荳蔻明誘導小鼠WEHI-3血癌細胞凋亡，增強血癌BALB / c小鼠的免疫反應並提高存活率

Cardamonin induces apoptosis in mice leukemia WEHI-3 cells, enhances immune responses and increases survival rate in leukemic BALB/c mice

廖年捷(Nien-Chieh Liao)

指導教授 ： 呂思潔

臺北醫學大學/醫學院/醫學科學研究所/博士(2020年)

摘要

白血病是一群不同白血球異常增生疾病的總稱，2017年在美國約有62,130新增白血病病例，並造成24,500例死亡。據2018年統計，白血病在台灣癌症死亡排名為第13位。白血病分為四種主要類型-急性淋巴性白血病（ALL）、急性骨髓性白血病（AML）、慢性淋巴性白血病（CLL）和慢性骨髓性白血病（CML）。其治療的方法包括化學療法、放射線療法、標靶療法、幹細胞移植和免疫療法。白血病的治療主要是以全身性藥物治療為主，同時合併化療及他種治療方式，有些還接受放射治療。在某些情況下，骨髓移植是有效的，但須找到HLA typing配對相符往往較為困難。荳蔻明（2',4'-二羥基-6'-甲氧基查爾酮）屬於一種查耳酮（Chalcone）衍生化合物，可從數種植物中分離出，包括Alpinia katsumadai 和 Alpinia conchigera等植物。由於人們發現它對人類健康有益，因此開始受到科學界越來越多的關注。目前實驗證實，荳蔻明具有多種藥理活性，例如抗發炎，抗腫瘤和血管鬆弛活性。儘管有許多研究報導荳蔻明具有抗腫瘤作用，但其對小鼠WEHI-3血癌細胞相關研究仍不清楚。我們利用體外實驗研究WEHI-3小鼠白血病細胞在荳蔻明作用下細胞週期停滯和相關凋亡分子作用機制，以及於體內實驗荳蔻明的抗腫瘤效果。利用流式細胞儀分析結果發現，荳蔻明通過誘導細胞的凋亡降低WEHI-3細胞存活率，引發細胞內ROS和Ca2 +產生增加，並降低粒線體的膜電位，造成WEHI-3細胞內caspase-3、-8和-9的活化。以西方墨點法分析荳蔻明作用下WEHI-3細胞中促進凋亡蛋白和抗凋亡蛋白的表現。實驗結果發現荳蔻明能降低WEHI-3細胞中抗凋亡蛋白Bcl-2表現，增加促凋亡蛋白Bax表現。荳蔻明也增加WEHI-3細胞中cytochrome c，AIF和Endo G的釋出，ER壓力相關的GRP78、caspase12、以及增加Fas、Fas-Ligand和FADD的表現，同時也促使與細胞凋亡途徑相關基因的表現增加。此外在動物實驗中，荳蔻明的治療不會影響BALB / c小鼠的體重，而在WEHI-3細胞白血病小鼠體內，於荳蔻明作用下造成CD3 (T細胞)、CD11b (單核球) 和Mac-3 (巨噬細胞)的數量減少，而CD19 (B細胞)數量增加，及增強白血病小鼠周邊血液單核細胞中巨噬細胞的吞噬能力，而在1 mg / kg荳蔻明治療下可提高白血病小鼠的存活率。因此荳蔻明能作為潛在有用治療白血病的藥物。

關鍵字

荳蔻明 ； WEHI-3血癌細胞 ； 細胞凋亡 ； 免疫反應

並列摘要

Generally, leukemia is used to describe a group of diseases characterized by abnormal leukocyte accumulation. In 2017, approximately 62,130 new cases of leukemia and 24,500 leukemia-related deaths were reported in the United States. In Taiwan, leukemia was the thirteenth leading cause of cancer-related deaths in 2018. The four main types of leukemia are acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML). Therapeutic strategies for leukemia include chemotherapy, radiation therapy, targeted therapy, stem cell transplantation, and immunotherapy. Most forms of leukemia are treated through multi-drug chemotherapy, while some cases are also treated through radiation therapy. Although bone marrow transplantation is an effective therapeutic strategy for leukemia, the need for a matching human leukocyte antigen type limits its application. Cardamonin (2',4'-dihydroxy-6'-methoxychalcone), a chalconoid found in several plants, including Alpinia katsumadai and Alpinia conchigera, has potential health benefits to humans. Previous studies have demonstrated that cardamonin exhibits diverse pharmacological activities, such as anti-inflammatory, antitumor, and vasorelaxant activities. Although cardamonin is reported to exhibit antitumor activity, the underlying molecular mechanisms involved in the induction of apoptosis and inhibition of metastasis have not been investigated in mice leukemia WEHI-3 cells. In this study, we investigated the molecular mechanisms underlying cardamonin-induced cell cycle arrest and apoptotic death in the murine WEHI-3 leukemic cells in vitro, as well as the in vivo antitumor activity of cardamonin using the WEHI-3 cell-based leukemic mouse model. Flow cytometric analysis revealed that cardamonin decreased the total viable WEHI-3 cell number through inducing apoptosis, enhancing reactive oxygen species production, increasing cellular Ca2+ levels, decreasing mitochondrial membrane potential, and stimulating caspase-3, -8, and -9 activities. Western blotting analysis revealed that cardamonin downregulated the expression of anti-apoptotic Bcl-2, but upregulated the expression of pro-apoptotic Bax in the WEHI-3 cells. Cardamonin enhanced the release of cytochrome c (cyt c), apoptosis-inducing factor, and endonuclease G, and upregulated the expression of endoplasmic reticulum stress-related GRP78 and caspase 12. Additionally, cardamonin upregulated the expression of Fas, Fas ligand, Fadd, and increased the gene expressions of apoptotic pathway-related genes, but did not affect the bodyweight of leukemic BALB/c mice. The populations of CD3 (T cells), CD11b (monocytes), and Mac-3 (macrophages) cells decreased, whereas those of CD19 cells (B cells) increased in the leukemic mice upon treatment with cardamonin. Cardamonin also enhanced the phagocytic abilities of macrophages isolated from the peripheral blood mononuclear cells (PBMCs) of leukemic mice. Furthermore, cardamonin (1 mg/kg bodyweight) improved the survival rate of leukemic mice. These findings indicate that cardamonin is a potential therapeutic agent for leukemia.

並列關鍵字

Cardamonin ； WEHI-3 cells ； apoptosis ； immune responses

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