在胃幽門螺旋桿菌誘發TRAIL引起人類胃上皮細胞凋亡中casein kinase 2 的角色

Abstract

胃幽門螺旋桿菌(Helicobacter pylori)是個常見的人類病原菌，感染胃幽門螺旋桿菌會造成慢性胃炎、胃潰瘍和胃癌。然而，目前對於胃幽門螺旋桿菌的致病機轉尚不清楚。研究指出在受到胃幽門螺旋桿菌感染時，可觀察到胃上皮細胞的細胞凋亡有增加的現象，此現象被認為對於胃炎的發生具有重要性。胃幽門螺旋桿菌所產生的毒力因子與宿主的免疫反應的活化皆可促成胃上皮的損害。在我們實驗室先前的研究中發現當胃幽門螺旋桿菌直接接觸到胃上皮細胞時，胃幽門螺旋桿菌可使人類胃上皮細胞對於Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)誘發的細胞凋亡產生感受性。胃幽門螺旋桿菌會調節人類胃上皮細胞裡TRAIL凋亡訊息傳導。在人類胃上皮細胞與胃幽門螺旋桿菌共同培養之後加入TRAIL，會促進caspase-8的活化，且此促進的caspase-8活化便足以造成下游Bid, caspase-9與caspase-3的活化，最終打破了胃上皮細胞對TRAIL引起細胞凋亡的耐受性。然而對於胃幽門螺旋桿菌如何去調節TRAIL凋亡訊息傳導的機制，目前尚不清楚。我們假設，當胃幽門螺旋桿菌與細胞接觸時，覆蓋於細胞表面的醣類物質可能在胃幽門螺旋桿菌誘發TRAIL感受性的過程中扮演著某個角色。為了要探討醣類在調節胃幽門螺旋桿菌誘發TRAIL感受性所扮演的角色，我們將不同的醣類分別加入體外共同培養系統。我們發現，heparan sulfate會抑制胃幽門螺旋桿菌誘發TRAIL感受性，但對胃幽門螺旋桿菌與細胞間的附著並沒有顯著影響。這些結果意味著，幽門螺旋桿菌可能藉與heparan sulfate交互作用來誘發胃上皮細胞對TRAIL產生感受性。 最近的研究發現一個普遍存在、高度保存且具固有活性的serine/threonine kinase, casein kinase Ⅱ (CK2)可以保護腫瘤細胞免於受到TRAIL所誘發的細胞凋亡。因為CK2的活性在對TRAIL具有感受性的癌症細胞株中是比較低的，然而在對TRAIL具耐受性的癌症細胞株中則是比較高的。當抑制CK2的活性，可以使腫瘤細胞對TRAIL誘發的細胞凋亡產生感受性，但對正常的細胞沒有影響。研究也指出，當CK2受到抑制時加入TRAIL，procaspase-8聚集到death-inducing signaling complex (DISC)的量會增加且迅速的誘發caspase-8、-9、-3的活化。於是我們假設，胃幽門螺旋桿菌可能透過調節CK2的活性來使胃上皮細胞對TRAIL所誘發的細胞凋亡產生感受性。為了要探討CK2在調節胃幽門螺旋桿菌誘發TRAIL感受性所扮演的角色，我們使用CK2的專一抑制劑DRB去抑制胃上皮細胞株AGS細胞內CK2的磷酸化作用。實驗結果顯示，抑制CK2的活性，可使AGS細胞對TRAIL所誘發的細胞凋亡產生感受性，這個結果也暗示CK2扮演一個保護AGS細胞免於受到TRAIL所誘發的細胞凋亡的角色。由西方墨點法的結果中發現，一併給予DRB與TRAIL可促進caspase-8活化，這個現象和與胃幽門螺旋桿菌共同培養時所觀察到的現象類似，使我們聯想胃幽門螺旋桿菌可能藉由調節CK2的活性來誘發AGS細胞對TRAIL引起的細胞凋亡產生感受性。使用西方墨點法與CK2激酶活性試驗來釐清胃幽門螺旋桿菌與CK2之間的交互作用。但是CK2的蛋白表現量與激酶活性在與胃幽門螺旋桿菌共同培養前後並沒有差異，這些結果顯示CK2並非參與調節胃幽門螺旋桿菌誘發TRAIL感受性的下游分子。

Helicobacter pylori (H. pylori), is a common pathogen of humans that causes chronic gastritis, peptic ulcer and gastric adenocarcinoma. However, the mechanisms by which H. pylori mediates pathogenesis are still unclear. Studies have shown that the enhanced gastric epithelial cell apoptosis observed during infection with H. pylori was significant in the pathogenesis of gastritis. The virulence factors produced by H. pylori and activation of immune response by host also contribute to gastric epithelium damage. Previous studies of our laboratory demonstrated that when direct contacting with gastric epithelial cells, H. pylori could sensitize human gastric epithelial cell line to be susceptible to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. TRAIL death signal transduction in human gastric epithelial cells was modulated by H. pylori. After co-cultured with H. pylori and treated with TRAIL, caspase-8 activation was enhanced and the enhanced caspase-8 activation was sufficient to lead downstream Bid, caspase-9 and caspase-3 cleavage and finally break the resistant to TRAIL-induced apoptosis. However, the mechanism of H. pylori modulate TRAIL death signaling is still not clear. We hypothesize that the carbohydrate molecules that cover the cell surface may play a role in H. pylori-induced TRAIL sensitivity during H. pylori contact to gastric epithelial cell surface. In order to study the role of cell surface carbohydrate molecules in regulation of H. pylori-induced TRAIL sensitivity, we use different carbohydrates adding to the in vitro co-culture system. We demonstrated that heparan sulfate could inhibit H. pylori-induced TRAIL sensitivity, but not the binding of H. pylori to cell surface. These results indicated that H. pylori sensitize gastric epithelial cells to TRAIL-induced apoptosis might through the interaction with cell surface heparan sulfate. Recent studies have shown casein kinase II (CK2), a highly conserved, ubiquitous and constitutively active serine/threonine kinase, could protect cancer cell from TRAIL-induced apoptosis. Since the CK2 activity is found low in TRAIL-sensitive cancer cell lines but high in TRAIL-resistant cancer cell lines. Inhibition of CK2 phosphorylation events result in dramatic sensitization of tumor cells to TRAIL-induced apoptosis but not normal cells. Studies also shown CK2 inhibition increase the level of recruitment of procaspase-8 to the death-inducing signaling complex (DISC) and induce rapid caspase-8,-9, -3 cleavage after TRAIL treatment. We hypothesize that H. pylori sensitize gastric epithelial cells to TRAIL-induced apoptosis may through modulation CK2 activity. In order to study the role of CK2 in regulation of H. pylori-induced TRAIL sensitivity, we use CK2 specific inhibitor, DRB, to inhibit the phosphorylation events of CK2 in human gastric epithelial cell line, AGS cells. We demonstrated that inhibition of CK2 activity could sensitize AGS to TRAIL-induced apoptosis, indicating a role of CK2 in protecting AGS cells from TRAIL-induced apoptosis. Western blotting also demonstrated that caspase-8 cleavage was enhanced by DRB and TRAIL treatment and this phenomenon was similar to co-culture with H. pylori, suggesting that H. pylori sensitize AGS cells to TRAIL-induced apoptosis through modulating CK2 activity. We use Western blotting and CK2 kinase assay to clarify the interaction between H. pylori and CK2. But the CK2 protein expression level and kinase activity did not show difference before and after co-cultured with H. pylori. These result demonstrated that CK2 is not the molecule that participates in the downstream of H. pylori-induced TRAIL sensitivity.