MITOCHONDRIAL DAMAGE AND APOPTOSIS INDUCTION OF QUINOLINIUMS ON TUMOR CELLS

喹啉对肿瘤细胞的线粒体损伤和凋亡诱导

• 批准号: 8360147
• 负责人: Beatriz Zayas
• 金额: $ 10.36万
• 依托单位: UNIVERSITY OF PUERTO RICO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360147
• 关键词: Antineoplastic Agents; Apoptosis; Apoptotic; Biomedical Research; Cancer cell line; Caspase; Cell Death; Cell Membrane Permeability; Cells; Charge; Cisplatin; DNA Fragmentation; Dose; Early identification; Endoplasmic Reticulum; Etoposide; Event; Fluorescence; Funding; Generations; Goals; Grant; Heterocyclic Compounds; Human; Induction of Apoptosis; Inhibitory Concentration 50; Lymphocyte; Mitochondria; Modification; Molecular; National Center for Research Resources; Nature; Normal Cell; Normal tissue morphology; Organelles; Outcome; Paclitaxel; Pathway interactions; Principal Investigator; Process; Puerto Rico; Reactive Oxygen Species; Research; Research Infrastructure; Resources; Salts; Site; Source; Staging; Stress; T-Lymphocyte; Therapeutic; Therapeutic Agents; Time; Toxic effect; Tumor Cell Line; United States National Institutes of Health; antitumor agent; cancer cell; caspase-3; caspase-8; cell growth; cost; cytochrome c; cytotoxic; cytotoxicity; endoplasmic reticulum stress; instrumentation; killings; lymphoblast; mitochondrial membrane; neoplastic cell; novel; research study; response; tumor

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The overall goal of this project is to further understand the mechanism of toxicity of Benzazolo[3,2-a]quinolinium Salts (BQs) in tumor cells and compare its cytotoxicity to the ones observed on normal T lymphocytes as surrogate of effect for other normal tissues. Within the cytotoxic effects, mitochondrial damage, apoptosis, ROS release and endoplasmic reticulum stress of BQs are included. Known anticancer drugs including etoposide and taxol often present the capability of multiple mechanisms of actions. Preliminary studies with A431 cells have indicated the capacity of BQs to inhibit cellular growth, interact with mitochondria and induce apoptosis similarly to Cisplatin. Additional studies however are necessary to further understand the cell death pathway of cells exposed to BQs. The compounds under study, are nitro and amino containing heterocyclic compounds possessing a positive charge that could facilitate their interaction with cell organelles specially mitochondria. Two of the compounds under study are amino derivatives with fluorescence activity facilitating the observation of their cellular site of action. The cationic nature of these BQs has been implicated in preferential intramitochondrial retention, which allows selective killing of cancer cells. In addition, substituent effects in these compounds may confer differential toxicity. Preliminary results suggested that the BQs can induce apoptosis on A431 cells through the intrinsic pathway involving mitochondrial membrane permeability and activating the effectors caspases 3 & 7. Results also suggest the inactivity of caspase 8 in the apoptotic pathway. In this study we will also analyze the toxicity of BQs on normal T lymphocytes as surrogate of effect for other normal cells. Comparison with normal cells is crucial in the process of identifying appropriate chemo therapeutic agents. Understanding how normal lymphocytes react to BQs' exposure as potential anti tumor agent will provide information regarding the selectivity of BQs toward tumor cells preferentially and the identification of early molecular events that could serve as predictive markers of BQs therapeutic outcomes. Hypothesis 1: Structural similarities with known anti tumor compounds suggest that the BQs understudy (NBQ-38, ABQ-38, NBQ-95, ABQ-95 and BQ-108) can induce apoptosis on A431 through an activation pathway that can include the following mechanisms: Mitochondrial damage; Cytochrome c release; Caspase activation; endoplasmic reticulum stress and or generation of reactive oxygen species. Aim 1: Determine the apoptosis induction and mitochondrial interaction of BQs in tumor cell lines. The mitochondrial Cytochrome c release on tumor cells will also be determined as confirmation of mitochondrial damage associated with apoptosis. Aim 2: Determine the activation of Caspases 3, 7 and 8 as a way of determining the extrinsic or intrinsic apoptotic pathway. Aim 3: Determine the generation of ROS as part of the mechanism of cell death on tumor cells treated with BQs. Aim 4: Determine the capacity of BQs to induce stress to the reticulum endoplasmic of tumor cells. Modifications have been made to the study aims. A new aim has been included at this stage:" To determine the DNA fragmentation capacity of BQs in tumor cells". This new aim was not included in the original proposal since we did not have the appropriate instrumentation at the time of submission. It is however relevant in the characterization of the mechanism of apoptosis induced by these novel substances since DNA fragmentation can be expexted as part of the apoptosis proces. The purpose of this experiment was to evaluate the capacity of BQs to induce DNA fragmentation in A431 tumor cells at the determined IC50. Hypothesis 2: BQs can induce higher toxicity to cancer cell lines than to normal lymphocytes cells through the induction of apoptosis and mitochondria! damage. Aim 5. Determine the cytotoxic dose response of BQs on human normal TK-6 lymphocytes. Aim 6. Determine the apoptosis induction and mitochondrial interaction of BQs in human TK6 lymphoblast cells in culture. Aim 7. Determine through statistical analysis the significant difference in dose response effect between tumor and normal cell exposed to BQs.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 该项目的总体目的是进一步了解苯佐佐洛[3,2-A]喹啉盐（BQ）在肿瘤细胞中的毒性机制，并将其细胞毒性与在正常T淋巴细胞上观察到的细胞毒性与其他正常组织的替代物进行比较。在细胞毒性作用中，包括BQ的线粒体损伤，凋亡，ROS释放和内质网应激。包括依托泊苷和紫杉醇在内的已知抗癌药物通常具有多种作用机理的能力。对A431细胞的初步研究表明，BQ抑制细胞生长，与线粒体相互作用并诱导凋亡的能力与顺铂相似。然而，有必要进一步了解暴露于BQ的细胞的细胞死亡途径。所研究的化合物是硝基和氨基，含有具有阳性电荷的杂环化合物，可以促进其相互作用 与细胞细胞器特别是线粒体。研究的两种化合物是氨基衍生物，具有荧光活性，促进了其细胞作用部位的观察。这些BQ的阳离子性质与优先的Intamochrial保留有关，这允许选择性杀死癌症 细胞。另外，这些化合物中的取代效应可能赋予差异毒性。初步结果表明，BQS可以通过涉及线粒体膜通透性的固有途径诱导A431细胞的凋亡，并激活效应子caspases 3＆7。结果还表明，在源途径中，caspase 8的不活跃。在这项研究中，我们还将分析BQ对正常T淋巴细胞的毒性，因为其他正常细胞的作用替代。在鉴定适当的化学治疗剂的过程中，与正常细胞的比较至关重要。理解正常淋巴细胞对BQS暴露的反应如何作为潜在的抗肿瘤剂，将优先提供有关BQ对肿瘤细胞的选择性的信息，并鉴定出可以用作BQS治疗结果预测标志物的早期分子事件的鉴定。 假设1：与已知抗肿瘤化合物的结构相似性表明，BQS研究（NBQ-38，ABQ-38，NBQ-95，ABQ-95，ABQ-95和BQ-108）可以通过激活途径诱导A431上的凋亡，该激活途径可以包括以下机制：线粒体损害损害；细胞色素C释放； caspase激活；内质网应激和反应性氧的产生。 目标1：确定肿瘤细胞系中BQ的凋亡诱导和线粒体相互作用。肿瘤细胞上的线粒体细胞色素C释放也将确定为确认与凋亡相关的线粒体损伤。 AIM 2：确定胱天蛋白酶3、7和8的激活，以确定外在或固有的凋亡途径。 AIM 3：确定ROS的产生，作为用BQ处理的肿瘤细胞机理的一部分。 AIM 4：确定BQ诱导肿瘤细胞内质的压力的能力。对研究的目标进行了修改。在此阶段已经包括了一个新的目标：“确定肿瘤细胞中BQ的DNA碎片能力”。这个新目标未包含在原始提案中，因为我们在提交时没有适当的仪器。但是，这与这些新型物质诱导的凋亡机理的表征相关，因为DNA片段化可以作为凋亡过程的一部分进行过渡。该实验的目的是评估BQ在确定的IC50处诱导A431肿瘤细胞中DNA碎片化的能力。 假设2：通过诱导细胞凋亡和线粒体，BQs可以诱导对癌细胞系的毒性高于正常淋巴细胞的毒性！损害。 目标5。确定BQ对人正常TK-6淋巴细胞的细胞毒性剂量反应。 目标6。确定人类TK6淋巴细胞在培养中BQS的凋亡诱导和线粒体相互作用。 目标7。通过统计分析确定肿瘤和暴露于BQ的正常细胞之间剂量反应效应的显着差异。