Illustrations
Figure 1
Mg status regulates intracellular calcium levels, oxidative stress, TRPM7 expression, and calpain activity, but not calpain 2 protein expression in CT26 colon cancer cells.
Schematic of Mg blocking intracellular Ca entry through Ca channels (A) . CT26 cells were grown under various conditions for 48 h: 100% Mg media, 50% Mg media, 10% Mg media with 100% Ca (i.e. varying Ca:Mg ratios) or with varying Ca levels (100% Mg/100% Ca, 50% Mg/50% Ca, 10% Mg/10% Ca, i.e. keeping Ca:Mg ratio at 1), as indicated (B, C, D, F). In addition, CT26 cells were grown in 10% Mg media followed by 90% Mg supplementation (MgS) (10% Mg + 90% Mg or MgD/MgS) for 48 h (B, C ). See Methods for details. (B) Intracellular Ca concentrations were analyzed using the intracellular Ca indicator Fluo-4, AM, as described in the Methods (n = 6 per condition). Data are shown as mean relative fluorescence units (RFU) (±SD). (C) Oxidative stress (±H2 O2 stimulation, 100 μM) was determined using the DCFH-DA assay, as described in the Methods (n = 6 per condition). Data are shown as mean percent control-oxidative stress (±SD). CT26 expression of TRPM7 and β-tubulin proteins (D-E) and calpain 2 and β-tubulin proteins (F-G) were assessed by western blotting; representative western blots are shown with quantitation of western blots shown above (mean ± SD; (n = 4-6 per condition). (H) Cell-associated calpain activity levels were assessed after treating CT26 cells with 100% Mg, 50% Mg, or 10% Mg with 100% Ca or 50% Mg/50% Ca and 10% Mg/10% Ca for 6 h or 24 h, as indicated. Data are shown as mean (±SD) relative calpain activity corrected for cell protein (n = 4 per condition). *** = p < 0.001 vs. 100% Mg; ††† = p < 0.001 vs. 100% Mg; ‡‡‡ = p < 0.001 vs. 10% Mg. * = p < 0.05 vs. 100% Mg.
Figure 1
Figure 2
Mg deficiency (MgD) promotes N-FAK, p53 degradation and cleavage of IκBα, p65 in CT26 cells; expression returns when Mg supplementation (MgS) follows MgD. No loss of p53, N-FAK, IκBα, or p65 expression occurs when Ca:Mg ratio is kept at 1.
CT26 cells were grown in various Mg and Ca-containing media (n = 3-5 per condition) as indicated (and described in figure 1 and the Methods section) for 6 h, 12 h, 18 h, 24 h, and 48 h, as indicated (A-B ). Representative western blots for N-FAK, p53, IκBα, p65, and β-tubulin proteins are shown for CT26 cells analyzed at 48 h post 100% Mg, 50% Mg, 10% Mg, and MgD/MgS (A). Representative western blots for N-FAK, p53, IκBα, p65, and β-tubulin proteins are shown for CT26 cells analyzed at 6 h, 12 h, 18 h, and 24 h post 100% Mg, 50% Mg or 10% MgD (B) . CT26 cells were grown in 100% Mg/100% Ca, 50% Mg/50% Ca, or 10% Mg/10% Ca (maintaining Ca:Mg ratio at 1) (n = 3-4 per condition) for 48 h and analyzed for N-FAK, p53, IκBα, p65, and β-tubulin protein levels by western blotting (C).
Figure 2
Figure 3
Mg deficiency (MgD) promotes E-cadherin cleavage in CT26 cells; no cleavage occurs when Ca:Mg ratio is kept at 1. MgD also promotes CT26 cell migration.
CT26 cells were grown in various Mg and Ca-containing media (n = 3-4 per condition) as indicated (and described in figure 1 and the Methods section) for 6 h, 12 h, 18 h, 24 h, and 48 h, as indicated (A-B ). Representative western blots for E-cadherin and β-tubulin proteins are shown for CT26 cells analyzed at 6 h, 12 h, 18 h, 24 h, and 48 h post 100% Mg, 50% Mg, 10% Mg, and MgD/MgS (A). CT26 cells were grown in 100% Mg/100% Ca, 50% Mg/50% Ca, or 10% Mg media/10% Ca (keeping Ca:Mg ratio at 1) (n = 3-4 per condition) for 48 h and analyzed for E-cadherin and β-tubulin protein levels by western blotting (B). For scratch/migration assays CT26 cells were grown in 100% Mg with 100% Ca until confluence (n = 3 per condition), when the scratch was made and then photographed (t = 0 h); cells were washed, the media was replaced with 100% Mg media or 50% Mg media or 10% Mg media with 100% Ca (as indicated), and the cells were incubated and then photographed 48 h later (t = 48 h) (C). Quantification of the migration assay is shown as percent wound closure (mean ± SD)(D ). *** = p < 0.001 vs. 100% Mg.
Figure 3
Figure 4
Culturing CT26 cells in MgD media with high Ca:Mg ratios increases aneuploidy.
CT26 cells were grown in 100% Mg media (A) , 50% Mg media (B), or 10% Mg (C) media for 48 h, with 100% Ca-containing media (i.e. increasing Ca:Mg ratios, n = 2-3 per condition). CT26 cells were analyzed for aneuploidy after 48 h by flow cytometry (A-C ). Quantification % aneuploidy from A-C is shown as mean (±SD) in (D ). A similar set of CT26 cell cultures was assessed for aneuploidy after exposure to 100% Mg/100% Ca (E ), 50% Mg/50% Ca (F ), and 10% Mg/10% Ca (G ), keeping Ca:Mg ratio at 1 (n = 2-3 per condition). CT26 cells were analyzed for aneuploidy after 48 h by flow cytometry (E-G ). Quantification of aneuploidy from E-G is shown as mean (±SD) in (H). *** = p < 0.001 vs. 100% Mg.
Figure 4
Figure 5
Summary: Mg deficiency with high Ca:Mg ratios increases intracellular Ca levels, calpain activity, oxidative stress, genomic instability, and cell migration by activating various signaling pathways in CT26 colon cancer cells.
Figure 5
Auteurs
1 Elmezzi Graduate School of Molecular Medicine, Northwell Health, 350 Community Drive, Manhasset, 11030, New York, United States
2 Institute of Molecular Medicine, Feinstein Institutes for Medical Research, Northwell Health, 350 Community Drive, Manhasset, 11030, New York, United States
3 Donald and Barbara Zucker School of Medicine at Hofstra/Northwell, 500 Hofstra Blvd, Hempstead, 11549, New York, United States