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.

全球诊断出的乳腺癌中约有15％被指定为三阴性乳腺癌（TNBC）。在这项研究中，我们研究了从堇青叶中纯化得到的天然化合物双（2-乙基己基）1H-吡咯-3,4-二羧酸酯（TCCP）对TNBC细胞系MDA-MB-231的影响。TCCP在MDA-MB-231上的促凋亡性质是通过评估各种凋亡标记物来确定的。ROS产生，细胞内钙，线粒体膜电位（ΔΨ米），荧光法测定MPTP，心磷脂过氧化和caspase活性。通过免疫印迹测定BAX，BCL-2，细胞色素c，胱天蛋白酶和p53蛋白的表达。此外，通过DNA片段化测定，膜联蛋白-V染色和细胞周期分析确定了TCCP对DNA和细胞死亡的影响。TCCP处理以浓度依赖的方式引起内源性ROS的产生，细胞内钙的增加和p53的磷酸化，这在用pifithrin-μ预处理后得以恢复。这导致Bcl-2和Bax蛋白的下游表达改变，线粒体膜去极化，MPTP和心磷脂过氧化。TCCP诱导细胞色素c释放到细胞质中，胱天蛋白酶激活，最终导致DNA断裂。进一步，₁人口。的体内艾氏腹水瘤（EAT）的小鼠的研究显示TCCP的有效性在降低肿瘤负荷，并导致1〜2倍的增加在小鼠的存活率以最小的肝肾毒性。总的来说，TCCP被证明可通过恢复MDA-MB-231细胞中的p53活性来诱导ROS和线粒体介导的凋亡，并在体内诱导EAT细胞死亡从而抑制肿瘤的增殖。