Activation of Casein Kinase II by Gallic Acid Induces BIK-BAX/BAK-Mediated ER Ca++-ROS-Dependent Apoptosis of Human Oral Cancer Cells

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, Ψ_m 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-X_L) 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.

Keywords: BAX/BAK; BIK; ER Ca++; ROS; casein kinase II; gallic acid.

Figure 1

Suppression of gallic acid (GA)-induced BCL-2-associated x protein (BAX)/BCL-2 antagonist killer 1 (BAK) oligomerization in the endoplasmic reticulum (ER), mitochondrial cytochrome c (Cyt c) release, Ψ_m loss, and apoptosis by 4,5,6,7-tetrabromobenzotriazole (TBB). (A,B) Cells were harvested 36 h after treatment with either vehicle, GA (300 μM), TBB (15 μM), or GA (300 μM) plus TBB (15 μM), and cell pellets were resuspended in hypotonic buffer. Crude homogenates were incubated with 5 mM bismaleimidohexane (BMH) in PBS for 30 min at room temperature and then subjected to subcellular fractionation to obtain the mitochondrial (Mt), ER/microsomal (Ms), and cytosolic (Cs) fractions. In total, 20 μg of total protein from the recovered fractions was analyzed by 10% SDS-PAGE and probed with specific antibodies, as indicated. (C–F) Cells were treated with either vehicle, GA (300 μM), TBB (15 μM), or GA (300 μM) plus TBB (15 μM) for for 36 h. The decrease in 3,3′-dihexyloxacarbocyanine fluorescence was measured by flow cytometry. The generation of cytosolic Ca⁺⁺ level and ROS were monitored by measuring increased fluorescence of Indo-1 and 2,7-dichlorodihydrofluorescein by flow cytometry. DNA fragmentation was determined using a Cell Death Detection ELISA kit. The values presented are the mean standard errors from three independent experiments. ^*Significantly different at p < 0.05.

Figure 2

GA-induced activity of Bcl-2-interacting killer (BIK) is responsible for gallic acid (GA)-induced BCL-2-associated x protein (BAX)/BCL-2 antagonist killer 1 (BAK) endoplasmic reticulum (ER) oligomerization, calcium (Ca⁺⁺) efflux from the ER, and cell apoptosis. At 12 h after transfection with control or BIK siRNA, the cells were treated with either vehicle or GA (300 μM) for an additional 36 h. (A,B) The levels of the indicated proteins in the lysates of the fractions of mitochondrial (Mt), ER/microsomal (Ms), and total cell (T) extracts were determined by Western blot analysis using specific antibodies. Cox-2, calnexin, and β-actin were used as internal controls for the mitochondria, ER, and cytosol, respectively. (C–E) The generation of cytosolic Ca⁺⁺ level and ROS were monitored by measuring increased fluorescence of Indo-1 and 2,7-dichlorodihydrofluorescein by flow cytometry. DNA fragmentation was determined using a Cell Death Detection ELISA kit. The values presented are the mean standard errors from three independent experiments. ^*Significantly different at p < 0.05.

Figure 3

Gallic acid (GA)-induced BCL-2-associated x protein (BAX)/BCL-2 antagonist killer 1 (BAK) apoptotic activity modulates the release of endoplasmic reticulum (ER)-associated calcium (Ca⁺⁺), mitochondrial Cyt c release, and apoptosis. At 12 h after transfection with control, BAX, or BAK siRNA, cells were treated with vehicle or GA (300 μM) for 36 h. (A,B) The levels of the indicated proteins in the lysates of the fractions of mitochondrial (Mt) and cytosolic (Cs) and total cell (T) extracts were determined by Western blot analysis using specific antibodies. Cox-2 and β-actin were used as internal controls for the mitochondria and cytosol, respectively. (C) The cytosolic levels of Ca⁺⁺, ROS, and DNA fragmentation were determined by measuring increased Indo-1 fluorescence and 2,7-dichlorodihydrofluorescein using flow cytometry and a Cell Death Detection ELISA kit, respectively. The values presented are the mean standard errors from three independent experiments. ^*Significantly different at p < 0.05.

Figure 4

Requirement of phospho-p53 (Ser 15) for targeting of BCL-2-associated x protein (BAX)/BCL-2 antagonist killer 1 (BAK) to the endoplasmic reticulum (ER) by gallic acid (GA). p53 shRNA cells were treated with vehicle or GA (300 μM) for 36 h. (A,B) The levels of the indicated proteins in the lysates of the fractions of mitochondrial (Mt) and ER/microsomal (Ms) extracts were determined by Western blot analysis using specific antibodies. Cox-2 and calnexin were used as internal controls for the mitochondria and ER, respectively. (C) The generation of cytosolic Ca⁺⁺ level and ROS were monitored by measuring increased fluorescence of Indo-1 and 2,7-dichlorodihydrofluorescein by flow cytometry. DNA fragmentation was determined by using a Cell Death Detection ELISA kit. The values presented are the mean standard errors from three independent experiments. ^*Significantly different at p < 0.05.

Figure 5

Involvement of decreased B-cell lymphoma 2 (BCL-2) protein level in GA-induced BCL-2-associated x protein (BAX)/BCL-2 antagonist killer 1 (BAK) oligomerization in the endoplasmic reticulum (ER). At 12 h after transfection with vector alone, FLAG-BCL-2, or FLAG-BCL-X_L, cells were treated with vehicle or GA (300 μM) for 36 h. (A,B) Expression levels of BCL-2 and BCL-X_L in lysates prepared from cells treated with GA or transfected with vector alone, FLAG-BCL-2, or FLAG-BCL-X_L. FLAG-BCL-2, FLAG-BCL-X_L, BCL-2, and BCL-X_L were detected with the antibodies shown. (C) The levels of the indicated proteins in the lysates of the fractions of mitochondrial (Mt) and ER/microsomal (Ms) and total cell (T) extracts were determined by Western blot analysis using specific antibodies. Cox-2 and calnexin were used as internal controls for the mitochondria and ER, respectively. (D) The generation of cytosolic Ca⁺⁺ level and ROS were monitored by measuring increased fluorescence of Indo-1 and 2,7-dichlorodihydrofluorescein by flow cytometry. DNA fragmentation was determined by using a Cell Death Detection ELISA kit. The values presented are the mean standard errors from three independent experiments. ^*Significantly different at p < 0.05.