扁桃斑鳩菊萃取物與大豆胜肽水解物誘導細胞凋亡及抑制癌細胞轉移之研究

Studies on the Induction of Apoptosis and Inhibition of Cancer Cell Metastasis by Vernonia amygdalina Extracts and Soybean Peptide Hydrolysates

賴珍珠(CHEN-CHU LAI)

指導教授 ： 劉炳嵐

朝陽科技大學/理工學院/應用化學系生化科技博士班/博士(2020年)

摘要

第一部分:
中文摘要
根據衛生福利部統計，肝癌在107年兩性十大癌症死因死亡率中位居台灣十大癌症死因的第2名，在過去的研究中，藥用植物萃取物是治療癌症的明日之星。扁桃斑鳩菊(Vernonia amygdalina)不僅具有抗氧化和抗發炎的能力，而且具有抗癌的作用。關於扁桃斑鳩菊萃取物(VA)的抗癌機制目前研究有限，故本研究旨在探討扁桃斑鳩菊萃取物對肝臟上皮細胞癌(Hep 3B)的抗癌效果及其作用機制。從MTT分析得知，扁桃斑鳩菊萃取物能抑制Hep 3B的增殖且呈時間和劑量依賴性。流式細胞儀分析發現，VA抑制Hep 3B細胞週期停滯在G1/S期，進一步利用Propidine iodide和Annexin-V進行雙染實驗，證實扁桃斑鳩菊會使Hep 3B細胞產生細胞凋亡。透過EMT主要調控轉錄因子如Snail及Slug等之蛋白表現實驗發現，VA除了誘導細胞凋亡，也會抑制癌細胞轉移。透過本研究證實VA確實有抗癌之功效，未來可望進一步發展成抗癌藥物。

第二部分:
中文摘要
乳癌是台灣女性罹患率最高的癌症，位居死亡率第四名。本研究以大豆胜肽水解物為研究題材，試著探討大豆胜肽水解物是否具有抑制癌細胞生長的能力。實驗以大豆胜肽水解物針對MCF-7、MDA-MB-453乳癌細胞株以及正常的人類乳房表皮細胞株HBL-100進行細胞生長抑制實驗，結果顯示大豆胜肽水解物對MCF-7與MDA-453細胞株的抑制效果較好，在100 μg/mL濃度下有抑制近50%生長率，顯示大豆胜肽水解物對於乳癌細胞有明顯的抑制生長效果。以流式細胞儀檢測大豆胜肽水解物對MCF-7細胞株細胞週期的影響，結果發現大豆胜肽水解物會使MCF-7細胞株之細胞週期停滯於G0/G1期，但是對於正常乳房細胞HBL-100沒有毒殺作用。另外，用西方墨點法分析P21、P27、P53 (與細胞週期停滯及凋亡相關之蛋白)、cyclin D1及CDK4這些與細胞週期中G0/G1相關的蛋白質表現情形，發現乳癌細胞中P21、P27、P53等蛋白在處理100 μg/mL大豆胜肽水解物後，表現量均有上升的趨勢，導致乳癌細胞分裂停留在G0/G1，而Cyclin D1及CDK4則是沒有變化。此外，大豆胜肽水解物可以抑制ERK1/2的磷酸化，但卻對JNK的磷酸化沒有影響，因此，若能對此機轉或是對其有效成分深入研究並評估其應用的可行性，或許能為乳癌治療帶來契機。

關鍵字

扁桃斑鳩菊萃取物 ； 肝癌 ； 細胞週期 ； 細胞凋亡 ； 大豆胜肽水解物 ； 乳癌

並列摘要

PART 1:
ABSTRACT
According to statistics from the Ministry of Health and Welfare, the liver cancer ranked second among the top ten cancer deaths in Taiwan in 2018. Previous studies have shown that the medicinal plant extracts are the rising stars in the treatment of cancer. Vernonia amygdalina not only has antioxidant and anti-inflammatory properties, and has anti-cancer effects. Yet, the research on the anti-cancer mechanism of V. amygdalus extract (VA) still limited, so this study aims to investigate the anti-cancer effects of VA on liver epithelial cell carcinoma (Hep 3B) and its mechanism of action. The MTT analysis suggested that the VA could inhibit the proliferation of Hep 3B in a time- and dose-dependent manner. Subsequently, flow cytometry analysis found that VA inhibited Hep 3B cell cycle arrest in the G1/S phase. Western blotting demonstrated VA-6 induced apoptosis through the inhibition of PI3K/Akt signaling pathway. Our data also showed that VA inhibited cancer migration and invasion through the inhibition of epithelial-mesenchymal transition (EMT). Interestingly, we found that VA-6 enhanced the sensitivity of Paclitaxel, and doxorubicin on the growth of Hep 3B cells. In conclusion, this study confirmed that VA does have anti-cancer effects, and is worth to be further developed into an anti-cancer drug.

PART 2:
ABSTRACT
Breast cancer is the cancer with the highest incidence of women in Taiwan, ranking fourth in mortality. In this study, soybean peptide hydrolysate was used as the research subject, and it tried to explore whether soybean peptide hydrolysate (SPH) has the ability to inhibit the growth of cancer cells. To test the cell growth inhibitors in cancer therapy, the SPH was employed against MCF-7, MDA-MB-453 breast cancer cell lines and the normal human breast epidermal cell line HBL-100. The results showed that the SPH had an effect on MCF-7 and MDA cells. Remarkable inhibitory result was found in MDA-MB-453 cell, with an inhibitory level of nearly 50% at a concentration of 100 μg/mL, demonstrated that the SPH has a significant inhibitory effect on breast cancer cells. Flow cytometry was used to examed the effect of SPH on the cell cycle of MCF-7 cell. It was found that SPH would arrest the cell cycle of MCF-7 cell in G0/G1 phase, no toxic effect was observed for normal breasts cell HBL-100. In addition, the western blot was used to analyze the expression of P21, P27, P53 (proteins related to cell cycle arrest and apoptosis), cyclin D1 and CDK4, which are related to G0/G1 in the cell cycle. Accordingly, the expression levels of P27, P53 and other proteins were increased, causing breast cancer cells to divide and stay at G0/G1, while Cyclin D1 and CDK4 remained unchanged after 100 μg/mL SPH treatment. Moreover, SPH can inhibit the phosphorylation of ERK1/2, but it has no effect on the phosphorylation of JNK. Consequently, if this mechanism or its active ingredients can be studied in depth and the feasibility of its application can be evaluated, it may bring opportunities for breast cancer treatment.

並列關鍵字

Vernonia amygdalina extract ； Liver cancer ； Cell cycle ； Apoptosis ； Soybean peptide hydrolysate ； Breast cancer

參考文獻

第一部分:

參考文獻

1. 王筱萱 (2018) 扁桃斑鳩菊對皮膚功效之評估。碩士論文，嘉南藥理大學。

2. 石界智 (2017) 養殖型紅藻海木耳(Sarcodia suieae)之生物活性研究。博士論文，國立中山大學。

3. 何美儀 (2015) 。扁桃斑鳩菊萃取物誘導肝癌細胞凋亡及抑制癌細胞轉移之研究。碩士論文，國立中興大學。