- 學位論文
植物倍半萜類化合物Deoxyelephantopin及其衍生物抗BRAF突變黑色素瘤之藥理活性與作用機制之研究
Pharmacological Effect and Underlying Molecular Insights of a Phytoagent Deoxyelephantopin and its Derivative against BRAF Mutant Melanoma
摘要
黑色素瘤為皮膚癌中最具致命性的癌症，且其臨床案例有逐年上升的趨勢。大約50~60%惡性黑色素瘤具有BRAF腫瘤蛋白質之突變基因。此突變所啟動之MAP kinase (RAF/RAS/MEK/ERK)訊息傳遞路徑之活化造成黑色素癌細胞不斷的增生。Vemurafenib (PLX4032)為專一性抑制BRAFV600E突變蛋白質的口服型藥物。此藥物在2011年通過美國FDA審核為治療末期黑色素瘤病患的抗癌藥物。不幸的是，接受vemurafenib治療的黑色素瘤病患普遍伴隨著抗藥性以及皮膚鱗狀細胞癌等副作用。目前研究結果顯示，重新活化MAP kinase訊息傳導路徑為產生抗藥性以及皮膚鱗狀細胞癌等副作用之主要原因。目前臨床上治療帶有BRAFV600E突變之黑色素癌病患以MEK抑制劑合併vemurafenib之方法為主要療法。雖然此合併療法顯示出較好的臨床數據，但病患所產生的副作用比例高於vemurafenib單藥療法，且腫瘤始終會復發。因此，尋找新穎抗黑色素瘤藥物或輔助vemurafenib療法之佐劑是非常重要且迫切的課題。我們研究室累積的實驗結果證實植物倍半萜類化合物deoxyelephantopin (DET)，可透過多重標的調控而達到抗發炎、保護肝臟和抗腫瘤的功效。這些研究成果支持DET具前導藥物的特色，值得進一步透過化學修飾或最佳化它的結構成為更好的癌症治療劑。因此，我們用半有機合成方法創造出60個DET衍生物 (DET derivatives, DETDs)，其中DETD-35在初步抗黑色素腫瘤細胞增生實驗中顯示出具有比DET更佳的抑制癌細胞生長之活性。體外實驗結果證明DETD-35可抑制A375（BRAFV600E突變）及A375-R（具vemurafenib抗藥性）之細胞增生。DETD-35與vemurafenib之合併使用對A375細胞的增生，群落形成以及誘導細胞凋亡具有協同作用。在A375異種腫瘤實驗中發現，利用腹腔注射方式給予DETD-35之抗BRAFV600E突變黑色素瘤之活性與當前臨床用藥vemurafenib相當。DETD-35有效地抑制87.1%腫瘤生成率以及降低70.5%的腫瘤重量，而vemurafenib則抑制87.4%腫瘤生成率以及降低71.9%的腫瘤重量。此外，DETD-35及vemurafenib之合併療法亦抑制91%腫瘤生成率以及降低72.3%的腫瘤重量。重要的是，在合併療法中，藥物施予小鼠的頻率只有DETD-35或vemurafenib單藥療法的一半。因此，這項結果證實了在A375異種腫瘤實驗中，DETD-35及vemurafenib合併療法亦產生協同效果。在A375-R異種腫瘤實驗中，vemurafenib對產生抗藥性的腫瘤不在具有活性，然而DETD-35明顯地抑制47.1%腫瘤生成率以及降低46.3%的腫瘤重量。值得注意的是，DETD-35及vemurafenib之合併療法顯示出最有效抑制腫瘤之活性，即抑制76.5%腫瘤生成率以及降低65.2%的腫瘤重量。藥理機制之結果顯示，DETD-35是經由抑制抗藥性細胞A375-R內所活化之MAP Kinase，Src-STAT3 以及 Akt等多種傳遞路徑而達到抑制腫瘤生成之效果。此外，DETD-35亦啟動活性氧化物(ROS)誘導之細胞凋亡機制而達到毒殺黑色素癌細胞之作用。另一方面，我們利用DMBA/TPA誘導之二階段小鼠皮膚致癌模式證實了DETD-35能降低使用vemurafenib所引起之小鼠皮膚鱗狀細胞癌之生長以及腫瘤數目。總而言之，此論文證實了DETD-35具有抑制帶有BRAFV600E突變的黑色素瘤，克服對vemurafenib所產生的抗藥性以及減少皮膚鱗狀細胞癌等副作用的活性。因此，DETD-35有潛力發展為抗BRAFV600E突變黑色素瘤之小分子藥物或癌症化療藥vemurafenib之佐劑。
並列摘要
Melanoma is the most lethal form of skin cancer and cases are increasing year by year. BRAFV600E mutation is found approximately in 50% of melanoma patients that drives constitutive activation of MAP kinase (RAF/RAS/MEK/ERK) signaling for cancer progression. Vemurafenib, an orally available small molecule that specifically targets BRAFV600E mutation, received FDA approval for treatment of late-stage melanoma in 2011. Unfortunately, patients treated with vemurafenib commonly developed drug resistance and diverse cutaneous side effects mainly through paradoxical activation of MAP kinase signaling pathway. Recent combination therapy such as MEK inhibitor in combination with vemurafenib shows improvement in major clinical end points but percentage of patients with adverse toxic events are higher compared to vemurafenib monotherapy, and most patients relapse ultimately. It is therefore an urgent need to develop new anti-melanoma drug and/or adjuvant agent for vemurafenib therapy. Previously, our laboratory isolated a bioactive phytochompound deoxyelephantopin (DET) from Elephantopus scaber L. and showed that DET possessed anti-inflammation, hepatoprotective and anti-mammary tumor activities. We considered DET as a novel lead compound for further modification or optimization of its structure to create a more potent drug lead for cancer management. Therefore, we created a total of sixty semi-organically modified derivatives of DET (DETDs). In this thesis study, I showed that DETD-35 suppressed both parental human BRAFV600E mutant melanoma (A375) and vemurafenib resistance melanoma (A375-R) cell proliferation in vitro. Moreover, DETD-35 showed synergism with vemurafenib in suppressing the proliferation, colony formation and inducing apoptosis of A375 melanoma cell. In A375 xenograft study, DETD-35 suppressed tumor growth with 87.1% TGI and reduced tumor mass by 70.5%, as effective as vemurafenib (87.4% TGI and 71.9% reduction in tumor mass), compared to tumor control group. Moreover, compound-drug combinational treatment with alternate administration of DETD-35 and vemurafenib in which the treatment frequency was only half of the single compound administration frequency also showed similar tumor growth inhibition efficacy (91.0% TGI and 72.3% reduction in tumor mass), suggesting in vivo synergistic or additive effect of DETD-35 and vemurafenib. In A375-R xenograft study, vemurafenib treatment showed little or no anti-tumor activity with similar tumor growth rate and sizes relative to the tumor control group, whereas DETD-35 suppressed A375-R tumor growth with 47.1% TGI and reduced tumor mass by 46.3%. Notably, the combination of DETD-35 and vemurafenib treatment exhibited the most significant effect with 76.5% TGI and 65.2% reduction in tumor mass. Mechanistic studies revealed the reactivation MAP Kinase, Src-STAT3 and Akt signaling pathways in the acquired vemurafenib resistance A375-R cells, and DETD-35 suppressed those reactivated multiple signaling pathways. In addition, DETD-35 triggered ROS-induced apoptotic cell death in both A375 and A375-R cells. Furthermore, through a DMBA/TPA-induced mouse skin carcinogenesis model mimicking cutaneous side effect caused by vemurafenib, I showed that DETD-35 delayed vemurafenib-induced acceleration of skin papillomas formation and also decreased total papillomas number in mice. Overall, results from my thesis study offers strong evidence that DETD-35 inhibits BRAFV600E mutant melanoma growth, overcomes acquired vemurafenib resistance and reduces cutaneous side effect of the BRAF inhibitor in mice. It is therefore suggested that DETD-35 might have great therapeutic or adjuvant potential in management of melanoma patients with BRAFV600E mutation.