材料(Materia)：
Anti-mouse 和 anti-rabbit IgG-conjugated horseradish peroxidase ， rabbit polyclonal antibodies 如 p-MEK、MEK、p-ERK、ERK、IKKα/β、
IκBα、p- IκBα、α5 integrin 購自 Santa Cruz Biotechnology (Sant Cruz, CA, USA.) 。PD98059、U0126、TPCK、PDTC 購買自 Calbiochem (San Diego, CA, USA.) 。 Rabbit polyclonal antibody specific for phspphor- IKKα/β(Ser180/181) 和 phspphor-p65(Ser536)購自 Cell Signaling (Danver, MA, USA) 。Luciferase assay kit 購自 Promega (Madison, MA, USA) 。其他所 有化學藥品購自 Sigma (Madison, MA, USA)。
細胞培養(Cell culture)：
人類軟骨瘤細胞(JJ012)由 Dr. Sean P Scully (University of Miami School of Medicine, Miami, FL)實驗室提供。細胞使用含 10％ Fetal bovine serum(FBS)之 DMEM/α-MEM，於 37℃、5% CO2 培養箱中培養。
細胞移行實驗 (Migration assay) :
細胞移行實驗需準備 transwell (Costar, NY; pore size, 8-μm)在 24 孔 盤。再準備進行細胞移行實驗前，細胞先使用不同濃度的抑制劑前處理 30 分鐘，包括 PD98059、U0126、TPCK、PDTC。近乎 1.5Ⅹ104 的細胞 量在 200μl serum- free JJ012 的培養液置於 transwell 的上層，用 300μl
serum-free JJ012 的培養液置於下層加入 50 ng/ml 的 VEGF，放到 37℃、
5% CO2 培養箱中培養 24 小時。然後再以 formaldehyde 固定 15 分鐘並用 0.05% crystal violet 染色 15 分鐘。上層的細胞以棉棒去除，再用 PBS 清 洗，細胞會移行到膜的下層吸附，再以顯微鏡計數細胞總量，每個實驗 操作三重複並於不同之時間點進行。
流式細胞技術分析(Flow Cytimetric Analysis)：
細胞分盤於六孔盤內。先用 PBS 清洗，以 37℃ trypsin 將細胞打起 分離。用 70%酒精固定細胞 10 分鐘。離心後再以 PBS 清洗，再把細胞與 α5 integrin 的 rabbit anti-human antibody 反應 1 小時；再將細胞洗淨後，
再與 anti-rabbit secondary IgG (1:100)反應一小時。接著使用流式細胞儀進 行分析，使用 FACSCalibur and Cellquest software(BD Biosciences)來分析 (Tang et al., 2006)
西方墨點法分析(Western blot analysis)：
細胞蛋白先在 SDS-PAGE 中分離，而後轉漬到 immobilon PVFD membrane 上；用 4%BSA 在室溫下作用一小時，然後用 p-MEK、MEK、
p-ERK、ERK、IKKα/β、IκBα、p- IκBα、p65、p-p65 的 rabbit anti-human antibody(1:1000) 作 用 一 個 小 時 ， 然 後 進 行 三 次 清 洗 ； 在 與 donkey anti-rabbit peroxidase-conjugated secondary antibody(1:1000)室溫下作用一
片。
及時定量聚合酶連鎖反應(Real time polymerase chain reaction, real-time PCR)
α5,sense CAGACCCTGCTCATCCAGAAT anti-sense GGCATTCTTGTCACCCAGGTAC GAPDH, sense AAGCCCATCACCATCTTCCAG anti-sense AGGGGCCATCCACAGTCTTCT
Transfection and reporter gene assay：
人類的腫瘤細胞 0.8 μg κB-luciferase plasmid 和 0.4 μg β-galactosidase 的表現載體(expression vector)做 co -transfection 送到細胞內。當腫瘤細胞
做 transfection 。首先將 DNA 和 LF2000 混合，在加入細胞反應 24 小時 後，加入 VEGF 再經過 24 小時；使用 100μL reporter lysis buffer (Promega,
Madison, WI) 加到每個 well 中，置於冰上 15 分鐘後將細胞刮取下來；再 以 13,000rpm 離心 15 分鐘後吸取上清液；每 20 μL 的細胞上清液中含有 等量(20-30 μL)的蛋白，將其置於不透光的 96 孔盤中，加入 80μL 的螢光 受質(luciferase substrate)加到所有的樣品中，螢光藉由 lumnometer 偵測。
形成雙體的蛋白質一般具有模組化的結構，在此蛋白質上可能有一部份
結果與討論 (Result and Discussion)
結果
B)；而 α2、β3、αvβ3、α2β1、α5β1、β1 integrin 都沒有明顯的增加(Fig.11A.)，
另外 VEGF 也可以增加 α5 integrin mRNA 的表現(Fig.11B)；所以，藉由 此結果可推論，α5 integrin 與 VEGF 造成軟骨肉瘤細胞的轉移可能有相關。
MEK/ERK signal pathway 參與 VEGF 增加 integrin 表現和人類軟骨肉 瘤細胞的移行
文獻當中 MEK/ERK 路徑的活化可由多種的生長因子引起，比如像是
胰島素或是與神經系統相關的生長因子，也包含了 VEGF；為了證明 行、α5 integrin 向上調節量上都有明顯的被抑制現象(Fig.12 B-D)；因此，
可以推論，MEK 的活化參與 VEGF 造成軟骨肉瘤細胞的轉移與 integrin
中常發現它的存在(Eli Pikarsky et al., 2004)；在人類癌症中也可發現它的 抑制劑（PDTC）或 IκB protease 抑制劑（TPCK）三十分鐘後進行流式細 胞儀分析及定量聚合酶連鎖反應分析，發現對於 VEGF 向上調節軟骨肉 mutant 轉染 JJ012 細胞 24 小時後，再進行移行試驗(Fig.15B)，發現 VEGF 造成軟骨肉瘤細胞的移行現象明顯的減少；所以 IKKα/β 的活化參與了
VEGF 調控軟骨肉瘤細胞的移行中。接著依照不同時間點對於 JJ012 細胞 給予 VEGF 也刺激了 IκBα 的磷酸化(Fig. 15A)。先前的研究即顯示出 p65 Ser536的磷酸化會增加 NF-κB 的轉錄活化(Transactivation)；依照不同時間
點對人類軟骨肉瘤細胞給予 VEGF 後，可以發現 p65 Ser536的磷酸化隨著 時間點不同而增加(Fig. 15A)；為了觀察軟骨肉瘤細胞被給予 VEGF 後
NF-κB 活性的變化，我們將軟骨肉瘤細胞轉染入 κB-luciferase 當作 NF-κB 活性的指示因子。實驗結果顯示，當給予軟骨肉瘤細胞 JJ012 24 小時 VEGF 後，會增加 κB-luciferase 的活性(Fig.16A)，此外，PD98059、U0126、
PDTC 及 TPCK 皆能降低由 VEGF 所誘發的 NF-κB 活性(Fig.16 B)；將 JJ012 細胞株一同轉染入 MEK、ERK、IKKα、IKKβ mutant (Fig.16 C )也 能抑制 NF-κB 促進因子的活性。綜合以上結果來看活化 MEK、ERK 訊 息傳導路徑在 VEGF 誘發軟骨肉瘤癌細胞 NF-κB 活化是需要的。
討論 合成 24 種不同的 heterodimericr 接受器，在細胞膜上不同的 Integrin 次單 位組合允許細胞去辨認與反應數種不同的 ECM(extracellular matrix)，包 括 fibronectin，laminin，collagen 和 vitronectin。因為 integrin 是細胞連接 到 ECM 的主要接受器；它們扮演著細胞訊息雙向傳遞的重要傳訊器的角 色；調節著細胞存活(survival)、分化(differentiation)、增生(proliferation) 、 轉移(migration)與組織重新製造(tissue remodeling)。(Giancotti et al., 1999)
但是， 因為 VEGF 對於人類軟骨肉瘤細胞表面的 integrin 表現情況還不 清楚。在實驗中我們發現，VEGF 增加人類軟骨肉瘤細胞（JJ012）表面 α5 integrin 的表達。在本研究中使用流式細胞儀，來分析 α5 integrin 對於 人類軟骨肉瘤細胞（JJ012）的移行現象的影響。 NF-κB-dependent 的轉錄。此外，p65 在 Ser536被磷酸化藉由不同的路徑，
來開始進行與 p65 相關路徑；TNF-α 會使 p65 的 Ser536 快速的被磷酸化 經由 IKK 路徑使得需依靠 p65 的轉錄可以被活化；在之前的實驗當中，
我們發現給予軟骨肉瘤細胞 VEGF 後，會造成 IKK、p65、IκBα 的磷酸 化增加；當我們使用 luciferease 轉染入軟骨肉瘤細胞作為 NF-κB 活化的 指示劑時，發現可以增加 NF-κB 的表現。在此研究可以發現轉入 MEK 與 ERK mutant 之後， NF-κB 的活性被降低了；加入 NF-κB 抑制劑(PDTC) 或 IκBα 抑制劑(TPCK)或送入 IKKα、IKKβ mutant 後，也可發現抑制了由 VEGF 調節的 NF-κB 的表現。
結論
由於軟骨肉瘤遠處轉移後病人的預後被認為是相當不樂觀的；因此，防 止人類軟骨肉瘤轉移是一個現在重要的課題。我們的研究提出的 VEGF 增加了α5 integrin 的活性經由 MEK、ERK、IKKα/β 和 NF-κB-dependent 的路徑 增加了人體軟骨肉瘤細胞的移行。此外，這一發現對 VEGF/
integrin/ NF-κB-dependent 路徑的發現幫助我們更加了解人類軟骨肉瘤的 轉移機制並且對治療及預防轉移上能更進一步。
Fig. 10. VEGF increased migration of chondrosarcoma cells.
JJ012 cells were incubated with various concentrations of VEGF, and in vitro migration activities measured with the Transwell after 24 hours. Results are expressed as the mean
±SE. *P < 0.05 compared with control.
Fig. 11 VEGF-directed migration of human chondrosarcoma cell involves up-regulation of α5 integrin(A) JJ012 cells were incubated with VEGF (50 ng/ml) for 24 hr, and the cell surface expression of α2, β3, αvβ3, α2β1, α5β1, α5β1 integrins were determined using flow cytometry. (B) JJ012 cells were incubated with various
concentrations of VEGF and the mRNA expression of α5 integrins was examined by qPCR.
Results are expressed as the mean ±SE. *P < 0.05 compared with control.
Fig. 12 MEK is involved in VEGF-mediated migration and integrin up-regulation in human chondrosarcoma cells. (A): JJ012 cells were
incubated with VEGF (50 ng/ml) for indicated time intervals, and p-MEK was examined by Western Blot analysis. (B): JJ012 cells were pretreated for 30 min with PD98059 and U0126 or (C): transfected with dominant negative (DN) mutant of MEK1 for 24 h
followed by stimulation with VEGF (50 ng/ml), and in vitro migration was measured with the Transwell after 24 h. .(D): JJ012 cells were transfected with dominant negative
(DN)mutant of MEK for 24 h and pretreated for 30 min with PD98059 and U0126
( followed by stimulation with VEGF(50 ng/ml) 24 hours ,and the mRNA expression of α5 integrins was examined by qPCR (E) : JJ012 cells were pretreated for 30 min with
PD98059 and U0126 followed by stimulation with VEGF (50 ng/ml), and the cell surface α5 integrin was measured by using flow cytometry. Results are expressed as the mean ±SE.
*P < 0.05 compared with control; #P < 0.05 compared with VEGF-treated group.
Fig. 13 ERK is involved in VEGF-mediated migration and integrin up-regulation in human chondrosarcoma cells. A: JJ012 cells were incubated with VEGF(50 ng/ml) for indicated time intervals, and p-ERK was examined by Western blot analysis. B: JJ012 cells were transfected with dominant negative (DN)mutant of ERK2 for 24 h followed by stimulation with VEGF(50 ng/ml), and in vitro migration was
measured with the Transwell after 24h.C: JJ012 cells were transfected with dominant negative (DN)mutant of ERK2 for 24 h followed by stimulation with VEGF(50 ng/ml) 24 h and the mRNA expression of α5 integrin was examined by qPCR D:, and the cell surface α5 integrin was measured by using flow cytometry. Results are expressed as the Mean ±SE. *P < 0.05 compared with control; #P < 0.05 compared with VEGF-treated group.
Fig. 14 VEGF induces cells migration and integrin up-regulation through NF-κB. (A): JJ012 cells were pretreated for 30 min with PDTC (10 μM) or TPCK (3 μM) followed by stimulation with VEGF (50 ng/ ml), and in vitro migration was measured with the Transwell after 24 h. (B): JJ012 cells were were pretreated for 30 min with PDTC (10 μM) or TPCK (3 μM) 24 hr, followed by stimulation with VEGF (50 ng/ ml),and the mRNA expression of α5 integrins was examined by qPCR ; (C): and the cell surface α5 integrin was measured by using flow cytometry. Results are expressed as the mean ±SE.
*P < 0.05 compared with control; #P < 0.05 compared with VEGF-treated group.
Fig. 15 VEGF induces IKK α/β phosphorylation, IκBα phosphorylation and p65 phosphorylation in chondrosarcoma cells. (A): JJ012 cells were incubated with VEGF (50 ng/ml) for indicated time intervals, and p- IKK α/β, p- IκBα and p-p65 expression was determined by Western blot analysis. (B): JJ012 cells were
transfected with dominant negative (DN) mutant of IKKα or IKKβ for 24h followed by stimulation with VEGF (50 ng/ml), and in vitro migration was measured with the
Transwell after 24 h. (C): JJ012 cells were transfected with DN mutant of IKKα, or IKKβ for24 h followed by stimulation with VEGF(50ng/ml), the mRNA expression of α5 integrins was examined by qPCR . (D):JJ012 cells were transfected with DN mutant of IKKα, or IKKβ for 24h followed by stimulation with VEGF(50ng/ml), and the cell surface α5 integrins was measured by using flow cytometry. Results are expressed as the Mean
±SE. *P < 0.05 compared with control; #P < 0.05 compared with VEGF-treated group.
Fig.16 MEK, ERK and IKKα/β pathway is involved in VEGF-mediated NF-κB activation. (A): JJ012 cells were incubated with various concentrations of VEGF, and NF-κB luciferase activity was examed. (B, C):JJ012 cells transiently
transfected with κB-luciferase plasmid for 24 h were either co-transfected with MEK, ERK, IKKα, and IKKβ mutants or pretreated with PD98059 (10μM),U0126 (10μM),TPCK (10 μM), or PDTC (3 μM) for 30min, before incubation with VEGF (50 ng/ml) for 24 h.
Luciferase activity was measured, and the results were normalized to the β-galactosidase activity. Results are expressed as the mean ±SE. *P < 0.05 compared with control; #P <
0.05 compared with VEGF-treated group.
參考文獻 (Reference)
參考文獻
Alam, N., H. L. Goel, et al. (2007). "The integrin - growth factor receptor duet." Journal of Cellular Physiology 213(3): 649-653.
Bachelder, R. E., M. A. Wendt, et al. (2002). "Vascular Endothelial Growth Factor Promotes Breast Carcinoma Invasion in an Autocrine Manner by Regulating the Chemokine Receptor CXCR4." Cancer Res 62(24):
7203-7206.
Boukerche, H., Z.-z. Su, et al. (2007). "mda-9/Syntenin Regulates the Metastatic Phenotype in Human Melanoma Cells by Activating Nuclear Factor-{kappa}B." Cancer Res 67(4): 1812-1822.
Chambers, A. F., A. C. Groom, et al. (2002). "Metastasis: Dissemination and growth of cancer cells in metastatic sites." Nat Rev Cancer 2(8): 563-572.
Chen, P.-S., M.-Y. Wang, et al. (2007). "CTGF enhances the motility of breast cancer cells via an integrin-{alpha}vbeta3-ERK1/2-dependent S100A4-upregulated pathway." J Cell Sci 120(12): 2053-2065.
Cheng, X., Z. Shen, et al. (2009). "ECRG2 Regulates Cell Migration/Invasion through Urokinase-type Plasmin Activator Receptor (uPAR)/β1 Integrin Pathway." Journal of Biological Chemistry 284(45): 30897-30906.
Chiu, Y.-C., D.-C. Shieh, et al. (2009). "Involvement of AdipoR receptor in adiponectin-induced motility and {alpha}2{beta}1 integrin upregulation in human chondrosarcoma cells." Carcinogenesis 30(10): 1651-1659.
Chung, B.-H., Y.-L. Cho, et al. (2009). "Promotion of direct angiogenesis in vitro and in vivo by Puerariae flos extract via activation of MEK/ERK-,
PI3K/Akt/eNOS-, and Src/FAK-dependent pathways." Phytotherapy Research 9999(9999): n/a.
Clark, P. E. (2009). "The role of VHL in clear-cell renal cell carcinoma and its relation to targeted therapy." Kidney Int 76(9): 939-945.
Ferrara, N. "Pathways mediating VEGF-independent tumor angiogenesis."
Cytokine & Growth Factor Reviews In Press, Corrected Proof.
Goel, H. L., J. Li, et al. (2008). "Integrins in prostate cancer progression."
Endocr Relat Cancer 15(3): 657-664.
Huang, C.-Y., C.-Y. Lee, et al. (2009). "Stromal cell-derived factor-1/CXCR4 enhanced motility of human osteosarcoma cells involves MEK1/2, ERK and NF-kappaB-dependent pathways." Journal of Cellular Physiology 221(1):
204-212.
Johnstone, S. and R. M. Logan (2006). "The role of vascular endothelial growth factor (VEGF) in oral dysplasia and oral squamous cell carcinoma."
Oral Oncology 42(4): 337-342.
Liotta, L. A. (1986). "Tumor Invasion and Metastases--Role of the
Extracellular Matrix: Rhoads Memorial Award Lecture." Cancer Res 46(1):
1-7.
Mundy, G. R. (2002). "Metastasis: Metastasis to bone: causes, consequences and therapeutic opportunities." Nat Rev Cancer 2(8): 584-593.
Pikarsky, E., R. M. Porat, et al. (2004). "NF-[kappa]B functions as a tumour promoter in inflammation-associated cancer." Nature 431(7007): 461-466.
Schwartz, M. A. (2001). "Integrin signaling revisited." Trends in Cell Biology 11(12): 466-470.
Steven M. Albelda, S. A. M., David E. Elder, RoseMary Stewart, Laszlo Damjanovich, Meenhard Herlyn, and a. C. A. Buck (1990). "Integrin
Distribution in Malignant Melanoma: Association of the ß3 Subunit with Tumor Progression." CANCER RESEARCH 50.
Su, B., Y. Bu, et al. (2009). "SSeCKS/Gravin/AKAP12 inhibits cancer cell invasiveness and chemotaxis by suppressing a PKC-RAF/MEK/ERK pathway." Journal of Biological Chemistry.
Viatour, P., M.-P. Merville, et al. (2005). "Phosphorylation of NF-[kappa]B
and I[kappa]B proteins: implications in cancer and inflammation." Trends in Biochemical Sciences 30(1): 43-52.
Woodhouse, E. C., R. F. Chuaqui, et al. (1997). "General mechanisms of metastasis." Cancer 80(S8): 1529-1537.
Yoshimura, K., K. F. Meckel, et al. (2009). "Integrin {alpha}2 Mediates Selective Metastasis to the Liver." Cancer Res 69(18): 7320-7328.