaCaT cells were exposed to blue light for 15–60 min. All measurements were obtained by flow cytometry. Data are represented as the means ± SEM (n = 6). Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05 and **p < 0.01 versus the control group. #p < 0.05, and ##p < 0.01 were considered significant

We assessed intracellular ROS levels in HaCaT cells, and observed that exposure to blue light alone for 60 min decreased ROS levels (Fig. 3b). Treatment with 5-ALA alone, similar to the B16F1 cells, did not result in any 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 using only blue light exposure. Blue light treatment consistently decreased ROS levels at all examined time points (Fig. 3c).

Following our assessment of intracellular ROS, we investigated ΔΨm as an indicator of early apoptotic events. Flow cytometry was conducted using the cell-permeable probe DiOC6, which selectively accumulates in mitochondria with an intact membrane potential. In B16F1 cells, neither blue light exposure nor 5-ALA treatment alone significantly changed ΔΨm (Fig. 4a). 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 the ΔΨm, suggesting a potential for the start of apoptosis (Fig. 4b).

Fig. 4.

Fig. 4

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Effect of blue light and 5-ALA on mitochondrial membrane potential in B16F1 and HaCaT cells. ΔΨm Low cells were determined using flowcytometry in B16F1 (a) and HaCaT cells (b). Cells were pretreated with or without 1 mM 5-ALA for 1 h, followed by exposure to blue light (50 W/m2) for 60 min. Data are represented as the means ± SEM (n = 6). Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05 and **p < 0.01 versus the control group. #p < 0.05, and ##p < 0.01 were considered significant

Additionally, we assessed cardiolipin peroxidation, a marker of mitochondrial oxidative stress and apoptosis. NAO preferentially binds non-oxidized cardiolipins; thus, a decrease in NAO fluorescence is indicative of cardiolipin peroxidation (Petrosillo et al. 2003; Zeno et al. 2009). As shown in Fig. 5a and b, co-treatment with blue light and 5-ALA significantly reduced the NAO fluorescence intensity in both cell lines.

Fig. 5.

Fig. 5

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Influence of blue light and 5-ALA on cardiolipin peroxidation in B16F1 and HaCaT cells. The proportion of cells exhibiting low nonyl acridine orange fluorescence, indicative of car-diolipin peroxidation, was determined by flow cytometry in B16F1 (a) and HaCaT cells (b). Cells were pre-treated with or without 1 mM 5-ALA for 1 h, followed by exposure to blue light (50 W/m2) for 60 min. Data are represented as the means ± SEM (n = 6). Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05 and **p < 0.01 versus the control group. #p < 0.05, and ##p < 0.01 were considered significant

We conducted these three experiments with cells co-treated for 30 min, which showed results similar to those of the 60 min co-treatment (Supplementary Figs. 1–3).

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 affect mitochondrial morphology. To investigate this, we visualized the mitochondria in live cells subjected to blue light and 5-ALA treatment using MitoTracker Green. As shown in Fig. 6a, B16F1 cells exposed only to blue light exhibited mitochondria with tubular and branched morphologies similar to the untreated controls. In cells co-treated with blue light and 5-ALA (0.2 and 1 mM), mitochondrial fluorescence appeared diffuse, and clear shapes were not discernible, suggesting potential mitochondrial disruption. In HaCaT cells (Fig. 6b), the mitochondria maintained their typical morphology under blue light exposure alone. In contrast, the cells treated with blue light and 0.2 mM 5-ALA showed numerous fragmented and rounded mitochondria. The 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.

Fig. 6.

Fig. 6

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Confocal microscopy for mitochondrial morphology assessment. The mitochondrial morphology of B16F1 (a) and HaCaT (b) cells was assessed using MitoTracker Green. The cells were pre-treated with or without 0.2 or 1 mM 5-ALA for 60 min, followed by exposure to blue light (50 W/m2) for 1 h. After treatment, cells were stained with MitoTracker and visualized under confocal microscope

We investigated the effects of co-exposure of B16F1 and HaCaT cells to blue light and 5-ALA using TEM. Cells were pre-treated with 0.02 or 0.2 mM 5-ALA for 1 h, followed by exposure to blue light for 1 h. As shown in Fig. 7, 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 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 membrane disruption and the disappearance of cristae.

Fig. 7.

Fig. 7

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Transmission electron microscopy (TEM) to detect mitochondrial damage. 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/m2) for another 1 h. After treatment, the cells were fixed and processed for TEM to visualize ultrastructural changes in the mitochondria. Arrows indicate sites of damaged in the mitochondria

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, MitoSOX Red was used for confocal microscopy. In B16F1 cells, exposure to blue light alone or co-treatment with 0.02 mM 5-ALA significantly increased mtROS levels (Fig. 8a, 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 the mtROS levels in HaCaT cells (Fig. 8c–d). Co-treatment with 0.2 mM 5-ALA in HaCaT cells slightly increased mtROS levels, in contrast to the results observed in B16F1 cells.

Fig. 8.

Fig. 8

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Effect of blue light and 5-ALA on mitochondrial ROS levels in B16F1 and HaCaT cells. Mitochondrial ROS levels were assessed in B16F1 (a, b) and HaCaT (c, d) cells after treatment with blue light with or without 5-ALA. Cells were pre-treated with or without 5-ALA (0.02 and 0.2 mM) and then exposed to blue light (50 W/m2) 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 the Zeiss Zen software. Data are represented as the means ± SEM. The experiments were performed four times, and for each experiment, the fluorescence intensity of at least 100 cells per treatment group was measured. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05 and **p < 0.01 versus the control group

Cell cycle analyses and SubG1 population cells

To assess the extent of DNA fragmentation, an indicator of apoptosis, we quantified the subG1 cell population in cells treated with blue light and 5-ALA. As shown in Fig. 9, co-treatment with blue light and 5-ALA significantly increased the subG1 population in both B16F1 and HaCaT cells. This increase was particularly pronounced after 60 min of exposure (a 60% increase in B16F1 cells). Furthermore, cell cycle analysis revealed that the 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. 10a). These results suggested 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 the different cell cycle phases after 30 min of blue light exposure, regardless of 5-ALA treatment (Supplementary Fig. 4). However, after 60 min of 5-ALA exposure, the number of cells in the G1 phaseb significantly increased, and the number of cells in the S and G2/M phases decreased (Fig. 10b). These results indicate that the combined treatment with blue light and 5-ALA affects cell cycle progression in response to checkpoint activation or DNA damage.

Fig. 9.

Fig. 9

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Population of B16F1 and HaCaT cells in subG1 phase. Effect of blue light and 5-ALA treatment on subG1 cell population and cell cycle distribution. B16F1 (a) and HaCaT (b) cells were pre-treated with 1 mM 5-ALA for 1 h, followed by exposure to blue light (50 W/m2) for 60 min. The subG1 population, which is indicative of apoptotic cells, was quantified using flow cytometry. Data are represented as the means ± SEM (n = 6). Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05 and **p < 0.01 versus the control group. #p < 0.05, and ##p < 0.01 were considered significant

Fig. 10.

Fig. 10

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Cell cycle analyses in B16F1 and HaCaT cells. Effect of blue light and 5-ALA treatment on the subG1 cell population and cell cycle distribution in B16F1 (a) and HaCaT cells (b). For cell cycle analysis, B16F1 and HaCaT cells were treated as described previously. Blue light exposure was performed for 60 min. After treatment, the proportion of cells in each phase of the cell cycle (G1, S, and G2/M) was determined using flow cytometry. Data are represented as the means ± SEM (n = 6). Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05 and **p < 0.01 versus the control group. #p < 0.05, and ##p < 0.01 were considered significant

Co-treatment with blue light and 5-ALA increases γ-H2AX expression

To further investigate DNA damage, we assessed γ-H2AX, a well-established marker of DNA DSBs (Redon et al. 2009). Previously, we performed a two-tailed comet assay to detect DSBs and SSBs in B16F1 and HaCaT cells and observed that co-treatment with blue light and 5-ALA-induced DSBs and SSBs (Sato et al. 2022). We thus attempted to quantify γH2AX expression by immunofluorescence. As shown in Fig. 11a–d, ultraviolet C (UVC) treatment, used as a positive control, significantly enhanced γH2AX fluorescence intensity in both cell lines. Co-treatment with blue light and 5-ALA increased γ-H2AX expression in both B16F1 and HaCaT cells, suggesting the induction of DNA damage.

Fig. 11.

Fig. 11

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Evaluation of γH2AX formation in response to blue light and 5-ALA treatment in B16F1 and HaCaT cells. Detection of immunofluorescence of γ-H2AX in B16F1 (a) and HaCaT (b) cells. Quantitative analyses of the γ-H2AX fluorescent intensity in B16F1 (c) and HaCaT (d) cells. The cells were pre-treated with or without 5-ALA and exposed to blue light for 1 h. After irradiation, the cells were stained with anti-γ-H2AX antibodies to detect DNA damage and visualized using a confocal microscope. Data are represented as the means ± SEM. The experiments were performed four times, and for each experiment, the fluorescence intensity of at least 100 cells per treatment group was measured. Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test

Co-treatment with blue light and 5-ALA did not enhance the formation of CPDs, 6-4PPs, or Dewar PPs

In a previous study, we established that visible light, including blue and red light, does not enhance CPD or 6-4PP formation (Nishio et al. 2022). In this study, we investigated the potential formation of CPD, 6-4PPs, and Dewar PPs in cells co-treated with blue light and 5-ALA by ELISA. As shown in Fig. 12a–d, co-treatment with blue light and 5-ALA did not enhance CPD or 6-4PP formation in either cell line. In contrast, the UVC treatment significantly increased CPD and 6-4PP production. 5-ALA did not increase the UVC-induced formation of these photoproducts. Furthermore, Dewar PP levels did not change in any of the treatment groups (Fig. 12e, f). We performed confocal laser scanning microscopy to visualize CPD formation in B16F1 and HaCaT cells (Fig. 12g, h). These microscopy results are consistent with the ELISA data, demonstrating that blue light and 5-ALA did not significantly induce CPD formation.

Fig. 12.

Fig. 12

Fig. 12

Fig. 12

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Assessment of photoproduct (PP) formation in B16F1 and HaCaT cells following blue light and 5-ALA treatment. Formation of CPD in B16F1 (a) and HaCaT (b) cells, respectively. Formation of 6–4 PPs in B16F1 (c) and HaCaT (d) cells. Formation of Dewar PPs in B16F1 (e) and HaCaT (f) cells. The 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 an enzyme-linked immunosorbent assay was performed to quantify these PPs. Detection of CPD in B16F1 (g) and HaCaT (h) cells using immunofluorescence. After treatments, the cells were observed via confocal microscopy. Data are represented as the means ± SEM (n = 10). Data were analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. *p < 0.05 and **p < 0.01 versus the control group

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 (Nishio et al. 2022). In this study, we investigated the effects of blue light and 5-ALA co-treatment on the transcription of a several genes in B16F1 and HaCaT cells.

B16F1 cells were exposed to blue light for 30 min in the presence of 0.2 mM 5-ALA (Fig. 13a). Time-point analyses of mRNA levels were conducted at 2, 4, 8, 16, and 24 h post-treatment. An increase in p21 mRNA, a well-known cyclin-dependent kinase inhibitor involved in cell cycle regulation (Bitar and Gali-Muhtasib 2019), peaking at 4 h, was observed after blue light exposure. The expression of survivin, a gene associated with cell survival and inhibition of apoptosis, was enhanced following irradiation. The transcription levels of Bax and Bcl-2, key genes in the apoptotic pathway, were assessed. Although Bax mRNA levels varied 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 (Qian et al. 2022). Caspase-3 mRNA levels increased 8 h post-irradiation.

Fig. 13.

Fig. 13

Fig. 13

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Real-time qRT-PCR analysis. Effect of combined blue light and 5-ALA on gene transcription in B16F1 melanoma cells (a). 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 2, 4, 8, 16, and 24 h post-treatment. Effect of combined blue light and 5-ALA on gene transcription in HaCaT cells (b). 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 2, 4, 8, 16, and 24 h post-treatment. Effect of blue light and co-treatment with 5-ALA on gene transcription in HaCaT cells (c). Cells were exposed to blue light for 15–120 min with or without 1 mM 5-ALA. After 3 h of incubation, mRNA levels were assessed using qRT-PCR. Data are represented as the means ± SEM (n = 6). Data were analyzed by one-way ANOVA followed by Dunnet’s test (a, b) or Tukey’s multiple comparison test (c). *p < 0.05 and **p < 0.01 versus the control group. #p < 0.05 and ##p < 0.01 versus the group treated with 5-ALA only.

HaCaT cells were exposed to blue light for 1 h in the presence of 0.2 mM 5-ALA (Fig. 13B). After treatment, we conducted a time-course analysis of mRNA levels at 2, 4, 8, 16, and 24 h. The mRNA levels of matrix metalloproteinase-1 (MMP-1), a gene associated with extracellular matrix remodeling and skin aging (Karadeniz et al. 2021), showed a gradual increase post-irradiation. p21 expression indicated an early response to treatment. Hyaluronan synthase 2 (HAS-2) mRNA levels significantly increased. Collagen type I, alpha 1 (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 that promotes cell survival in response to DNA damage and is associated with enhanced cellular longevity and stress resistance (Kume et al. 2006; Luo et al. 2001). 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 the 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 at 2 h post-irradiation and subsequently decreased.

HaCaT cells were exposed to blue light for different durations (15, 30, 60, and 120 min) in the presence or absence of 1 mM 5-ALA. Following irradiation, the cells were incubated for an additional 3 h before quantification by qRT-PCR. Figure 13c 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; however, 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, in a time-dependent manner. 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 increased modestly after exposure to blue light. 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 (Sato et al. 2013). Additionally, we observed that co-treatment with 5-ALA severely damaged the DNA in both B16F1 and HaCaT cells (Sato et al. 2022). This study aimed to elucidate the cellular mechanisms by which these effects are exerted, including their impact on mitochondrial morphology, and gene transcription.

First, we assessed the effects of 5-ALA on B16F1 and HaCaT cell proliferation. In B16F1 cells, we observed the suppression of cell proliferation during short-term treatment. No significant inhibition of HaCaT cell proliferation was observed. Prolonged treatment (72 h) with 5-ALA significantly suppressed the proliferation of both cell lines. Kumar et al. (2023) reported similar findings, where 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. (2013) reported that 5-ALA suppressed the viability of B16F1 cells. Grigalavicius et al. (2022) suggested that 5-ALA may act as a lactate dehydrogenase inhibitor to induce cancer cell death. These results suggest that 5-ALA or its intermediates can affect the cell cycle and cell proliferation. We investigated the ameliorative effects of NAC on the inhibition of cell proliferation induced by co-treatment with blue light and 5-ALA. NAC did not rescue the inhibition of cell 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 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 with 5-ALA co-treatment. ROS levels were enhanced in HaCaT cells after co-treatment. Blue light alone decreased the ROS levels in HaCaT cells, suggesting a potential cell-protective effect of blue light in certain cell types. Sutterby et al. (2022) demonstrated that monochromatic visible light (yellow, orange, and red light) induced proliferation and wound healing in HaCaT cells. The effect of blue light was not assessed; therefore, this question requires further investigation. Neither blue light nor 5-ALA alone affected ΔΨm in either cell line. However, co-treatment resulted in the depolarization of ΔΨm in both cell lines. As ΔΨm depolarization is an early indicator of apoptosis (Nabekura et al. 2022), this result suggests that co-treatment promotes cell death. We assessed cardiolipin peroxidation, a process that weakens cytochrome c retention in the inner mitochondrial membrane and triggers apoptosis (Zhang et al. 2010). Our findings showed that neither blue light nor 5-ALA alone induced cardiolipin peroxidation in either cell line. However, the co-treatment led to significant cardiolipin peroxidation. These observations suggest that co-treatment with blue light and 5-ALA induces significant cell death in both B16F1 and HaCaT cells, primarily by increasing ROS levels and promoting mitochondrial dysfunction. A previous report indicated that the combination of ultrasound and 5-ALA increases ROS levels, promoting subsequent cell death in a human leukemia cell line (Sun et al. 2018).

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 the 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 the elongated mitochondria to become increasingly smaller and more granular. Such mitochondrial fragmentation, often linked to ΔΨm depolarization (Machiela et al. 2020), may aid in removing damaged mitochondria and help cells recover from stress (Fernandes et al. 2021). This effect was even more evident with 1 mM 5-ALA, which possibly 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 in the presence of 1 mM 5-ALA, where the mitochondria appeared to be dispersed. Reports have indicated that perinuclear clustering of mitochondria occurs in response to intracellular ROS accumulation and several other types of cellular stress (Bolfer et al. 2020; Al-Mehdi et al. 2012). These observations suggest that co-treatment with blue light and 5-ALA can induce significant mitochondrial stress and morphological changes.

TEM provided intriguing insights into mitochondrial damage in response to various treatments. 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 high concentration of 5-ALA (0.2 mM). This exacerbated damage is 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 exposure to blue light and low-concentration 5-ALA co-treatment. However, similar to the B16F1 cells, HaCaT cells exhibited 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. (2023) reported that mitochondrial DNA DSBs result in the loss of membrane potential and abnormal or missing cristae. The alterations in mitochondrial morphology observed in our study could be a consequence of similar DNA damage, potentially affecting both the mitochondrial and nuclear DNA.

We assessed mtROS levels in B16F1 and HaCaT cells following exposure to blue light 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 attributed to severe mitochondrial damage. In contrast, HaCaT cells did not show a significant increase in mtROS levels with either blue light alone or low 5-ALA concentrations. 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.

Additionally, we explored the effects of blue light and 5-ALA co-treatment on the subG1 cell population and cell cycle progression. Co-treatment markedly increased the number of subG1-phase cells 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 (Lim et al. 2021). In contrast, HaCaT cells exposed to blue light for 60 min showed an increase in the number of cells in the G1 phase and a decrease in the number of cells in the G2/M phase. These findings suggest that the co-treatment may induce cell cycle arrest in response to DNA damage. Consequently, we assessed multiple markers of DNA damage following co-treatment.

To investigate DNA damage, we first assessed γ-H2AX, a marker of DNA DSBs. In both B16F1 and HaCaT cells, γ-H2AX levels increased following UVC irradiation. In B16F1 cells, exposure to blue light alone elevated γ-H2AX levels. Co-treatment with blue light and 5-ALA increased γ-H2AX levels in both cell lines. These findings are consistent with those of our previous study, which indicated the induction of DSBs and SSBs under similar experimental conditions (Sato et al. 2022). In HaCaT cells, blue light alone decreased γ-H2AX levels compared with those in the control cells. This observation suggests a potential reduction in cellular stress. With respect to CPD and 6-4PPs, key markers of DNA base damage, neither blue light exposure nor co-treatment with 5-ALA induced an increase in either cell lines. Shorter wavelengths of visible light may cause the formation of CPD and other photoproducts (Lawrence et al. 2018). In contrast, UVC irradiation significantly increased CPD and 6-4PP levels in both cell lines. However, co-treatment with 5-ALA and UVC did not affect the production of these PPs. No significant changes were observed under any treatment condition with Dewar PPs, which are formed through photoisomerization of 6-4PPs under UVA light (Douki 2020). These results suggest that co-treatment with 5-ALA does not enhance base damage in DNA. Further studies are required to fully elucidate these mechanisms.

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 and downregulation of Bcl-2. These changes in gene expression suggested the initiation of apoptosis. In contrast to our previous findings, survivin exhibits variable responses, with long-term blue light exposure significantly inhibiting survivin transcription in B16F1 cells (Nishio et al. 2022). 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 increase MMP-1 levels, leading to collagen fiber degradation and contributing to skin aging and wrinkling (Hwang et al. 2014; Park et al. 2018). MMPs play a role in cancer cell invasion (Akter et al. 2015; Soto-Guzman et al. 2010; Rattanasinchai et al. 2017). 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 (Liu et al. 2019; Engeland 2022). p21 has been implicated in cellular responses to stress and can confer protection against apoptosis under certain conditions (Karimian et al. 2016). 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. Further studies are required to fully elucidate these complex changes. Nevertheless, our findings provide foundational data for future research in this field.

5-ALA accumulates intensively in tumor tissues (Mahmoudi et al. 2019). Gorbenko et al. (2021) assessed the intracellular PPIX levels in cancer cell lines (HeLa and A549) and immortalized normal fibroblast cells (3T3). They revealed that PPIX levels in cancer cell lines were strongly enhanced after 5-ALA treatment compared to those in normal fibroblasts. However, they suggested that baseline PPIX levels in fibroblasts were higher than those in cancer cell lines in the absence of 5-ALA. This indicates that PPIX accumulation is not exclusive to cancer cells but can occur in normal cells. We also confirmed the enhancement of intracellular PPIX levels in B16F1 and HaCaT cells after 2 h of treatment with 1 mM 5-ALA (data not shown). These suggest that the observed phototoxic effects may not be entirely cancer-specific and that normal cells can be susceptible to damage under 5-ALA and light treatment.

This study elucidated the effects of co-treatment with blue light and 5-ALA on B16F1 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 the induction of apoptosis-related gene expression were observed following treatment with 5-ALA and blue light. Although these results advance our understanding of the cellular responses to blue light and 5-ALA, they raise concerns about the potential adverse effects of 5-ALA in normal cells. This study highlights the need for the cautious application of 5-ALA in clinical settings, considering its potential to induce damage to target cancer cells as well as normal cells.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 364 KB) (363.5KB, pdf)

Acknowledgements

We would like to thank Prof. Dr. Tetsuhiko Sasaki for his technical advice on fluorescent imaging techniques. We are also grateful to Prof. Dr. Keiko Ohashi-Kaneko for her significant contributions to the genetic analysis.

Author Contributions

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.

Funding

Not applicable.

Availability of data and materials

No datasets were generated or analysed during the current study.

Declarations

Conflict of interests

The authors declare no competing interests.

Ethical approval

Not applicable.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

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Supplementary Materials

Supplementary file1 (PDF 364 KB) (363.5KB, pdf)

Data Availability Statement

No datasets were generated or analysed during the current study.

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