Abstract
The calcineurin B subunit (CNB) has antitumor activity. We showed previously that recombinant human CNB (rhCNB) also had strong anti-tumor activity in vivo, and was thus a promising candidate anti-tumor drug. It appeared to kill tumor cells via immunomodulation. Here, we show that rhCNB inhibits the proliferation of human hepatoma HepG-2 cells, resulting in their apoptosis. Exogenous CNB was found to localize to mitochondria in tumor cells and activate the mitochondrial apoptosis pathway, as indicated by a decrease of mitochondrial transmembrane potential, release of cytochrome C and activation of caspase-9, which then activates caspase-3. At the same time Bcl-2 &Bcl-xL expression decreased, Bim expression increased, and Bax was activated. Interaction between rhCNB and Bcl-xL was detected, which may inhibit the function of Bcl-xL. Long-term tumor targeting was also observed in nude mice. These data deepened our understanding of the anti-tumor mechanism of rhCNB and provided guidance for its drug development.
Highlights
Calcineurin (CN) is a calcium- and calmodulindependent serine/threonine protein phosphatase [1, 2], whose function and structure have been widely studied
To examine the antiproliferative activity of recombinant human CNB (rhCNB), HepG-2 cells were exposed to various concentrations of rhCNB for 48h, after which the extent of cell death was monitored by CCK8 assay
A dose-dependent increase in the percentage of apoptotic HepG-2 cells from 7.40 ± 1.35% to 62.86 ± 4.72% was observed after treatment with 5-35μM of rhCNB (Figure 1C) determined by Annexin V binding, suggesting that most of the antiproliferative activity of rhCNB is mediated by apoptosis
Summary
Calcineurin (CN) is a calcium- and calmodulindependent serine/threonine protein phosphatase [1, 2], whose function and structure have been widely studied. It plays a central role in activating immune cells and is the target of the much-used immunosuppressive agents cyclosporin A (CsA) and tacrolimus (FK506) [3, 4]. The B subunit (CNB) is a 19 KD regulatory subunit with four Ca2+ binding sites [7,8,9] These regions and sites are essential for Ca2+ regulation of CN activity
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