Reappraisal of the fundamental mechanisms of the sHA14-1 molecule as a Bcl-2/Bcl-XL ligand in the context of anticancer therapy: A cell biological study.

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HA14-1 is a small-molecule, stable B-cell lymphoma 2 (Bcl-2) antagonist that promotes apoptosis in malignant cells through an incompletely-defined mechanism of action. Bcl-2 and related anti-apoptotic proteins, such as B-cell lymphoma-extra-large [Bcl-XL]), are predominantly localized to the outer mitochondrial membrane, where they regulate cell death pathways. However, the notably short half-life of HA14-1 in vitro limits its potential therapeutic application. To address this limitation, a more stable analog, ethyl-2-amino-6-phenyl-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (sHA14-1), was developed. This study investigated the relationship between sHA14-1 and Bcl-2/Bcl-XL. The sHA14-1 molecule acts as a hormetic substance. Therefore, it is crucial to determine whether the hormetic zone corresponds to a putative therapeutic window, that is, the optimal concentration at which sHA14-1 selectively kills cancer cells overexpressing Bcl-2 or Bcl-XL while causing minimal damage to normal cells. Using classical cell biology and flow cytometry, we examined the main signaling pathways involving Bcl-2 or Bcl-XL, and their modification in the presence of sHA14-1. We showed that sHA14-1 exerted a dual effect on mitochondria: (i) it sensitized cells to increased permeability, and (ii) it inhibited adenosine diphosphate-stimulated respiration and uncoupled respiration. At relatively low concentrations, sHA14-1 induced mitochondrial swelling, reminiscent of "pore opening" but with distinct characteristics. Over 30 μM, sHA14-1 caused mitochondrial transition depolarization independent of permeability transition and cell death that resembled secondary necrosis (i.e., occurring after maximal mitochondrial permeability) rather than apoptosis. The balance between apoptotic and necrotic cell death induced by sHA14-1 was also evaluated. Our results suggested that sHA14-1 plays a multifunctional role, involving both mitochondria and the endoplasmic reticulum. Its actions are more complex than its originally intended role in targeting anti-apoptotic Bcl-2 family members, which may complicate its potential application as an anticancer therapy.