肥胖是罹患前列腺癌的風險之一，而瘦體素是與肥胖十分相關的 物質。瘦體素的功能除了與新陳代謝、能量消耗和食慾控制的功能之 外，之前的研究顯示出瘦體素的表現也會增加人類腫瘤細胞的生成及 轉移，但是關於人類前列腺細胞的入侵機制尚未清楚(Gainsford et al., 1996) (Vona-Davis et al., 2007)。本研究顯示出瘦體素誘導細胞移行的其
中一個機制是經由αvβ3 integrins 調節訊號傳遞，並且是經由活化 OBRl 接受器、IRS-1、PI3K、Akt 及 NF-κB 的訊息傳遞路徑(Fig. 14)。
瘦體素是屬於組織介素(Cytokine)的一員(Campfield et al., 2000)，
而且在之前的研究中已發現人類前列腺癌細胞會表現 OBRl 及 OBRs 兩種接受器(Bergström et al., 2001) (Ishikawa et al., 2004)，但關於其在 人體中的表現，仍是尚未釐清的部份。
Integrins 連接細胞外基質與細胞內骨架，並且與分子細胞傳遞以及 調節細胞內的生理功能，包括：附著(adhesion)、訊號傳遞(signaling)
、細胞的運動(motility)、細胞的生存(survival)、基因的表現、細胞生長 和細胞分化(Giancotti et al., 1999)。當我們進行流式細胞分析實驗後發 現瘦體素會增加在轉移中扮演重要角色的αvβ3 integrins 之表現。因此
，我們使用αvβ3 integrins 的抗體來探討 αvβ3 integrins 作用並發現它會
抑制瘦體素所造成的細胞移行現象。而且，當我們給予細胞 RGD 及 用p85 的 dominant-negative mutant 進行實驗後，也發現其瘦體素所誘 導的細胞移行能力及αvβ3 integrins 的表現皆被抑制，這結果進一步的 證實PI3K 參與在由瘦體素調控增加前列腺癌細胞的轉移的過程中。
PI3K 的活化會造成下游標的物 Akt 的磷酸化，Akt 在調節細胞生 長、細胞分化、細胞附著及發炎反應中扮演重要的角色(Hirsch et al., 2000)。本篇中我們證明使用 Akt 抑制劑時，瘦體素所誘導的細胞移行
能力及integrins 的表現皆會被明顯的抑制。此外，dominant-negative Akt mutant 的使用也會造成瘦體素所誘導的細胞移行能力及 integrins 的表 現被抑制，所以在人類前列腺癌細胞中 Akt 是會被瘦體素活化的。這 結果指出，藉由Akt 的活化可證明 IRS-1/PI3K 這條訊息傳遞路徑參予 在由瘦體素調控增加前列腺癌細胞的轉移的過程中。
已有研究指出，NF-κB 的活化與 integrins 的表現及腫瘤的轉移有 關(Wen et al., 2006)。在本研究我們證明，瘦體素所誘導的細胞移行能 會被釋放入核內，進而活化基因的表現(Vermeulen et al., 2003)。在之前
的實驗我們發現當給予前列腺癌細胞株 PC3 瘦體素後，會增加 IKK、
IκBα 及 p65 的磷酸化。當我們使用 κB-luciferase 短暫的轉染入細胞當 作測試NF-κB 活性的指示劑時，我們發現瘦體素也會誘導增加 NF-κB
的活性。在本研究我們發現瘦體素所誘導的 IKK 及 p65 磷酸化會被 PI3K 的抑制劑 Ly294002 及 Akt 抑制劑所抑制，這結果顯示 PI3K 及 Akt 會透過活化 IKKα/β 造成 p65 的磷酸化，並增加 NF-κB 的轉錄活性
。
我們的研究得到的結果指出：瘦體素增加αvβ3 integrins 的活性是 經由OBRl-、IRS1-、PI3K-、Akt-、IKKα/β-及 NF-κB-訊息傳遞路徑所 造成的，並且進而增加人類前列腺癌細胞的細胞移行能力(Fig. 14)。此 外，瘦體素誘導之訊號傳的路徑的發現可以幫助我們了解人類前列腺 癌細胞轉移的機制，這或許可以引導我們在未來發展出更有效的治療 方法。
結論
在本研究中我們得到了關於瘦體素誘導前列腺癌細胞移行的新 機制是藉由上調節αv 及 β3 integrins。瘦體素會增加細胞的移行能力及 αvβ3 integrins 的表現是經由活化 IRS1、PI3K、Akt、IKKα/β 及 NF-κB 訊 息傳遞路徑(Fig. 14)。
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Fig. 5. Leptin-directed migration of human prostate cancer cells.
Fig. 5. Leptin-directed migration of human prostate cancer cells. PC3, DU145 and LNCaP cells were incubated with various concentrations of leptin, and in vitro migration activities measured with the Transwell after 24 h. Results are expressed as the mean ± SEM. *p<0.05 compared with control.
Fig. 6 . Leptin-directed chemoinvasion of human prostate cancer cells involves up-regulation of αvβ3 integrins
Fig. 6. Leptin-directed chemoinvasion of human prostate cancer cells involves up-regulation of αvβ3 integrins. (A) PC3 cells were incubated with leptin 1μM for indicated time intervals, and the cell surface expression of α2, α5, β1, β3, αvβ3 or α5β1 integrin were determined using flow cytometry. (B) PC3 cells were pretreated with αvβ3 monoclonal antibody ( 10ng/ml ), cyclic RGD (100nM) or cyclic RAD ( 100nM ) for 30min followed by stimulation with leptin 1μM. The in vitro migration activity measured after 24 h showed that αvβ3 monoclonal antibody and cyclic RGD but not cyclic RAD could inhibit the activity. Results are expressed as the mean ± SEM. *p<0.05 compared with control;
#p<0.05 compared with leptin-treated group.
Fig. 7. OBRl activation is involved in leptin-mediated migration and integrin up-regulation in human prostate cancer cells.
Fig. 7. OBRl activation is involved in leptin-mediated migration and integrin up-regulation in human prostate cancer cells. PC3 cells were transfected with OBRl or control siRNA for 24 h followed by stimulation with leptin, and in vitro migration (A) and cell surface αvβ3 integrin (B) were measured with the Transwell and flow cytometry after 24 h. Results are expressed as the mean ± SEM. *p<0.05 compared with control; #p<0.05 compared with leptin-treated group.
Fig. 8. IRS-1 pathway is involved in leptin-mediated migration and integrin up-regulation in human prostate cancer cells.
Fig. 8. IRS-1 pathway is involved in leptin-mediated migration and integrin up-regulation in human prostate cancer cells. (A) PC3 cells were incubated with leptin for indicated time intervals and IRS1 phosphorylation was examined by Western blot analysis. Cells were transfected with IRS-1 or control siRNA for 24 h followed by stimulation with leptin, and in vitro migration (B) and cell surface αvβ3 integrin (C) were measured with the Transwell and flow cytometry after 24 h. Results are expressed as the mean ± SEM.
*p<0.05 compared with control; #p<0.05 compared with leptin-treated group.
Fig. 9. PI3K pathway is involved in leptin-mediated migration and integrin up-regulation in human prostate cancer cells.
Fig. 9. PI3K pathway is involved in leptin-mediated migration and integrin up-regulation in human prostate cancer cells. (A) PC3 cells were incubated with leptin for various time intervals and pp85 phosphorylation was examined by western blog analysis.
(B) PC3, DU145 and LNCaP Cells were pretreated for 30 min with Ly294002 (10μM) and (C) PC3 cells transfected with dominant-negative (DN) mutant of p85 for 24 h followed by stimulation with leptin, and in vitro migration were measured with the Transwell after 24 h. (D&E) PC3 Cells were pretreated for 30 min with Ly294002 and transfected with dominant-negative (DN) mutant of p85 for 24 h followed by stimulation with leptin, and cell surface αvβ3 integrin were measured by flow cytometry. Results are expressed as the mean
± SEM. *p<0.05 compared with control; #p<0.05 compared with leptin-treated group.
Fig. 10. Akt pathway is involved in leptin-mediated migration and integrin up-regulation in human prostate cancer cells.
Fig.10. Akt pathway is involved in leptin-mediated migration and integrin up-regulation in human prostate cancer cells. (A) PC3 cells were incubated with leptin for various time intervals, and Akt phosphorylation was determined by immunoblotting using phospho-Akt-specific antibody. (B) PC3, DU145 and LNCaP cells were pretreated for 30 min with Akt inhibitor (20μM) and (C) PC3 cells transfected with dominant-negative (DN) mutant of Akt for 24 h followed by stimulation with leptin, and in vitro migrations were measured with the Transwell after 24 h. (D&E) PC3 cells were pretreated for 30 min with Akt inhibitor and transfected with dominant-negative (DN) mutant of Akt for 24 h followed by stimulation with leptin, and cell surface αvβ3 integrin were measured by flow cytometry. Results are expressed as the mean ± SEM. *p<0.05 compared with control; #p<0.05 compared with leptin-treated group.
Fig. 11. Leptin induces cells migration and integrin up-regulation through NF-κB.
Fig. 11. Leptin induces cells migration and integrin up-regulation through NF-κB. (A) PC3, DU145 and LNCaP cells were pretreated for 30 min with PDTC (10μM) or TPCK (1μM) followed by stimulation with leptin, and in vitro migration was measured with the Transwell after 24 h. (B) PC3 cells were pretreated for 30 min with PDTC (10μM) or TPCK (3μM) followed by stimulation with leptin for 24 h, and the cell surface αvβ3 integrin was measured by flow cytometry. Results are expressed as the mean ± SEM. *p<0.05 compared with control; #p<0.05 compared with leptin-treated group.
Fig. 12. Leptin induces cells migration and integrin up-regulation through NF-κB.
Fig. 12. Leptin induces cells migration and integrin up-regulation through NF-κB. (A) PC3 cells were incubated with leptin for indicated time intervals, and p-IKKα/β, p-IκBα and p-p65 expression was determined by western blot analysis. (B) PC3 cells were transfected with dominant-negative (DN) mutant of IKKα or IKKβ for 24 h followed by stimulation with leptin, and in vitro migration were measured with the Transwell after 24 h. Results are expressed as the mean ± SEM. *p<0.05 compared with control; #p<0.05 compared with leptin-treated group.
Fig. 13. Leptin induces cells migration and integrin upregulation through NF-κB.
Fig. 13. Leptin induces cells migration and integrin upregulation through NF-κB. (A&B) PC3 cells transiently transfected with κB-luciferase plasmid for 24 h and then co-transfection with OBRl-siRNA, IRS-1-siRNA, and (C) pretreated with Ly294002, Akt inhibitor, PDTC and TPCK for 30 min or (D) co-transfection with p85, Akt, IKKα and IKKβ mutant, before incubation with leptin for 24 h. Luciferase activity was measured, and the results were normalized to the β-galactosidase activity. Results are expressed as the mean ± SEM. *p<0.05 compared with control; #p<0.05 compared with leptin-treated group. μ
Fig. 14. Schematic presentation of the signaling pathways involved in leptin-induced migration and integrins expression of prostate cancer cells.
Fig. 14. Schematic presentation of the signaling pathways involved in leptin-induced migration and integrins expression of prostate cancer cells. Leptin activates PI3K and Akt pathway , which in turn induces IKKα/β phosphorylation, p65 Ser536 phosphorylation, and NF-κB activation, which leads to αvβ3 integrins expression and increases the migration of human prostate cancer cells.