丙烯醯胺誘導U-1240 MG星狀膠細胞株傷害及其調控

Abstract

丙烯醯胺（acrylamide, ACR）廣泛應用於工業及實驗室電泳膠體，對職業工人與實驗動物造成神經系統的影響。1994年International Agency for Research on Cancer將丙烯醯胺歸類為“人類可能致癌物”（Group 2A），2002年食物中發現丙烯醯胺存在後，更引發食品安全的疑慮。流行病學、實驗動物與細胞實驗證據顯示，丙烯醯胺影響神經系統中的神經元細胞，然而神經膠細胞的影響則不清楚。本研究因此針對丙烯醯胺引發星狀膠細胞基因傷害的反應，依相關調控分子與機制，進行體外實驗，探討細胞週期與細胞凋亡的相關影響。 首先評估星狀膠細胞（U-1240 MG）與神經元細胞（SH-SY5Y）在丙烯醯胺處理下的細胞毒性影響，包括細胞存活率、乳糖去氫酶活性和細胞中小片段DNA的表現量。U-1240 MG與SH-SY5Y細胞經丙烯醯胺處理後皆有細胞毒性產生，但U-1240 MG細胞需要較高的丙烯醯胺處理濃度與較長的處理時間。進一步分析U-1240 MG細胞之p53蛋白磷酸化及以glial fibrillary acidic protein（GFAP）於細胞中累積評估膠樣變性反應，顯示2 mM丙烯醯胺處理24 h後，p53蛋白磷酸化有顯著上升，而星狀膠細胞於丙烯醯胺處理下亦引發膠樣變性反應。接著，觀察MTT assay 在1與2 mM丙烯醯胺分別處理36 h與24 h以後細胞生長受到抑制，Ki-67蛋白在2 mM丙烯醯胺處理48 h較控制組少45.9％，彗星偵測結果在0.5、1和2 mM丙烯醯胺處理48 h後DNA傷害現象隨著處理濃度的增加而上升，此證實丙烯醯胺造成U-1240 MG細胞生長受到抑制與DNA傷害。 流式細胞儀分析U-1240 MG細胞週期發現，丙烯醯胺處理後細胞週期停滯在G0/G1時期。進一步以丙烯醯胺和咖啡因（抑制ATM與ATR作用）共處理U-1240 MG細胞，分析G0/G1時期的調控蛋白（包括pp53、p53、p21、p27、cyclin D1及Cdk2），結果顯示丙烯醯胺透過ATM/ATR途徑的訊息傳遞，造成pp53、p53和p21增加、cyclin D1降低，使細胞週期停滯G0/G1時期。另外，比較U-1240、U-251和U-87 MG細胞之G0/G1時期調控蛋白，發現pp53和Cdk2皆顯著增加、p27和cyclin D1皆顯著降低，然p53與p21的表現在U-1240和U-87 MG細胞中皆增加，而U-251 MG細胞則是不變與降低；進一步分析發現U-251 MG細胞經丙烯醯胺處理後，細胞週期停滯在G2/M時期。因此，不同神經膠細胞經丙烯醯胺處理造成不同的細胞週期影響。 根據流式細胞儀結果、Pro-caspase 3和PARP蛋白的表現及細胞中小片段DNA的表現量，顯示U-1240 MG細胞經2 mM丙烯醯胺處理48 h後造成細胞凋亡。2 mM丙烯醯胺處理U-1240 MG細胞48與72 h，ATR蛋白顯著上升而ATM蛋白下降，由於ATM/ATR途徑是丙烯醯胺造成U-1240 MG細胞基因傷害的訊息傳遞途徑之一，因此，ATR蛋白為U-1240 MG細胞經丙烯醯胺處理的反應蛋白。細胞凋亡調控蛋白Bax、Bad和bcl-2在2 mM處理72 h後表現量顯著降低，bcl-XL無影響，Bax/bcl-2、Cyt c和Caspase 8則顯著增加。進一步分析咖啡因抑制細胞凋亡訊號傳遞蛋白，包括pp53、p53、Bax、Bad、bcl-2、Cyt c、Pro-caspase 3和Caspase 8蛋白的表現與Bax/bcl-2比值，結果顯示U-1240 MG細胞經丙烯醯胺處理係由粒線體調控細胞凋亡。穿透性電子顯微鏡觀察丙烯醯胺處理U-1240 MG細胞，在48 h時，粒線體內形成分隔並有局部腫脹且細胞質囊泡增加，處理72 h使核染色質有凝集（chromatin condensation）、核固縮（pyknosis）與細胞腫脹（swelling），粒線體結構改變形成空泡化致結構瓦解。因此，丙烯醯胺暴露造成粒線體顯微結構變化，進而引發細胞凋亡。 綜合以上結果，丙烯醯胺處理除了影響神經元細胞外亦會影響星狀膠細胞，包括引發星狀膠細胞膠樣變性、生長抑制、基因傷害、細胞週期停滯與粒線體調控細胞凋亡。而丙烯醯胺造成U-1240 MG細胞基因傷害係透過ATR途徑引發G0/G1時期停滯及細胞凋亡。不同的星狀膠細胞可能有不同的訊息調控途徑與影響，此亦鼓勵其他細胞株與初代細胞的後續比較研究。

Acrylamide (ACR) has many applications in industries and for protein analysis. ACR is a neurotoxin to humans and laboratory animals, and has been classified as “2A, probably human carcinogen” by International Agency for Research on Cancer since 1994. The fact that ACR in starchy food after high temperature processing was found in 2002 raises food safety concerns in general population. Epidemiological, in vivo and in vitro studies conclude that ACR influences normal neurological functions. However, toxic effects of ACR on glia cells remain unsolved. This study focused on the determination of the regulatory molecules and functional mechanisms involved in ACR-induced cytotoxicity, including DNA damage responses, cell-cycle arrest and apoptosis in astroglial cells in vitro. ACR-induced cytotoxic effects on astrocytes (U-1240 MG) and neuronal cells (SH-SY5Y) were examined by measuring the cell viability, lactate dehydrogenase leakage (LDH) assay and oligonucleosomal DNA fragmentation. Both U-1240 MG and SH-SY5Y cells showed cytotoxicity after ACR treatment, however, U-1240 MG cells were less sensitive to ACR than SH-SY5Y cells. ACR-induced cytotoxicity in U-1240 MG cells was further confirmed by detecting the phosphorylated p53 (pp53, DNA damage marker protein) and glial fibrillary acidic protein (GFAP, chemical-induced astrogliotic response marker). Increasing pp53 after 2 mM ACR treatment for 24 h and accumulation of GFAP after 2 mM ACR treatment for 48 h indicated that ACR induced astrogliosis in U-1240 MG cells. U-1240 MG cells proliferation were suppressed at 1 mM ACR for 36 h or 2 mM ACR for 24 h by MTT assay, and also decreased 45.9% of Ki-67 protein expression when treated with 2 mM ACR for 48 h than control. ACR increased DNA damage of U-1240 MG cells in a dose-dependent manner with 48 h exposure, as evaluated by comet assay. Therefore, ACR inhibited cell proliferation and caused DNA damage in U-1240 MG cells. Analysis of cell-cycle arrest by flow cytometry revealed that the ACR treatment resulted in significant increases in the G0/G1-arrested cells in U-1240 MG cells. Expression of DNA damage-associated/checkpoint-related signaling molecules (pp53, p53, p21, p27, Cdk2, and cyclin D1) were parallelly analyzed by immunoblotting in three human astrocytoma cell lines (U-1240 MG, U-251 MG, and U-87 MG). Under 2 mM ACR treated for 48 h, three cell lines caused marked increases in pp53 and Cdk2, as well as decreases in cyclin D1 and p27. Moreover, increases in p53 and p21 were detected in both U-1240 and U-87 MG cells, whereas no marked change in p53 and a decrease in p21 were observed in U-251 MG cells. Cell-cycle arrest of U-251 MG cells was found in G2/M phase with 2 mM ACR treatment for 48 h which reveals a different response to ACR toxicity than other two cell lines in G0/G1 arrest. To address the involvement of ataxia telangiectasia mutated/ATM-Rad3-related (ATM/ATR) kinase in signaling of ACR-induced G0/G1 arrest, caffeine was used to block the ATM/ATR pathway in U-1240 MG cells. The results showed caffeine significantly attenuated the ACR-induced G0/G1 arrest as well as the expression of DNA damage-associated/checkpoint-related signaling molecules in a dose-dependent manner. ACR causes apoptosis (with 2 mM treatment for 48 h) by analyses of sub-G1 group arrest in cell cycle, protein expressions of pro-caspase 3 and poly ADP ribose polymerase (PARP), and oligonucleosomal DNA fragmentation. Analysis of apoptosis signaling molecules in U-1240 MG cells, including Bax, Bad, bcl-2, bcl-XL, Bax/bcl-2, Cyt c and Caspase 8, further supported the above observations. Treatment of 2 mM ACR for 72 h caused marked decreases in Bax, Bad and bcl-2 expressions, as well as increases in the Bax/bcl-2 ratio, Cyt c and Caspase 8 expressions. However, no marked change in bcl-XL was observed. The above protein expressions indicated that ACR induced mitochondria-mediated apoptosis in U-1240 MG cells. To address the involvement of ATM/ATR kinase in the signaling of ACR-induced apoptosis, caffeine was applied to block the ATM/ATR pathway in U-1240 MG cells. Caffeine significantly attenuated the ACR-induced expression of apoptosis-related signaling molecules, including pp53, p53, Bax, Bad, bcl-2, Bax/bcl-2, Cyt c, Pro-caspase 3 and Caspase 8, in a dose-dependent manner. These results were concordant with the evaluation of ATM and ATR protein expressions and the observation of the high expression of ATR protein by fluorescence microscopy in ACR-treated U-1240 MG cells. The ultrastructure alterations induced by ACR in U-1240 MG cells were observed by Transmission Electron Microscope. Mitochondria with vesicular matrix compartments and cytoplasmic vacuole formation were noticed while U-1240 MG cells treated with 2 mM ACR for 48 h and cells with chromatin condensation, pyknosis, and swollen mitochondria were observed at 72 h. These results suggest that ACR exposure leads to astrogliosis, proliferation inhibition, DNA damage, G0/G1 phase arrest and mitochondria-dependent apoptosis in human astrocytoma cells in vitro. These studies clearly demonstrate the critical role of ATR in signaling the ACR-induced cell-cycle arrest and apoptosis in U-1240 MG cells. Whether ACR causes different cytotoxic effects and mechanisams on different astrocytic cell lines and primary cells is suggested for future investigation.