Curcumin chemo-sensitizes intrinsic apoptosis through ROS-mediated mitochondrial hyperpolarization and DNA damage in breast cancer cells.

Abstract

Background: Curcumin (CUR), a polyphenol phyto-compound extracted from turmeric rhizome (Curcuma longa), suppresses cancer by inducing apoptosis while also limiting cell survival and proliferation. This in vitro and in silico research work focuses on the synergistic sensitization of Doxorubicin (DOXO) on regulating ROS-mediated apoptosis of the breast cancer cells (MDA-MB-231 and MCF-7) in DOXO-CUR co-treatment. We observed dose-dependent cytotoxicity, increased ROS production, and mtDNA fragmentation by reduced membrane potential. The combined molecular docking of Bcl2, Bax, and Caspase3 proteins with DOXO and CUR with lower binding energies proves the stable interactions of protein-ligand complexes in the combination doses.

Methodology: Cell survival was measured by MTT and flow cytometry assays. Mitochondrial ROS production, mtDNA condensation, and MMP depletion were documented using fluorescence micrographs. The enrichment analysis of the ROS pathway genes by RT-qPCR (relative fold change) indicates the activation of Caspase3-mediated intrinsic apoptosis. Autodock 4.2 and Gromac 2022.4 were performed for in silico binding interaction and stability analysis.

Result & conclusion: Our study calculates the DOXO and CUR combination (0.33 + 33 μM in MDA-MB-231 and 0.14 + 14 μM in MCF-7) shows maximum growth inhibition (70-75 %) by elevated oxidative stress and reduced membrane potential, which suggests that CUR could be a potential therapeutic agent for treating breast cancers in near future. The method of apoptosis was further analyzed, where elevated cellular ROS level by CUR + DOXO combination therapy depleted mitochondrial membrane potential and enhanced the DNA condensation. The mitochondrial pro-apoptotic genes BAX, BAK, BIM, CASPASE9, and CASPASE3 and anti-apoptotic BCL2 gene expressions depicted triggering intrinsic apoptosis pathway, co-relating with the in silico molecular docking, simulation, and MM-PBSA energy calculations. The synergism between CUR and DOXO was also validated by increased binding affinity and reduced inhibitory constant against key proteins Bcl2, Bax, and Caspase3. Bcl2-DOXO showed BE: -5.03 and Bcl2-DOXO-CUR showed BE: -4.7. Whereas, Bax-DOXO binding energy was -5.49 and Bax-DOXO-CUR binding was -3.83. The most preferable synergistic binding was found with Caspase3 protein, where Caspase3-DOXO docking energy was -1.63 but Caspase3-DOXO-CUR combined docking energy was -3.51. The stability of protein-ligand complexes was accessed with MD simulations and binding free energy calculations, Bcl2-CUR-DOXO combination complex showed ∆G: -25.62, Bax-DOXO-CUR complex showed the maximum ∆G: -34.18, with Caspase-CUR-DOXO complex (∆G: -15.18), indicating the proteins most stable conformation while interacting with CUR + DOXO combination. The correlation between the in vitro and in silico analysis of apoptosis pathway components widens the further research scope for proteomics and organoid studies, which might be worthy for successful clinical trials.