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<p>Many studies on the activation of the intrinsic apoptosis pathway often generalize findings across various cell lines without taking into account the potential cell line dependent variability at the molecular level. This study aims to highlight significant variations in caspase activation and inhibition by Inhibitor of Apoptosis Proteins (IAPs) across three human cell lines of HeLa, HEK293T, and A549 as examples. The induction of the intrinsic apoptosis is mimicked in the cell extracts by the addition of cytochrome c and dATP, and caspase activation was assessed by monitoring the procaspase cleavages using the Western blot. In addition, BIR3 domains of XIAP, NAIP and its C338S variant were included in the assays to compare caspase-9 responsiveness to inhibition by these proteins. Furthermore, inhibition of endogenous caspase-9 and its pure form by the above-mentioned BIR3 domains was compared. Our observations revealed dramatic variations in caspase activation and inhibitor sensitivity among the three cell lines. HeLa cells exhibited normal caspase activation as well as inhibition by XIAP-BIR-3, while being unaffected by NAIP-BIR-3 and its variant. A549 cells displayed no caspase activation upon apoptosis induction, while HEK293T cells demonstrated normal caspase activation but resistance to inhibitors. BIR3 domain of XIAP was found to be a much stronger inhibitor of caspase-9 as compared to NAIP. Our findings clearly suggest significant variations in the intrinsic apoptosis pathway with respect to both caspase-9 and -3 activation and IAP inhibition thus carry potential implications for toxicological studies aimed at apoptosis activation in cancer therapy.</p>

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Compromised Integrity of the Initial Stage of the Intrinsic Apoptosis Pathway in HEK293T and A549 but not HeLa Cells

  • 期刊论文

  • 发表日期：

    2025年9月17日

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  • Elham Vahdati,
  • Azra Rabbani-Chadegani,
  • Sina Fallah Kohan,
  • Bahman Zeynali,
  • Jamshid Davoodi

摘要

Many studies on the activation of the intrinsic apoptosis pathway often generalize findings across various cell lines without taking into account the potential cell line dependent variability at the molecular level. This study aims to highlight significant variations in caspase activation and inhibition by Inhibitor of Apoptosis Proteins (IAPs) across three human cell lines of HeLa, HEK293T, and A549 as examples. The induction of the intrinsic apoptosis is mimicked in the cell extracts by the addition of cytochrome c and dATP, and caspase activation was assessed by monitoring the procaspase cleavages using the Western blot. In addition, BIR3 domains of XIAP, NAIP and its C338S variant were included in the assays to compare caspase-9 responsiveness to inhibition by these proteins. Furthermore, inhibition of endogenous caspase-9 and its pure form by the above-mentioned BIR3 domains was compared. Our observations revealed dramatic variations in caspase activation and inhibitor sensitivity among the three cell lines. HeLa cells exhibited normal caspase activation as well as inhibition by XIAP-BIR-3, while being unaffected by NAIP-BIR-3 and its variant. A549 cells displayed no caspase activation upon apoptosis induction, while HEK293T cells demonstrated normal caspase activation but resistance to inhibitors. BIR3 domain of XIAP was found to be a much stronger inhibitor of caspase-9 as compared to NAIP. Our findings clearly suggest significant variations in the intrinsic apoptosis pathway with respect to both caspase-9 and -3 activation and IAP inhibition thus carry potential implications for toxicological studies aimed at apoptosis activation in cancer therapy.