Ca2+ protein alpha 1D of CaV1.3 regulates intracellular calcium concentration and migration of colon cancer cells through a non-canonical activity

Figures

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  Figure 1. Immunohistochemical analysis of α1D protein expression in colon tissues microarrays. (A) Proportion of samples showing no (white), low (light gray), medium (black grey) or strong (black) α1D staining in normal mucosa, compared with adenoma and adenocarcinoma samples. The number of samples per condition is indicated in brackets. α1D staining was stronger in adenoma (χ 2, p < 0.04) and adenocarcinoma tissues (χ 2, p < 0.001) compared to normal tissues and in adenoma tissues compare to adenocarcinoma tissues (χ 2, p < 0.03). (B) α1D protein of CaV1.3 expression in normal mucosa (left), adenoma (center) and adenocarcinoma (right) from a same sample (1:100 dilution, Red revelation, Hematoxylin counter-coloration). Magnification, ×100 (top), ×300 (bottom). (C) Proportion of samples showing no (white), low (light gray), medium (black grey) or strong (black) α1D staining according to the state of differentiation (right) or stage (left) of adenocarcinoma samples. No difference was observed according the state of differentiation of adenocarcinoma samples (Kruskall Wallis test, p = 0.5). An influence of pT stages was observed on α1D staining (Kruskall Wallis test, p = 0.008).

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  Figure 2. α1D protein regulates HTC116 cancer cell migration. (A) Representative cropped western blot of α1D protein expression in colon cancer cell lines (N = 3). Full-length western blots are included in the Supplementary Fig. 2A. Lower panel, levels of α1D proteins in colon cancer lines were determined by densitometry scanning to generate the values shown in the bar graph. Results are expressed as mean ± SEM. *p < 0.05 and **p < 0.01, (N = 3, Holm-Sidak test). (B) Histograms showing the effect of 10 µM verapamil and 10 µM nifedipine on HCT116 cell migration using inserts cell migration assays. The normalized cell number corresponds to the ratio of total number of cells in presence of drugs/total number of migrating cells in control experiments. Results are expressed as mean ± S.E.M. *Significantly different from control (p = 0.01, N = 3, n = 3, Mann Whitney test). (C) Histograms showing the effect of 10 µM verapamil (left), 10 µM nifedipine (middle) and 25 µM NK571 or 5 µM Mitotane (MDR blockers) (right) on HCT116 cell migration ability using a monolayer wound-healing assay. Results are expressed as mean ± S.E.M. *Significantly different from control (nifedipine: p = 0.001, N = 3, n =…

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  Figure 3. Lack of detectable voltage-dependent Ca2+ currents and cellular localization of α1D protein. (A,B) Current records during 600 ms pulses in the range of −70 to 110 mV from holding potential of −100 mV. Representative examples of current recording in presence of barium (20 mM) and Bayk 8644 (50 µM) in broken (A) and perforated (B) patch configurations. Whole cell currents are obtained in normal condition (B.1) and during the migration process (B.2). Arrows indicates zero current. (C) Fluorescence-activated cell sorter (FACS) analysis of α1D protein in HCT116 cell line permeabilized or not. Control staining is shown in light grey, and dark grey represent staining with anti-CaV1.3. Graph represents the percentage of cells (N = 6) expressing α1D protein in permeabilized (Total) and not permeabilized cells (Plasma membrane cell surface). (D) Subcellular distribution of α1D protein in HCT116 cell line. A primary anti-CaV1.3 antibody was used with a secondary AlexaFluor 488-conjugated anti-rabbit (green color). The short arrows indicate the location α1D protein in the edges membranes, and the long arrows show the presence of α1D protein in the cytosol.

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  Figure 4. α1D protein regulated basal cytosolic Ca2+ concentration of HCT116 cells. (A) left, example of measurement of F340/F380 recorded in control condition and in cell in which α1D was silenced. Middle, histograms showing mean ± S.E.M of F340/F380 with a significant differences between control and siα1D#1 conditions (F340/F380: p = 0.001, N = 5, n = 89 si-control; N = 6, n = 97 siα1D, Mann Whitney tests). Right, representative traces recorded in the presence or not of verapamil in control condition and in cell in which α1D was silenced (N = 3, n = 6). (B) Histograms showing mean ± S.E.M of [Ca2+]c with a significant differences between control and siα1D#1 conditions (p = 0.001, N = 3, n = 47 si-control; N = 3, n = 58 siα1D#1, Mann Whitney tests). (C) left, effect of high external concentrations of K+ (4 + 56 mM: K60 and 4 + 76 mM: K80) with or without 20 µM CyPPA on [Ca2+]c (N = 3, n = 17) and of high external concentrations of Na+ as osmotic control solutions (140 + 56: Na196 and 140 + 76: Na216). None of the changes induced a significant effect (N = 3, n = 10). (D) Time dependent measurements of [Ca2+]c with (2 Ca) or without (0 Ca) external Ca2+ solutions. (E) left, time dependent…

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  Figure 5. α1D protein regulated cytosolic Ca2+ concentration by inhibiting NCX. (A) left, the schema represents NCX working in its forward mode driving Na+ entry and Ca2+ efflux. Middle, Histograms showing the effect of 10 µM SEA0400 or 30 µM KBR7943 on F340/F380 ratios. Results are normalized to CT conditions and are expressed as mean ± S.E.M. *Significantly different from control (p = 0.05, N = 3, n = 12, Kruskal-Wallis one way analysis of variance. Right, Representative cropped Western blot of NCX1 and NCX3 in HCT116 colon cancer cells (N = 3). Full-length Western blots are included in the Supplementary Fig. 6. (B) Histograms showing the effect of 10 µM SEA0400 on TG slope, the TG area, the TG peak Ca2+ responses and the TG relaxation slope. Results are normalized to CT conditions and are expressed as mean ± S.E.M. *Significantly different from control (p = 0.001, N = 3, n = 12, Mann Whitney test). (C) Time dependent measurements of [Ca2+]c in the presence of 140 mM (Na140) or 10 mM (Na10) of external Na+ concentration. The schema represents NCX working in its reverse mode driving Na+ efflux and Ca2+ entry. Histograms showing the effect of reducing external Na+ concentration to 10 mM on…