Thorpe Jeffery A, Christian Perry A, Schwarze Steven R
Department of Molecular and Cellular Biochemistry and Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536, USA.
Prostate. 2008 Feb 1;68(2):200-9. doi: 10.1002/pros.20706.
Proteasome inhibition through the administration of Velcade is a viable chemotherapeutic strategy that is approved to treat multiple myeloma and is being evaluated for efficacy against prostate cancer. Currently, the apoptotic pathways that contribute to this anticancer response are poorly understood. Our goal is to test the extent to which proteasome inhibition modulates apoptosis through death receptor pathways.
Several prostate cancer cell lines and primary prostate epithelial cells (PrECs) were used as models. The death receptor pathway was activated by the expression of Fas ligand (FasL) or addition of TNF-related apoptosis-inducing ligand (TRAIL) in the presence or absence of proteasome inhibitors. The apoptotic response was quantified by annexin V, TUNEL and nuclear condensation assays. Western blot analysis was conducted to quantify protein levels and enzyme assays were used to measure caspase activity.
Proteasome inhibition markedly sensitized prostate cancer cells to apoptosis initiated by Fas ligand (FasL) or TRAIL. In the presence of either death ligand, procaspase-8 processing occurred, but led to minimal amounts of active caspase-8. The addition of Velcade, however, led to robust active caspase-8 protein abundance and activity. In the presence of Velcade the caspase-8 p18 subunit half-life increased from 22 min to over 2 hr.
These findings demonstrate that proteasome inhibition can sensitize cells to apoptosis elicited by tumor necrosis factor ligands and retarding caspase-8 degradation provides one explanation for this activity. This study suggests that the clinical efficacy of Velcade may result, at least in part, from the activity of death ligands.
通过使用万珂抑制蛋白酶体是一种可行的化疗策略，已被批准用于治疗多发性骨髓瘤，目前正在评估其对前列腺癌的疗效。目前，对于促成这种抗癌反应的凋亡途径了解甚少。我们的目标是测试蛋白酶体抑制通过死亡受体途径调节凋亡的程度。
使用几种前列腺癌细胞系和原代前列腺上皮细胞（PrECs）作为模型。在存在或不存在蛋白酶体抑制剂的情况下，通过Fas配体（FasL）的表达或添加肿瘤坏死因子相关凋亡诱导配体（TRAIL）来激活死亡受体途径。通过膜联蛋白V、TUNEL和核浓缩测定法对凋亡反应进行定量。进行蛋白质印迹分析以定量蛋白质水平，并使用酶测定法测量半胱天冬酶活性。
蛋白酶体抑制显著增强了前列腺癌细胞对由Fas配体（FasL）或TRAIL引发的凋亡的敏感性。在存在任何一种死亡配体的情况下，发生了前半胱天冬酶-8的加工，但产生的活性半胱天冬酶-8量极少。然而，添加万珂导致活性半胱天冬酶-8蛋白丰度和活性显著增强。在存在万珂的情况下，半胱天冬酶-8 p18亚基的半衰期从22分钟增加到超过2小时。
这些发现表明，蛋白酶体抑制可使细胞对肿瘤坏死因子配体引发的凋亡敏感，而延缓半胱天冬酶-8降解为这种活性提供了一种解释。这项研究表明，万珂的临床疗效可能至少部分源于死亡配体的活性。
