背景:肽基(蛋白质)精氨酸脱亚胺酶(PAD)在钙的存在下提供肽基精氨酸向肽基瓜氨酸的转化,并进行翻译后修饰。失调的PAD活性对包括癌症在内的太多疾病起重要作用。在这项研究中,目的是确定PAD抑制剂氯脒(Cl-amidine)的潜在细胞毒性和凋亡活性,最近在人胶质母细胞瘤细胞系Uppsala87恶性胶质瘤(U-87MG)的体外和体内研究中显示了其有效性,形成了多形性胶质母细胞瘤(GBM)的体外模型,该模型在脑肿瘤中最具侵略性且死亡率最高。
方法:在研究中,研究了Cl-脒对GBM癌症模型的抗增殖和凋亡作用。在第24和48小时,通过4-[3-(4-碘苯基)-2-(4-硝基苯基)-2H-5-四唑]-1,3-苯二磺酸法测定了Cl-脒对U-87MG细胞的抗增殖作用。通过膜联蛋白V和碘化丙啶染色分析凋亡效应,caspase-3激活,和线粒体膜极化(5,5',6,6'-四氯-1,1',流式细胞术中的3,3'四乙基苯并咪唑基碳花青碘化物)方法。
结果:已经确定Cl-脒以时间和浓度依赖性方式在U-87MG细胞系上表现出显著的抗增殖特性,如通过4-[3-(4-碘苯基)-2-(4-硝基苯基)-2H-5-四唑]-1,3-苯二磺酸盐测定所测定。通过膜联蛋白V和碘化丙啶染色和5,5',6,6'-四氯-1,1',3,3'四乙基苯并咪唑基碳花青碘化物方法显示出显着的功效,特别是在24小时的暴露期之后。已经观察到Cl-脒通过增强线粒体去极化诱导细胞凋亡,独立于caspase-3的激活。此外,关于它对健康细胞的影响,已经证明,与卡莫司汀相比,Cl-脒显示出更低的细胞毒性作用,胶质母细胞瘤的重要治疗剂。
结论:这项研究的结果表明,Cl-脒在治疗GBM中显示出作为抗癌药物的重要潜力。这一结论是基于其在U-87MG细胞中观察到的值得注意的抗增殖和凋亡作用。以及与现有治疗相比,其对健康细胞的细胞毒性降低。我们建议应通过更广泛的癌细胞类型进一步研究Cl-脒的抗肿瘤特性。此外,我们认为,研究Cl-脒与单一或联合治疗的协同相互作用有望发现新型抗癌药物。
BACKGROUND: Peptidyl (protein) arginine deiminases (PADs) provide the transformation of peptidyl arginine to peptidyl citrulline in the presence of calcium with posttranslational modification. The dysregulated PAD activity plays an important role on too many diseases including also the cancer. In this study, it has been aimed to determine the potential cytotoxic and apoptotic activity of chlorine-amidine (Cl-amidine) which is a PAD inhibitor and whose effectiveness has been shown in vitro and in vivo studies recently on human glioblastoma cell line Uppsala 87 malignant glioma (U-87 MG) forming an in vitro model for the glioblastoma multiforme (GBM) which is the most aggressive and has the highest mortality among the brain tumors.
METHODS: In the study, the antiproliferative and apoptotic effects of Cl-amidine on GBM cancer model were investigated. The antiproliferative effects of Cl-amidine on U-87 MG cells were determined by 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate method at the 24th and 48th hours. The apoptotic effects were analyzed by Annexin V and Propidium iodide staining, caspase-3 activation, and mitochondrial membrane polarization (5,5\', 6,6\'-tetrachloro-1,1\', 3,3\' tetraethyl benzimidazolyl carbocyanine iodide) methods in the flow cytometry.
RESULTS: It has been determined that Cl-amidine exhibits notable antiproliferative properties on U-87 MG cell line in a time and concentration-dependent manner, as determined through the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate assay. Assessment of apoptotic effects via Annexin V and Propidium iodide staining and 5,5\', 6,6\'-tetrachloro-1,1\', 3,3\' tetraethyl benzimidazolyl carbocyanine iodide methods has revealed significant efficacy, particularly following a 24-hour exposure period. It has been observed that Cl-amidine induces apoptosis in cells by enhancing mitochondrial depolarization, independently of caspase-3 activation. Furthermore, regarding its impact on healthy cells, it has been demonstrated that Cl-amidine shows lower cytotoxic effects when compared to carmustine, an important therapeutic agent for glioblastoma.
CONCLUSIONS: The findings of this study have shown that Cl-amidine exhibits significant potential as an anticancer agent in the treatment of GBM. This conclusion is based on its noteworthy antiproliferative and apoptotic effects observed in U-87 MG cells, as well as its reduced cytotoxicity toward healthy cells in comparison to existing treatments. We propose that the antineoplastic properties of Cl-amidine should be further investigated through a broader spectrum of cancer cell types. Moreover, we believe that investigating the synergistic interactions of Cl-amidine with single or combination therapies holds promise for the discovery of novel anticancer agents.