Fig 1: The inhibitory effect of BCF is mediated through Cav3.2 T-type channel and CAMKII/p38 MAPK pathway. (A) 231BrM and SKBrM3 cells were treated with Sham or random frequencies (RCF) or BCF for 7 days followed by thymidine incorporation cell proliferatin assay (n = 5/group). (B) SKBrM3 cells were treated with 1 h, 3 h and 6 h-per day for 7 days and cell proliferation was examined at day 7 by thymidine incorporation assay. (C) Various cell lines were seeded on 96-well plates at day 7 after Sham or BCF treatment, and cell proliferation was examined at day 1, 3 and 5 by MTS assay (n = 8/group). (D) 231BrM and SKBrM3 cells were exposed to Sham or BCF daily for 7 days in the presence of ethosuximide or vehicle, and cell proliferation was quantified by thymidine incorporation at day 7 (n = 5/group). Results are normalised to Sham group. (E-F) The T-type voltage-gated calcium channel subunit genes, Cav3.1, Cav3.2 or Cav3.3, were knocked down in 231BrM or SKBrM3 cells by shRNAs, and they were treated with BCF for 7 days followed by quantifying cell proliferation at day 7. (G) SKBrM3shCav3.2 cells were intracardially injected to NOD/SCID mice. At Day 30, ex vivo tumour signal in brain was quantified by bioluminescence. Right panel shows representative brain images. (H) Cells treated with Sham or BCF were stained with Fluo-4 calcium dye and the level of intracellular calcium level was examined using flow cytometry. The representative histogram is shown. (I) SKBrM3 cells treated with Sham or BCF in the presence of vehicle or ethosuximide were stained with Fluo-4-am dye and cytoplasmic calcium level was quantified by flow cytometry (n = 6/group). (J) SKBrM3 cells cultured in media with or without calcium and examined for cytoplasmic calcium as in I. (K) The expression of total and activated p38 and phosphorylated JNK was examined in 231BrM cells treated with Sham or BCF for 7 days by western blot. α-tubulin was used as a loading control. (L) 231-BrM cells were treated with Sham or BCF or BCF in the presence of KN93 (5 μM) and protein levels of phospho-CAMKII, total CAMKII, phopho-p38 and total p38 were examined by western blot. (M) SKBrM3 cells treated with Sham or BCF for 7 days were subjected to cell cycle analysis using FACS. % of population in G1, S and G2 phase are shown (n = 5/group). (N) Enrichment of biocarta P38MAPK signature in breast cancer patients with or without brain metastasis incidence was analysed by GSEA. (O) p38WT or p38 D176A (active mutation) gene or vector control was ectopically expressed in SKBrM3 cells using retrovirus and expression, and cells were seeded on 96-well plate (n = 500/well) and subjected to cell proliferation assay using MTS reagent at Day 5 (n = 8/group). *, P-value<.05, ** P-value<.01 and ***, P-value<.0001.
Fig 2: HCCMF antiproliferative effects on HCC cells and downregulation of CSCs are mediated by Cav 3.2 T-type voltage gated calcium channels (CACNA1H). a, Ca2+ chelation abrogates AM RF EMF-mediated inhibition of Huh7 cell proliferation. Huh7 cells were exposed to either randomly chosen (RCF) or hepatocellular carcinoma-specific (HCCMF) AM RF EMF 3 h daily for seven days prior to cell proliferation assays with tritiated thymidine incorporation. ANOVA followed by Tukey post hoc-test: [Anova: F = (3, 20) 8·258, p = 0·0009] showed that proliferation of cells exposed to HCCMF was significantly lower than cells exposed to RCF [Post-Hoc Tukey Test p = 0·0023]. b, T-type voltage gated calcium channel blockade with ethosuximide abrogates HCCMF inhibition of Huh7 cell proliferation. Huh7 cells were exposed to HCCMF 3 h daily for seven days prior to cell proliferation assays. Huh7 cells not exposed to AM RF EMF (SHAM) were used as controls. Experiments were performed in the presence or absence of ethosuximide (Ethos). ANOVA followed by Tukey Post Hoc-Test [Anova: F = (3, 36) = 13·06, p < 0·0001] indicates that only HCCMF block Huh7 cell proliferation [Post-Hoc Tukey Test: SHAM vs HCCMF p < 0·0001]. c, Inhibition of cell proliferation by HCCMF in Huh7 and Hep3B cells with selective knockdown of Cav 3·1, 3·2 and 3·3 T-type voltage gated calcium channels. The three T-type voltage-gated calcium channels Cav 3·1, Cav 3·2, and Cav 3·3 were selectively knocked down using siRNA targeting the CACNA1G (sh3·1), CACNA1H (sh3·2), and CACNA1I (sh3·3) genes, respectively. CACNA1I (sh3·3) expression in Hep3B cells was not detectable by qPCR, as noted in Cav isoform relative expression graph, hence knockdown was not attempted. Cell proliferation was assessed after exposure to HCCMF 3 h daily for seven days. Huh7 data (LEFT): shScramble (N = 5 for both groups) [Student's two-tailed t-test p value: 0·0092]; sh3·1 (N = 6 for both groups) [Student's two tailed t-test p value: < 0·0001]; sh3·2 (N = 6 for both groups) [Student's two-tailed t-test p value: 0·4948]; Sh3·3 (N = 6 for both groups) [Student's two-tailed t-test p-value: 0·0071]. Hep3B data (RIGHT): shScramble (N = 5 for SHAM and N = 6 for HCCMF) [Student's two-tailed t-test p-value: 0·0493]; sh3·1 (N = 6 for SHAM and N = 5 for HCCMF) [Student's two-tailed t-test p-value: 0·0443]; sh3·2 (N = 6 for both groups) [Student's two-tailed t-test p-value: 0·1691]. (Lower): Basal expression levels of T-type voltage-gated calcium channels isoforms Cav 3·1, Cav 3·2, and Cav 3·3 in multiple cell lines (RED circles signify targets with no detectable expression via qRT-PCR). d, Antiproliferative effects of HCCMF on HBV positive HCC cells. Cell proliferation was assessed in HCC cells exposed to HCCMF using a [3H] thymidine incorporation assay as described before. e, Effect of HCCMF on HCC cancer stem cells. The cancer stem cell population of Huh7 (upper panel) and Hep3B (lower panel) cells was assessed after one-week exposure to HCCMF. Control cells were not exposed to EMF. The population of cancer stem cells was significantly lower following exposure to HCCMF. (UPPER PAIR) Huh7 %CSC Data: SHAM (N = 4) and HCCMF (N = 4); [Student's two-tailed t-test p-value = 0·0091]. Huh7 Sphere formation: SHAM (N = 5) and HCCMF (N = 5); [Student's two-tailed t-test p-value = 0·0011]. (LOWER PAIR) Hep3B %CSC Data: SHAM (N = 4) and HCCMF (N = 3); [Student's two-tailed t-test p value = 0·0091]. Hep3B Sphere formation: SHAM (N = 7) and HCCMF (N = 6); [Student's two-tailed t-test p-value = 0·0011]. f, Effect of HCCMF on Cav 3·2 knockdown HCC cancer stem cells. The cancer stem cell population of Huh7 Cav3·2 knockdown (upper panel) and Hep3B Cav 3·2 knockdown (lower panel) cells was assessed after one week of exposure to HCCMF. Control cells were not exposed to EMF. The population of cancer stem cells was equal to or greater than the control group following exposure to HCCMF. (UPPER) Huh7 Cav 3·2 knockdown sphere formation: SHAM (N = 5) and HCCMF (N = 5); [Student's two-tailed t-test p-value = 0·2364]. (LOWER) Hep3B Cav 3.2 knockdown sphere formation: SHAM (N = 6) and HCCMF (N = 7); [Student's two-tailed t-test p-value = 0·0034]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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