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  • br A br Sham br BCF br RCF

    2020-08-28


    A
    Sham
    BCF
    RCF
    H-Thymidine
    D
    Sham 231BrM
    BCF ns
    3H-ThymidineCPM (normalizedtoSham)
    3H-ThymidineCPM(normalizedtoSham)
    Vehicle Ethosuximide
    (normalizedtoSham)
    %decrease -10 ns
    hrs/day s/day
    hr
    Sham SKBrM3
    BCF
    ns
    3H-Thymidine CPM
    Vehicle Ethosuximide
    C
    % inhibition
    E
    (normalized
    to
    (Normalized
    Sham 231BrM
    BCF
    ns
    MCF7shXIST
    F
    3H-Thymidine(CPM)(normalizedtoSham)
    I
    Meanfluorescenceintensity(NormalizedtoSham) 1.6
    Sham SKBrM3
    BCF
    ns
    G
    SKBrM3shCAV3.2 Sham BCFH
    Count
    Sham BCF
    m
    m F F
    J
    a a
    Kh C h C
    Fluo-4 staining
    Fluo-4 staining S B S B
    Meanfluorescenceintensity(NormalizedtoSham) 1.6
    ns
    ns
    p-JNK
    Sham BCF Sham BCF 0.8
    Sham BCF Sham BCF
    -tubulin
    + Calcium - Calcium
    media
    media shScr sh3.2
    SKBrM3
    BCF
    Sham
    L ShamBCF
    BCF
    CamKII
    -tubulin
    M
    BCF
    N
    Enrichment Score  P38 MAPK signature
    O
    time as in clinical and in vivo studies [15,17], for a period of 7 days. A sig-nificant reduction in cell proliferation was observed at day 7 when cells were treated with BCF but not by RCF (Fig. 3A). We have previously determined that 3 h and 6 h but not 1 h daily exposure for one week re-sults in inhibition of hepatocellular cancer cell proliferation [16]. Similar inhibition was obtained for SKBrM3 cells treated with BCF 3 h or 6 h daily with no inhibition observed with 1 h daily (Fig. 3B). BCF treatment also decreased colony forming ability of Paclitaxel metastatic cells (Supple-mentary Fig. 1G). In addition, BCF inhibited growth of highly brain-tropic cell lines; MCF7 and ZR-75-1 in which XIST expression was knocked-down [23] (Supplementary Fig. 1H). Similarly, the growth in-hibitory effect of BCF was evident in multiple breast cancer cell lines (Fig. 3C). In contrast, treatment of normal breast cell, HMEC, brain-resident microglia, HMC3, and non-malignant immortalised breast cells, MCF10A, did not show any growth suppressive effect, providing strong support for the breast cancer-specific effect of BCF (Supplemen-tary Fig. 1I). The decrease in cell proliferation was not associated with cell death, as BCF treatment did not induce apoptosis in brain metastatic breast cancer cells (Supplementary Fig. 1 J).
    We previously reported that cellular calcium flux was affected by low energy electromagnetic fields, which were amplitude-modulated at specific frequencies [24–26]. In our study of hepatocellular carci-noma, we discovered that T-type voltage gated calcium channels (VGCC) mediate hepatocellular carcinoma specific AM RF EMF cell growth inhibition of hepatocellular carcinoma cell lines and xenografts [13]. Therefore, we treated breast cancer cells with ethosuximide, a pan T-type VGCC inhibitor, and found that the growth suppressive effect of BCF was significantly abrogated (Fig. 3D). We then knocked-down T-type channels by shRNAs (Supplementary Fig. 2A) and examined the ef-fect of BCF. As shown in Fig. 3E and F, knock down of CACNA1H (Cav3.2 isoform), but not CACNA1G (Cav3.1 isoform) or CACNA1 (Cav3.3 iso-form), rescued the suppressive effect of BCF in 231-BrM and SKBrM3 cells. Knockdown of CACNA1H also abrogated in vivo inhibitory effect of BCF when cells were injected systemically (Fig. 3G). T-type channels are transiently activating plasma membrane channels that mediates cal-cium influx upon their activation [27]. Concomitantly, BCF treatment in-duced intracellular calcium influx, which was nullified in the presence of ethosuximide or when cells were cultured in calcium free media, Paclitaxel in-dicating that BCF mediates calcium influx through T-type channels (Fig. 3H-J). This effect was specific to BCF as RCF treatment showed no effect in intracellular calcium level (Supplementary fig. 2B). Addition-ally, treatment with BCF did not alter intracellular calcium level of nor-mal glia (HMC3) and muscle cells (Supplementary 2C). These results suggest that BCF specifically target breast cancer CACNA1H resulting in calcium influx restricted to the breast cancer cells.
    Intracellular and extracellular calcium level is precisely controlled in cells and it is involved in regulating various pathways as well as in stress signalling [28]. Calcium influx disrupts intracellular calcium homeosta-sis and induces various stress-responding protein kinases, such as, p38 MAPK and JNK that are known to play a critical role in cell cycle arrest and growth inhibition [29,30]. Therefore, we examined signalling from