The GHIRH-R mediated signaling pathways in regulating cell death of retinoblastoma cells

Ph.D.Retinoblastoma (RB) is the most common primary intraocular cancer in young children, and 95% tumors are diagnosed before the age of 5 years old. Late diagnose and poor treatment methods may lead to cancer metastasis and fatality. The incidence of retinoblastoma in the world is one case among 15,000 to 20,000 livebirths with an estimation of about 8000 children every year.Growth hormone-releasing hormone receptor (GHRH-R) is highly expressed in various types of cancer, and recently it has been found highly and specifically expressed in retinoblastoma other than normal retinal cells. GHRH antagonists, MIA- 602 and MIA-690 have been shown to inhibit proliferation and induce apoptosis in RB cells. In our study, we used a retinoblastoma cell line (Y79) to identify the signaling mechanism mediated by GHRH-R, meanwhile the retinal pigment epithelial cells (ARPE-19) were applied as normal control.The results of flow cytometry and TUNEL assay showed that apoptotic cell population in Y79 cells were significantly increased, while the ARPE-19 cells were not sensitive to treatment of GHRH antagonists. Moreover, the expression level of apoptosis and proliferation related proteins, such as p-AKT, cleaved PARP, P53 and cytochrome c, were measured by Western Blot. The results showed that AKT phosphorylation level was decreased, whereas P53 was activated and cytochrome c was released into cytosol after MIA-602 and MIA-690 treatment. We further checked the changes of apoptosis related genes, such as Caspase-3, Caspase-8, Caspase-9, Bad and P53. We found that intrinsic caspase family and P53 genes were activated after blockage of GHRH receptor. All the results suggested that AKT and intrinsic apoptosis signaling pathways played important roles in the GHRH antagonists induced cell death.Besides, we used PrePPI and FpClass prediction software to identify proteins that could interact potentially with GHRH-R. We found insulin receptor substrate 1 (IRS- 1) as a candidate, which played an important role in PI3K-AKT pathway. By using immunoprecipitation (IP), we confirmed the interaction between GHRH-R and IRS-1, suggesting a potential role of IRS-1 in the GHRH-R signaling pathway mediating proliferation and apoptosis in retinoblastoma cells.Finally, IRS-1 inhibitor NT157 was used to treat Y79 cells, and it could greatly enhance the cell apoptosis of retinoblastoma cells. All the results provided new evidence that the combination therapy with GHRH antagonist and IRS-1 inhibitor might be a potential option for retinoblastoma treatment.視網膜母細胞瘤（RB）是幼兒中最常見的原發性眼內癌，並且95％的腫瘤在5歲之前診斷出。 晚期診斷和不良治療方法可能導致癌症轉移和死亡。 世界上視網膜母細胞瘤的發病率是15 000 至20 000 例活產中的一例，估計每年約有8000 名兒童。生長激素釋放激素受體（GHRH-R）在各種類型的癌症中高度表達，並且最近發現它在視網膜母細胞瘤中高度特異性地表達，而不在正常的視網膜細胞中。研究已經顯示GHRH-R 拮抗劑MIA-602 和MIA-690 在RB 細胞中抑制增殖並誘導細胞凋亡。在我們的研究中，我們選擇視網膜母細胞瘤細胞（Y79）為實驗對象，視網膜色素上皮細胞（ARPE-19）作為對照，以鑑定GHRH-R 介導的信號傳導機制。流式細胞術和TUNEL 法檢測結果顯示，在GHRH-R 拮抗劑處理之後，Y79 細胞凋亡明顯增多，而ARPE-19 細胞對GHRH-R 拮抗劑的治療不敏感。此外，通過蛋白質印跡法測量凋亡和增殖相關蛋白的表達水平，例如磷酸化的AKT，切割的PARP，P53 和細胞色素c。結果顯示，在MIA-602 和MIA-690 處理後，AKT 磷酸化水平降低，而P53 被激活，細胞核c 被釋放到細胞質中。我們進一步檢查了凋亡相關基因水平的變化，如Caspase-3，Caspase-8，Caspase-9，Bad和P53。我們發現內源性caspase 家族和P53 在GHRH 受體阻斷後被激活。所有結果表明，AKT 信號通路和內源性凋亡信號通路在GHRH-R 拮抗劑誘導的RB 細胞死亡中起重要作用。此外，我們使用PrePPI 和FpClass 預測軟件來鑑定可能與GHRH-R 相互作用的蛋白質。 我們發現胰島素受體底物1（IRS-1）作為候選物，其在PI3K-AKT 和AMPK-ERK 通路中起重要作用。 通過使用免疫沉澱（IP），我們證實了GHRH-R 和IRS-1 之間的相互作用，表明IRS-1 在GHRH-R 信號傳導途徑中介導視網膜母細胞瘤細胞中增殖和凋亡的潛在作用。最後，IRS-1 抑製劑NT157 用於治療Y79 細胞，可顯著增強視網膜母細胞瘤細胞的凋亡。 所有結果提供了新的證據，即GHRH 拮抗劑和IRS-1 抑製劑的聯合治療可能是視網膜母細胞瘤治療的潛在選擇。Chen, Binbin.Thesis Ph.D. Chinese University of Hong Kong 2019.Includes bibliographical references (leaves 83-98).Abstracts also in Chinese.Title from PDF title page (viewed on 13, May, 2021)