Empagliflozin enhances cisplatin activity in chemo-resistant EJ138 bladder cancer cells: The importance of anti-diabetic medications in cancer treatment

Empagliflozin enhances cisplatin activity in chemo-resistant EJ138 bladder cancer cells: The importance of anti-diabetic medications in cancer treatment | 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 Empagliflozin enhances cisplatin activity in chemo-resistant EJ138 bladder cancer cells: The importance of anti-diabetic medications in cancer treatment Saeedeh Shariati, Shokooh Mohtadi, Shahrzad Molavinia, Maryam Salehcheh, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4634713/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Anti-diabetic medications has been found to reduce chemotherapy resistance. This study sought to investigate the role of Empagliflozin (Empa) as an anti-diabetic medication in reversing Cisplatin (Cis) resistance in EJ138 bladder cancer (BC) cells. Materials and Methods The EJ138 cell line was cultured and divided into four groups: control, Cis-treated, Empa-treated, and Cis + Empa-treated groups. The effects of Cis and/or Empa on cell viability were determined using the MTT technique. The level of ROS produced by cells was evaluated using the green fluorescent dye dichloro-dihydro fluorescein (DCF). The expression of proteins involved in glucose transport, proliferation, apoptosis, cell cycle control, and invasion was evaluated by Western blotting. The Data were analyzed using GraphPad prism software and a One-way ANOVA test. All experiments were repeated three times. Data were presented as Mean ± SEM. The significant difference between groups was calculated based on P < 0.05. Results IC50 was calculated equal to 16 mM for Cis and 72 µg/ml for Empa. Treatment with Cis caused a significant increase in SGLT2 expression (p < 0.001). Conversely, the group treated with 72 µg/ml Empa showed a significant decrease in SGLT2 compared with the control group (P < 0.001). ROS generation was significantly elevated after treatment with Cis, Empa, and their combination (P < 0.001). Treatment with Cis and/or Empa downregulated AKT, PI3K, mTOR, Bax, MMP-2, and MMP-9 expression (P < 0.001). However, Bcl2, P21, and P53 expression showed a significant increase following Cis and/or Empa treatment (P < 0.001). Protein expression differed significantly across the Cis-treated group and all other groups. Conclusion Empa exhibits beneficial anti-cancer activity against EJ138 cells. Empa boosts the anti-cancer activity of Cis in EJ138 BC cancer cells through SGLT2 inhibition. Empagliflozin Cisplatin Bladder cancer Chemotherapy Resistance Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Bladder cancer (BC) accounts for 3% of worldwide cancer diagnoses [ 1 ]. BC is one of the most prevalent cancers among the elderly [ 2 ]. The development of BC is often associated with certain risk factors such as smoking, exposure to certain chemicals, chronic bladder infections, and a family history of the disease [ 3 ]. Symptoms of BC may include blood in the urine, frequent urination, pain during urination, and lower back pain [ 4 ]. Early detection and prompt treatment can improve the prognosis and outcome for individuals with bladder cancer [ 5 ]. In BC, alterations in the PI3K/Akt/mTOR pathway are commonly observed [ 6 ]. The dysregulated PI3K/Akt/mTOR pathway has implications for cancer cell proliferation, invasion, metastasis, and resistance to therapy [ 7 ]. Cisplatin (Cis) is a chemotherapy drug commonly used in the treatment of various types of cancer [ 8 ]. Cis works by interfering with the DNA in cancer cells, preventing their ability to divide and grow [ 9 ]. Depending on the particular cancer type and stage, Cis may be used alone or in conjunction with other chemotherapy medications [ 10 ]. Treatment of BC is significantly hampered by resistance to Cis-based chemotherapy [ 9 ]. In this context, several strategies are being investigated to improve Cis's effectiveness [ 11 ]. Previous research demonstrated sodium-glucose co-transporter 2 (SGLT2) upregulation among Cis-resistance hepatocarcinoma cells [ 12 ]. It has been proposed that SGLT2 inhibitors reduce cancer cells' chemotherapy resistance by restricting glucose uptake. Fujiyoshi et al employed Dapagliflozin, an SGLT2 inhibitor, to overcome Cis resistance in hepatoblastoma cells [ 12 ]. Several combination treatments are being researched to counteract the resistance of BC cells to Cis [ 13 – 15 ]. Empagliflozin (Empa) is frequently given medication for the treatment of type 2 diabetes mellitus as an SGLT2 inhibitor [ 16 ]. It has been demonstrated that Empa has anticancer effects in cervical, breast, and hepatic cancer cells. Empa targets the kidney's SGLT2 protein to prevent glucose reabsorption and promote glucose excretion through urine. Research shows that SGLT2 is expressed in pancreatic, prostate, and glioblastoma cancer cells. Generally, cancer cells are reprogrammed to express additional glucose transporters to facilitate glucose influx into the cytoplasm. Glucose influx into cancer cells activates the β-catenin-Wnt pathway, leading to cyclin D1 and TRPC6 transcription and cell proliferation. SGLT inhibitors reduce the progression of cancer by inhibiting the activation of β-catenin. Currently, researchers are investigating the idea of utilizing specific SGLT-2 inhibitors to decrease glucose uptake by cancer cells. However, the impact of Empa on BC cells has not yet been investigated. Previous studies show a link between diabetes and BC development. Therefore, exploring the dual use of Empa for the concurrent treatment of diabetes and BC is an attractive category. The current study evaluated the therapeutic efficacy of Empa and Cis for EJ138 BCs. Material and Methods Cell Culture Protocol This experimental study was approved by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (Ethical code: IR.AJUMS.REC.1400.460). The BC cell line EJ138 (C429) was purchased from the Pasteur Institute Resource Center (Iran). EJ138 cells were cultured in RPMI medium (Sigma, USA) containing 20% FBS (Sigma, USA) and 1% penicillin/streptomycin (Sigma, USA). The cells were incubated at 37°C with 5% CO2. The cells were passaged every two days. MTT Assay The cells were cultured at 104 cells/well density in a 96-well culture plate and incubated at 37°C with 5% CO2 for 24 h. Five wells were allocated Within each experimental group. The cells were treated with varying concentrations of Cis (0, 6.25, 12.25, 25, 50, and 100 mM) (Sigma, USA) and Empa (0, 6.25, 12.25, 25, 50, and 100 µg/ml) (Sigma, USA) [ 17 ]. Afterward, the culture medium was replaced with 100 µL of 0.5 mg/mL MTT (Sigma, USA) solution, and the plates were incubated for 4 h at 37°C with 5% CO2 in the dark. Then each well received 100 µL of Dimethyl sulfoxide (DMSO) (Sigma, USA), and the plates were shaken for 15 min. The absorbance of the samples was measured at 570 nm using an ELISA reader (Bio-Rad, USA). The viability of the cells was calculated using the formula: 100-(absorbance test/absorbance control) × 100 [ 18 ]. The IC50 values were calculated using GraphPad Prism software (GraphPad Software, USA). Study Design The cancer cells were categorized into four groups. Group I (control group) consisted of EJ138 cells that received no treatment. Group II was EJ138 cells that received Cis. Group III consisted of EJ138 cells that received Empa. Group IV consisted of EJ138 cells treated with a combination of Cis and Empa. All groups were incubated at 37°C with 5% CO2 for 24 h. Western blot The cells were harvested and lysed using SDS lysis buffer (Sigma, USA), followed by centrifugation to separate the cellular components. The proteins were then solubilized in an SDS-PAGE loading buffer and subjected to denaturation by heating at 95°C for 10 minutes. Subsequently, the denatured protein samples were transferred onto a nitrocellulose membrane (Sigma, USA). To minimize non-specific binding, the membrane was blocked using a solution comprising 5% nonfat milk (Sigma, USA) in Tris-buffered saline (TBS) (Sigma, USA) supplemented with 0.05% Tween 20 (Sigma, USA). Following blocking, the membrane was incubated for 2 h at room temperature with primary antibodies specific to SGLT2 (sc-393350) (SANTA CRUZ, USA), PI3K (ab302958), AKT (ab38449), mTOR (ab134903), p21 (ab109520), p53 (ab32049), MMP-2 (ab92536), MMP-9 (ab76003), BAX (ab32503), and Bcl-2 (ab182585) (Abcam, USA) diluted 1:500 in blocking solution. The membranes were washed three times with Tris-buffered saline with Tween 20. The suitable secondary antibody (NB500-420, Novus Biologicals, USA) coupled to horseradish peroxidase and diluted 1:1000 in blocking solution was incubated on the membranes for two hours at room temperature. Once three washes were completed, protein reactivity was evaluated with an ECL detection kit (ParsTous, Iran). GAPDH was used to normalize the protein loading (D16H11; Cell Signaling Technology, USA). The CLIQS 1D program (TotallLab, UK) was used to analyze optical density [ 19 ]. Measuring reactive oxygen species (ROS) The levels of ROS production by the cells were determined using the Oxi SelectTM assay kit based on green fluorescent dye dichloro-dihydro fluorescein (DCF) production. The cells were grown on 96-well plates in triplicate. The cells were incubated for 24 h at 37°C. Afterward, the supernatant was discarded, and 100 µL of DA-DCFH 1X was added. After 30 min incubation at 37°C and three times washing, 100 µL of culture medium and 100 µL of lysing buffer were added and the plates were incubated for 5 min at 37°C. The amount of fluorescence was measured at 530 nm using a Dako flow cytometer (Troy, USA). Data were analyzed using Flow Jo software (Flow Jo, USA) [ 20 ]. Statistical analysis The statistical analysis was conducted using GraphPad Prism 9 and Excel software. All experiments were performed in triplicate, ensuring the robustness and reliability of the results. To assess the differences between multiple means, a one-way analysis of variance (ANOVA) followed by Tukey's post hoc analysis was employed. All experimental data were presented as the mean ± SD. A statistically significant difference between distinct experimental groups was indicated as follows: #,*p < 0.05, ##, **p < 0.01, ###, *** p < 0.001, **** p < 0.0001. Results Combining Cisplatin with Empagliflozin resulted in reduced cell viability EJ138 BC cells were treated with Cis, Empa, or a combination of Cis and Empa for 24 hours. The vitality of EJ138 cells correlated inversely with the concentrations of Cis and Empa. The IC50 values were determined to be 16 mM for the Cis-treated group and 72 µg/ml for the Empa-treated group (Fig. 1 ). The impact of Empa and/or on SGLT2 expression SGLT2 enhances glucose re-absorption and tumour cell growth. According to Western Blot results, Cis-therapy showed a direct positive influence on SGLT2 expression. The expression of SGLT2 showed a significant decrease following Empa treatment (P < 0.001). Empa attenuated the effect of Cis on SGLT2 expression (P < 0.001) (Fig. 2 ). The effects of Cisplatin and/or Empagliflozin on the proliferation-dependent pathway AKT, PI3K, and mTOR promote cell growth, proliferation, and survival. According to Western b138lot results, the expression of AKT, PI3K, and mTOR was down-regulated by Cis and Empa administration. The groups treated with Cis, Empa or a combination of Cis and Empa showed significantly decreased levels of AKT, PI3K, and mTOR compared to the control group (P < 0.001 for all groups). However, the group treated with a combination of Cis and Empa had the lowest level of AKT, PI3K, and mTOR expression. The difference between Cis and Cis + Empa groups was statistically significant (P < 0.001) (Fig. 3 ). The effects of Cisplatin and/or Empagliflozin on cell cycle regulator proteins P53 regulates downstream proteins such as p21 to promote cell cycle arrest, apoptosis, and DNA repair. The results of the present study show an increase in p53 and p21 expression following exposure to Cis, Empa or a combination of Cis and Empa, compared to the control group. However, the difference in p21 expression between the Empa group and the control group was not significant (P < 0.001, P = 0.002, P < 0.001, for p53) and (P < 0.001, P = 0.88, P < 0.001 for p21). EJ138 cells treated with a combination of Cis and Empa presented statistically increased levels of p53 and p21 compared to the group treated with Cis alone (P < 0.001) (Fig. 4 ). The effects of Cis and/or Empa on the expression of apoptotic-related proteins The EJ138 cells subjected to treatment with Cis, Empa, or their combination revealed notable alterations in the expression of key apoptosis-regulating genes. Specifically, the groups treated with Cis or Empa alone showed a significant decrease in the expression of prominent anti-apoptotic protein Bcl2, coupled with a marked increase in the expression of pro-apoptotic protein Bax (P < 0.001 for all groups). The group that received a combination of Cis and Empa showed a great increase in Bax and a significant decrease in Bcl2 expression compared to Cis group (P < 0.001) (Fig. 5 ). The influence of Cis and/or Empa on ROS production The outcomes demonstrated that oxidative stress caused by Cis, Empa, and their combined treatment had a discernible cytotoxic effect on BC cells. The groups treated with Cis, Empa, or a combination of Cis and Empa, exhibited a substantial increase in ROS generation compared to the control group (P < 0.001 for all groups). The combination of Cis and Empa significantly increased the formation of ROS Compared to the administration of Cis alone (P < 0.001) (Fig. 6 ). The effects of Cis and/or Empa on invasion-related protein expression To investigate the potential of each treatment on EJ138 cancer cell invasion and metastasis, the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) proteins was investigated. There was a decrease in MMP-2 and MMP-9 in the groups treated with Cis, Empa, or a combination of Cis and Empa, compared to the control group (P < 0.001, P = 0.52, P < 0.001 for MMP-2) and (P < 0.001, P = 0.04, P < 0.001 for MMP-9). The group received a combination of Cis and Empa revealed a decrease in MMP-2 and MMP-9 expression compared to the groups that were treated with Cis alone (P = 0.99, P = 0.02) (Fig. 7 ). Discussion In this study, chemotherapy-resistant EJ138 BC cells revealed an unavoidable increase in SGLT2 expression. However, Empa therapy attenuated the Cis effect on SGLT2 expression. Concurrent treatment with Cis and Empa led to upregulation of Bax, P21, and P53 expression and enhanced ROS activity in EJ138 cells. The treatment resulted in downregulation of Bcl2 expression along with a reduction in the PI3K/AKT/mTOR and MMP-2/MMP-9 pathways activity. Also, the use of Cis and Empa strengthened the Cis anticancer effect. Consequently, this combined treatment effectively inhibited the proliferation and invasion of BC cells. Cisplatin has been shown to affect several critical biological processes, including cell cycle regulation, apoptosis, cell proliferation, DNA repair mechanisms, and energy metabolism pathways [ 21 ]. Despite its effectiveness, one of the major challenges in Cisplatin-based cancer therapy is the development of resistance in cancer cells [ 22 ]. Cancer cells can acquire various mechanisms to evade Cisplatin-induced cell death, leading to reduced treatment efficacy and disease recurrence [ 23 ]. Overcoming Cisplatin resistance remains a significant hurdle in cancer treatment and continues to be an active area of research in oncology [ 24 , 25 ]. Several mechanisms can contribute to Cisplatin resistance in bladder cancer [ 9 ]. Cancer cells can develop mechanisms to decrease the uptake of Cis, reducing the drug's concentration within the cells and limiting its effectiveness [ 26 ]. Besides, cancer cells can enhance the efflux of Cis through various transporters, such as ATP-binding cassette (ABC) transporters. This efflux mechanism reduces the intracellular drug concentration and diminishes its cytotoxic effects [ 27 ]. According to research, Cis enhances oxidative stress and inflammation in the kidneys [ 28 ]. The study by Eslamlou et al showed that Cis therapy results in TGF-β and IL-1β overexpression in rats [ 28 ]. Evidence indicates that the usage of Cis enhances the expression of the SGLT22 receptor in kidney cells. Cancer cells require high levels of glucose for growth and development. Consequently, some research administered SGLT2 inhibitors to reverse glucose influx and chemoresistance in cancer cells [ 28 ]. Fujiyoshi et al. found that dapagliflozin, an SGLT2 inhibitor, had a similar effect in reducing chemoresistance in hepatocarcinoma cells [ 12 ]. Our study showed a substantial increase in SGLT2 expression following Cis administration. This finding supports the glucose-dependent mechanism of Cis resistance in EJ138 BC cells. In the subsequent stage, the application of Empa in combination with Cis modulated SGLT2 expression in EJ138 cells. Thus, suppression of the SGLT2 receptor can be viewed as an effective method for reducing resistance to cis in BC cells. Empa is an FDA-approved topical medication for type 2 diabetes that demonstrates non-insulin-dependent glycemic control capabilities. Therefore, diabetics getting Empa treatment do not experience hypoglycemia. Empa represents some anti-inflammatory, anti-oxidative stress and cardio-protective qualities. Thus, Empa is considered a safe medicine with no secondary side effects for diabetes type 2 treatment. Some investigations have found that Empa-type medicines may have a role in epithelial-mesenchymal transition (EMT) and cell cycle arrest suppression in cancer cells. Previous study reveals that Empa potentiates AMPK activation to inhibit mTOR and NFκB. Lower expression of NFκB prevents cancer cells from proliferating, angiogenesis, metastasis, and inflaming. Furthermore, Empa contributes to the suppression of ERK1/2 and AKT expression and stops the growth of cancer cells. The primary site of Empa expression is renal proximal tubules. Empa causes glucose to be released and excreted into urine by inhibiting the kidney's SGLT2 receptor. According to research, Empa suppresses the SGLT2 receptor in lung, brain, liver, and breast cancer tissues. Empa-induced glucose restriction via SGLT2 inhibition leads to cancer cell death. Given the strong link between diabetes and BC, as well as the resistance of BC cells to chemotherapy, we evaluated the potential effect of empagliflozin on EJ138 BC cells. However, no research is available on the existence of the SGLT2 receptor in BC cells. Interestingly, our research revealed a substantial decrease in SGLT2 expression in EJ138 cells following Empa treatment. This finding suggests that Empa directly suppresses SGLT2 in EJ138 cells. AKT (also known as protein kinase B), PI3K (phosphoinositide 3-kinase), and mTOR (mechanistic target of rapamycin) are all key proteins involved in the PI3K/AKT/mTOR signaling pathway, which plays a crucial role in regulating various cellular processes, including cell growth, proliferation, survival, and metabolism [ 29 ]. We found that a combination of Cis and Empa downregulated the expression of AKT, PI3K, and mTOR. This downregulation may occur through various mechanisms, including direct inhibition of protein synthesis or indirect modulation of signalling pathways that regulate the expression of these proteins. This indicates that the combined treatment may have a synergistic effect on inhibiting the PI3K/AKT/mTOR pathway, potentially enhancing the therapeutic efficacy of both treatments. Zhang et al. demonstrated that hyperactivation of PI3K/Akt pathway was closely associated with Cisplatin resistance by regulating the Bax-mitochondria-mediated apoptosis pathway in human lung cancer. Inhibition of PI3K/Akt activity in A549/DDP cells and H460/DDP cells could reverse Cisplatin resistance by enhancing the effect of Cisplatin on Bax oligomerization and release of Cytochrome C, allowing activation of the caspase-mediated apoptosis pathway. Cisplatin resistance of lung cancer could be reversed via the inhibition of the PI3K/Akt signaling pathway. Therefore, both PI3K and Akt may be potential targets for overcoming cisplatin resistance in lung cancer [ 30 ]. Recent research findings have shed light on the synergistic effects of Empagliflozin and Doxorubicin in suppressing the survival of triple-negative breast cancer cells through the targeting of the mTOR pathway [ 31 ]. We found that co-treatment of Cis and Empa led to an increase in the expression levels of p53. p53 is a tumour suppressor protein that plays a crucial role in regulating cell growth, DNA repair, and apoptosis. When cells are exposed to Cis therapy-related DNA damage, p53 levels typically increase to initiate DNA repair or induce cell death. the levels of p21 protein are increased in response to p53 up-regulation. P21 inhibits the activity of cyclin-dependent kinases (CDKs), which are enzymes involved in regulating the cell cycle. CDKs halt cell cycle progression, allowing time for DNA repair or triggering necessary apoptosis [ 32 , 33 ]. The alterations in the expression levels of apoptosis-regulating genes, Bax and Bcl2, in our study suggest significant impacts of Cis and Empa combined administration on the apoptotic pathways in EJ138 cells. Bax accelerates cell death by targeting mitochondrial outer membrane permeabilization and facilitating the release of apoptotic factors. Bcl2 plays a key role in suppressing cell death by preventing the release of cytochrome c from mitochondria and subsequent stimulation of caspases [ 34 ]. Our study revealed a notable elevation in the activity of ROS within the EJ138 cells following Cis and Empa administration. It is well-established that Cis exerts its anticancer effects by inducing oxidative stress. This oxidative stress triggers an upsurge in the production of ROS within the cancer cells. The increased ROS levels play a critical role in the cytotoxicity of Cis, contributing to the disruption of cellular processes and ultimately leading to cell death. This mechanism highlights the importance of oxidative stress in the anticancer efficacy of Cis/Empa combination therapy and provides insights into its potential therapeutic applications [ 35 ]. A recent study reported that the co-treatment of β-ELE (beta-element) and Cis resulted in increased accumulation of ROS and activation of 5'AMP-activated protein kinase (AMPK), ultimately leading to apoptosis [ 36 ]. Choi et al. discovered a notable association between Cisplatin treatment and the production of mitochondrial ROS. Cisplatin was found to induce mitochondrial dysfunction, resulting in fragmentation and collapse of the mitochondrial membrane potential, which was attributed to an increase in mitochondrial ROS generation. Additionally, they demonstrated that Cisplatin activated p53 and inhibited glycolysis. These findings shed light on the intricate mechanisms by which Cisplatin affects mitochondrial function, ROS generation, and cellular metabolism, providing insight into its potential therapeutic implications [ 37 ]. Studies have shown that Empa can induce oxidative stress in various cell types, including cancer cells, by altering cellular metabolism and increasing ROS production [ 38 ]. Wu et al. conducted a study that revealed the potential benefits of co-treatment with ursolic acid and Empagliflozin in reducing inflammation, oxidative stress, and renal fibrosis in diabetic nephropathy. Their research showed that the combined administration of these compounds resulted in significant improvements in these pathological processes associated with diabetic kidney disease. By reducing inflammation, suppressing oxidative stress, and mitigating renal fibrosis, the co-treatment with ursolic acid and Empagliflozin holds promise as a therapeutic strategy for managing diabetic nephropathy and improving renal function in individuals with diabetes [ 39 ]. Furthermore, we found a noticeable decrease in MMP-2/MMP-9 activity following Cis/Empa combination therapy. MMP-2 and MMP-9 are enzymes involved in the breakdown of extracellular matrix components, and their activity is associated with BC cell invasion and metastasis The results suggest that the combination therapy could have a greater impact on impairing the invasive properties of cancer cells. By diminishing the invasive properties of cancer cells, combined therapy may help to confine the disease within the primary tumour site and limit its spread to other organs or lymph nodes. This could potentially increase the chances of successful surgical removal or localized treatment of the tumor, leading to improved patient outcomes [ 40 ]. Shi et al. documented that the administration of Cis led to the downregulation of MMP-2/MMP-9 and PI3K/AKT signalling pathways in melanoma cells. This downregulation was associated with metastasis in melanoma cells. The results highlighted the potential of Cis as a therapeutic approach to impede the progression and spread of melanoma [ 41 ]. Another study revealed that treatment with Empa led to a decrease in MMP-2/MMP-9 activity. Empa treatment resulted in a decrease in the protein expression of MMP-9, thereby contributing to improved cardiovascular outcomes following myocardial infarction. Empa exhibited potential cardioprotective effects by reducing the levels of MMP-9 [ 42 ]. The findings of this study suggest that Empa reduced EJ138 cells' resistance to Cis treatment by inhibiting SGLT2 expression. Overall, Empa enhanced the likelihood of apoptosis and complemented the effects of Cis in suppressing the growth, invasion, and proliferation in EJ138 cells. It is worth noting that further research is needed to fully elucidate the precise mechanisms underlying the synergistic effects of Cis and Empa on EJ138 BC cells. Conclusion This study revealed SGLT2 receptors in EJ138 BC cells. Furthermore, Empa caused a significant decrease in SGLT2 expression and Cis-resistance in EJ-138 BC cells. Empa demonstrated a synergic action with Cis to induce apoptosis in EJ138 BC cells. Empa enhanced Cis' ability to decrease EJ138 BC growth and invasion. Our findings suggest a probable use of Empa alone or in combination with Cis for BC therapy. However, the specific connection between Empa and SGLT2 needs to be elucidated. It is suggested to design research to investigate the effect of Empa on animal models of BC in vivo. Declarations Ethics approval and consent to participate This experimental study was approved by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (Ethical code: IR.AJUMS.REC.1400.460). Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare no conflict of interest. Consent for publication Not applicable. Authors' contributions S.Sh. and Sh.M. carried out the experiment, Sh.M. and M.S. and planned the experiments, performed the analytic calculations, and performed the numerical simulations, D.D. and M.F contributed to the interpretation of the results and wrote the manuscript. All authors read and approved the final manuscript. Funding This manuscript was financially supported by the Cellular and Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran: (Grant No: CMRC-0046). Acknowledgment The practical stages of the project have been performed at the Cellular and Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. The authors thank Avin Stem Gen Bio Health Inc. for technical support performing. References Saginala K, Barsouk A, Aluru JS, Rawla P, Padala SA, Barsouk A: Epidemiology of Bladder Cancer. Med Sci (Basel) 2020, 8 . Mignot F, Fabiano E, Xylinas E, Alati A, Méjean A, Masson‐Lecomte A, et al: Clinical outcomes of adapted hypofractionated radiotherapy for bladder cancer in elderly patients. 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Theile D, Wizgall P: Acquired ABC-transporter overexpression in cancer cells: transcriptional induction or Darwinian selection? Naunyn Schmiedebergs Arch Pharmacol 2021, 394: 1621-1632. Farrokh-Eslamlou N, Momtaz S, Niknejad A, Hosseini Y, Mahdaviani P, Ghasemnejad-Berenji M, et al: Empagliflozin protective effects against cisplatin-induced acute nephrotoxicity by interfering with oxidative stress and inflammation in Wistar rats. Naunyn-Schmiedeberg's Archives of Pharmacology 2024 : 1-10. He Y, Sun MM, Zhang GG, Yang J, Chen KS, Xu WW, et al: Targeting PI3K/Akt signal transduction for cancer therapy. Signal Transduction and Targeted Therapy 2021, 6: 425. Zhang Y, Bao C, Mu Q, Chen J, Wang J, Mi Y, et al: Reversal of cisplatin resistance by inhibiting PI3K/Akt signal pathway in human lung cancer cells. Neoplasma 2016, 63: 362-370. Eliaa SG, Al-Karmalawy AA, Saleh RM, Elshal MF: Empagliflozin and doxorubicin synergistically inhibit the survival of triple-negative breast cancer cells via interfering with the mTOR pathway and inhibition of calmodulin: in vitro and molecular docking studies. ACS Pharmacology & Translational Science 2020, 3: 1330-1338. Kciuk M, Gielecińska A, Mujwar S, Mojzych M, Kontek R: Cyclin-dependent kinases in DNA damage response. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer 2022, 1877: 188716. Tran AP, Tralie CJ, Reyes J, Moosmüller C, Belkhatir Z, Kevrekidis IG, et al: Long-term p21 and p53 dynamics regulate the frequency of mitosis events and cell cycle arrest following radiation damage. Cell Death & Differentiation 2023, 30: 660-672. Qian S, Wei Z, Yang W, Huang J, Yang Y, Wang J: The role of BCL-2 family proteins in regulating apoptosis and cancer therapy. Front Oncol 2022, 12: 985363. Xue D-F, Pan S-T, Huang G, Qiu J-X: ROS enhances the cytotoxicity of cisplatin by inducing apoptosis and autophagy in tongue squamous cell carcinoma cells. The international journal of biochemistry & cell biology 2020, 122: 105732. Gan D, He W, Yin H, Gou X: β‑elemene enhances cisplatin‑induced apoptosis in bladder cancer cells through the ROS‑AMPK signaling pathway. Oncol Lett 2020, 19: 291-300. Choi YM, Kim HK, Shim W, Anwar MA, Kwon JW, Kwon HK, et al: Mechanism of Cisplatin-Induced Cytotoxicity Is Correlated to Impaired Metabolism Due to Mitochondrial ROS Generation. PLoS One 2015, 10: e0135083. Uthman L, Li X, Baartscheer A, Schumacher CA, Baumgart P, Hermanides J, et al: Empagliflozin reduces oxidative stress through inhibition of the novel inflammation/NHE/[Na(+)](c)/ROS-pathway in human endothelial cells. Biomed Pharmacother 2022, 146: 112515. Wu X, Li H, Wan Z, Wang R, Liu J, Liu Q, et al: The combination of ursolic acid and empagliflozin relieves diabetic nephropathy by reducing inflammation, oxidative stress and renal fibrosis. Biomedicine & Pharmacotherapy 2021, 144: 112267. Kudelski J, Tokarzewicz A, Gudowska-Sawczuk M, Mroczko B, Chłosta P, Bruczko-Goralewska M, et al: The Significance of Matrix Metalloproteinase 9 (MMP-9) and Metalloproteinase 2 (MMP-2) in Urinary Bladder Cancer. Biomedicines 2023, 11 . Shi H, Wu Y, Wang Y, Zhou M, Yan S, Chen Z, et al: Liquiritigenin potentiates the inhibitory effects of cisplatin on invasion and metastasis via downregulation MMP-2/9 and PI3 K/AKT signaling pathway in B16F10 melanoma cells and mice model. Nutrition and cancer 2015, 67: 761-770. Goerg J, Sommerfeld M, Greiner B, Lauer D, Seckin Y, Kulikov A, et al: Low-dose empagliflozin improves systolic heart function after myocardial infarction in rats: regulation of MMP9, NHE1, and SERCA2a. International Journal of Molecular Sciences 2021, 22: 5437. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4634713","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322624644,"identity":"8b7064ff-3efa-48f8-aecf-378981fd53d4","order_by":0,"name":"Saeedeh Shariati","email":"","orcid":"","institution":"Ahvaz Jundishapur University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Saeedeh","middleName":"","lastName":"Shariati","suffix":""},{"id":322624645,"identity":"7cc1bf31-b533-4cf1-8d26-1a835ca8945e","order_by":1,"name":"Shokooh Mohtadi","email":"","orcid":"","institution":"Ahvaz Jundishapur University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shokooh","middleName":"","lastName":"Mohtadi","suffix":""},{"id":322624646,"identity":"c0b99bb7-04fc-41b4-bad9-9c498ca7d19a","order_by":2,"name":"Shahrzad Molavinia","email":"","orcid":"","institution":"Ahvaz Jundishapur University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Shahrzad","middleName":"","lastName":"Molavinia","suffix":""},{"id":322624647,"identity":"6692393f-a766-46c4-b890-ed6487db4249","order_by":3,"name":"Maryam Salehcheh","email":"","orcid":"","institution":"Ahvaz Jundishapur University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Salehcheh","suffix":""},{"id":322624648,"identity":"458b1417-7dbe-47ba-bd41-1b5e7f0f027a","order_by":4,"name":"Dian Dayer","email":"","orcid":"","institution":"Ahvaz Jundishapur University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Dian","middleName":"","lastName":"Dayer","suffix":""},{"id":322624649,"identity":"69cdf55f-710e-4f4f-b250-469c8bc1a1c7","order_by":5,"name":"Maryam Farzaneh","email":"data:image/png;base64,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","orcid":"","institution":"Imam Khomeini Hospital, Ahvaz Jundishapur University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Farzaneh","suffix":""}],"badges":[],"createdAt":"2024-06-25 08:16:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4634713/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4634713/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60788976,"identity":"79bc6f9f-69f6-49cf-992c-beb5ed5d692c","added_by":"auto","created_at":"2024-07-22 06:36:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":24016,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Empagliflozin (Empa) or Cisplatin (Cis) on EJ138 BC cell's viability. The EJ138 cells were exposed to 0, 6.25, 12.25, 25, 50, and 100 mM of Cis (A) or 0, 6.25, 12.25, 25, 50, and 100 µg/ml of Empa (B) for 24 h. Cell viability was measured using the MTT test. The results are mean ± SEM for triplicated tests. The data are normalized to untreated EJ138 cells grown in baseline media. The viability of EJ138 cells showed a reverse correlation with the concentration of Cis and Empa.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-4634713/v1/6341402d306ba32bb4770a23.png"},{"id":60788971,"identity":"3ee68cf4-b72c-42ab-962e-ac6c7d1d99e3","added_by":"auto","created_at":"2024-07-22 06:36:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":27144,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Empagliflozin (Empa) or Cisplatin (Cis) on SGLT2 expression. EJ138 BC cells showed a significant increase in SGLT2 protein expression when treated with 16 mM Cis. Treatment with 72 µg/ml Empa resulted in SGLT2 downregulation. GAPDH was employed as the housekeeping gene. Results are presented as the mean ± SEM of three different sets of experiments. *** P\u0026lt;0.001 compared to the control group, ### P\u0026lt;0.001 compared to the Cis-treated group.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-4634713/v1/d5134b912d1db905166650bb.png"},{"id":60788978,"identity":"ec7e36b2-3edd-4d53-8ded-de6682757cf4","added_by":"auto","created_at":"2024-07-22 06:36:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":39125,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Empagliflozin (Empa) or Cisplatin (Cis) on EJ138 BC cell's proliferation-associated proteins expression. The effects of 24 h exposure to 72 µg/ml Empa and/or 16 mM Cis on AKT, PI3K, and mTOR expression in EJ138 BC cells. GAPDH was employed as the housekeeping gene. Results are presented as the mean ± SEM of three different sets of experiments.*** P\u0026lt;0.001 compared to the control group, ### P\u0026lt;0.001 compared to the Cis-treated group.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-4634713/v1/ffb7fc46c2f015285a485248.png"},{"id":60789599,"identity":"29bc4014-858c-4326-9eee-1d2bf8c9ae55","added_by":"auto","created_at":"2024-07-22 06:44:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":29965,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Empagliflozin (Empa) or Cisplatin (Cis) on EJ138 BC cell's cell cycle-associated proteins expression. The effects of 24 h exposure to 72 µg/ml Empa and/or 16 mM Cis on P21 and P53 expression in EJ138 BC cells. GAPDH was employed as the housekeeping gene. Results are presented as the mean ± SEM of three different sets of experiments. ** P\u0026lt;0.01, *** P\u0026lt;0.001 compared to the control group, ### P\u0026lt;0.001 compared to the Cis-treated group.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-4634713/v1/18bd6baa01fbc91918b7ca48.png"},{"id":60788977,"identity":"11025dc5-552d-4e36-a629-b3ba4e3f6696","added_by":"auto","created_at":"2024-07-22 06:36:06","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31220,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Empagliflozin (Empa) or Cisplatin (Cis) on EJ138 BC cell's apoptosis-associated proteins expression. The effects of 24 h exposure to 72 µg/ml Empa and/or 16 mM Cis on Bax and Bcl2 expression in EJ138 BC cells. GAPDH was employed as the housekeeping gene. Results are presented as the mean ± SEM of three different sets of experiments. *** P\u0026lt;0.001 compared to the control group, ### P\u0026lt;0.001 compared to the Cis-treated group.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-4634713/v1/a780043c800edb81e914df99.png"},{"id":60788973,"identity":"e9279b18-82f6-4928-a98f-9c47bc4910c8","added_by":"auto","created_at":"2024-07-22 06:36:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":12148,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Empagliflozin (Empa) or Cisplatin (Cis) on EJ138 BC cell's ROS production. The effects of 24 h exposure to 72 µg/ml Empa and/or 16 mM Cis on ROS production in EJ138 BC cells. GAPDH was employed as the housekeeping gene. Results are presented as the mean ± SEM of three different sets of experiments. *** P\u0026lt;0.001 compared to the control group, ### P\u0026lt;0.001 compared to the Cis-treated group.\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-4634713/v1/8c53c6bb20fdf414309a0e6b.png"},{"id":60788974,"identity":"5de4e6ac-6df7-4acf-8fe5-df2df301d5b1","added_by":"auto","created_at":"2024-07-22 06:36:04","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":26107,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Empagliflozin (Empa) or Cisplatin (Cis) on EJ138 BC cell's invasion-associated proteins expression. The effects of 24 h exposure to 72 µg/ml Empa and/or 16 mM Cis on MMP-2 and MMP-9 expression in EJ138 BC cells. GAPDH was employed as the housekeeping gene. Results are presented as the mean ± SEM of three different sets of experiments. *** P\u0026lt;0.001, ** P\u0026lt;0.01 compared to the control group,# P\u0026lt;0.05 ### P\u0026lt;0.001 compared to the Cis-treated group.\u003c/p\u003e","description":"","filename":"Slide7.png","url":"https://assets-eu.researchsquare.com/files/rs-4634713/v1/0207fb8ffe112fa0dfbb677b.png"},{"id":60808517,"identity":"b34bdd28-b929-4050-8d97-a389561c9bff","added_by":"auto","created_at":"2024-07-22 10:33:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1753141,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4634713/v1/c1d2af59-1144-4199-99c5-bb32e9a5c6ca.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Empagliflozin enhances cisplatin activity in chemo-resistant EJ138 bladder cancer cells: The importance of anti-diabetic medications in cancer treatment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBladder cancer (BC) accounts for 3% of worldwide cancer diagnoses [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. BC is one of the most prevalent cancers among the elderly [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The development of BC is often associated with certain risk factors such as smoking, exposure to certain chemicals, chronic bladder infections, and a family history of the disease [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Symptoms of BC may include blood in the urine, frequent urination, pain during urination, and lower back pain [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Early detection and prompt treatment can improve the prognosis and outcome for individuals with bladder cancer [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn BC, alterations in the PI3K/Akt/mTOR pathway are commonly observed [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The dysregulated PI3K/Akt/mTOR pathway has implications for cancer cell proliferation, invasion, metastasis, and resistance to therapy [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCisplatin (Cis) is a chemotherapy drug commonly used in the treatment of various types of cancer [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Cis works by interfering with the DNA in cancer cells, preventing their ability to divide and grow [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Depending on the particular cancer type and stage, Cis may be used alone or in conjunction with other chemotherapy medications [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Treatment of BC is significantly hampered by resistance to Cis-based chemotherapy [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In this context, several strategies are being investigated to improve Cis's effectiveness [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Previous research demonstrated sodium-glucose co-transporter 2 (SGLT2) upregulation among Cis-resistance hepatocarcinoma cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. It has been proposed that SGLT2 inhibitors reduce cancer cells' chemotherapy resistance by restricting glucose uptake. Fujiyoshi et al employed Dapagliflozin, an SGLT2 inhibitor, to overcome Cis resistance in hepatoblastoma cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Several combination treatments are being researched to counteract the resistance of BC cells to Cis [\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEmpagliflozin (Empa) is frequently given medication for the treatment of type 2 diabetes mellitus as an SGLT2 inhibitor [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It has been demonstrated that Empa has anticancer effects in cervical, breast, and hepatic cancer cells. Empa targets the kidney's SGLT2 protein to prevent glucose reabsorption and promote glucose excretion through urine. Research shows that SGLT2 is expressed in pancreatic, prostate, and glioblastoma cancer cells. Generally, cancer cells are reprogrammed to express additional glucose transporters to facilitate glucose influx into the cytoplasm. Glucose influx into cancer cells activates the β-catenin-Wnt pathway, leading to cyclin D1 and TRPC6 transcription and cell proliferation. SGLT inhibitors reduce the progression of cancer by inhibiting the activation of β-catenin. Currently, researchers are investigating the idea of utilizing specific SGLT-2 inhibitors to decrease glucose uptake by cancer cells. However, the impact of Empa on BC cells has not yet been investigated.\u003c/p\u003e \u003cp\u003ePrevious studies show a link between diabetes and BC development. Therefore, exploring the dual use of Empa for the concurrent treatment of diabetes and BC is an attractive category. The current study evaluated the therapeutic efficacy of Empa and Cis for EJ138 BCs.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Culture Protocol\u003c/h2\u003e \u003cp\u003eThis experimental study was approved by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (Ethical code: IR.AJUMS.REC.1400.460). The BC cell line EJ138 (C429) was purchased from the Pasteur Institute Resource Center (Iran). EJ138 cells were cultured in RPMI medium (Sigma, USA) containing 20% FBS (Sigma, USA) and 1% penicillin/streptomycin (Sigma, USA). The cells were incubated at 37\u0026deg;C with 5% CO2. The cells were passaged every two days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMTT Assay\u003c/h2\u003e \u003cp\u003eThe cells were cultured at 104 cells/well density in a 96-well culture plate and incubated at 37\u0026deg;C with 5% CO2 for 24 h. Five wells were allocated Within each experimental group. The cells were treated with varying concentrations of Cis (0, 6.25, 12.25, 25, 50, and 100 mM) (Sigma, USA) and Empa (0, 6.25, 12.25, 25, 50, and 100 \u0026micro;g/ml) (Sigma, USA) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Afterward, the culture medium was replaced with 100 \u0026micro;L of 0.5 mg/mL MTT (Sigma, USA) solution, and the plates were incubated for 4 h at 37\u0026deg;C with 5% CO2 in the dark. Then each well received 100 \u0026micro;L of Dimethyl sulfoxide (DMSO) (Sigma, USA), and the plates were shaken for 15 min. The absorbance of the samples was measured at 570 nm using an ELISA reader (Bio-Rad, USA). The viability of the cells was calculated using the formula: 100-(absorbance test/absorbance control) \u0026times; 100 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The IC50 values were calculated using GraphPad Prism software (GraphPad Software, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design\u003c/h2\u003e \u003cp\u003eThe cancer cells were categorized into four groups. Group I (control group) consisted of EJ138 cells that received no treatment. Group II was EJ138 cells that received Cis. Group III consisted of EJ138 cells that received Empa. Group IV consisted of EJ138 cells treated with a combination of Cis and Empa. All groups were incubated at 37\u0026deg;C with 5% CO2 for 24 h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eThe cells were harvested and lysed using SDS lysis buffer (Sigma, USA), followed by centrifugation to separate the cellular components. The proteins were then solubilized in an SDS-PAGE loading buffer and subjected to denaturation by heating at 95\u0026deg;C for 10 minutes. Subsequently, the denatured protein samples were transferred onto a nitrocellulose membrane (Sigma, USA). To minimize non-specific binding, the membrane was blocked using a solution comprising 5% nonfat milk (Sigma, USA) in Tris-buffered saline (TBS) (Sigma, USA) supplemented with 0.05% Tween 20 (Sigma, USA). Following blocking, the membrane was incubated for 2 h at room temperature with primary antibodies specific to SGLT2 (sc-393350) (SANTA CRUZ, USA), PI3K (ab302958), AKT (ab38449), mTOR (ab134903), p21 (ab109520), p53 (ab32049), MMP-2 (ab92536), MMP-9 (ab76003), BAX (ab32503), and Bcl-2 (ab182585) (Abcam, USA) diluted 1:500 in blocking solution. The membranes were washed three times with Tris-buffered saline with Tween 20. The suitable secondary antibody (NB500-420, Novus Biologicals, USA) coupled to horseradish peroxidase and diluted 1:1000 in blocking solution was incubated on the membranes for two hours at room temperature. Once three washes were completed, protein reactivity was evaluated with an ECL detection kit (ParsTous, Iran). GAPDH was used to normalize the protein loading (D16H11; Cell Signaling Technology, USA). The CLIQS 1D program (TotallLab, UK) was used to analyze optical density [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMeasuring reactive oxygen species (ROS)\u003c/h2\u003e \u003cp\u003eThe levels of ROS production by the cells were determined using the Oxi SelectTM assay kit based on green fluorescent dye dichloro-dihydro fluorescein (DCF) production. The cells were grown on 96-well plates in triplicate. The cells were incubated for 24 h at 37\u0026deg;C. Afterward, the supernatant was discarded, and 100 \u0026micro;L of DA-DCFH 1X was added. After 30 min incubation at 37\u0026deg;C and three times washing, 100 \u0026micro;L of culture medium and 100 \u0026micro;L of lysing buffer were added and the plates were incubated for 5 min at 37\u0026deg;C. The amount of fluorescence was measured at 530 nm using a Dako flow cytometer (Troy, USA). Data were analyzed using Flow Jo software (Flow Jo, USA) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe statistical analysis was conducted using GraphPad Prism 9 and Excel software. All experiments were performed in triplicate, ensuring the robustness and reliability of the results. To assess the differences between multiple means, a one-way analysis of variance (ANOVA) followed by Tukey's post hoc analysis was employed. All experimental data were presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. A statistically significant difference between distinct experimental groups was indicated as follows: #,*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ##, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ###, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCombining Cisplatin with Empagliflozin resulted in reduced cell viability\u003c/h2\u003e \u003cp\u003eEJ138 BC cells were treated with Cis, Empa, or a combination of Cis and Empa for 24 hours. The vitality of EJ138 cells correlated inversely with the concentrations of Cis and Empa. The IC50 values were determined to be 16 mM for the Cis-treated group and 72 \u0026micro;g/ml for the Empa-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eThe impact of Empa and/or on SGLT2 expression\u003c/h2\u003e \u003cp\u003eSGLT2 enhances glucose re-absorption and tumour cell growth. According to Western Blot results, Cis-therapy showed a direct positive influence on SGLT2 expression. The expression of SGLT2 showed a significant decrease following Empa treatment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Empa attenuated the effect of Cis on SGLT2 expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eThe effects of Cisplatin and/or Empagliflozin on the proliferation-dependent pathway\u003c/h2\u003e \u003cp\u003eAKT, PI3K, and mTOR promote cell growth, proliferation, and survival. According to Western b138lot results, the expression of AKT, PI3K, and mTOR was down-regulated by Cis and Empa administration. The groups treated with Cis, Empa or a combination of Cis and Empa showed significantly decreased levels of AKT, PI3K, and mTOR compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for all groups). However, the group treated with a combination of Cis and Empa had the lowest level of AKT, PI3K, and mTOR expression. The difference between Cis and Cis\u0026thinsp;+\u0026thinsp;Empa groups was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eThe effects of Cisplatin and/or Empagliflozin on cell cycle regulator proteins\u003c/h2\u003e \u003cp\u003eP53 regulates downstream proteins such as p21 to promote cell cycle arrest, apoptosis, and DNA repair. The results of the present study show an increase in p53 and p21 expression following exposure to Cis, Empa or a combination of Cis and Empa, compared to the control group. However, the difference in p21 expression between the Empa group and the control group was not significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;=\u0026thinsp;0.002, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, for p53) and (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;=\u0026thinsp;0.88, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for p21). EJ138 cells treated with a combination of Cis and Empa presented statistically increased levels of p53 and p21 compared to the group treated with Cis alone (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eThe effects of Cis and/or Empa on the expression of apoptotic-related proteins\u003c/h2\u003e \u003cp\u003eThe EJ138 cells subjected to treatment with Cis, Empa, or their combination revealed notable alterations in the expression of key apoptosis-regulating genes. Specifically, the groups treated with Cis or Empa alone showed a significant decrease in the expression of prominent anti-apoptotic protein Bcl2, coupled with a marked increase in the expression of pro-apoptotic protein Bax (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for all groups). The group that received a combination of Cis and Empa showed a great increase in Bax and a significant decrease in Bcl2 expression compared to Cis group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eThe influence of Cis and/or Empa on ROS production\u003c/h2\u003e \u003cp\u003eThe outcomes demonstrated that oxidative stress caused by Cis, Empa, and their combined treatment had a discernible cytotoxic effect on BC cells. The groups treated with Cis, Empa, or a combination of Cis and Empa, exhibited a substantial increase in ROS generation compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for all groups). The combination of Cis and Empa significantly increased the formation of ROS Compared to the administration of Cis alone (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eThe effects of Cis and/or Empa on invasion-related protein expression\u003c/h2\u003e \u003cp\u003eTo investigate the potential of each treatment on EJ138 cancer cell invasion and metastasis, the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) proteins was investigated. There was a decrease in MMP-2 and MMP-9 in the groups treated with Cis, Empa, or a combination of Cis and Empa, compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;=\u0026thinsp;0.52, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for MMP-2) and (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, P\u0026thinsp;=\u0026thinsp;0.04, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for MMP-9). The group received a combination of Cis and Empa revealed a decrease in MMP-2 and MMP-9 expression compared to the groups that were treated with Cis alone (P\u0026thinsp;=\u0026thinsp;0.99, P\u0026thinsp;=\u0026thinsp;0.02) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, chemotherapy-resistant EJ138 BC cells revealed an unavoidable increase in SGLT2 expression. However, Empa therapy attenuated the Cis effect on SGLT2 expression. Concurrent treatment with Cis and Empa led to upregulation of Bax, P21, and P53 expression and enhanced ROS activity in EJ138 cells. The treatment resulted in downregulation of Bcl2 expression along with a reduction in the PI3K/AKT/mTOR and MMP-2/MMP-9 pathways activity. Also, the use of Cis and Empa strengthened the Cis anticancer effect. Consequently, this combined treatment effectively inhibited the proliferation and invasion of BC cells.\u003c/p\u003e \u003cp\u003eCisplatin has been shown to affect several critical biological processes, including cell cycle regulation, apoptosis, cell proliferation, DNA repair mechanisms, and energy metabolism pathways [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Despite its effectiveness, one of the major challenges in Cisplatin-based cancer therapy is the development of resistance in cancer cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Cancer cells can acquire various mechanisms to evade Cisplatin-induced cell death, leading to reduced treatment efficacy and disease recurrence [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Overcoming Cisplatin resistance remains a significant hurdle in cancer treatment and continues to be an active area of research in oncology [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral mechanisms can contribute to Cisplatin resistance in bladder cancer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Cancer cells can develop mechanisms to decrease the uptake of Cis, reducing the drug's concentration within the cells and limiting its effectiveness [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Besides, cancer cells can enhance the efflux of Cis through various transporters, such as ATP-binding cassette (ABC) transporters. This efflux mechanism reduces the intracellular drug concentration and diminishes its cytotoxic effects [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to research, Cis enhances oxidative stress and inflammation in the kidneys [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The study by Eslamlou et al showed that Cis therapy results in TGF-β and IL-1β overexpression in rats [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Evidence indicates that the usage of Cis enhances the expression of the SGLT22 receptor in kidney cells. Cancer cells require high levels of glucose for growth and development. Consequently, some research administered SGLT2 inhibitors to reverse glucose influx and chemoresistance in cancer cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Fujiyoshi et al. found that dapagliflozin, an SGLT2 inhibitor, had a similar effect in reducing chemoresistance in hepatocarcinoma cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Our study showed a substantial increase in SGLT2 expression following Cis administration. This finding supports the glucose-dependent mechanism of Cis resistance in EJ138 BC cells. In the subsequent stage, the application of Empa in combination with Cis modulated SGLT2 expression in EJ138 cells. Thus, suppression of the SGLT2 receptor can be viewed as an effective method for reducing resistance to cis in BC cells.\u003c/p\u003e \u003cp\u003eEmpa is an FDA-approved topical medication for type 2 diabetes that demonstrates non-insulin-dependent glycemic control capabilities. Therefore, diabetics getting Empa treatment do not experience hypoglycemia. Empa represents some anti-inflammatory, anti-oxidative stress and cardio-protective qualities. Thus, Empa is considered a safe medicine with no secondary side effects for diabetes type 2 treatment. Some investigations have found that Empa-type medicines may have a role in epithelial-mesenchymal transition (EMT) and cell cycle arrest suppression in cancer cells. Previous study reveals that Empa potentiates AMPK activation to inhibit mTOR and NFκB. Lower expression of NFκB prevents cancer cells from proliferating, angiogenesis, metastasis, and inflaming. Furthermore, Empa contributes to the suppression of ERK1/2 and AKT expression and stops the growth of cancer cells. The primary site of Empa expression is renal proximal tubules. Empa causes glucose to be released and excreted into urine by inhibiting the kidney's SGLT2 receptor. According to research, Empa suppresses the SGLT2 receptor in lung, brain, liver, and breast cancer tissues. Empa-induced glucose restriction via SGLT2 inhibition leads to cancer cell death. Given the strong link between diabetes and BC, as well as the resistance of BC cells to chemotherapy, we evaluated the potential effect of empagliflozin on EJ138 BC cells. However, no research is available on the existence of the SGLT2 receptor in BC cells. Interestingly, our research revealed a substantial decrease in SGLT2 expression in EJ138 cells following Empa treatment. This finding suggests that Empa directly suppresses SGLT2 in EJ138 cells.\u003c/p\u003e \u003cp\u003eAKT (also known as protein kinase B), PI3K (phosphoinositide 3-kinase), and mTOR (mechanistic target of rapamycin) are all key proteins involved in the PI3K/AKT/mTOR signaling pathway, which plays a crucial role in regulating various cellular processes, including cell growth, proliferation, survival, and metabolism [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe found that a combination of Cis and Empa downregulated the expression of AKT, PI3K, and mTOR. This downregulation may occur through various mechanisms, including direct inhibition of protein synthesis or indirect modulation of signalling pathways that regulate the expression of these proteins. This indicates that the combined treatment may have a synergistic effect on inhibiting the PI3K/AKT/mTOR pathway, potentially enhancing the therapeutic efficacy of both treatments.\u003c/p\u003e \u003cp\u003eZhang et al. demonstrated that hyperactivation of PI3K/Akt pathway was closely associated with Cisplatin resistance by regulating the Bax-mitochondria-mediated apoptosis pathway in human lung cancer. Inhibition of PI3K/Akt activity in A549/DDP cells and H460/DDP cells could reverse Cisplatin resistance by enhancing the effect of Cisplatin on Bax oligomerization and release of Cytochrome C, allowing activation of the caspase-mediated apoptosis pathway. Cisplatin resistance of lung cancer could be reversed via the inhibition of the PI3K/Akt signaling pathway. Therefore, both PI3K and Akt may be potential targets for overcoming cisplatin resistance in lung cancer [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Recent research findings have shed light on the synergistic effects of Empagliflozin and Doxorubicin in suppressing the survival of triple-negative breast cancer cells through the targeting of the mTOR pathway [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe found that co-treatment of Cis and Empa led to an increase in the expression levels of p53. p53 is a tumour suppressor protein that plays a crucial role in regulating cell growth, DNA repair, and apoptosis. When cells are exposed to Cis therapy-related DNA damage, p53 levels typically increase to initiate DNA repair or induce cell death. the levels of p21 protein are increased in response to p53 up-regulation. P21 inhibits the activity of cyclin-dependent kinases (CDKs), which are enzymes involved in regulating the cell cycle. CDKs halt cell cycle progression, allowing time for DNA repair or triggering necessary apoptosis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe alterations in the expression levels of apoptosis-regulating genes, Bax and Bcl2, in our study suggest significant impacts of Cis and Empa combined administration on the apoptotic pathways in EJ138 cells. Bax accelerates cell death by targeting mitochondrial outer membrane permeabilization and facilitating the release of apoptotic factors. Bcl2 plays a key role in suppressing cell death by preventing the release of cytochrome c from mitochondria and subsequent stimulation of caspases [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur study revealed a notable elevation in the activity of ROS within the EJ138 cells following Cis and Empa administration. It is well-established that Cis exerts its anticancer effects by inducing oxidative stress. This oxidative stress triggers an upsurge in the production of ROS within the cancer cells. The increased ROS levels play a critical role in the cytotoxicity of Cis, contributing to the disruption of cellular processes and ultimately leading to cell death. This mechanism highlights the importance of oxidative stress in the anticancer efficacy of Cis/Empa combination therapy and provides insights into its potential therapeutic applications [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA recent study reported that the co-treatment of β-ELE (beta-element) and Cis resulted in increased accumulation of ROS and activation of 5'AMP-activated protein kinase (AMPK), ultimately leading to apoptosis [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Choi et al. discovered a notable association between Cisplatin treatment and the production of mitochondrial ROS. Cisplatin was found to induce mitochondrial dysfunction, resulting in fragmentation and collapse of the mitochondrial membrane potential, which was attributed to an increase in mitochondrial ROS generation. Additionally, they demonstrated that Cisplatin activated p53 and inhibited glycolysis. These findings shed light on the intricate mechanisms by which Cisplatin affects mitochondrial function, ROS generation, and cellular metabolism, providing insight into its potential therapeutic implications [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStudies have shown that Empa can induce oxidative stress in various cell types, including cancer cells, by altering cellular metabolism and increasing ROS production [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Wu et al. conducted a study that revealed the potential benefits of co-treatment with ursolic acid and Empagliflozin in reducing inflammation, oxidative stress, and renal fibrosis in diabetic nephropathy. Their research showed that the combined administration of these compounds resulted in significant improvements in these pathological processes associated with diabetic kidney disease. By reducing inflammation, suppressing oxidative stress, and mitigating renal fibrosis, the co-treatment with ursolic acid and Empagliflozin holds promise as a therapeutic strategy for managing diabetic nephropathy and improving renal function in individuals with diabetes [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, we found a noticeable decrease in MMP-2/MMP-9 activity following Cis/Empa combination therapy. MMP-2 and MMP-9 are enzymes involved in the breakdown of extracellular matrix components, and their activity is associated with BC cell invasion and metastasis The results suggest that the combination therapy could have a greater impact on impairing the invasive properties of cancer cells. By diminishing the invasive properties of cancer cells, combined therapy may help to confine the disease within the primary tumour site and limit its spread to other organs or lymph nodes. This could potentially increase the chances of successful surgical removal or localized treatment of the tumor, leading to improved patient outcomes [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Shi et al. documented that the administration of Cis led to the downregulation of MMP-2/MMP-9 and PI3K/AKT signalling pathways in melanoma cells. This downregulation was associated with metastasis in melanoma cells. The results highlighted the potential of Cis as a therapeutic approach to impede the progression and spread of melanoma [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Another study revealed that treatment with Empa led to a decrease in MMP-2/MMP-9 activity. Empa treatment resulted in a decrease in the protein expression of MMP-9, thereby contributing to improved cardiovascular outcomes following myocardial infarction. Empa exhibited potential cardioprotective effects by reducing the levels of MMP-9 [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe findings of this study suggest that Empa reduced EJ138 cells' resistance to Cis treatment by inhibiting SGLT2 expression. Overall, Empa enhanced the likelihood of apoptosis and complemented the effects of Cis in suppressing the growth, invasion, and proliferation in EJ138 cells. It is worth noting that further research is needed to fully elucidate the precise mechanisms underlying the synergistic effects of Cis and Empa on EJ138 BC cells.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study revealed SGLT2 receptors in EJ138 BC cells. Furthermore, Empa caused a significant decrease in SGLT2 expression and Cis-resistance in EJ-138 BC cells. Empa demonstrated a synergic action with Cis to induce apoptosis in EJ138 BC cells. Empa enhanced Cis' ability to decrease EJ138 BC growth and invasion. Our findings suggest a probable use of Empa alone or in combination with Cis for BC therapy. However, the specific connection between Empa and SGLT2 needs to be elucidated. It is suggested to design research to investigate the effect of Empa on animal models of BC in vivo.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis experimental study was approved by the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran (Ethical code: IR.AJUMS.REC.1400.460).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.Sh. and Sh.M. carried out the experiment, Sh.M. and M.S. and planned the experiments, performed the analytic calculations, and performed the numerical simulations, D.D. and M.F contributed to the interpretation of the results and wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis manuscript was financially supported by the Cellular and Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran: (Grant No: CMRC-0046).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe practical stages of the project have been performed at the Cellular and Molecular Research Center, Medical Basic Sciences Research Institute, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. The authors thank Avin Stem Gen Bio Health Inc. for technical support performing.\u0026emsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSaginala K, Barsouk A, Aluru JS, Rawla P, Padala SA, Barsouk A: \u003cstrong\u003eEpidemiology of Bladder Cancer.\u003c/strong\u003e \u003cem\u003eMed Sci (Basel) \u003c/em\u003e2020, \u003cstrong\u003e8\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eMignot F, Fabiano E, Xylinas E, Alati A, M\u0026eacute;jean A, Masson‐Lecomte A, et al: \u003cstrong\u003eClinical outcomes of adapted hypofractionated radiotherapy for bladder cancer in elderly patients.\u003c/strong\u003e \u003cem\u003eBJU international \u003c/em\u003e2023.\u003c/li\u003e\n\u003cli\u003evan Hoogstraten LM, Vrieling A, van der Heijden AG, Kogevinas M, Richters A, Kiemeney LA: \u003cstrong\u003eGlobal trends in the epidemiology of bladder cancer: challenges for public health and clinical practice.\u003c/strong\u003e \u003cem\u003eNature Reviews Clinical Oncology \u003c/em\u003e2023, \u003cstrong\u003e20:\u003c/strong\u003e287-304.\u003c/li\u003e\n\u003cli\u003eKaram AM, Alshrouf MA, Albandi AM, Alhanbali AE, Abu-Obeida WH, Tarbiah MM, et al: \u003cstrong\u003eAwareness of Bladder Cancer Symptoms and Risk Factors in Jordan: A Nationwide Study.\u003c/strong\u003e \u003cem\u003eAsia Pacific Journal of Public Health \u003c/em\u003e2023, \u003cstrong\u003e35:\u003c/strong\u003e69-72.\u003c/li\u003e\n\u003cli\u003eKamecki H, Dębowska M, Poleszczuk J, Demkow T, Przewor A, Nyk Ł, et al: \u003cstrong\u003eIncidental Diagnosis of Urothelial Bladder Cancer: Associations with Overall Survival.\u003c/strong\u003e \u003cem\u003eCancers (Basel) \u003c/em\u003e2023, \u003cstrong\u003e15\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003ePeng Y, Wang Y, Zhou C, Mei W, Zeng C: \u003cstrong\u003ePI3K/Akt/mTOR Pathway and Its Role in Cancer Therapeutics: Are We Making Headway?\u003c/strong\u003e \u003cem\u003eFront Oncol \u003c/em\u003e2022, \u003cstrong\u003e12:\u003c/strong\u003e819128.\u003c/li\u003e\n\u003cli\u003eSathe A, Nawroth R: \u003cstrong\u003eTargeting the PI3K/AKT/mTOR Pathway in Bladder Cancer.\u003c/strong\u003e \u003cem\u003eMethods Mol Biol \u003c/em\u003e2018, \u003cstrong\u003e1655:\u003c/strong\u003e335-350.\u003c/li\u003e\n\u003cli\u003eBrown A, Kumar S, Tchounwou PB: \u003cstrong\u003eCisplatin-Based Chemotherapy of Human Cancers.\u003c/strong\u003e \u003cem\u003eJ Cancer Sci Ther \u003c/em\u003e2019, \u003cstrong\u003e11\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eLi F, Zheng Z, Wei C, Zhang H, Li D, Zhu Y, et al: \u003cstrong\u003eRegulation of cisplatin resistance in bladder cancer by epigenetic mechanisms.\u003c/strong\u003e \u003cem\u003eDrug Resistance Updates \u003c/em\u003e2023\u003cstrong\u003e:\u003c/strong\u003e100938.\u003c/li\u003e\n\u003cli\u003eBhat A, Verma S, Chander G, Jamwal RS, Sharma B, Bhat A, et al: \u003cstrong\u003eCisplatin-based combination therapy for cancer.\u003c/strong\u003e \u003cem\u003eJournal of Cancer Research and Therapeutics \u003c/em\u003e2023.\u003c/li\u003e\n\u003cli\u003eKuan F-C, Li J-M, Huang Y-C, Chang S-F, Shi C-S: \u003cstrong\u003eTherapeutic Potential of Regorafenib in Cisplatin-Resistant Bladder Cancer with High Epithelial\u0026ndash;Mesenchymal Transition and Stemness Properties.\u003c/strong\u003e \u003cem\u003eInternational Journal of Molecular Sciences \u003c/em\u003e2023, \u003cstrong\u003e24:\u003c/strong\u003e17610.\u003c/li\u003e\n\u003cli\u003eFujiyoshi S, Honda S, Ara M, Kondo T, Kobayashi N, Taketomi A: \u003cstrong\u003eSGLT2 is upregulated to acquire cisplatin resistance and SGLT2 inhibition reduces cisplatin resistance in hepatoblastoma.\u003c/strong\u003e \u003cem\u003eJournal of Hepato‐Biliary‐Pancreatic Sciences \u003c/em\u003e2024, \u003cstrong\u003e31:\u003c/strong\u003e223-233.\u003c/li\u003e\n\u003cli\u003eElahi Najafi MA, Yasui M, Teramoto Y, Tatenuma T, Jiang G, Miyamoto H: \u003cstrong\u003eGABBR2 as a Downstream Effector of the Androgen Receptor Induces Cisplatin Resistance in Bladder Cancer.\u003c/strong\u003e \u003cem\u003eInternational Journal of Molecular Sciences \u003c/em\u003e2023, \u003cstrong\u003e24:\u003c/strong\u003e13733.\u003c/li\u003e\n\u003cli\u003eYang C, Ou Y, Zhou Q, Liang Y, Li W, Chen Y, et al: \u003cstrong\u003eMethionine orchestrates the metabolism vulnerability in cisplatin resistant bladder cancer microenvironment.\u003c/strong\u003e \u003cem\u003eCell Death \u0026amp; 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This study sought to investigate the role of Empagliflozin (Empa) as an anti-diabetic medication in reversing Cisplatin (Cis) resistance in EJ138 bladder cancer (BC) cells.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eThe EJ138 cell line was cultured and divided into four groups: control, Cis-treated, Empa-treated, and Cis\u0026thinsp;+\u0026thinsp;Empa-treated groups. The effects of Cis and/or Empa on cell viability were determined using the MTT technique. The level of ROS produced by cells was evaluated using the green fluorescent dye dichloro-dihydro fluorescein (DCF). The expression of proteins involved in glucose transport, proliferation, apoptosis, cell cycle control, and invasion was evaluated by Western blotting. The Data were analyzed using GraphPad prism software and a One-way ANOVA test. All experiments were repeated three times. Data were presented as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. The significant difference between groups was calculated based on P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIC50 was calculated equal to 16 mM for Cis and 72 \u0026micro;g/ml for Empa. Treatment with Cis caused a significant increase in SGLT2 expression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Conversely, the group treated with 72 \u0026micro;g/ml Empa showed a significant decrease in SGLT2 compared with the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). ROS generation was significantly elevated after treatment with Cis, Empa, and their combination (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Treatment with Cis and/or Empa downregulated AKT, PI3K, mTOR, Bax, MMP-2, and MMP-9 expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, Bcl2, P21, and P53 expression showed a significant increase following Cis and/or Empa treatment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Protein expression differed significantly across the Cis-treated group and all other groups.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eEmpa exhibits beneficial anti-cancer activity against EJ138 cells. Empa boosts the anti-cancer activity of Cis in EJ138 BC cancer cells through SGLT2 inhibition.\u003c/p\u003e","manuscriptTitle":"Empagliflozin enhances cisplatin activity in chemo-resistant EJ138 bladder cancer cells: The importance of anti-diabetic medications in cancer treatment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-22 06:35:56","doi":"10.21203/rs.3.rs-4634713/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f271e4af-479d-4558-aa62-c233435ede8a","owner":[],"postedDate":"July 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-22T10:25:12+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-22 06:35:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4634713","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4634713","identity":"rs-4634713","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}