Obstet Gynecol Sci.  2022 Nov;65(6):531-541. 10.5468/ogs.22108.

Triphenyl phosphate activates estrogen receptor α/NF-κB/ cyclin D1 signaling to stimulate cell cycle progression in human Ishikawa endometrial cancer cells

• Kwon HY
• Park SB
• Han M
• Park JW
• Lee Y
• Han Sj
• Kwon Y
• Cho YJ

• KMID: 2535961
• DOI: http://doi.org/10.5468/ogs.22108

Abstract

Objective
Triphenyl phosphate (TPHP) is one of the most commonly used organophosphorus flame retardants that may accumulate in the environment. However, its effects on human reproductive organs have not been well studied. We aimed to investigate the in vitro effects of TPHP in human Ishikawa endometrial cancer cells to elucidate how TPHP exposure disrupts intracellular signaling and cell proliferation in reproductive tissues.
Methods
Human Ishikawa endometrial cancer cells were exposed to TPHP.
Results
Exposure to TPHP elevated the levels of estrogen receptor (ER) α and progesterone receptor-B and reduced ER β in human Ishikawa endometrial cancer cells. TPHP stimulated phosphoinositide 3-kinase/protein kinase B and mitogenactivated protein kinase/ extracellular signal-regulated kinases 1/2 kinase signaling, which may contribute to the activation of ER function and induce nuclear translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in human Ishikawa endometrial cancer cells. Activated ER and NF-κB stimulate the expression of cyclin D1/ cyclin-dependent kinase (CDK) 4/CDK6, indicating cell cycle progression and proliferation.
Conclusion
This report may provide new information on the molecular mechanisms underlying how TPHP exposure dysregulates the cellular physiology of the human endometrium.

Keyword

Triphenyl phosphate; Estrogen receptor; Cyclin D1; Cell cycle; Human Ishikawa endometrial cancer cells

Figure

• 

  Fig. 1 Expression of hormone receptors in triphenyl phosphate (TPHP)-treated human Ishikawa endometrial cancer cells. (A) Effects of TPHP on estrogen receptor (ER) α, ER β, PR-A, and PR-B protein expression, as evaluated by western blotting. (B) Proteins were quantitated by densitometry and normalized to their respective β-actin loading controls. Data were quantified using ImageJ software (Bethesda, MD, USA). (C) Confocal microscopy of ER α immunofluorescence staining. acquired at ×50. Green: ER protein stained with an anti ER α antibody and secondary antibody labeled with Alexa-488. Blue: nucleus stained with 4′-6′diamidino-2-phenylindole dihydrochloride (DAPI). The data are presented as means±standard error of means from at least three separate experiments. An asterisk (*) indicates significant difference from the control (P ≤0.05). PR, progesterone receptor.

• 

  Fig. 2 Expression of cell signaling proteins in triphenyl phosphate (TPHP)-treated human Ishikawa endometrial cancer cells. (A) Proteins (ERK, protein kinase B [AKT], p38, polyclonal phosphoinositide 3-kinase [PI3K]) were quantitated by densitometry and normalized to their respective β-actin loading controls. Data were quantified using ImageJ software. (B) Proteins (nuclear factor kappa-light-chain-enhancer of activated B cells [NF-κB], inhibitor of κB [I-κB]) were quantitated by densitometry and normalized to their respective β-actin loading controls. Data were quantified using ImageJ software. (Bethesda, MD, USA). (C) Confocal analysis of immune fluorescence staining of NF-κB. acquired at ×500. Red: NF-κB protein stained with an anti-NF-κB antibody and secondary antibody labeled with Texas Red. Blue: nucleus stained with 4′-6′diamidino-2-phenylindole dihydrochloride (DAPI). The data are presented as means±standard error of means from at least three separate experiments. An asterisk (*) indicates significant difference from the control (P ≤0.05).

• 

  Fig. 3 mRNA expression of cell cycle markers in triphenyl phosphate (TPHP)-treated endometrial cancer cells. Real-time polymerase chain reaction showing cyclin A2, B1, B2, D1, and D3, and cyclin-dependent kinase (CDK) 1, 2, and 4 expression levels in dimethyl sulfoxide- or TPHP-treated human Ishikawa endometrial cancer cells. (A) Cyclin D/CDK4 is required for G1/S cell cycle transition and can also be used as a G2/M checkpoint marker; (B) cyclin A/CDK2 have the highest abundance at the G2 phase of the cell cycle, and (C) cyclin B/CDK1 are highest at the M phase of the cell cycle. Data were normalized to the internal mRNA levels of glyceraldehyde 3-phosphate dehydrogenase. The data are presented as means±standard error of means from at least three separate experiments. An asterisks (*) indicates significant difference from the control (P ≤0.05).

• 

  Fig. 4 Protein expression of cell cycle markers in triphenyl phosphate (TPHP)-treated human Ishikawa endometrial cancer cells. (A) Proteins (cyclin D1, cyclin D3) were quantitated by densitometry and normalized to their respective β-actin loading controls. Data were quantified using ImageJ software. (Bethesda, MD, USA) (B) Effects of TPHP on cell cycle distribution in human Ishikawa endometrial cancer cells were analyzed using flow cytometric analysis. The cells were incubated with dimethyl sulfoxide or TPHP for 48 hours. Control and drug-treated cells were ethanol-fixed and stained with propidium iodide solution. (C) G0/G1, (D) S, and (E) G2/M phases. The data are presented as means±standard error of means from at least three separate experiments. An asterisk (*) indicates significant difference from the control (P≤0.05).

• 

  Fig. 5 A model of triphenyl phosphate (TPHP) activation pathway in human Ishikawa endometrial cancer cell. TPHP stimulates estrogen receptor (ER) α, PRs and triggers PI3K/AKT complex assembly. TPHP treatment induces ERK activity and the phosphorylation of p38 and then reduces PPAR-γ and I-κB. This results in an increase in the level of NF-κB, which up-regulates cyclin D1, leading to cell cycle progression. NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; PI3K, polyclonal phosphoinositide 3-kinase; AKT, protein kinase B; PR, progesterone receptor; PPAR-γ, peroxisome proliferator-activating receptor-γ; I-κB, inhibitor of κB.

Reference

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