Necroptosis triggered by ROS accumulation and Ca overload, partly explains the inflammatory responses and anti-cancer effects associated with 1Hz, 100 mT ELF-MF in vivo.

Abstract

Whereas the anti-neoplastic activity of extremely low frequency magnetic fields (ELF-EMF) is well-documented in literature, little is known about its underlying anti-cancer mechanisms and induced types of cell death. Here, for the first time, we reported induction of necroptosis, a specific type of programed necrotic cell death, in MC4-L2 breast cancer cell lines following a 2 h/day exposure to a 100 Hz, 1 mT ELF-EMF for five days. For in vivo assessment, inbred BALB/c mice bearing established MC-4L2 tumors were exposed to 100 mT, 1 Hz ELF-EMF 2 h daily for a period of 28-day, following which tumors were dissected and fixed for evaluation of tumor biomarkers expression and types of cell death induced using TUNEL assay, Immunohistochemistry and H&E staining. Peripheral blood samples were also collected for assessing pro-inflammatory cytokine profile following exposure. An exaggerated proinflammatory response evident form enhancement of IFN-γ (4.8 ± 0.24 folds) and TNF-α (3.1 ± 0.19 folds) and number of tumors infiltrating lymphocytes (TILs), specially CD8 T cells (~20 folds), proposed occurrence of necroptosis in vivo. Meanwhile, exposure could effectively suppress tumor growth and expression of Ki-67, CD31, VEGFR2 and MMP-9. In vitro studies on ELF-EMF exposed MC-4L2 cells demonstrated a meaningful increase in phosphorylation of RIPK1/RIPK3/MLKL proteins and cleavage of caspase-9/caspase-3, confirming occurrence of both necroptosis and apoptosis. Complementary in vitro studies by treating ELF-EMF exposed MC-4L2 cells with verapamil (a calcium channel inhibitor), N-acetyl cysteine (a ROS scavenger) or calcium chloride confirmed the role of elevated intracellular calcium and ROS levels in ELF-EMF induced necroptosis.

AI evidence extraction

Main findings

In vitro, ELF-EMF exposure (100 Hz, 1 mT; 2 h/day for 5 days) increased RIPK1/RIPK3/MLKL phosphorylation and caspase-9/caspase-3 cleavage, consistent with necroptosis and apoptosis. In vivo, BALB/c mice with MC-4L2 tumors exposed to 1 Hz, 100 mT ELF-EMF (2 h/day for 28 days) showed suppressed tumor growth and reduced expression of Ki-67, CD31, VEGFR2 and MMP-9, alongside increased IFN-γ and TNF-α and increased tumor-infiltrating lymphocytes (notably CD8 T cells). Modulation with verapamil, N-acetyl cysteine, or calcium chloride supported roles for intracellular calcium and ROS in ELF-EMF–associated necroptosis in vitro.

Outcomes measured

• Necroptosis markers (RIPK1/RIPK3/MLKL phosphorylation)
• Apoptosis markers (caspase-9/caspase-3 cleavage; TUNEL)
• Tumor growth
• Tumor biomarker expression (Ki-67, CD31, VEGFR2, MMP-9)
• Pro-inflammatory cytokines (IFN-γ, TNF-α)
• Tumor infiltrating lymphocytes (including CD8 T cells)
• Intracellular calcium involvement (verapamil/calcium chloride modulation)
• ROS involvement (N-acetyl cysteine modulation)

Suggested hubs

• occupational-exposure (0.2)
  Study involves ELF magnetic field exposure parameters, but no specific occupational setting is described.

{
    "study_type": "animal",
    "exposure": {
        "band": "ELF",
        "source": null,
        "frequency_mhz": null,
        "sar_wkg": null,
        "duration": "In vitro: 2 h/day for 5 days; In vivo: 2 h/day for 28 days"
    },
    "population": "In vitro MC4-L2 breast cancer cell line; in vivo inbred BALB/c mice bearing established MC-4L2 tumors",
    "sample_size": null,
    "outcomes": [
        "Necroptosis markers (RIPK1/RIPK3/MLKL phosphorylation)",
        "Apoptosis markers (caspase-9/caspase-3 cleavage; TUNEL)",
        "Tumor growth",
        "Tumor biomarker expression (Ki-67, CD31, VEGFR2, MMP-9)",
        "Pro-inflammatory cytokines (IFN-γ, TNF-α)",
        "Tumor infiltrating lymphocytes (including CD8 T cells)",
        "Intracellular calcium involvement (verapamil/calcium chloride modulation)",
        "ROS involvement (N-acetyl cysteine modulation)"
    ],
    "main_findings": "In vitro, ELF-EMF exposure (100 Hz, 1 mT; 2 h/day for 5 days) increased RIPK1/RIPK3/MLKL phosphorylation and caspase-9/caspase-3 cleavage, consistent with necroptosis and apoptosis. In vivo, BALB/c mice with MC-4L2 tumors exposed to 1 Hz, 100 mT ELF-EMF (2 h/day for 28 days) showed suppressed tumor growth and reduced expression of Ki-67, CD31, VEGFR2 and MMP-9, alongside increased IFN-γ and TNF-α and increased tumor-infiltrating lymphocytes (notably CD8 T cells). Modulation with verapamil, N-acetyl cysteine, or calcium chloride supported roles for intracellular calcium and ROS in ELF-EMF–associated necroptosis in vitro.",
    "effect_direction": "mixed",
    "limitations": [],
    "evidence_strength": "low",
    "confidence": 0.7399999999999999911182158029987476766109466552734375,
    "peer_reviewed_likely": "yes",
    "keywords": [
        "extremely low frequency magnetic field",
        "ELF-EMF",
        "ELF-MF",
        "breast cancer",
        "MC4-L2",
        "BALB/c mice",
        "necroptosis",
        "apoptosis",
        "RIPK1",
        "RIPK3",
        "MLKL",
        "ROS",
        "calcium overload",
        "IFN-γ",
        "TNF-α",
        "tumor infiltrating lymphocytes",
        "CD8 T cells"
    ],
    "suggested_hubs": [
        {
            "slug": "occupational-exposure",
            "weight": 0.200000000000000011102230246251565404236316680908203125,
            "reason": "Study involves ELF magnetic field exposure parameters, but no specific occupational setting is described."
        }
    ]
}

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