All reagents were purchased from Sinopharm (Shanghai, China) unless otherwise indicated. Venlafaxine hydrochloride (Cat. #V129637), SP600125 (Cat. #S125267) and PD98059 (Cat. #P126620) were purchased from Aladdin (Shanghai, China).

Human melanoma (MV3) cells were purchased from the China center for type culture collection (Cat. #GDC0615). MV3 cells were cultured in DMEM medium supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin in humidified 5% CO₂ atmosphere at 37°C until 80% confluence. MV3 cells were then treated with venlafaxine (0–100 μM), SP600125 (.5 μM) and PD98059 (15 μM) (a MEK/ERK inhibitor that can inhibit MEK activation and subsequent ERK phosphorylation) for 30 min, or human Nur77 siRNA (50 nM), JNK1/2 siRNA (Cell signaling, Cat. #6232S, 50 nM), ERK1/2 siRNA (Cell signaling, Cat. #6560S, 50 nM) and HiPerfect transfection reagent (Qiagen, 301704, United States) for 12 h, followed by incubation at 37°C for 0–72 h (Li et al., 2018). The siRNA sequences were described below:

Control siRNA: 5′-GCGCGCUUUGTAGGAUUCGdTdT-3′

Nur77 siRNA: 5′-CAGUCCAGCCAUGCUCCUCdTdT-3′

Cell viability was measured at 72 h, using the cell counting kit-8 (CCK8) according to the manufacturer’s instructions (Liu J et al., 2022; Qin et al., 2022). MV3 cells were collected and incubated with a CCK-8 reagent (Dojindo, Cat. #CK04, 5 mg/ml, 10 μl) at 37°C for 60 min. The OD values of the reaction mixture were measured at the wavelength of 450 nm. The cell viability was calculated using the following formula: Cell viability (%) = OD (control) − OD (test)/OD (control) − OD (blank).

Apoptosis assay was conducted at 0, 4 and 8 h, using a dead cell apoptosis kit with Annexin V-FITC and propidium iodide (PI) (Thermo, Cat. #V13241) according to the manufacturer’s instructions (Chen et al., 2019; Li et al., 2022). MV3 cells were washed with PBS, resuspended in binding buffer, incubated with Annexin V-FITC and PI for 15 min according to the kit protocol, and analyzed immediately by cytoFLEX Flow Cytometry System (Beckman-Coulter) using FITC and PerCP.

The animal experiments were approved by the Animal Care and Use Committee of Hainan Medical University [Approval No. Med-Eth-Re (2022) 736]. Female BALB/c nude mice (20–25 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. Mice were maintained under specific pathogen-free conditions, group-housed in ventilated cages with controlled temperature (25°C ± 1°C) and relative humidity (55% ± 10%). Standard mouse chow and tap water were provided ad libitum. The nude mice were anesthetized by intravenous injection of pentobarbital sodium (25 mg/kg), followed by subcutaneous transplantation of MV3 or Nur77 knockout (Nur77 KO) MV3 melanoma cells in the right posterior axillary line (Liu Y. X et al., 2022). The dose of venlafaxine for the mice was based on the preliminary experiments and the references (Zhang et al., 2019; Madrigal-Bujaidar et al., 2021). Mice were treated with venlafaxine (20 mg/kg, i.p.) or its .1% DMSO-containing saline vehicle once daily after tumor size grew up to 50–100 mm³. Body weight and tumor volume were measured every 3 days. Mice were sacrificed by CO₂ inhalation after 15-day drug treatment and the tumors were stripped for various assessments (Chen et al., 2019). Nur77 knockout (KO) MV3 cells were generated by CRISPR/Cas9 system using a previously reported method (Chen et al., 2019). The gRNA targeting sequence of Nur77 is 5′-ACC​TTC​ATG​GAC​GGC​TAC​AC-3′. Protein lysates were prepared from tumors with previously reported method (Chen et al., 2015). Tumors were homogenized in cold 1× RIPA lysis buffer and centrifuged at 15,000 × g for 15 min at 4°C. The supernatants were collected and the protein concentration of sample was measured by Pierce BCA protein assay kit (Thermo, Cat. #23225). Ultimately, all samples were normalized to the same total protein concentration of 2 mg/ml. Frozen tumor tissues were thawed and then homogenated in Trizol reagent. Total RNA was extracted with chloroform, isopropanol, and 75% ethanol. The RNA concentration was measured with a spectrophotometer (Beckman Coulter, United States).

Western blots were performed using the standard sodium dodecyl sulfate (SDS)-PAGE polyacrylamide gel electrophoresis method (Li et al., 2021a). The protein of cell or tumor lysates was prepared and measured by Pierce BCA protein assay kit (Thermo, Cat. #23225) (Xie et al., 2022a). The total protein (50 μg) was separated by 10% SDS-PAGE gels and transferred to a nitrocellulose membrane (Amersham Biosciences, Shanghai, China). Membranes were blocked in 5% (w/v) nonfat milk for 1 h at room temperature, washed with saline buffer (containing .05% tween-20) and then incubated at 4°C overnight with the primary antibody: Nur77 (Cell signaling, Cat. #3960S, dilution 1:500), cleaved caspase-3 (Cell signaling, Cat. #9661S, dilution 1:300), PARP (Santa Cruz, Cat. #sc-8001, dilution 1:500), p38 (Novus, Cat. #AF8691, dilution 1:500), p-p38 (Santa Cruz, Cat. #sc-166182, dilution 1:300), JNK1/2 (Santa Cruz, Cat. #sc-137019, dilution 1:800), p-JNK1/2 (R&D, Cat. #AF1205, dilution 1:400), c-Jun (Novus, Cat. #MAB8930, dilution 1:500), p-c-Jun (Cell signaling, Cat. #3270S, dilution 1:300), ERK1/2 (Cell signaling, Cat. #68303SF, dilution 1:600), p-ERK1/2 (Cell signaling, Cat. #9101S, dilution 1:300), GAPDH (Santa Cruz, Cat. #sc-47724, dilution 1:1000). The membranes were then incubated for 1 h at room temperature with horseradish peroxidase (HRP)-linked anti-rabbit IgG antibody (Santa Cruz, Cat. #sc-2004, dilution 1:5000) and detected with an electrochemiluminescence plus kit (Amersham Biosciences).

Total RNAs were extracted by Trizol (Invitrogen) and complemental DNA was synthesized using RevertAid First‐Strand cDNA Synthesis Kits (Fermentas). RT-PCR was performed using SYBR Green dye and the Roche LightCycler^® 480 II system following the manufacturer’s instructions on a 7300 real-time PCR system (Applied Biosystems) using respective primers (Hu et al., 2021):

Nur77: 5′-ACC​CAC​TTC​TCC​ACA​CCT​TG-3′ (forward), 5′- ACTTGGCGTTTTTCT GCACT-3′ (reverse).

β-Actin: 5′-AGA​GCT​ACG​AGC​TGC​CTG​AC-3’ (forward), 5′-AGCACTGTGTTG GCGTACAG-3′ (reverse).

Expression data were normalized to β-Actin mRNA expression.

Tumor tissues were excised, sectioned and fixed in 10% (w/v) formalin for 24 h, followed by embedding in paraffin. The specimen was embedded in paraffin and cut into 5 μm sections for further assessments (Xie et al., 2022b).

In vitro–cultured MV3 cells were fixed with 4% paraformaldehyde, permeabilized with .1% Triton X-100 in PBS for 20 min, and then blocked with goat serum in .3 M glycine in PBS at 25°C for 1 h. Sections were then incubated at 4°C overnight with the primary antibody: Nur77 (Cell signaling, Cat. #3960S, dilution 1:500) and Bcl2 (Abcam, Cat. #ab692, dilution 1:500). Sections were rinsed with .1 M PBS and exposed to donkey secondary antibodies conjugated with Alexa Fluor 488 or 647 (Abcam, dilution 1:1,000) at room temperature for 2 h. After an additional rinse, cells were then counterstained with 4′, 6-diamidino-2-phenylindole (DAPI) for nuclear labelling (Wang et al., 2022). Fluorescence images were captured with a confocal microscope.

The paraffin-embedded tumor sections were incubated with the following primary antibodies at 4°C overnight: Nur77 (Cell signaling, Cat. #3960S, dilution 1:500), cleaved caspase-3 (Cell signaling, Cat. #9661S, dilution 1:300), Ki-67 (Abcam, Cat. #ab15580, dilution 1:800). After incubation, sections were rinsed with .1 M PBS and exposed to goat secondary antibodies conjugated with Alexa Fluor 488 or 647 (Abcam, dilution 1:1,000) at room temperature for 1 h (Li et al., 2021b). After an additional rinse, sections were then counterstained with DAPI for nuclear labelling. Fluorescence images were captured with a confocal microscope.

Reactive oxygen species (ROS) were stained with cellular ROS assay kit (deep red) (Abcam, Cat. #ab186029) according to the manufacturer’s instructions. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were carried out according to the manufacturer’s instructions (Promega, Cat. #G7131). The number of apoptotic cells was counted by Image J.

Data are presented as means ± SEM. Analyses were performed with GraphPad Prism 9.0.5. Three or more different groups were analyzed by one-way ANOVA with Dunnett’s post hoc multiple comparison tests. p < .05 was considered statistically significant.

We first investigated whether venlafaxine could regulate the cellular viability of MV3 cells. As shown in Figure 1B, venlafaxine showed a great inhibition on the growth of MV3 cells with an LD₅₀ = 9.01 ± .97 μM. To investigate whether the cytotoxicity effect of venlafaxine was associated with its induction of Nur77 expression, we then examined the effect of venlafaxine on Nur77 expression in MV3 cells. Based on the LD₅₀ data of venlafaxine, MV3 cells were treated with venlafaxine at a concentration of 10 μM. qRT-PCR (Figure 1C) and western blot (Figure 1D) analyses showed that the mRNA and protein levels of Nur77 were low in MV3 cells, but were persistently elevated after venlafaxine treatment. Together, these results suggested that venlafaxine reduced the cell proliferation and introduced Nur77 expression in MV3 cells.

Given that the death effect of Nur77 is associated with its induction of apoptosis, which has been observed in other studies (Zhang, 2007), we speculate that venlafaxine may exert an anti-proliferative effect through activating the Nur77-dependent apoptotic pathway. As a proof of concept, we examined the expression of BAX-2, cleaved caspase 3 and cleaved PARP, the indicators of apoptosis, in MV3 cells treated with venlafaxine (10 μM). Western-blot analysis showed that venlafaxine treatment persistently induced caspase 3 and PARP cleavage and BAX-2 expression in MV3 cells (Figure 2A). We further assessed the effect of venlafaxine on cell death using flow cytometry-based Annexin V/Propidium iodide (PI) apoptosis assay. As shown in Figure 2B, approximately 30% of MV3 cells were apoptotic when treated with venlafaxine for 8 h, whereas only about 2% of cells were apoptotic in vehicle control group. Moreover, TUNEL assay revealed extensive apoptosis in venlafaxine-treated MV3 cells (Figure 2C). Together, these results revealed that venlafaxine induces apoptosis in MV3 cells.

Nur77 can induce apoptosis by translocating to mitochondria where it binds to Bcl-2 to trigger cytochrome c release and ROS production (Lin et al., 2004; Chen et al., 2019). Thus, we further examined whether venlafaxine could induce Nur77 translocation from the nucleus to mitochondria. Immunofluorescence assay showed that Nur77 was mainly localized in the nucleus of MV3 cells, while treatment of venlafaxine promoted the mitochondrial translocation of this protein (Figure 3A). Furthermore, following Nur77 mitochondrial translocation, the production of ROS in MV3 cells was also enhanced by venlafaxine (Figure 3B). Together, these results revealed that venlafaxine induced Nur77 mitochondrial targeting and ROS production in MV3 cells.

We next determined whether venlafaxine-induced apoptosis was Nur77-dependent. MV3 cells were transfected with Nur77 siRNA and subjected to venlafaxine treatment for 8 h. We found that Nur77 knockdown decreased venlafaxine-induced expressions of BAX-2, cleaved caspase 3 and cleaved PARP were in MV3 cells (Figure 4A). Furthermore, venlafaxine-induced apoptosis was rescued by transfecting with Nur77 siRNA, but not control siRNA (Figures 4B, C). Additionally, venlafaxine induced lower levels of ROS in Nur77-knockdown cells than control MV3 cells (Figure 4D). Together, these data indicated that Nur77 expression was necessary for venlafaxine-induced apoptosis.

Given that MAPKs play an important role in the regulation of melanoma cell survival and Nur77 post-translational modifications (Liu et al., 2017; Huo et al., 2020; Hu et al., 2021), we further studied whether venlafaxine affects Nur77 expression through MAPKs signaling. MV3 cells expressed relatively low levels of phosphorylated MEK (p-MEK), p-JNK1/2, p-c-Jun, and p-ERK. After treatment with venlafaxine, the levels of p-MEK, p-JNK1/2, p-c-Jun, and p-ERK were increased in MV3 cells (Figure 5A). To further study the roles of MAPK signaling in venlafaxine-induced apoptosis, MV3 cells were co-treated with venlafaxine and different inhibitors of MAPK kinases, including MEK/ERK signaling inhibitor PD98059 and JNK1/2 inhibitor SP600125. CCK8 assay showed that inhibition of JNK1/2 activity by SP600125 impaired venlafaxine-induced cell death effects, while PD98059 had no such effects (Figure 5B). Moreover, knockdown of JNK1/2 but not ERK increased cell viability in venlafaxine-treated MV3 cells (Figure 5C). These data demonstrated that JNK1/2 signaling is involved in venlafaxine-induced cell death effects. Next, we examined whether venlafaxine affects Nur77 expression in MV3 cells through JNK1/2 signaling. As shown in Figures 5D, E, both genetic inactivation and pharmacological administration of JNK1/2 inhibitor SP600125 significantly suppressed venlafaxine-induced Nur77 expression, indicating that venlafaxine increases Nur77 expression in MV3 cells through activation of JNK1/2 signaling. Furthermore, blockade of JNK1/2 signaling also suppressed mitochondrial translocation of Nur77 (Figure 5F). Taken together, these data suggested that venlafaxine induced MV3 cells death through activation of JNK1/2 signaling and induction of Nur77 expression.

Encouraged by the in vitro activity of venlafaxine, we further investigated whether the induction of Nur77 by venlafaxine contribute to its growth inhibitory effect in vivo. BALB/c nude mice were inoculated subcutaneously with MV3 cells, and were treated with venlafaxine (20 mg/kg, i.p., once daily for 15 days) when the average tumor size grew up to 50–100 mm³. Consistent with the in vitro results, treatment with venlafaxine for 15 days promoted expressions of Nur77 and activation of JNK1/2 in tumor tissues (Figure 6A). Furthermore, at the 15 days after implantation, the average tumor volume of the vehicle control group was 1,732 mm³; and that of venlafaxine treatment group was 630 mm³, with the inhibition ratio of tumor growth was 64% (Figure 6B). These results demonstrated that venlafaxine induced Nur77 expression and inhibit the growth of MV3 xenograft tumor in vivo.

To study whether venlafaxine inhibit the growth of MV3 xenograft tumor associated with its induction of apoptosis. We examined the expression of BAX-2, cleaved caspase 3 and cleaved PARP in tumor tissues. Western-blot assay and immunostaining showed that venlafaxine treatment significantly increased the level of these protein (Figures 7A, B). Additionally, immunostaining assay demonstrated that venlafaxine enhanced apoptotic cell death, as assessed by TUNEL staining, and suppressed cell proliferation, as assessed by Ki-67 immunostaining, in tumor tissues (Figure 7C). When combined, these results suggested that venlafaxine potently inhibited the growth of melanoma cells in animals through its apoptotic cell death effects.

Furthermore, we also studied the role of Nur77 in the anti-cancer effects of venlafaxine in vivo. We used the CRISPR/Cas9 technology to generate Nur77 knockout (KO) MV3 cells, and subcutaneously injected Nur77 KO MV3 melanoma cells into the BALB/c nude mice. At the 15 days after implantation, we found that the average tumor volume of the Nur77 KO MV3 group was similar to that of MV3 group, indicating Nur77 is not required for the growth of MV3 xenograft tumor. Furthermore, treatment with venlafaxine could not inhibit the growth of Nur77 KO MV3 xenograft tumor (Figures 6B, C), as well as the expression of BAX-2, cleaved caspase 3 and cleaved PARP in tumor tissues (Figures 7A, B). Moreover, treatment with venlafaxine had no effects on the numbers of TUNEL-positive and Ki-67-positive cells (Figure 7C). Taken together, these results suggested that venlafaxine suppresses the growth of melanoma cells through Nur77-dependent apoptotic pathway.