Abstract

Purpose

   Melanomas are highly malignant and rapidly develop drug resistance due
   to dysregulated apoptosis. Therefore, pro-apoptotic agents could be
   effective for the management of melanoma. Hydrogen sulfide is
   ubiquitous in the body, and exogenous hydrogen sulfide has been
   reported to show inhibitory and pro-apoptotic effects on cancer cells.
   However, whether high concentrations of exogenous hydrogen sulfide have
   pro-apoptotic effects on melanoma and its mechanisms remain unknown.
   Hence, this study aimed to explore the pro-apoptotic effects and
   mechanisms of exogenous hydrogen sulfide on the A375 melanoma cell line
   treated with a hydrogen sulfide donor (NaHS).

Methods

   The cell proliferation test, flow cytometric analysis, Hoechst 33258
   staining, and Western blotting of B-cell lymphoma 2 and cleaved
   caspase-3 were used to explore the pro-apoptotic effects of hydrogen
   sulfide on A375 cells. The transcriptional profile of NaHS-treated A375
   cells was further explored via high-throughput sequencing. Western
   blotting of phosphorylated inositol-requiring enzyme 1α (p‐IRE1α),
   phosphorylated protein kinase R-like ER kinase (p-PERK), phosphorylated
   eukaryotic translation initiation factor 2α (p-eIF2α), C/EBP homologous
   protein, glucose-regulating protein 78, IRE1α, PERK, and eIF2α was
   performed to verify the changes in the transcriptional profile.

Results

   NaHS inhibited A375 melanoma cell proliferation and induced apoptosis.
   The endoplasmic reticulum stress unfolded protein response and
   apoptosis-associated gene expression was upregulated in NaHS-treated
   A375 melanoma cells. The overactivation of the unfolded protein
   response and increase in endoplasmic reticulum stress was verified at
   the protein level.

Conclusion

   Treatment with NaHS increased endoplasmic reticulum stress, which
   triggered the overactivation of the unfolded protein response and
   ultimately lead to melanoma cell apoptosis. The pro-apoptotic effect of
   NaHS suggests that it can be explored as a potential therapeutic agent
   in melanoma.

   Keywords: hydrogen sulfide, melanoma, apoptosis, unfolded proteins
   response, endoplasmic reticulum stress

Introduction

   Melanoma is caused by the malignant transformation of melanocytes and
   is a fatal type of skin cancer that is resistant to chemo- and
   radiotherapy.[30]^1 Each year, approximately 232,100 individuals are
   diagnosed with cutaneous melanomas worldwide.[31]^2 About half of the
   patients diagnosed with melanomas have BRAF mutations.[32]^3 Combined
   treatment with BRAF inhibitors (such as dabrafenib and vemurafenib) and
   mitogen-activated protein kinase (MAPK) inhibitors (such as trametinib
   and cobimetinib) improves the treatment response and overall survival
   in patients with cutaneous melanoma.[33]^4 Recently, the development
   and application of immune checkpoint inhibitors, such as anti-cytotoxic
   T lymphocyte antigen-4 and anti-programmed cell death-1 antibodies,
   have been shown to prolong the overall survival of patients with
   melanoma.[34]^5 Despite the initial response, genetic mutations in
   multiple pathway molecules and intrinsic tumor factors or
   microenvironment features lead to a high proportion of melanoma
   patients developing resistance to targeted drugs and immune checkpoint
   inhibitors after a few months of treatment.[35]^6–9 Due to the high
   rate of disease recurrence and treatment resistance, melanoma patients
   succumb to the disease within a few months, resulting in approximately
   55,500 melanoma-attributed deaths worldwide each year.[36]^2^,[37]^10
   The dysregulation of apoptosis is a major factor contributing to drug
   resistance in melanoma. A significant decrease in the rate of apoptosis
   was reported in melanoma cells resistant to chemotherapy and targeted
   drugs.[38]^11^,[39]^12 The frequent anti-apoptotic phenomenon observed
   in melanoma patients implies that exploring pro-apoptotic agents might
   be a promising anti-cancer strategy.[40]^13–16 Therefore, further
   improvements, especially the development of highly effective drugs to
   induce a pro-apoptotic effect in melanoma, are warranted to improve the
   overall survival of patients with cutaneous melanoma.

   Hydrogen sulfide (H[2]S) is the third most common gasotransmitter in
   the body apart from carbon monoxide (CO) and nitric oxide (NO).[41]^17
   The three enzymes that can catalyze the generation of H[2]S in human
   cells are: 3-mercaptopyruvate sulfotransferase, cystathionine
   beta-synthase, and cystathionine gamma-lyase.[42]^18 H[2]S possesses
   functions in an assortment of physiological and pathological processes
   in human cells. It can relax the blood vessels and protect the neurons
   and myocardium from oxidative stress.[43]^19^,[44]^20 Different doses
   of H[2]S have various effects on cancer. Low concentrations of H[2]S
   promotes cancer progression by increasing the expression of vascular
   endothelial growth factor and promoting angiopoiesis; preventing tumor
   cell apoptosis by decreasing the intracellular reactive oxygen species
   (ROS) levels; reducing DNA damage caused by mitochondrial disruption;
   and accelerating the cell cycle of cancer cells by regulating the
   phosphorylation level of the protein kinase B/Akt.[45]^21^,[46]^22 In
   contrast, higher concentrations of exogenous H[2]S exhibited an
   inhibitory effect on cancer cells — it promoted uncontrolled
   intracellular acidification and accelerated apoptosis by inducing cell
   cycle arrest in human colon cancer cells.[47]^23 The H[2]S donor,
   ATB-346, significantly downregulated the expression of anti-apoptotic
   genes (eg, Fas-associated death domain-like interleukin-1β-converting
   enzyme inhibitory protein and B-cell lymphoma 2 [Bcl-2]), which lead to
   a pro-apoptotic effect in the cancer cells.[48]^24^,[49]^25 Exogenous
   H[2]S increments the capacity of the anion and sodium exchangers to
   promote the generation of metabolic acids, which can lead to cancer
   cell death.[50]^23 The potential pro-apoptotic activity of H[2]S donors
   leads to the question of whether high concentrations of exogenous H[2]S
   exerts a pro-apoptotic effect on melanoma, and by which mechanism(s) is
   this accomplished.

   Hence, this study aimed to investigate the pro-apoptotic effect of
   exogenous H[2]S on melanoma cells and its mechanism of action to
   provide new insights regarding alternative strategies for melanoma
   treatment. The effects of H[2]S on the proliferation and apoptosis of
   melanoma cells and the alterations in the transcriptome and
   corresponding biological processes in melanoma cell lines were examined
   after administering exogenous H[2]S. Furthermore, changes in the
   transcriptome of cells treated with H[2]S were verified at the protein
   level.

Materials and Methods

Cell Culture and Treatment

   The human melanoma cell line, A375, was acquired from the Institute of
   Basic Medicine, Chinese Academy of Medical Sciences (Beijing, China).
   Cells were cultivated in Dulbecco’s Modified Eagle Medium (DMEM;
   11995040, Gibco, NY, USA) supplemented with 10% fetal bovine serum
   (10099-141, Gibco, NY, USA) at 37°C in a humidified incubator with 5%
   CO[2] and 95% air conditions. Salt of sodium hydrosulfide (NaHS;
   S106641, Aladdin, Shanghai, China) was dissolved in the cell culture
   medium (DMEM; 50 mL) to make the stock solution (concentration of 400
   mM). The temperature and time at which the solution was stored was 4°C
   and one week. Immediately before experiments, the NaHS stock solution
   was diluted with DMEM to concentrations of 0, 0.5, 5, 10, 20, and 50
   mM, and the A375 cells were treated for 24 h.

Cell Proliferation Analysis

   A cell counting kit-8 (CCK-8, CK04, Dojindo Laboratory, Kyushu, Japan)
   was used according to the manufacturer’s instructions to evaluate cell
   proliferation at the indicated time points. NaHS-treated A375 cells and
   control cells were seeded at a density of 5×10^3 cells/well in 96-well
   plates in quintuplicate. The optical absorbance value of each well was
   determined at 450 nm on a microplate reader (iMark Reader, Bio-Rad,
   USA) after incubating with CCK-8 for 1 h at 37°C. The formula used to
   calculate the cell proliferation rate was: relative cell proliferation
   rate% = (450 nm sample – 450 nm background) / (450 nm control – 450 nm
   background) ×100%.

Hoechst 33258 Staining

   Paraformaldehyde (4%) was used to fix 20 mM NaHS-treated cells and
   control cells for 10 min. The cells were then stained with 5 mg/L
   Hoechst 33258 for 10 min, after which they were observed under an
   Olympus fluorescent microscope (Tokyo, Japan). Hoechst 33258 (H266330,
   Aladdin, Shanghai, China) staining revealed changes in chromatin
   morphology. The apoptotic cell nuclei showed condensed, fractured
   fluorescence representing chromosomal condensation and fragmentation,
   while normal cells exhibited uniform fluorescence and a completely
   rounded nucleus.

Cell Apoptosis Analysis

   NaHS-treated (20 mM) cells and control cells were harvested via
   trypsinization after treatment with NaHS for 24 h. The cells were
   resuspended in the binding buffer after washing twice with
   phosphate-buffered saline (PBS). Annexin V-FITC and propidium iodide
   solutions (C1067S, Beyotime Institute of Biotechnology, Shanghai,
   China) were added to the cell suspension. The cells were examined using
   flow cytometry (BD, Becton, USA) after incubating for 20 min at room
   temperature.

RNA Isolation and Sequencing

   Total RNA from the NaHS-treated and control cells was isolated using
   the Qiagen RNeasy Plus Mini kit (74134, Hilden, Germany) according to
   the manufacturer’s instructions. The quality of the RNA was assessed
   using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). The
   Illumina^® (NEB, USA) sequencing libraries were generated using a
   NEBNext^® Ultra^TM RNA Library Prep Kit following the manufacturer’s
   instructions. The constructed libraries were loaded and sequenced on a
   Hiseq 4000 platform (Illumina, USA). RNA isolation and sequencing were
   conducted by the Majorbio Institute, Shanghai, China. Finally,
   paired-end reads of length approximately 150 bp were generated.

Assembly of Transcriptome and Gene Function Annotation

   Raw reads generated in FASTQ format were processed in several steps,
   including removing adapter sequences, poly-N, and low-quality reads to
   obtain high-quality clean reads. TopHat2 was used to align the data to
   the human reference genome (hg38). The quality of the sequencing data
   was evaluated by FastQC. Gene mapping of the reads was conducted by
   using FeatureCounts v1.5.0-p3. The transcripts per million values were
   used to estimate gene expression levels.

Differential Gene Expression and Functional Enrichment Analyses

   The expression pattern of each sample was analyzed using a principal
   component analysis (PCA). The differential gene expression (DGE)
   analysis was performed using the R DESeq2 package (version 3.2.3). The
   p-value and fold change (FC) of the comparison between samples and
   controls were calculated. The differentially expressed genes (DEGs)
   were filtrated based on a FC of <0.5 or >2 and a p-value of <0.05.
   Volcano plots of the DEGs were generated using the R ggplot2 package
   (version 3.3.3). The Kyoto Encyclopedia of Genes and Genomes (KEGG) and
   Gene Ontology (GO) databases ([51]https://david.ncifcrf.gov/) were
   utilized to conduct biological function and pathway enrichment analysis
   of the DEGs. Inter-cluster similarities and intra-cluster redundancies
   were evaluated using the Metascape online database
   ([52]http://metascape.org).

Western Blot

   Protein lysates extracted from A375 cells were separated through sodium
   dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
   transferred to polyvinylidene difluoride (PVDF) membranes (88585,
   Thermo Fisher Scientific, MA, USA). The PVDF membranes were blocked
   using 5% bovine serum albumin, and then incubated with the following
   primary antibodies at 4°C overnight: Bcl-XL (dilution 1:2000,
   10783-1-AP, Proteintech, Wuhan, China), CHOP (dilution 1:1000,
   15204-1-AP, Proteintech, Wuhan, China), GRP78 (dilution 1:2000,
   1587-1-AP, Proteintech, Wuhan, China), anti-β-actin (dilution 1:2000,
   20536-1-AP, Proteintech, Wuhan, China), cleaved Caspase-3 (dilution
   1:1000, AF7022, Affinity, Changzhou, China), p-IRE1α (dilution 1:2000,
   AF7150, Affinity, Changzhou, China), IRE1α (dilution 1:2000, DF7709,
   Affinity, Changzhou, China), p-PERK (dilution 1:2000, DF7576, Affinity,
   Changzhou, China), PERK (dilution 1:2000, AF5304, Affinity, Changzhou,
   China), p-eIF2α (dilution 1:2000, AF3087, Affinity, Changzhou, China),
   and eIF2α (dilution 1:2000, AF6087, Affinity, Changzhou, China), and
   then incubated with HRP-conjugated secondary antibodies (dilution
   1:5000, ZB-5301, ZSBIO, Beijing, China) for 1 h and detected using the
   ECL System (NEL104001EA, PerkinElmer, Shanghai, China).

Statistical Analysis

   All data are presented as the mean ± standard deviation (S.D.). SPSS
   (version 26.0; NY, USA) was used for the data analysis. Images were
   drawn using GraphPad Prism 8.0 (San Diego, CA, USA). One-way analysis
   of variance (ANOVA) and non-parametric tests were performed to analyze
   the differences between groups. p < 0.05 was considered statistically
   significant.

Results

NaHS Suppresses Cell Proliferation and Induces Apoptosis of A375 Melanoma
Cells

   A375 cells were treated with different concentrations of NaHS to
   explore the effects of NaHS. Treatment with NaHS (0, 20, and 50 mM)
   resulted in morphological changes in the A375 cells, including cell
   roundness and detachment from the cell culture plate ([53]Figure 1A).
   CCK8 assay was performed to determine the cell proliferation rate. NaHS
   treatment (0, 0.5, 5, 10, 20, and 50 mM) inhibited the proliferation of
   A375 cells in a dose-dependent manner ([54]Figure 1B). Hochest 33258 is
   a blue, fluorescent dye that penetrates cell membranes and is used to
   detect apoptosis. Hoechst 33258 staining showed that the nuclei of A375
   cells subjected to NaHS (20 mM) exposure for 5 h were fragmented and
   densely stained compared with the control (0 mM; [55]Figure 1C).
   Moreover, NaHS (20 mM) induced apoptosis in A375 cells, as confirmed by
   flow cytometry ([56]Figure 1D). Caspase-3 and Bcl-XL are the key
   proteins involved in apoptosis. The decreased Bcl-XL and up-regulated
   cleaved Caspase-3 expression in 20 mM NaHS-treated A375 cells were
   further confirmed by Western blotting ([57]Figure 1E)
   ([58]Supplementary Material 1). Therefore, NaHS suppressed cell
   proliferation and induced apoptosis by providing exogenous H[2]S in
   human A375 melanoma cells.

Figure 1.

   [59]Figure 1
   [60]Open in a new tab

   NaHS suppressed cell proliferation and induced apoptosis of A375 cells.
   (A) Morphological changes of A375 cells caused by treatment at
   different concentrations of NaHS (0, 20, and 50 mM). (B) CCK-8 assay at
   different concentrations of NaHS-treated A375 cells. (C) Hoechst 33258
   staining of A375 cells subjected to NaHS (20 mM) exposure and control
   cells (0 mM). (D) Cytometric analysis of NaHS (20 mM) treated A375
   cells and control cells (0 mM). (E) Western blotting analysis of
   cleaved Caspase-3 and Bcl-XL in NaHS (20 mM) treated A375 cells and
   control cells (0 mM). **P < 0.01, ***P < 0.001 compared with negative
   control (NC).

Transcriptional Profiles of NaHS-Treated A375 Melanoma Cells Showed Increased
Endoplasmic Reticulum Stress and Overactivation of the Unfolded Protein
Response

   To understand transcriptome changes after NaHS treatment,
   high-throughput sequencing was performed on 20 mM NaHS-treated A375
   cells. The quality of the sequencing data was verified, and the mapping
   rates were 96.25% to 96.51%. The data were then dimensionally reduced,
   and the noise was removed by PCA to demonstrate the relationship
   between the samples, resulting in distinct clustering of both the
   treated and untreated samples ([61]Figure 2A). Correlation analysis
   between samples was based on the gene expression matrix. Correlation
   analysis showed that the treated and untreated samples were positively
   correlated with biological replicates ([62]Figure 2B).

Figure 2.

   [63]Figure 2
   [64]Open in a new tab

   Transcriptional profiles showed increased endoplasmic reticulum stress
   and unfolded protein response in NaHS-treated A375 cells. (A)
   Principal-component analysis (PCA) of the samples. (B) Correlation
   analysis of the samples. (C) Volcano plot of the differential
   expression of genes between NaHS (20 mM) treated A375 cells and control
   cells (0 mM). (D) Heat map of the differentially expressed genes
   (DEGs). (E) GO biological process enrichment analysis of the
   up-regulated DEGs. (F) KEGG pathway analysis of the up-regulated DEGs.
   (G) Metascape network showed inter-cluster enriched GO terms for the
   up-regulated DEGs.

   The R DESeq2 package (version 3.2.3) was used to perform DGE analysis.
   A total of 5752 DEGs were found between the treated and untreated
   cells. As shown on the volcano plot, 2208 upregulated and 3544
   downregulated genes were found in the NaHS-treated cells compared to
   the untreated cells ([65]Figure 2C). Unsupervised hierarchical
   clustering based on the similarities in the expression of these DEGs
   distinguished the treated cells from the untreated cells on the heat
   map ([66]Figure 2D). Furthermore, biological process enrichment
   analysis was performed using the GO and KEGG databases to understand
   the functions of these DEGs. The DEGs were related to the regulation of
   response to unfolded proteins, response to the endoplasmic reticulum
   (ER) stress, negative regulation of cellular processes, and regulation
   of the cell cycle, according to the GO database analysis ([67]Figure
   2E). The KEGG pathway analysis revealed that protein processing in the
   ER, apoptosis, ferroptosis, MAPK signaling pathway, and other cell
   stress-associated pathways were enriched in the NaHS-treated A375 cells
   ([68]Figure 2F). The Metascape database was also used to perform a
   network enrichment analysis. The findings revealed that the unfolded
   protein response (UPR) and ER stress-associated processes were
   prominent in the transcriptome and induced a series of related
   biological processes with a radial distribution ([69]Figure 2G). Our
   findings indicate that the unfolded protein response, endoplasmic
   reticulum stress, and apoptosis-associated gene expression signatures
   were prominent in the NaHS-treated A375 melanoma cells.

Increase in Unfolded Protein Response and Endoplasmic Reticulum Stress in the
NaHS-Treated A375 Cells

   Treatment with NaHS led to an increase in the number of unfolded
   proteins inside the ER lumen as well as an increase in ER stress.
   Considering that UPR and ER stress was prominently observed during the
   transcriptional phase of 20 mM NaHS-treated A375 cells, Western
   blotting was conducted to verify these changes. CHOP and GRP78 were
   considered markers for ER stress. As shown in [70]Figure 3A, the
   increased expression levels of CHOP and GRP78 in 20 mM NaHS-treated
   A375 cells indicates a significant increase in ER stress. When the
   expression levels of unfolded proteins reach the threshold values, the
   cells initiate UPR to restore homeostasis. If the reaction is mild,
   cellular homeostasis is restored, and the cells remain alive. When the
   expression level of unfolded proteins is excessively high and ER stress
   triggers the apoptotic process, the cell dies. IRE1α, PERK, and eIF2α
   are critical molecules in the UPR pathway. The UPS was increased in the
   20 mM NaHS-treated A375 cells based on the results of Western blotting,
   and the ratio of p‐PERK to PERK protein, p‐IRE1α to IRE1α protein, and
   p‐eIF2α to eIF2α protein significantly increased ([71]Figure 3B)
   ([72]Supplementary Material 1). As a result of high ER stress and
   overreaction of UPR, the application of 20 mM NaHS induced cancer cell
   apoptosis ([73]Figure 1D). These findings indicate that NaHS induced
   the apoptosis of melanoma cells by increasing the ER stress levels and
   UPR.

Figure 3.

   [74]Figure 3
   [75]Open in a new tab

   The increased protein expression levels of unfolded proteins response
   and endoplasmic reticulum stress in NaHS-treated A375 cells. (A)
   Western blot of CHOP and GRP78 in NaHS (20 mM) treated A375 cells and
   control cells (0 mM). (B) Western blot of p‐IRE1α, IRE1α protein,
   p‐PERK, PERK protein, p‐eIF2α, and eIF2α in NaHS (20 mM) treated A375
   cells and control cells (0 mM). ***P < 0.001 compared with negative
   control (NC).

Discussion

   The present study showed that exposure of A375 human melanoma cells to
   NaHS, an exogenous H[2]S donor, suppressed cell proliferation and
   increased cell apoptosis. The ER stress and UPS showed remarkable
   transcriptional changes, identified using high-throughput sequencing.
   These changes were verified at the protein level. Thus, this study’s
   findings broadened the understanding of ER stress and UPS mechanisms
   involved in the antitumor effect of exogenous H[2]S.

   Melanoma is highly malignant, and patients with advanced tumors have a
   poor prognosis, with a mortality rate of over 90%.[76]^26 Systemic
   chemotherapy has been applied for the treatment of advanced melanoma
   for a long time.[77]^27 Dacarbazine is commonly used as an FDA-approved
   single agent to treat metastatic melanoma, and clinical trials of
   dacarbazine in combination with other drugs are currently underway for
   the treatment of malignant melanoma.[78]^28 Carboplatin plus paclitaxel
   was another widely used first- and second-line combination chemotherapy
   treatment.[79]^29 But unfortunately, in patients with advanced disease,
   conventional chemotherapy treatments are ineffective in preventing
   disease recurrence, and several patients with various cancer types
   develop resistance within a few months of treatment.[80]^30 In tumors
   resistant to chemotherapy, cancer cells resist apoptosis, and cells
   survive and overproliferate.[81]^16 Cancer cell’s evasion of and
   resistance to apoptosis often emerges in conventional chemotherapeutic
   treatment, resulting in poor clinical outcomes.[82]^31 Therefore, the
   induction of cancer cell apoptosis is expected to be a promising and
   effective strategy for the treatment of melanoma.

   Recently, several studies have confirmed the involvement of H[2]S in
   cancer development, although these studies have reported conflicting
   results on the pro- and anticancer effects of H[2]S. Wu et al treated
   thyroid cancer cells with NaHS for 24 h; the cells showed reduced ROS
   levels, the PI3K/Akt/mTOR signaling pathway was activated, and cancer
   cell proliferation and migration were promoted.[83]^22 However, some
   studies revealed that H[2]S shows an inhibition effect on the
   proliferation and viability of cancer cells.[84]^32 Lu et al reported
   that cell cycle arrest was triggered by the downregulation of cell
   cycle protein D1 in the HepG2 cells within 24 h of treatment with the
   H[2]S donor, GYY4137.[85]^33 Interestingly, studies proved that H[2]S
   has a selective toxic effect on cancer cells while not apparently
   damaging to normal cells.[86]^32^,[87]^34 Under the same conditions,
   CA9-22 cancer cells treated with H[2]S for 48–72 h showed a significant
   increase in apoptosis, while no pro-apoptotic effect was observed on
   the normal human gingival keratin-forming cells.[88]^35 Therefore,
   exposure to a high concentration of exogenous H[2]S selectively
   suppresses the proliferation of cancer cells and induces
   apoptosis.[89]^36 Here, we demonstrated the ability of NaHS to inhibit
   melanoma cell proliferation and induce apoptosis in vitro by exposing
   A375 cells to the H[2]S donor, NaHS, at higher concentrations for 24 h.
   A previous study reported that H[2]S inhibits NF-κB activity in human
   melanoma cells and decreases the expression level of Bcl-2 to induce
   spontaneous apoptosis.[90]^25 This was confirmed in our study, where
   Bcl-XL expression was downregulated, while cleaved-caspase-3 was
   upregulated in NaHS-treated A375 cells. Exogenous H[2]S exerts
   anti-cancer effects by enhancing autophagy and promoting apoptosis in
   human melanoma cells by regulating the PI3K/Akt/mTOR signaling
   pathway.[91]^37 Our study demonstrated the promotion of apoptosis in
   NaHS-treated cells. Moreover, the unfolded proteins were significantly
   increased in NaHS-treated cells, thus triggering ER stress and
   eventually apoptosis, which expanded the antitumor mechanism of high
   concentration H[2]S.

   Cells broadly alter their transcriptional profile accordingly when
   facing changes in their environment or specific stimuli to achieve
   homeostasis.[92]^38^,[93]^39 This is especially true for
   stress-responsive pathways, where transcription must be consistently
   altered to adapt to the environment. Our results, using transcriptome
   sequencing, indicate that treatment of melanoma with high
   concentrations of H[2]S significantly upregulated the UPR-related and
   ER stress-related pathway signals. UPR is an element of the integrative
   stress response activated under ER stress conditions and aims to
   achieve cellular and tissue homeostasis.[94]^40^,[95]^41 The ER is an
   essential site of protein biosynthesis, post-translational
   modifications, and newly synthesized protein folding and
   assembly.[96]^42 As a membrane compartment, the ER is highly sensitive
   to changes affecting its structure, integrity, and function, which can
   disrupt the protein folding process.[97]^43 Treatment with high
   concentrations of H[2]S may lead to impaired protein synthesis in the
   ER and increased accumulation of unfolded or misfolded proteins. When
   the pathological proteins are overloaded, UPR is initiated.[98]^44
   Three receptor proteins control the classical UPR: PERK, IRE1α, and
   ATF6.[99]^45 Our results showed a significant upregulation of PERK and
   IRE1α. The UPR, triggered by the activation of stress sensors, aimed to
   reduce the unfolded protein load, an adaptive response that helps
   maintain cell viability and function.[100]^46 When the UPR fails to
   adapt to ER stress, the cells are unable to achieve homeostasis,
   leading to apoptosis.[101]^47^,[102]^48 Our study found that in the
   presence of high concentrations of exogenous H[2]S, the UPR of melanoma
   cells was significantly enhanced, ER stress increased, cellular
   homeostasis was not achieved, apoptotic processes were activated, and
   apoptotic pathway signals were significantly upregulated.

Conclusion

   This study shows that the exogenous H[2]S donor, NaHS, enhances the
   apoptotic process in melanoma cells. This study was the first to report
   that the protein synthesis process in the ER was affected in treated
   cells, with a significant increase in unfolded proteins, triggering ER
   stress and ultimately leading to apoptosis. The present study findings
   help expand the understanding of the mechanism of the antitumor effect
   of H[2]S treatment at high concentrations.

Funding Statement

   Research reported in this publication was supported by the Traditional
   Chinese Medicine Science and Technology Project of Shandong Province
   (No. 2021Q093), the Doctoral Startup Fund of Affiliated Hospital of
   Weifang Medical University (No. 2021BKQ02), and Shandong Province
   Natural Science Foundation (No. ZR2020QH138).

Abbreviations

   Bcl-2, B-cell lymphoma 2; Bcl-XL, B-cell lymphoma 2 -extra large;
   CCK-8, cell counting kit-8; CHOP, C/EBP homologous protein; DMEM,
   Dulbecco’s modified eagle medium; FITC, fluorescein isothiocyanate;
   GRP78, glucose-regulating protein 78; mTOR, mammalian target of
   rapamycin; NaHS, sodium hydrosulfide; p-eIF2α, phosphorylated
   eukaryotic translation initiation factor 2α; PI, propidium iodide;
   p‐IRE1α, phosphorylated inositol-requiring enzyme 1α; p-PERK,
   phosphorylated protein kinase R-like ER kinase; UPR, unfolded protein
   response.

Data Sharing Statement

   Data supporting this study’s findings are available from the
   corresponding author upon reasonable request.

Author Contributions

   All authors made a significant contribution to the work reported,
   whether that is in the conception, study design, execution, acquisition
   of data, analysis, and interpretation, or all these areas; took part in
   drafting, revising, or critically reviewing the article; gave final
   approval of the version to be published; have agreed on the journal to
   which the article has been submitted; and agree to be accountable for
   all aspects of the work.

Disclosure

   The authors report no conflicts of interest in this work.

References