反8,反10,顺12-十八碳三烯酸和反8,反10,反12-十八碳三烯酸诱导膀胱癌细胞凋亡

Apoptosis induction in bladder cancer cells by t8,t10,c12-octadecatrienoic acid and t8,t10,t12-octadecatrienoic acid

反8,反10,顺12十八碳三烯酸(t8,t10,c12-18:3)和反8,反10,反12十八碳三烯酸(t8,t10,t12-18:3)是来源于金盏花属植物的共轭亚麻酸异构体,其对膀胱癌细胞的影响及作用机理尚未阐明。本实验以膀胱癌T24细胞为体外细胞模型,分别对细胞活力和乳酸脱氢酶(LDH)活性检测,分析了t8,t10,c12-18:3和t8,t10,t12-18:3对相对细胞活力的影响;利用AV/PI双染流式细胞术、蛋白免疫印迹技术对谷胱甘肽(GSH)、活性氧(ROS)和丙二醛(MDA)进行检测;通过测定活性氧清除剂(NAC)、抗氧化剂α-生育酚和PPARγ抑制剂T0070907的抑制作用,初步探讨了t8,t10,c12-18:3和t8,t10,t12-18:3诱导膀胱癌细胞凋亡的作用机理。结果表明,t8,t10,c12-18:3和t8,t10,t12-18:3能够显著抑制T24细胞的细胞活力,增加LDH的释放,并呈时间和剂量依赖性。两种脂肪酸都能够明显诱导细胞凋亡,引起Caspase-3/Caspase-9表达增加和Bax/Bcl-2表达比例的升高,并导致ROS和MDA的积累以及GSH减低。NAC和α-生育酚能够明显拮抗t8,t10,c12-18:3和t8,t10,t12-18:3对T24细胞活力的抑制作用。t8,t10,c12-18:3和t8,t10,t12-18:3能够上调T24细胞PPARγ蛋白表达,T0070907可以拮抗两种脂肪酸对细胞活力的影响,并下调凋亡相关蛋白Caspase-3和Caspase-9的表达。因此,t8,t10,c12-18:3和t8,t10,t12-18:3对膀胱癌T24细胞的凋亡诱导作用可能与ROS积累和脂质过氧化及凋亡相关蛋白和PPARγ表达的调控有关。

The t8,t10,c12-octadecatrienoic acid(t8,t10,c12-18:3)and t8,t10,t12-octadecatrienoic acid(t8,t10,t12-18:3)are conjugated linolenic acid isomers derived from calendula plants.The effects on cell apoptosis of human bladder cancer cells remained unclear.T24 cells of bladder cancer were used as the cell model in vitro to detect the cell activity and lactate dehydrogenase(LDH)activity,and the effects of t8,t10,c12-18:3 and t8,t10,t12-18:3 on the relative cell activity were analyzed.Glutathione(GSH),reactive oxygen species(ROS)and malondialdehyde(MDA)were detected by AV/PI double staining flow cytometry and Western Blot.The inhibitory effect of active oxygen scavenger(NAC),antioxidantα-tocopherol and the inhibitor of PPARγT0070907,and the mechanism of t8,t10,c12-18:3 and t8,t10,t12-18:3 inducing apoptosis of bladder cancer cells were discussed.The results showed that t8,t10,c12-18:3 and t8,t10,t12-18:3 could significantly inhibit the cell viability of T24 cells and increase the release of LDH in a time and dose dependent manner.Both t8,t10,c12-18:3 and t8,t10,t12-18:3 can significantly induce apoptosis,increase the expression of Caspase-3/Caspase-9 and the expression ratio of Bax/Bcl-2,and lead to the accumulation of ROS and MDA and the decrease of GSH.NAC andα-tocopherol can obviously antagonize the inhibitory effect of t8,t10,c12-18:3 and t8,t10,t12-18:3 on the activity of T24 cells.T8,t10,c12-18:3 and t8,t10,t12-18:3 can up-regulate PPARγof T24 cells protein expression,T0070907 can antagonize the effects of two fatty acids on cell viability,and down regulate the expression of apoptosis related proteins Caspase-3 and Caspase-9.Therefore,the apoptosis induction of t8,t10,c12-18:3 and t8,t10,t12-18:3 on T24 cells of bladder cancer may be related to ROS accumulation,lipid peroxidation,apoptosis related proteins and PPARγexpression regulation.