Induction of apoptosis by collinin from Zanthoxylum schinifolium is mediated via mitochondrial pathway in human Jurkat T cells

• An apoptogenic ingredient collinin was isolated from the leaves of Zanthoxylum schinifolium.
• Multidomain pro-apoptotic Bcl-2 proteins Bak and Bax were activated by collinin.
• Δψm loss and caspase cascade activation, leading to PARP degradation, was induced by collinin.
• Collin-induced apoptotic events were abrogated by overexpression of Bcl-2.
• Either FADD-deficiency or caspase-8-deficiency failed to affect the sensitivity of Jurkat T cells to collinin-induced apoptosis.

Collinin, which was isolated from the leaves of Zanthoxylum schinifolium, could exert cytotoxic effect on various human tumor cells with IC50 values in the range of 38.1–111.6 μM, whereas the IC50 value for human normal mammary epithelial MCF-10A cells was 124.4 μM. To examine the contribution of apoptosis to the cytotoxicity of collinin toward tumor cells, collinin-induced apoptotic events of Jurkat T cells transfected with vector (JT/Neo) were compared with those of Jurkat T cells transfected with Bcl-2 gene (JT/Bcl-2). Treatment of JT/Neo cells with collinin (30–60 μM) resulted in induction of sub-G1 peak representing apoptotic cells along with activation of Bak and Bax, mitochondrial membrane potential (Δψm) loss, activation of caspase-9, -3, -8, and -7, degradation of PARP, and DNA fragmentation dose-dependently, but these apoptotic events were abrogated by overexpression of Bcl-2, which could prevent the induced activation of Bak and Bax, and subsequent mitochondrial damage. Under these conditions, necrosis was not accompanied. Pretreatment of JT/Neo cells with the pan-caspase inhibitor z-VAD-fmk completely blocked collinin-induced apoptotic sub-G1 cells and caspase cascade activation, whereas it failed to suppress Bak activation and Δψm loss. Neither FADD-deficiency nor caspase-8-deficiency affected the susceptibility of Jurkat T cells to collinin-induced cytotoxicity and apoptotic cell death. These results demonstrate that the apoptogenic activity of collinin was mediated by the intrinsic mitochondrial apoptotic pathway which was preceded by activation of pro-apoptotic multidomain Bcl-2 family members Bak and Bax, mitochondrial damage, and resultant activation of caspase cascade, leading to PARP degradation, which could be regulated by Bcl-2.