Effect of CaS/DMSO dispersions in the cytoskeleton and focal adhesion points of human skin adherent benign and melanoma cells
Authors: Rodriguez Olga M. , Narváez Ramos Michelle A. , Diffoot Nanette , Castro Miguel E. 
Published: May 2022

CaS/DMSO has been the subject of work in our group because it dissociates spontaneously in proton rich environments to produce Ca2+ ions and H2S. Extracellular Ca2+ concentrations in the neighbourhood of 500nM can induce apoptosis. Smaller concentrations of H2S can also induce apoptosis. We present here our efforts to establish the effect of CaS clusters in the cytoskeleton and focal adhesion points of human skin adherent benign and melanoma cells. We hypothesize that if the CaS nanoclusters can induce programmed death, then we will observe a significant impact in the focal adhesion points of cancer cells. We have studied the effect of DMSO and diluted CaS/DMSO on the focal adhesion points and cytoskeleton of cancer and benign cells using a digital confocal microscope. Anti‐vinculin monoclonal antibody and FITC‐conjugated secondary antibodies were used to detect vinculin in the cell environment. Vinculin expression was found to increase in benign cells in the first 24 and 48 hours of incubation with 1% DMSO. Comparison with control experiments the DMSO appears to localize vinculin expression around benign cell nuclei as well as the boundaries of the cytoskeleton. The CaS/DMSO increase the focal adhesion points of the adherent benign cells. In contrast, vinculin expression decreased in human melanoma fibroblasts cells incubated with 1% DMSO‐ as compared to the corresponding melanoma control cells‐ in the first 24 hours. In contrast, vinculin expression of the melanoma cells increased and de‐localized to the cell boundary edges in the first 24 hours when incubated with the CaS/DMSO. The vinculin expression in the melanoma cells incubated with 1% DMSO was found to increase significantly 48 hours as compared to the control. As with the benign cells, the melanoma cells vinculin expression was found to be more localized around the nuclei at the 48‐hour mark as compared to the first 24 hours. Vinculin expression could barely be detected in the fluorescence microscope following incubation of the melanoma cells with the CaS dispersion for 48 hours. Thus, in contrast to the benign cells, the CaS/DMSO reduce the focal adhesion points of the adherent melanoma cell. TRITC conjugated phalloidin antibody was employed to study the effect of DMSO in the F‐actin associated with the cytoskeleton of human benign and melanoma adherent skin cells. The F‐actin expression in the benign cells incubated with 1 % DMSO was found to decrease in the first 24 hours and then to increase significantly 48 hours following incubations as compared to the control. The CaS/DMSO does not seem to have any noticeable effect in the benign cells following 24 or 48 hours of incubation as compared to the control. We are led to control that the CaS/DMSO does not affect the benign skin cells cytoskeleton. The F‐actin expression changed from a protein with linear‐like to branch morphology in the melanoma cells incubated with DMSO. The CaS/DMSO increased the F‐actin branching but reduced its overall expression. In summary the results indicate that CaS does not affect adversely the normal cell cytoskeleton and focal adhesion point expression in benign cells, but it significantly reduces the vinculin expression in human melanoma adherent cells. Furthermore, as these effects are not observed in the benign cells, we are led to conclude that the difference in extracellular pH between human skin benign and melanoma cancer cells results in the observed selectivity to affect the focal adhesion point.