郭應誠臺灣大學：獸醫學研究所陳家真Chen, Chia-chenChia-chenChen2007-11-282018-07-092007-11-282018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/59923摘要 瘦體素為脂肪細胞主要分泌的內泌素之一，可能參與肥胖相關的癌生成與進展，也被視為許多類型癌細胞的生長因子，除了內分泌方式，也可能以旁分泌方式作用於標的組織。較少文獻探討瘦體素於乳癌、卵巢癌與肝癌細胞的作用，本研究乃針對此三類型的癌細胞，研究瘦體素的作用與其傳訊途徑與機制。在所有測試的三類型癌細胞，瘦體素都促進細胞的增生與抗凋亡作用。在促進細胞增生上，瘦體素普遍刺激cyclin D1的表現、進而加速細胞週期的運轉；而在兩種婦癌細胞，一種cyclin D1的轉錄因子c-Myc其表現也被瘦體素刺激。此外，在乳癌細胞中，抑癌因子p53與細胞素依賴性激酶抑制因子p21WAF/CIP1的表現則被瘦體素所減弱，顯示瘦體素也能藉由減弱p53與p21WAF/CIP1的表現以推促細胞週期的運轉。在兩種婦癌細胞細的抗凋亡上，瘦體素主要刺激抗凋亡因子Mcl-1的表現，而在肝癌細胞，則減少凋亡因子Bax的表現；顯然瘦體素抗凋亡的作用機制有細胞類型的差別。在所有測試的三類型癌細胞中，瘦體素的作用皆由JAK2所連接的訊息途徑所傳達，而在JAK2下游必有MEK1/2與ERK1/2（一般構成所謂MEK/ERK1/2的傳訊單位）的參與，形成JAK2-MEK/ERK1/2的傳訊路徑。在肝癌細胞，於JAK2-MEK/ERK1/2的傳訊路徑間另有PI3K與Akt（一般構成所謂PI3K/Akt的傳訊單位）的參與，形成JAK2-PI3K/Akt-MEK/ERK1/2的傳訊途徑；而在乳癌細胞，瘦體素作用的傳訊途徑則為JAK2-PI3K-EK/ERK1/2，瘦體素並不活化Akt。有趣的是，瘦體素分別活化卵巢癌細胞中JAK2-MEK/ERK1/2與PI3K/Akt兩條分明的路徑，雖然PI3K/Akt與JAK2-MEK/ERK1/2之間有訊息交接，彼此卻不為上下游的關係。本研究顯示活化cyclin D1以加速細胞週期運轉為瘦體素促進乳癌、卵巢癌與肝癌細胞增生作用的共同機制，而瘦體素抗凋亡的作用機制則因細胞類型略有不同。在乳癌、卵巢癌與肝癌細胞中，瘦體素作用的的傳訊途徑明顯呈現細胞類型的特異性。Abstract Leptin, one of the major hormones synthesized by adipocytes, is involved in the development of obesity associated cancers. It plays as a paracrine/ autocrine regulator mediating growth of various types of cancer cells. Effects of leptin in breast, ovarian and liver cancer cells were less studied, therefore in the present study, we used the three cancer cells to investigate leptin effects and its signaling pathways and mechanisms. In all three examined cancer cells, we found leptin induced cell proliferation and anti-apoptosis. Leptin-stimulated cell proliferation was achieved by provoking expression of cyclin D1 that speeds up cell-cycle progression. In breast and ovarian cancer calls, the expression of c-Myc, one of the transcription factors of cyclin D1 promoter, was enhanced by leptin. In addition, we observed leptin also down-regulated tumor suppressor p53 and cyclin-depedent kinase (CDK) inhibitor p21WAF1/CIP1 to promote cell-cycle progression. In both gynaecological cancer cells, leptin attenuated cell apoptosis by up-regulating expression of antiapoptotic MCL-1. In hepatocellular carcinoma (HCC) cells, the anti-apoptotic effects of leptin were mediated by decreasing expression of proapoptotic Bax. The results indicated the mechanism of leptin-inhibited anti-apoptosis is cell type-specific. In all three cancer cells, the intracellular signals of leptin were transduced by a Janus kinase 2 (JAK2)-linked pathways and at the downstream of JAK2, MEK1/2 and ERK1/2 are involved in the signaling cascade. Besides of JAK2, MEK1/2 and ERK1/2, in HCC cells, PI3K and Akt were participated in the leptin activated signaling pathway. We define that the signaling cascade of leptin in HCC is a JAK2-PI3K/Akt-MEK/ERK1/2. In breast cancer cells, not like in HCC, leptin has no influence on phosphorylation of Akt and leptin-induced signaling pathway is JAK2-PI3K-MEK/ERK1/2. Interestingly, in ovarian cancer cells, crosstalk between two signaling pathways, JAK2-MEK/ERK1/2 and PI3K/Akt, were activated by leptin. Our study demonstrated that in the three examined cancer cells, leptin promotes cell proliferation is generally mediated by stimulating the expression of cyclin D1 to accelerate cell-cycle progression. However the mechanism of leptin on inhibiting apoptosis is cell-specific. The signaling pathways activated by leptin are cell type-dependent.Index 摘要………………………………………………………………...1 Abstract…………………………………………………………...2 Introduction……………………………………………………...4 Leptin………………………………………………………...………………...4 Regulation of leptin secretion…………………………………………………4 Leptin receptors………………………..………………………..…………….5 Leptin signaling………………………………………..…………..………..…8 Leptin and obesity……………………………………..……….…………..….8 Leptin and cancers………………………………….......……………..………9 Material & Methods………………………………………………….11 Reagents and antibodies……………..………..…...…………………………11 Cell lines and culture……………..…..……………….……………………....11 RNA extraction and reverse transcription (RT)…………..……...….……...12 Polymease chain reaction (PCR)..……………………..………..…….……...12 MTT assay..…………………….…………………………….…..……………14 Cell number counting…………………………………….…………………...14 Flow cytometry………………………….….………………………………….14 Western blotting analysis………………….………………………………….14 Construction of pCD-1 Luc plasmid………….……………………………...15 Transfection and luciferase activity assay……….…………………………...15 Statistical analysis……………………………………..……………………….16 Part I Effects of leptin in breast cancer cells………..……..17 Introduction………………………………………………………..19 I.1: Leptin-induced growth of human ZR-75-1 breast cancer cells is associated with up-regulation of cyclin D1 and c-Myc and down-regulation of tumor suppressor p53 and p21 WAF1/CIP1………………………………………..…..22 Results…………………………………………………………………..22 Leptin autoregulated expressions of itself and its receptor……..………………..22 Leptin stimulated cell proliferation………………………………..………………22 Leptin stimulated cyclin D1 expression…………………………………..………..23 Leptin inhibited expression of cyclin-dependent kinase inhibitor p21WAF1/CIP1..23 Leptin down-regulated expression of tumor suppressor p53…………….……...23 Leptin up-regulated proto-oncogene c-Myc expression……………….…………24 Leptin stimulated cell proliferation through cross-talk of multiple pathways...24 Effect of cooperation between leptin and estrogen on cell growth……….……..24 Leptin reduced the proportion of G0/G1-phase cell population…………….…..25 Figures……………………………………………………………...…..26 Figure 1. Autoregulation and proliferative effect of leptin………………………26 Figure 2. Leptin regulated expression of cyclin D1, p21 WAF1/CIP1, tumor suppressor p53 and c-Myc………………………………………………………..28 Figure 3. JAK2 inhibitor prevented leptin stimulation……….…………………30 Figure 4. MEK/ERK1/2 inhibitor prevented leptin stimulation..……………….31 Figure 5. PI3K inhibitor prevented leptin stimulation…………..………………32 Figure 6. Effect of cooperation between leptin and estrogen……..……………..33 Figure 7. Leptin reduced G0/G1-phase cell population…………………………34 Figure 8. Hypothesized model of leptin signaling on the growth of human breast cancer cells…………………………………………………………………..…....36 Discussion………..……………………………………..…….……….37 I.2: Leptin stimulates cell growth by inducing expressions of c-Myc and cyclin D1 and prevents apoptosis by inducing antiapoptotic Mcl-1 expression through a JAK2-PI3K-MEK/ERK1/2 signaling pathway…...…43 Results………………………………………………………...………...44 Leptin stimulates MCF-7 and T-47D cell growth.……………………...………....44 Leptin attenuates serum-deprivation induced apoptosis…………………..…….44 Leptin up-regulates c-Myc and cyclin D1 expressions.............................….…….45 Leptin stimulates anti-apoptotic Mcl-1 expression……………………….……....45 Leptin stimulates cell growth through activations of JAK2, PI3K and MEK/ERK1/2……………………………….…………………………………...…45 Figures……………………………..…………………………………..48 Figure 1. Leptin stimulates MCF-7 and T-47D cell growth.……………..……...48 Figure 2. Leptin protect MCF-7 cells from serum deprivation induced apoptosis…………………………………………………………………………..49 Figure 3. Leptin regulates expressions of c-Myc, cyclin D1 and Mcl-1……......50 Figure 4. Pharmacological inhibitors of JAK2, PI3K/Akt and MEK/ERK1/2 prevent MCF-7 growth from leptin induction………………………….…….…52 Figure 5. Leptin triggers a JAK2- MEK/ERK1/2 signaling pathway…….…....54 Discussion……………………………………………………….….....56 Part II Effects of leptin in hepatocarcinoma cells……....…...58 Leptin induces proliferation and anti-apoptosis in human hepatocarcinoma cells by up-regulating cyclin D1 and down-regulating Bax via a Janus kinase 2-linked pathways…………………………………………………………….58 Introduction……………………………………..…………………...60 Results………………………………………………………………..62 Hepatic malignant and non-malignant cell lines express leptin receptors.…………………………………………………………………………..62 Leptin promotes cell-cycle progression to stimulate growth of hepatic cells….63 Leptin induces cyclin D1 expression……………………………………………..64 Leptin stimulates cell growth through activations of JAK2, PI3K/Akt and MEK/ERK1/2……………………………………………………………………..65 Leptin activates signaling crosstalks among JAK2, PI3K/Akt and MEK/ERK1/2………………………………………………………………...…...66 JAK2, PI3K/Akt and MEK/ERK1/2 mediate leptin-induced cyclin D1 -dependent cell-cycle progression…………………………..…………..……..…67 Leptin inhibits TGF- b1-induced apoptosis by down-regulating Bax expression………………………………………………………………………….68 Anti-apoptotic effect of leptin goes through activation of JAK2, PI3K/Akt and MEK/ERK1/2………………………...…………………………………………...69 Figures & Tables…...…………………………………………………..70 Figure 1. Expressions of leptin receptors in hepatic cells………………………..70 Figure 2. Leptin stimulates hepatic cell growth………………………………......71 Table 1. Leptin promotes cell cycle progression in Hep3B cells…….……………73 Figure 3. Leptin induces hepatic cyclin D1 expression…………………...………74 Figure 4. Leptin activates phophorylations of JAK2, Akt and ERK1/2 in hepatic cells.............................................................................................................................76 Figure 5. Pharmacological inhibitors of JAK2, PI3K/Akt and MEK/ERK1/2 prevent hepatic cell growth from leptin induction……………………………….78 Figure 6. Leptin triggers JAK2-linked PI3K/Akt and MEK/ERK1/2 signaling pathways…………………………………………………………………………….80 Figure 7. Pharmacological inhibitors of JAK2, PI3K/Akt and MEK/ERK1/2 prevent hepatic cyclin D1 expression from leptin induction…………………….82 Table 2. Inhibitors of JAK2, PI3K/Akt and MEK/ERK1/2 prevent leptin-activated hepatic cell cycle progression…..……...………………...…..84 Figure 8. Leptin protects HCC cells from TGF-b1-induced apoptosis by down-regulating apoptotic Bax expression……………………………….…...85 Figure 9. Inhibitors of JAK2, PI3K/Akt and MEK/ERK1/2 withdraw leptin rescue of TGF-b1-induced HCC cell death…………………………..…...…….87 Figure 10. Leptin passes through a JAK2-PIK3/Akt-MEK/ERK1/2 signaling cascade to partially reverse the TGF-b1-reduced Bcl-2/Bax ratio and thus prevent HCC apoptosis…………..……………………………………….……...89 Figure 11. A hypothetic mode underlying somatotropic effect of leptin on HCC cells………………….………………………………………………………..……91 Discussion………..……………………………………………………92 Part III Effects of leptin in ovarian cancer cells......………..99 Leptin stimulates cell growth and anti-apoptosis by inducing expressions of c-Myc, cyclin D1 and Mcl-1 through activations of JAK2- -MEK/ERK1/2 and PI3K/Akt signaling pathways in OVCAR-3 cells………………………99 Introduction…………………………..………………………………...99 Results…………………………………..……………………………...102 Leptin stimulates growth of ovarian cancer cells….………………….…………102 Leptin reduces serum-deprivation induced apoptosis………………….……….102 Leptin induced expressions of cyclin D1, c-Myc and Mcl-1.................................103 Activations of JAK2, PI3K/Akt and MEK/ERK1/2 pathways mediate leptin-stimulated cell growth in OVCAR-3……………………………….…….104 Leptin activates crosstalks among JAK2, PI3K/Akt and MEK/ERK1/2 signaling pathways…………………………….…………………………………………….104 Figures……………………………………………………………...106 Figure 1. Leptin stimulates growth of ovarian cancer cells....……..………..106 Figure 2. Leptin protects OVCAR-3 cells from serum deprivation induced apoptosis………………………………………...………………………….…..107 Figure 3. Leptin up-regulates expressions of cyclin D1 and Mcl-1 …..…..…107 Figure 4. Leptin acutely induces c-Myc expression in OVCAR-3 cells..…....108 Figure 5. Leptin induces Mcl-1 expression in two phases in OVCAR-3 cells.109 Figure 6. Leptin activates phophorylations of JAK2, Akt and ERK1/2 in OVCAR-3 cells…………………………………………………..……………...110 Figure 7. Pharmacological inhibitors of JAK2, PI3K/Akt and MEK/ERK1/2 prevent ovarian cancer cell growth from leptin induction……………....…....112 Figure 8. Leptin triggers JAK2-MEK/ERK1/2 and PI3K/Akt signaling pathways..........................114 Discussion…..………………………………………………..………116 Conclusions…………………………………….………….……....118 References…………………………………………………….…....1203632786 bytesapplication/pdfen-US瘦體素乳癌肝腫瘤卵巢癌細胞生長leptinbreast cancerhepatocellular carcinomaovarian cancercell growth[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/59923/1/ntu-96-F87629001-1.pdf