Part I 木樨草素加強太平洋紫杉醇誘導人類乳癌細胞凋亡之研究 Part II 桑白皮素抑制人類肝癌細胞入侵性生長之研究

Part I Promotion Effect of Luteolin on Paclitaxel-Induced Cell Death in MDA-MB-231 Cell Lines Part II Morusin Inhibits Invasive Growth in SK-Hep-1 Hepatoma Cells

陳乃鳳(Nai-Fang Chen)

指導教授 ： 曾翠華 ； 呂鋒洲

中山醫學大學/健康管理學院/應用化學系碩士班/碩士(2011年)

摘要

Part I 木樨草素加強太平洋紫杉醇誘導人類乳癌細胞凋亡之研究

目前利用太平洋紫杉醇 (Paclitaxel) 治療乳癌患者的效果一直無法突破並且有所限制是因為其所伴隨的神經毒性、骨髓毒性等等，以及乳癌腫瘤細胞容易對紫杉醇產生抗藥性。因此，科學家致力於尋找能與太平洋紫杉醇搭配治療的天然抗癌藥劑來達到降低太平洋紫杉醇的劑量而減輕毒性與副作用，並且還需要提升太平洋紫杉醇所產的的治療效果。木樨草素 (Luteolin, 3’, 4’, 5, 7-tetrahydroxyflavone)，廣泛存在於菊科的藥用植物內，屬於類黃酮中的黃酮，有多數研究證實木樨草素在多種疾病中具有抗發炎和抗癌的功效。先前的實驗結果指出35 μM木樨草素會有效使人類乳癌細胞MDA-MB-231 cell lines的細胞生長週期停滯，75 μM以上的木樨草素則造成細胞凋亡。本研究先初步將木樨草素與太平洋紫杉醇結合處理癌細胞之後分別進行細胞毒性、DAPI染色以及Annexin V-FITC/PI雙染法等實驗發現木樨草素會提升太平洋紫杉醇所誘導的凋亡現象。接著透過西方墨點法探討其中的分子機制發現到15 μM木樨草素與0.04 μM太平洋紫杉醇結合處理之後會促進caspase-9和caspase-3的活化，並且負調控Bcl-2 family，抑制效果比0.12 μM太平洋紫杉醇來得好。另外，太平洋紫杉醇會因為Stat 3過度表現而造成抗藥性的反應，木樨草素與太平洋紫杉醇合併處理之後會顯著性地抑制住Stat 3 Tyr705磷酸化表現，進而抑制Stat3 signaling pathway。此外還發現到木樨草素單獨處理會抑制MAPK，PI3K/Akt以及NF-κB的磷酸化表現，木樨草素與太平洋紫杉醇合併處理更有加乘的效果。更進一步利用三種MAPK kinase抑制劑 (PD98059、SB20219、SB600125)、PI3K/Akt抑制劑 (Wortannin) 和NF-κB抑制劑 (PDTC) 分別與太平洋紫杉醇結合處理，除了PDTC之外的抑制劑對Stat 3 Tyr705磷酸化表現也會得到與木樨草素結合處理類似的抑制效果，證實因為木樨草素抑制住MAPK，PI3K/Akt，使Stat3訊息路徑的活化被抑制住。最後利用Stat3抑制劑以及NF-κB抑制劑確認木樨草素會透過抑制Stat3以及NF-κB訊息路徑來增強太平洋紫杉醇所誘發的細胞凋亡。

Part II 桑白皮素抑制人類肝癌細胞入侵性生長之研究

桑白皮素 (morusin) 是桑科植物Morus alba L.的桑根皮內組成分之一，根據文獻初步了解桑白皮素具有抗發炎、減少超氧陰離子的形成、血小板生長因子產生、NF-κB的活化等能力並且也會抑制腫瘤生長如大腸結腸癌。由於目前只清楚桑白皮素會使癌症產生細胞凋亡，其他抗癌活性仍然不清楚。因此，本研究擬探討桑白皮素對惡性肝癌hepatocellular carcinoma (HCC)入侵轉移作用之影響及分子機制。首先利用細胞毒性分析發現到桑白皮素皆有效抑制SK-Hep 1惡性肝癌細胞的生長，接著透過細胞週期測定的結果顯示桑白皮素 (5 μM、10 μM、15 μM) 會造成細胞生長停滯，再觀察移動入侵的相關實驗結果，表示皆具有不錯的抑制效果。另外，癌細胞產生移動的原因有可能是因為一些生長因子的刺激或欠缺養分使癌細胞產生偽足與觸角，產生移動與吸附能力，接著亦會造成血管新生促使癌細胞更容易入侵到其他器官，因此再藉由F-actin免疫螢光染色、細胞-基質吸附、capillary tubule形成分析等實驗觀察結果發現處理桑白皮素會明顯地產生抑制作用。更進一步利用西方墨點法去了解桑白皮素抑制肝癌細胞入侵之相關分子機制，首先發現桑白皮素會抑制FAK、PAK、integrins與vimentin，而促進E-cadherin的表現，再利用凝膠酶譜法和西方墨點法發現對MMP-2與MMP-9的活性和蛋白表現也有的抑制效果。接著發現到桑白皮素會對FAK、PAK、ERK、Akt、Stat3與NF-κB磷酸化產生抑制作用，再透過處理ERK、Akt與NF-κB的抑制劑發現會進而抑制FAK、PAK與integrins，此外還發現到ERK、Akt與NF-κB也會影響Stat3，證明桑白皮素會經由對ERK、Akt、Stat3與NF-κB相互作用產生抑制來減少附著蛋白、integrins與vimentin，促進E-cadherin的表現進而降低惡性肝癌產生偽足、移動、附著等能力。細胞生長因子與細胞生長因子接受器被啟動活化之後會連續活化一連串的訊息傳遞作用，因此再透過實驗發現桑白皮素會影響EGFR與c-Met的磷酸化，對VEGF和HIF-1α也會產生抑制。因為Stat3會調控轉錄因子Twist影響vimentin與負調控E-cadherin的表現，而利用Stat3抑制劑的結果表示的確會抑制Twist。經過本研究了解桑白皮素會抑制住SK-Hep 1惡性肝癌細胞的入侵性生長是抑制了細胞膜上的生長因子及其下游訊息路徑的不正常活化，進而抑制多種侵略性相關蛋白表現。

關鍵字

木樨草素 ； 太平洋紫杉醇 ； 乳癌 ； 桑白皮素 ； 肝癌

並列摘要

Part I Promotion Effect of Luteolin on Paclitaxel-Induced
Cell Death in MDA-MB-231 Cell Lines

The effective of paclitaxel chemotherapy in breast cancer patients is limited by myelotoxicity and neurotoxicity, and tumor cells tend to acquire resistance to cytotoxic paclitaxel. Combination of natural anti-cancer agents with paclitaxel that are considerably with a capability of activating apoptotic signals may provide a rational molecular basis for novel chemotherapeutic strategies. Luteolin, 3’, 4’, 5, 7-tetra hydroxyflavone, exists in medicinal herbs of Compositae family and demonstrates anti-inflammatory and anti-cancer potential previously. In the present study, by MTT assay, it showed combination of luteolin with paclitaxel significantly increased cell death as compared to paclitaxel treated alone in human breast cancer MDA-MB-231 cells. In addition, flow cytometry (FACS) analysis via Annexin V-FITC/PI staining was used to demonstrate combination of luteolin with paclitaxel significantly increased paclitaxel-induced apoptosis of MDA-MB-231 cells. Meanwhile, immunoblotting analysis showed that co-administration of luteolin and paclitaxel could promote activation of intrinsic apoptosis pathway including activation of caspase 9 and caspase 3, and down regulation of Bcl-2 and Bcl-xL. It has been reported that signaling pathways such as JAK/Stat3, MAPK, and PI3K/Akt , and NF-κB signaling pathway may modulate the efficacy of paclitaxel. In this study, MAPK kinase inhibitors (PD98059、SB20219、SB600125) and PI3K/Akt inhibitor (Wortannin) combined with paclitaxel could inhibit Stat3 Tyr705 phosphorylation, which demonstrated luteolin can enhance the effect of paclitaxel-induced apoptosis in MDA-MB-231cells by suppressed Stat3 signaling. Stat3 inhibitor (S3I-201) or NF-κB inhibitor (PDTC) combined with paclitaxel could enhance paclitaxel-induced apoptosis. Therefore, we clarified the signal pathway with the augmentation of luteolin in paclitaxel-induced apoptosis in human breast cancer MDA-MB-231cells by suppressed MAPK, PI3K/Akt — Stat 3 and NF-κB signaling pathway.

Part II Morusin Inhibits Invasive Growth in SK-Hep-1
Hepatoma Cells

Currently, Cortex Mori has been used in traditional Chinese medicine. Morusin, a prenylated flavonoid in Cortex Mori, which exhibited inhibitory effection in inflammation, superoxide anion formantion, platelet-derived growth factor aggregation and NF-κB activation, etc. Morusin has been demonstrated to induce apoptotic effect in some cancers such as liver cancer, but other anticancer action is not well known. Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide, despite advances in diagnostic and therapeutic techniques, the prognosis of HCC patients remains poor because of the high incidence of hematogenous intrahepatic metastasis after initial treatment. Therefore, in the present study, we found morusin exhibited cytotoxicity and antimigration effects in SK-Hep 1 cells. Further, 5, 10 and 15 μM of morusin reduced cell mobility, migration and invasion by analysis wound healing assay and boyden chamber assay in SK-Hep 1 cells. Cell migration, and adhesion as well as angiogenesis require the formation of a leading pseudopodium, angiogenesis growth factor. It demonstrated morusin significantly inhibited lamellipodia and filopodia formation, cell-matrix adhesion and angiogenesis. Furthermore, morusin could inhibit growth factors and FAK phosphorylation, and integrins and VEGF expression. By zymography assay and western blot assay, morusin inhibitd MMP-2 and MMP-9 activation expression. Final, we found morusin inhibited c-Met and EGFR activation, and down stream activation inhibited ERK, Akt, NF-κB, FAK, and Stat3. All result demonstrated morusin inhibits invasive growth of malignant liver through signaling inhibition. Morusin possesses antitumor progression potential.

並列關鍵字

Luteolin ； Paclitaxel ； MDA-MB-231 Cell Lines ； Morusin ； SK-Hep-1

參考文獻

Part I 木樨草素加強太平洋紫杉醇誘導人類乳癌細胞凋亡之研究

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