Tert-Butyl Hydroperoxide Stimulated Apoptosis Independent of Prostaglandin E2 and IL-6 in the HTR-8/SVneo Human Placental Cell Line

Abstract

Significant gaps exist in our knowledge of how cellular redox status, sometimes referred to as oxidative stress, impacts placental trophoblasts. The present study used tert-butyl hydroperoxide (TBHP) as a known generator of reactive oxygen species (ROS) in the extravillous trophoblast cell line HTR-8/SVneo to examine the role of cellular redox disruption of prostaglandin E2 (PGE2) and the cytokine IL-6 in cell death. Cells were exposed to 0, 12.5, 25, or 50 μM TBHP for 4, 8, and 24 h to ascertain effects on cell viability, caspase 3/7 activity, PGE2 release, PTGS2 mRNA expression, and IL-6 release. Experiments with inhibitors included the cyclooxygenase inhibitor indomethacin, mitogen-activated protein kinase inhibitors (PD169316, U0126, or SP600125), or treatments to counter expected consequences of TBHP-stimulated generation of ROS (deferoxamine [DFO], butylated hydroxyanisole [BHA], and N,N′-diphenyl-1,4-phenylenediamine [DPPD]) using 24-h exposure to 50 μM TBHP. Cell viability, measured by ATP content, decreased 24% relative to controls with a 24-h exposure to 50 μM TBHP, but not at lower TBHP concentrations nor at earlier time points. Exposure to 50 μM TBHP increased caspase 3/7 activity, an indicator of apoptosis, after 8 and 24 h. Antioxidant treatment markedly reduced TBHP-stimulated caspase 3/7 activity, PGE2 release, and IL-6 release. TBHP-stimulated IL-6 release was blocked by PD169316 but unaltered by indomethacin. These data suggest that TBHP-stimulated IL-6 release and caspase 3/7 activation were independent of PGE2 yet were interrupted by treatments with known antioxidant properties, providing new insight into relationships between PGE2, IL-6, and apoptosis under conditions of chemically induced cellular oxidation.

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Acknowledgments

We thank Dr. Peter Mancuso and Dr. Jeff Martens for their constructive criticism and advice regarding this work, Joel Whitfield of the University of Michigan Immunologic Monitoring Core for cytokine analysis, and Faith Bjork for supportive laboratory assistance. Thank you to members of the Loch-Caruso lab for providing a thoughtful sounding board, especially Dr. Lauren Tetz.

Funding

This work was supported by the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), with a research project to RL-C (P42ES017198) training grant fellowship support to CSK and KAH (T32ES007062), and supplementary project support from the Michigan Center for Lifestage Environmental Exposure and Disease (P30ES017885). Additional fellowship support for KAH was from the Michigan Institute for Clinical & Health Research, funded by the National Center for Advancing Translational Sciences (NCATS), NIH (UL1TR000433). Support from a University of Michigan Rackham Graduate Student Research Grant is also gratefully acknowledged. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIEHS, NCATS, NIH, or the University of Michigan.

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Correspondence to Rita Loch-Caruso.

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Loch-Caruso, R., Korte, C.S., Hogan, K.A. et al. Tert-Butyl Hydroperoxide Stimulated Apoptosis Independent of Prostaglandin E2 and IL-6 in the HTR-8/SVneo Human Placental Cell Line. Reprod. Sci. (2020). https://doi.org/10.1007/s43032-020-00231-5

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Keywords

  • Oxidative stress
  • Reactive oxygen species
  • Trophoblasts
  • Apoptosis
  • Prostaglandins