CAIX-HIF-1α Inhibition by 2-(4-Chlorophenyl)-3-(4-Ethoxyphenyl) Thiazolidin-4-one (BCS12) Activates Caspase Cascade and PARP Cleavage, Driving Mitochondrial Apoptosis in Hypoxic Breast Cancer

Introduction: Hypoxia is a hallmark of aggressive breast cancers, particularly Triple- Negative Breast Cancer (TNBC), where stabilization of Hypoxia-Inducible Factor-1α (HIF-1α) upregulates Carbonic Anhydrase IX (CAIX), thereby enabling tumor pH homeostasis, proliferation, and resistance to therapy. Selective CAIX inhibition, therefore, offers a promising therapeutic strategy. In this study, a thiazolidine derivative was identified as a selective inhibitor of CAIX with the use of integrated computational, in vitro, and in vivo studies to develop targeted therapy against TNBC.

Methods: A series of thiazolidinone derivatives was subjected to in silico molecular docking against human Carbonic Anhydrase isoforms. The most active compound, 2-(4-chlorophenyl)-3- (4-ethoxyphenyl)thiazolidin-4-one (BCS12), was tested and analyzed using 200 ns MD simulations to evaluate the stability of the complexes. Pharmacokinetic parameters were predicted using ADME profiling. Cytotoxicity was evaluated in MDA-MB-231 and MDA-MB-468 breast Cancer cells, with IC50 values of 15.49 μM and 18.81 μM, compared to 121.6 μM in noncancerous MCF-10A cells (n = 3, p < 0.05). Free Radical generation, mitochondrial membrane depolarization, and expression of Apoptosis markers (Bax, Bcl-2, Caspase 9, Caspase 7, and Caspase 3, and PARP) were quantified using mechanistic studies in hypoxia-adapted MDA-MB- 231 cells by RT-qPCR, western blotting, and immunofluorescence. The number of apoptotic cells was also determined by flow cytometry. The in vivo activity was assessed against DMBAinduced breast tumor-bearing rats by histopathology and TUNEL staining.

Results: BCS12 showed strong docking affinity as well as stable binding with active-site residues of CAIX in simulations. It preferentially suppressed TNBC cell viability, promoted oxidative stress, impaired the integrity of mitochondria, and activated an intracellular Apoptosis pathway. In vivo, BCS12 treatment induces an increase of TUNEL-positive nuclei in a dosedependent manner compared with the control, with significantly higher contrast index values at 20 mg/kg and 40 mg/kg (p < 0.0001). The apoptotic response was strongest in the 40 mg/kg group, and was associated with a marked decrease in tumor burden and restoration of tissue architecture. The lead compound reduced tumor mass, improved histological parameters, and increased TUNEL-positive apoptotic nuclei with an associated decrease in expression of CAIX and activation of pro-apoptotic proteins.

Discussion: Our findings suggest that BCS12 disrupts hypoxia-induced survival pathways through inhibition of CAIX to enhance TNBC cell sensitivity to Apoptosis. These data reveal a therapeutic benefit of selective CAIX inhibition to counteract hypoxia-induced therapy resistance and further insights into the mechanism underlying hypoxia-targeted therapies for aggressive breast Cancer.

Conclusion: BCS12 selectively inhibits CAIX, leading to the effective impairment of hypoxic adaptation and Apoptosis in TNBC. The in silico, in vitro, and pre-clinical model data together provide evidence for the candidacy of this compound as a lead candidate for hypoxia-targeted therapy of breast Cancer. Due to its unique chemical structure and therapeutic characteristics, BCS12 may be a candidate for patent filing for future translational development.