探討缺氧環境下osteopontin對結腸直腸癌細胞的調節作用

Abstract

人類結腸直腸癌是全球第三常見的癌症，而且在癌症中的死亡率排名全球第四位，根據統計每年大約有一百萬病人罹患大腸癌，而大約有五十萬位大腸癌病人病逝，手術治療是目前最主要治療大腸癌的方式，而且對於早期的大腸癌病患有較好的治療效果。骨調素(osteopontin)是一種含有RGD序列的分泌性磷酸化醣蛋白，骨調素會大量表現在許多不同種類的癌症上，而大量的骨調素表現通常會伴隨著腫瘤惡化的情形發生。有許多的文獻指出骨調素在癌細胞中扮演了很重要的角色，包括抑制免疫細胞的功能、增加細胞的存活、細胞的增生、細胞遷移、細胞侵入與血管新生，癌症病人中骨調素的表現量增加會降低病患的存活率。在許多的人類腫瘤中會有缺氧的情形發生，而細胞會透過缺氧誘導轉錄因子(HIF)去克服缺氧的環境。除此之外，在缺氧環境下骨調素的表現會上升，且骨調素被認為具有作為腫瘤缺氧環境下指標的潛力。 在本篇的研究中我們發現在給予一種誘發化學性缺氧的化合物氯化鈷(CoCl2)之後，人類大腸癌細胞HT-29的骨調素蛋白表現量會有所增加，接著我們探討骨調素在HT-29細胞處於缺氧環境下所扮演的角色，發現到外給骨調素會促進缺氧誘導轉錄因子的蛋白表現，但卻不會影響缺氧誘導轉錄因子mRNA的表現。除此之外，我們利用西方點墨法分析核內蛋白證實外給骨調素會促進缺氧誘導轉錄因子轉移到細胞核內。此外，我們也發現外加骨調素增加缺氧誘導轉錄因子的增加現象會被αvβ5以及CD44抗體所拮抗。 我們接著探討骨調素增加缺氧誘導轉錄因子表現的訊息傳遞機制，我們發現骨調素增加缺氧誘導轉錄因子表現的作用會被PF573228 (FAK抑制劑)、LY294002 (PI3K抑制劑)及PD98059 (MEK抑制劑)所拮抗，進一步我們也發現外給骨調素會增加細胞中FAK、Akt及ERK的磷酸化。當我們轉殖一段具有專一性拮抗骨調素mRNA的RNA使細胞內骨調素的表現量降低，進而發現骨調素表現量缺乏的HT-29細胞在氯化鈷(CoCl2)的刺激之下和正常細胞比較會表現較少的缺氧誘導轉錄因子蛋白。 我們更進一步地去探討骨調素在缺氧環境下對於葡萄糖轉運子(GLUT)表現的影響，在本篇的研究結果顯示骨調素在氯化鈷的作用下會促進葡萄糖轉運子三(GLUT3)的mRNA及蛋白的表現。然而，骨調素並不會影響葡萄糖轉運子一、二及四的表現。我們還探討了骨調素在活體中所扮演的角色，但是我們發現骨調素表現量缺乏的HT-29細胞和正常細胞對於腫瘤的生長並沒有明顯的影響。 總結本篇的研究結果，我們發現在人類大腸癌細胞給予氯化鈷的刺激會增加骨調素的表現，而骨調素則會促進缺氧誘導轉錄因子蛋白的表現和轉移到細胞核，這個現象會被αvβ5 and CD44的抗體所拮抗。此外我們也證實了人類大腸癌細胞在缺氧的環境下骨調素與缺氧誘導轉錄因子之間的關係，骨調素可能會藉由αvβ5 integrin、CD44 receptor、FAK、PI3K/Akt和ERK這些訊息傳遞路徑去調控缺氧誘導轉錄因子的表現。骨調素無論是在生物體外或活體內都會促進葡萄糖轉運子三的表現，但是抑制骨調素的表現卻對於腫瘤的生長沒有影響。

Human colorectal carcinoma is the third most common cancer and the fourth most cause cancer death all over the world. It is estimated that more than 1,000,000 new cases of colon cancer develop each year, and around 500,000 patients die. Surgery is the primary form of therapy for this disease, and it is beneficial to cure in early-stage CRC. Osteopontin (OPN), a secreted RGD-containing phosphoglycoprotein, is abundantly expressed in a number of cancers and concordant with tumor stage. Previous studies have indicated that OPN in cancer cells plays an important role in the inhibition of immune cell function and increase of cell survival, cell proliferation, migration, invasion and angiogenesis. Increase of OPN in cancer patients is associated with poor survival rate. Hypoxia occurs in most solid human tumors, and cells respond to hypoxia is through hypoxia-inducible transcription factor 1 (HIF-1). In addition, OPN is up-regulated in hypoxia condition and considered to be a potential marker of tumor hypoxia. In the present study, we found that the protein expression of osteopontin was elevated by CoCl2 treatment in HT-29 human colorectal adenocarcinoma cells. We thus further investigated the role of osteopontin in HT-29 cells under hypoxia. Exogenous application of osteopontin enhanced HIF-1α protein expression under CoCl2 treatment, whereas, the mRNA expression of HIF-1α was not affected by the application of osteopontin (3 ng/ml). In addition, it was found that exogenous application of osteopontin enhanced HIF-1α nuclear translocation using Western blotting analysis of nuclear extract. Furthermore, increase of HIF-1α by exogenous osteopontin was antagonized by the co-treatment with αvβ5 and CD44 monoclonal antibody. We then investigated the signaling mechanism by which osteopontin increased CoCl2-induced HIF-1α protein expression. Our data showed that HIF-1α protein expression was inhibited by the pretreatment of PF573228 (an inhibitor of FAK), LY294002 (an inhibitor of PI3K/Akt) and PD98059 (an inhibitor of MEK).Furthermore, exogenous application of osteopontin increased the phosphorylation levels of FAK, Akt and ERK. We further transfected with the specific osteopontin small hairpin RNA (shRNA) in HT-29 cells, and found that osteopontin knockdown shRNA caused a significant reduction of HIF-1α protein expression under CoCl2 treatment. We further examined the effect of osteopontin on glucose transporter (GLUT) expression under hypoxic condition. It was shown that glucose transporter 3 (GLUT3) mRNA and protein expression was increased by osteopontin under CoCl2 treatment. However, it exerted no effect on GLUT1, GLUT2 and GLUT4 expression. We finally explored the role of osteopontin in tumor growth. However, knockdown of osteopontin did not affect the growth of tumor. Whether osteopontin knockdown affects tumor metastasis needs further investigation. In conclusion, elevation of osteopontin after CoCl2 treatment enhanced HIF-1α protein expression and accumulation in nucleus, which could be antagonized by pretreatment of αvβ5 and CD44 antibody. In addition, the signaling pathways between osteopontin and HIF-1α in HT-29 cells under hypoxia were demonstrated. The regulation of HIF-1α protein expression by osteopontin may act through αvβ5 integrin, CD44 receptor, FAK, PI3K/Akt, and ERK signaling pathways. Moreover, osteopontin enhanced GLUT3 expression both in vitro and in vivo. However, knockdown of osteopontin in HT-29 did not influence the growth of tumor.