| 摘要: | 胃癌在世界上癌症發生率高居第二位。依組織型態胃癌分為腸型 (intestinal type) 與瀰漫型 (diffuse type)。臨床上證實瀰漫型胃癌細胞會轉移至腹腔及肝臟，是造成胃癌死亡的主要原因。在先前的研究中已經利用Transwell invasion assay的篩選方式，建立具有高轉移能力的MKN45胃癌細胞亞株。與MKN45-GFP相較，MKN45-GFP-12具有較高的轉移能力，且此現象在動物模式中獲得證實；接著利用cDNA microarray及real time PCR分析MKN45-GFP及MKN45-GFP-12細胞total mRNA，結果顯示相較於 MKN45-GFP，MKN45-GFP-12 的催產素受體(Oxytocin receptor, OXTR) mRNA表現量皆下降，然而在催產素受體在胃癌轉移中所扮演的角色仍不清楚。催產素受體為G protein receptor (GPCR)的一員，GPCR是細胞膜上主要受體之ㄧ，負責細胞內訊息傳遞，進而調控細胞內多種生理功能。許多疾病，例如癌症皆因GPCR功能減弱或增強而造成。過去文獻中指出，oxytocin能透過催產素受體抑制卵巢癌細胞的遷移。而本研究的主軸在於探討催產素受體在胃癌侵襲中所扮演的角色。本實驗利用Transwell系統，發現200-400 nM oxytocin能有效的抑制 MKN45-GFP-12 侵襲能力，但不影響細胞正常的貼附、死亡與增生，顯示oxytocin透過抑制細胞的motility進而抑制了MKN45-GFP-12侵襲。oxytocin 所造成MKN45-GFP-12 侵襲能力的抑制作用可被OXTR inhibitor抑制劑Atosiban 所阻斷。這實驗結果顯示oxytocin抑制invasion 的現象是藉由活化OXTR所引起。利用Western Blot去偵測所分離 MKN45-GFP-12的質膜蛋白中，我們發現，oxytocin會抑制Rho A 與Rac 由細胞質轉移至細胞膜上的量。Over-expresison RhoA V14則恢復了由oxytocin抑制invasion 的現象。同時也發現給予oxytocin增加了Src的磷酸化，利用 Src 抑制劑PP2 抑制 Src 活性時，同時也影響了由oxytocin 所抑制的invasion。綜合以上實驗我們發現oxytocin可藉由活化cSrc來抑制RhoA 與Rac的活性，進而抑制胃癌細胞遷移。
Gastric cancer is the second most common cause of cancer death in the world. According to histological classification, gastric cancer can be divided into intestinal and diffuse type. Metastases to the liver and peritoneum are the main cause of the death of the diffuse type of gastric cancer. Using the Transwell system, Dr. Chiung-Tong Chen’s Lab has identified that MKN45-GFP-12 is a highly metastatic gastric cancer cell line. Using cDNA microarray and real time PCR, they also demonstrated a lowest expression level of oxytocin receptor in MKN45-GFP-12 among the MKN45-GFP cell sublines examined. However, the role of oxytocin receptor in gastric cancer metastases is still unknown. Oxytocin receptor is a member of G protein couple receptor (GPCR). It has been suggested that GPCRs can regulate a variety of physiological functions within the cell. Many diseases caused by enhanced or reduced the function of GPCRs. It has been demonstrated that oxytocin can inhibit ovarian cancer cell migration through an oxytocin receptor-mediated pathway. The aim of this study was to study the role of oxytocin receptor in gastric cancer cell invasion. Using the Transwell assay, we detected that treatment with 200-400 nM oxytocin significantly decreased the invaded number of MKN45-GFP-12 cells. At these treatment conditions, the cell adhesion to collagen, cell proliferation and cell death were not affected by oxytocin, suggesting that the oxytocin-induced invasion inhibition is through reducing the cell motility. The oxytocin-induced migration inhibition was prevented by treating the cell with an oxytocin receptor inhibitor, Atosiban, indicating that oxytocin-induced migration inhibition through an oxytocin receptor-mediated pathway. To delineate the signaling pathway involved in the oxytocin-induced migration inhibition in MKN45-GFP-12, the membrane translocations of RhoA and Rac-1, an indicator for RhoA and Rac-1 activity, respectively, were examined. Our data show that oxytocin inhibited translocation of Rac and RhoA from the cytoplasm to the plasma membrane, suggesting that oxytocin could suppress the RhoA and Rac-1 activity. Over-expression of a constitutively active form RhoA construct (RhoA V14) abolished oxytocin-induced migration inhibition in MKN45-GFP-12. Moreover, oxytocin treatment increased the levels of phosphorylated cSrc, and pretreatment with a cSrc inhibitor, PP2, prevented the oxytocin-induced migration inhibition in MKN45-GFP-12 cells. Taken together, our results suggest that oxytocin might activate cSrc, which in turn suppress the activities of RhoA and Rac-1, and eventually inhibiting migration in MKN45-GFP-12 cells. |
