Approximately 15% of globally diagnosed breast
cancers are designated as
triple negative breast cancer (TNBC). In this study, we investigated the effect of the natural compound, Bis(2- ethyl hexyl) 1H-pyrrole-3,4-dicarboxylate (TCCP), purified from Tinospora cordifolia on MDA-MB-231, a TNBC cell line. The pro-apoptotic nature of TCCP on MDA-MB-231 was determined by assessing various apoptotic markers. ROS generation, intracellular
calcium, mitochondrial membrane potential (ΔΨm),
MPTP,
cardiolipin peroxidation and
caspase activity were determined fluorometrically. BAX, BCL-2,
cytochrome c,
caspases, and p53
protein expressions were determined by immunoblotting. Further, the effect of TCCP on
DNA and cell death was determined by DNA fragmentation assay,
annexin-V staining, and cell cycle analysis. TCCP treatment caused endogenous ROS generation, increase in intracellular
calcium and phosphorylation of p53 in a concentration-dependent manner, which was reverted upon pre-treatment with
pifithrin-μ. This led to the downstream altered expression of Bcl-2 and Bax
proteins, mitochondrial membrane depolarization,
MPTP, and
cardiolipin peroxidation. TCCP induced
cytochrome c release into the cytosol,
caspase activation, ultimately resulting in DNA fragmentation. Further, induction of apoptosis and morphological alterations were evident from the
phosphatidylserine externalization and increase in sub G1 population. The in vivo
Ehrlich ascites tumor (EAT) mouse study revealed the effectiveness of TCCP in reducing the
tumor burden and resulted in a ~2 fold increase in mice survival with minimal hepato-renal toxicity. Overall, TCCP was shown to be efficient in inducing ROS and mitochondrial-mediated apoptosis by restoring p53 activity in MDA-MB-231 cells and also induced EAT cell death in vivo thereby inhibiting
tumor proliferation.