At the time of the first diagnosis, BCa, the second most prevalent malignancy in the urinary system is predominantly identified as NMIBC. The use of tobacco is a major factor that contributes significantly, along with exposure to hydrocarbons in the workplace, genetic vulnerability, and the consumption of drinking water contaminated with arsenic for NMIBC, the majority of patients experience recurrence but progression to MIBC is rare. As a result, TURBT is widely regarded as the primary therapy for NMIBC, with the potential addition of adjuvant bladder instillation therapy [20]. Conversely, individuals identified with MIBC experience a reduced five-year survival rate and diminished quality of life in contrast to those diagnosed with NMIBC[3]. Chemotherapy is a crucial component in the management of BCa. A research conducted by Liu and colleagues. The study showed that the use of immune checkpoint inhibitors, antibody-drug conjugates, and VEGF inhibitors in combination with chemotherapy drugs has the ability to improve both the rate of response and the overall survival of patients with BCa[21]. However, tumor cells can develop drug resistance due to drug stimulation and reduce the efficacy. Preoperative chemotherapy, also referred to as neoadjuvant chemotherapy (NAC), has the potential to eradicate micrometastases, lower the TNM stage of the tumor, enhance the likelihood of bladder-sparing surgery,boost patient survival rates, and decrease the risk of mortality[22]. Nevertheless, NAC may result in a delay in surgery for BCa patients who do not respond favorably to NAC, potentially increasing the risk of mortality. Hence, it is crucial to prioritize the creation of efficient and personalized treatment approaches to enhance the outlook for individuals with BCa.
Dixon et al. initially identified ferroptosis as a unique type of programmed cell death. Furthermore, it has been linked to the development of numerous human illnesses, such as cancers[11]. Ferroptosis is often characterized by the presence of excessive iron buildup within cells and the oxidation of lipids, which are widely acknowledged as significant aspects. In terms of morphology, cells undergoing ferroptosis display changes in the structure of mitochondria, such as decreased size, heightened density of the mitochondrial membrane, and the breakdown or lack of mitogpndrial cristae. In terms of biochemistry, ferroptosis is distinguished by the buildup of reactive oxygen species (ROS),increased amounts of lipid peroxides and Fe2+, and the suppression of GPX4 and the cystine/glutamate antiporter system (xCT) [23, 24]. GPX4, belonging to the glutathione peroxidase group, is a protein associated with ferroptosis and can be suppressed by RSL3, an inducer of ferroptosis. By catalyzing the conversion of GSH to its oxidized form, GSSG, GPX4 prevents ferroptosis by inhibiting lipid peroxidation and transforming dangerous lipid peroxides into harmless lipid alcohols, as well as converting H2O2 to H2O[25]. SLC7A11, a component of the xCT system, is also closely associated with ferroptosis. Multiple studies in BCa have shown that the dysregulation of GPX4 and SLC7A11 plays a role in triggering ferroptosis, as evidenced by previous research[26–28]. The distinguishing factors for ferroptosis, in comparison to other forms of regulated cell death, are these characteristic features.
Extensive research has been conducted on brusatol, a natural herbal medicine extract for the treatment of different types of tumors such as non-small cell lung cancer (NSCLC), glioblastoma, and pituitary adenomas[29–31]. brusatol demonstrates strong antineoplastic properties in these studies, either by directly impacting cancer cells or augmenting the efficacy of chemotherapy drugs. In NSCLC, a study identified 793 brusatol-binding potential proteins and found that brusatol could directly bind with Skp1.Consequently, brusatol-Skpl hindered the functioning of the Skp2-SCF E3 ligase and β-TRCP-SCF E3 ligase by interfering with the interaction between Skpl and the F box protein Skp2[29]. Moreover, the suppressive effect on cell growth and ability to spread to other parts of the body by brusatol has been linked to the buildup of p27 and E-cadherin. A different investigation indicated that brusatol inhibited the growth of glioblastoma cells by negatively controlling the expression of EMC1. Additionally, reducing EMC1 further increased the suppressive impact of brusatol[30]. Brusatol has demonstrated the ability to hinder the proliferation of pituitary adenoma cells and trigger apoptosis in both laboratory and living organisms when considering pituitary adenomas. The modulation of the mTORC1 signaling pathway and the buildup of ROS [31] mediate these impacts. Moreover, the pairing of brusatol and cabergoline has demonstrated an augmented antitumor impact in both pituitary tumor cells and nude mice. Brusatol has shown the capacity to hinder cell growth in laryngeal cancer by triggering apoptosis and halting the cell cycle in the S phase. Moreover, brusatol has been found to inhibit the process of EMT by suppressing the JAK2/STAT3 signaling pathway. [32]. Additionally, brusatol functions as a crucial Nrf2 inhibitor in the process of ferroptosis[33]. Nevertheless, the complete impact and fundamental mechanism of brusatol in BCa remain unclear and necessitate additional exploration. The study observed that brusatol caused a decrease in viability of T24 and 5637 BCa cell lines, which was dependent on its concentration. Furthermore, the use of brusatol effectively suppressed the growth and invasiveness of these cells. The results indicate that brusatol may have the capability to efficiently suppress the growth and spread of BCa cells. In order to examine the possible triggering of ferroptosis by brusatol, different concentrations of brusatol were administered to T24 and 5637 cells for investigation. The results demonstrated a dose-dependent induction of ferroptosis by brusatol. In particular, the administration of brusatol resulted in: 6 significant reduction in the cellular expression of GPX4, causing the buildup of reactive oxygen species (ROS), iron ions (Fe2+), and malondialdehyde (MDA), whereas the concentration of glutathione (GSH) noticeably declined. Moreover, the combination of brusatol with RSL3 intensified the effects, while the use of ferrostatin-1 rescued cell viability that was compromised by brusatol treatment. Furthermore, the findings demonstrated that brusatol caused a gradual reduction in mitochondrial cristae, which was dependent on the dosage. Taken together, these findings indicate that brusatol is capable of inducing ferroptosis in BCa cells in vitro.
Chacl belongs to the gamma-glutamyl cyclotransferase family, which specifically breaks down reduced GSH into cysteinylglycine and 5-oxoproline, while not affecting GSSG. Chacl possesses r-glutamyl cyclotransferase activity and serves as an antioxidant molecule by eliminating GSH[34]. Therefore, Chacl can affect ferroptosis in humans. Chacl plays an important physiological role in individual development. Furthermore, elevated expression of the Chacl gene was detected in various types of tumors and showed a positive association with unfavorable prognosis[19, 35]. Wang et al. discovered that artesunate has the potential to act as a potent growth inhibitor and induce ferroptosis in human Burkitt's lymphoma this effect is mediated through the ATF4-CHOP-Chacl pathway[36]. Moreover, the activation of the ATF4-Chacl pathway was observed in glioma cells when exposed to sevoflurane[35]. Dihydroartemisinin was found to trigger cell demise by inducing ferroptosis in primary liver cancer, and the treatment directly boosted the Chac1 promoter's effectiveness [37]. These researches suggested that Chac1 also has an important pathophysiological role. The study found that brusatol had a significant impact on increasing the expression of Chacl in BCa cells. According to the authors' understanding, this discovery is being reported here for the first time. Subsequently, an experimental cell line with reduced Chacl expression was employed to investigate the exact mechanism. Knocking down the expression of Chacl caused a notable rise in the expression of GPX4 and SLC7A11. This rescue of cell viability decline caused by brusatol also resulted in elevated ROS levels. This implies that Chacl plays a role in controlling ferroptosis. Moreover, it is widely recognized that Nrf2, a regulatory protein, has a vital function in the mechanism of ferroptosis. Several research studies have indicated that brusatol has the ability to function as a suppressor of Nrf2. However, the relationship between brusatol and Nrf2 in BCa has yet to be explored[38, 39]. Therefore, the expression level of Nrf2 was examined after treatment with brusatol to verify this hypothesis. The obtained results showed a decrease in the expression of Nrf2 after brusatol treatment. The reduction in Nrf2 expression was accompanied by a simultaneous decrease in the level of Chacl expression. Furthermore, Nrf2 has been documented to control the manifestation of SLC7A11.Therefore, it appears likely that brusatol activated the Chac1/Nrf2/SLC7A11 pathway to trigger ferroptosis
In order to validate the findings of the current research, experiments were conducted on BALB/c nude mouse xenografts to ascertain the impact of brusatol. The study findings revealed that the brusatol-treated group exhibited notably reduced tumor volume in comparison to the control group. This suggests that brusatol has a significant inhibitory effect on tumor growth in vivo. In order to evaluate cell proliferation in various groups, an IHC analysis was conducted, which demonstratet that brusatol significantly inhibited the proliferation of BCa cells in vivo. After treatment with brusatol, the expression of Chacl was observed to have increased, whereas the level of GPX4 decreased in contrast to the control group. These findings further support the hypothesis that brusatol can induce ferroptosis in vivo.