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.