Investigating drug targets and improving drug delivery for anti-cancer treatment of osteosarcoma

研究药物靶点并改善骨肉瘤抗癌治疗的药物输送

• 批准号: 10599098
• 负责人: Shahana Sultana Mahajan
• 金额: $ 39万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10599098
• 关键词: ABL1 gene; Alanine; Amyotrophic Lateral Sclerosis; Animals; Antineoplastic Agents; Apoptosis; Apoptotic; B-Lymphocytes; Binding; Biodistribution; Biological Assay; Bioluminescence; Cell Nucleus; Cell Proliferation; Cells; Child; Collaborations; Cytoplasm; DNA Damage; Data; Diagnosis; Drug Delivery Systems; Drug Targeting; FDA approved; Genes; Genetic Transcription; Glutamates; Growth; Human; Hyperthermia; Implant; In Vitro; Induction of Apoptosis; Knowledge; Lateral; Literature; Luciferases; Magnetic Resonance Imaging; Magnetism; Malignant - descriptor; Malignant Bone Neoplasm; Malignant Childhood Neoplasm; Malignant Neoplasms; Maps; Measures; Mediating; Metastatic Osteosarcoma; Methods; Modeling; Molecular; Monitor; Mouse Cell Line; Mus; Nanosphere; Nanotechnology; Neoplasm Metastasis; Nuclear; Nude Mice; Oncogenes; Organ; Pathway interactions; Patients; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Predisposition; Proliferating; Proteins; Publishing; Receptor Signaling; Reporter; Riluzole; Role; Serine; Site; Small Interfering RNA; Sorting; Stress; Survival Rate; Tail; Testing; Therapeutic; Time; Transcription Coactivator; Treatment outcome; Veins; Work; Xenograft procedure; animal imaging; anticancer treatment; autocrine; bioluminescence imaging; drug repurposing; effective therapy; efficacy evaluation; experimental study; glutamatergic signaling; improved; in vivo; inhibitor; iron oxide; knock-down; metabotropic glutamate receptor 5; mouse model; mutant; nanobiotechnology; nanocage; nanoparticle; novel therapeutic intervention; osteosarcoma; prevent; primary bone cancer; promoter; receptor; siRNA delivery; small hairpin RNA; transcription factor; tumor; tumor growth; young adult

Osteosarcoma is cancer of bone, most common in children and young adults. Metastasis is either present at the time of diagnosis or develops later during the course of treatment in most patients. The survival rate with metastatic osteosarcoma is very low, therefore new therapeutic interventions are needed. Our lab has demonstrated that Riluzole, a glutamate release inhibitor, is effective in inhibiting proliferation and inducing apoptosis both in human and mouse osteosarcoma. Furthermore, we have demonstrated that Riluzole blocks the activity of the mGluR5 receptor signaling to inhibit growth in osteosarcoma cells. Yes associated protein (YAP) is a transcription co-activator involved in cell proliferation. The evidence in literature shows that YAP phosphorylated at serine 127 facilitates cytoplasmic sequestration and degradation of YAP. Furthermore, YAP is phosphorylated at Y357 by C-Abl kinase under DNA damage-induced stress. Interestingly, phosphorylation of YAP at Y357 promotes strong interaction with p73, a transcription co-activator, to induce transcription of pro- apoptotic genes. Our data has shown that Riluzole decreased phosphorylation of YAP at serine 127 and increased nuclear localization of YAP. Furthermore, Riluzole also changed the localization of a YAP mutant, YAPS5A (five serine residues at 61, 109, 127, 164, and 397 changed to alanine) from cytoplasm to nucleus suggesting that change is localization is independent of phosphorylation at these sites. We hypothesize that the increase in nuclear localization of YAP facilitates transcription of pro-apoptotic genes. We want to determine if YAP is directly involved in Riluzole-induced apoptosis and if YAP is regulated by C-Abl to activate pro-apoptotic genes in osteosarcoma cells. We will use human metastatic osteosarcoma cells, LM7, and mouse cells lines, OS482, to study the effect of: a) Riluzole on phosphorylation of YAP at Y357 b) C-Abl inhibitors on phosphorylation of YAP at Y357 and Riluzole-induced apoptosis c) Riluzole on osteosarcoma cells with YAP knockdown or C-Abl knockdown to confirm the role of YAP and C-Abl in apoptosis d) Riluzole on YAP and P73 binding and on transcription of Bax promoter in a luciferase reporter assay. Our recent in vitro data has demonstrated that Riluzole released from the iron oxide nanocage is more effective in inducing apoptosis in LM7 cells compared to free Riluzole or Riluzole released from the nanosphere. We hypothesize that Riluzole released from Riluzole-loaded nanocage will be more effective, compared to free Riluzole, in reducing metastasis in a nude mouse model. We will implant osteosarcoma cells, LM7.eGFP.ffLuc, in the tail vein of nude mouse. We will randomly sort the animals in 4 groups and carry out the following treatments. 1) no treatment, 2) free Riluzole, 3) nanocage alone, 4) Riluzole-loaded nanocage. We will treat the animals and monitor metastasis using bioluminescence imaging in all groups. We will perform whole animal magnetic resonance imaging (MRI) for biodistribution of iron oxide nanocage carriers and then use quantitative susceptibility mapping (QSM) to quantify the concentration of iron oxide nanocages at metastasis sites. Our data has demonstrated that glutamate signaling via mGluR5 is important in colony forming ability of LM7 cells. In Aim 3, we will assess the efficiency of mGluR5 siRNA delivery to decrease tumor size in a xenograft mouse model. We will use mGluR5 siRNA-loaded iron oxide nanocage and induce siRNA release using magnetic hyperthermia in mice. We will perform whole animal MRI/QSM and ex vivo MRI/QSM to determine the nanocage concentration in each organ and tumor site. We will perform and analyze the experiments in Aim 2 and Aim 3 in collaboration with my colleague, Dr. Hiroshi Matsui, who is an expert in bionanotechnology. In brief, we expect to: a) demonstrate a direct role of YAP in Riluzole-induced apoptosis of osteosarcoma cells b) assess the efficacy of the delivery method of Riluzole in preventing metastasis in xenograft nude mouse model c) deliver mGluR5 siRNA via nanoparticle to prevent tumor establishment in a xenograft mouse model. Riluzole is used as a FDA approved therapeutic drug for Amyotrophic Lateral Sclerosis (ALS). This study is a drug- repurposing study in which Riluzole is used as an anti-cancer agent for osteosarcoma. !

骨肉瘤是骨骼癌，最常见于儿童和年轻人。转移是存在于 大多数患者在治疗过程中诊断或在后期发展。与 转移性骨肉瘤非常低，因此需要新的治疗干预措施。我们的实验室有 证明Riluzole是一种谷氨酸释放抑制剂，可有效抑制增殖和诱导 人和小鼠骨肉瘤中的凋亡。此外，我们已经证明了riluzole块 MGLUR5受体信号传导的活性抑制骨肉瘤细胞的生长。是相关的蛋白质 （YAP）是参与细胞增殖的转录共激活剂。文献中的证据表明yap 在丝氨酸127上磷酸化促进了YAP的细胞质隔离和降解。此外，是的 在DNA损伤诱导的应激下，通过C-ABL激酶在Y357处磷酸化。有趣的是，磷酸化 在Y357处的YAP促进了与转录共激活因子P73的强烈相互作用，以诱导促进的转录 凋亡基因。我们的数据表明，riluzole在丝氨酸127和 YAP的核定位增加。此外，riluzole还改变了yap突变体的定位， YAPS5A（61、109、127、164和397的五个丝氨酸残基变为丙氨酸）从细胞质变为核 表明变化是本地化的独立于这些位点的磷酸化。我们假设这一点 YAP核定位的增加促进了促凋亡基因的转录。我们想 确定YAP是否直接参与riluzole诱导的细胞凋亡，以及是否通过C-ABL调节YAP以激活 骨肉瘤细胞中的凋亡基因。我们将使用人类转移性骨肉瘤细胞，LM7和 小鼠细胞系OS482，研究：a）riluzole对Y357 yap磷酸化的影响 Y357和riluzole诱导凋亡的YAP磷酸化的抑制剂c）骨肉瘤上的riluzole 具有YAP敲低或C-ABL敲低的细胞确认YAP和C-ABL在凋亡中的作用d）riluzole 在Yap和p73结合以及在荧光素酶报告基因测定中的BAX启动子的转录上。 我们最近的体外数据表明，从氧化铁纳米叶中释放的瑞唑更多是 与自由riluzole或Riluzole相比，有效诱导LM7细胞凋亡 纳米球。我们假设从riluzole负载的纳米层释放的里卢佐尔将更有效， 与游离riluzole相比，在减少裸鼠模型中的转移中。我们将植入骨肉瘤细胞， lm7.egfp.ffluc，在裸鼠的尾部静脉中。我们将以4组的方式随机分类动物，然后进行 以下治疗方法。 1）无治疗，2）游离riluzole，3）单独使用纳米，4）riluzole负载的纳米腔。 我们将使用所有组中的生物发光成像来治疗动物并监测转移。我们将表演 全动物磁共振成像（MRI）用于氧化铁纳米载体的生物分布，然后 使用定量敏感性映射（QSM）来量化氧化铁纳米的浓度 转移部位。 我们的数据表明，通过MGLUR5通过MGLUR5信号传导在LM7的菌落形成能力中很重要 细胞。在AIM 3中，我们将评估MGLUR5 siRNA递送的效率，以减少异种移植物中的肿瘤大小 鼠标模型。我们将使用MGLUR5 siRNA负载的氧化铁纳米层，并使用 小鼠的磁性体温。我们将执行全动物MRI/QSM和EX VIVO MRI/QSM来确定 每个器官和肿瘤部位的纳米含量。我们将执行并分析AIM中的实验 2和AIM 3与我的同事Hiroshi Matsui博士合作，他是Bionanotechnology的专家。 简而言之，我们期望：a）证明YAP在riluzole诱导的骨肉瘤细胞凋亡中的直接作用 b）评估riluzole递送方法预防异种移植小鼠转移的功效 C）通过纳米颗粒传递MGLUR5 siRNA，以防止异种移植小鼠模型中的肿瘤建立。 riluzole用作FDA批准的肌萎缩性侧索硬化症（ALS）的治疗药物。这项研究是 riluzole用作骨肉瘤的抗癌剂的药物重复研究。 呢

项目成果

Delivery of mGluR5 siRNAs by Iron Oxide Nanocages by Alternating Magnetic Fields for Blocking Proliferation of Metastatic Osteosarcoma Cells.

• DOI: 10.3390/ijms23147944
• 发表时间: 2022-07-19
• 期刊: International journal of molecular sciences
• 影响因子: 5.6

Riluzole-induced apoptosis in osteosarcoma is mediated through Yes-associated protein upon phosphorylation by c-Abl Kinase.

• DOI: 10.1038/s41598-021-00439-8
• 发表时间: 2021-10-25
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Raghubir M;Azeem SM;Hasnat R;Rahman CN;Wong L;Yan S;Huang YQ;Zhagui R;Blyufer A;Tariq I;Tam C;Lhamo S;Cecilio L;Chowdhury Y;ChandThakuri S;Mahajan SS
• 通讯作者: Mahajan SS