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

Abstract

Anti-diabetic medications have 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. The cells were cultured and divided into Cis-treated, Empa-treated, and Cis + Empa-treated cells. The effects of Cis and/or Empa on cell viability were determined using the MTT technique. The levels of ROS produced by cells were evaluated using the green fluorescent dye dichloro-dihydro fluorescein (DCF). The colorimetric method was used to measure Caspase 3/7 activity. The expression of proteins involved in glucose transport, proliferation, apoptosis, cell cycle control, and invasion was evaluated by Western blotting. The IC50 values for Cis and Empa were determined at 16 µM and 160 µM, respectively. ROS generation was significantly elevated after treatment with Cis, Empa, and their combination. Treatment with Cis caused a significant increase in SGLT-2 expression. Conversely, the group treated with Empa showed a significant decrease in SGLT-2 compared with the control group. The combination of Cis and Empa downregulated the expression of SGLT-2, AKT, PI3K, mTOR, Bcl-2, MMP-2, and MMP-9. However, Bax, P21, and P53 expression and caspase 3/7 enzyme activity showed a significant increase following Cis and Empa combination therapy. Empa exhibits beneficial anti-cancer activity against EJ138 cells. Empa boosts SGLT-2 inhibition and anti-cancer activity of Cis in EJ138 BC cancer cells.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-49196-6.

Introduction

Bladder cancer (BC) accounts for 3% of the global cancer diagnoses¹. BC is one of the most common cancers among the elderly². Certain risk factors, such as smoking, exposure to specific chemicals, persistent bladder infections, and a family history of the disease, are frequently linked to the development of bladder cancer³. BC symptoms may include blood in the urine, frequent urination, pain while urinating, and lower back pain⁴. Early detection and rapid treatment can improve BC prognosis⁵.

Cisplatin (Cis) is known to be used extensively in BC treatment⁶. Cis interferes with the DNA in cancer cells, preventing their ability to divide and grow⁷. Treatment of BC is severely hindered by Cis-based chemotherapy resistance^7,8. In this regard, several ways are being examined to improve the effectiveness of Cis-therapy⁹.

Sodium-glucose cotransporter-2 (SGLT-2) inhibitors are proposed as potential BC cell treatments. These medications improve insulin sensitivity, reduce hyperglycemia, and promote weight loss, all of which can contribute to lowering cancer risk¹⁰. Zhou et al. examined the effects of Dapagliflozin and Canagliflozin on the AMPK/mTOR pathway in breast cancer cells. They discovered that SGLT-2 inhibitors suppress the AMPK/mTOR signaling pathway in cancer cells¹¹. The dysregulated PI3K/Akt/mTOR pathway has implications for cancer cell proliferation, invasion, metastasis, and chemotherapy resistance¹².Furthermore, reduced PI3K/AMPK pathway activity suppresses glycolysis and reduces cancer cell proliferation¹². Research suggests that SGLT-2 inhibitors may improve chemotherapy resistance in cancer cells by reducing glucose intake. Fujiyoshi et al. employed Dapagliflozin, an SGLT-2 inhibitor, to overcome Cis resistance in hepatoblastoma cells¹³.

Empagliflozin (Empa) is a well-known SGLT-2 inhibitor recommended for diabetes control. It has been demonstrated that Empa has anticancer effects in cervical, breast, and hepatic cancer cells^14,15. Additional evidence suggests that Empa protects neurons from doxorubicin-induced damage in rat¹⁶. It has been proposed that Empa can play a dual role by promoting apoptosis in cancer cells while also protecting healthy cells from chemotherapy-induced destruction¹⁵.

However, the role of SGLT-2 inhibitors in cancer is controversial. Some studies have found a direct link between Empa and increased risk of BC¹⁷ in patients who suffered from diabetes. According to certain research, patients with a history of BC should utilize SGLT-2 inhibitors with caution¹⁸. An international multisite cohort study found comparable short-term BC risk between SGLT-2 inhibitor users and patients receiving other glucose-lowering therapies¹⁹. Yet, no significant correlation between SGLT-2 inhibitors and BC has been found in other research¹⁹. Further investigation is required to elucidate the exact role of SGLT-2 inhibitors in BC management^20,21.

The effect of Empa on BC cells has yet to be fully studied, exposing a study gap in the link between diabetes medicines and cancer. Exploring these effects could provide new insights into using Empa concurrently to treat diabetes and BC. Although direct experimental data in BC cell lines are still insufficient, the present evidence suggests that SGLT-2 inhibition alters metabolic and signaling pathways linked with BC chemoresistance, warranting additional exploration in BC-specific preclinical models²². We investigated the Empa effects on Cis activity in chemo-resistant EJ138 BC cells which are derived from a human bladder tumor carrying an activating mutation in Harvey Rat Sarcoma Oncogene Homolog (HRAS).

Results

The impact of Empa and/or Cis on cell viability

EJ138 BC cells were treated with Cis, Empa, or a combination of Cis and Empa for 72 h. According to data generated from triplicate biological and technical experiments, as Cis or Empa concentrations elevated, EJ138 cell viability decreased in a dose-dependent pattern (P < 0.05). The IC50 values for the cells treated with Cis and Empa were determined at 16 µM²³ and 160 µM²⁴ respectively. The IC50 of Empa was ten times higher than that of Cis (Fig. 1A and B).

Fig. 1.

Effects of Empa or Cis on EJ138 BC cell viability. (A) The EJ138 cells were exposed to 0, 1.62, 3.25, 7.5, 15, and 30 µM of Cis for 72 h. (B) The EJ138 cells were exposed to 0, 12.5, 25, 50, 100, and 200 µM of Empa for 72 h. Cell viability was measured using the MTT method. The data were normalized to untreated EJ138 cells grown in baseline media. All experiments were performed in triplicate.

The effects of Cis and/or Empa on the PI3K/Akt/mTOR pathway

According to data generated from triplicate biological and technical experiments, the expression levels of Akt, PI3K, and mTOR were downregulated by the Cis and Empa combination. The groups treated with Cis showed significantly reduced Akt, PI3K, and mTOR expression compared to the control group (P < 0.001) (Fig. 2A-C). Treatment with Empa presented a similar significant reduction in Akt, PI3K, and mTOR expression compared to the control group (P < 0.001) (Fig. 2A-C). The group treated with Cis and Empa combination significantly lowered Akt, PI3K, and mTOR expression compared to the Cis group (P < 0.001) (Fig. 2A-C). Representative. Western blot images are shown in Fig. 2E.

Fig. 2.

The effects of Cis, Empa or Cis+Empa on the proliferation-dependent and SGLT-2 protein expression in EJ138 BC cells. Western blot results are shown for Akt (A), PI3K (B), mTOR (C) and SGLT-2 (D). Significant difference compared to the control group (** P < 0.01, *** P < 0.001). Significant difference compared to the Cis group (### P < 0.001). Significant difference compared to the Empa group ($$$ P < 0.001). Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test.

The effects of Cis and/or Empa on SGLT-2 expression

According to data generated from triplicate biological and technical experiments, Cis increased SGLT-2 expression (P < 0.001) (Fig. 2D). SGLT-2 expression significantly reduced following Empa treatment (P < 0.01) (Fig. 2D). Treatment with Cis and Empa combination induced a significant reduction in SGLT-2 levels compared to the control group (P < 0.001) (Fig. 2D). Representative Western blot images are shown in Fig. 2E.

The effects of Cis and/or Empa on cell cycle regulator proteins

According to data generated from triplicate biological and technical experiments, there was a significant increase in P21 expression following exposure to Cis (P < 0.001) (Fig. 3A). However, the increase in P21 expression following Empa treatment was not significant (P > 0.05) (Fig. 3A). There was an increase in the expression of P53 following the exposure of Cis or Empa, compared to the control group (P < 0.001 and P < 0.01 conversely) (Fig. 3B). Also, Cells treated with a combination of Cis and Empa showed significantly increased expression of P53 and P21 compared to the control group (P < 0.001 in both) (Fig. 3A and B). Representative Western blot images are shown in Fig. 3E.

Fig. 3.

The effects of Cis, Empa or Cis+Empa on the cell cycle-regulator and invasion-dependent protein expression in EJ138 BC cells. Western blot results are shown for P21 (A), P53 (B), MMP-2 (C) and MMP-9 protein expression (D). Significant difference compared to the control group (*P < 0.05, *** P < 0.001). Significant difference compared to the Cis group (#<0.05, ##<0.01, ### P < 0.001). Significant difference compared to the Empa group ($$$ P < 0.001). Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test.

The effects of Cis and/or Empa on invasion-related proteins

According to data generated from triplicate biological and technical experiments, the expression of MMP-9 and MMP-2 was lowered by Cis and Empa treatment (P < 0.001 for both) (Fig. 3C and D). Treatment with Empa induced a significant reduction in the expression of MMP-9 (P < 0.05) (Fig. 3D), while the reduction in MMP-2 was not significant (P > 0.05) (Fig. 3C). The group treated with Cis and Empa combination showed significantly decreased levels of MMP-9 and MMP-2 compared to the control group (P < 0.001 for both) (Fig. 3C and D). The group that received both Cis and Empa revealed a decrease in MMP-2 and a significant decrease in MMP-9 expression compared to the group that was treated with Cis alone (P > 0.05, P < 0.05) (Fig. 3C and D). Representative Western blot images are shown in Fig. 3E.

The effects of Cis and/or Empa on apoptosis- related proteins

The EJ138 cells subjected to treatment with Cis, Empa, or their combination revealed significant changes in the expression of key apoptosis-regulating proteins. According to data generated from triplicate biological and technical experiments, the groups treated with Cis or Empa alone showed a significant increase in the expression of Bax and a significant decrease in the expression of Bcl-2 compared to the control group (P < 0.001 for both) (Fig. 4A and B). Cotreatment with Cis and Empa resulted in significantly higher expression of Bax compared to the Cis group (P < 0.001) (Fig. 4A). However, there was a significant decrease in Bcl-2 expression (P < 0.01) (Fig. 4B). Representative Western blot images are shown in Fig. 4D.

Fig. 4.

The effects of Cis, Empa or Cis+Empa on the apoptosis-dependent protein expression and Caspase 3/7 activity in EJ138 BC cells. Western blot results are shown for Bax (A) and Bcl2 (B). The ELISA results are presented for Caspase 3/7 (C). Significant difference compared to the control group (*** P < 0.001). Significant difference compared to Cis group (## P < 0.01, ### P < 0.001). Significant difference compared to the Empa group ($$$ P < 0.001). Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test.

The effects of Cis and/or Empa on apoptotic-related enzymes

According to data generated from triplicate biological and technical experiments, the groups treated with Cis or Empa alone showed a significant increase in the levels of caspase 3/7 compared to the control group (P < 0.001 for both) (Fig. 4C). The group that was treated with a combination of Cis and Empa showed significantly elevated caspase 3/7 compared to the Cis group (P < 0.001) (Fig. 4C).

The influence of Cis and/or Empa on ROS production

According to data generated from triplicate biological and technical experiments, the groups treated with Cis or Empa exhibited a substantial increase in ROS generation compared to the control group (P < 0.001 for both) (Fig. 5). The combination of Cis and Empa significantly increased ROS formation compared to the Cis-treated group (P < 0.001) (Fig. 5).

Fig. 5.

The effects of Cis, Empa or Cis+Empa on ROS production in EJ138 BC cells. Significant difference compared to the control group (*** P < 0.001). Significant difference compared to the Cis group (### P < 0.001). Significant difference compared to the Empa group ($$$ P < 0.001). Data were analyzed by one-way ANOVA followed by Tukey’s post hoc test.

Discussion

Cis resistance is a major issue in BC therapeutic management^6,25,26. Cis resistance may be triggered by several processes⁹. Cancer cells can create defenses against Cis absorption, which lowers the drug’s intracellular concentration and limits its efficacy²⁷. Additionally, cancer cells can use ATP-binding cassette (ABC) transporters to increase Cis efflux²⁸. Cis-resistant cancer cells adapt through increased antioxidant capacity, metabolic reprogramming, and modified survival pathways to avoid ROS-mediated death²⁹. Research suggests that Cis-therapy promotes SGLT-2 receptor expression, resulting in glucose uptake and chemoresistance in cancer cells³⁰.

Empa, a well-known diabetes medication, mitigates the risk of hypoglycemia in diabetic patients. Previous research has shown that Empa suppresses SGLT-2 expression^24,31.

In this project, we examined whether Empa alters the EJ138 BC cell line’s Cis resistance in vitro. We found that Empa increases the cytotoxic and anti-invasive properties of Cis. The results were linked to impairment of a glucose-dependent metabolic adaptation and coordinated stimulation of pro-apoptotic signaling.

The anti-cancer role of SGLT-2 inhibitors is controversial. Fujiyoshi et al. discovered that dapagliflozin effectively lowered chemoresistance in cancer cells¹³. On the contrary, a case report study claimed that Empa can increase the risk of BC among diabetic patients¹⁷. Some other studies demonstrated no significant association between SGLT-2 inhibitors and the risk of urothelial carcinoma³². A meta-analysis study of 27 clinical trials reported that diabetic patients treated with SGLT-2 inhibitors had no noteworthy rise in BC progression^33–35.

We propose that Empa’s chemical sensitizing impact comes from its interaction with stress-dependent metabolic reprogramming. Resistant cells are projected to increase glucose intake to satisfy the higher calorie needs and biosynthetic needs for DNA repair, redox maintenance, and pro-survival signaling during chemotherapeutic stress^6,34. In our research, Cis monotherapy decreased SGLT2 expression in EJ138 cells. Subsequently, Empa suppressed the overexpression of SGLT2 produced by Cis-therapy. This impact is consistent with Empa’s documented role as an SGLT-2 inhibitor. Empa reduces SGLT-2 expression through post-transcriptional feedback¹⁵. This synergy was characterized by a considerable increase in intracellular ROS levels. Treatment with SGLT-2 inhibitors causes oxidative stress and ROS accumulation, which leads to increased Bax/Bcl2 expression and tumor cell apoptosis³². Some studies, however, reported that Empa protects normal cells from chemotherapy cytotoxic effects. In this regard, Empa may play a dual role in cancer management^32–35.

In this investigation, Cis and Empa caused dose-dependent cytotoxicity in EJ138 BC cells. The Cis-treated cells had IC50 values of 16 µM ²³. The IC50 of Empa was calculated equal to 160 µM. This result is comparable to the IC50 = 177 µM for Empa in Naunyn et al. study²⁴.

The Cis/Empa combination therapy increased ROS levels, which can lead to DNA damage. Based on previous knowledge, DNA destruction promotes P53 activation while simultaneously downregulating the PI3K/Akt/mTOR axis³⁶. Activation of p53 begins a transcriptional program to boost P21 expression enhancing Bax/Bcl2 expression. and cell cycle arrest. The p53-mediated shift in the Bax/Bcl-2 ratio causes mitochondrial outer membrane permeabilization, cytochrome C release, and apoptosis. The increase in Caspase-9 activates Caspase-3/7 and promotes apoptosis^37,38. Our results indicate a suppression of the PI3K/Akt/mTOR pathway, activation of p53 and p21, an elevated Bax/Bcl‑2 ratio, and increased activity of caspases-3/7. The findings imply that the Cis/Empa combination therapy may suppress cell proliferation while promoting apoptotic activation in BC cells. The simultaneous reduction in MMP-2 and MMP-9 activity justifies cellular invasion prevention. Abdelhamid et al. reported that the Empa/metformin combination therapy inhibits hepatocellular carcinoma progression by suppressing MMP-2³⁹.

Our findings propose that SGLT2 could be a metabolic target in BC. The observed effects of Empa, including apoptosis activation and PI3K/Akt suppression, are consistent with the known anti-cancer effects of SGLT-2 inhibitors⁴⁰. Our findings suggest that SGLT-2 may be regarded as targetable objective in the Cis-resistant BC model, which is a new and potentially therapeutically beneficial aspect of our study²⁴.

The validity of our results appears to enhance by repeating the experiments in multiple BC cell lines as well as through in vivo studies. Furthermore, genetic approaches, such as SGLT2 knockdown in EJ138 cells are needed. The in vivo confirmation of the Cis/Empa combination effect on BC animal models is an important next step.

Conclusion

Empa enhances the anti-proliferative and anti-invasive characteristics of Cis in the Cis-resistant EJ138 BC cell line. These in vitro findings support the need for further preclinical and in vivo studies. SGLT2 inhibition may help overcome Cis resistance in preclinical models.

Materials and methods

Preparation and handling of chemical reagents

Cis

A 3.3 mM stock solution was prepared by dissolving Cis powder in sterile NaCl solution to a final concentration of 1 mg/ml. The stock was stored in the dark at − 20° C. Working concentrations (0, 1.62, 3.25, 7.5, 15, and 30 µM) were freshly diluted in cell culture medium immediately before each experiment.

Empa

Working concentrations (0, 12.5, 25, 50, 100, and 200 µM) were prepared by diluting a 50 mM stock in cell culture medium.

MTT

5 mg of MTT powder was dissolved in PBS and stored at − 20 °C. Immediately before use, the stock was diluted in serum-free medium to a final working concentration of 0.5 mg/ml.

DCFH-DA

A 10 mM stock solution was prepared and stored at − 80° C protected from light and moisture. On the day of the assay, an aliquot was thawed and diluted in warm, serum-free, phenol red-free HBSS to a final working concentration of 10 µM.

RIPA

A base solution was prepared from 50 mM Tris-HCl,150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS was prepared. The buffer was supplemented with 1x protease inhibitor cocktail and 1x phosphatase inhibitor cocktail to create the complete lysis buffer.

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 containing 20% FBS and 1% penicillin/streptomycin (Pen/Strep). The cells were incubated at 37° C containing 5% CO₂.

Cell viability 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 72 h. The cells were treated with varying concentrations of Cis (0, 1.62, 3.25, 7.5, 15, and 30 µM)²³ and Empa (0, 12.5, 25, 50, 100, and 200 µM)²⁴. Afterwards, the culture medium was replaced with 100 µL of 0.5 mg/ml MTT solution, and the plates were incubated for 4 h with 5% CO₂ in the dark. Then, each well received 100 µL of DMSO 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: % Viability = (absorbance test/absorbance control) × 100⁴¹. The IC50 values were calculated using GraphPad Prism software (GraphPad prism 9 Software, USA).

Study design

EJ138 cancer cells were divided into four experimental groups. Group I served as the untreated control. Group II treated with Cis. Group III was exposed to 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. The lysis buffer was added. Cell lysates were solubilized in an SDS-PAGE loading buffer. After heating at 95 °C for 10 min, the denatured proteins were transferred onto a nitrocellulose membrane blocked by 5% nonfat milk. The membrane was incubated with primary antibodies specific to SGLT-2 (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) for 2 h at room temperature. The membranes were washed three times with Tris-buffered saline. The secondary antibody (NB500-420) was incubated on the membranes for 2 h 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 density⁴².

Measurement of caspase 3/7 activity

Caspase 3/7 activity was determined by the Kiazist kit (Iran). The cells were trypsinized and incubated with lysis buffer at 4º C for 20 min. The samples were centrifuged at 4º C for 15 min. The supernatant was collected and used for the subsequent stages. The samples were incubated with DEVD-pNA at 37º C for 2 h. The absorbance of the samples was measured at 405 nm using an ELISA reader (Bio-Rad, USA). To draw the standard curve, concentrations of 0, 10, 20, 30, 40, and 50 nM were prepared from the standard sample and their OD was determined. The final concentrations of the samples were calculated using the standard curve.

ROS production assay

The levels of ROS production by the cells were determined using the Oxi Select TM assay kit (Sigma, USA) based on green fluorescent dye dichloro-dihydro fluorescein (DCF) production. The cells were grown on 96-well plates for 24 h at 37° C. Afterwards, the supernatant was discarded, and the samples were incubated with DA-DCFH at 37° C for 30 min. Then, 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)⁴³.

Statistical analysis

The statistical analysis was conducted using SPSS 26 software. GraphPad Prism 9 software was used to draw the figures. All experiments were performed in triplicate, ensuring the robustness and reliability of the results. Normal distribution was checked. Data were analyzed by one-way ANOVA (for normal symmetric distribution) followed by Tukey’s post hoc test (for asymmetric distribution). All experiments were performed in triplicate. Data are presented as Mean ± SD.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Author contributions

DD, SS and SM designed the research. S.MN and SS conducted experiments. DD, SS, and MF analyzed data. DD and MS wrote the manuscript. DD, MS and SM revised the manuscript. All authors read and approved the 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).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author.

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).

Competing interests

The authors declare no competing interests.