We confirmed that Cl− channel blockers enhanced the cytocidal effects of hypotonic shock using a range of bladder cancer cells. Thus, intravesical therapy using sterile water with a Cl− channel blocker may be a feasible treatment option for NMIBC patients, thereby avoiding radical surgery.
We have previously revealed cytocidal effects in bladder cancer cells [9, 10]. However, the phenotypes were different in each bladder cancer cell line. Moreover, clinical research has demonstrated no significant differences in the recurrence-free rates between patients undergoing sterile water irrigation and control patients [11]. Thus, simply irrigating with sterile water may not be effective in clinical application.
Recent reports have shown the roles of ion transporters in cancer cells; numerous types of ion transporters affect various organs in cancer patients [12, 13, 14]. Cl− channels/transporters are important in cancer cells. Iitaka et al. reported that NPPB increases cell volume by inhibiting regulatory volume and enhancing the cytocidal effects of the hypotonic solution in gastric cancer cells [15]. The activation of Cl− channels is key in regulating the cell volume of several gastric cancer cells. The inhibition of Cl− channels during hypotonic shock enhances cell swelling, thereby enabling its cytocidal effects.
Mechanisms underlying the regulation of cell volume have been studied upon exposing cells to abrupt changes in extracellular osmolarity. Cells exposed to hypotonic extracellular fluids initially swell as more or less perfect osmometers but approach the original cell volume based on regulatory cell volume decrease (RVD). Cells exposed to hypertonic extracellular fluids initially shrink like almost perfect osmometers but approach original cell volume by regulatory cell volume increase. Ion transport across the cell membrane is the most efficient and rapid way of altering cellular osmolarity [16].
Several ion transport systems are activated by cell swelling during RVD. RVD is caused by the outflow of water accompanying the extracellular discharge of KCl. K+ channel, Cl− channel, or K+/Cl− co-transporter are the most frequently utilized transport systems for the release of KCl. Intracellular Cl− activity is important for the regulation of intracellular osmolarity. Intracellular Cl− activity decreases during osmotic cell swelling. However, osmotic cell shrinkage is expected to increase intracellular Cl− activity. Thus, Cl− transport plays an important role in human cells.
Therefore, this study investigated whether Cl− channel blockers enhance the effect of sterile water on bladder cancer cells. As in previous studies, bladder cancer cells differed in their sensitivity to sterile water-induced hypotonic shock. RT112 cells demonstrated limited hypotonic swelling with less cell rupture. However, the cytocidal effect of sterile water was enhanced in cells after treatment with the Cl− channel blocker. RT112 cells treated with the Cl− channel blocker showed faster swelling and rupture upon exposure to sterile water as compared to the phenotypes observed in control cells. Moreover, the percentage of live cells after exposure to sterile water with Cl− channel blocker decreased as compared to the control cells. These findings provide experimental evidence that the combination of sterile water with Cl− channel blocker was more effective in imparting hypotonic shock-induced cytocidal effects on cultured bladder cancer cells otherwise resistant to sterile water.
The high recurrence rate in NMIBC is attributed to the adhesion of free-floating tumor cells during TURBT. The most significant potential use of this treatment is in early post-operative instillation since floating tumor cells have the greatest contact area with sterile water. We believe that single bladder irrigation directly after TURBT is the most efficient. Thus, we plan on conducting a pilot study for single bladder irrigation with Cl− channel blocker and sterile water for 30 min each to achieve successful clinical application.
The major limitation of the present study is that the effect of sterile water and Cl− channel blocker on normal urothelium was not investigated. Nonetheless, our observations of the novel cytocidal effect of sterile water with the Cl− channel blocker against bladder cancer cell indicate that this is a potentially effective therapy useful for reducing the risk of intravesical recurrence in patients with NMIBC.