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.

Free radical biology & medicine · 2021 · Mixed · Low evidence

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MixedLow evidencePaper 5695

Study snapshot

PublicationFree radical biology & medicine · 2021

Study typeanimal

Reported directionMixed

Evidence ratingLow

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.

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Study typeanimal

Effect directionmixed

PopulationIn vitro MC4-L2 breast cancer cell line; in vivo inbred BALB/c mice bearing established MC-4L2 tumors

Sample sizeNot supplied

ExposureELF · In vitro: 2 h/day for 5 days; In vivo: 2 h/day for 28 days

Evidence strengthLowConfidence: 74% · Peer reviewed: yes

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)

View raw extracted JSON

{
  "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": 0.74,
  "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": 0.2,
      "reason": "Study involves ELF magnetic field exposure parameters, but no specific occupational setting is described."
    }
  ]
}

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AI-extracted fields are generated from the abstract and metadata and may be incomplete or incorrect. This material is for informational purposes and is not medical advice.

Identifiers and source

DOI: 10.1016/j.freeradbiomed.2021.04.002
PubMed: 33857627

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