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研究生:陳筱婷
研究生(外文):Hsiao-Ting Chen
論文名稱:蟾蜍靈Bufalin在人類乳癌細胞中調控蛋白激酶C所誘發的第二型環氧酵素與介白素第八的表現
論文名稱(外文):Regulation of PKC-induced COX2 and IL-8 expression by bufalin in human breast cancer cells
指導教授:吳鈺琳
指導教授(外文):Yuh-Lin Wu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生理學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:102
中文關鍵詞:發炎反應乳癌蟾蜍靈
外文關鍵詞:inflammationbreast cancerbufalin
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慢性發炎反應在癌症發展的過程中扮演相當重要的角色。許多研究中發現慢性發炎反應所媒介的相關介質,如生合成前列腺素E2 (PGE2) 的重要酵素-第二型環氧化酵素 (COX2)與介白素第八 (IL-8)都能促進癌細胞增生、遷移與增加血管新生的能力。先前研究顯示在各種發炎反應刺激下,第二型環氧酵素與介白素第八會被蛋白激酶C調控;而蛋白激酶C也曾被報導過在腫瘤發展過程中會被大量表現。蟾蜍靈 (Bufalin)是由傳統中藥蟾酥所萃取出的主要活化成分之一,曾經有文獻指出其能夠抑制淋巴T細胞增生。近年來發現蟾蜍靈在前列腺癌、肝癌與血癌中都具有抑制腫瘤的作用。然而蟾蜍靈在發炎反應所扮演的角色與其對於癌症的影響尚未釐清。因此本研究目的為利用蛋白激酶C的促效劑 (TPA),探討蟾蜍靈是否會影響蛋白激酶C所誘發的第二型環氧化酵素與介白素第八的表現,並且了解其中間參與的機制調控為何。首先我們發現在人類乳癌細胞株 (MB231 與 MCF-7 細胞株)與人類單核球細胞株 (THP-1 細胞株)中,蟾蜍靈都能促進蛋白激酶C所誘導第二型環氧酵素及介白素第八的蛋白表現,並且增加前列腺素E2和介白素第八的分泌,但是蟾蜍靈對於人類正常乳腺上皮細胞 (H184細胞株)中卻沒有明顯的影響。同時,也發現蟾蜍靈能夠增加蛋白激酶C所誘導的第二型環氧酵素及介白素第八的核糖核酸 (mRNA)表現。為了釐清蟾蜍靈透過何種訊息傳遞路徑調控第二型環氧酵素及介白素第八的蛋白表現,我們發現在加入MAPK (p38, JNK和ERK)與NF-B抑制劑後,能夠有效阻斷蟾蜍靈促進蛋白激酶C所誘發的第二型環氧酵素及介白素第八的蛋白表現。另外,我們的實驗結果也顯示蟾蜍靈能夠增進蛋白激酶C所促進的p38, JNK和ERK磷酸化與NF-B轉移至細胞核內的作用。進一步探討蟾蜍靈所誘發的前列腺E2在乳癌細胞的增生與遷移所扮演的角色,發現前列腺E2能夠增加乳癌細胞增生與遷移的能力。除此之外,我們也觀察到在蛋白激酶C活化下,曾經被報導過會受到前列腺E2所調控並且對於乳癌轉移與發展調控很重要的基質金屬蛋白激酶3 (MMP3)的核糖核酸與蛋白表現會被蟾蜍靈顯著地誘發。綜合以上結果,我們認為蟾蜍靈可能夠會透過活化p38, JNK, ERK和NF-B的調控路徑增加蛋白激酶C所誘發的第二型環氧化酵素與介白素第八的核糖糖酸與蛋白表現,並且促進前列腺E2與介白素第八的分泌;誘發的前列腺E2則能夠促進乳癌細胞增生與遷移能力,這樣的結果可能會加劇乳癌的病程。
Chronic inflammation plays a significant role in tumor development. It has been well-recognized that chronic inflammation-associated mediators, such as cyclooxygenase 2 (COX2), a key rate-limiting step in the biosynthesis of prostaglandins (PGs), and interleukin-8 (IL-8), both of which can promote cancer cell proliferation, migration and angiogenesis. Several studies have revealed that induction of COX2 and IL-8 could be mediated by protein kinase C (PKC) signaling upon various inflammatory stimuli. In addition, it is also reported that PKC is highly activated in tumorigenesis. Bufalin, a major active component in the traditional Chinese medicine Chan Su, has been demonstrated to inhibit T cell proliferation. Recent studies have shown that bufalin exerts anti-cancer activity to induce prostate cancer, hepatocellular carcinoma and leukemia cell apoptosis. However, the specific role of bufalin in the inflammatory cascades and the associated impact in cancers remains mostly unclear. Thus, the aim of this study was to clarify whether and how bufalin may affect 12-O-tetradecanoyl-phorbol-13-acetate (TPA), a PKC activator, -mediated COX2 and IL-8 expression in MB231 human breast cancer cells. First, we discovered that bufalin could enhance TPA-induced COX2 and IL-8 protein expression, as well as the PGE2 and IL-8 secretion in MB231 cells, MCF-7 human breast cancer cells and THP-1 human acute monocytic leukemia cells, but not in H184 normal human mammary epithelial cells. We also observed that bufalin could elevate TPA-mediated COX2 and IL-8 mRNA expression in MB231 cells. We further clarified that the combined TPA and bufalin treatment-induced COX2 and IL-8 expression would be attenuated by inhibitors of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-B). Furthermore, bufalin appeared to increase TPA-induced MAPKs phosphorylation and NF-B nuclear translocation. We also endeavored to examine the role of COX2-derived PGE2 in breast cancer cell proliferation or migration and we noted that PGE2 could elevate breast cancer cell proliferation and migration. In addition, we discovered that the mRNA and protein expression of matrix metalloproteinase 3 (MMP3) which has been shown to promote breast tumor cell invasion and mammary carcinogenesis and to be regulated by microsomal PGE synthase-1 (mPGES-1), an enzyme to synthesize PGE2 were further increased by bufalin in combination with TPA treatment. Our current findings suggest that bufalin can elevate TPA-mediated COX2 and IL-8 expression, as well as PGE2 and IL-8 secretion, possibly at least in part by targeting to MAPKs phosphorylation and NF-B translocation to potentially participate in the promotion of breast cancer cell migration and cancer progression.
Index
Abstract 1
中文摘要 3
Chapter 1. Introduction 5
1-1. Inflammation and cancer 5
1-2. Significance of cyclooxygenase 2 (COX2) 7
1-3. Significant of interleukin-8 (IL-8) 9
1-4. Protein kinase C (PKC) 11
1-5. Bufalin 13
1-6. Objectives 14
Chapter 2. Materials and methods 16
2-1. Materials 16
Table 1. Chemicals and reagents 16
Table 2. Antibodies 20
Table 3. Oligonucleotide primers for RT-PCR 21
2-2. Cell cultures 22
2-3. Microarray analysis 23
2-4. Preparation of whole cell lysates and western blotting 23
2-5. Preparation of nuclear and cytosolic proteins 25
2-6. Measurement of PGE2, IL-8 production and PKC kinase activity by enzyme-linked immunosorbent assay (ELISA) 25
2-7. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) 26
2-8. Determination of cell viability 27
2-9. Evaluation of COX2 and IL-8 promoter as well as NF-kB and AP-1 reporter activities 28
2-10. Wound healing assay 29
2-11. Statistical analysis 29
Chapter 3. Results 30
3-1. Gene expression array for TPA- and bufalin-regulated genes in MB231 cells 30
3-2. The effects of bufalin on TPA-enhanced COX2 and IL-8 protein expression, as well as PGE2 and IL-8 secretion and secretion in MB231 cells 31
3-3. No cytotoxic effect of bufalin on MB231 cells 32
3-4. The regulation of COX2 and IL-8 protein expression by bufalin in MCF-7, H184 and THP-1 cells 32
3-5. Regulation of TPA-induced COX-2 and IL-8 mRNA expression and promoter activity by bufalin in MB231 cells 33
3-6. No effect on regulation of PKC activity by bufalin in MB231 cells 34
3-7. Understanding of MAPK and AP-1 signaling pathways on bufalin-regulated TPA-induced COX2 and IL-8 expression in MB231 cells 34
3-8. Involvement of NF-kB signaling pathway in bufalin-regulated TPA-induced COX2 and IL-8 expression in MB231 cells 36
3-9. The parallel pathways of MAPK and NF-kB in MB231 cells 37
3-10. Enhanced cell proliferation by PGE2 in MB231 cells 377
3-11. Regulation of TPA-promoted MMP3 by bufalin in MB231 cells 38
3-12. Promotion of cell migration by PGE2 in MB231 cells 39
Chapter 4. Discussion 40
Chapter 5. References 48
Tables and figures 65
Table 4. Gene expression array for TPA- and bufalin-regulated genes in MB231 human breast cancer cells 66
Figure 1. Bufalin structure. 67
Figure 2. TPA-induced COX-2 and IL-8 expression through PKC pathway in MB231 cells. 69
Figure 3. Enhancement of TPA-promoted COX2 and IL-8 protein expression, PGE2 and IL-8 secretion by bufalin in MB231 cells. 72
Figure 4. Non-cytotoxic effect of bufalin on the cell viability of MB231 cells…. 74
Figure 5. The effects of bufalin-induced TPA-increased COX2 and IL-8 protein expression in MCF-7 cells. 76
Figure 6. Regulation of COX2 and IL-8 protein expression by bufalin in combination with TPA in H184 cells. 78
Figure 7. Regulation of TPA- or LPS-mediated COX2 and IL-8 protein expression by bufalin in THP-1 cells 81
Figure 8. Increase of TPA-induced COX-2 and IL-8 mRNA expression by bufalin in MB231 cells 83
Figure 9. Regulation of TPA-mediated COX2 and IL-8 promoter activity by bufalin in MB231 cells. 85
Figure 10. The effects of bufalin on PKC activity by in MB231 cells ..86
Figure 11. Identification of MAPK and NF-kB signaling pathways mediating TPA-induced COX2 and IL-8 expression by bufalin in MB231 cells... 88
Figure 12. Promotion of TPA-enhanced p38, ERK and JNK phosphorylation by bufalin in MB231 cells. 90
Figure 13. The regulation of bufalin-affected TPA-mediated AP-1 reporter activity in MB231 cells. 91
Figure 14. Identification of NF-kB signaling pathways mediating TPA-induced COX2 and IL-8 expression by bufalin in MB231 cells... 93
Figure 15. Promotion of TPA-induced NF-kB nuclear translocation by bufalin in MB231 cells. 95
Figure 16. The effects of bufalin on TPA-regulated NF-kB reporter activity in MB231 cells. 96
Figure 17. The parallel pathways of p38 and NF-kB in MB231 cells 97
Figure 18. Proliferation of MB231 cells induced by PGE2. 98
Figure 19. Elevation of TPA-induced MMP3 mRNA and protein expression by bufalin in MB231 cells. 100
Figure 20. Promotion of cell migration by PGE2 in MB231 cells. 102


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