2025 Volume 50 Issue 7 Pages 351-359
It is well known that apoptosis is triggered by arsenic. Meanwhile, recent evidence has demonstrated that arsenic also induces a non-canonical form of regulated cell death (RCD) called parthanatos that is triggered by the overactivation of poly (ADP-ribose) polymerase-1 (PARP-1). Here, we provide evidence of a novel mechanistic link between parthanatos and apoptosis induced by arsenic. Exposure to sodium arsenite clearly induced parthanatos in typical cancer cell lines such as HeLa and HT1080 cells, without activation of the apoptotic cascade, including the caspase-3 activation. Of note, we observed aggregation of caspase-3 in response to sodium arsenite, which was abolished by treatment with 4-phenylbutyrate (4-PBA), a chemical chaperone that prevents protein aggregation. Interestingly, in the presence of 4-PBA, sodium arsenite induced apoptosis rather than parthanatos. These findings suggest that the disaggregation of caspase-3 allows arsenic to induce the caspase-3 activation, and subsequent apoptosis. Thus, our results show that the degree of the caspase-3 aggregation may be a critical determinant of the selectivity of sodium arsenite-induced cell death.
Inorganic arsenic, including sodium arsenite, is an environmental pollutant that is a well-known human carcinogen (Rossman et al., 2004; Chung et al., 2014). Basically, exposure to inorganic arsenic initiates cytotoxic stress, such as reactive oxygen species (ROS) production, DNA damage, and genetic and epigenetic changes (Rossman, 2003). Therefore, toxicological studies of inorganic arsenic will no doubt contribute to a better understanding of arsenic-induced carcinogenesis. Programmed or regulated cell death is an anti-carcinogenic mechanism that actively eliminates cells with DNA damage caused by arsenic exposure, and to investigate arsenic-induced cell death leads to elucidation of mechanisms of carcinogenesis and establishment of its risk assessment.
It is generally believed that arsenic induces apoptosis by activating the tumor suppressor p53 (Ding et al., 2005; Chowdhury et al., 2009; Chou and Huang, 2002). However, accumulating evidence has shown that long-term exposure to arsenic inactivates pro-apoptotic factors such as p53 and caspase-3, leading to apoptosis resistance (Komissarova and Rossman, 2010; Pi et al., 2005). Therefore, it is controversial whether the cell death that is induced during arsenic exposure is apoptosis. Meanwhile, arsenic induces non-apoptotic types of cell death, such as parthanatos and ferroptosis (So and Oh, 2023; Wei et al., 2020). Parthanatos is non-apoptotic cell death mediated by the hyperactivation of PARP-1 in response to DNA damage (David et al., 2009; Venderova and Park, 2012). Under steady-state conditions, PARP-1 works to repair the DNA, but persistent DNA damage induces overactivation of PARP-1, leading to the activation of apoptosis-inducing factor (AIF) that causes cell death accompanied by extensive DNA fragmentation (Lee et al., 2014). Thus, although previous reports have demonstrated that arsenic induces both apoptosis and parthanatos, the mechanism that determines the selectivity between apoptosis and parthanatos induced by arsenic remains unknown.
In this study, we found that arsenic exposure induces parthanatos accompanied by caspase-3 aggregation in cancer cells, such as HeLa and HT1080 cells. However, interestingly, arsenic exposure induces apoptosis under conditions in which the caspase-3 aggregation is dissolved by the chemical chaperone 4-PBA. Thus, these observations suggest that the caspase-3 aggregation determines the selectivity between apoptosis and parthanatos induced by arsenic.
HT1080 cells and HeLa cells were grown in Dulbecco's Modified Eagle Medium (DMEM), 10% heat-inactivated fetal bovine serum (FBS), and 1% penicillin-streptomycin solution, at 37°C under a 5% CO2 atmosphere.
Reagents and antibodiesAll reagents were obtained from commercial sources. Sodium arsenite (NaAsO2) and rucaparib were purchased from Sigma-Aldrich (USA). z-VAD-fmk was purchased from Peptide Institute (Osaka, Japan). Cisplatin was purchased from Wako (Osaka, Japan). Thapsigargin was purchased from Santa Cruz (USA). 4-PBA was purchased from Tokyo Chemical Institute (Tokyo, Japan). The antibodies used were against PARP-1, PAR, AIF, p62, cleaved caspase-3, and K48-linked ubiquitin (Cell Signaling, Massachusetts, USA), and β-actin, caspase-3, G3BP1, and Lamin A/C (Santa Cruz).
Generation of knockout cell linesPARP-1 and p62 KO cells were generated using the CRISPR/Cas9 system (Hamano et al., 2024; Noguchi et al., 2018). Guide RNAs were designed to target a region exon 1 of PARP-1 gene (5′-GAGTCGAGTACGCCAAGAGC-3′), and that in the exon 3 of p62 gene (5’-AGACTACGACTTGTGTAGCG-3’) using CRISPRdirect. Guide RNA-encoding oligonucleotide was cloned into lenti- CRISPRv2 plasmid (addgene), and KO cells were established and characterized as previously described (Tsuchida et al., 2020). To determine the mutations of PARP-1 and p62 in cloned cells, genomic sequence around the target region was analyzed by PCR-direct sequencing using extracted DNA from each clone as a template and the following primers used. For determination of PARP-1 mutation; forward: 5′-GCATCAGCAATCTATCAG-3′, reverse: 5′-CTTCCCGGACACAGTTAA-3′. For determination of p62 mutation; 5′-GAGGACTTTAGGGGGTCCCA-3′ and 5′-AGGAATTAGCAGAGCGGCAG-3′.
Colorimetric cell viability assayCells were seeded on 96-well plates. After the indicated stimulation or treatment, cell viability was determined using Cell Titer 96 Cell Proliferation Assay (Promega, Wisconsin, USA), according to the manufacturer's protocol (Suzuki et al., 2020). The absorbance was read at 492 nm using a microplate reader. Data are normalized to control (100%) without stimulus, unless noted otherwise.
FACS AnalysisFACS analysis was performed as described previously (Yamada et al., 2023). For propidium iodide (PI) staining, HT1080 cells were labeled with PI for 15 min. Fluorescent cells were detected by CytoFLEX (Beckman Coulter, California, USA), and apoptotic cells were analyzed by using CytoExpert (Beckman Coulter).
ImmunoblotCells were lysed with DISC lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 7.4), 1% protease inhibitor cocktails (Nacalai Tesque, Kyoto, Japan), and 10% glycerol). After centrifugation at 15,000 rpm for 15 min, the cell extracts were resolved by SDS-PAGE, and subjected to immunoblot analysis as previously described (Sekiguchi et al., 2019).
Nuclear extractionNuclear extraction was performed as described previously (Hirata et al., 2021). Cells were lysed in ice-cold lysis buffer containing 10 mM Hepes (pH 7.5), 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, and 1% protease inhibitor cocktails (Nacalai Tesque) for 15 min. Cell lysates were added 1% NP-40 and then centrifuged at 4°C at 2500 rpm for 3 min. After the supernatants containing cytoplasmic fraction were removed, the pellets were suspended in ice-cold lysis buffer containing 20 mM Hepes (pH 7.5), 400 mM NaCl, 1 mM EGTA, 1 mM DTT, and 1% protease inhibitor cocktails for 15 min vortexed every 5 min. Cell lysates were then centrifuged at 4°C at 15,000 rpm for 15 min, and then the supernatants were collected as nuclear fraction.
Immunofluorescence stainingHT1080 cells were fixed with 3.7% formaldehyde, permeabilized with 0.5% Triton X-100, blocked with 3% bovine serum albumin-PBS, and incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies (p62: goat anti-rabbit Alexa Fluor488, caspase-3 and G3BP1: goat anti-mouse Alexa Fluor 555, Invitrogen) for 1 hr at room temperature. The immunostained samples were enclosed with Fluoro-KEEPER Antifade Reagent, Non-Hardening Type with DAPI (Nacalai), and observed using BZ-X800L fluorescence microscope. (Kagi et al., 2025).
Statistical analysisThe value was expressed as the mean ± standard error of the mean (S.E.M.) using Prism software (GraphPad). All experiments were repeated at least three independent times. Two groups were compared using Student’s t-test. Data were considered significant when *p < 0.05, **p < 0.01, ***p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.001.
We first examined which type of cell death is induced by sodium arsenite in cancer cell lines such as HeLa and human fibrosarcoma HT1080 cells, both of which are widely used models for analyzing programmed cell death. As shown in Fig. 1A and 1B, reduced cell viability upon exposure to sodium arsenite was significantly restored by PARP inhibitor rucaparib but not pan-caspase inhibitor z-VAD. PI staining assays also revealed that rucaparib reduces PI-positive (dead) cells during exposure to sodium arsenite (Fig. 1C). Additionally, it is revealed that PARP-1 KO cells have resistance to sodium arsenite-induced cell death by both cell viability and PI staining assay, suggesting that sodium arsenite induces PARP-1-dependent cell death, so-called parthanatos, in HT1080 cells (Fig. 1D-1F). Furthermore, sodium arsenite induced both the production of poly ADP-ribose (PAR) by PARP-1, which is essential for parthanatos, and the nuclear translocation of apoptosis-inducing factor (AIF), a key executor of parthanatos (Fig. 1G and 1H). On the other hand, pan-caspase inhibitor z-VAD did not inhibit cell death, but rather enhanced it (Fig. 1C and 1F). Sodium arsenite did not induce activation of caspase-3 at all compared to cisplatin, which is a DNA-damaging agent (Fig. 1I). Collectively, these observations suggest that sodium arsenite only induces parthanatos but not apoptosis in these cancer cells.
Sodium arsenite induces parthanatos but not apoptosis in HT1080 and HeLa cells. (A) HT1080 cells were treated with NaAsO2 (0, 2.5, 5, 7.5, 10 μM) and z-VAD (0, 20 μM) and/or rucaparib (0, 2 μM) for 24 hr. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t-test; ** p < 0.01 (vs. untreated cells), ### p < 0.001 (vs. NaAsO2-treated cells). (B) HeLa cells were treated with NaAsO2 (0, 8, 12, 16, 20 μM) and z-VAD (0, 20 μM) and/or rucaparib (0, 2 μM) for 48 hr. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t-test; * p < 0.05, *** p < 0.001 (vs. untreated cells), # p < 0.05, ## p < 0.01, ### p < 0.001 (vs. NaAsO2-treated cells). (C) HT1080 cells were treated with NaAsO2 (0, 10 μM) and z-VAD (0, 20 μM) or rucaparib (0, 2 μM) for 24 hr. Dead cells were labeled with PI for 15 min, and analyzed by FACS. Quantification of the percentage of PI-positive cells is shown as Cell Death (%). Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t-test; *** p < 0.001 (vs. untreated cells), ### p < 0.001, (vs. NaAsO2-treated cells). (D) Immunoblot analysis of PARP-1 in HT1080 cells. Cell lysates were subjected to immunoblotting with the indicated antibodies. β-actin was used as a loading control. (E) WT and PARP-1 KO HT1080 cells were treated with NaAsO2 (0, 2.5, 5, 7.5, 10 μM) for 24 hr. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t-test; ** p < 0.01, *** p < 0.001 (vs. untreated WT cells), ## p < 0.01, ### p < 0.001 (vs. NaAsO2-treated WT cells). (F) WT and PARP-1 KO HT1080 cells were treated with NaAsO2 (0, 10 μM) and z-VAD (0, 20 μM) for 24 hr. Dead cells were labeled with PI for 15 min, and analyzed by FACS. Quantification of the percentage of PI-positive cells is shown as Cell Death (%). Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t-test; ** p < 0.01, *** p < 0.001 (vs. WT cells). (G) HT1080 cells were treated with NaAsO2 (0, 2.5, 5, 7.5 μM) for 24 hr, and then cell lysates were subjected to immunoblotting with the indicated antibodies. (H) HT1080 cells were treated with NaAsO2 (0, 5, 10 μM) for 24 hr. Cytoplasm or nucleus fractions were subjected to immunoblotting with the indicated antibodies. (I) HT1080 cells were treated with NaAsO2 (0, 2, 4, 6, 8 μM) for 24 hr or cisplatin (25 μM) for 8 hr, and then cell lysates were subjected to immunoblotting with the indicated antibodies.
A recent report has demonstrated that sodium arsenite promotes formation of cytoplasmic stress granules (SGs), including caspase-3 (Fujikawa et al., 2023). We speculated that the inclusion of caspase-3 in SGs prevents the caspase-3 activation, resulting in failure to induce apoptosis in HT1080 cells. To test this possibility, we investigated the behavior of SGs and caspase-3 in sodium arsenite-treated cells. Unpredictably, sodium arsenite failed to accumulate Ras GTPase-activating protein-binding protein 1 (G3BP1), an essential component of SGs, even though Thapsigargin, an inducer of SGs, promoted the SG formation (Fig. 2A). The formation of SGs is usually observed in the short-term treatment of sodium arsenite with high concentrations of 100 μM or more. Therefore, under the conditions of this study, where treatment was performed at a maximum concentration of 10 μM for 24 hr, it is considered that SGs were not formed. Interestingly, although the SG formation was not observed, the caspase-3 punctate formation was observed during exposure to sodium arsenite (Fig. 2B). Therefore, it seems that sodium arsenite promotes the caspase-3 puncta formation without SGs. Thus, we next speculated that the aggregation of unfolded proteins might be necessary for caspase-3 aggregation, because several lines of evidence have demonstrated that protein aggregates, such as α-synuclein, TDP-43, and STSQM1/p62 droplets are associated with the induction of parthanatos (Martire et al., 2013; Kam et al., 2018; McGurk et al., 2018; Noguchi et al., 2018). Indeed, the disaggregating agent 4-PBA not only abolished the accumulation of K48-linked polyubiquitinated proteins indicating unfolded proteins, but also abolished the caspase-3 puncta induced by sodium arsenite (Fig. 2C and 2D). These observations suggest that sodium arsenite induces caspase-3 aggregation along with the unfolded protein aggregates. It has been reported that low concentrations of sodium arsenite cause the accumulation of the autophagy receptor p62/SQSTM1 (Sanchez-Martin et al., 2020), which led us to examine p62 as a potential mediator of caspase-3 aggregation. Interestingly, we found that p62 and caspase-3 colocalize and form puncta during sodium arsenite treatment (Fig. 2E). Consistently, similar to 4-PBA treatment, p62 deficiency suppressed both the aggregation of K48-polyubiquitinated proteins and the formation of caspase-3 puncta induced by sodium arsenite (Fig. 2F and 2G). These results suggest that p62 is required for the aggregation of both caspase-3 and the K48-polyubiquitinated proteins in response to low concentration of sodium arsenite.
Sodium arsenite promotes caspase-3 aggregation during parthanatos. (A) HT1080 cells were treated with NaAsO2 (10 μM) for 24 hr or Thapsigargin (10 μM) for 1 hr, then immunofluorescence staining with the anti-G3BP1 antibody and DAPI nuclear staining were performed. Scale bar represents 10 μm. (B) HT1080 cells were treated with NaAsO2 (0, 2.5, 5, 10 μM) for 24 hr, then immunofluorescence staining with the anti-caspase-3 antibody and DAPI nuclear staining were performed. Scale bar represents 10 μm. (C) HT1080 cells were treated with NaAsO2 (0, 2.5, 5, 7.5, 10 μM) and 4-PBA (0, 3 mM) for 18 hr, and then cell lysates were subjected to immunoblotting with the indicated antibodies. (D) HT1080 cells were treated with NaAsO2 (0, 5, 10 μM) and 4-PBA (0, 3 mM) for 24 hr, then immunofluorescence staining with the anti-caspase-3 antibody and DAPI nuclear staining were performed. Scale bar represents 10 μm. (E) HT1080 cells were treated with NaAsO2 (10 μM) for 24 hr, then immunofluorescence staining with the anti-caspase-3 antibody and anti-p62 antibody and DAPI nuclear staining were performed. Scale bar represents 10 μm. (F) WT and p62 KO HT1080 cells were treated with NaAsO2 (0, 5, 10, 15 μM) for 18 hr, and then cell lysates were subjected to immunoblotting with the indicated antibodies. (G) WT and p62 KO HT1080 cells were treated with NaAsO2 (10 μM) for 24 hr, then immunofluorescence staining with the anti-caspase-3 antibody and anti-p62 antibody and DAPI nuclear staining were performed. Scale bar represents 10 μm.
Given that caspase-3 is released into cytosol by treatment with 4-PBA or p62 deficiency, sodium arsenite might promote the caspase-3 activation and subsequent apoptosis induction. Interestingly, in the presence of 4-PBA, sodium arsenite activated caspase-3 and promoted caspase-3-dependent cleavage of PARP-1, suggesting that 4-PBA can restore sodium arsenite-induced caspase-3 activation (Fig. 3A). Moreover, both rucaparib and z-VAD failed to inhibit sodium arsenite-induced cell death in the presence of 4-PBA (Fig. 3B and 3C). However, co-treatment with rucaparib and z-VAD strongly inhibited sodium arsenite-induced cell death in the presence of 4-PBA (Fig. 3B and 3C). Similar results were obtained in HeLa cells (Fig. 3D). These observations suggest that the treatment with 4-PBA increased sensitivity to apoptosis, and sodium arsenite can induce apoptosis when the induction of parthanatos is inhibited. Of note, in HeLa cells, the effect of the co-treatment with rucaparib and z-VAD was much weaker than HT1080 cells (Fig. 3D). As to the causes of this difference, it is possible that the effect of the co-treatment with rucaparib and z-VAD was difficult to observe, because 4-PBA is toxic to HeLa cells, as shown in Fig. 3E. On the other hand, in p62-deficient HT1080 cells, sodium arsenite induced caspase-3 activation and caspase-3-dependent cleavage of PARP-1, similar to the effects observed with 4-PBA treatment (Fig. 3F and 3G). Consistently, sodium arsenite-induced cell death in p62-deficient cells was not suppressed by either z-VAD or rucaparib alone, whereas co-treatment with both inhibitors almost completely abolished cell death (Fig. 3H and 3I). These findings suggest that inhibition of caspase-3 aggregation, either by 4-PBA or by genetic ablation of p62, restores caspase-3 activation and sensitizes cells to apoptosis under sodium arsenite exposure.
Disaggregation of caspase-3 increases sensitivity to apoptosis. (A) HT1080 cells were treated with NaAsO2 (0, 2.5, 5 μM) and 4-PBA (0, 3 mM) for 24 hr, and then cell lysates were subjected to immunoblotting with the indicated antibodies. (B) HT1080 cells were treated with 4-PBA (3 mM) and NaAsO2 (0, 2.5, 5, 7.5, 10 μM) and z-VAD (0, 20 μM) and/or rucaparib (0, 2 μM) for 24 hr. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t-test; *** p < 0.001 (vs. control cells). (C) HT1080 cells were treated with 4-PBA (3 mM) and NaAsO2 (0, 5 μM) and z-VAD (0, 20 μM) and/or rucaparib (0, 2 μM) for 24 hr. Dead cells were labeled with PI for 15 min, and analyzed by FACS. Quantification of the percentage of PI-positive cells is shown as Cell Death (%). Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t-test; *** p < 0.001 (vs. control cells). (D) HeLa cells were treated with 4-PBA (3 mM) and NaAsO2 (20 μM) and z-VAD (0, 20 μM) and/or rucaparib (0, 2 μM) for 48 hr. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t-test; * p < 0.05, N.S. p > 0.05 (vs. control cells). (E) HT1080 cells were treated with 4-PBA (0, 3 mM) for 24 hr, and HeLa cells were treated with 4-PBA (0, 3 mM) for 48 hr. Cell viability was determined by PMS/MTS assay. (F) WT and p62 KO HT1080 cells were treated with NaAsO2 (0, 5, 10 μM) for 24 hr, and then cell lysates were subjected to immunoblotting with the indicated antibodies. (G) WT and p62 KO HT1080 cells were treated with NaAsO2 (0, 5, 10, 15 μM) for 24 hr, and then cell lysates were subjected to immunoblotting with the indicated antibodies. (H) p62 KO HT1080 cells were treated with NaAsO2 (0, 4, 6, 8, 10 μM) and z-VAD (0, 20 μM) and/or rucaparib (0, 2 μM) for 24 hr. Cell viability was determined by PMS/MTS assay. Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t-test; ** p < 0.01, *** p < 0.001 (vs. control cells). (I) p62 KO HT1080 cells were treated with NaAsO2 (0, 5 μM) and z-VAD (0, 20 μM) and/or rucaparib (0, 2 μM) for 24 hr. Dead cells were labeled with PI for 15 min, and analyzed by FACS. Quantification of the percentage of PI-positive cells is shown as Cell Death (%). Data shown are the mean ± SD (n = 3). Statistical significance was tested using an unpaired Student’s t-test; *** p < 0.001 (vs. control cells).
In this study, we demonstrated that sodium arsenite cannot induce apoptosis in the cancer cells, whereas apoptosis was induced when the aggregation of caspase-3 is inhibited by 4-PBA or p62-deficiency (Fig. 4). These observations show that aggregation status of caspase-3 is a critical determinant of the selectivity of sodium arsenite-induced cell death. Moreover, our results raise the possibility that parthanatos works as an alternative cell death machinery when the ability of cells to induce apoptosis is impaired. If so, ensuring the induction of parthanatos when apoptotic machinery is impaired is important for preventing arsenic-induced carcinogenesis. Thus, toxicological studies of arsenic-induced parthanatos will lead to a better understanding of arsenic-induced carcinogenesis.
Schematic model to explain our study. When exposed to sodium arsenite, apoptosis is suppressed by the aggregation of caspase-3, and parthanatos is alternatively induced. On the other hand, disaggregation of caspase-3 induced by 4-PBA allows the caspase-3 activation, and then increases sensitivity to apoptosis.
We thank all members of Lab of Health Chemistry for helpful discussions. This work was supported by JSPS KAKENHI Grant Numbers JP21H02691, JP21H02620, JP24KJ0428, JP24K02237, JP24K22011 and JP24K02173.
Conflict of interestThe authors declare that there is no conflict of interest.
