- 學位論文
檳榔鹼抑制第二穀氨醯胺酶的表現並降低細胞的抗氧化作用
Arecoline Inhibits Glutaminase 2 Expression And Reduces Cellular Antioxidation Function
摘要
檳榔鹼 (arecoline)是檳榔子中主要的生物鹼，具有基因毒性並且能夠消耗還原型的穀胱甘肽 (glutathione, GSH)和增加活性氧 (reactive oxygen species, ROS)產生。然而，檳榔鹼影響穀胱甘肽以及活性氧的產生之機制目前還不清楚。第二穀氨醯胺酶 (Glutaminase 2, GLS2)參與穀氨醯胺水解得到產物穀氨酸 (glutamate, GLU)，其可作為穀胱甘肽的前驅產物。因此，第二穀氨醯胺酶能夠增加細胞內穀胱甘肽的含量。在氧化壓力增加的情況下，p53能轉錄活化第二穀氨醯胺酶的表現量來增加細胞內抗氧化能力。我們先前的研究指出，檳榔鹼可以抑制p53基因的轉錄活化能力。在本篇研究中，我們假設檳榔鹼也能夠抑制第二穀氨醯胺酶表現量並導致還原態穀胱甘肽降低和活性氧增加。實驗結果顯示：在HEp-2和293細胞中，檳榔鹼以劑量依賴性的方式抑制第二穀氨醯胺酶的表現量。過表達的p53或組蛋白去乙醯化酶抑製劑(HDACi)的處理能夠恢復檳榔鹼所抑制之第二穀氨醯胺酶的表現量，這意味著p53和HDAC的參與。從我們實驗的結果發現，檳榔鹼能藉由抑制第二穀氨醯胺酶，來導致還原態穀胱甘肽降低和活性氧增加，但是沒有改變穀氨酸、α酮戊二酸、還原態的煙酰胺腺嘌呤二核苷酸磷酸的表現量，穀胱甘肽及還原態的煙酰胺腺嘌呤二核苷酸磷酸生合成相關的基因，在mRNA的表現量也並未改變。
並列摘要
Arecoline is the major alkaloid in areca nut, the principal constitute of betel quid (BQ). Arecoline is genotoxic and is able to deplete reduced glutathione (GSH) and induce reactive oxygen species (ROS). However, the mechanism underlying arecoline-mediated dysregulation of GSH level as well as ROS generation is unclear. Glutaminase 2 (GLS2) is involved in glutaminolysis, which results in the production of glutamate, the precursor of GSH. Therefore, GLS2 is able to increase the cellular level of GSH. Upon oxidative stress, expression of GLS2 is transcriptionally upregulated by p53 to enhance cellular antioxidation function. Previously we have reported that arecoline can inhibit p53’s transactivation function. In this study, we hypothesize that arecoline also inhibits GLS2 expression and leads to GSH reduction and ROS generation. The results showed that arecoline suppressed GLS2 expression in HEp-2 and 293 cells in a dose-dependent manner. Overexpression of p53 or administration of histone deacetylase (HDAC) inhibitor was able to restore arecoline-mediated repression of GLS2 expression, suggesting that p53 and HDAC were involved. In this study, we found that arecoline inhibited GLS2 expression to decrease the intracellular levels of GSH and ROS generation, but did not change the levels of glutamate, α-ketoglutarate, NADP/NADPH, and mRNA levels of GSH/NADPH generation-related genes.