本研究首次表示了 adiponectin 調控前列腺腫瘤細胞的移行。幫助我們了解 人類前列腺細胞的轉移機制，強調其中一個機制是藉由活化AdipoR1 receptor、
AMPK、p38 和 NF-κB 訊息傳導路徑，增加 α5β1 integrins 的轉錄調控 (Fig.9)。
以上推論能需要做進一步的研究與探討，以找出其真正的機制，在未來或許可以 應用在治療癌症上。
未來展望與建議
台灣的前列腺癌發生率少於歐美國家，根據行政院衛生署癌症死亡統計數據 指出，國人罹患前列腺癌近年來有增加的趨勢。由於前列腺癌初期沒有明顯的症 狀， 腫瘤進展速度緩慢，所以在台灣無法得知較確切的發生率。近年來前列腺 癌之生物行徑 ( biologic behavior ) 慢慢被研究，基因治療逐漸進步成熟，渴望 這方面前瞻性的發展對台灣前列腺癌的治療與預後有實質上的幫助。
Fig. 6 Adiponectin-directed migration of human prostate cancer cells.
PC3, DU145 and LNCaP cells were incubated with various concentrations of adiponectin, and in vitro migration activities measured with the Transwell after 24 hr showed all supported the cells migrations in a concentration-dependent way. The migration activity was analyzed by Transwell assay. Results are expressed as the mean±S.E.M. *, p <0.05 compared with control. #, p <0.05 as compared with adiponectin -treated group.
Fig. 7
Fig. 7 Adiponectin-directed migration of human prostate cancer cells involves up-regulation of α5β1 integrin.
(A) PC3 cells were incubated with adiponectin (0.3μg/ml) for 24 hr, and the cell surface expression of α2, α5, β1, β3, α2β1, or α5 integrins were determined using flow cytometry. (B) Cells were incubated with adiponectin (0.3μg/ml) for 24 hr, and the mRNA level of α2, α5, αv, β1 or β3 integrins were determined using qPCR. (C) Cells were pretreated for 30 min with α5β1 integrin followed by stimulation with adiponectin (0.3μg/ml) and in vitro migration activitie measured with the Transwell after 24 hr showed α5β1 integrin supported the cells migration. Results are expressed as the mean ± S.E.M. *, p < 0.05 compared with control；#, p < 0.05 compared with adiponectin-treated group.
Fig. 8 AdipoR receptor is mediated adiponectin-mediated cell migration.
(A) Total RNA were extracted from prostate cancer cell lines, and subjected to qPCR analysis for AdipoR1 and AdipoR2. (B) PC3 cells were transfected with AdipoR1, AdipoR2 or control siRNA for 24 hr followed by stimulation with adiponectin. The in vitro migration measured with the Transwell after 24 hr showed the suppressed migration activity by AdipoR1 siRNA. Results are expressed as the mean ± S.E.M. *, p < 0.05 compared with control; #, p < 0.05 compared with adiponectin-treated group.
Fig. 9
Fig. 9 P38 is involved in adiponectin-mediated cell migration and integrin up-regulation in human prostate cancer cells.
(A) PC3 cells were incubated with adiponectin for indicated time intervals, and phosphor -p38 expression was determined by Western blot analysis (upper panel). The p38 activity was also examined by p38 activity kit (lower panel). Cells were pretreated for 30 min with SB203580 (10 μM) (B) or transfected with dominant negative (DN) mutant of p38 (C) for 24 hr followed by stimulation with adiponectin, and in vitro migration (B&C) cells surface α5β1 integrin (D) and mRNA expression of α5β1 integrin were measured with the Transwell and flow cytometry or qPCR (E) after 24 hr. Results are expressed as the mean ± S.E. *, p < 0.05 compared with control; #, p < 0.05 compared with adiponectin-treated group.
Fig. 10
Fig. 10 AMPK pathway is involved in adiponectin-mediated integrin up-regulation in human prostate cancer cells.
(A) PC3 cells were incubated with adiponcetin (0.3μg/ml) for indicated time intervals, and phosphor-AMPK Thr172 (upper panel) or AMPK (lower panel) expression was determined by Western blot analysis. Cells were pretreated with AraA (0.5 mM) and compound C (10μM) for 30 min followed by stimulation with adiponectin or treated with 5-Aminoimidazole-4-caroxamide-1-β-D-ribofuranoside [AICAR (1 mM)] for 24 hr. The in vitro migration activity and migration expression was examined by Transwell (B) flow cytometry (D) and qPCR (E). (C) Cells were transfected with AMPKα1, AMPKα2 or control siRNA for 24 hr followed by stimulation with adiponectin. The in vitro migration measured with the Transwell after 24 hr. (F) Cells were pretreated with SB203580 (10μM) for 30 min followed by stimulation with adiponectin (0.3μg/ml) for 60 min, and phosphor-AMPK Thr172 expression was determine by Western Blot analysis (G) Cells were pretreated with AraA (0.5 mM) and Compound C (10μM) for 30 min followed by stimulation with adiponectin (0.3μg/ml) for 60 min, and p38 activity were then examined by using p38 activity kit.
Results are expressed as the mean± S.E.M. *, p < 0.05 compared with control; #, p <
0.05 compared with adiponectin–treated group.
Fig. 11
Fig. 11 Adiponectin induces cell migration and integrins up-regulation through NF-κB.
(A) Cells were pretreated for 30 min with PDTC (60μM) or TPCK (3μM) followed by stimulation with adiponectin (0.3μg/ml), and in vitro migration was measured with the Transwell after 24 hr. (B) PC3 cells were pretreated for 30min with PDTC (60μM) or TPCK (3μM) followed by stimulation with adiponectin (0.3μg/ml) for 24hr, and the cell surface α5β1 integrin was measured by flow cytometry. (C) PC3 cells were pretreated for 30min with PDTC (60μM) or TPCK (3μM) followed by stimulation with adiponectin (0.3μg/ml) for 24hr, and mRNA expression of α5 and β1 integrin was measured by qPCR. (D) PC3 cells were incubated with adiponectin (0.3μg/ml) for indicated time intervals, and p-IKKα/β, p-IκBα, and p-p65 expression was determined by Western blot analysis. (E) PC3 cells were transfected with dominant negative (DN) mutant of IKKα or IKKβ for 24 hr followed by stimulation with adiponectin (0.3μg/ml), and in vitro migration was measured with the Transwell after 24 hr. Results are expressed as the mean± S.E.M. *, p < 0.05 compared with control;
#, p < 0.05 compared with adiponectin–treated group.
Fig. 12 P38, AMPK and NF-κB inhibitors antagonized the adiponectin-induced κB luciferase activity in human prostate cancer cells.
(A) PC3 cells transiently transfected with κB-luciferase plasmid for 24 hr were pretreated with Compound C ( 10μM ), AraA ( 0.5 mM ), SB203580 ( 10μM ), PDTC ( 60μM ) and TPCK ( 3μM ) for 30 min, before incubation with adiponectin ( 0.3μg/ml ) for 24 hr. (B) Cells were cotransfected with DN- IKKα, IKKβ or p38 mutant and κB-luciferase plasmid for 24 hr. Luciferase activity was measured, and the results were normalized to the β-galactosidase activity. Results are expressed as the mean± S.E.M. *, p < 0.05 compared with control；#, p < 0.05 compared with adiponectin -treated group.
Fig. 13 Schematic diagram of the signaling pathways involved in adiponectin- induced cell migration in human prostate cancer cell.
Adiponectin increases migration activity by binding to the AdipoR1 receptor and activation of p38, AMPKα1, IKKα/β phosphorylation, which enhances binding of p65 to the NF-κB site, lead to α5β1 integrin expression and increases the migration of human prostate cancer cell.
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