Bladder cancer is a common tumor type of the urinary system, which has high levels of morbidity and mortality. The first-line treatment is cisplatin-based combination chemotherapy, but a significant proportion of patients relapse due to the development of drug resistance. Therapy-induced senescence can act as a ‘back-up’ response to chemotherapy in cancer types that are resistant to apoptosis-based anticancer therapies. The circadian clock serves an important role in drug resistance and cellular senescence. The aim of the present study was to investigate the regulatory effect of the circadian clock on paclitaxel (PTX)-induced senescence in cisplatin-resistant bladder cancer cells. Cisplatin-resistant bladder cancer cells were established via long-term cisplatin incubation. PTX induced apparent senescence in bladder cancer cells as demonstrated via SA-β-Gal staining, but this was not observed in the cisplatin-resistant cells. The cisplatin-resistant cells entered into a quiescent state with prolonged circadian rhythm under acute PTX stress. It was identified that the circadian protein cryptochrome1 (CRY1) accumulated in these quiescent cisplatin-resistant cells, and that CRY1 knockdown restored PTX-induced senescence. Mechanistically, CRY1 promoted p53 degradation via increasing the binding of p53 with its ubiquitin E3 ligase MDM2 proto-oncogene. These data suggested that the accumulated CRY1 in cisplatin-resistant cells could prevent PTX-induced senescence by promoting p53 degradation.

The acidic tumor microenvironment stands as a major obstacle to the efficient elimination of tumor cells. Norcantharidin (NCTD) is a powerful antitumor agent with multiple bioactivities. However, the effect of NCTD under acidic conditions is still unclear. Here, we report that NCTD can efficiently kill bladder cancer (BC) cells in acidic culture, and more intriguingly, NCTD can induce immunogenic cell death (ICD), thereby promoting antitumor immunity. In NCTD-treated BC cells, the surface-exposed calreticulin (ecto-CALR) was significantly increased. Consistently, co-culture with these cells promoted dendritic cell (DC) maturation. The NCTD-induced ICD is autophagy dependent, as autophagy inhibition completely blocked the NCTD-induced ecto-CALR and DC maturation. In addition, the DC showed a distinct maturation phenotype (CD80high CD86low) in acidic culture, as compared to that in physiological pH (CD80 high CD86high). Finally, the NCTD-induced ICD was validated in a mouse model. NCTD treatment significantly increased the tumor-infiltrating T lymphocytes in MB49 bladder cancer mice. Immunizing mice with NCTD-treated MB49 cells significantly increased tumor-free survival as compared to control. These findings demonstrate that NCTD could induce ICD in an acidic environment and suggest the feasibility to combine NCTD with anticancer immunotherapy to treat BC.

Shikonin (SK) is the major bioactive component extracted from the roots of Lithospermum erythrorhizon with anticancer activity. SK could inhibit the epithelial-to-mesenchymal transition (EMT) of cancer cells. However, the underlying mechanism is elusive. In the present study, the inhibitory activities of SK on proliferation, invasion and migration were examined in bladder cancer (BC) cells. SK potently decreased the viabilities of BC cells but showed less cytotoxicity to normal bladder epithelial cells. Moreover, SK reversed the EMT, suppressed the migration and invasion of BC cells. Intriguingly, NHE1, the major proton efflux pump, was dramatically down-regulated by SK. The EMT-inhibitory effect of SK was mediated by NHE1 down-regulation, as NHE1-overexpress alleviated while Cariporide (NHE1 inhibitor) enhanced this effect. Further, enforced alkalinization of intracellular pH (pHi) reversed the EMT-inhibitory effect of SK, indicating a key role of acidic pHi in this process. Finally, elevated NHE1 expression was observed in human bladder cancer tissues. Collectively, this research reveals a supportive effect of NHE1 and alkaline pHi on EMT. SK can suppress EMT through inhibiting NHE1 and hence inducing an acidic pHi.

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