Effects of acrylamide on signal transduction

以人神经母细胞瘤SH-SY5Y细胞为研究对象，观察了丙烯酰胺对细胞内P53蛋白和细胞内信号转导通路的影响。丙烯酰胺增加P53、磷酸化P53和P53相关蛋白小鼠双分钟2(MDM2)。P53的磷酸化是Ser15位点特异性的。在丝裂原活化蛋白激酶(MAPKs)中，丙烯酰胺可引起细胞外信号调节蛋白激酶(ERK)和p38的磷酸化，但不能引起c-jun氨基末端的磷酸化。然而，LL-Z1640-2阻断p38通路并不能抑制P53在Ser15位的磷酸化。相反，ERK的特异性抑制剂(U0126或PD98059)可以消除P53的积聚和磷酸化。U0126也取消了MDM2的上调。磷脂酰肌醇3-激酶相关激酶(Pikk)通路的抑制剂(Wortmannin)可抑制P53蛋白及其磷酸化的增加。因此，丙烯酰胺通过ER…增加P53蛋白及其在Ser15的磷酸化更多的K和/或Pikk路径。另一方面，U0126和PD98059在一定程度上抑制了台盼蓝拒染法和乳酸脱氢酶漏出法测定的丙烯酰胺的细胞毒性，而LL-Z1640-2和Wortmannin均不能有效地抑制丙烯酰胺的细胞毒性。因此，ERK通路似乎在引起SH-SY5Y细胞中P53的磷酸化和丙烯酰胺的细胞毒性中发挥作用。丙烯酰胺(1-5 mM)剂量依赖性地降低SH-SY5Y细胞的存活率。Caspase-3活性和亚G1期细胞数在3 mM丙烯酰胺作用下升高并达到峰值，而在较高剂量(4 mM和5 mM)时两者均较低。PAN-caspase抑制剂Z-VAD-fmk可降低丙烯酰胺的表观细胞毒性。U0126抑制Caspase-3活性的升高，同时抑制亚G1期细胞的活性。因此，虽然可能涉及caspase依赖的细胞凋亡以外的机制，但丙烯酰胺对SH-SY5Y细胞的毒性可能是通过ERK途径和caspase-3的激活而发生的。较少

Using human neuroblastoma SH-SY5Y cells, effects of acrylamide on p53 protein and intracellular signal transducting pathways were examined. Acrylamide increased p53, phosphorylated p53, and p53-associated protein murine double minute 2 (MDM2). The phosphorylation of p53 was specific for the Ser15 site. Among mitogen-activated protein kinases (MAPKs), acrylamide caused phosphorylation of extracellular signal-regulated protein kinase (ERK) and p38 but not c-Jun NH2-terminal kinase. Nevertheless, blocking p38 pathway by LL-Z1640-2 did not suppress the phosphorylation of p53 at Ser15. In contrast, a specific inhibitor of ERK kinase (U0126 or PD98059) could abolish the accumulation as well as the phosphorylation of p53. Elevation of MDM2 was also abolished by U0126. An inhibitor of phosphatidylinositol 3-kinase-related kinase (PIKK) pathway (wortmannin) suppressed the increase of p53 and its phosphorylation. Hence, acrylamide increases p53 protein and its phosphorylation at Ser15 through ER … More K and/or PIKK pathways. On the other hand, U0126 and PD98059 suppressed to some extent the cytotoxicity of acrylamide evaluated by trypan blue exclusion and lactate dehydrogenase (LDH) leakage, whereas neither LL-Z1640-2 nor wortmannin was effective in suppressing the toxicity. Thus, ERK pathway seems to play a role both in causing the phosphorylation of p53 and in the cytotoxicity of acrylamide in SH-SY5Y cells.Acrylamide (1-5 mM) dose-dependently decreased cell viability in SH-SY5Y cells. The caspase-3 activity and cell population in sub-G1 phase were elevated and peaked on exposure to 3 mM acrylamide, while both were less so at higher dose (4 and 5 mM). Z-VAD-fmk, a pan-caspase inhibitor, lowered the apparent cytotoxicity of acrylamide. U0126 suppressed the elevation of caspase-3 activities as well as that of sub-G1 population. Thus, although mechanisms other than caspase-dependent apoptosis may be involved, apoptotic process seems to take place in the genesis of toxicity of acrylamide in SH-SY5Y cells through ERK pathway and activation of caspase-3. Less