Bacillus Calmette–Guérin (BCG) is widely used in the clinic to effectively treat superficial urinary bladder cancer. However, a significant proportion of patients who fail to respond to BCG risk cystectomy or death. Though more than 3 million cancer treatments with BCG occur annually, surprisingly little is known about the initial signaling cascades activated by BCG. Here, we report that BCG induces a rapid intracellular Ca²⁺ (calcium ion) signal in bladder cancer cells that is essential for activating the transcription factor nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) and for synthesizing and secreting proinflammatory cytokines, including interleukin 8 (IL‐8). A similar Ca²⁺ response was observed when cells were exposed to the supernatant of BCG. Studying cellular molecular mechanisms involved in the BCG signaling event, we found pivotal roles for phospholipase C and the Toll‐like receptor 4. Further assessment revealed that this signaling pathway induces synthesis of IL‐8, whereas exocytosis appeared to be controlled by global Ca²⁺ signaling. These results shed new light on the molecular mechanisms underlying BCG treatment of bladder cancer, which can help in improving therapeutic efficacy and reducing adverse side effects.

Background: Polycystin-2 (PC2), encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), functions as a calcium (Ca²⁺) permeable ion channel. Considerable controversy remains regarding the subcellular localization and signaling function of PC2 in kidney cells. Methods: We investigated the subcellular PC2 localization by immunocytochemistry and confocal microscopy in primary cultures of human and rat proximal tubule cells after stimulating cytosolic Ca²⁺ signaling. Plasma membrane (PM) Ca²⁺ permeability was evaluated by Fura-2 manganese quenching using time-lapse fluorescence microscopy. Results: We demonstrated that PC2 exhibits a dynamic subcellular localization pattern. In unstimulated human or rat proximal tubule cells, PC2 exhibited a cytosolic/reticular distribution. Treatments with agents that in various ways affect the Ca²⁺ signaling machinery, those being ATP, bradykinin, ionomycin, CPA or thapsigargin, resulted in increased PC2 immunostaining in the PM. Exposing cells to the steroid hormone ouabain, known to trigger Ca²⁺ oscillations in kidney cells, caused increased PC2 in the PM and increased PM Ca²⁺ permeability. Intracellular Ca²⁺ buffering with BAPTA, inositol 1,4,5-trisphosphate receptor (InsP₃R) inhibition with 2-aminoethoxydiphenyl borate (2-APB) or Ca²⁺/Calmodulin-dependent kinase inhibition with KN-93 completely abolished ouabain-stimulated PC2 translocation to the PM. Conclusions: These novel findings demonstrate intracellular Ca²⁺-dependent PC2 trafficking in human and rat kidney cells, which may provide new insight into cyst formations in ADPKD.

Radiation therapy is employed in treating various cancer types. However, depending on the type and stage of cancer, its benefits can be limited, and it may cause serious side effects that detrimentally affect the quality of life (QOL) for the patient. Preventing the side effects of radiation therapy can enhance the response to cancer treatment and improve the patient’s QOL. Independently, vitamin C and hydrogen have demonstrated potential in reducing the side effects of anticancer drugs and radiation therapy. Furthermore, both have been postulated to possess direct anticancer properties. However, the effects of their combined therapy remain underexplored. This study investigates the hypothesis that combining vitamin C and hydrogen efficiently prevents radiation-induced injuries. Survival rates were investigated in cancer cell lines (MDA-MB231 and GL261) and normal cells (HUVEC) treated with hydrogen, vitamin C and irradiation. Apoptosis was assessed using the FLICA test, and EMT gene expression was elucidated via the qPCR technique. In the normal cell line, the introduction of vitamin C and hydrogen to the culture medium boosted survival rates, exhibiting a radioprotective effect upon irradiation. In contrast, cancer cells showed a decreased survival rate with the introduction of vitamin C and hydrogen, which was further diminished with irradiation. Treated cancer cells showed signs of apoptosis. Caspase activity in viable cells was reduced by the combination treatment with vitamin C and hydrogen. Additionally, glioblastoma cells treated with this combination showed reduced EMT gene expression. This study reveals that a combined therapy of hydrogen and vitamin C offers radioprotective effects on normal cells and exerts direct anticancer effects on cancer cells, while also amplifying the anticancer effects of radiation. Importantly, this combined therapy attenuated the radiation-induced EMT signature in the GL261 murine glioma cell line, suggesting potential in diminishing treatment resistance and tumor invasion.

Background: Radiation therapy is widely used to treat cancer but frequently causes normal tissue toxicities that compromise patients’ quality of life, highlighting the need for strategies that protect healthy tissue without reducing antitumor efficacy. Here, we investigated whether a redox-modulating intervention combining vitamin C and molecular hydrogen could mitigate radiation-induced injury while preserving or enhancing anticancer effects. Methods: Normal endothelial cells together with breast cancer and glioma cell lines were treated with vitamin C and hydrogen before and/or after irradiation, followed by assessments of cell viability, apoptosis, reactive oxygen species (ROS), and epithelial–mesenchymal transition (EMT)-associated gene expression. To explore the potential clinical relevance of these findings, two patients undergoing radiotherapy received the combination treatment as supportive care. Results: In normal cells, the combination treatment increased post-irradiation viability and reduced intracellular ROS levels, consistent with a cytoprotective antioxidant effect. In contrast, cancer cells exhibited reduced viability and increased apoptosis following the combined treatment, effects that were further enhanced by irradiation. In glioma cells, the combination treatment also suppressed the expression of EMT-associated genes. In the two clinical cases, improvements in radiation-associated toxicities were observed, including dermatitis, oral mucositis, gastrointestinal symptoms, and hair loss, along with improved treatment tolerability. Conclusions: Collectively, these exploratory findings suggest that the combination of vitamin C and hydrogen produces distinct responses among the cell models examined, with increased viability and reduced ROS in HUVECs and reduced viability, increased apoptosis, and altered EMT-associated gene expression in the cancer cell lines. These findings warrant further investigation of this combination as a potential adjunct to radiotherapy.