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. 2026 May 13;31:115. doi: 10.1186/s11658-026-00945-5

H2S induces apoptosis of tumors with high IDO1 expression via NR4A1-BCL-2 and SOCS3 pathways

Zhen Ning Tony He 1,#, Fangzhou Meng 1,#, Xiaoyang Qian 1, Yuying Liu 1, Xin Fang 1, Dan Yang 2,✉, Qing Yang 1,✉
PMCID: PMC13374331  PMID: 42129631

Abstract

Background

Hydrogen sulfide (H2S) has been reported to exert both protumor and antitumor functions. It is worthy to clarify the condition under which H2S exerts antitumor effects and its underlying mechanism. Our previous study connected the immune checkpoint indoleamine 2,3-dioxygenase 1 (IDO1) and H2S by revealing that H2S downregulates IDO1 expression, leading to our hypothesis that antitumor effect of H2S is associated with IDO1 expression in tumor cells.

Methods

Apoptosis, cellular distribution of NR4A1, phosphorylation of IDO1, and the binding of phosphorylated IDO1 and SOCS3 are examined both in vitro and in vivo.

Results

In this study, we confirm our hypothesis by showing that H2S significantly reduces proliferation and induces apoptosis of tumor cells with high IDO1 expression. In tumor cells with high IDO1 expression, H2S promotes the translocation of NR4A1 out of nucleus and its binding with BCL-2, as well as the phosphorylation of IDO1 and the codegradation of phosphorylated IDO1 and SOCS3. Such mechanism by which H2S induces apoptosis of tumors with high IDO1 expression is also elucidated in tumor-bearing mice, where H2S shows great therapeutic effect against tumors with high IDO1 expression.

Conclusions

H2S induces apoptosis of tumor cells with high expression of IDO1 by promoting NR4A1-BCL-2 apoptotic pathway and the codegradation of phosphorylated IDO1 and SOCS3. Our study provides new theoretical and experimental evidence for a strategy for tumor therapy with H2S.

Supplementary Information

The online version contains supplementary material available at 10.1186/s11658-026-00945-5.

Keywords: Hydrogen sulfide; Indoleamine 2,3-dioxygenase 1; Apoptosis; Tumor therapy

Introduction

Gaseous signaling molecule hydrogen sulfide (H2S) exerts various physiological and pathological functions in mammals [1]. Dysregulation of H2S is related to various diseases, including cancer, in which all three enzymes producing H2S, cystathionine-γ-lyase, cystathionine-β-synthase, and 3-mercaptopyruvate sulfurtransferase, are reported to be upregulated [1, 2]. Both protumor effects and antitumor effects of H2S have both been reported [2–10]. However, the condition under which H2S exerts antitumor effects remains to be further explored.

A bell-shaped model has been proposed suggesting that whether H2S exerts pro- or antitumor effects is related to the concentration of H2S in the microenvironment: A moderate amount of H2S promotes tumor growth, while excessive or insufficient H2S restrains tumor progression [1]. Apart from this, whether the antitumor effect of H2S is related to some endogenous molecules in tumor cells remains to be determined. Previously, we have reported that H2S can downregulate the expression of immune checkpoint indoleamine 2,3-dioxygenase 1 (IDO1) in hepatocellular carcinoma, and H2S donors effectively restrict the tumor development in H22 hepatocellular carcinoma (HCC)-bearing mice via downregulating IDO1 expression [11]. IDO1 is a monomeric enzyme catalyzing the first and rate-limiting step of kynurenine pathway (KP), converting tryptophan (Trp) into kynurenine (Kyn) and subsequent metabolites [12, 13]. IDO1 and KP have also been reported to be associated with tumor growth and apoptosis. In oral squamous cell carcinoma, IDO1 inhibition leads to the upregulation of pro-apoptotic protein BCL2A1 [14]. In diffuse large B cell lymphoma, IDO1 inhibition induces cell cycle arrest and apoptosis via MDM2-p53 pathway [15]. IDO1 inhibition induces apoptosis in colorectal cancer by downregulating CDC20 and promoting mitochondrial injury [16]. KP metabolite Kyn can lead to activation of β-catenin and proliferation of human colon cancer cells, and IDO1 knockout can reduce proliferation of the cells [17]. In addition, Kyn and its receptor aryl hydrocarbon receptor (AhR) mediate neuronal apoptosis by mitochondrial death and free-radical accumulation via RhoA/BAX signaling pathway [18]. We hypothesize that the mechanism by which H2S exerts antitumor effects is related to IDO1 expressed in tumor cells.

IDO1 protein consists mainly of a large heme-binding catalytic domain, and a small regulatory domain containing two immunoreceptor tyrosine-based inhibition motifs (ITIM1 and ITIM2) and a YENM motif [19–22]. IDO1 contributes to tumor immune escape via its enzymatic function, and its upregulation correlates to poor patient prognosis [23–26]. IDO1 is expressed in tumors and immune cells and leads to a decrease in Trp level and an increase in Kyn level in tumor microenvironment. Trp depletion results in the activation of the general control nonderepressible 2 pathway and inhibition of mammalian target of rapamycin pathway, causing T cell anergy and natural killer cell dysfunction [27–29]. Kyn accumulation activates the AhR pathway, leading to the proliferation of immunosuppressive cells, including regulatory T cells and myeloid-derived suppressor cells [29, 30]. Owing to its key role in tumor immune escape, inhibiting IDO1 activity has been a widely studied strategy in cancer immune therapy, with many IDO1 inhibitors designed and treatment strategies, either used alone or in combination with other agents, tested [12, 29, 30].

The nonenzymatic functions of IDO1 are less studied and are considered as novel functions. First studied in mouse plasmacytoid dendritic cells (pDCs), IDO1 becomes phosphorylated via phosphorylation of ITIMs under different stimulations, and then binds to different molecular partners [31]. Specially, ITIM2 is phosphorylated by the proinflammatory cytokine interleukin 6 (IL-6), which leads to the binding of IDO1 and suppression of cytokine signaling 3 (SOCS3), resulting in the ubiquitination and proteasomal degradation of the protein complex [32]. Both ITIMs are phosphorylated by immunosuppressive cytokine transforming growth factor-β‌ (TGF-β), resulting in the binding of IDO1 and tyrosine phosphatases SHP1 and SHP2 as well as the activation of anti‐inflammatory nuclear factor kappa-B pathway, which upregulates the expression of TGF-β, along with IDO1, forming a positive feedback and bestowing an immunosuppressive phenotype [13, 31]. A recent report has revealed that in mouse mastocytoma tumor cells, IDO1 inhibitor epacadostat enhances the binding of IDO1 and SHPs and the subsequent immunosuppressive signal, suggesting that the nonenzymatic and enzymatic functions of IDO1 might compensate each other [33].

In this study, we aimed to examine the effect of H2S on the proliferation and apoptosis of tumor cells with different expression levels of IDO1 and clarify the underlying mechanism. Our study could pave the way for future H2S-related tumor therapies.

Materials and methods

Cell culture

Human breast cancer cell line MCF-7 (RRID:CVCL_0031), gastric cancer cell line SGC-7901 (RRID:CVCL_0520), lung cancer cell line A549 (RRID:CVCL_0023), rat pheochromocytoma cell line PC12 (RRID:CVCL_0481), mouse glioma cell line GL261 (RRID:CVCL_Y003), fibroblast cell lines L (RRID:CVCL_4536), and L-929 (RRID:CVCL_0462) were purchased from the Shanghai Cell Bank of Chinese Academy of Sciences. IDO1 stably overexpressing GL261 strain (GL261 IDO1-OE) was constructed by our laboratory as previously reported [34]. Cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Gibco, Thermo Fisher Scientific, MA, USA, cat. no. 12800017) supplemented with 10% fetalbovine serum (FBS; Capricorn Scientific, Germany, cat. no. FBS-12A), 100 U/mL penicillin (Aladdin, China, cat. no. P431514) and 100 μg/mL streptomycin (Aladdin, cat. no. S432673). Cells were cultured in a humidified incubator at 37 °C under 5% CO2, and regularly checked for mycoplasma contamination.

Animal

Female, 6-week-old, C57BL/6 mice were purchased from Shanghai Jiesijie Experiment Animal Co., Ltd.

Reagents

H2S donors NaHS (Aladdin, cat. no. S106641), GYY4137 (Aladdin, cat. no. G275260), proteasome inhibitor MG132 (Lablead, China, cat. no. 474790), Kyn (Sigma Aldrich, MI, USA, cat. no. K8625), and IDO1 inhibitor 1-methyl-l-tryptophan (1-MT, Sigma-Aldrich, cat. no. 447439) were purchased from the respective manufacturers. IDO1 inhibitor RY103 was designed and synthesized in our laboratory.

Plasmids, small interfering RNA (siRNA), and transfections

The pcDNA3.1(+) empty vectors were purchased from Thermo Fisher Scientific. The pcDNA3.1(+)-hIDO1 plasmids were constructed in our laboratory as previously described [35]. The pcDH-GFP-Puro-3xFLAG empty vectors and pcDH-GFP-Puro-3xFLAG-hIDO1 plasmids, along with their variants with mutated IDO1, were constructed by Fenghbio Inc., China. Small interfering RNA (siRNA) was synthesized by GenePharma, China. Sequences of the siRNA that were used are presented in Supplementary Table S1. Plasmids and siRNAs were transfected into the cells using Lip2000 transfection reagent (Biosharp, China, cat. no. BL623B) following the manufacturer’s protocol. After transfections, cells were cultured for 24 h.

MTT assay and cell counting kit-8 (CCK-8) assay

Cell viability was evaluated using MTT Assay Kit (Servicebio, China, cat. no. G4101) and CCK-8 Assay Kit (Servicebio, cat. no. G4103) following the manufacturer’s protocol.

Clone formation assay

A clone formation assay was performed as previously described [34].

Apoptosis detection

Cell apoptosis was detected by Annexin V-FITC/PI Apoptosis Detection Kit (Lablead, cat. no. AF2020) following the manufacturer’s protocol and analyzed using Gallios Flow Cytometer (Beckman Coulter, CA, USA).

Protein extraction and western blot (WB)

Protein extraction from whole-cell lysate or tissues and WB analysis were performed according to standard protocol. Extraction of nuclear and cytoplasmic proteins was performed using Nuclear Protein Extraction Kit (Solarbio, China, cat. no. R0050). Antibodies used are listed as follows: GADPH (Bosterbio, cat. no. a00227-1, RRID:AB_2813842), IDO1 (Proteintech, cat. no. 66528-1-Ig, RRID:AB_2881891), BAX (Proteintech, cat. no. 50599-2-Ig, RRID:AB_2061561), BCL-2 (ABclonal, cat. no. A20777, RRID:AB_3678973), SOCS3 (Proteintech, cat. no. 66797-1-lg, RRID:AB_2882141), NR4A1 (ABclonal, cat. no. A24016, RRID: AB_3678975), pIDO1 targeting phosphorylated ITIM2 (Huabio, custom-made), HRP-conjugated anti-mouse secondary antibody (Epizyme, cat. no. LF101, RRID:AB_3083706), and anti-rabbit secondary antibody (Epizyme, cat. no. LF102, RRID:AB_3083707).

Reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time PCR (qPCR)

RNA extraction, RT-PCR, and qPCR were performed according to standard protocol. Primers used are presented in Supplementary Table S2.

Coimmunoprecipitation (co-iP)

Co-iP was performed with DYKDDDDK-Magnetic Immunoprecipitation Kit (Lablead, cat. no. PFM025) and Immunoprecipitation Kit with Protein A + G Magnetic Beads (Beyotime, cat. no. P2179M) following the manufacturer’s protocol.

Fluorescence staining and immunofluorescence

Mitochondria were stained with Mito-Tracker Red CMXRos (Beyotime, cat. no. C1035) following the manufacturer’s protocol.

Immunofluorescence was performed according to standard protocol. Antibodies used are listed as follows: pIDO1, SOCS3, and cytochrome c (Proteintech, cat. no. 66264-1-Ig, RRID:AB_2716798), Alexa Fluor 488-conjugated goat-anti-rabbit (Servicebio, cat. no. GB25303, RRID:AB_2910224), and Cy3-conjugated goat-anti-mouse (Servicebio, cat. no. GB21301, RRID:AB_2923552). The cell nuclei were then stained with DAPI (Beyotime, cat. no. C1002). The cell slices were examined using FV3000 confocal microscope (Olympus, Japan).

Mice treatment

Mice were randomized into GL261 and GL261 IDO1-OE groups. Corresponding glioma cells (2 × 106 cells per mouse) were injected subcutaneously into the right forelimb. In each group, mice were further divided into control, NaHS, and GYY4137 groups randomly. Treatment was initiated 7 days after tumor implantation. NaHS and GYY4137 groups received 100 mg/m2 of the reagents intratumorally according to the tumor area (calculated by long diameter × short diameter), dissolved in 0.1 mL normal saline every 24 h. Control groups received 0.1 mL normal saline every 24 h intratumorally. Mice were sacrificed after 8 days of treatment, and tumors and serum from the mice were collected.

Experiments on mice were approved by the Animal Ethics Committee of Fudan University and performed in compliance with ARRIVE guidelines (approval no. JS-006, 4 March 2019). Tumor sizes in this study did not exceed 2000 mm3, the maximum size permitted by the ethics committee.

High-performance liquid chromatography (HPLC) analysis of Trp and Kyn

HPLC analysis was performed as reported previously [35].

Immunohistochemistry

Immunohistochemistry analysis was performed according to standard protocol. Antibodies used are listed as follows: IDO1, pIDO1, SOCS3, goat anti-rabbit secondary antibody (Servicebio, cat. no. GB23303), and goat anti-mouse secondary antibody (Servicebio, cat. no. GB23301).

Terminal deoxynucleotidyl transferase-mediated dUTP Nick-end labeling (TUNEL) assay

TUNEL assays were performed using TUNEL kit (CF488, Servicebio, cat. no. G1504), following the manufacturer’s protocol, and examined under FV3000 confocal microscope.

Statistics

All experiments were repeated independently three times. No statistical methods were used to predetermine sample size. Data were analyzed and visualized using GraphPad Prism 9 software, and were expressed as means ± standard deviation (SD). One-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test was used to compare among several groups. Student’s t-test was used to compare between two groups. Significance values were set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Results

H2S reduced the proliferation of tumor cells with high IDO1 expression

We first examined the expression of endogenous IDO1 in different cells, including tumor and nontumor origins. MCF-7 and SGC-7901 were considered as cells with high IDO1 expression, while the others as cells with low IDO1 expression (Supplementary Fig. S1A). The effect of H2S on the proliferation ability of these cells was investigated. MTT analysis showed that H2S donor (NaHS and GYY4137) treatment significantly reduced the proliferation of MCF-7 and SGC-7901 cells, and had no effects on other cells (Fig. 1A, B). The result was confirmed by a clone formation assay (Fig. 1C, D). Furthermore, in IDO1 knockdown MCF-7 or SGC-7901 cells (Supplementary Fig. S1B, C), H2S treatment had no significant effect on the proliferation ability (Fig. 1E). Among the cells overexpressing IDO1(Supplementary Fig. S1D), only A549, a tumor cell line of human origin, showed reduced proliferation after H2S treatment (Fig. 1F). Our results showed that H2S selectively reduced proliferation of tumor cells with high IDO1 expression, either endogenously or through plasmid transfection.

Fig. 1.

Fig. 1

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Effects of H2S on the proliferation ability of cells with different expression level of IDO1. Cells were treated with different concentrations of NaHS or GYY4137 for 24 h. A, B Proliferations of cells detected by MTT assay. C, D Proliferations of cells detected by clone formation assay. E Proliferations of IDO1 knockdown MCF-7 or SGC-7901 cells detected by MTT assay. F Proliferations of IDO1-overexpressing L, L-929, or A549 cells detected by MTT assay. ANOVA test followed by Dunnett’s post hoc test was conducted. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

H2S induced the apoptosis of tumor cells with high IDO1 expression

We speculated that the effect of H2S treatment on proliferation ability of tumor cells was associated with apoptosis, so we investigated whether H2S had an impact on apoptosis in tumor cells. Flow cytometry analysis revealed that H2S significantly induced the apoptosis of SGC-7901 cells but not A549 cells (Fig. 2A, B), which was consistent with what we observed from proliferation assays. Similarly, H2S treatment was less effective in inducing apoptosis in IDO1 knockdown MCF-7 and SGC-7901 cells (Fig. 2C, E), but more effective in IDO1-overexpressing A549 cells (Fig. 2D, F), compared with their respective wild-type groups. IDO1 inhibitors 1-MT and RY103 mitigated the elevated level of H2S-induced apoptosis in MCF-7 and SGC-7901 cells (Fig. 3A–D). To summarize, these results indicated that H2S induced the apoptosis of tumor cells with high IDO1 expression.

Fig. 2.

Fig. 2

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Effects of H2S on the apoptosis of tumor cells with different expression level of IDO1. Apoptosis of cells was detected by flow cytometry analysis. A, B SGC-7901 (A) and A549 (B) cells treated with different concentrations of NaHS or GYY4137 for 24 h. C, E IDO1 knockdown MCF-7 or SGC-7901 cells treated with NaHS (2 mM) or GYY4137 (400 μM) for 24 h. D, F IDO1-overexpressing A549 cells treated with NaHS (2 mM) or GYY4137 (400 μM) for 24 h. Control: cells transfected with nontargeting siRNA (siNC) (C, E) or empty vector (D, F). ANOVA test followed by Dunnett’s post hoc test was conducted. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 3.

Fig. 3

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Effects of H2S on the apoptosis of tumor cells with different IDO1 activity. Apoptosis of cells was detected by flow cytometry analysis. MCF-7 and SGC-7901 cells were pretreated with IDO1 inhibitors 1-MT (500 μM, A, C) or RY103 (2 μM, B, D) for 24 h then incubated with NaHS (2 mM) or GYY4137 (400 μM) for 24 h. Control: cells not treated with IDO1 inhibitors. ANOVA test followed by Dunnett’s post hoc test was conducted. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

H2S-induced intrinsic apoptosis of tumor cells with high IDO1 expression was mediated by NR4A1

We first explored the potential pathway by which H2S-induced apoptosis of tumor cells with high IDO1 expression was mediated. Mitochondria membrane potential and cytochrome c (Cyt C) release were used to assess the degree of cell apoptosis [36]. Fluorescence staining revealed that the membrane potential of mitochondria significantly dropped after H2S treatment in MCF-7 cells, but stayed the same in IDO1 knockdown cells (Fig. 4A, B). This result was confirmed by immunofluorescence analysis of Cyt C released into the cytosol in MCF-7 cells upon H2S treatment. The green signals of Cyt C appeared diffused in the cells, rather than constrained within certain boundaries, and also overlapped with blue signals of nuclei after treatment (Fig. 4C). This phenomenon is consistent with the characteristics of intrinsic apoptosis. BCL-2 and BAX are BCL-2 family proteins related to mitochondrial outer membrane perforation and lead to Cyt C release. BCL-2 is considered an anti-apoptotic protein in the intrinsic apoptotic pathway, while BAX is regarded as a pro-apoptotic protein [37]. The expression of BAX increased while that of BCL-2 decreased in MCF-7 cells upon H2S treatment. Such changes were not found in IDO1 knockdown MCF-7 cells treated with H2S. After H2S treatment, the expression of BAX increased while that of BCL-2 decreased in IDO1-overexpressing A549 cells. However, the expressions of these two apoptotic proteins were not markedly changed in A549 cells upon H2S treatment (Fig. 4D and Supplementary Fig. S2). In conclusion, in tumor cells with high IDO1 expression, H2S treatment led to a decrease in BAX expression and an increase in BCL-2 expression, while such effect was not observed in tumor cells with low IDO1 expression.

Fig. 4.

Fig. 4

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Effects of H2S on NR4A1-mediated intrinsic apoptosis of tumor cells with different expression level of IDO1. Cells were treated with NaHS (2 mM) or GYY4137 (400 μM) for 24 h. A, B Mitochondria in IDO1 knockdown MCF-7 cells detected by fluorescence staining. Magnification: 400×; scale bars represent 50 μm. C CF488-labeled Cyt C (green) in MCF-7 cells upon different treatments recorded by fluorescence microscopy. DAPI (blue) was used for nuclear staining. Representative image of three independent experiments is shown. Scale bars represent 10 μm. D Protein expressions of IDO1, BCL-2, and BAX in IDO1 knockdown MCF-7 cells and IDO1-overexpressing A549 cells detected by WB. Statistical plots are shown in Supplementary Fig. S2. E–G Apoptosis of NR4A1 knockdown MCF-7, SGC-7901, or A549 cells detected by flow cytometry analysis. Control: cells transfected with siNC (A, D, E, F, G, left) or empty vector (D, right). ANOVA test followed by Dunnett’s post hoc test was conducted. n = 3. **p < 0.01, ***p < 0.001, and ****p < 0.0001

Further, we explored the molecular mechanism of H2S-induced intrinsic apoptosis of tumor cells with high IDO1 expression. It has been known that orphan nuclear receptor NR4A1 is involved in cell apoptosis pathway by interacting with BCL-2 [38]. Previous study has shown that the expression of NR4A1 is upregulated in cells treated by IDO1 inhibitor, suggesting its association with IDO1 and KP [39]. Therefore, we investigated the effect of NR4A1 knockdown on H2S-induced intrinsic apoptosis of tumor cells with high IDO1 expression. As shown in Fig. 4E–G, in NR4A1 knockdown MCF-7 and SGC-7901 cells, H2S-induced apoptosis of cells was quite lower than that of control groups. H2S did not induce the apoptosis of A549 cells with low IDO1 expression, which was not affected by the knockdown of NR4A1 in A549 cells. To summarize, our results showed that H2S induced apoptosis of tumor cells with high IDO1 expression via intrinsic pathway mediated by NR4A1.

H2S promoted nuclear translocation of NR4A1 in tumor cells with high IDO1 expression

Mainly localized in the nucleus, NR4A1 has both genomic and nongenomic functions [38]. It can only bind to BCL-2 after translocated from the nucleus to the mitochondria [38, 40]. Thus, we next investigated whether H2S affected the nuclear translocation of NR4A1 in tumor cells with high IDO1 expression. Immunofluorescence and WB analyses revealed H2S treatment increased cytosolic NR4A1 distribution and decreased nuclear NR4A1 distribution in cells with high IDO1 expression (Fig. 5A–C and Supplementary Fig. S3). Furthermore, the messenger RNA (mRNA) expression level of Cartpt, a target gene of NR4A1 [41], was examined to provide additional clues to the subcellular location of NR4A1. As expected, the expression of Cartpt in cells with high IDO1 expression that received H2S treatment significantly dropped, indicating that nuclear NR4A1 expression decreased, while this phenomenon was not observed in cells with low IDO1 expression (Fig. 5D). Co-iP assay further confirmed that the binding of NR4A1 and BCL-2 in MCF-7 cells had increased upon H2S treatment (Fig. 5F). These results indicated that in tumor cells with high IDO1 expression, H2S promoted translocation of NR4A1 out of nucleus.

Fig. 5.

Fig. 5

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Effects of H2S on NR4A1 nuclear translocation in tumor cells with different expression level of IDO1. Cells were treated with NaHS (2 mM, A–F), GYY4137 (400 μM, A–F), or Kyn (150 μM, G–H) for 24 h. A, B CF488-labeled NR4A1 (green) in MCF-7, SGC-7901, and A549 cells upon different treatments recorded by fluorescence microscopy. DAPI (blue) was used for nuclear staining. Magnification: 400×, scale bars represent 50 μm. C NR4A1 protein levels in cytosolic and nuclear fractions in MCF-7, SGC-7901, and A549 cells detected by WB. Total cellular NR4A1 (total) was used as internal control. Statistical plots are shown in Supplementary Fig. S3. D mRNA expression of Cartpt in MCF-7, SGC-7901, and A549 cells detected by qPCR. E Kyn levels in MCF-7, SGC-7901, and A549 cells detected by HPLC. F Binding of NR4A1 and BCL-2 in MCF-7 cells detected by co-iP. G NR4A1 protein levels in cytosolic and nuclear fractions in A549 cells detected by WB. Total cellular NR4A1 (total) was used as internal control. H mRNA expression of Cartpt in MCF-7 and SGC-7901 cells detected by qPCR. Student’s t-test and ANOVA test followed by Dunnett’s post hoc test were conducted. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Our previous study has shown that H2S treatment decreased IDO1 expression [11], which could also be observed in the current study (Fig. 4D). Consistent with this observation, Fig. 5E shows that H2S treatment significantly decreased Kyn level in cells with high IDO1 expression. Both WB analysis of NR4A1 protein expression in different cellular fractions in A549 cells, and mRNA expression of Cartpt in SGC-7901 and MCF-7 cells indicated that Kyn treatment led to increased nuclear NR4A1 expression, and decreased cytosolic NR4A1 expression (Fig. 5G, H). This result showed that the effect of Kyn on NR4A1 was opposite to that of H2S, where supplementation of Kyn increased nuclear NR4A1 expression.

In summary, our results suggested that in tumor cells with high IDO1 expression, H2S downregulated IDO1, resulting in a decrease in Kyn level, and thus promoted the translocation of NR4A1 out of nucleus and its binding to BCL-2. This effect was not observed in tumor cells with low IDO1 expression, as IDO1 expression or Kyn level did not drop significantly when H2S treatment was received.

H2S promoted proteasomal codegradation of SOCS3 and phosphorylated IDO1 in tumor cells with high IDO1 expression

As mentioned above, our previous study has shown that H2S downregulated IDO1 expression [11]. We then aimed to verify whether the decrease in IDO1 expression resulted from increased protein degradation. Interestingly, WB analysis showed that H2S promoted IDO1 phosphorylation only in MCF-7 cells and not in A549 cells (Fig. 6A). We thus designed pcDH-GFP-Puro-3xFLAG-hIDO1 plasmids expressing wild-type and mutated IDO1 with either tyrosine-111, tyrosine-249, or both residues replaced by glutamate (Y111E, Y249E, and Y111E + Y249E, respectively) to mimic its conformation with phosphorylated ITIMs, similar to the previous report [20]. The binding between phosphorylated IDO1 (pIDO1) and SOCS3 in A549 cells transfected with these designed plasmids was then confirmed by immunofluorescence and co-iP analyses using anti-Flag (Fig. 6B, C and Supplementary Figs. S4 and S5). Subsequent co-iP assays revealed that H2S promoted the binding of pIDO1 and SOCS3 in MCF-7 or A549 cells transfected with a plasmid expressing wild-type IDO1. The signal was stronger in the groups treated with MG132, a proteasome inhibitor (Fig. 6D, E). MG132 inhibited the codegradation of pIDO1 and SOCS3 in H2S-treated MCF-7 cells in a concentration- and time-dependent manner (Supplementary Fig. S6). WB analysis revealed that SOCS3 knockdown in both MCF-7 and SGC-7901 cells increased pIDO1 levels, further indicating the codegradation of the two proteins (Fig. 6F). Since such binding and degradation reduced the levels of both pIDO1 and SOCS3, and neither apoptosis nor the binding and degradation of pIDO1 and SOCS3 was promoted in A549 cells treated by H2S, we speculated that SOCS3 in A549 cells remained at a high level and exerted anti-apoptosis functions. As expected, after SOCS3 knockdown, H2S-induced apoptosis of A549 cells increased (Fig. 6G, H).

Fig. 6.

Fig. 6

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Effects of H2S on phosphorylation of IDO1 and codegradation of phosphorylated IDO1 (pIDO1) and SOCS3. Cells were treated with NaHS (2 mM) or GYY4137 (400 μM) for 24 h (A, H), or with MG132 (10 μM) for 4 h then incubated with GYY4137 (400 μM) for 20 h (D, E). A Phosphorylation of IDO1 in MCF-7 and A549 cells detected by WB. B Colocalization of wild-type or mutated IDO1 (red) and SOCS3 (green) in A549 cells detected by immunofluorescence. WT: cells expressing wild-type IDO1. Y111E, Y249E, and Y111E + Y249E: cells expressing corresponding mutated IDO1. Magnification: 400×; scale bars represent 50 μm. Statistical plots are shown in Supplementary Fig. S5. C–E Binding of pIDO1 or mutated IDO1 and SOCS3 in MCF-7 and A549 cells transfected with plasmid encoding wild-type or mutated IDO1 detected by co-iP. F Protein levels of pIDO1 in SOCS3 knockdown MCF-7 or SGC-7901 cells detected by WB. Representative image of three different experiments is shown. G CF488-labeled Cyt C (green) in A549 cells recorded by fluorescence microscopy. DAPI (blue) was used for nuclear staining. Representative image of three independent experiments is shown. Scale bars represent 10 μm. H H2S-induced apoptosis of SOCS3 knockdown A549 cells detected by flow cytometry analysis. Control: cells transfected with siNC. Student’s t-test and ANOVA test followed by Dunnett’s post hoc test were conducted. n = 3. *p < 0.05, and **p < 0.01

Our results suggested that H2S treatment in tumor cells with high IDO1 expression reduced the level of anti-apoptotic SOCS3 via proteasomal codegradation along with pIDO1. In tumor cells with low IDO1 expression, H2S did not promote degradation of SOCS3, which remained inhibitory against apoptosis.

H2S retarded growth and induced apoptosis of tumors with high IDO1 expression in vivo

We next sought to find out whether H2S treatment had similar effects in vivo. Tumor-bearing mice were constructed using wild-type and IDO1 stably overexpressing (IDO1-OE) GL261 glioma cells developed previously in our laboratory [34]. Effect of H2S on proliferation of these cells was confirmed to be consistent with previous in vitro experiments before mice model construction (Fig. 7A). H2S reduced the size and weight of the tumors (Fig. 7B and Supplementary Fig. S7A), and decreased IDO1 activity in serum in mice bearing IDO1-OE tumors (Fig. 7C), but not their body weight (Supplementary Fig. S7B). TUNEL assays and WB analyses revealed that H2S promoted apoptosis of IDO1-OE tumors (Fig. 7D, E).

Fig. 7.

Fig. 7

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Effects of H2S on mice bearing wild-type (GL261) or IDO1-overexpressing GL261 (GL261 IDO1-OE) glioma. A Proliferations of GL261 IDO1-OE cells and GL261 cells upon treatment with NaHS (0–800 μM) or GYY4137 (0–800 μM) for 24 h detected by CCK-8 assay. n = 3. B Tumor weight. C Concentration of Trp, Kyn, and IDO1 activity (represented by Kyn/Trp*100) in mice serum detected by HPLC. D Apoptosis of tumors detected by TUNEL assay. CF488: green fluorescence signals for apoptotic cells. DAPI (blue) was used for nuclear staining. Magnification: 200×; scale bars represent 100 μm. E Protein levels of pIDO1, IDO1, BAX, and BCL-2 in tumor cells detected by WB. F NR4A1 protein levels in cytosolic and nuclear fractions in tumor cells detected by WB. Total cellular NR4A1 (total) was used as internal control. G Colocalization of IDO1 and SOCS3 in tumors detected by immunofluorescence analysis. Scale bars represent 50 μm. For panel D–G, representative images of six independent experiments are shown. ANOVA test followed by Dunnett’s post hoc test was conducted. n = 6. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

H2S also promoted the translocation of NR4A1 out of nucleus in IDO1-OE tumors, as shown in WB analysis (Fig. 7F). In addition, H2S promoted phosphorylation of IDO1 as well as colocalization of pIDO1 and SOCS3, and resulted in decrease in SOCS3 expression in IDO1-OE tumors (Fig. 7E, G and Supplementary Fig. S8). These effects were not significant in mice bearing wild-type tumors, which was consistent with our previous in vitro results. These results suggested H2S induced tumor apoptosis via NR4A1 and SOCS3 both in vitro and in vivo.

Discussion

Endogenous H2S has been reported to be favorable to tumor, and high level of H2S from exogenous donors exerts an antitumor function [6, 42]. It would be worth investigating whether other conditions exists under which H2S exerts antitumor effects. Our present study first demonstrated that H2S reduced proliferation and induced apoptosis of tumors with high IDO1 expression. Most studies on IDO1 mainly focus on its role in tumor immune evasion [29, 30] and tumor apoptosis, which associates with its enzymatic activity [14–16]. Our pioneering study, being the first study on the association between IDO1 and H2S since our previous report in 2019, examined the role of IDO1 in H2S-induced tumor apoptosis, and further expanded knowledge in this less-studied field [11].

Nonenzymatic functions of IDO1 are far less studied than its enzymatic functions [13]. While it has been reported that pIDO1 is proteasomally degraded when bound to SOCS3, this effect is less well characterized and mostly studied in mouse pDCs [13, 32, 43]. This study expanded our knowledge on this moonlighting function beyond mouse pDCs by demonstrating that H2S, other than previously reported IL-6 [32], could induce such codegradation, and showing the apoptosis-promoting effect of codegradation of SOCS3 and pIDO1 in both human and mouse tumor cells (Figs. 6 and 7). While previous reports have demonstrated that phosphorylation of ITIMs of IDO1 is mediated by Src family kinases Fyn and Src, these studies are limited to mouse immune cells [13, 19]. The kinases that mediate H2S-promoted IDO1 phosphorylation in tumors still require clarification.

NR4A1 is one of the most studied orphan nuclear receptors, and its nuclear export can be promoted by various agents [38]. In our study, we have found that H2S promoted the nuclear export of NR4A1, followed by the binding between NR4A1 and BCL-2, which activated apoptosis, by downregulating Kyn (Fig. 5). Since retinoid signal and insulin-like growth factor binding protein 3 signal have been reported to promote NR4A1 translocation to the mitochondria [38], H2S-promoted nuclear export of NR4A1 may be mediated via the same downstream pathways to these signals, which requires further exploration. Our results connected the translocation of the nuclear receptor and the metabolism of an essential amino acid, Trp, which also conferred an immunoregulatory effect, and might inspire further studies on NR4A1 in the field of tumor metabolism and tumor immune evasion.

In this study, we clarified the role of IDO1 in H2S-induced apoptosis of tumor cells with high IDO1 expression. In tumors with high IDO1 expression, H2S downregulated IDO1 and KP activity, thus promoting nuclear export of NR4A1 and its binding with BCL-2. H2S also promoted phosphorylation of IDO1 and the codegradation of IDO1 and SOCS3 (Fig. 8). These effects did not exist in tumor cells with low IDO1 expression. Our study was not perfect as we did not examine the kinases mediating the nuclear export of NR4A1 or those responsible for the phosphorylation of IDO1. Future study may further explore the mechanism of IDO1 phosphorylation in tumor, that by which the loss of SOCS3 contributes to the activation of intrinsic apoptotic pathway in tumors, and that of the H2S-induced, Kyn-associated nuclear export of NR4A1, including the kinases mediating these processes. Since there have been reports on the effect of phosphorylation of IDO1 on its enzymatic activity [20, 33], whether phosphorylation of IDO1 has any effect on NR4A1 nuclear export in tumors would also be an interesting topic for future studies.

Fig. 8.

Fig. 8

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A schematic overview of the effect of H2S on NR4A1-BCL-2 pathway and the codegradation of pIDO1 and SOCS3 in tumor cells with high or low IDO1 expression, which resulted in apoptosis of tumor cells with high IDO1 expression

Conclusions

This study demonstrated the mechanism by which H2S induced apoptosis of tumor with high expression of IDO1. Our results elucidate a novel role of IDO1 in tumor apoptosis, providing mechanistic insight into H2S-induced intrinsic apoptosis of tumor cells; further translational and clinical studies will be required to determine whether these findings can be developed into H2S-related therapeutic strategies for immune-evasive cancers with high IDO1 expression.

Supplementary Information

Additional file 1. (15.9MB, docx)

Acknowledgements

We are grateful to Professor Chunxiang Kuang of Tongji University for his help in the synthesis of IDO1 inhibitor RY103.

Abbreviations

AhR

Aryl hydrocarbon receptor

BAX

BCL-2-associated X

BCL-2

B cell lymphoma 2

CCK-8

Cell counting kit-8

co-ip

Coimmunoprecipitation

Cyt C

Cytochrome c

DMEM

Dulbecco’s modified Eagle medium

FBS

Fetal bovine serum

H2S

Hydrogen sulfide

IDO1

Indoleamine 2,3-dioxygenase 1

IL-6

Interleukin 6

ITIM

Immunoreceptor tyrosine-based inhibition motif

KP

Kynurenine pathway

Kyn

Kynurenine

NR4A1

Nuclear receptor 4A1

pDC

Plasmacytoid dendritic cell

qPCR

quantitative real-time PCR

SHP

Src homology 2 domain tyrosine phosphatase

siRNA

Small interfering RNA

SOCS3

Suppressor of cytokine signaling 3

TGF-β

Transforming growth factor-β

Trp

Tryptophan

TUNEL

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling

WB

Western blot

Author contributions

ZNTH: conceptualization, investigation, data curation, and writing—original draft; FM: investigation, data curation, and visualization; XQ: investigation, data curation, and visualization; YL: investigation and data curation; XF: investigation and data curation; DY: conceptualization and investigation; QY: conceptualization, supervision, and writing—review and editing. All authors read and approved the final manuscript.

Funding

This work is supported by State Key Laboratory of Druggability Evaluation and Systematic Translational Medicine, Tianjin Medical University Cancer Institute & Hospital (no. QZKF24-4), National Natural Science Foundation of China (no. 82002839), and Training Program for Emerging Clinical Scientists at Shanghai Children’s Hospital (no. 2024QK04).

Data availability

No datasets were generated or analyzed during the current study.

Declarations

Ethics approval and consent to participate

Experiments on mice were approved by the Animal Ethics Committee of Fudan University (approval no. JS-006, 4 March 2019) and performed in compliance with ARRIVE and ICLAS guidelines. Tumor sizes in this study did not exceed 2000 mm3, the maximum size permitted by the ethics committee.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

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

Zhen Ning Tony He and Fangzhou Meng contributed equally to this work.

Contributor Information

Dan Yang, Email: yangdan@shchildren.com.cn.

Qing Yang, Email: yangqing68@fudan.edu.cn.

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Associated Data

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Supplementary Materials

Additional file 1. (15.9MB, docx)

Data Availability Statement

No datasets were generated or analyzed during the current study.

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