Fig. 5.

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.)