探討第二型己糖激酶調控氯化鎳誘發肺支氣管上皮細胞自體吞噬與凋亡之機制

鎳化合物已被認定為致癌物質且可能造成人類支氣管上皮細胞的損害。細胞自體吞噬被認為是一種保護的機制，但過多的自噬可能造成細胞衰亡。Hexokinase-II (HK-II, HK2) 為糖解作用中的第一步驟酵素，其能將葡萄糖磷酸化為G-6P且能調控細胞自噬，已知氯化鎳透過HIF-1α增加HK2表現進而促進癌化過程。然而，氯化鎳是否活化HK2並調控細胞自噬發生的機制還尚未釐清，因此本研究想探討在人類支氣管上皮細胞中，氯化鎳是否誘導HK2蛋白及酵素活性增加進而調控細胞自噬，以及細胞自噬對細胞存活率的影響。本實驗室過去使用Agilent SurePrint G3 Human V2 GE 8×60K微陣列檢測，發現氯化鎳處理的細胞增加約38倍的HK2 mRNA表現量。因此我們先利用BEAS-2B與WI-38細胞處理氯化鎳，確認HK2的mRNA表現上升。接著以HK2抑制劑Metformin及2-deoxy-D-glucose (2-DG)，降低氯化鎳所誘導的HK2 mRNA、蛋白表現及酵素活性，並發現在Metformin與2-DG處理下會降低氯化鎳誘發的自噬標記蛋白LC3-II及凋亡標記蛋白Cleaved caspase-7表現量。以流式細胞儀定量及共軛焦顯微鏡觀察，Metformin抑制氯化鎳誘發細胞內酸性囊狀胞器及自噬小體的產生。使用Annexin V/PI結果也顯示Metformin 能減少氯化鎳造成的細胞凋亡。最後由VSV-G pseudotyped lentivirus-shRNA系統抑制HK2表現後皆減少氯化鎳誘發的細胞自噬及凋亡。另外，我們使用溶酶體抑制劑Chloroquine、LC3 shRNA及Atg5−/− mouse embryonic fibroblast細胞株進行實驗，皆證實氯化鎳誘發自體吞噬進而促進細胞凋亡。綜合以上，我們推論氯化鎳造成的細胞損傷其中可能透過活化HK2造成人類肺支氣管上皮細胞細胞自噬並導致凋亡。

關鍵字

氯化鎳；細胞自體吞噬；細胞凋亡；第二型己糖激酶Metformin

並列摘要

Nickel compounds are classified as carcinogen and have shown that exposure to nickel compounds may cause lung injury. Autophagy is generally thought of as a survival mechanism though the excessive levels of autophagy can also promote cell death. Hexokinase-II (HK-II, HK2) catalyzes glycolysis and regulates autophagy. Studies have been shown that nickel chloride up-regulate HK2 expression in HIF-1α-dependent pathway. However, the mechanism of HK2 in NiCl2-elicted autophagy and apoptosis is still unclear. The aim of this study is to investigate the role of NiCl2-induced HK2 in autophagy and cytotoxicity in lung bronchial cells. Our previous research showed that NiCl2 induced a 38-fold increase in HK2 mRNA expression using Agilent SurePrint G3 Human V2 GE 8×60K microarray analysis. In this study, NiCl2 induced HK2 mRNA expression in both BEAS-2B and WI-38 cells. Metformin and 2-deoxy-glucose (2-DG), HK2 inhibitors, decreased NiCl2-induced HK2 in the mRNA, protein and enzyme activity level by Q-PCR, Western blot and hexokinase activity assay. NiCl2-elicited expressions of LC3-II and cleaved caspase-7 were reversed via inhibition of HK2 through metformin, 2-DG and HK2 shRNA. Treatment with metformin decreased the NiCl2-stimulated GFP-LC3 puncta formation by confocal microscopy. Using acridine orange staining and flow cytometry, NiCl2-induced acidic vesicular organelles (AVOs) development was inhibited by metformin. Metformin also alleviated NiCl2-induced apoptosis on annexin V/PI staining and flow cytometry analysis. Chloroquine, a lysosome inhibitor, mitigated the NiCl2-mediated caspase-7 activation. LC3 knockdown Beas-2B cells and Atg5−/− mouse embryonic fibroblasts were demonstrated that inhibition of NiCl2-induced autophagy abolished apoptotic cell death. Our results demonstrated that NiCl2-induced apoptosis partially by autophagy via HK2 activation in human bronchial epithelial cells.

並列關鍵字

Nickel chloride ； Autophagy ； Apoptosis ； Hexokinase-II ； Metformin

參考文獻

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探討第二型己糖激酶調控氯化鎳誘發肺支氣管上皮細胞自體吞噬與凋亡之機制

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