Medium (RPMI-1640), Lipofectamine 3000, and Lipofectamine RNAiMAX were obtained from Thermo Fisher Scientific, Inc. Fetal bovine serum (FBS) was purchased from AusGeneX. Trypsin-EDTA and isoproterenol (ISO) were obtained from Sigma-Aldrich (Merck KGaA). Sorafenib, cabozantinib, lenvatinib, rapamycin and HIF-2α inhibitor PT2385 were purchased from LC Laboratories. Ferroptosis inhibitor ferrostatin-1 (Fer-1), selective ferroptosis inducer RSL3 and p38 inhibitor SB203580 were purchased from Cayman Chemical Company. Apoptosis inhibitor Z-VAD-FMK (z-VAD), autophagy inhibitor 3-methyladenin (3-MA), necroptosis inhibitor necrostatin-1 (Nec-1), ERK1/2 pathway inhibitor PD0325901 and JNK inhibitor SP600125 were purchased from Adipogen Life Sciences. Ferroptosis inducer erastin was purchased from ChemScene LLC. Propranolol (PRO) hydrochloride was obtained from Sumitomo Pharma.

Human RCC cell lines A-498 (VHL mutant RCC; HIF-1α-negative, HIF-2α-positive), 769-P (VHL mutant RCC; HIF-1α/HIF-2α-negative), 786-O (VHL mutant RCC; HIF-1α negative, HIF-2α-positive), Caki-1 (VHL null RCC; HIF-1α/HIF-2α-negative), Caki-2 (VHL null papillary RCC, HIF-1α/HIF-2α-negative) and ACHN (VHL null RCC, HIF-1α/HIF-2α-negative) were obtained from the American Type Culture Collection. Cells were cultured in RPMI medium supplemented with 50 μg/ml kanamycin and 10% fetal bovine serum in a 5% CO₂, 37°C incubator. All cell lines were verified to be free of mycoplasma contamination.

The present study was approved by the Ethics Committee of Yamagata University (Yamagata, Japan; approval no. 2020-425). Written informed consent for the use of clinical samples was obtained from all patients. The present study investigated the expression of ADRB1 and ADRB2 in primary tumors of 35 patients (26 male and 9 female) diagnosed with metastatic RCC who underwent nephrectomy at Yamagata University Hospital (Yamagata, Japan) between January 2010 and December 2016 using IHC staining. Inclusion criteria were as follows: i) Diagnosis of metastatic disease and ii) age >17 years. Exclusion criteria were: 1) withdrawal of research participation, and 2) insufficient tumor volume of surgical specimen for analysis. Because the number of available cases was limited, no formal statistical power calculation was performed and all samples were included to maximize the descriptive value of the IHC assessment. All IHC staining procedures were conducted in a single batch using identical protocols and reagent lots to minimize technical variability. The median age was 64 (range, 43-77) years. The excised specimens were fixed in 10% formalin at room temperature for 24-48 h and paraffin-embedded. The samples were cut into 3 μm thick sections, placed on Cyanoacrylate®-coated slides (Dako; Agilent Technologies, Inc.) and baked 43°C for 1 h. Following three 5 min washes with xylene and four 5 min washes with 100% ethanol, paraffin was removed and the samples were dehydrated and hydrated. The samples were subjected to an antigen inactivation procedure at 120°C for 20 min using 10 mM Tris, 1 mM EDTA (pH 9.0) solution. To inhibit endogenous peroxidase, samples were immersed in 3% hydrogen peroxide-containing methanol solution for 10 min. Rabbit anti-ADRB1 (cat. no. 12271s) and rabbit anti-ADRB2 polyclonal antibody (both 1:50, cat. no. 8513; both Cell Signaling Technology, Inc.) were incubated overnight at 4°C in a humid environment, then washed with PBS. Sections were treated with HRP-conjugated anti-rabbit IgG (cat. no. P0448, Dako; Agilent Technologies, Inc.; 1:500) for 1 h at room temperature and then washed with PBS. Histofine Simple Stain MAX-PO® (Nichirei Biosciences, Inc.) was incubated for 30 min at room temperature, then the color was developed by reacting with DAB (cat. no. D5905; Sigma-Aldrich; Merck KGaA) for 20 min. Hematoxylin staining was performed for nuclear staining at room temperature. Immunohistochemical staining for ADRB1 and ADRB2 was evaluated by light microscope using a three-tier scoring system based on staining intensity and the approximate percentage of positive tumor cells. Staining intensity was evaluated by comparing the RCC region with the normal renal tubules on the same histological slide. 'No expression' was defined as absent or trace staining in <5% of tumor cells. 'Low expression' was defined as staining in 5-50% of tumor cells. 'High expression' was defined as staining in >50% of tumor cells. All samples were independently reviewed by two investigators and discrepancies were resolved by consensus.

RNA was extracted from cells using the Reliaprep RNA Cell Miniprep System (Promega Corporation) and cDNA was synthesized using the High Capacity cDNA RT kit (Applied Biosystems, Thermo Fisher Scientific Inc.), according to the manufacturer's instructions. RT-qPCR was performed using Power Track SYBR Green Master Mix (Thermo Fisher Scientific, Inc.) and CFX Connect Real-Time System (Bio-Rad Laboratories, Inc.). The thermocycling conditions were as follows: Initial denaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 1 min. The expression of mRNA was determined using the ΔΔCq method, relative to GAPDH, which was used as an endogenous control (31). The expression levels in each cell line were compared with those in 769P cells. Primers (Integrated DNA Technologies, Inc.) were as follows: ADRB1 forward, 5′-TAC GGC TCC TTC TTC TGC GA-3′ and reverse, 5′-TGC ATG AGG ATG GGC AGG AA-3′; ADRB2 forward, 5′-ATG GTG TGG ATT GTG TCA GGC-3′ and reverse, 5′-TGG AAG CGG CCC TCA GAT TT-3′ and GAPDH forward, 5′-GCA CCG TCA AGG CTG AGA AC-3′ and reverse, 5′-TGG TGA AGA CGC CAG TGG A-3′.

Western blot analysis was performed as previously described (25). Western Blot Hyper HRP Substrate and Western Blot Ultra HRP Substrate^® (both Takara Bio, Inc.) were used. Image acquisition was performed with a Light Capture AE-6981 system^® and analysis was performed with CS Analyzer ver 3.0 for Windows^® (both ATTO Corporation). For the detection of β-receptor proteins, ADRB1 (cat. no. 28323-1-AP; Proteintech Group, Inc.) and ADRB2 (both 1:1,000; 2 h at room temperature; cat. no. 8513; Cell Signaling Technology Japan) antibodies were used. The following antibodies were used to detect intracellular signaling molecules: p38 MAPK (p38; cat. no. 8690), phosphorylated (p-)p38 (T180/Y182; cat. no. 4511), JNK (all 1:1,000 and incubated for 2 h at room temperature; cat. no. 9252), p-JNK (T183/Y185; cat. no. 4668), Erk1/2 (cat. no. 9102), p-Erk1/2 (T202/Y204; cat. no. 4370), Akt (cat. no. 9272) and p-Akt (S473; cat. no. 4060; all Cell Signaling Technology Japan). α-tubulin (all 1:1,000 and incubated for 2 h at room temperature; cat. no. 017-25031) was purchased from FUJIFILM Wako Pure Chemical Corporation. Secondary antibodies were anti-mouse IgG, HRP-conjugated (diluted 1:10,000 and incubated for 1 h at room temperature; cat. no. NA931V, GE Healthcare) and the anti-rabbit IgG, HRP-conjugated (diluted 1:500 and incubated for 1 h at room temperature; cat. no. P0448, Dako; Agilent Technologies, Inc.).

siRNA was used to knockdown ADRB1 and ADRB2. Human-specific siRNA targeting ADRB1 or ADRB2 (10 nM) was transfected using Lipofectamine RNAiMAX. Kanamycin and FBS-free RPMI medium were used during transfection. After 24 at 37°C, the medium was replaced with RPMI medium containing 10% FBS. Cells were cultured for 24 h at 37°C before experiments. For ADRB1 knockdown, siADRB1(1) (cat. no. s1118; sense target sequence, 5′-GCU UAU UUA UGU AUC AAU Att-3′; anti-sense target sequence, 5′-UAU UGA UAC AUA AAU AAG Ctg-3′), siADRB1(2) (cat. no. s1119; sense target sequence, 5′-GAA AGA CUU GUU UAU AUU Att-3′; anti-sense target sequence, 5′-UAA UAU AAA CAA GUC UUU Cta-3′) were used. For ADRB2 knockdown, siADRB2(1) (cat. no. s1122; sense target, 5′-CCA GAA GAU UGA CAA AUC Utt-3′; anti-sense, 5′-AGA UUU GUC AAU CUU CUG Gag-3′), siADRB2(2) (cat. no. s1123; sense, 5′-CUU CUU CAU CGU UAA CAU Utt-3′, anti-sense, 5′-AAU GUU AAC GAU GAA GAA Ggg-3′) were used. Silencer Negative Control siRNA #1 (cat. no. AM4611; sequence not available) was used as a negative control. ADRB and negative control siRNA were purchased from Thermo Fisher Scientific, Inc.

Cell viability was measured using MTS assay. ACHN and A-498 cells were seeded in 96-well plates at a density of 1,000 cells/well and cultured at 37°C for 24 h. The medium was removed and 100 μl ISO (10 μM) was added to each well. After 24 h, the medium was removed and 100 μl ISO (10 μM) was added to each well., and the cells were cultured for another 24 h at 37°C. Cell viability was measured using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Corporation). Absorbance at 490 nm was measured using an iMark Microplate Reader (Bio-Rad Laboratories, Inc.) as previously described (32).

Cells were pretreated in the presence or absence of 10 μM ISO for 10 min at 37°C. Subsequently, sorafenib (0.00, 1.25, 2.50, 5.00 or 10.00 μM), cabozantinib (0.00, 1.25, 2.50, 5.00 or 10.00 μM), lenvatinib (0.00, 1.25, 2.50, 5.00, or 10.00 μM), and rapamycin (0.0, 0.1, 1.0, 10.0, or 100.0 nM)was administered to the cells, and the cells were cultured for 48 h at 37°C.

Cells were pretreated in the presence or absence of 10 μM ISO for 10 min at 37°C. Subsequently, ACHN cells were treated with erastin (0, 1, 2, 4 or 8 μM) or RSL3 (0, 25, 50, 100, or 200 nM), and cultured for 48 h at 37°C.

To determine whether ISO acts via ADRB1 or ADRB2 to influence the effect of sorafenib, ACHN cells were treated with siRNA and cultured for 24 h in serum-free medium at 37°C. The medium was replaced with medium containing 10 μM ISO, 5 μM sorafenib, and 10% FBS. After 24 h at 37°C, the medium was replaced with medium containing 10 μM ISO, 5 μM sorafenib, and 10% FBS, and the cells were cultured for 24 h at 37°C.

To investigate whether ADRB inhibitor PRO counteracts the effect of ISO, cell viability was assessed in ACHN cells treated with PRO (0, 1 or 10 μM), sorafenib (0 or 5 μM), and ISO (0 or 10 μM). Sorafenib and PRO were added concurrently with ISO.

To investigate the mechanism underlying sorafenib-induced suppression of cell viability, ACHN and A-498 cells were treated with sorafenib in combination with cell-death inhibitors, including the ferroptosis inhibitor Fer-1 (5 μM), the apoptosis inhibitor z-VAD (20 μM), the autophagy inhibitor 3-MA (20 μM) and the necroptosis inhibitor Nec-1 (20 μM).

GloSensor cAMP 20F (Promega Corporation) consists of the cAMP-binding domain of protein kinase A and a variant of firefly luciferase. When these components bind cAMP, a structural change occurs that leads to an increase in luminescence (33). ACHN and A-498 cells were transfected with GloSensor cAMP plasmid (100 ng DNA/well) using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C. After 24 h transfection, the cells were harvested and incubated with 2 mM endotoxin-free luciferin (Promega Corporation) at 37°C for 2 h. The cells were then treated with 10 μM ISO. Luciferase luminescence activity was measured using a luminometer (GLOMAX 20/20 LUMINOMETER, Promega Corporation) at baseline and at 1, 3, 5, 10 and 15 min and then every 15 min up to 120 min.

ADRB2 knockdown was performed using a second-generation lentiviral system consisting of pLKO.1-puro, psPAX2, and pVSV-G. Lentiviral particles were generated by transfecting HEK293T cells (kindly provided by Professor Hironobu Asao, Department of Immunology, Yamagata University Faculty of Medicine, Yamagata, Japan) in 6-well plates. For each well, 930 ng of shRNA vector was transfected together with packaging and envelope plasmids at 1:1:1 molar ratio using Lipofectamine 3000 at 37°C. The culture supernatant containing lentiviral particles was harvested 48 h post-transfection and filtered through a 0.45-μm membrane. ACHN cells were infected with the virus at a multiplicity of infection of 10 for 24 h at 37°C in the presence of 8 μg/ml polybrene. Cells were selected with puromycin (2 μg/ml) for ~2 weeks before subsequent experiments. The same puromycin concentration was used for both selection and maintenance. The expression of ADRB2 in the infected cells was validated by RT-qPCR and western blotting, as aforementioned. Lentivirus vectors containing the interfering sequence of ADRB2 (shADRB2: SHCLNV MISSION shRNA TRCN0000008085; target sequence, 5′-CCT CAA GAC GTT AGG CAT CAT-3′) and mock (SHC001 MISSION pLKO.1-puro Empty vector control plasmid DNA) was purchased from Sigma-Aldrich (Merck KGaA).

All procedures were performed using female BALB/c nude mice (age, 6-7 weeks; weight, 20-25 g; CLEA Japan Inc.) according to the animal welfare regulations of Yamagata University Faculty of Medicine based on the 'Guidelines for the implementation of animal experiments' established by the Ministry of Education, Culture, Sports, Science, and Technology of Japan (34). A total of 20 mice were maintained at 20-26°C and 40-60% humidity on a 12-h light/dark cycle and were provided with sterilized water and standard rodent feed ad libitum, except for stress group during periods of stress induction. All animal studies were approved by the institutional review board of Yamagata University (approval no. R5063, Animal Care and Committee of Yamagata University, Yamagata, Japan).

ACHN cells with ADRB2 knocked down by shRNA (1x10⁷ cells) were injected with Matrigel (Corning Life Science) into the subcutaneous left flank, while mock-transfected ACHN (1x10⁷ cells) were injected into the right side. When the tumor volume of negative control ACHN reached ~100 mm³, the mice were randomly divided into the following groups (all n=5): Control + vehicle, control + sorafenib, stress + vehicle and stress + sorafenib. Blinding was not performed. The mice were housed in metabolic cages for 2 weeks, and then in normal cages for the next 2 weeks. Their urine was collected in a tube containing hydrochloric acid for 3 days (day 10-12) and stored at -80°C until analysis. The stress groups were placed in a well-ventilated 50 ml conical tube for 6 h/day, as previously described (21). The mice were treated with oral sorafenib (10 mg/kg body weight in saline) or vehicle (saline) once/day. The drug administration and stress induction were conducted 5 days/week for 4 weeks. Tumor growth was measured every 2-3 day and the volume of the xenograft tumors were calculated as length × width × thickness × 0.5. The humane endpoint was tumor volume >1,500 mm³. However, no mice reached this threshold (maximum volume, 1,487 mm³). The sample size was calculated on a tumor volume difference of 110 mm³ between sorafenib-treated stressed and non-stressed mice, with a standard deviation of 60 mm³, a significance level of 0.05 and a statistical power of 0.8.

After 4 weeks, mice were anesthetized by intraperitoneal administration of medetomidine (0.3 mg/kg), midazolam (4 mg/kg) and butorphanol (5 mg/kg) (35) and euthanized via cervical dislocation. Death was confirmed by the absence of respiration and heartbeat. Blood samples were collected from the inferior vena cava, into EDTA-coated tubes and centrifuged at 2,000 × g for 15 min at 4°Cto obtain plasma and stored at -80°C until analysis. The measurement of adrenaline, noradrenaline and dopamine in acidic urine, as well as the measurement of cortisol in plasma, was performed by NIKKEN SEIL Co., Ltd.

MDA levels were used to assess the level of ferroptosis. Cells were treated with 10 μM ISO and/or 1 μM erastin at 37°C for 1 h and collected using a 0.08% Trypsin-0.3 mM EDTA at 37°C for 2 min. Cell lysate was prepared by sonication for 2 min using 4 cycles of 30 min on and 10 min off, at 20 kHz, while maintained on ice, and MDA concentration was assessed using MDA assay kit (cat. no. M496, Dojindo Laboratories, Inc.) according to the manufacturer's instructions. Fluorescence was measured using a fluorescence plate reader (varioskan LUX, Thermo Fisher Scientific, Inc.).

The present study detected ROS levels to assess the level of ferroptosis. Cells were incubated with RPMI medium containing 5 μM CellRox Deep Red (cat. no. C10422, Thermo Fisher Scientific, Inc.) at 37°C for 3 h. Next, cells were treated with 10 μM ISO and/or 1 μM erastin at 37°C for 1 h and collected using 0.08% Trypsin-0.3mM EDTA at 37°C for 2 min. Following fixation with PBS containing 4% paraformaldehyde at room temperature for 15 min, intracellular ROS levels were assessed using CellROX Deep Red Reagent (Themo Fisher Scientific),. Fluorescence intensity of the cells was measured using a flow cytometer (FACS Melody, BD Biosciences) and analyzed using FlowJo software version 10.8.1 (BD Biosciences). Each sample was measured in triplicate. The rate of increase in intracellular ROS was calculated as follows: ROS increase %=100 × (fluorescence intensity after treatment-fluorescence intensity of untreated cells)/fluorescence intensity of untreated cells.

Comparisons between immunostaining levels and clinical data were performed using cross tabulation and χ² or Fisher's exact tests. Kaplan-Meier survival curve analysis and a log-rank test were used to estimate cause-specific survival. Continuous variables are presented as the median or mean ± SD of three independent experimental repeats. Normality was assessed using the Kolmogorov-Smirnov test, and all data sets showed P-values >0.05. Homogeneity of variance was evaluated using the F-test, and all data showed P-values >0.05. Group comparisons of means were analyzed using unpaired t-tests or one- or two-way ANOVA with followed by Dunnett and Bonferroni post hoc tests. P<0.05 was considered to indicate a statistically significant difference. Statistical analyses were performed with GraphPad Prism 9.1.1 software (Dotmatics) and EZR version 1.68 (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R version 4.5.2 (The R Foundation for Statistical Computing) designed to add statistical functions frequently used in biostatistics (36). Cancer-specific survival (CSS) was defined as the time from the date of diagnosis of metastatic RCC to death from RCC, with censoring at the last follow-up or death from other causes. CSS was estimated using Kaplan-Meier method, and the values were compared using the log-rank test.

In primary RCC tissue from patients with metastases, expression of both ADRB1 and ADRB2 was confirmed in all cases. Higher ADRB1 expression compared with normal tubules was observed in 29/35 cases (82.9%), while higher ADRB2 expression was detected in 28/35 cases (80.0%). No significant association was found between ADRB expression and clinical factors, including grade, stage and cause-specific survival (Fig. S1A, Table SI).

In ccRCC tissue, there was a mixture of areas that expressed ADRB and areas that did not. No clear association was observed between high-expression regions and specific anatomical locations, such as near the pseudo-capsule, tumor vessels or necrotic areas. In papillary RCC tissue, both ADRB1 and ADRB2 were highly expressed throughout the lesion (Fig. S1B).

mRNA expression levels of ADRB1 and ADRB2 in RCC cell lines were quantified relative to ADRB2 expression in 769-P cells. ADRB1 mRNA levels were comparable among A-498, Caki-1 and Caki-2 but were notably lower in 769-P cells. By contrast, ACHN cells exhibited 11.7-fold higher ADRB1 compared with ADRB2 expression in 769-P cells. ADRB2 mRNA levels were 6.2-fold higher in A-498, 100-fold higher in Caki-1 and 640-fold higher in ACHN than in 769-P cells compared with those in 769P cells (Fig. 1B; Table SII).

Western blot analysis confirmed high protein expression of both ADRB1 and ADRB2 in ACHN cells (Figs. 1C and S2). While ADRB1 was detected in all examined cell lines, ADRB2 expression was minimal except in ACHN cells. Notably, mRNA and protein expression levels were not associated (Figs. 1B and C and S2).

No association was observed between ADRB2 levels and VHL gene mutation in the present study (Figs. 1B and C and S2). Based on these findings, ACHN, a RCC cell line with high ADRB1/2 expression, and A-498, a typical ccRCC cell line with VHL gene mutation, were selected for subsequent experiments.

To assess the response of RCC cells to ADRB, cAMP levels were measured in ACHN and A-498 cells following ISO treatment using the GloSensor cAMP 20F. In both cell lines, ISO treatment led to an increase in cAMP levels (Fig. 2A). Viability was evaluated in ACHN and A-498 cells treated with ISO using the MTS assay; however, no significant changes were observed (Fig. 2B). To examine the potential association between ADRB stimulation and drug resistance, an MTS assay was performed in ACHN and A-498 with clinically relevant MTDs, including sorafenib (0-10 μM), cabozantinib (0-20 μM), lenvatinib (0-20 μM) and rapamycin (0-100 nM). The assay was performed under ISO-free conditions and under conditions mimicking physiological ISO concentrations [10 nM (blood levels) and 10 μM (neuronal terminal levels)] (37). Cell viability ratios were calculated relative to MTD-free conditions. No significant difference in cell viability was observed with 10 nM ISO compared with ISO-free conditions for sorafenib treatment. However, at 10 μM ISO, the efficacy of sorafenib was attenuated in ACHN cells. By contrast, in A-498 cells,10 μM ISO did not affect the sorafenib-induced suppression of viability. Additionally, ISO did not influence the drug-induce viability suppression in either ACHN or A-498 cells (Fig. 2C).

To determine whether ADRB1/2 knockdown reverses the attenuation of sorafenib efficacy by ISO, cell viability was assessed in ADRB1/2 knockdown ACHN cells treated with 5 μM sorafenib, in the presence or absence of 10 μM ISO (Figs. 3A and B and S3; Table SIII). While sorafenib treatment decreased cell viability, the addition of ISO attenuated its effect. ADRB2, but not ADRB1, knockdown decreased the attenuating effect of ISO on sorafenib efficacy (Fig. 3C). Furthermore, to explore the potential therapeutic application of ADRB antagonism, cell viability was examined following co-administration of a non-specific ADRB antagonist (PRO). Although 10 μM PRO, which is not clinically achievable, slightly decreased cell viability, low concentrations of PRO did not reverse ISO-induced attenuation of sorafenib efficacy (Fig. 3D).

To investigate the impact of stress-induced catecholamine stimulation on sorafenib resistance and the role of ADRB2 in an in vivo model, ADRB2 knockdown ACHN cells were generated using the shRNA method. ADRB2 mRNA and protein levels decreased in ADRB2 knockdown ACHN cells (Fig. S4A).

Under stress conditions, mice treated with sorafenib exhibited a significant increase in urinary adrenaline and dopamine levels (Fig. 4A), whereas cortisol levels remained unchanged (Fig. 4B).

In control mice inoculated with mock ACHN cells, sorafenib effectively inhibited tumor growth. In the vehicle group, stress exposure did not significantly affect tumor volume. However, in sorafenib-treated mice, the stress group demonstrated a significantly greater increase in tumor volume compared with the control group. By contrast, in mice with shADRB2 ACHN tumors, no significant differences in tumor volume were observed between the control and stress-exposed groups under sorafenib treatment, with both groups showing suppressed tumor growth (Fig. 4C and D). These findings suggested that stress-induced ADRB2 activation attenuated the anti-tumor effect of sorafenib.

To investigate the mechanisms underlying sorafenib-induced suppression of cell viability, the present study assessed its effects using the ferroptosis inhibitor Fer-1, the apoptosis inhibitor z-VAD, the autophagy inhibitor 3-MA and the necroptosis inhibitor Nec-1. In ACHN cells, Fer-1 counteracted the decrease in cell viability caused by sorafenib, whereas z-VAD, 3-MA and Nec-1 had no impact on sorafenib-induced suppression of cell viability (Fig. 5A). These findings suggested that sorafenib decreased viability in ACHN cells via ferroptosis induction. However, in A498 cells, Fer-1 did not reverse sorafenib-induced suppression of cell viability, indicating that sorafenib-induced ferroptosis exhibited cell line-dependent variability.

The present study examined the effects of ISO on ferroptosis inducers erastin and RSL3. Although these inducers decreased cell viability, ISO mitigated their effects (Fig. 5B). Additionally, erastin increased levels of the lipid peroxidation marker MDA but this increase was inhibited by ISO (Fig. 5C). Similarly, erastin treatment led to increased ROS levels, which were suppressed by ISO (Fig. 5D). These results indicated that ISO suppressed ferroptosis induced by erastin and RSL3.

The present study investigated sorafenib-induced ferroptosis in additional RCC cell lines, including Caki-1, Caki-2, 769-P and 786-O. Fer-1 rescued sorafenib-induced loss of viability in Caki-1 cells, but had no protective effect in Caki-2, 769-P and 786-O cells (Fig. 5E). Sorafenib did not decrease cell viability at clinically achievable concentrations (<5 μM). Because VHL-mutant cell lines did not exhibit sorafenib-induced ferroptosis and loss of VHL leads to stabilization of HIF-2α (38), the present study examined whether ferroptosis susceptibility was influenced by HIF-2α, the key oncogenic driver in ccRCC. In HIF-2α-positive, VHL mutant A-498 and 786-O cells, HIF2α inhibitor PT2385 failed to reverse erastin-indued loss of viability, indicating that HIF-2α did not modulate ferroptosis under these conditions (Fig. 5F).

A previous study demonstrated that adrenaline increases dual-specificity phosphatase 1 (DUSP1) expression, which inhibits the phosphorylation of MAPKs (39). Additionally, multiple studies have shown that MAPK, ERK1/2, p38, and JNK pathways enhance ferroptosis induction (12,40,41). To investigate the mechanisms by which ISO modulates ferroptosis, the present study assessed the effect of ISO on DUSP1 expression and MAPK phosphorylation. ISO upregulated DUSP1. Phosphorylation of JNK and ERK was inhibited by both 10 nM and 10 μM ISO, whereas inhibition of p38 phosphorylation was observed only at the higher concentration (Figs. 6A and S5A). The present study assessed cell viability following treatment with ferroptosis inducers and MAPK inhibitors. The JNK inhibitor SP600125 and ERK1/2 inhibitor PD0325901 attenuated the effects of sorafenib and erastin. Moreover, simultaneous inhibition ERK1/2, p38 or JNK suppressed ferroptosis induction (Figs. 6B and C and S5B and C).