Chowdhury Parimal
University of Arkansas for Medical Sciences, Little Rock, AR, USA
Ann Clin Lab Sci. 2017 Sep;47(5):572-580.
Aminoguanidine (AG), a diamine oxidase and a nitric oxide synthase inhibitor, was used in diabetes, thyroid follicular carcinoma, hepatocellular carcinoma, pancreatic cancer xenografts and in breast cancer research. The effects of AG on these pathologic conditions may be related to its regulatory effects on cell proliferation, angiogenesis, and expression of antioxidant enzymes. However, its role as pro and/or anti-oxidant affecting signaling and function in pancreatic tumor cell lines has not been studied. The current study tested the hypothesis that exposure of AR42J cells to aminoguanidine will induce pro-oxidant effects that may lead to increased proliferation and growth of these cells.
AR42J cells were grown in F-12 nutrient medium in 5% CO at 37°C to attain over 90% confluency before being treated with 20 uM hydrogen peroxide (HO) for 20 min and 100 uM AG for 30 min separately and in combination. Cell lysates collected from these experiments were measured for formation of lipid peroxides by malondialdehyde (MDA) assay and for activation of phospho-ERK 1/2 signal transduction by Western blotting. The activation of ERK signaling was further confirmed by immunohistochemical analysis. Effect of ERK1/2 on cell proliferation in response to AG and HO was evaluated by MTT assay while the functional status of AR42J cells was determined by release of amylase following CCK-8 stimulation.
MDA concentration in cells treated with AG was not different from untreated cells. However, treatment with HO either alone or in combination with AG increased MDA significantly (<0.05). AG treatment alone induced 3.5 fold activation of pERK-1/2, as compared to 2.5 fold increase with HO alone (<0.05) as compared to untreated control. The results of ERK activation were confirmed further by its co-localization employing FITC-conjugated ERK antibody. AG -induced maximal cell proliferation occurred at 48 hr. incubation (<0.05); these values were not significantly different from that of HO treated and control cells. Cell function (CCK-stimulated amylase release) was significantly enhanced by AG (<0.05).
These data suggest that in an in-vitro system, AG acts as a pro-oxidant on AR42J cell proliferation and possibly affects the resulting function.
氨基胍（AG）是一种二胺氧化酶和一氧化氮合酶抑制剂，已用于糖尿病、甲状腺滤泡癌、肝细胞癌、胰腺癌异种移植以及乳腺癌研究。AG对这些病理状况的影响可能与其对细胞增殖、血管生成和抗氧化酶表达的调节作用有关。然而，其作为促氧化剂和/或抗氧化剂对胰腺肿瘤细胞系信号传导和功能的影响尚未得到研究。本研究检验了以下假设：AR42J细胞暴露于氨基胍会诱导促氧化作用，可能导致这些细胞的增殖和生长增加。
AR42J细胞在含5%二氧化碳的F-12营养培养基中于37°C培养，直至汇合度超过90%，然后分别用20 μM过氧化氢（H₂O₂）处理20分钟和100 μM AG处理30分钟，以及联合处理。从这些实验中收集的细胞裂解物通过丙二醛（MDA）测定法测量脂质过氧化物的形成，并通过蛋白质印迹法测量磷酸化ERK 1/2信号转导的激活。ERK信号传导的激活通过免疫组织化学分析进一步证实。通过MTT测定法评估ERK1/2对AG和H₂O₂刺激下细胞增殖的影响，而AR42J细胞的功能状态通过CCK-8刺激后淀粉酶的释放来确定。
用AG处理的细胞中MDA浓度与未处理的细胞无差异。然而，单独用H₂O₂处理或与AG联合处理均显著增加了MDA（<0.05）。与未处理的对照相比，单独用AG处理诱导pERK-1/2激活3.5倍，单独用H₂O₂处理增加2.5倍（<0.05）。通过使用FITC偶联的ERK抗体共定位进一步证实了ERK激活的结果。AG诱导的最大细胞增殖发生在孵育48小时时（<0.05）；这些值与H₂O₂处理的细胞和对照细胞无显著差异。AG显著增强了细胞功能（CCK刺激的淀粉酶释放）（<0.05）。
这些数据表明，在体外系统中，AG对AR42J细胞增殖起促氧化剂作用，并可能影响其最终功能。
