Inhibition of Regulatory Volume Decrease Enhances the Cytocidal Effect of Hypotonic Shock in Hepatocellular Carcinoma

Abstract

Background: Hypotonic shock induces cytocidal effects through cell rupture, and cancer therapy based on this mechanism has been clinically administered to hepatocellular carcinoma patients. We herein investigated the effectiveness of hypotonic shock combined with the inhibition of regulatory volume decrease as cancer therapy for hepatocellular carcinoma.Methods: Morphological changes in human hepatocellular carcinoma cell lines were observed under a differential interference contrast microscope connected to a high-speed digital video camera. Cell volume changes under hypotonic shock with or without chloride, potassium, or water channel blockers were observed using a high-resolution flow cytometer. In order to investigate cytocidal effects, the number of surviving cells was compared after exposure to hypotonic solution with and without each channel blocker (re-incubation experiment).Results: Video recordings showed that cells exposed to distilled water rapidly swelled and then ruptured. Cell volume measurements revealed regulatory volume decrease under mild hypotonic shock, whereas severe hypotonic shock increased the number of broken fragments as a result of cell rupture. Moreover, regulatory volume decrease was inhibited in cells treated with each channel blocker. Re-incubation experiments showed the cytocidal effects of hypotonic shock in cells exposed to hypotonic solution, and additional treatments with each channel blocker enhanced these effects.Conclusion: The inhibition of regulatory volume decrease with chloride, potassium, or water channel blockers may enhance the cytocidal effects of hypotonic shock in hepatocellular carcinoma. Hypotonic shock combined with the inhibition of regulatory volume decrease was a more effective therapy than hypotonic shock alone.

The Proposal of Molecular Mechanisms of Weak Organic Acids Intake-Induced Improvement of Insulin Resistance in Diabetes Mellitus via Elevation of Interstitial Fluid pH

“…Bodily and cellular functions are controlled by various factors including ions such as Na + , K + , Ca 2+ , Mg 2+ , Zn 2+ , and Cl − [ 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 , 85 , 86 , 87 , 88 , 89 , 90 , 91 , 92 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 ]. On the one hand, pH (−log[H + ]) is recognized as one of classical items considered to be already fully understood in roles and mechanisms of homeostasis.…”

Effect of low temperature on the regulation of cell volume after hypotonic shock in gastric cancer cells

“…Several in vitro and in vivo studies have investigated the cytocidal effects of hypotonic stress on cancer cells (6)(7)(8)(9). We examined the changes in the morphology and volume of cancer cells subjected to hypotonic shock using several methods, such as a differential interference contrast microscope connected to a digital video camera, and revealed that DW exposure rapidly increases cell volume followed by cell rupture in esophageal, gastric, colonic, pancreatic and liver cancer cell lines (12)(13)(14)(15)(16). Our research group also determined the therapeutic effects of a peritoneal injection of DW into nude mice for the treatment of peritoneal dissemination of GC (21).…”

“…Measurement of cell volume changes in GC cells after hypotonic shock using a high-resolution flow cytometer. Cell volume measurements were performed using a high-resolution flow cytometer (Cell Lab Quanta; Beckman Coulter, Inc.), as previously described (12)(13)(14)(15)(16). This flow cytometer was designed to measure the electronic volume (EV) of a cell, and EV data of >10,000 cells were collected and analyzed using Quanta control software (Beckman Coulter, Inc.).…”

“…cells with 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a Clchannel blocker, to increase cell volume by inhibiting RVD, and found that NPPB enhanced the cytocidal effects of hypotonic shock on various cancer cells, including GC cells (12)(13)(14)(15)(16). However, NPPB, which blocks multiple types of chloride channels, is neurotoxic in vivo; therefore, there is a need for development of a more specific, safe and simple method to inhibit RVD.…”

Methods for cell volume measurement

“…Cells are assumed to be approximately spherical; their diameters d or cross‐sectional areas are estimated and used to calculate the volume as ⅙πd 3 . Imaging is usually done in transmitted light but fluorescence can also be used . It is obvious that representing even a loosely attached cell with a sphere (rather than a spheroid or some other shape) requires a certain leap of faith from the experimenter, and direct testing of cell sphericity is rarely done .…”