攝護腺癌細胞在缺氧環境下調節osteopontin表現之研究

Regulation of osteopontin expression by hypoxia in prostate cancer cells

黃韋勳(Wei-Hsun Huang)

指導教授 ： 符文美

國立臺灣大學/醫學院/藥理學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.00050

摘要

癌症的轉移是造成病情惡化以及導致治療失敗的主要原因，而癌細胞進行轉移首先會侵犯周圍的組織，之後再進一步的跟隨循環系統到達新的組織，這步驟之中是由許多調控因子和一連串複雜的訊息傳遞所組成的。攝護腺癌是造成男性跟癌症相關死亡排名的第二位，因攝護腺癌而死亡的病人有超過85%的有伴隨著骨頭轉移的情形。骨調素(Osteopontin) 是一種高度磷酸化和醣化的分泌型蛋白質，參與很多不同的生理功能，在先前的研究中也指出骨調素在癌症的侵入以及轉移中扮演了重要的角色。如果在癌症病人體內的骨調素有不正常增加表現的情形發生時，往往代表病人有較差的預後以及較低的存活率。而癌症的微環境常因為癌細胞的過度增生而呈現缺氧的情形，癌細胞在此環境之中會增加其侵入性以及轉移的能力。在本篇的研究當中發現在缺氧環境下或給予一種誘發化學性缺氧的化合物CoCl2，攝護腺癌細胞PC-3的骨調素mRNA以及蛋白表現有增加的情形，並且透過免疫螢光染色的方法也觀察到在缺氧環境下攝護腺癌細胞膜的周圍以及細胞質當中增加了骨調素的表現，之後更進一步的去檢視究竟增加骨調素的表現會不會影響細胞遷移(migration)的能力，研究結果顯示在缺氧環境之下會增加PC-3細胞遷移的能力，而這增加的情形會在減少骨調素基因表現的PC-3細胞中同樣的有被抑制。先前的研究中有提到骨調素影響癌細胞的遷移是透過其受體αvβ3 integrin，因此在我們的研究中利用αvβ3 integrin的抗體有顯著的抑制缺氧環境所促進癌細胞遷移的情形，這樣的結果指出αvβ3 integrin參與了攝護腺癌細胞的遷移行為。此外我們更進一步利用從Rhodostomin之中改變RGD domain所獲得的衍生物，並且結合了C34S-HSA來避免HSA蛋白發生dimmer情形的αvβ3-selective disintegrin HSA(C34S)-ARLDDL，發現此disintegrin有抑制癌細胞缺氧下所促進的遷移以及侵入(invasion)的情形。這些結果顯示αvβ3在缺氧下促進遷移和侵入行為中扮演著重要的角色。然而無論是αvβ3的抗體或是disintegrin都無法完全的抑制缺氧環境下所促進癌細胞遷移的能力，因此可能有其他的調控因子參與，而在先前的研究中指出趨化素受體(chemokine receptor)也參與了癌症的轉移，因此檢視了並發現在缺氧環境之下CXCR4蛋白表現有增加，並且這增加的CXCR4的表現會更進一步的促進攝護腺癌細胞遷移至其配體SDF-1α，並且在缺氧環境之下給與CXCR4抑制劑AMD3100也同樣的抑制了其遷移的能力。
　　總結本篇的研究發現在缺氧的環境之下有增加攝護腺癌細胞的骨調素表現並且參與了缺氧下所促進遷移的能力，並且更進一步的提供了disintegrin中帶有RGD-containing或是類似物可以用來做為癌症治療的候選藥物。

關鍵字

osteopontin ； hypoxia ； prostate cancer ； integrin ； disintegrin

並列摘要

Tumor invasion and metastasis consist of multiple and complicated processes, which are the main cause of therapeutic failure and death for cancer patients. Prostate cancer is the second leading cause of cancer-related deaths in males. More than 85% of those who die of prostate carcinoma have bone metastases. Osteopontin (OPN), a secreted glycosylated phosphoprotein, is abundantly expressed in bone matrix and is involved in numerous physiological functions and associated with the late stage of various cancers. OPN plays an important role in metastatic and invasive ability of tumor cells. Increase of osteopontin expression in cancer patients is associated with poor outcome and low survival rate. Hypoxia is also an important factor to decrease the effectiveness of conventional chemotherapy and radiotherapy. In the present study, it was found that the mRNA and protein expression levels of osteopontin were upregulated by hypoxia or CoCl2 treatment. Immunofluorescence analysis also indicated that OPN expression was upregulated around the cell membrane and the cytoplasm after exposure to hypoxia. ROS was involved in the hypoxia-induced OPN expression. We thus further examined whether OPN influenced the migration ability of PC-3 cells. It was found that hypoxia treatment increased the migration of PC-3 cells and osteopontin knockdown antagonized the hypoxia-induced migration activity. Previous reports have shown that osteopontin affects cell migration through its receptor αvβ3 integrin. In our study, it was found that αvβ3 integrin antibody markedly inhibited hypoxia-induced tumor cell migration, indicating the involvement of αvβ3 integrin in hypoxia-induced migration activity. In addition, we used an αvβ3-selective disintegrin HSA(C34S)-ARLDDL, which is derived from the mutation of neighborhood of RGD domain of Rhodostomin and is modified to prevent HSA protein dimmer formation by conjugation with C34S-HSA. It was found that HSA(C34S)-ARLDDL also antagonized hypoxia-induced migration and invasion activity. The results indicate that αvβ3 plays an important role in hypoxia-induced migration and invasion. Since αvβ3 integrin monoclonal antibody or disintegrin cannot completely inhibit hypoxia-induced cancer cell migration, other factors may be involved in hypoxia-induced action. It has been previously shown that chemokine receptors participate in tumor metastasis. We thus further examined the chemokine receptor expression in hypoxia condition and found that CXCR4 was upregulated by hypoxia treatment. It was also found that hypoxia-induced CXCR4 expression increased the migration activity toward its ligand SDF-1α and CXCR4 inhibitor AMD3100 antagonized hypoxia-induced migration activities.
In conclusion, we found that osteopontin was upregulated by hypoxia treatment and was involved in hypoxia-induced migration of prostate cancer cell line. Moreover, we also provide the evidence that RGD-containing or mimetics of disintegrin is a potential therapeutic drug candidate for cancer treatment.

並列關鍵字

osteopontin ； hypoxia ； prostate cancer ； integrin ； disintegrin

參考文獻

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