Novel Platinum (Pt)-Vandetanib Hybrid Compounds: Design, Synthesis and Investigation of Anti-cancer Activity and Mechanism of Action

• Other Authors: Fei, Rong (author.)
• Format: Others
• Language: English; Chinese
• Published: 2016
• Online Access: http://repository.lib.cuhk.edu.hk/en/item/cuhk-1292417

實驗目的： === 肺癌已經成為造成人類的惡性腫瘤死亡原因的一大成因，其中非小細胞肺癌（NSCLC）占全部肺癌的80-85%。約70%的非小細胞肺癌患者攜帶有表皮生長因子受體絡氨酸激酶（EGFR）編碼外顯子的變異，從而使得酶活性發生改變。抑制劑（EGFR-TKI）已經成為一種有效的NSCLC的治療手段，已經被FDA認證的一代抑制劑，是專門針對治療EGFR敏化突變的靶向治療藥物。但是EGFR的二次突變會造成一代抑制劑的抗藥性。凡德他尼作為一個多靶向抑制劑，被引入到臨床治療中，然而并沒有能夠克服EGFR T790M導致的抗藥性。因而發展新型針對性的小分子抗癌藥物十分迫切。本研究中凡德他尼與二價鉑通過化學鍵形成雜合物，結合兩種藥物的共同作用克服抗藥性，展現新的生化性質。 === 實驗方法： === 新型鉑-凡德他尼雜合物通過 “鉑-生物活性分子”化合物合成手段合成，并由HPLC，NMR，質譜等進行特征表徵。雜合物對目標受體EGFR抑製作用通過多種途徑測試。對於雜合物的抗藥性，分別從（1）HSCLC細胞系，（2）EGFR變異蛋白兩種模型進行研究。在雜合物作用機理的研究中，除去EGFR的作用，同時還測試其作為鉑類藥物對DNA斷裂的貢獻。在順鉑抗藥性課題中，研究雜合物對轉運蛋白和CIP2A治病蛋白的作用，研究抗癌機理。 === 實驗結果： === 鉑-凡德他尼雜合物通過合成并純化，測試發現雜合物保持穩定。雜合物與5’-GMP及GSH反應，形成穩定的單配位化合物。相較順鉑，雜合物表現出低腎毒性。雜合物在D800等位與EGFR形成有效氫鍵，對其磷酸化保持抑制活性。在HSCLC細胞中，能夠有效降低藥物的交叉抗藥性。雜合物作用機理研究表明雖然對EGFR T790M抑制不足，但是雜合物成功導致DNA斷裂和細胞凋亡。通過兩條路徑的共同作用，雜合物能夠有效克服EGFR突變導致的耐藥性。 === 鉑-凡德他尼雜合物在順鉑耐藥細胞KB CP20中表現出極低的耐藥性。順鉑的CP20細胞累積相較於KB3.1細胞顯著降低，然而雜合物沒有發生積累減弱。細胞外流性轉運蛋白基因在CP20細胞中過度表達，導致順鉑低累積，但是雜合物表現出的轉運惰性說明對於轉運蛋白“非受體”。另外，雜合物能夠在KB細胞中能成功引發DNA斷裂和細胞凋亡，而順鉑失敗了。致癌蛋白CIP2A的過度表達也是順鉑耐藥性的原因，凡德他尼和雜合物能夠有效抑制其表達，從而表明引入凡德他尼對鉑類藥物克服耐藥性很有助益。 === 總結： === 新型鉑-凡德他尼雜合物表現出克服EGFRT 790M變異導致的耐藥性，通過（1）抑制EGFR磷酸化及後續信號通路，（2）引發DNA斷裂和細胞凋亡通路。同時雜合物表現出有效的克服順鉑的抗藥性，表現為變成外流性轉運蛋白的“非受體”，且對致癌蛋白非正常表達的有效抑制。雜合物是一個有潛力的新型藥物手段。 === Purpose: Lung cancer is one of the most common cancers and non-small cell lung cancer (NSCLC) accounts for 80-85% of lung cancers. 70% of individuals with NSCLC harboring somatic mutations in exons of the epidermal growth factor receptor (EGFR) gene that encode tyrosine kinase domain. EGFR tyrosine kinase inhibitors (TKIs) are promising molecular targeted therapy for NSCLC with sensitizing EGFR mutations. However, secondary mutation of EGFR after treatment of TKIs develops resistance. Vandetanib is introduced to overcome erlotinib resistance as a multi-targeted TKI. However, its anticancer effect is still compromised by EGFR T790M mutation. Therefore, new molecular anticancer strategies are necessarily needed. In this study, vandetanib is incorporated with Pt-based anticancer agents as hybrid compounds, aiming to circumvent TKI resistance. Furthermore, hybrid compounds are investigated in cisplatin resistant problem to expect to overcome resistance by introduction of vandetanib. === Methods: Three novel Pt-vandetanib hybrid compounds were synthesized and its physicochemical properties were characterized. Anticancer activity and cytotoxicity were evaluated by sulforhodamine B assay and lactate dehydrogenase release. Docking simulation was performed to investigate the interaction of compounds with EGFR harboring different mutations. Inhibition efficacy of hybrids to kinases was evaluated by kinase inhibition profiling service and cell-free kinase inhibition assay. Mechanistic studies on cytotoxicity activity of the hybrid compounds were carried out. DNA damage response of hybrid compounds was further investigated in KB cells. The cytotoxicity of hybrids was tested in cisplatin resistant KB CP20 cells. Mechanistic of anticancer activity was studied to test inhibition on oncoprotein CIP2Aand DNA damage. === Results: Platinum-vandetanib hybrid compounds were synthesized and test to be stable under extracellular condition. Hybrids reacted with 5’-GMP2- and glutathione, and both of them formed mono-dentate adducts. Moreover, hybrid compounds exhibited low toxicity in human normal kidney cells. Compounds maintained the inhibition selectivity towards EGFR from the results of kinase inhibition profiling and cell-free kinase inhibition assay. Hybrids formed strong H-bond at D800 on EGFR. Pt-vandetanib hybrids were highly effective against HCC827 cells harboring sensitizing EGFR mutation. Importantly, relative resistant rate of hybrids were much smaller than vandetanib in H1975 cells. Western blot analysis results revealed that the hybrid compounds could efficiently inhibit EGFR phosphorylation in a dose dependent manner in HCC827. While, inhibition of p-EGFR was not as good as the original TKI in H1975 cells. However, the hybrid compounds induced DNA damage and caused apoptosis of the NSCLC cells. Both of the two pathways were contributed to cancer cell death and overcome vandetanib resistance. === Pt-vandetanib hybrids showed little resistance in cisplatin resistant cell line KB-CP20. Drug accumulation evaluation revealed that cisplatin accumulation in CP20 cells decreased to one eighth of that in the parental KB3.1 cells. While hybrids maintained similar drug accumulation extent in both cells lines. Mechanistic study showed that hybrid compounds could induce DNA damage and cause apoptosis, whereas cisplatin failed to cause DNA damage in KB-CP20 cells. Oncoprotein CIP2A was overexpressed in CP20 cell and was ascribed to CDDP resistance. The hybrids inhibited CIP2A expression and downstream AKT phosphorylation. It was hypothesized that downregulation of CIP2A contributed to circumvention platinum resistance. === Conclusion: Novel Pt-vandetanib hybrid compounds were able to overcome vandetanib resistance in H1975 cells by maintaining inhibition to the EGFR and inducing DNA damage and apoptosis. Moreover, Pt-vandetanib hybrid compounds behaved low toxicity and overcome cisplatin resistance by being “non-substrate” to efflux transporter and successfully causing DNA damage. Hybrids were found to downregulate oncogene CIP2A expression level. The novel Pt-vandetanib hybrid compounds are potent for further development. === Fei, Rong. === Thesis Ph.D. Chinese University of Hong Kong 2016. === Includes bibliographical references (leaves ). === Abstracts also in Chinese. === Title from PDF title page (viewed on …). === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only.