本研究主要想證實生長因子受體的訊息傳遞調如何促進子宮頸癌生長和侵襲，以及證實electroneutral K-Cl cotransport (簡稱KCC) 於子宮頸癌中所扮演的角色。我們首先證實了第一型類胰島素生長因子 (insulin like growth factor-1，簡稱IGF-1) 於50 ng/ml的濃度下可以顯著促進子宮頸癌細胞的侵襲與增生的能力，此促進效果可以被IGF-1 receptor (簡稱IGF-1R) 以及alphav beta3 integrin的抗體阻斷。從time course以及細胞中Src homorgy 2-containing phosphotyrosine phosphotase (簡稱SHP-2) 表現的結果證實其訊息傳遞路線是受到alphav beta3 integrin經由調節SHP-2傳送到IGF-1R的速率而影響IGF-1R的活化，接著影響了IGF-1R下游的訊息傳遞分子，包括IRS-2 (insulin receptor substrate 2)，phosphatidylinositol-3 kinase (簡稱PI3K) 的p85 subunit以及Akt的活化，當我們利用PI3K的抑制劑 (LY294002與wortamanin) 可發現有劑量依賴性的抑制子宮頸癌細胞的侵襲與增生，證實IGF-1R會經由活化PI3K/Akt訊息傳遞路徑而調控子宮頸癌細胞的侵襲與增生能力。在in vivo 的實驗中，我們將腫瘤細胞打入SCID老鼠皮下，待腫瘤形成後，固定時間投予IGF-1R 與alphav beta3 integrin的抗體，實驗結果顯示，投予IGF-1R的抗體能有效的抑制腫瘤的成長，證實了IGF-1R於子宮頸癌的癌化過程中扮演著相當重要的調控角色。我們又更進一步從臨床取得71個早期子宮頸癌病患的組織切片，由染色實驗的結果發現，隨著侵襲或蔓延情形的增加，IGF-1R的表現也會隨之大量表現，其病患的預後相對較差，因此或許可以藉由利用阻斷 alphav beta3 integrin與IGF-1R 的訊息傳遞路線的方式以達到有效治療子宮頸癌的效果。於實驗室之前的研究發現，KCC3不單只有扮演體積調節的功能，還具有促進子宮頸癌細胞生長的功能，因此很有興趣想要了解在IGF-1所促進的子宮頸癌化過程中，KCC所扮演的角色。由實驗結果證實，IGF-1具有劑量依賴性與時間依賴性的促進KCC活性和mRNA 的表現量進而促使細胞調節體積減少(簡稱RVD)能力的增加。IGF-1刺激之下會使得phosphatidylinositol 3 kinase (PI3K) 和 mitogen-activated protein kinase (MAPK) 啟動進而分別誘導Akt 和extracellular signal-regulated kinase1/2 (Erk1/2)的活化作用。活化的Erk1/2 MAPK和PI3K信號路徑具有調節KCC polypeptides生合成的程度上差異。利用SiRNA的方式專一性地減少Erk1/2蛋白質表現量會導致IGF-1所促進的KCC活性會受到抑制。利用藥理學抑制劑和基因修飾的方式改變KCC活性的實驗顯示，KCC於IGF-1所誘導的癌細胞侵襲與擴散的過程扮演必需的角色。在臨床組織切片的染色中發現IGF-1和KCC的表現在子宮頸癌 (n=28) 的樣本中均表現在相同位置，證實IGF-1可能透過autocrine或paracrine 的方式促進KCC的表現。我們的結果顯示出IGF-1的確可以促進KCC的活化作用，並且證實KCC於IGF-1訊息傳遞路徑中扮演著促進子宮頸癌生長和侵襲的重要角色。

　　This study is aimed to identify the specific growth factors which are critically involved in cervical cancer cell invasion and proliferation, and test the hypothesis that adhesion receptors of integrin family and growth factor receptor signaling may cooperate functionally to promote cancer invasion and proliferation. The results demonstrate that insulin like growth factor-1 (IGF-1) is a potent stimulator of cervical cancer cell invasion and proliferation. The IGF-1-stimulating effects are completely inhibited by antagonistic antibody against IGF-1 receptor (IGF-1R), whereas the effects was unaffected by either IgG or blocking antibody to insulin receptor (IR). Blocking ligand occupancy of alphav beta3, but not alpha2, alpha3, alpha4, alpha6, beta1, beta4 or alpha2 beta1 integrin, result in attenuation of cervical cancer cell response to IGF-1 stimulation. The immunofluorescent stains of surgical specimens show that cervical cancer cell with a strong tendency to invade or metastasize have higher expression of IGF-1/IGF-1R than those with a low ability to do so. Furthermore, IGF-1R overexpression correlates with poor clinical outcome in early stage of cervical cancer. Blockade of alphav beta3 integrin and IGF-1R signalings may provide novel strategies for the treatment of invasive phenotypes of cervical cancer. The mechanisms by which insulin-like growth factor 1 (IGF-1) cooperates with　membrane ion transport system to modulate epithelial cell motility and proliferation remain poorly understood. Here, we investigated the role of electroneutral K-Clcotransport (KCC), in IGF-1-dependent invasiveness and proliferation of cervical cancer cells. IGF-1 increased KCC activity and mRNA expression in a dose-and time-dependent manner in parallel with the enhancement of regulatory volume decrease. IGF-1 treatment triggers phosphatidylinositol 3-kinase (PI3K) and　mitogen-activated protein kinase (MAPK) cascades leading to the activation of Akt and　extracellular signal-regulated kinase1/2 (Erk1/2), respectively. The activated Erk1/2MAPK and PI3K signaling pathways are differentially required for IGF-1-stimulated biosynthesis of KCC polypeptides. IGF-1 stimulated cellular invasiveness and proliferation are abolished by the KCC inhibitor, 50 mM DIOA. It demonstrates that KCC is necessary for IGF-1-induced cancer cell invasiveness and proliferation. IGF-1 and KCC colocalize in the surgical specimens of cervical cancer (n=28), suggesting autocrine or paracrine IGF-1 stimulation of KCC production. Taken together, our results indicate that KCC activation by IGF-1 plays an important role in IGF-1 signaling to promote growth and invasion of cervical cancer cells.

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