15d-PGJ2 enhances cisplatin cytotoxicity via mitochondrial ROS-mediated cell death in human bladder cancer

15d-PGJ2 enhances cisplatin cytotoxicity via mitochondrial ROS-mediated cell death in human bladder cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 15d-PGJ2 enhances cisplatin cytotoxicity via mitochondrial ROS-mediated cell death in human bladder cancer Chien-Rui Lai, Ying Chen, Tzu-Min Chen, Yu-Ling Tsai, Wen-Chiuan Tsai, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9134319/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Background Bladder cancer (BC) is a common urologic malignancy with high recurrence and progression rates, and outcomes for advanced disease remain poor. Peroxisome proliferator-activated receptor gamma (PPARγ) is involved in tumor metabolism and progression, but its role in BC is not fully defined. Methods We investigated the effects of 15dPGJ2, alone or in combination with cisplatin, on bladder cancer cells using a series of functional assays in T24 and RT4 cell lines and further validated the antitumor effects in a xenograft mouse model. Results Analysis of The Cancer Genome Atlas (TCGA) dataset revealed that higher PPARγ expression is associated with improved overall survival in BC patients. Treatment with 15-deoxy-Δ¹²,¹⁴-prostaglandin J₂ (15d-PGJ2), a natural PPARγ ligand, significantly suppressed cell viability and colony formation in T24 and RT4 bladder cancer cells. Mechanistically, 15d-PGJ2 induced mitochondrial oxidative stress, as demonstrated by increased mitochondrial reactive oxygen species (ROS), elevated JC-1 green/red fluorescence ratios, and mitochondrial depolarization and fragmentation. These changes were accompanied by reduced mitochondrial length and downregulation of mitochondrial dynamics and respiratory proteins, including Tom20, MFF, COX IV, and ATP5A, along with increased p-Drp1 and cleaved PARP expression. Combined treatment with cisplatin and 15d-PGJ2 further enhanced mitochondrial dysfunction and ROS production, resulting in synergistic inhibition of cell viability. In a T24 xenograft mouse model, 15d-PGJ2 alone or in combination with cisplatin significantly reduced tumor growth, as confirmed by IVIS imaging, histological analysis, and immunohistochemistry, with tumor tissues showing increased apoptosis and suppression of mitochondrial-related proteins. Conclusion 15d-PGJ2 inhibits bladder cancer growth by inducing mitochondrial oxidative stress and dysfunction and represents a promising mitochondrial-targeting chemosensitizer for bladder cancer therapy. PPARγ 15d-PGJ2 cisplatin mitochondria ROS bladder cancer apoptosis mitochondria membrane potential Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Urothelial carcinoma represents one of the most frequently diagnosed malignancies of the urinary system and remains a substantial global public health concern. In Western countries, bladder cancer (BC) ranks as the fourth most common cancer among men, accounting for approximately 6% of newly diagnosed malignancies in 2023 [1, 2], and contributes to 4% of cancer-related mortality in this population, with a median age at diagnosis of approximately 70 years [3]. At initial presentation, 70–75% of patients exhibit non-muscle-invasive bladder cancer (NMIBC), 20–25% present with muscle-invasive bladder cancer (MIBC), and approximately 5% are diagnosed with metastatic disease [4]. NMIBC is characterized by a high recurrence rate and heterogeneous progression risk, with five-year recurrence and progression rates ranging from one-third to three-fourths and 1% to 45%, respectively [5]. Despite definitive surgical management, approximately 30% of patients experience disease recurrence, and prognosis remains poor in those with distant metastases, reflected in persistently low five-year overall survival rates [6]. Therapeutically, NMIBC continues to present considerable challenges due to its propensity for recurrence and progression. Bacillus Calmette-Guérin (BCG) immunotherapy remains the mainstay for intermediate- and high-risk disease; however, a significant proportion of patients ultimately develop BCG-unresponsive tumors for which no universally effective therapy is available [7, 8]. Recently, intravesical gene therapy, immune checkpoint inhibitors [9], and novel drug delivery platforms have shown promise in this context, yet long-term outcomes remain under investigation [10]. Similarly, despite advancements in surgical techniques and perioperative care, MIBC and advanced disease continue to confer poor survival outcomes [11]. Collectively, these challenges underscore the urgent need for innovative therapeutic strategies and precision medicine approaches to improve outcomes across the bladder cancer disease spectrum. Among the emerging molecular strategies aimed at addressing tumor progression and therapeutic resistance, bioactive lipid mediators, particularly 15-deoxy-Δ¹²,¹⁴-prostaglandin J₂ (15d-PGJ2), a natural ligand for peroxisome proliferator-activated receptor gamma (PPARγ) [12], have attracted significant attention. 15d-PGJ2 is known to exert a range of biological effects, including anti-inflammatory, pro-apoptotic, and anti-proliferative activities [13]. A growing body of evidence suggests that 15d-PGJ2 influences various signaling pathways associated with tumorigenesis, such as the inhibition of NF-κB, the induction of oxidative stress, and the regulation of mitochondrial function. Its capacity to induce apoptosis, autophagy, and cell cycle arrest has been documented in multiple cancer types, including breast, lung, prostate, and colorectal cancers [14–17]. Although these findings are encouraging, the therapeutic role and mechanistic actions of 15d-PGJ2 in bladder cancer have yet to be fully elucidated. Given the urgent need for novel treatment strategies, particularly for cisplatin-resistant and advanced-stage urothelial carcinoma, elucidating the antitumor mechanisms of 15d-PGJ2 represents a critical direction for future research. Methods Cell culture Two urothelial carcinoma cell lines (RT4 and T24), which exhibit distinct epithelial morphologies, and a normal human uroepithelial cell line (SV‑HUC‑1) were sourced from the Bioresource Collection and Research Center (BCRC) in Taiwan. RT4 and T24 cells were propagated in McCoy’s 5A medium supplemented with 1% sodium pyruvate, 1% L‑glutamine (Corning, NY, USA), and 10% fetal bovine serum (FBS; Thermo Fisher Scientific, MA, USA). SV‑HUC‑1 cells were maintained in Ham’s F‑12 medium (Sigma‑Aldrich, St. Louis, MO), also supplemented with 1% sodium pyruvate, 1% L‑glutamine, and 7% FBS. All cultures were incubated at 37°C in a humidified atmosphere with 5% CO₂. Drugs and reagents Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), coomassie brilliant blue G-250 were purchased from Sigma–Aldrich (St. Louis, MO, USA). 15-Deoxy-Δ-12,14-prostaglandin J 2 (15d-PGJ2), cisplatin, N-acetylcysteine (NAC) and Mito-TEMPO were purchased from MedChemExpress (Princeton, NJ, USA). MTT assay RT4 and T24 cells were seeded in 96-well plates at densities of 5 × 10³ and 2 × 10³ cells separately per well. After the treatments, MTT solution (0.5 mg/ml) was added to each well and incubated for 3 hours. The formazan crystals formed were then dissolved in DMSO, and the absorbance was measured at 590 nm. Colony formation assay One hundred RT4 and T24 cells were seeded in 6-well plates and incubated with either DMSO or 15d-PGJ2 for 10 days. Next, cells were fixed with 4% paraformaldehyde and stained with Coomassie Brilliant Blue G-250 (Sigma-Aldrich, Burlington, MA, USA) to visualize the colonies. Finally, the colonies in each well were imaged and counted. Total ROS assay RT4 (3 × 10 5 ) and T24 (2 × 10 5 ) cells are cultured in 6-well plates and treated with 15d-PGJ2 and cisplatin for 24 h. Subsequently, ROS staining was performed using the Total ROS Detection Kit (ENZ-51011, Enzo Life Sciences, Plymouth, MA) following the manufacturer's protocol. After incubating for 1 hour, the fluorescence was measured using a BioTek Fluorescence Microplate Reader (Agilent Technologies, Santa Clara, CA, USA). MitoSox Red staining RT4 and T24 cells were cultured on coverslips and treated with DMSO, 10 µM 15d-PGJ2, or a combination of 10 µM 15d-PGJ2 and 5 µM cisplatin for the indicated time periods. Mitochondrial ROS levels were assessed using the mitochondrial-specific superoxide indicator MitoSOX™ Red (Invitrogen, MA, USA). Following treatment, cells were incubated with 1 µM MitoSOX Red for 15 minutes at 37°C to ensure adequate staining. After incubation, cells were washed twice with PBS and analyzed for mitochondrial ROS by using the Attune NxT Flow Cytometer (Thermo Fisher Scientific). MitoTracker staining Mitochondrial length was assessed using MitoTracker staining. Following the completion of the experimental procedures, cells were incubated with 400 nM MitoTracker for 1 hour at 37°C in the dark. Imaging was performed using the Thunder Imaging System (Leica Wetzlar, Germany), and mitochondrial length was quantified using ImageJ software. JC-1 staining Mitochondrial membrane potential (ΔΨm) was evaluated using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-iodocarbocyanine iodide (JC-1, Thermo Fisher). JC-1 exhibits potential-dependent accumulation in mitochondria, shifting from green fluorescence (monomeric form) at low membrane potential to red fluorescence (aggregated form) at high membrane potential. Following treatment, cells were incubated with 2 µM JC-1 for 30 minutes at 37°C in the dark. Fluorescence signals were analyzed using the Thunder Imaging System (Leica Wetzlar, Germany), and quantitative analysis of JC-1 fluorescence was performed by the Attune NxT Flow Cytometer (Thermo Fisher Scientific) to evaluate changes in mitochondrial membrane potential. Adenosine Triphosphate (ATP) Assay Intracellular ATP levels were quantified using a commercially available ATP assay kit (A-22066, Molecular Probes, OR, USA). Following treatment, cells were washed with PBS and incubated with 1 mL of trypsin for 2 minutes. After detachment, cells were collected and lysed in an appropriate volume of RIPA buffer, followed by mixing and incubating for 20 minutes. The lysates were then centrifuged at 16,000 × g for 15 minutes at 4°C. The resulting supernatants were harvested and analyzed to ATP quantification according to the manufacturer’s instructions. Western blotting Cells were lysed in Mammalian Protein Extraction Buffer (GE Healthcare) supplemented with phosphatase and protease inhibitors (MedChemExpress). Proteins were separated on 11% SDS–PAGE gels, transferred to nitrocellulose membranes (Bio-Rad), and blocked with BlockPro™ protein-free blocking buffer (Visual Protein) for 20 min. Membranes were incubated overnight at 4°C with primary antibodies, followed by HRP-conjugated secondary antibodies (1:5000; Cell Signaling Technology) for 1 h at room temperature. Chemiluminescence was detected using an ECL substrate (Bio-Rad) and imaged with an Xplorer system (SPOT Imaging). Band intensities were quantified using ImageJ, normalized to GAPDH, and expressed relative to controls. Antibody details are listed in Supplementary Table 1. Xenograft mouse model All animal experiments were conducted with approval from the Laboratory Animal Center of the National Defense Medical Center, Taiwan. Eight-week-old male BALB/c AnN.Cg-Foxnlnu/CrlNarl nude mice were obtained from the National Laboratory Animal Center (NARLabs, Taipei, Taiwan). During all procedures, mice were anesthetized using a mixture of isoflurane and oxygen. A total of 3 × 10⁶ T24 bladder cancer cells were subcutaneously implanted into the right flank of each mouse. Three weeks after implantation, the mice were randomly divided into four groups (n = 6 per group): control, 2 mg/kg 15d-PGJ2, 2.5 mg/kg cisplatin, and 15d-PGJ2 + cisplatin combination treatment. Both 15d-PGJ2 and Cisplatin were administered via intraperitoneal injection every two days for five weeks. Tumor progression was monitored using the IVIS Spectrum™ in vivo imaging system (PerkinElmer, Waltham, MA, USA) every three days throughout the 5-week treatment. At the experimental endpoint, tumors and organs (heart, liver, lungs, and kidneys) were harvested, fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned at 3 µm thickness. Tissue sections were subjected to hematoxylin and eosin (H&E) staining and immunohistochemical analysis. Tumor tissues were also collected and homogenized for subsequent Western blot analysis. H&E and IHC staining Tumor tissues were excised from xenograft-bearing mice and fixed in 10% formalin solution, followed by paraffin embedding and sectioning into 5 µm thick sections. Hematoxylin and eosin (H&E) staining was conducted for histological analysis. The expression levels of CK7, Tom20, and p-Drp1 in the tumor tissues were determined using immunohistochemical staining. IHC staining was conducted using the Ventana BenchMark ULTRA system (Roche, Basel, Switzerland). The primary antibodies were diluted using the Antibody Dilution Buffer (Ventana). Antigen retrieval was conducted using the manufacturer’s standard protocol. Secondary anti-rabbit IgG (goat anti-rabbit; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used for the experiment. Expression of CK7, Tom20, and p-Drp1 was evaluated, and images were randomly taken from each group. TUNEL assay For assessment of apoptosis in xenograft tumor tissue, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used. Xenograft tumor tissue sections (5 µm) fixed in paraffin were deparaffinized and subjected to TUNEL staining according to the manufacturer’s protocol. The stained sections were observed under a fluorescence microscope after nuclear staining with DAPI. Photomicrographs were taken at ×200 and ×400 magnifications to evaluate apoptotic cell death in each group. Statistical analysis All experiments were conducted with a minimum of five independent replicates. Data are presented as mean ± standard error of the mean (SEM). Statistical comparisons were performed using the Kruskal–Wallis test, followed by the Mann–Whitney U test for post hoc pairwise comparisons. A p-value of less than 0.05 was considered statistically significant. Results High PPARγ expression correlates with poor prognosis and is elevated in bladder cancer tissues To investigate the clinical significance of PPARγ in bladder cancer (BC), we performed survival analysis by utilizing The Cancer Genome Atlas (TCGA) datasets. Kaplan-Meier survival curves indicated that high PPARG expression was significantly associated with better overall survival rate (Fig. 1 A) (p = 0.0023; HR = 0.57), and although the difference in disease-free survival did not reach statistical significance, a similar favorable trend was observed in the high-expression group (Fig. 1 B). Subsequently, the protein expression levels of PPARγ were assessed using Western blotting techniques. The results indicated that PPARγ expression was higher in the low-grade bladder cancer cell line RT4 and in normal bladder epithelial cells (SV-HUC-1) than in the high-grade bladder cancer cell line T24 (Fig. 1 C). PPARγ activation by 15d-PGJ2 reduces cell viability and clonogenic potential in bladder cancer cells In response to these clinical observations, we investigated the effects of PPARγ activation on bladder cancer cell viability. T24 and RT4 cell lines were subjected to treatment with varying concentrations of the endogenous PPARγ ligand, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2; 5, 10, 30 µM) over periods of 24, 48, and 72 hours. MTT assays revealed a significant, dose- and time-dependent decrease in cell viability in both cell lines, with T24 cells exhibiting greater sensitivity across all concentrations, particularly at 48 and 72 hours (Fig. 1 D). Instead, RT4 cells showed a significant reduction in viability only at 72 hours with 30 µM 15d-PGJ2 (Fig. 1 E). 15d-PGJ2 significantly suppressed colony formation in both T24 and RT4 cells, as shown in Fig. 1 F and quantified in Fig. 1 G. These findings indicate that PPARγ activation by 15d-PGJ2 reduces both short-term cell viability and long-term clonogenic capacity in bladder cancer cells. 15d-PGJ2 induces mitochondrial ROS accumulation and disrupts mitochondrial membrane potential in bladder cancer cells To further elucidate the mechanism underlying 15d-PGJ2–induced cytotoxicity, we assessed total ROS, mtROS, and ΔΨm. ROS levels were elevated between 60 and 120 minutes after 15d-PGJ2 treatment, with a peak at 120 minutes in T24 and at 60 minutes RT4 cells. (Fig. 2 A, B). Mitochondrial membrane potential was assessed by JC-1 staining. MitoSOX Red staining revealed increased mitochondrial ROS accumulation in both T24 and RT4 cells at 6 hours and 24 hours (not shown in data) following 15d-PGJ2 treatment (Fig. 2 C). Flow cytometry staining of MitoSOX red quantitative analysis showed comparable elevations in mtROS levels between the two cell lines under the same conditions (Fig. 2 D). Consistent with these observations, Confocal microscopy revealed decreased red (JC-1 aggregates) and increased green (JC-1 monomer) fluorescence in both T24 and RT4 cells following 15d-PGJ2 treatment, consistent with mitochondrial depolarization (Fig. 2 E). Flow cytometry staining of JC-1 quantitative analysis confirmed a significant reduction in the red/green fluorescence ratio after 6 hours (not shown in data) and 24 hours of treatment (Fig. 2 F), indicating progressive disruption of ΔΨm. These findings indicate that 15d-PGJ2 induces mitochondrial oxidative stress and membrane depolarization in bladder cancer cells, suggesting the involvement of mitochondrial dysfunction in its cytotoxic effects. 15d-PGJ2 alters cellular ATP levels and sensitizes bladder cancer cells to cisplatin in association with mitochondrial dysfunction Given the role of mitochondrial function in cellular energy metabolism, we examined the effects of 15d-PGJ2 on ATP levels in bladder cancer cells. In T24 cells, 15d-PGJ2 induced a time-dependent alteration in total ATP levels, with a transient increase at early time points followed by a significant reduction at later time points, whereas ATP levels in RT4 cells remained relatively stable (Fig. 3 A and 3 B). Co-treatment with 15d-PGJ2 and cisplatin significantly reduced cell viability and increased apoptotic cells ratio compared with either agent alone especially in T24 cells (Fig. 3 C and 3 D). Immunoblotting analysis further showed that combination treatment was associated with altered expression of mitochondrial structural and fission-related proteins, including Tom20, MFF (Mitochondrial fission factor), and phosphorylated Drp1 (p-Drp1), along with changes in mitochondrial respiratory proteins COX IV and ATP5A (Fig. 3 E). Enhanced PARP cleavage further supported the induction of apoptotic signaling (Fig. 3 E and 3 F). Collectively, these results indicate that 15d-PGJ2 modulates cellular ATP homeostasis and enhances cisplatin-induced cytotoxicity, accompanied by changes in mitochondrial dynamics and apoptosis in bladder cancer cells. 15d-PGJ2 enhances cisplatin-induced oxidative stress and mitochondrial dysfunction in bladder cancer cells To further elucidate the mitochondrial basis of the enhanced cytotoxicity induced by combined 15d-PGJ2 and cisplatin treatment, mitochondrial ROS production, morphology, and membrane potential were examined in bladder cancer cells. Confocal microscopy showed that either agent alone caused a modest increase in MitoSOX Red fluorescence, whereas combined treatment markedly enhanced mitochondrial ROS accumulation in both T24 and RT4 cells ; In MitoTracker staining, combined treatment markedly reduced mitochondrial distribution especially in T24 cells Consistently ; JC-1 staining demonstrated a decrease in red fluorescence with a concomitant increase in green fluorescence, indicating mitochondrial membrane depolarization (ΔΨm loss) in both cell lines (Fig. 4 A and 4 B). Supporting these observations, quantitative analyses showed transient increases in total ROS at early time points following combination treatment (Fig. 4 C and D). Flow cytometric analysis further confirmed that combined treatment significantly reduced MitoTracker Red positive cells (Fig. 4 E) and decreased the red/green fluorescence ratio of JC-1 staining (Fig. 4 F). Together, these results indicate that 15d-PGJ2 enhances cisplatin-induced mitochondrial oxidative stress, accompanied by mitochondrial fragmentation and membrane depolarization in both T24 and RT4 bladder cancer cells. Inhibition of mitochondrial ROS attenuates 15d-PGJ2-mediated chemo sensitization and mitochondrial injury in bladder cancer cells To determine whether mitochondrial ROS contributes to the chemo sensitizing effect of 15d-PGJ2, we examined whether the antioxidant NAC or the mitochondria-targeted ROS scavenger MitoTEMPO could restore cell viability and mitochondrial integrity. NAC co-treatment partially rescued 15d-PGJ2-induced loss of cell viability in both T24 and RT4 cells, with an approximate recovery of 30% and 10%, respectively (Fig. 5 A), indicating that ROS contributes to 15d-PGJ2-mediated cytotoxicity. Similarly, MitoTEMPO restored cell viability by approximately 25% in T24 cells (Fig. 5 B), suggesting a prominent role for mitochondrial ROS in this response. At the molecular level, MitoTEMPO reversed the 15d-PGJ2-induced downregulation of mitochondrial structural and fission-related proteins, including Tom20, MFF, COX IV, and phosphorylated Drp1 (p-Drp1), in both bladder cancer cell lines (Fig. 5 C). In parallel, PARP cleavage was markedly reduced following MitoTEMPO treatment, indicating attenuation of apoptotic signaling. Quantitative analysis further confirmed that scavenging mitochondrial ROS restored mitochondrial protein expression and reduced cleaved PARP accumulation (Fig. 5 D). Collectively, these findings indicate that mitochondrial ROS plays a critical role in mediating the cytotoxic and chemo sensitizing effects of 15d-PGJ2, and that mitochondrial oxidative stress is closely associated with mitochondrial dysfunction and apoptosis in bladder cancer cells. 15d-PGJ2 enhances cisplatin antitumor efficacy in vivo through mitochondrial disruption To further validate the chemo sensitizing potential of 15d-PGJ2 in vivo, a T24 subcutaneous xenograft model was established and treated with vehicle, 15d-PGJ2, cisplatin, or their combination. One week after tumor implantation, mice were randomized into four treatment groups, and tumor growth was longitudinally monitored using an IVIS imaging system for up to three weeks (Fig. 6 A). Combination treatment significantly suppressed tumor growth, as evidenced by a marked reduction in bioluminescent signal compared with vehicle or single-agent-treated groups (Fig. 6 B, D, and E). Importantly, no significant differences in body weight were observed among the treatment groups, indicating the absence of overt systemic toxicity (Fig. 6 C). Consistent with the imaging results, tumors harvested from the combination-treated group were significantly smaller than those from control or monotherapy groups (Fig. 7 A). Histopathological analysis using H&E staining further revealed extensive tumor degeneration and reduced cellularity in tumors from the combination cohort (Fig. 7 B). TUNEL assay revealed a progressive increase in apoptotic cells across the experimental groups. At both ×200 and ×400 magnifications, the high-signal group showed more abundant TUNEL positive cells than the control and single treatments groups, indicating increased apoptosis under the experimental condition (Fig. 7 C). At the molecular level, tumor lysates from combination-treated mice showed reduced Drp1 phosphorylation and decreased expression of mitochondrial marker COX IV, compared with tumors from control groups (Fig. 7 D, 7 E). Immunohistochemical analysis showed that bladder cancer cells specific antibody CK7 expression was reduced in the combination groups compared with the control and single treatments groups (Fig. 8 A). Tom20 expression showed a similar pattern, being markedly decreased in the 15dPGJ2 and combination groups, whereas relatively stronger staining was retained in the cisplatin group (Fig. 8 B). In addition, p-Drp1 staining was attenuated particularly in the combination group, compared with single treatments groups (Fig. 8 C). The above findings suggest that 15dPGJ2, especially in combination with cisplatin, suppresses tumor marker expression and is associated with reduced mitochondrial/fission-related signaling in vivo. Collectively, these results demonstrate that 15d-PGJ2 enhances the antitumor efficacy of cisplatin in vivo, accompanied by alterations in mitochondrial markers and Drp1-associated stress responses, without apparent systemic toxicity, thereby supporting the mitochondria-associated chemosensitizing effects observed in vitro. Discussion PPARγ has recently come to be as a context-dependent modulator of urothelial carcinomas[18–20]. Despite the growing evidence for its role in urothelial carcinomas, the precise functional and therapeutic implications of this pathway continue to be incompletely understood. In our research, we combined correlative studies with multiple layers of functional analysis to clarify a mitochondria-centric pathway by 15d-PGJ2. We accomplished on the basis of the following three main findings. First, there was a favorable correlation between PPARγ and overall survival in bladder cancers. This may indicate that the marker of this pathway represents a lower hazard form of the disease process. Second, there was a reduction in the clonogenic and viability effects mediated by 15d-PGJ2 in T24 and RT4 cells. Third and most importantly 15d-PGJ2 cooperated with cisplatin to enhance tumor cell apoptosis and inhibit tumor growth in vivo. A striking feature of our data is the heterogeneity in the levels of PPARγ expression across urothelial models. RT4 cells expressed higher PPARγ protein than T24, while SV-HUC-1 expressed intermediate levels. This pattern is in broad agreement with the idea that PPARγ is enriched in more differentiated, luminal-like urothelial carcinoma, while more aggressive phenotypes may downregulate PPARγ programs [20]. Considering that 15d-PGJ2 can act both as a PPARγ ligand and as an electrophilic lipid mediator capable of modifying cellular redox circuits [21], our findings are consistent with the possibility that both PPARγ-mediated transcription and receptor-independent redox effects contribute to the observed phenotypes. Mechanistically, our findings suggested that a rapid accumulation of oxidative stress with a pronounced mitochondrial involvement. 15d-PGJ2 increased total ROS in 30–120 min and increased mitochondrial superoxide signals detected by MitoSOX Red staining. Concomitantly, JC-1 assays demonstrated a loss of mitochondrial membrane potential consistent with the acute disruption of mitochondrial bioenergetics. These early shifts in redox status and bioenergetics were followed by a drop in ATP levels with a series of mitochondrial dynamics proteins change, Tom20, MFF, as well as activated Drp1. In addition to changes in respiratory chain and ATP synthase subunits, COX IV and ATP5A. Notably, the mitochondria-associated cytotoxic phenotypes observed in this study are consistent with previous reports demonstrating that activation of PPARγ or treatment with its ligands suppresses bladder cancer cell proliferation and induces programmed cell death. Specifically, PPARγ agonists such as rosiglitazone and pioglitazone have been shown to exert antiproliferative and pro-apoptotic effects in bladder cancer models, accompanied by significant tumor growth suppression in vivo [22, 23]. In addition, troglitazone has been reported to induce apoptosis and other forms of programmed cell death in urothelial and bladder cancer models[24]. Collectively, these findings support a conserved role of PPARγ activation in mediating mitochondrial dysfunction and cytotoxicity in urothelial malignancies, in agreement with our current observations [24, 25]. The above data suggest a temporal cascade in which 15d-PGJ2 triggers mitochondrial ROS accumulation, mitochondrial membrane potential loss, energetic failure, and ultimately activation of PARP-mediated apoptosis through cleavage. In collaborative therapy, our studies indicate that 15d-PGJ2 also makes the lower apoptotic threshold to cisplatin. Cisplatin is recognized to cause DNA damage and could secondarily enhance ROS production and mitochondrial injury [26]. In T24 and RT4 cells, a combined treatment with 15d-PGJ2 and cisplatin showed a dramatic synergistic effect in reducing viability and increasing the number of apoptotic cells compared to each agent alone. Moreover, both mitochondrial stress and mitochondria membrane potential were further changed in cells that received a combination of agents. Interestingly, using MitoSOX Red, MitoTracker Red, and JC-1 staining, imaging also indicated that mitochondrial injury was further exacerbated in combination-treated groups. Most importantly, the in vivo experiments were also shown to be relevant, bioluminescent signals were most reduced in groups that received a combination treatment, with no obvious body-weight loss. Further pharmacological development to mitigate toxicity could be needed, but a 15d-PGJ2-mediated mitochondrial injury apparently holds a high potential to serve as a cisplatin sensitizer in urothelial carcinoma. Notably, the application of rescue strategies to support causality is essential to our study design. The non-specific antioxidant NAC and mito-specific antioxidant Mito-TEMPO partially reversed cellular viability and changes in mitochondrial protein levels caused by 15d-PGJ2 supplementation. The ability of a mitochondria-targeted ROS to rescue the phenotype supports the idea that mitoROS is an upstream driver of apoptosis and loss of viability. This is crucial because redox signals frequently appear as secondary effects in apoptosis models [27], and an antioxidant rescue does not automatically establish a direct causal link to the apoptotic or bioenergetic outcomes. Several limitations should be acknowledged. First, although our data support a mitochondria-centric and potentially PPARγ-independent mechanism, we did not genetically validate this using PPARγ knockdown or knockout approaches. However, pharmacological inhibition of PPARγ with GW9662 in T24 and RT4 cells partially restored cell viability and colony formation and increased the expression of proliferation- and mitochondria-related proteins, including Ki67, Tom20, and COX IV (Supplementary Fig. X), suggesting partial involvement of PPARγ signaling. Second, while this study employed extensive mitochondrial assessments, including ROS production, mitochondrial membrane potential (ΔΨm), ATP levels, and multiple mitochondrial markers, further evaluation of mitochondrial bioenergetics, such as electron transport chain capacity using Seahorse analysis, would strengthen the mechanistic interpretation. Third, in vivo validation was limited to a single subcutaneous xenograft model. Future studies should incorporate additional models, including cisplatin-resistant or orthotopic bladder cancer models, and include comprehensive toxicity assessments, particularly renal function and hematological parameters. Conclusion Our study identifies 15d-PGJ2 as a potent inducer of mitochondrial ROS–associated mitochondrial dysfunction in urothelial carcinoma cells and demonstrates that engagement of this mitochondrial stress response enhances tumor sensitivity to cisplatin. These findings link clinical associations of PPARγ expression to a functionally actionable mitochondrial vulnerability and support the further development of mitochondrial redox modulation as a therapeutic strategy for bladder cancer. Declarations Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work was supported by Tri-Service General Hospital (TSGH_D_115094), and the Teh-Tzer Study Group for Human Medical Research Foundation (B1141017 and B1151012). Author Contribution Chien-Rui Lai: Investigation, Writing – original draft, Methodology, Ying Chen: Validation, Visualization, Writing – original draft, Writing – review & editing. Tzu-Min Chen: Data curation, Investigation, Validation. Yu-Ling Tsai: Investigation. Wen-Chiuan Tsai: Investigation. 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Frontiers in physiology 2021, 12 :640374. Clay CE, Namen AM, Atsumi G-i, Willingham MC, High KP, Kute TE, Trimboli AJ, Fonteh AN, Dawson PA, Chilton FH: Influence of J series prostaglandins on apoptosis and tumorigenesis of breast cancer cells . Carcinogenesis 1999, 20 (10):1905-1911. Shin S-W, Seo C-Y, Han H, Han J-Y, Jeong J-S, Kwak J-Y, Park J-I: 15d-PGJ2 induces apoptosis by reactive oxygen species–mediated inactivation of Akt in leukemia and colorectal cancer cells and shows in vivo antitumor activity . Clinical Cancer Research 2009, 15 (17):5414-5425. Slanovc J, Mikulčić M, Jahn N, Wizsy NGT, Sattler W, Malle E, Hrzenjak A: Prostaglandin 15d-PGJ2 inhibits proliferation of lung adenocarcinoma cells by inducing ROS production and activation of apoptosis via sirtuin-1 . Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 2024, 1870 (1):166924. Kaikkonen S, Paakinaho V, Sutinen P, Levonen A-L, Palvimo JJ: Prostaglandin 15d-PGJ2 inhibits androgen receptor signaling in prostate cancer cells . Molecular Endocrinology 2013, 27 (2):212-223. Sanchez DJ, Missiaen R, Skuli N, Steger DJ, Simon MC: Cell-intrinsic tumorigenic functions of PPARγ in bladder urothelial carcinoma . Molecular Cancer Research 2021, 19 (4):598-611. Tate T, Xiang T, Wobker SE, Zhou M, Chen X, Kim H, Batourina E, Lin C-S, Kim WY, Lu C: Pparg signaling controls bladder cancer subtype and immune exclusion . Nature communications 2021, 12 (1):6160. Peng T, Wang G, Cheng S, Xiong Y, Cao R, Qian K, Ju L, Wang X, Xiao Y: The role and function of PPARγ in bladder cancer . Journal of Cancer 2020, 11 (13):3965. Surh YJ, Na HK, Park JM, Lee HN, Kim W, Yoon IS, Kim DD: 15-Deoxy-Δ¹²,¹⁴-prostaglandin J₂, an electrophilic lipid mediator of anti-inflammatory and pro-resolving signaling . Biochem Pharmacol 2011, 82 (10):1335-1351. Xu X, Wang J, Jiang H, Meng L, Lang B: Rosiglitazone induces apoptosis on human bladder cancer 5637 and T24 cell lines . International Journal of Clinical and Experimental Pathology 2017, 10 (10):10197. Mansure JJ, Nassim R, Kassouf W: Peroxisome proliferator-activated receptor γ in bladder cancer: a promising therapeutic target . Cancer biology & therapy 2009, 8 (7):575-584. Yan S, Yang X, Chen T, Xi Z, Jiang X: The PPARγ agonist Troglitazone induces autophagy, apoptosis and necroptosis in bladder cancer cells . Cancer Gene Therapy 2014, 21 (5):188-193. Lv S, Wang W, Wang H, Zhu Y, Lei C: PPARγ activation serves as therapeutic strategy against bladder cancer via inhibiting PI3K-Akt signaling pathway . BMC cancer 2019, 19 (1):204. Tang C, Livingston MJ, Safirstein R, Dong Z: Cisplatin nephrotoxicity: new insights and therapeutic implications . Nature Reviews Nephrology 2023, 19 (1):53-72. Circu ML, Aw TY: Reactive oxygen species, cellular redox systems, and apoptosis . Free radical biology and medicine 2010, 48 (6):749-762. Additional Declarations No competing interests reported. Supplementary Files FigureLegend.jpg Figure Legend Schematic model showing the proposed mechanism of the inhibitory effect of 15dPGJ2 on the growth of bladder cancer. In vitro, 15dPGJ2 has antiproliferative activity, causing cell viability loss, as well as mitochondrial dysfunction, as indicated by increased levels of mitochondrial ROS, loss of membrane potential, reduced expression of mitochondrial markers including phospho-DRP1, Tom20, and COX IV, as well as increased levels of PARP cleavage and apoptosis. In vivo, 15dPGJ2 alone or in combination with cisplatin inhibits T24 xenograft growth, as well as the expression of mitochondrial markers. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 18 May, 2026 Reviews received at journal 26 Apr, 2026 Reviews received at journal 22 Apr, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviewers agreed at journal 14 Apr, 2026 Reviewers agreed at journal 13 Apr, 2026 Reviewers invited by journal 13 Apr, 2026 Editor assigned by journal 21 Mar, 2026 Submission checks completed at journal 21 Mar, 2026 First submitted to journal 16 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9134319","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626491273,"identity":"f94bd1fd-8637-4c15-bbaa-043f927177b0","order_by":0,"name":"Chien-Rui Lai","email":"","orcid":"","institution":"National Defense Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chien-Rui","middleName":"","lastName":"Lai","suffix":""},{"id":626491275,"identity":"c8ae823d-1c04-482a-ae3b-e5aaae0036a5","order_by":1,"name":"Ying Chen","email":"","orcid":"","institution":"National Defense Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Chen","suffix":""},{"id":626491277,"identity":"ca0496f3-629d-4f87-9243-3eb45a77274f","order_by":2,"name":"Tzu-Min Chen","email":"","orcid":"","institution":"National Defense Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tzu-Min","middleName":"","lastName":"Chen","suffix":""},{"id":626491278,"identity":"96428823-7c60-44b6-a0d2-766a9ba3f7e7","order_by":3,"name":"Yu-Ling Tsai","email":"","orcid":"","institution":"Tri-Service General Hospital and National Defense Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yu-Ling","middleName":"","lastName":"Tsai","suffix":""},{"id":626491280,"identity":"12b3d993-0dff-40e9-9da2-7f8838b7b95b","order_by":4,"name":"Wen-Chiuan Tsai","email":"","orcid":"","institution":"Tri-Service General Hospital and National Defense Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wen-Chiuan","middleName":"","lastName":"Tsai","suffix":""},{"id":626491282,"identity":"f405e6b4-7b79-4861-a9a3-44a63ad57580","order_by":5,"name":"Sheng-Tang Wu","email":"","orcid":"","institution":"Tri-Service General Hospital and National Defense Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sheng-Tang","middleName":"","lastName":"Wu","suffix":""},{"id":626491283,"identity":"87d47535-e2b2-48ca-93df-117e1570b8e5","order_by":6,"name":"Chien-Chang Kao","email":"data:image/png;base64,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","orcid":"","institution":"Tri-Service General Hospital and National Defense Medical University","correspondingAuthor":true,"prefix":"","firstName":"Chien-Chang","middleName":"","lastName":"Kao","suffix":""}],"badges":[],"createdAt":"2026-03-16 07:24:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9134319/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9134319/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107519780,"identity":"f54cef7d-49c6-4310-a6a6-9eb4838374b6","added_by":"auto","created_at":"2026-04-22 08:57:53","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":216101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElevated PPARγ expression predicts poor prognosis and sensitizes bladder cancer cells to 15d-PGJ2-induced cytotoxicity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Kaplan-Meier survival analysis of bladder cancer patients stratified by PPARγ expression levels (TCGA dataset). (B) Kaplan–Meier analysis of disease-free survival according to PPARG expression status. (C) The expression levels of PPARγ in bladder cancer cell lines were assessed using Western blot analysis, with GAPDH serving as a loading control. A statistically significant difference was observed, with p \u0026lt; 0.05 when compared to the RT4 group. (D–E) T24 and RT4 bladder cancer cells were treated with increasing concentrations of 15d-PGJ2 (1, 5, 10, 30 μM) for 24, 48, and 72 hours. Cell viability was assessed by MTT assay, and relative viability is presented. Data represent mean ± SEM from n = 6 independent experiments. (F) Representative images of colony formation assays in T24 and RT4 cells treated with 15d-PGJ2 (10 μM) and vehicle control (CTL). Cells were allowed to form colonies over 10 days, followed by crystal violet staining. (G) Quantification of relative colony numbers in T24 and RT4 cells following 15d-PGJ2treatment. Data are expressed as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Dunnett’s test, ***p \u0026lt; 0.001 versus CTL.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9134319/v1/40e7e4f16b4e402d21d2a903.jpg"},{"id":107519924,"identity":"c1466188-8f24-484f-8554-17a73c160327","added_by":"auto","created_at":"2026-04-22 08:58:08","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":239423,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e15d-PGJ2 induces mitochondrial ROS accumulation and disrupts mitochondrial membrane potential in bladder cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A–B) T24 and RT4 cells were treated with 15d-PGJ2 (10 μM) for the indicated time points (30–240 min), and total ROS levels were quantified. Data represent mean ± SEM from n = 6 independent experiments. (C) Representative confocal microscopy images of JC-1-stained T24 and RT4 cells treated with 15d-PGJ2 (10 μM) for 6 h. Scale bar = 50 μm. (D) Quantification of JC-1 green fluorescence ratio in T24 and RT4 cells following 15d-PGJ2 treatment at 6 h. (E) Representative immunofluorescence images of MitoSOX Red staining in T24 and RT4 cells after 24 h treatment with 15d-PGJ2 (30 μM). Nuclei were counterstained with DAPI. Scale bar = 50 μm. (F) Quantification of MitoSOX Red fluorescence intensity in T24 and RT4 cells. Data are expressed as mean ± SEM from n = 6 independent experiments. Statistical significance was determined by one-way ANOVA test; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus CTL.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9134319/v1/9e82a78ec7ffa946568a0972.jpg"},{"id":107519779,"identity":"9372c7fe-9007-49c4-a77a-a4a2d3cd640e","added_by":"auto","created_at":"2026-04-22 08:57:53","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":228151,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e15dPGJ2 enhances cisplatin-induced cytotoxicity, mitochondrial dysfunction, and apoptosis\u003c/strong\u003e\u003cbr\u003e\n (A, B) Total intracellular ATP levels in T24 (A) and RT4 (B) cells treated with 15dPGJ2 for the indicated time periods. ATP levels were progressively reduced after treatment, especially in T24 cells. (C) Cell viability of T24 and RT4 cells treated with vehicle, 15dPGJ2, cisplatin, or the combination of 15dPGJ2 and cisplatin were measured by MTT assay. \u0026nbsp;(D) Quantification of apoptotic cells after treatment with vehicle, 15dPGJ2, cisplatin, or the combination, showing enhanced apoptosis in the combination group. (E) Representative immunoblots of mitochondrial dynamics- and apoptosis-related proteins, including Tom20, MFF, phospho-DRP1, COX IV, ATP5A, and PARP, in T24 and RT4 cells under the indicated treatment conditions. GAPDH served as the loading control. (F) Densitometric quantification of the immunoblots shown in (E). Data represent mean ± SEM from n = 6 independent experiments. Statistical significance is indicated in each panel. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus CTL. #p \u0026lt; 0.05, ##p \u0026lt; 0.01 versus 15dPGJ2.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9134319/v1/d57d7c83b71759d991c81193.jpg"},{"id":107519927,"identity":"a61ae0ea-2eef-4ec8-8153-e52bd2d1a154","added_by":"auto","created_at":"2026-04-22 08:58:09","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":225447,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCombined 15dPGJ2 and cisplatin treatment exacerbates oxidative stress and mitochondrial injury in bladder cancer cells.\u003c/strong\u003e\u003cbr\u003e\n (A,B) Representative fluorescence images of T24 (A) and RT4 (B) cells treated with vehicle, 15dPGJ2, cisplatin, or the combination, followed by staining with MitoSOX Red, MitoTracker, or JC-1, as indicated. Scale bar = 50µm. (C, D) Time-course quantification of total ROS levels in T24 (C) and RT4 (D) cells after treatment with vehicle, 15dPGJ2, cisplatin, or the combination. (E)Flow cytometry of MitoTracker Red-positive signal was performed to evaluate mitochondrial signal intensity after combination treatment. (F) Flow cytometry of the JC-1 red/green fluorescence ratio was performed to evaluate mitochondrial membrane potential. Data represent mean ± SEM from n = 6 independent experiments. Statistical significance is indicated in each panel. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus CTL. #p \u0026lt; 0.05, ##p \u0026lt; 0.01 ###p \u0026lt; 0.001 versus 15dPGJ2.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9134319/v1/143e81e72e1d7e773ddb14b0.jpg"},{"id":107519772,"identity":"5c50c494-4d8e-4064-85c9-37c1c469bc8e","added_by":"auto","created_at":"2026-04-22 08:57:53","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":204459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntioxidants attenuate 15dPGJ2-induced cytotoxicity and restore mitochondrial proteins. \u003c/strong\u003e\u003cbr\u003e\n (A) Cell viability of T24 and RT4 cells treated with 15dPGJ2 in the presence or absence of the ROS scavenger N-acetyl-L-cysteine (NAC). (B) Cell viability of T24 and RT4 cells treated with 15dPGJ2 in the presence or absence of the mitochondria-targeted antioxidant MitoTempo. (C) Representative immunoblots of Tom20, phospho-DRP1, MFF, COX IV, ATP5A, and PARP in T24 and RT4 cells treated with 15dPGJ2 and/or MitoTempo. GAPDH was used as the loading control. (D) Densitometric quantification of the immunoblots shown in (C). Data represent mean ± SEM from n = 6 independent experiments. Statistical significance is indicated in each panel. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus CTL. #p \u0026lt; 0.05, ##p \u0026lt; 0.01 ###p \u0026lt; 0.001 versus 15dPGJ2.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9134319/v1/40dd2f807b7f4778dd317f3d.jpg"},{"id":107519981,"identity":"00a1b992-6542-4e92-a388-31958e0b1be6","added_by":"auto","created_at":"2026-04-22 08:58:18","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":191038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vivo study design and antitumor efficacy by IVIS imaging\u003c/strong\u003e\u003cbr\u003e\n (A) Schematic overview of the in vivo xenograft study. T24 cells were subcutaneously injected, mice were randomized into control, 15dPGJ2, cisplatin, and combination groups, and tumor burden was monitored serially by IVIS imaging during treatment. Tumors were harvested at the experimental endpoint for downstream analyses.\u003cbr\u003e\n(B) Representative IVIS bioluminescence images from each treatment group at baseline for reduced tumor signal in the 15dPGJ2, cisplatin, and combination groups. (C) Body weight curves during treatment, indicating the general tolerability of the treatment regimens. (D) Longitudinal quantification of bioluminescence signal intensity over time. (E, F) Scatter plots showing tumor bioluminescence signal at baseline day 0 (E) and at the endpoint day 21(F). Data represent mean ± SEM from n = 8 independent experiments. Statistical significance is indicated in each panel. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus CTL. #p \u0026lt; 0.05, ##p \u0026lt; 0.01 ###p \u0026lt; 0.001 versus 15dPGJ2.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9134319/v1/381d9b46599c1e8ff6cc3163.jpg"},{"id":107519982,"identity":"de4b352c-c7d6-45c5-b959-b7b8e99b96e2","added_by":"auto","created_at":"2026-04-22 08:58:18","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":385233,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEx vivo tumor analyses confirm enhanced tumor suppression and apoptosis\u003c/strong\u003e\u003cbr\u003e\n (A) Representative images of excised tumors from each treatment group at the study endpoint. (B) Representative hematoxylin and eosin (H\u0026amp;E) staining of tumor sections at 100X and 200X magnification, showing histopathologic changes across treatment groups. (C) Representative TUNEL staining of tumor sections at 200X and 400X magnification, demonstrating increased apoptotic cell death in treated tumors, particularly in the combination group. (D) Representative immunoblots of phospho-DRP1, Tom20, and COX IV in tumor lysates from the indicated treatment groups, with GAPDH as the loading control. (E) Densitometric quantification of the immunoblots shown in (D). Treatment decreased the expression of mitochondrial proteins and phospho-DRP1 in vivo. Data represent mean ± SEM from n = 8 independent experiments. Statistical significance is indicated in each panel. ***p \u0026lt; 0.001 versus CTL.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9134319/v1/48e86a2538ee0acc4e134a5c.jpg"},{"id":107520022,"identity":"4c4a0d10-beff-4ae2-badd-281602ee97f5","added_by":"auto","created_at":"2026-04-22 08:58:21","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":417430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIHC validation of tumor identity and mitochondrial changes in vivo\u003c/strong\u003e\u003cbr\u003e\n (A) Representative immunohistochemical staining for CK7 in xenograft tumors from the control, 15dPGJ2, cisplatin, and combination groups at low and high magnification. CK7 staining confirmed the urothelial phenotype of the tumor tissues. (B) Representative immunohistochemical staining for Tom20 in tumor sections from the indicated groups. (C) Representative immunohistochemical staining for phospho-DRP1 in tumor sections from the indicated groups. Scale bars were indicated at 100X and 200X magnification.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9134319/v1/c7275f6083cbcc057c84edd1.jpg"},{"id":107520218,"identity":"c0b06f0f-c8dd-40c3-a7a6-7bf8ae229cdb","added_by":"auto","created_at":"2026-04-22 08:59:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3490872,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9134319/v1/e3d11b7d-43d8-4254-a136-9575d4608503.pdf"},{"id":107519771,"identity":"bb74cf90-5e48-44df-90a4-17a682e53d1a","added_by":"auto","created_at":"2026-04-22 08:57:51","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":97430,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure Legend\u003c/strong\u003e\u003cbr\u003e\n Schematic model showing the proposed mechanism of the inhibitory effect of 15dPGJ2 on the growth of bladder cancer. In vitro, 15dPGJ2 has antiproliferative activity, causing cell viability loss, as well as mitochondrial dysfunction, as indicated by increased levels of mitochondrial ROS, loss of membrane potential, reduced expression of mitochondrial markers including phospho-DRP1, Tom20, and COX IV, as well as increased levels of PARP cleavage and apoptosis. In vivo, 15dPGJ2 alone or in combination with cisplatin inhibits T24 xenograft growth, as well as the expression of mitochondrial markers.\u003c/p\u003e","description":"","filename":"FigureLegend.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9134319/v1/f8d65fc67bd04e440a1ab7c9.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"15d-PGJ2 enhances cisplatin cytotoxicity via mitochondrial ROS-mediated cell death in human bladder cancer","fulltext":[{"header":"Background","content":"\u003cp\u003eUrothelial carcinoma represents one of the most frequently diagnosed malignancies of the urinary system and remains a substantial global public health concern. In Western countries, bladder cancer (BC) ranks as the fourth most common cancer among men, accounting for approximately 6% of newly diagnosed malignancies in 2023 [1, 2], and contributes to 4% of cancer-related mortality in this population, with a median age at diagnosis of approximately 70 years [3]. At initial presentation, 70\u0026ndash;75% of patients exhibit non-muscle-invasive bladder cancer (NMIBC), 20\u0026ndash;25% present with muscle-invasive bladder cancer (MIBC), and approximately 5% are diagnosed with metastatic disease [4]. NMIBC is characterized by a high recurrence rate and heterogeneous progression risk, with five-year recurrence and progression rates ranging from one-third to three-fourths and 1% to 45%, respectively [5]. Despite definitive surgical management, approximately 30% of patients experience disease recurrence, and prognosis remains poor in those with distant metastases, reflected in persistently low five-year overall survival rates [6].\u003c/p\u003e \u003cp\u003eTherapeutically, NMIBC continues to present considerable challenges due to its propensity for recurrence and progression. Bacillus Calmette-Gu\u0026eacute;rin (BCG) immunotherapy remains the mainstay for intermediate- and high-risk disease; however, a significant proportion of patients ultimately develop BCG-unresponsive tumors for which no universally effective therapy is available [7, 8]. Recently, intravesical gene therapy, immune checkpoint inhibitors [9], and novel drug delivery platforms have shown promise in this context, yet long-term outcomes remain under investigation [10]. Similarly, despite advancements in surgical techniques and perioperative care, MIBC and advanced disease continue to confer poor survival outcomes [11]. Collectively, these challenges underscore the urgent need for innovative therapeutic strategies and precision medicine approaches to improve outcomes across the bladder cancer disease spectrum.\u003c/p\u003e \u003cp\u003eAmong the emerging molecular strategies aimed at addressing tumor progression and therapeutic resistance, bioactive lipid mediators, particularly 15-deoxy-Δ\u0026sup1;\u0026sup2;,\u0026sup1;⁴-prostaglandin J₂ (15d-PGJ2), a natural ligand for peroxisome proliferator-activated receptor gamma (PPARγ) [12], have attracted significant attention. 15d-PGJ2 is known to exert a range of biological effects, including anti-inflammatory, pro-apoptotic, and anti-proliferative activities [13]. A growing body of evidence suggests that 15d-PGJ2 influences various signaling pathways associated with tumorigenesis, such as the inhibition of NF-κB, the induction of oxidative stress, and the regulation of mitochondrial function. Its capacity to induce apoptosis, autophagy, and cell cycle arrest has been documented in multiple cancer types, including breast, lung, prostate, and colorectal cancers [14\u0026ndash;17]. Although these findings are encouraging, the therapeutic role and mechanistic actions of 15d-PGJ2 in bladder cancer have yet to be fully elucidated. Given the urgent need for novel treatment strategies, particularly for cisplatin-resistant and advanced-stage urothelial carcinoma, elucidating the antitumor mechanisms of 15d-PGJ2 represents a critical direction for future research.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eTwo urothelial carcinoma cell lines (RT4 and T24), which exhibit distinct epithelial morphologies, and a normal human uroepithelial cell line (SV‑HUC‑1) were sourced from the Bioresource Collection and Research Center (BCRC) in Taiwan. RT4 and T24 cells were propagated in McCoy\u0026rsquo;s 5A medium supplemented with 1% sodium pyruvate, 1% L‑glutamine (Corning, NY, USA), and 10% fetal bovine serum (FBS; Thermo Fisher Scientific, MA, USA). SV‑HUC‑1 cells were maintained in Ham\u0026rsquo;s F‑12 medium (Sigma‑Aldrich, St. Louis, MO), also supplemented with 1% sodium pyruvate, 1% L‑glutamine, and 7% FBS. All cultures were incubated at 37\u0026deg;C in a humidified atmosphere with 5% CO₂.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDrugs and reagents\u003c/h3\u003e\n\u003cp\u003eDimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), coomassie brilliant blue G-250 were purchased from Sigma\u0026ndash;Aldrich (St. Louis, MO, USA). 15-Deoxy-Δ-12,14-prostaglandin J\u003csub\u003e2\u003c/sub\u003e (15d-PGJ2), cisplatin, N-acetylcysteine (NAC) and Mito-TEMPO were purchased from MedChemExpress (Princeton, NJ, USA).\u003c/p\u003e\n\u003ch3\u003eMTT assay\u003c/h3\u003e\n\u003cp\u003eRT4 and T24 cells were seeded in 96-well plates at densities of 5 \u0026times; 10\u0026sup3; and 2 \u0026times; 10\u0026sup3; cells separately per well. After the treatments, MTT solution (0.5 mg/ml) was added to each well and incubated for 3 hours. The formazan crystals formed were then dissolved in DMSO, and the absorbance was measured at 590 nm.\u003c/p\u003e\n\u003ch3\u003eColony formation assay\u003c/h3\u003e\n\u003cp\u003eOne hundred RT4 and T24 cells were seeded in 6-well plates and incubated with either DMSO or 15d-PGJ2 for 10 days. Next, cells were fixed with 4% paraformaldehyde and stained with Coomassie Brilliant Blue G-250 (Sigma-Aldrich, Burlington, MA, USA) to visualize the colonies. Finally, the colonies in each well were imaged and counted.\u003c/p\u003e\n\u003ch3\u003eTotal ROS assay\u003c/h3\u003e\n\u003cp\u003eRT4 (3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) and T24 (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e) cells are cultured in 6-well plates and treated with 15d-PGJ2 and cisplatin for 24 h. Subsequently, ROS staining was performed using the Total ROS Detection Kit (ENZ-51011, Enzo Life Sciences, Plymouth, MA) following the manufacturer's protocol. After incubating for 1 hour, the fluorescence was measured using a BioTek Fluorescence Microplate Reader (Agilent Technologies, Santa Clara, CA, USA).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMitoSox Red staining\u003c/h2\u003e \u003cp\u003eRT4 and T24 cells were cultured on coverslips and treated with DMSO, 10 \u0026micro;M 15d-PGJ2, or a combination of 10 \u0026micro;M 15d-PGJ2 and 5 \u0026micro;M cisplatin for the indicated time periods. Mitochondrial ROS levels were assessed using the mitochondrial-specific superoxide indicator MitoSOX\u0026trade; Red (Invitrogen, MA, USA). Following treatment, cells were incubated with 1 \u0026micro;M MitoSOX Red for 15 minutes at 37\u0026deg;C to ensure adequate staining. After incubation, cells were washed twice with PBS and analyzed for mitochondrial ROS by using the Attune NxT Flow Cytometer (Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMitoTracker staining\u003c/h3\u003e\n\u003cp\u003eMitochondrial length was assessed using MitoTracker staining. Following the completion of the experimental procedures, cells were incubated with 400 nM MitoTracker for 1 hour at 37\u0026deg;C in the dark. Imaging was performed using the Thunder Imaging System (Leica Wetzlar, Germany), and mitochondrial length was quantified using ImageJ software.\u003c/p\u003e\n\u003ch3\u003eJC-1 staining\u003c/h3\u003e\n\u003cp\u003eMitochondrial membrane potential (ΔΨm) was evaluated using 5,5\u0026prime;,6,6\u0026prime;-tetrachloro-1,1\u0026prime;,3,3\u0026prime;-tetraethylbenzimidazolyl-iodocarbocyanine iodide (JC-1, Thermo Fisher). JC-1 exhibits potential-dependent accumulation in mitochondria, shifting from green fluorescence (monomeric form) at low membrane potential to red fluorescence (aggregated form) at high membrane potential. Following treatment, cells were incubated with 2 \u0026micro;M JC-1 for 30 minutes at 37\u0026deg;C in the dark. Fluorescence signals were analyzed using the Thunder Imaging System (Leica Wetzlar, Germany), and quantitative analysis of JC-1 fluorescence was performed by the Attune NxT Flow Cytometer (Thermo Fisher Scientific) to evaluate changes in mitochondrial membrane potential.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAdenosine Triphosphate (ATP) Assay\u003c/h2\u003e \u003cp\u003eIntracellular ATP levels were quantified using a commercially available ATP assay kit (A-22066, Molecular Probes, OR, USA). Following treatment, cells were washed with PBS and incubated with 1 mL of trypsin for 2 minutes. After detachment, cells were collected and lysed in an appropriate volume of RIPA buffer, followed by mixing and incubating for 20 minutes. The lysates were then centrifuged at 16,000 \u0026times; g for 15 minutes at 4\u0026deg;C. The resulting supernatants were harvested and analyzed to ATP quantification according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eCells were lysed in Mammalian Protein Extraction Buffer (GE Healthcare) supplemented with phosphatase and protease inhibitors (MedChemExpress). Proteins were separated on 11% SDS\u0026ndash;PAGE gels, transferred to nitrocellulose membranes (Bio-Rad), and blocked with BlockPro\u0026trade; protein-free blocking buffer (Visual Protein) for 20 min. Membranes were incubated overnight at 4\u0026deg;C with primary antibodies, followed by HRP-conjugated secondary antibodies (1:5000; Cell Signaling Technology) for 1 h at room temperature. Chemiluminescence was detected using an ECL substrate (Bio-Rad) and imaged with an Xplorer system (SPOT Imaging). Band intensities were quantified using ImageJ, normalized to GAPDH, and expressed relative to controls. Antibody details are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eXenograft mouse model\u003c/h2\u003e \u003cp\u003e All animal experiments were conducted with approval from the Laboratory Animal Center of the National Defense Medical Center, Taiwan. Eight-week-old male BALB/c AnN.Cg-Foxnlnu/CrlNarl nude mice were obtained from the National Laboratory Animal Center (NARLabs, Taipei, Taiwan). During all procedures, mice were anesthetized using a mixture of isoflurane and oxygen. A total of 3 \u0026times; 10⁶ T24 bladder cancer cells were subcutaneously implanted into the right flank of each mouse. Three weeks after implantation, the mice were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;6 per group): control, 2 mg/kg 15d-PGJ2, 2.5 mg/kg cisplatin, and 15d-PGJ2\u0026thinsp;+\u0026thinsp;cisplatin combination treatment. Both 15d-PGJ2 and Cisplatin were administered via intraperitoneal injection every two days for five weeks. Tumor progression was monitored using the IVIS Spectrum\u0026trade; in vivo imaging system (PerkinElmer, Waltham, MA, USA) every three days throughout the 5-week treatment. At the experimental endpoint, tumors and organs (heart, liver, lungs, and kidneys) were harvested, fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned at 3 \u0026micro;m thickness. Tissue sections were subjected to hematoxylin and eosin (H\u0026amp;E) staining and immunohistochemical analysis. Tumor tissues were also collected and homogenized for subsequent Western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eH\u0026amp;E and IHC staining\u003c/h2\u003e \u003cp\u003eTumor tissues were excised from xenograft-bearing mice and fixed in 10% formalin solution, followed by paraffin embedding and sectioning into 5 \u0026micro;m thick sections. Hematoxylin and eosin (H\u0026amp;E) staining was conducted for histological analysis. The expression levels of CK7, Tom20, and p-Drp1 in the tumor tissues were determined using immunohistochemical staining. IHC staining was conducted using the Ventana BenchMark ULTRA system (Roche, Basel, Switzerland). The primary antibodies were diluted using the Antibody Dilution Buffer (Ventana). Antigen retrieval was conducted using the manufacturer\u0026rsquo;s standard protocol. Secondary anti-rabbit IgG (goat anti-rabbit; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used for the experiment. Expression of CK7, Tom20, and p-Drp1 was evaluated, and images were randomly taken from each group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTUNEL assay\u003c/h2\u003e \u003cp\u003eFor assessment of apoptosis in xenograft tumor tissue, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used. Xenograft tumor tissue sections (5 \u0026micro;m) fixed in paraffin were deparaffinized and subjected to TUNEL staining according to the manufacturer\u0026rsquo;s protocol. The stained sections were observed under a fluorescence microscope after nuclear staining with DAPI. Photomicrographs were taken at \u0026times;200 and \u0026times;400 magnifications to evaluate apoptotic cell death in each group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were conducted with a minimum of five independent replicates. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical comparisons were performed using the Kruskal\u0026ndash;Wallis test, followed by the Mann\u0026ndash;Whitney U test for post hoc pairwise comparisons. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eHigh PPARγ expression correlates with poor prognosis and is elevated in bladder cancer tissues\u003c/h2\u003e \u003cp\u003eTo investigate the clinical significance of PPARγ in bladder cancer (BC), we performed survival analysis by utilizing The Cancer Genome Atlas (TCGA) datasets. Kaplan-Meier survival curves indicated that high PPARG expression was significantly associated with better overall survival rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) (p\u0026thinsp;=\u0026thinsp;0.0023; HR\u0026thinsp;=\u0026thinsp;0.57), and although the difference in disease-free survival did not reach statistical significance, a similar favorable trend was observed in the high-expression group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Subsequently, the protein expression levels of PPARγ were assessed using Western blotting techniques. The results indicated that PPARγ expression was higher in the low-grade bladder cancer cell line RT4 and in normal bladder epithelial cells (SV-HUC-1) than in the high-grade bladder cancer cell line T24 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePPARγ activation by 15d-PGJ2 reduces cell viability and clonogenic potential in bladder cancer cells\u003c/h2\u003e \u003cp\u003eIn response to these clinical observations, we investigated the effects of PPARγ activation on bladder cancer cell viability. T24 and RT4 cell lines were subjected to treatment with varying concentrations of the endogenous PPARγ ligand, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2; 5, 10, 30 \u0026micro;M) over periods of 24, 48, and 72 hours. MTT assays revealed a significant, dose- and time-dependent decrease in cell viability in both cell lines, with T24 cells exhibiting greater sensitivity across all concentrations, particularly at 48 and 72 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Instead, RT4 cells showed a significant reduction in viability only at 72 hours with 30 \u0026micro;M 15d-PGJ2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). 15d-PGJ2 significantly suppressed colony formation in both T24 and RT4 cells, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF and quantified in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG. These findings indicate that PPARγ activation by 15d-PGJ2 reduces both short-term cell viability and long-term clonogenic capacity in bladder cancer cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003e15d-PGJ2 induces mitochondrial ROS accumulation and disrupts mitochondrial membrane potential in bladder cancer cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further elucidate the mechanism underlying 15d-PGJ2\u0026ndash;induced cytotoxicity, we assessed total ROS, mtROS, and ΔΨm. ROS levels were elevated between 60 and 120 minutes after 15d-PGJ2 treatment, with a peak at 120 minutes in T24 and at 60 minutes RT4 cells. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Mitochondrial membrane potential was assessed by JC-1 staining. MitoSOX Red staining revealed increased mitochondrial ROS accumulation in both T24 and RT4 cells at 6 hours and 24 hours (not shown in data) following 15d-PGJ2 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Flow cytometry staining of MitoSOX red quantitative analysis showed comparable elevations in mtROS levels between the two cell lines under the same conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsistent with these observations, Confocal microscopy revealed decreased red (JC-1 aggregates) and increased green (JC-1 monomer) fluorescence in both T24 and RT4 cells following 15d-PGJ2 treatment, consistent with mitochondrial depolarization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Flow cytometry staining of JC-1 quantitative analysis confirmed a significant reduction in the red/green fluorescence ratio after 6 hours (not shown in data) and 24 hours of treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), indicating progressive disruption of ΔΨm.\u003c/p\u003e \u003cp\u003eThese findings indicate that 15d-PGJ2 induces mitochondrial oxidative stress and membrane depolarization in bladder cancer cells, suggesting the involvement of mitochondrial dysfunction in its cytotoxic effects.\u003c/p\u003e \u003cp\u003e \u003cb\u003e15d-PGJ2 alters cellular ATP levels and sensitizes bladder cancer cells to cisplatin in association with mitochondrial dysfunction\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGiven the role of mitochondrial function in cellular energy metabolism, we examined the effects of 15d-PGJ2 on ATP levels in bladder cancer cells. In T24 cells, 15d-PGJ2 induced a time-dependent alteration in total ATP levels, with a transient increase at early time points followed by a significant reduction at later time points, whereas ATP levels in RT4 cells remained relatively stable (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Co-treatment with 15d-PGJ2 and cisplatin significantly reduced cell viability and increased apoptotic cells ratio compared with either agent alone especially in T24 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Immunoblotting analysis further showed that combination treatment was associated with altered expression of mitochondrial structural and fission-related proteins, including Tom20, MFF (Mitochondrial fission factor), and phosphorylated Drp1 (p-Drp1), along with changes in mitochondrial respiratory proteins COX IV and ATP5A (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Enhanced PARP cleavage further supported the induction of apoptotic signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Collectively, these results indicate that 15d-PGJ2 modulates cellular ATP homeostasis and enhances cisplatin-induced cytotoxicity, accompanied by changes in mitochondrial dynamics and apoptosis in bladder cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e15d-PGJ2 enhances cisplatin-induced oxidative stress and mitochondrial dysfunction in bladder cancer cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further elucidate the mitochondrial basis of the enhanced cytotoxicity induced by combined 15d-PGJ2 and cisplatin treatment, mitochondrial ROS production, morphology, and membrane potential were examined in bladder cancer cells. Confocal microscopy showed that either agent alone caused a modest increase in MitoSOX Red fluorescence, whereas combined treatment markedly enhanced mitochondrial ROS accumulation in both T24 and RT4 cells ; In MitoTracker staining, combined treatment markedly reduced mitochondrial distribution especially in T24 cells Consistently ; JC-1 staining demonstrated a decrease in red fluorescence with a concomitant increase in green fluorescence, indicating mitochondrial membrane depolarization (ΔΨm loss) in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Supporting these observations, quantitative analyses showed transient increases in total ROS at early time points following combination treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and D). Flow cytometric analysis further confirmed that combined treatment significantly reduced MitoTracker Red positive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE) and decreased the red/green fluorescence ratio of JC-1 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Together, these results indicate that 15d-PGJ2 enhances cisplatin-induced mitochondrial oxidative stress, accompanied by mitochondrial fragmentation and membrane depolarization in both T24 and RT4 bladder cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of mitochondrial ROS attenuates 15d-PGJ2-mediated chemo sensitization and mitochondrial injury in bladder cancer cells\u003c/h2\u003e \u003cp\u003eTo determine whether mitochondrial ROS contributes to the chemo sensitizing effect of 15d-PGJ2, we examined whether the antioxidant NAC or the mitochondria-targeted ROS scavenger MitoTEMPO could restore cell viability and mitochondrial integrity. NAC co-treatment partially rescued 15d-PGJ2-induced loss of cell viability in both T24 and RT4 cells, with an approximate recovery of 30% and 10%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), indicating that ROS contributes to 15d-PGJ2-mediated cytotoxicity. Similarly, MitoTEMPO restored cell viability by approximately 25% in T24 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), suggesting a prominent role for mitochondrial ROS in this response. At the molecular level, MitoTEMPO reversed the 15d-PGJ2-induced downregulation of mitochondrial structural and fission-related proteins, including Tom20, MFF, COX IV, and phosphorylated Drp1 (p-Drp1), in both bladder cancer cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In parallel, PARP cleavage was markedly reduced following MitoTEMPO treatment, indicating attenuation of apoptotic signaling. Quantitative analysis further confirmed that scavenging mitochondrial ROS restored mitochondrial protein expression and reduced cleaved PARP accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Collectively, these findings indicate that mitochondrial ROS plays a critical role in mediating the cytotoxic and chemo sensitizing effects of 15d-PGJ2, and that mitochondrial oxidative stress is closely associated with mitochondrial dysfunction and apoptosis in bladder cancer cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e15d-PGJ2 enhances cisplatin antitumor efficacy in vivo through mitochondrial disruption\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further validate the chemo sensitizing potential of 15d-PGJ2 in vivo, a T24 subcutaneous xenograft model was established and treated with vehicle, 15d-PGJ2, cisplatin, or their combination. One week after tumor implantation, mice were randomized into four treatment groups, and tumor growth was longitudinally monitored using an IVIS imaging system for up to three weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Combination treatment significantly suppressed tumor growth, as evidenced by a marked reduction in bioluminescent signal compared with vehicle or single-agent-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, D, and E). Importantly, no significant differences in body weight were observed among the treatment groups, indicating the absence of overt systemic toxicity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsistent with the imaging results, tumors harvested from the combination-treated group were significantly smaller than those from control or monotherapy groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Histopathological analysis using H\u0026amp;E staining further revealed extensive tumor degeneration and reduced cellularity in tumors from the combination cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). TUNEL assay revealed a progressive increase in apoptotic cells across the experimental groups. At both \u0026times;200 and \u0026times;400 magnifications, the high-signal group showed more abundant TUNEL positive cells than the control and single treatments groups, indicating increased apoptosis under the experimental condition (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). At the molecular level, tumor lysates from combination-treated mice showed reduced Drp1 phosphorylation and decreased expression of mitochondrial marker COX IV, compared with tumors from control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Immunohistochemical analysis showed that bladder cancer cells specific antibody CK7 expression was reduced in the combination groups compared with the control and single treatments groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Tom20 expression showed a similar pattern, being markedly decreased in the 15dPGJ2 and combination groups, whereas relatively stronger staining was retained in the cisplatin group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). In addition, p-Drp1 staining was attenuated particularly in the combination group, compared with single treatments groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). The above findings suggest that 15dPGJ2, especially in combination with cisplatin, suppresses tumor marker expression and is associated with reduced mitochondrial/fission-related signaling in vivo.\u003c/p\u003e \u003cp\u003eCollectively, these results demonstrate that 15d-PGJ2 enhances the antitumor efficacy of cisplatin in vivo, accompanied by alterations in mitochondrial markers and Drp1-associated stress responses, without apparent systemic toxicity, thereby supporting the mitochondria-associated chemosensitizing effects observed in vitro.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePPARγ has recently come to be as a context-dependent modulator of urothelial carcinomas[18\u0026ndash;20]. Despite the growing evidence for its role in urothelial carcinomas, the precise functional and therapeutic implications of this pathway continue to be incompletely understood. In our research, we combined correlative studies with multiple layers of functional analysis to clarify a mitochondria-centric pathway by 15d-PGJ2. We accomplished on the basis of the following three main findings. First, there was a favorable correlation between PPARγ and overall survival in bladder cancers. This may indicate that the marker of this pathway represents a lower hazard form of the disease process. Second, there was a reduction in the clonogenic and viability effects mediated by 15d-PGJ2 in T24 and RT4 cells. Third and most importantly 15d-PGJ2 cooperated with cisplatin to enhance tumor cell apoptosis and inhibit tumor growth in vivo.\u003c/p\u003e \u003cp\u003eA striking feature of our data is the heterogeneity in the levels of PPARγ expression across urothelial models. RT4 cells expressed higher PPARγ protein than T24, while SV-HUC-1 expressed intermediate levels. This pattern is in broad agreement with the idea that PPARγ is enriched in more differentiated, luminal-like urothelial carcinoma, while more aggressive phenotypes may downregulate PPARγ programs [20]. Considering that 15d-PGJ2 can act both as a PPARγ ligand and as an electrophilic lipid mediator capable of modifying cellular redox circuits [21], our findings are consistent with the possibility that both PPARγ-mediated transcription and receptor-independent redox effects contribute to the observed phenotypes.\u003c/p\u003e \u003cp\u003eMechanistically, our findings suggested that a rapid accumulation of oxidative stress with a pronounced mitochondrial involvement. 15d-PGJ2 increased total ROS in 30\u0026ndash;120 min and increased mitochondrial superoxide signals detected by MitoSOX Red staining. Concomitantly, JC-1 assays demonstrated a loss of mitochondrial membrane potential consistent with the acute disruption of mitochondrial bioenergetics. These early shifts in redox status and bioenergetics were followed by a drop in ATP levels with a series of mitochondrial dynamics proteins change, Tom20, MFF, as well as activated Drp1. In addition to changes in respiratory chain and ATP synthase subunits, COX IV and ATP5A. Notably, the mitochondria-associated cytotoxic phenotypes observed in this study are consistent with previous reports demonstrating that activation of PPARγ or treatment with its ligands suppresses bladder cancer cell proliferation and induces programmed cell death. Specifically, PPARγ agonists such as rosiglitazone and pioglitazone have been shown to exert antiproliferative and pro-apoptotic effects in bladder cancer models, accompanied by significant tumor growth suppression in vivo [22, 23]. In addition, troglitazone has been reported to induce apoptosis and other forms of programmed cell death in urothelial and bladder cancer models[24]. Collectively, these findings support a conserved role of PPARγ activation in mediating mitochondrial dysfunction and cytotoxicity in urothelial malignancies, in agreement with our current observations [24, 25]. The above data suggest a temporal cascade in which 15d-PGJ2 triggers mitochondrial ROS accumulation, mitochondrial membrane potential loss, energetic failure, and ultimately activation of PARP-mediated apoptosis through cleavage.\u003c/p\u003e \u003cp\u003eIn collaborative therapy, our studies indicate that 15d-PGJ2 also makes the lower apoptotic threshold to cisplatin. Cisplatin is recognized to cause DNA damage and could secondarily enhance ROS production and mitochondrial injury [26]. In T24 and RT4 cells, a combined treatment with 15d-PGJ2 and cisplatin showed a dramatic synergistic effect in reducing viability and increasing the number of apoptotic cells compared to each agent alone. Moreover, both mitochondrial stress and mitochondria membrane potential were further changed in cells that received a combination of agents. Interestingly, using MitoSOX Red, MitoTracker Red, and JC-1 staining, imaging also indicated that mitochondrial injury was further exacerbated in combination-treated groups. Most importantly, the in vivo experiments were also shown to be relevant, bioluminescent signals were most reduced in groups that received a combination treatment, with no obvious body-weight loss. Further pharmacological development to mitigate toxicity could be needed, but a 15d-PGJ2-mediated mitochondrial injury apparently holds a high potential to serve as a cisplatin sensitizer in urothelial carcinoma.\u003c/p\u003e \u003cp\u003eNotably, the application of rescue strategies to support causality is essential to our study design. The non-specific antioxidant NAC and mito-specific antioxidant Mito-TEMPO partially reversed cellular viability and changes in mitochondrial protein levels caused by 15d-PGJ2 supplementation. The ability of a mitochondria-targeted ROS to rescue the phenotype supports the idea that mitoROS is an upstream driver of apoptosis and loss of viability. This is crucial because redox signals frequently appear as secondary effects in apoptosis models [27], and an antioxidant rescue does not automatically establish a direct causal link to the apoptotic or bioenergetic outcomes.\u003c/p\u003e \u003cp\u003eSeveral limitations should be acknowledged. First, although our data support a mitochondria-centric and potentially PPARγ-independent mechanism, we did not genetically validate this using PPARγ knockdown or knockout approaches. However, pharmacological inhibition of PPARγ with GW9662 in T24 and RT4 cells partially restored cell viability and colony formation and increased the expression of proliferation- and mitochondria-related proteins, including Ki67, Tom20, and COX IV (Supplementary Fig. X), suggesting partial involvement of PPARγ signaling. Second, while this study employed extensive mitochondrial assessments, including ROS production, mitochondrial membrane potential (ΔΨm), ATP levels, and multiple mitochondrial markers, further evaluation of mitochondrial bioenergetics, such as electron transport chain capacity using Seahorse analysis, would strengthen the mechanistic interpretation. Third, in vivo validation was limited to a single subcutaneous xenograft model. Future studies should incorporate additional models, including cisplatin-resistant or orthotopic bladder cancer models, and include comprehensive toxicity assessments, particularly renal function and hematological parameters.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study identifies 15d-PGJ2 as a potent inducer of mitochondrial ROS\u0026ndash;associated mitochondrial dysfunction in urothelial carcinoma cells and demonstrates that engagement of this mitochondrial stress response enhances tumor sensitivity to cisplatin. These findings link clinical associations of PPARγ expression to a functionally actionable mitochondrial vulnerability and support the further development of mitochondrial redox modulation as a therapeutic strategy for bladder cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eDeclaration of Competing Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by Tri-Service General Hospital (TSGH_D_115094), and the Teh-Tzer Study Group for Human Medical Research Foundation (B1141017 and B1151012).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eChien-Rui Lai: Investigation, Writing \u0026ndash; original draft, Methodology, Ying Chen: Validation, Visualization, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing. Tzu-Min Chen: Data curation, Investigation, Validation. Yu-Ling Tsai: Investigation. Wen-Chiuan Tsai: Investigation. Sheng-Tang Wu: Supervision, Validation, Visualization, Writing \u0026ndash; review \u0026amp; editing. Chien-Chang Kao: Funding acquisition, Supervision.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNierengarten MB: \u003cstrong\u003eCancer Statistics 2024: Deaths drop, incidences increase, prevention needed\u003c/strong\u003e. 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in bladder cancer: a promising therapeutic target\u003c/strong\u003e. \u003cem\u003eCancer biology \u0026amp; therapy \u003c/em\u003e2009, \u003cstrong\u003e8\u003c/strong\u003e(7):575-584.\u003c/li\u003e\n\u003cli\u003eYan S, Yang X, Chen T, Xi Z, Jiang X: \u003cstrong\u003eThe PPAR\u0026gamma; agonist Troglitazone induces autophagy, apoptosis and necroptosis in bladder cancer cells\u003c/strong\u003e. \u003cem\u003eCancer Gene Therapy \u003c/em\u003e2014, \u003cstrong\u003e21\u003c/strong\u003e(5):188-193.\u003c/li\u003e\n\u003cli\u003eLv S, Wang W, Wang H, Zhu Y, Lei C: \u003cstrong\u003ePPAR\u0026gamma; activation serves as therapeutic strategy against bladder cancer via inhibiting PI3K-Akt signaling pathway\u003c/strong\u003e. \u003cem\u003eBMC cancer \u003c/em\u003e2019, \u003cstrong\u003e19\u003c/strong\u003e(1):204.\u003c/li\u003e\n\u003cli\u003eTang C, Livingston MJ, Safirstein R, Dong Z: \u003cstrong\u003eCisplatin nephrotoxicity: new insights and therapeutic implications\u003c/strong\u003e. \u003cem\u003eNature Reviews Nephrology \u003c/em\u003e2023, \u003cstrong\u003e19\u003c/strong\u003e(1):53-72.\u003c/li\u003e\n\u003cli\u003eCircu ML, Aw TY: \u003cstrong\u003eReactive oxygen species, cellular redox systems, and apoptosis\u003c/strong\u003e. \u003cem\u003eFree radical biology and medicine \u003c/em\u003e2010, \u003cstrong\u003e48\u003c/strong\u003e(6):749-762.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"PPARγ, 15d-PGJ2, cisplatin, mitochondria, ROS, bladder cancer, apoptosis, mitochondria membrane potential","lastPublishedDoi":"10.21203/rs.3.rs-9134319/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9134319/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eBladder cancer (BC) is a common urologic malignancy with high recurrence and progression rates, and outcomes for advanced disease remain poor. Peroxisome proliferator-activated receptor gamma (PPARγ) is involved in tumor metabolism and progression, but its role in BC is not fully defined.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe investigated the effects of 15dPGJ2, alone or in combination with cisplatin, on bladder cancer cells using a series of functional assays in T24 and RT4 cell lines and further validated the antitumor effects in a xenograft mouse model.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAnalysis of The Cancer Genome Atlas (TCGA) dataset revealed that higher PPARγ expression is associated with improved overall survival in BC patients. Treatment with 15-deoxy-Δ\u0026sup1;\u0026sup2;,\u0026sup1;⁴-prostaglandin J₂ (15d-PGJ2), a natural PPARγ ligand, significantly suppressed cell viability and colony formation in T24 and RT4 bladder cancer cells. Mechanistically, 15d-PGJ2 induced mitochondrial oxidative stress, as demonstrated by increased mitochondrial reactive oxygen species (ROS), elevated JC-1 green/red fluorescence ratios, and mitochondrial depolarization and fragmentation. These changes were accompanied by reduced mitochondrial length and downregulation of mitochondrial dynamics and respiratory proteins, including Tom20, MFF, COX IV, and ATP5A, along with increased p-Drp1 and cleaved PARP expression. Combined treatment with cisplatin and 15d-PGJ2 further enhanced mitochondrial dysfunction and ROS production, resulting in synergistic inhibition of cell viability. In a T24 xenograft mouse model, 15d-PGJ2 alone or in combination with cisplatin significantly reduced tumor growth, as confirmed by IVIS imaging, histological analysis, and immunohistochemistry, with tumor tissues showing increased apoptosis and suppression of mitochondrial-related proteins.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003e15d-PGJ2 inhibits bladder cancer growth by inducing mitochondrial oxidative stress and dysfunction and represents a promising mitochondrial-targeting chemosensitizer for bladder cancer therapy.\u003c/p\u003e","manuscriptTitle":"15d-PGJ2 enhances cisplatin cytotoxicity via mitochondrial ROS-mediated cell death in human bladder cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-22 08:55:42","doi":"10.21203/rs.3.rs-9134319/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-19T03:05:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-26T05:01:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-22T15:29:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"251898808422856707788734052269420434127","date":"2026-04-20T16:38:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"262592205017668045143910807332025295106","date":"2026-04-14T07:24:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"124768398698253611670998669665911988635","date":"2026-04-14T03:58:15+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-14T03:10:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-21T11:07:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-21T11:06:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Cell International","date":"2026-03-16T07:19:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f2acccd0-ee0d-42bb-a14f-6a5e551346d8","owner":[],"postedDate":"April 22nd, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-19T03:05:31+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T03:10:14+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-22 08:55:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9134319","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9134319","identity":"rs-9134319","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}