Abstract
Induction of the generation of endoplasmic reticulum (ER) calcium (Ca++)-mediated reactive oxygen species (ROS) by gallic acid (GA) has been implicated in the mitochondrial apoptotic death of human oral cancer (OC) cells, but the molecular mechanism by which GA causes ER Ca++ release of OC cells to undergo cell death remains unclear. Here, we report that GA-induced phosphorylation of B-cell lymphoma 2 (BCL-2)-interacting killer (BIK) (threonine (Thr) 33/Serine (Ser) 35) and p53 (Ser 15 and Ser 392), Bcl-2-associated x protein (BAX)/BCL-2 antagonist killer 1 (BAK) oligomerization on the ER and mitochondria, rising of cytosolic Ca++ and ROS, cytochrome c (Cyt c) release from the mitochondria, Ψm loss, and apoptosis were suppressed in cells co-treated with a specific inhibitor of casein kinase II (CK II) (4,5,6,7-tetrabromobenzotriazole). Small interfering RNA (siRNA)-mediated suppression of BIK inhibited GA-induced oligomeric complex of BAX/BAK in the ER and mitochondria, increase of cytosolic Ca++ and ROS, and apoptosis, but did not attenuate the increase in the level of Ser 15-phosphated p53 induced by GA. Blockade of p53 expression by short hairpin RNA suppressed BAX/BAK oligomerization and ER Ca++–ROS-associated apoptosis induced by GA but did not affect GA-induced phospho-BIK (Thr 33/Ser 35) levels. Induction of mitochondrial Cyt c release and ROS generation, increased cytosolic Ca++ level, and apoptosis by GA was attenuated by expression of the BAX or BAK siRNA. Over-expression of BCL-2 (but not BCL-XL) inhibited formation of ER oligomeric BAX/BAK by GA. Our results demonstrated that activation of the CK II by GA is required for the BIK-mediated ROS-dependent apoptotic activity of ER-associated BAX/BAK.
Highlights
Calcium (Ca++) is an important second messenger responsible for a variety of the control of cellular processes, including cell growth and survival (Loughery et al, 2014)
We first investigated the ability of gallic acid (GA) to modulate casein kinase II (CK II) activity, which has been shown to play a key role in targeting of BCL2-associated x protein (BAX)/BCL-2 antagonist killer 1 (BAK) to endoplasmic reticulum (ER) and the increase of ER Ca++ depletion (Verma et al, 2001; Mathai et al, 2005)
Western blot analysis revealed that treatment of oral cancer (OC) cells with GA resulted in increased in Bcl-2-interacting killer (BIK) (Thr 33/Ser 35) and p53 (Ser 15 and Ser 392) phosphorylation but had no effect on the expression level of CK II protein (Figure 1A)
Summary
Calcium (Ca++) is an important second messenger responsible for a variety of the control of cellular processes, including cell growth and survival (Loughery et al, 2014). CKII-Mediated BIK-BAX/BAK Signaling Induced Apoptosis is regulated by the B-cell lymphoma 2 (BCL-2) family of proteins, which can be identified as either pro-apoptotic BCL2-associated x protein (BAX)/BCL-2 antagonist killer 1 (BAK) or anti-apoptotic BCL-2/BCL-XL proteins (Unger et al, 1993; Cory et al, 2003). In addition to the anti-apoptotic function of BCL-2 in the mitochondria, this protein has been reported to modulate ER Ca++ homeostasis by the ER targeting (Pinton and Rizzuto, 2006). The protection of ER-targeted BCL-2 against BAX-induced apoptosis has suggested that BCL-2 exerts its anti-apoptotic function to BAX by targeting ER (Wang et al, 2001)
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