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Zinc depletion induced apoptosis through Ca²⁺-dependent mitochondrial apoptotic pathway in human breast cancer MDA-MB-231 cells Lin, Yi-Hsuan


Breast cancer is the most frequently diagnosed cancer among Canadian women. Despite the use of advanced therapeutics, breast cancer remains the second leading cause of cancer death among Canadian women. Therefore, the development of novel and effective therapeutics to treat breast cancer remains an important goal. Defective or inhibited apoptosis is a major causative factor in the development and progression of cancer. Zinc is considered an apoptotic regulator. Further, previous work has also shown that there is an association between zinc depletion-induced apoptosis and an elevated intracellular Ca²⁺ level in human breast cancer MDA-MB-231 cells. Ca²⁺ is a known mediator of the mitochondrial apoptotic pathway. The overall objective of my thesis research was to investigate the role of intracellular Ca²⁺ and its involvement of the mitochondria in zinc depletion-induced apoptosis in human breast cancer MDA-MB-231 cells. MDA-MB-231 cells were cultured in DMEM containing FBS (10%) followed by depletion of intracellular zinc using N,N,N’,N’-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN; 20 µM) with or without the presence of intracellular Ca²⁺ chelator, 1,2-bis (2-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid acetoxymethyl ester (BAPTA-AM; 10 or 20 µM). Apoptosis was assessed by caspase-9 and -3 activities using corresponding fluorogenic substrates and the proportion of cells with fragmented DNA using PI-staining flow cytometry assay. Intracellular Ca²⁺ was assessed using Fura-2 assay. Mitochondrial membrane potential was assessed by DiOC₆-staining flow cytometry assay. Cytochrome c release was detected by Western blot. Addition of TPEN resulted in an increase of caspase-9 and -3 activities, an increase in the proportion of cells with fragmented DNA, and a prolonged increase in intracellular Ca²⁺ level. TPEN treatment also reduced mitochondrial membrane potential and induced cytochrome c release. Zinc replenishment (10 – 40 µM) prevented TPEN-induced apoptosis. Intracellular Ca²⁺ chelation with BAPTA-AM suppressed TPEN-induced apoptosis, mitochondrial membrane potential loss, and cytochrome c release. Collectively these results showed that zinc depletion-induced apoptosis was mediated through the Ca²⁺-dependent mitochondrial apoptotic pathway in human breast cancer MDA-MB-231 cells.

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