Effect of Benzo[a]pyrene on Cellular Senescence in MCF7 Breast Cancer Cells

Abstract

Studies on the effect of environmental chemicals on cancer has primarily focused on the early stages, while the impact on later stages, particularly cancer progression, has been less explored. Cellular senescence, a response to stress such as DNA damage and oxidative stress, has been reported to play a crucial role in cancer progression. Environmental chemicals are suspected to promote cancer progression, yet it is unclear whether this is mediated through cellular senescence. Benzo[a]pyrene (BP) is known to induce DNA damage, a key trigger of senescence, but whether it directly induces senescence in cancer cells remains uncertain. This study aims to evaluate the senescence-inducing potential of BP in breast cancer cells to better understand its role in cancer progression. We examined the effects of BP, a polycyclic aromatic hydrocarbon primarily generated via incomplete combustion of organic matter and commonly found in water, soil, automobile exhaust, and tobacco smoke, on MCF7 breast cancer cells. BP enters the human body via inhalation, ingestion, and dermal contact. Here, as indicated by multiple senescence markers, including nuclear elongation, senescence-associated β-galactosidase activity, DNA damage, and increased p21 expression, BP induced cellular senescence in MCF7 cells. As cellular senescence is associated with malignant cell transformation, our results might suggest that BP contributes to cancer progression by inducing cellular senescence.

INTRODUCTION

The effect of environmental chemicals on cancer research mainly focuses on genotoxicity and carcinogenicity, both in vitro and in vivo. However, most studies have concentrated on the early stages of cancer, such as initiation and development.^1–3) The impact of chemical substances on later stages, especially cancer progression, has not been well-explored. Since these stages are crucial for cancer metastasis and recurrence, which are the leading causes of cancer-related deaths,⁴⁾ it is essential to consider the effects of chemicals. From the perspective of cancer progression, cellular senescence has been reported to play a crucial role in this process.^5–9)

Cellular senescence is a cell response induced by various stresses, including DNA damage and oxidative stress, that leads to stable cell cycle arrest.⁶⁾ It was discovered in the 1960s by Hayflick and Moorhead in human fibroblast cell lines that had exhausted their replicative capacity.^10,11) Subsequent studies revealed that cells possess a limited capacity for division before entering into an irreversible replicative arrest, known as replicative senescence, and defined the phenomenon of premature senescence.⁶⁾

In addition to cell cycle arrest, senescence-associated secretory phenotype (SASP) is a key feature of cellular senescence. SASP allows senescent cells to secrete various cytokines, chemokines, and growth factors, thereby affecting both close and distant cells and tissues. SASP components vary depending on the cellular senescence trigger.⁶⁾

Cellular senescence plays important physiological roles in various processes, such as embryonic development, wound healing, and tumor suppression.⁵⁾ However, prolonged senescence leads to tumor progression, chronic inflammation, and immune deficiency.⁵⁾ Therefore, although senescence is essential for maintaining biological homeostasis, it is also associated with various age-related diseases and conditions, including atherosclerosis and type 2 diabetes.¹¹⁾

In the context of cancer, cellular senescence is typically considered to prevent the malignant transformation of cancer cells by irreversibly halting the cell cycle, thereby suppressing tumor growth.^6,10) However, recent studies have suggested that senescence characteristics, especially SASP, which secretes various cytokines, chemokines, and growth factors, also promote tumor development.^7,8) In relation to environmental chemicals, several reports suggest that they may promote cancer progression.¹²⁾ For instance, it has been shown that benzo[a]pyrene (BP) enhances the migratory ability of cancer cells in vitro,¹³⁾ and that PM2.5 accelerates the progression of lung cancer.¹⁴⁾ However, whether these effects are mediated through cellular senescence remains unclear and requires further investigation.

Among the numerous chemicals to which humans may be exposed, BP is a well-known environmental chemical that contributes to tumor development by inducing DNA damage.¹⁵⁾ In addition to mutagenic and teratogenic effects, BP exerts non-carcinogenic effects and causes reproductive toxicity, immunotoxicity, and neurotoxicity.^16,17) BP is primarily produced via incomplete combustion of organic compounds and is found in water, soil, automobile exhaust, and tobacco smoke.¹⁶⁾ People are exposed to BP via various routes, including inhalation, ingestion, and dermal contact. Average daily intake of BP is estimated as 0.004 μg/kg/d, making it a common chemical exposed to humans via food and air.¹⁶⁾ Although BP is a potent inducer of DNA damage, a key cellular senescence trigger, whether it directly induces cellular senescence remains unclear. Therefore, in this study, we aimed to evaluate the senescence-inducing potential of BP in breast cancer cells.

MATERIALS AND METHODS

Cell Culture

MCF7 cells (estrogen receptor-positive breast cancer cell line) purchased from the Japanese Collection of Research Bioresources (JCBR0134, Osaka, Japan) were cultured in the Dulbecco’s modified Eagle’s medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (Biosera, lot: S00HA, Cholet, France) and 1% antibiotic–antimycotic solution (FUJIFILM Wako Pure Chemical Corporation) at 37°C in 95% air and 5% CO₂. All experiments were performed with cells after less than 20 passages. Mycoplasma contamination was assessed using a commercially available kit (EZ-PCR Mycoplasma Detection Kit; Biological Industries, Beit Haemek, Israel).

Cell Viability Assay

MCF7 cells were seeded in a 96-well plate (5000 cells; Thermo Fisher Scientific, Waltham, MA, U.S.A.) and incubated with BP for 72 h. Then, cell viability was measured using 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma-Aldrich, Saint Louis, MO, U.S.A.), according to the manufacturer’s instructions, and normalized to that of the untreated cells.

Immunoblotting

MCF7 cells were exposed to 0.1 and 1 μM of BP (Sigma-Aldrich) for three days, and whole cell lysates were prepared for immunoblotting. Whole cell lysates were collected using the RIPA buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1% NP40, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate, and 1 mM EDTA) with the Halt Protease and Phosphatase Inhibitor Cocktail Kit (Thermo Fisher Scientific). Protein was mixed with the 6× sample buffer (0.375M Tris [pH 6.8], 12% SDS, 60% glycerol, 0.6 M dithiothreitol, and 0.06% bromophenol blue) and boiled at 95°C for 5 min and separated via SDS-polyacrylamide gel electrophoresis. Protein Ladder One Triple-color (Broad Range) for SDS-PAGE (Nacalai Tesque, Kyoto, Japan) was used as the standard. The proteins were electrotransferred onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, U.S.A.) and incubated overnight at 4°C with the rabbit anti-p21 (1 : 2000; #2947S; Cell Signaling Technology, Danvers, MA, U.S.A.), rabbit anti-γH2AX (1 : 2000; #2947S; Cell Signaling Technology), rabbit anti-p53 (1 : 2000; #ab179477; Abcam), and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (1 : 20000; #2118S; Cell Signaling Technology) primary. Subsequently, the membranes were incubated with the anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase-conjugated (1 : 2000; Cell Signaling Technology) and anti-mouse IgG (Fab-specific)-peroxidase-conjugated (1 : 50000 and 1 : 10000; Sigma-Aldrich) secondary antibodies. Finally, protein bands were detected using ImmunoStar LD (FUJIFILM Wako Pure Chemical Corporation) and visualized using the ImageQuant LAS 4000 Mini Biomolecular Imager (GE Healthcare, Tokyo, Japan).

Senescence-Associated β-Galactosidase (SA-β Gal) Staining

Cellular Senescence Detection Kit (SPiDER-βGal) was purchased from Dojindo Laboratories (Kumamoto, Japan). For SA-β gal staining, the MCF7 cells were mixed with bafilomycin A1 for 1 h and stained with the SPiDER-β Gal working solution at 37°C for 30 min. Flow cytometric analysis was performed using the BD MACSQuant Analyzer X (#130-105-100; Miltenyi Biotec, Bergisch Gladbach, Germany). Fluorescence intensity of SA-β-gal was measured using the fluorescein isothiocyanate (FITC) channel, and side scatter (SSC-A) was also recorded. The proportion of cells with high SA-β-gal activity was calculated based on the fluorescence distribution. The experiment was independently repeated four times, and are shown in the representative histogram. Quantified fluorescence intensity and SSC-A values from the four independent experiments were expressed as relative values compared with the control group.

Real-Time Reverse Transcription Polymerase Chain Reaction (Real-Time RT-PCR)

MCF7 cells were exposed to 0.1 and 1 μM of BP (Sigma-Aldrich) for three days, and total RNA was extracted by using a FastGene RNA Kit (Nippon Genetics, Tokyo, Japan) and reverse-transcribed into cDNA by using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, U.S.A.). A PCR mixture was prepared containing the above cDNA as a template and primers for the genes, interleukin-6 (IL-6) (forward, 5′-CTTCGGTCCAGTTGCCTTCT-3′ and reverse, 5′-TGGAATCTTCTCCTGGGGGT-3′), vascular endothelial growth factor A (VEGFA) (forward, 5′-CGCAGCTACTGCCATCCATT-3′ and reverse, 5′-GTGAGGTTTGATCCGCATAATCT-3′), Intercellular Adhesion Molecule 1 (ICAM1) (forward, 5′-TGACAGTGAAGTGTGAGGCC-3′ and reverse, 5′-GCCATACAGGACACGAAGCT-3′), MKI67 (forward, 5′-CGTCCCAGTGGAAGAGTTGT-3′ and reverse, 5′-CGACCCCGCTCCTTTTGATA-3′) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward, 5′-GAAGGTGAAGGTCGGACTC-3′ and reverse, 5′-GAAGATGGTGATGGGATTTC-3′) (Eurofins Genomics, Tokyo, Japan), as well as GeneAce SYBR qPCR Mix α Low ROX (Nippon Gene, Tokyo, Japan). RT-PCR was performed using a CFX-384 Real-Time PCR Detection System (BioRad Laboratories, Hercules, CA, U.S.A.). The expression level of each gene was normalized to that of GAPDH.

Immunofluorescence Assay

MCF7 cells were seeded in a 96-well black plate (Thermo Fisher Scientific) and incubated with BP for 72 h. Then, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) (FUJIFILM Wako Pure Chemical Corporation) for 30 min at room temperature (20–25°C), washed thrice with PBS, and incubated with 0.1% Triton X-100 (Nacalai Tesque) in PBS for 15 min at room temperature. The cells were blocked with PBS containing 1% bovine serum albumin for 30 min at room temperature and incubated with primary antibodies in PBS containing 1% bovine serum albumin overnight. After washing thrice with PBS, the cells were incubated with secondary antibodies for 2 h at room temperature and washed with PBS. NucSpot [R] Live 650 (100 μM; #40082; Nacalai Tesque) was used to stain the nucleus, and phalloidin–rhodamine (#R415; Thermo Fisher Scientific) was used to stain F-actin. Both primary (rabbit anti-p21 #2947S, 1 : 500; and rabbit anti-Phospho-Histone H2A.X (Ser139) (20E3) #9718; 1 : 100; Cell Signaling Technology, MA, U.S.A.) and secondary (Goat anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 #A-11008; Invitrogen, MA, U.S.A.) antibodies were used for the assay.

Statistical Analyses

Statistical analyses were conducted using the Prism 10 software for MacOS (GraphPad Software, San Francisco, CA, U.S.A.). Data are represented as the mean ± standard deviation. p-Values were calculated via one-way ANOVA followed by Dunnett’s test or Kruskal–Wallis test followed by Dunn’s test. Statistical significance was set at p < 0.05.

RESULTS

Effect of BP Exposure on MCF7 Cell Viability and Proliferation

MCF7 cells were used as model breast cancer cells as they express CYP family 1 subfamily A member 1 (CYP1A1), a metabolic enzyme for BP.¹⁸⁾ MCF7 cells were exposed to various concentrations of BP (0.008–125 μM) for 72 h, and a significant decrease in cell viability was observed starting from 0.2 μM (Fig. 1A). For further assessments, 0.1 and 1 μM of BP were selected as they suppressed cell viability after 72 h exposure. Notably, exposure of MCF7 cells to the selected concentrations of BP for 72 h over 3 days decreased the cell proliferation in a concentration-dependent manner (Fig. 1B). A hallmark of cellular senescence is the sustained arrest of cell proliferation even after removal of the inducing stimulus.¹⁹⁾ To investigate this, MCF7 cells were treated with BP for 72 h and subsequently cultured in BP-free medium. A cell proliferation assay revealed that the proliferative capacity of MCF7 cells remained suppressed following BP withdrawal (Supplementary Fig. S1). Overall, 72-h BP exposure decreased the viability and proliferation of MCF7 cells.

Fig. 1. Viability and Proliferation of MCF7 Cells after BP Exposure

(A) MCF7 cells (5000 cells/well) were exposed to BP for 72 h, and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to measure their viability. Data are represented as the mean ± standard deviation (S.D.; n = 6). This experiment was repeated twice with similar results. ^****p < 0.0001 and ^**p < 0.01 via one-way ANOVA, followed by Dunnett’s multiple-comparison test. (B) MCF7 cells exposed to 0.1 and 1 μM BP for 72 h were seeded to new 96–well plates. After 24, 48, and 72 h of seeding, MTT assay was performed to evaluate cell proliferation. Data are represented as the mean ± S.D. (n = 6). This experiment was repeated thrice with similar results. ^***p < 0.001 and ^*p < 0.05 via Kruskal–Wallis test, followed by Dunn’s multiple-comparison test. BP: benzo[a]pyrene.

BP-Induced Changes in Senescence Markers

Next, several morphological and molecular senescence markers were assessed to evaluate whether BP exposure induces senescence in MCF7 cells. As senescent cells exhibit nuclear enlargement,²⁰⁾ nuclear size was evaluated after BP treatment. Notably, nuclear size was increased in both 0.1 and 1 μM BP-treated cells compared with that in the non-treated cells (Figs. 2A, 2B). Additionally, flow cytometry was used to evaluate the expression of SA-β-gal, a widely used cellular senescence marker.⁸⁾ BP increased the mean fluorescent intensity of SA-β-gal in a concentration-dependent manner (Figs. 2C, 2D). Furthermore, intracellular complexity, often used as one of the markers of cellular senescence, was assessed by side scatter. The results showed that side scatter also increased with BP treatment (Figs. 2C, 2E). Next, we evaluated the expression levels of p21 and p53, cell cycle-associated proteins often used as cellular senescence markers,⁸⁾ via Western blotting. Overexpression of p21 and p53 contributes to cell cycle arrest. Transcription factor p53 is activated by sustained DNA damage responses, promoting the transcription of downstream cyclin-dependent kinase inhibitor 1 and inducing p21 expression. Subsequently, p21 inhibits cyclin-dependent kinases, thereby preventing cell cycle progression at the G1/S checkpoint.¹⁷⁾ Here, BP increased the expression levels of these molecules, which halt the cell cycle, in a concentration-dependent manner (Fig. 3A). Expression of the DNA damage marker, γH2AX, was also increased following BP exposure (Fig. 3A). Furthermore, to determine whether components of SASP, a key feature of cellular senescence, are altered by BP exposure, we performed real-time RT-PCR analysis. As a result, the expression of various SASP factors, including IL-6, ICAM1 and VEGFA was increased after BP exposure (Fig. 2B). We also evaluated the expression levels of several molecules via immunofluorescence assay. Immunostaining revealed increased γH2AX expression and foci following BP treatment (Fig. 3C). γH2AX foci count in nuclei was also increased in a BP concentration-dependent manner, with the expression observed with 1 μM BP being comparable to that observed with doxorubicin, which induces cellular senescence¹⁸⁾ (Fig. 3D). Expression of p21, which functions in the nucleus, also increased following BP and doxorubicin treatments (Fig. 3E). Nuclear fluorescence intensity analysis revealed a significant dose-dependent increase in fluorescence intensity upon BP exposure (Fig. 3F). In addition, BP treatment resulted in a concentration-dependent decrease in MKI67 expression, a gene encoding Ki-67, which is a well-established marker of cell proliferation. This reduction was comparable to that observed in the positive control group treated with 2 μM etoposide (Fig. 3G). Collectively, changes in multiple cellular senescence markers confirmed that BP induced senescence in MCF7 cells.

Fig. 2. Changes in Cell Morphology after BP Exposure

(A) MCF7 cells were exposed to 0, 0.1, and 1 μM of BP for 72 h, and the nucleus was stained with NucSpot [R] Live 650. Scale bars, 100 and 20 μm (zoom). (B) Image analysis of (A) was performed to measure the nuclear size. Subsequently, nuclear size was plotted as a relative value compared with that of the control group. Data are represented as the mean ± S.D. This experiment was repeated thrice with similar results. ^**p < 0.01 and ^****p < 0.0001 via Kruskal–Wallis test, followed by Dunn’s multiple-comparison test. (C) MCF7 cells were seeded in a 6-well plate, exposed to 0, 0.1, and 1 μM of BP, and stained with SPiDER β-galactosidase (β-gal) to evaluate the senescence-associated (SA)-β-gal activity. Then, SA-β-gal intensity (fluorescein isothiocyanate [FITC]) and SSC were measured via flow cytometry, and the proportion of cells with high SA-β-gal activity was calculated. This experiment was independently repeated four times, and the data shown are representative of these experiments. (D) Quantification of SA-β-gal fluorescence intensity from four independent experiments, as shown in (C). Data are presented as the mean ± S.D., expressed as relative values compared with the control group. p < 0.01, one-way ANOVA followed by Dunnett’s multiple comparison test. (E) SSC-A values from the same assay shown in (C) are also presented as the mean ± S.D. from four independent experiments, expressed as relative values compared with the control. BP: benzo[a]pyrene; SSC: side scatter.

Fig. 3. BP-Induced Changes in Cellular Senescence Markers

(A) Proteins were extracted from MCF7 cells exposed to 0, 0.1, and 1 μM BP for 72 h, and expression changes in cellular senescence markers were evaluated via Western blotting. This experiment was repeated thrice with similar results. (B) MCF7 cells exposed to 0, 0.1, and 1 μM BP for 72 h, and mRNA expression in SASP factors were evaluated via real time RT-PCR. This experiment was repeated twice with similar results. Data are represented as the mean ± S.D. This experiment was repeated twice with similar results. ^*p < 0.05, and ^***p < 0.001 via one-way ANOVA followed by Dunnett’s multiple comparison test. (C) MCF7 cells were exposed to 0, 0.1, and 1 μM of BP for 72 h, and γH2AX (green) expressed in MCF7 cells was stained via immunofluorescence assay. Additionally, nucleus and F-actin were stained with NucSpot (cyan) and phalloidin–rhodamine (magenta), respectively. Scale bar, 100 μm. (D) γH2AX foci in the nucleus was quantified via image analysis of (C) and plotted on a graph. Data are represented as the mean ± S.D. ^****p < 0.0001 via Kruskal–Wallis test, followed by Dunn’s multiple-comparison test. (E) MCF7 cells were exposed to 0, 0.1, and 1 μM of BP for 72 h, and p21 (green) expressed in MCF7 cells was stained via immunofluorescence assay. Additionally, nucleus and F-actin were stained with NucSpot (cyan) and phalloidin–rhodamine (magenta), respectively. Scale bar, 100 μm. (F) Fluorescence intensity of p21 in the nucleus was quantified via image analysis of (D) and plotted on a graph. Data are represented as the mean ± S.D. ^****p < 0.0001, ^***p < 0.001 via Kruskal–Wallis test, followed by Dunn’s multiple-comparison test. (G) MCF7 cells exposed to 0, 0.1, and 1 μM BP for 72 h, and mRNA expression in MKI67 (ki-67) was evaluated via real time RT-PCR. This experiment was repeated twice with similar results. Data are represented as the mean ± S.D. This experiment was repeated twice with similar results. ^*p < 0.05 and ^**p < 0.01 via one-way ANOVA followed by Dunnett’s multiple comparison test. BP: benzo[a]pyrene.

DISCUSSION

Chemical substance safety has been evaluated in various contexts. Specifically, cancer-related studies have assessed genotoxicity and carcinogenicity of chemical substances in vitro and in vivo.⁹⁾ However, most studies regarding chemical substances safety have mainly focused on the early stages, such as the initiation and development stages, of cancer.⁹⁾ By contrast, effects of chemical substances on the later stages, particularly the progression and malignant transformation stages, of cancer with high malignancy have not been reported. Progression and malignant transformation are critical stages associated with cancer metastasis and recurrence, which are the primary causes of cancer-related deaths.⁴⁾ Therefore, potential contributions of these stages must be evaluated in chemical safety assessments to appropriately regulate chemical use. Since cellular senescence has gained attention for its association with malignant transformation,^5,6) in this study, we investigated the effects of chemical substances on cellular senescence, using MCF7 cancer cells.

Given the physiological relevance, the concentrations of BP primarily used in this study (0.1 and 1 μM) are higher than those typically encountered in environmental exposure scenarios. The primary objective of our study was to examine the cellular effects of acute BP exposure (72 h), with a particular focus on the induction of cellular senescence. These concentrations were selected to ensure detectable and interpretable cellular responses within a short exposure period. However, to better reflect real-world exposure conditions, it will be essential for future studies to investigate the effects of lower, environmentally relevant concentrations of BP exposure over extended periods. Such investigations will be crucial to fully evaluate the characteristics of BP-induced senescence, including SASP factor secretion, and to determine how these changes may contribute to tumor malignant phenotypes.

In this study, exposure to BP, a representative chemical substance, induced cellular senescence in breast cancer cells (Figs. 2, 3). BP-induced DNA damage possibly acts as a trigger for cellular senescence.¹⁷⁾ BP diol epoxide, a metabolic intermediate of BP, forms DNA adducts, inducing DNA damage and causing genotoxicity.^15,16) Here, BP exposure induced cellular senescence, possibly via DNA damage or interactions between downstream molecules via activation of various pathways, such as the aryl hydrocarbon receptor pathway. Future studies should investigate the roles of CYP1A1 in MCF7 cells and assess the senescence-inducing potential of BP diol epoxide to clarify the differences between BP and BP diol epoxide in inducing cellular senescence in cancer cells.

A previous study revealed that BP exposure induces cellular senescence in MCF7 cells, primarily focusing on the DNA damage response pathway by examining p21 and pRb expression.²¹⁾ In this point, our study provides a more comprehensive characterization of BP-induced cellular senescence from multiple perspectives, including molecular markers (Figs. 2C, 3A, 3C–3F), cellular phenotypic changes (Figs. 2A, 2C), and the expression of secreted SASP factors (Fig. 3B). In particular, we investigated the effects of BP on SASP expression, which is a hallmark of cellular senescence²²⁾ and plays a pivotal role in promoting tumor malignancy through the secretion of cytokines and other inflammatory mediators.²³⁾ Several cytokines (IL-6, IL-1β and VEGFA) previously associated with tumor malignancy were^24–26) upregulated upon BP exposure (Fig. 3B), suggesting that senescence triggered by environmental chemicals may contribute to cancer progression.

Estrogen receptor-positive MCF7 breast cancer cells²⁰⁾ were used in this study. Breast cancer comprises various subtypes, including luminal types accounting for over 70% of cases worldwide, human epidermal growth factor receptor-2-positive breast cancer, and triple-negative breast cancer.²⁰⁾ Moreover, given the involvement of either the p16–pRB or p53–p21 pathway in the DNA damage response, it is important to note that the expression levels and pathway dependency vary across different cell lines.²⁷⁾ Considering this heterogeneity, future studies should examine other breast cancer subtype cell lines in addition to the luminal-type MCF7 cell line. Furthermore, different cancer cell lines must be evaluated to clarify the role of BP in cellular senescence. As BP and similar environmental chemicals affect humans via various exposure routes,^3,16) future studies should assess other chemicals with a high likelihood of causing cancer and verify the senescence-inducing potential of BP in vivo.

This study demonstrated that BP exposure induced cellular senescence. As cellular senescence is closely associated with malignant transformation,^7,8) our findings suggest that BP may contribute to cancer malignancy through the induction of senescence. However, further investigation is required to determine how senescence affects the malignant properties of these cells, particularly through the evaluation of specific phenotypic changes in senescence-induced cancer cells. Previous studies have reported that senescent cells secrete various cytokines and chemokines that enhance cellular invasiveness and stemness, suggesting that senescent cells contribute to malignancy via autocrine or paracrine effects.^7,8) Further elucidation of the mechanisms by which BP induces senescence and its contribution to the malignant transformation of cancer cells can facilitate the prediction of the effects of other chemical substances on cancer malignancy. Such evaluations will aid in the prediction of various environmental chemical risks and regulation of chemical substances to prevent cancer malignancy.

Acknowledgments

This work was supported by Japan Society for the Promotion of Science KAKENHI (Grant Nos.: 22K15275 to YH and 23K27342 to YT), and Health Labor Sciences Research Grant from the Ministry of Health, Labor, and Welfare of Japan (Grant No.: 22KA3006 to YH).

This work was partially supported by the Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research) from AMED (Grant No.: JP24ama121054).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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