Targeting GSTM3 for therapeutic potential in advanced prostate cancer

Abstract

Prostate cancer (PCa) represents one of the most prevalent malignancies among men worldwide. When diagnosed at an early, localized stage, PCa is generally associated with a favorable prognosis and is often amenable to curative treatment modalities. However, the management of advanced PCa continues to pose significant therapeutic challenges despite the availability of conventional treatment modalities such as androgen deprivation therapy (ADT), salvage radiotherapy, and systemic chemotherapy. This underscores the urgent need to identify novel molecular targets that can enhance therapeutic efficacy and improve clinical outcomes in advanced disease. Glutathione S-transferase mu 3 (GSTM3), a detoxification enzyme involved in redox regulation and cellular homeostasis, has emerged as a potential contributor to PCa progression. The present study investigates the expression profile and functional significance of GSTM3 in advanced PCa, with the aim of elucidating its role in disease pathobiology and therapeutic targeting. GSTM3 expression levels were analyzed using public datasets from GEO and UALCAN. Quantitative RT-PCR was employed to validate expression levels in DU-145 and PC-3 cell lines, as well as in tumorsphere models. GSTM3 silencing was achieved using siRNA. ROS and mtMP were measured using H2DCFDA and Rhodamine assays, respectively. The impact on the cell cycle was assessed via PI staining, apoptosis and necrosis were evaluated using flow cytometry. Analysis of previous datasets and current experimental data revealed that GSTM3 was overexpressed in advanced PCa cells. Tumorsphere models exhibited even higher GSTM3 expression compared to conventional cell lines. In GSTM3-silenced cells, mtMP increased, while ROS levels showed a slight decrease. Silencing GSTM3 also led to cell cycle arrest in the G0/G1 phase, with a significant increase in necrosis and a modest rise in apoptosis. Our findings demonstrate the functional role of GSTM3 in advanced PCa, highlighting its potential as a therapeutic target. The consistent data suggest that targeting GSTM3 could offer new avenues for PCa treatment.

Introduction

PCa is a serious health concern for men worldwide, characterized by its complexity and increasing prevalence with age. It is most commonly diagnosed in men aged 45 to 60, with the incidence rising sharply in those over 65 years old [1]. The 5-year survival rate of the localized and regional malignancy is nearly high, but it decrease dramatically for advanced-stage tumors [2]. PCa risk is influenced by various factors, including age, family history, race, and lifestyle choices (diet and microbiome) [3, 4]. The Gleason grading system is a classification tool employed to assess the severity of PCa considering the microscopic diagnosis of prostate tissue samples. A cancer grade of 6 or below is typically characterized as low-grade, having a slow growth rate and a lower likelihood of spreading, whereas Gleason score 7 indicates an intermediate stage. Gleason scores between 8 and 10 implies high grade cancer, which is typically aggressive and prone to rapid dissemination [5, 6].

PCa commonly metastasis to specific sites within the body such as bone, brain, lymph nodes and lung. Recent data indicate a significant upward trend in the incidence of metastatic prostate cancer (mPCa) over the past decade. Between 2008 and 2016, the incidence of mPCa rose by 72%, contrasting with a 37% decline in the incidence of low-risk PCa during the same period [7]. 15% of PCa patients are pronounced at an advanced stage or with metastasis. Among these metastatic cases, 45% are de novo, while the remaining are recurrent PCa [8].

Treatment strategies for regional PCa include active surveillance, radical prostatectomy and ablative radiotherapy [9]. For patients who experience disease relapse or metastatic phase of the cancer, the therapeutic approach expands to include salvage radiotherapy, androgen deprivation therapy (ADT), and systemic chemotherapy. Current evidence suggests that combining treatment modalities yields superior outcomes compared to monotherapy [10]. Nevertheless, despite advancements in therapeutic options, PCa remains an incurable disease, underscoring the need for continued research and innovation in its management. Targeted therapies represent an evolving and critical approach in the treatment of PCa, particularly for advanced or treatment-resistant cases.

The Glutathione S-transferase mu 3 (GSTM3) gene, a member of the GST family, plays a pivotal role in the detoxification of carcinogens [11]. rs7483 polymorphism on the GSTM3 has emerged as a potential biomarker for predicting treatment outcomes in PCa patients undergoing ADT. Research suggests that this genetic variant may influence individual responses to ADT, thereby serving as a valuable tool for stratifying patients based on their likelihood of therapeutic success. Furthermore, in the context of metastatic PCa, the presence of the GSTM3 rs7483 polymorphism has been significantly associated with an increased risk of disease progression [12]. These findings highlight the potential clinical utility of GSTM3 rs7483 as both a prognostic and predictive marker in the management of PCa.

The expression level of the GSTM3 gene varies significantly across different cancer types, reflecting its complex role in tumor biology. Low GSTM3 expression has been reported in several cancers, including esophageal squamous cell carcinoma [13], pancreas cancer [14], and renal cell carcinoma [15]. Conversely, elevated GSTM3 expression has been reported in various malignancies, such as colon [16], bladder [17], breast cancer [18] and hepatocellular carcinoma [19]. This dual expression pattern suggests that GSTM3 may act as either a tumor suppressor or promoter, depending on the cancer type and cellular context. Notably, a comprehensive study analyzing the genomic, transcriptomic, and epigenomic profiles of PCa in the American population revealed that GSTM3 expression was significantly overexpressed in African American individuals [20]. The present study aims to elucidate the functional role of the GSTM3 gene in advanced PCa, focusing on its contributions to tumor progression.

Material methods

GSTM3 gene expression across multiple cancers

To determine GSTM3 gene expression across multiple cancers, we used a web based tool, Correlation AnalyzeR. Correlation AnalyzeR was run with default parameters. Single gene section was used to profile the GSTM3 level in multiple cancers. The analysis integrates key metrics such as log2 fold change, base mean and statistical significance obtained from differential expression gene (DEG) analysis. The corrplot package of the analysis leads to visualization of the results [21].

Prostate cancer dataset and Geo2R analysis

To identify the genes accountable for the development of PCa, we obtained two distinct datasets from the GEO database. The array GSE200879 contains 9 samples of healthy tissue and 115 samples of tumor prostate tissue. The analysis was conducted using 30 patients of the advanced Gleason score, which ranges from 8 to 9. Another dataset, GSE210205, includes benign prostate cells BPH1 as well as DU-145 and PC-3 cells.

GEO2R is an analytical tool designed to examine gene expression data retrieved from the GEO database. It is particularly valuable for conducting comparative analyses of gene expression across different experimental conditions or groups. Using GEO2R, we employed DEG analysis to identify differentially expressed genes in tumor cells compared with benign control cells, with a false discovery rate of 0.001. The tool’s instructions were followed using default parameters, which involved using raw counts to assess gene expression through DeSeq2, with adjustments made for sequencing depth and RNA composition variability by standardizing median ratios. Normalization for sequence depth had a negligible effect on the samples derived from a particular study. The analysis incorporated volcano plots. The volcano plot displays differentially expressed genes by correlating the statistical significance of changes (the negative logarithm of the p-value, expressed as -log10(p value) with the magnitude of gene expression change (the logarithm base 2 of the fold change, or log2(fold change)), thereby providing an intuitive representation of genes with both statistical significance and substantial expression variations. The genes exhibiting differential expression were pinpointed by filtering the expression data, with an adjusted p-value threshold set at < 0.001 and a log2 fold change of ≥ 0.5, utilising default parameters and the findings of previous studies, as well as a consideration of the size of the resultant gene set identified as differentially expressed between tumor and benign cells.

To evaluate the expression pattern of GSTM3 across various cancer types, we utilized the UALCAN online database (http://ualcan.path.uab.edu). The “TCGA Gene Analysis” module was used to query GSTM3 expression levels across multiple tumor types compared to their corresponding normal tissues. Expression data were visualized as boxplots, and statistical significance between tumor and normal samples was assessed automatically by the UALCAN platform.

Cell culture

The DU145 cell line, derived from brain metastatic site and the PC3 cell line, representing bone metastatic PCa whereas the PNT1A cells, which are non-tumorigenic prostate epithelial cells, were cultured in RPMI medium. All cell lines were regularly confirmed to be mycoplasma free. Cells were maintained in RPMI medium supplemented with 10% FBS and 1% Penicillin-Streptomycin, and incubated at 37°C in a 5% CO2 atmosphere. The medium was refreshed three times a week, and cells were passaged via trypsinization upon reaching 80% confluence.

Sphere formation assay

To evaluate the sphere-forming potential of PCa cells, single-cell suspensions were prepared, and 15,000 cells were seeded well in by using 24 well ultra low attachment plates (Corning Inc., Corning, NY, USA). The cells were cultured in RPMI medium supplemented with 10 ng/mL basic fibroblast growth factor (bFGF) and 10 ng/mL epidermal growth factor (EGF) (GIBCO, USA), 2% B27 supplement (GIBCO, USA), 1% N2 supplement (GIBCO, USA), and 1% penicillin-streptomycin. The spheroids were incubated for 7 days and subsequently imaged. The cell pellet was then collected for RNA isolation.

SiRNA transfection

siRNA transfection was applied based on the manufacturer’s protocol. DU145 and PC3 cells were transfected with 10 nM GSTM3-specific siRNAs (Catalog Number: 4392420, ID: s6271) or control siRNA (Catalog Number: 4390847) (Ambion/Applied Biosystems) through Lipofectamine (Invitrogen) in OPTI-MEM medium (Gibco). The efficiency of gene silencing was assessed 48 h post-transfection to evaluate the functional effects of the knockdown.

Cell viability

Cell viability was assessed using the MTS assay. CTRL siRNA and GSTM3-silenced cells were placed in 96-well plates at 2000 cells per well and incubated at 37°C overnight. After 24–48–72 h of treatment, MTS solution was added to each well (CellTiter 96^® AQueous One Solution, Promega). The plate was incubated for an additional 1–4 h, and absorbance was measured at 490 nm using a microplate reader.

RNA isolation and cDNA synthesis

The extraction of total RNA from each cell pellet was meticulously performed using the TRIzol Reagent (Invitrogen, Life Technologies), adhering rigorously to the manufacturer’s protocol to ensure consistency and reliability. The purity and concentration of the extracted RNA were then evaluated with precision using the NanoDrop ND-1000 spectrophotometer, Subsequently, cDNA synthesis was carried out with 1 µg of total RNA, carefully reverse transcribed in a 20 µL reaction volume. This process utilized the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Rotkreuz, Switzerland) following the provided guidelines.

Quantitative real-time PCR (qPCR) analysis

Quantitative PCR was performed using TaqMan Gene Expression Assays and TaqMan Universal PCR Master Mix (Applied Biosystems) on a StepOne Plus Real-Time PCR System (Applied Biosystems). The cycling conditions were as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.

Based on initial observations of mitochondrial membrane potential and ROS levels, GPX4 gene expression was evaluated to investigate whether antioxidant response pathways were activated following GSTM3 silencing. GPX4, a key regulator of lipid peroxidation and ferroptosis, was selected due to its known role in redox homeostasis and its potential functional relationship with GSTM3.

GSTM3 (Assay ID: Hs00356079_m1), GPX4 (Assay ID: Hs00989766_g1), ACTB (Assay ID: Hs99999903_m1) as the endogenous control were used in the study. Relative gene expression levels were calculated using the ΔΔCt method. All reactions were performed in triplicate, and expression data were normalized to ACTB.

Determination of ROS levels

Intracellular reactive oxygen species (ROS) levels were assessed using the oxidative conversion of the cell-permeable probe 2′,7′-dichlorofluorescein diacetate (H2DCFDA) (Thermo Fisher Scientific, USA), which is measured by flow cytometer. Cells (1 × 10⁵ per well) were seeded in 6-well plates and allowed to adhere overnight. After rinsing with PBS, cells were incubated with 10 µM H2DCFDA for 30 min in the dark at 37°C. Following incubation, cells were detached, washed with PBS, and analyzed for DCF fluorescence using a BD FACS Aria III flow cytometer with excitation at 488 nm and emission at 535 nm.

Detection of mitochondrial membrane potential

MtMP (mitochondrial membrane potential) was utilized using a cationic dye, Rhodamine 123. 10⁵ per well cells were seeded in 6-well plates. Prior to and following treatment, cells were rinsed with PBS. Subsequently, cells were incubated with 10 µM Rhodamine 123 for 30 min in the dark at 37°C. After the incubation, cells were detached and washed with PBS. Mitochondrial membrane potential was then quantified by measuring the fluorescence intensity of Rhodamine 123 in the cells using a BD FACSAria III flow cytometer, with an excitation wavelength of 507 nm and emission wavelength of 523 nm.

Cell apoptosis assay

In order to investigate the potential role of GSTM3 in apoptosis, an Apoptosis and Necrosis Kit (Abcam) was utilized in accordance with the manufacturers instructions. Briefly, prostate cells were harvested and centrifuged at 200 × g for 5 min to collect the cell pellet. The cells were then resuspended in Apoptosis Assay Buffer containing Apopxin Green Indicator to label apoptotic cells, 7-AAD to stain necrotic cells, and CytoCalcein Indicator to identify healthy, live cells. The prepared samples were incubated in a dark environment for 60 min at room temperature to allow optimal staining. Following incubation, the samples were analyzed using a flow cytometer. Apoptotic cells were detected based on fluorescence at excitation/emission wavelengths of 490/525 nm, necrotic cells at 546/647 nm, and live cells at 405/450 nm.

Cell cycle analysis

1 × 10⁵ cells were seeded per well in 6 well plates and placed incubator to allow attachment. Cells were centrifuged at 300 × g for 5 min, and the supernatant was discarded. The cell pellet was resuspended in PBS and fixed in 70% ice-cold ethanol for at least 2 h at 4°C or overnight. Fixed cells were then washed twice with PBS to remove excess ethyl alcohol. The fixated cells were resuspended in PBS and treated with RNase A (50 µg/mL) for 30 min at 37°C to remove RNA interference. After incubation, cells were stained with Propidium Iodide (PI) (50 µg/mL) for 30 min at room temperature in a dark room. A minimum of 10,000 events for each sample were counted. The DNA content of the cells was assessed by measuring the fluorescence emitted from PI at an excitation wavelength of 488 nm and emission wavelength of 617 nm. The flow cytometer data was analyzed using ModFit software, and the cell cycle distribution were determined. DNA damage was assessed by quantifying the sub-G1 cell population using flow cytometry with propidium iodide (PI) staining, which serves as an indicator of DNA fragmentation.

Statistical analysis

All statistical analyses were performed using the either the R programme with appropriate standard packages or Graphpad. Group comparisons were carried out using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons, or unpaired t-tests where applicable. Statistical significance was applied at a threshold of p < 0.05, indicating a rejection of the null hypothesis that group means are equal. Each experiment was performed in triplicate to ensure data reliability and reproducibility.

Results

Expression of GSTM3 levels in prostate cancer

A significant elevation in GSTM3 expression was detected in various cancers compared to normal tissues, including those of the pancreas, brain, respiratory system, liver, bone, and prostate (Fig. 1). Analysis of differential gene expression in PCa datasets revealed notable findings. In the GSE210205 dataset, which includes two biological replicates of healthy prostate cell lines alongside PC3 and DU145 PCa cell lines, 1581 DEGs were identified (p adj.<0.001). Similarly, in the GSE200879 dataset, which comprises samples from nine healthy prostate tissues and 30 advanced PCa patients with a Gleason score exceeding 8, a total of 944 DEGs were detected (padj.<0.001). The results are visually represented in the volcano plots shown in Fig. 2a and b. GSTM3 was identified as one of the top 10 DEGs in the GSE210205 dataset and demonstrated significantly elevated expression levels in the GSE200879 dataset (p < 0.001). The distribution of GSTM3 expression between tumor and healthy samples, as well as its variation within tumor samples, are illustrated in Fig. 3a and b, respectively. To validate the dataset findings, GSTM3 gene expressions were also measured in the PNT1A healthy prostate cell line and the DU145 and PC3 PCa cell lines. A significant upregulation of GSTM3 expression was observed in the tumor cell lines compared to the healthy cell line, corroborating the dataset results and suggesting a potential role for GSTM3 in PCa progression (Fig. 4). Tumorsphere formation assays were performed to highlight the aggressive characteristics of tumor cells (Fig. 5a). Significantly higher levels of GSTM3 expression were observed in tumorspheres compared to cell line-derived samples, suggesting a potential role for GSTM3 in promoting aggressive tumor phenotypes and enhanced tumorigenic capacity Fig. 5b.

Fig. 1.

Expression of GSTM3 in various cancers compared to healthy tissues

Fig. 2.

The volcano plots illustrate the differential gene expression profiles of two transcriptome datasets. A Represents the 1581 differentially expressed genes (DEGs) identified in the GSE210205 dataset. B Depicts the 944 DEGs identified in the GSE200879 dataset. Significant upregulation is highlighted in red, while significantly downregulated genes are shown in blue (p < 0.001)

Fig. 3.

Expression levels of GSTM3 across different datasets: data are presented as TPM (Transcripts Per Million) read counts. A Depicts the expression levels of GSTM3 in the GSE210205 dataset, which includes both tumor and healthy prostate cell lines. B GSTM3 expression in the GSE200879 dataset, comprising 9 healthy prostate tissue samples and 30 advanced PCa tissue samples

Fig. 4.

Expression of GSTM3 in cell lines. High level of GSTM3 is observed in PC3 and DU-145 cell lines compared to the PNT1A.(*:p<0.05, **:p<0.01)

Fig. 5.

Tumorsphere formation from prostate cancer cells and GSTM3 expression levels. A Representative images of tumorspheres derived from DU145 and PC3 cell lines. B GSTM3 mRNA expression levels in parental cell lines and their corresponding tumorspheres

Functional effects of GSTM3 silencing in prostate cancer

The functional role of the GSTM3 gene was also investigated in PCa. To do so, we utilized gene-specific siRNA to knockdown its expression. This resulted in a significant reduction of GSTM3 levels in the silenced cell lines, as shown in Fig. 6a. Cell viability was monitored over a period of three days, and a decrease in the viability of GSTM3-silenced cells was observed, indicating that the loss of GSTM3 expression negatively affects the survival or growth of the cells (Fig. 6b). The evaluation of mitochondrial membrane potential’s function was also undertaken. The level of mtMP was found to be higher in cells where GSTM3 was silenced compared to control cells, with a notable increase seen in DU145 cells (Fig. 7). Conversely, the levels of reactive oxygen species (ROS) were reduced in GSTM3-silenced cells relative to the controls (Fig. 8). Given the increased mitochondrial membrane potential and the unexpected reduction in ROS levels observed upon GSTM3 silencing, the mRNA expression of GPX4 was evaluated. GPX4 levels were found to be upregulated in GSTM3-silenced cells compared to controls, indicating a potential compensatory antioxidant response (Fig. 9). The cell cycle analysis comparing control cells to GSTM3-silenced cells revealed a notable arrest in the G0/G1 phase within the GSTM3-silenced group, especially in DU-145 cells (Fig. 10a). In addition, GSTM3 silencing was associated with a significant increase in DNA damage, further highlighting its impact on cellular processes associated with DNA stability (Fig. 10b). Flow cytometry analysis demonstrated GSTM3 silencing resulted with a significant increase in the apoptotic cell population. The data showed that both early apoptosis (Q1) and late apoptosis (Q2) were elevated in GSTM3-silenced cells compared to controls. Moreover, the population of necrotic cells, represented by Q3, also showed a notable increase. These findings suggest that GSTM3 plays a crucial role in regulating apoptotic processes and maintaining cell viability, with its silencing driving cells toward programmed cell death and necrosis (Fig. 11).

Fig. 6.

Silencing GSTM3 gene expression. A Significant downregulation in PC3 and DU-145 cells was illustrated B Effect of GSTM3 Knockdown on Cell Viability compared to control siRNA at 72 h (*:p < 0.05, **:p < 0.01)

Fig. 7.

MtMP in GSTM3- Silenced Cells. GSTM3-silenced prostate cancer cells exhibit a significant increase in mitochondrial membrane potential (*: p < 0.05)

Fig. 8.

ROS Levels in Silenced Cells. GSTM3-silenced cells show a decrease in ROS levels

Fig. 9.

GPX4 mRNA expression following GSTM3 knockdown (KD) in PCa cells

Fig. 10.

Cell cycle analysis of prostate cancer cells. A The illustration highlights the effects of GSTM3 silencing on cell cycle dynamics in PCa cells. Cell population within the G0/G1 phase is observed upon GSTM3 silencing, indicating cell cycle arrest at this stage. B GSTM3 silencing is associated with a significant rise in DNA damage, emphasizing its potential role in genome stability. Statistical significance is denoted as follows: *p = 0.05, **p < 0.01

Fig. 11.

Impact of GSTM3 silencing on apoptosis. A rise in the apoptotic cell population following GSTM3 silencing. The data reveals that silencing GSTM3 leads to an increase in both early and late apoptotic cells, as indicated by quadrants Q1 and Q2, respectively and Q3 highlights the proportion of necrotic cells.

Discussion

In this study, we aimed to investigate the significance of the GSTM3 gene in advanced PCa. To explore its expression, we leveraged metadata that highlighted the expression levels of GSTM3 across multiple cancers and their corresponding healthy tissues. Additionally, two datasets derived from the Gene Expression Omnibus (GEO) were analyzed, both of which corroborated that PCa tissues with high Gleason scores and metastatic PCa cell lines exhibited significantly elevated GSTM3 expression compared to their healthy tissue or cell line counterparts. Previous studies provide further context for our findings. For instance, a comparative analysis demonstrated that African American men exhibited significantly higher GSTM3 expression levels compared to other populations [22]. Furthermore, screening the genomic profiles of the American population and specifically analyzing PCa cases revealed that African American men have elevated GSTM3 expression levels compared to European Americans [20]. These population-level differences suggest a potential genetic or epigenetic basis for GSTM3 dysregulation in certain demographic groups. To corraborate and expand these findings in the laboratory setting, we examined GSTM3 expression in metastatic PCa cells and compared them to the benign prostate epithelial cell line PNT1A. Consistent with the metadata and previous studies, our experimental results confirmed a significant increase in GSTM3 expression in metastatic PCa cells. This experimental validation underscores the potential of GSTM3 as a biomarker for aggressive PCa and provides a foundation for future research into its mechanistic role in PCa progression and racial disparities in disease outcomes. To further elucidate the potential of role GSTM3 in the tumor aggressiveness, we analyzed its expression in spheroid models of metastatic PCa cells, which represent an aggressive cancer phenotype enriched with stemness properties. Human skin-derived hepatic progenitor cells express the GSTM3 enzyme alongside other key biotransformation-related genes such as CYP1B1, FMO1, and GSTA4, underscoring the metabolic energy demands associated with biotransformation processes [23]. Similarly, our findings demonstrated that spheroids exhibit significantly elevated GSTM3 expression, suggesting its critical role in enhancing stemness and driving the aggressive phenotype in PCa. This upregulation may be linked to the activation of pathways involved in cell survival, proliferation, and metastasis, as well as the enrichment of cancer stem cell-like characteristics that contribute to tumor progression. These observations highlight GSTM3 as a key player in both normal and pathological contexts, bridging its roles in biotransformation and the regulation of stemness and malignancy.

The reduction of the expression of the GSTM3 gene significantly impacted reduced cell viability, with pronounced effects being observed especially in PC3 cells within 72 h, exhibiting a time-dependent pattern. The temporal nature of this response suggests that GSTM3 plays a critical role in maintaining cellular survival over time, potentially through mechanisms involving cell cycle regulation or stress response pathways. Literature research revealed that a similar trend was observed in T98G brain tumor cells, where GSTM3 knockdown not only reduced cell viability but also enhanced sensitivity to temozolomide. This finding highlights the potential dual role of GSTM3 in tumor cell survival. The increased sensitivity to temozolomide suggests that GSTM3 may influence pathways involved in DNA repair or drug metabolism, making it a promising target for combination therapies aimed at overcoming chemoresistance in aggressive cancers [24]. Interestingly, silencing GSTM3 in PCa cells revealed notable mitochondrial and oxidative changes. GSTM3 silencing resulted in an increase in mtMP but also in a paradoxical decrease in ROS levels. A similar effect was observed in GSTM3-silenced tamoxifen-resistant MCF-7 breast cancer cells, highlighting the potential role of GSTM3 in mediating ROS defense [18]. We interpret this as a compensatory response, wherein oxidative stress is buffered by increased expression of GPX4, a master regulator of lipid peroxide detoxification and ferroptosis resistance. The upregulation of GPX4 likely limits the accumulation of ROS despite mitochondrial stress, serving as a cellular adaptation to GSTM3 loss. A similar phenomenon has been described in other cancer models, including cutaneous squamous cell carcinoma, where GSTM3 negatively regulates GPX4 expression and ferroptosis susceptibility [25]. This adaptive antioxidant shift may protect cells from immediate oxidative damage but could also make them more vulnerable to GPX4-targeted therapies in the long term. Notably, previous studies have shown that reducing ROS production can be an effective strategy against PCa cells [26]. However, the observed inconsistency between mtMP and ROS levels suggests the activation of adaptive antioxidant pathways. To investigate this compensatory mechanism, we analyzed the expression of the GPX4 gene, which encodes Glutathione Peroxidase 4, a critical regulator of antioxidant metabolism. The observed upregulation of GPX4 in GSTM3-silenced cells supports our hypothesis that GPX4 counteracts oxidative stress in the absence of GSTM3. In accordance with our findings, recent studies in cutaneous squamous cell carcinoma have revealed a negative correlation between GSTM3 and GPX4 expression, highlighting their interplay in regulating ferroptosis [25]. These results emphasize the intricate balance between ROS production, GSTM3, and GPX4 in oxidative stress management and its implications for cell fate decisions.

Cell cycle analysis revealed that silencing the GSTM3 gene led to G0/G1 cell cycle arrest, with a significant increase in DNA damage. Although GPX4 levels were elevated as a compensatory defense mechanism, this upregulation appears insufficient to mitigate the observed DNA damage. Supporting our findings, previous studies have demonstrated that among the GST family members, GSTM3 plays a pivotal role in protecting cells from electrophilic DNA damage by catalyzing the conjugation of reactive oxygen species (ROS) with glutathione [14, 27]. These results underscore the critical function of GSTM3 in maintaining genomic integrity under oxidative stress. The slight increase in apoptotic cells, coupled with a substantial rise in necrotic cells, suggests a shift from regulated cell death mechanisms to necrosis. This shift may be driven by severe cellular stress and energy depletion resulting from GSTM3 loss. Notably, GSTM3 silencing has been shown in U251 and U87 cells to be associated with increased apoptosis and reduced proliferation, mediated through the regulation of LncRNA GAS5 [28]. Another cell death mechanism linked to the GSTM3 gene is ferroptosis [13, 25, 29] which also should be investigated for further studies in PCa. However, our study also revealed a significant increase in necrosis in GSTM3-silenced cells, at least in part, by the accumulation of DNA damage. A study suggesting that GSTM3 is a tumor suppressor in gastric cancer revealed that GSMT3 deficiency decreased DNA mismatch repair gene expression and increase mutagenesis through CAND1/NRF2 [30]. This highlights the multifaceted role of GSTM3 in regulating cell death pathways and maintaining genomic integrity. All these findings emphasize the multifaceted role of GSTM3 in modulating cell fate and stress responses.

Silencing of GSTM3 in advanced PCa cells revealed multifaceted downstream effects that implicate this gene in critical cellular processes beyond detoxification. Notably, the observed increase in mtMP despite a reduction in ROS levels suggests a decoupling of mitochondrial activity and oxidative stress, potentially driven by a compensatory upregulation of GPX4. As a key antioxidant enzyme involved in lipid peroxide detoxification and ferroptosis regulation, GPX4 induction may represent an adaptive mechanism to buffer redox imbalance in the absence of GSTM3 [18]. This interplay highlights the involvement of GSTM3 in maintaining mitochondrial redox homeostasis. In parallel, GSTM3 silencing resulted in cell cycle arrest at the G0/G1 phase, accompanied by a marked increase in DNA damage, suggesting impaired genomic integrity and disruption of cell cycle progression [27]. These findings point toward a possible regulatory role of GSTM3 in DNA damage response or checkpoint control pathways. Moreover, the simultaneous elevation of apoptotic and necrotic cell populations indicates that GSTM3 is essential for sustaining cellular viability under stress conditions. The convergence of mitochondrial dysfunction, redox dysregulation, and compromised DNA stability upon GSTM3 knockdown supports its role as a molecular safeguard against multiple stressors. Together, these observations suggest that GSTM3 functions at the intersection of oxidative stress regulation, mitochondrial health, and genome maintenance, underscoring its potential as a mechanistically relevant therapeutic target in aggressive PCa.

Most studies on GSTM3 have focused on its genetic polymorphisms. However, its expression and functional significance in PCa warrant further research. Our findings underscore GSTM3 as a critical regulator of oxidative stress, mitochondrial function, and cell death pathways in PCa cells. These results position GSTM3 as a pivotal factor in maintaining cellular homeostasis and suggest its potential as a therapeutic target in PCa.

Authors’ contributions

Conceptualization: DS, Methodology: DS, Formal analysis and investigation: DS; Writing - original draft preparation: DS, Writing - review and editing: DS, ABD, ÖFB.

Funding

This research was supported by Yeditepe Research Funding.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

This study utilized commercially available cell lines; therefore, no ethical approval was required.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.