臺灣博碩士論文加值系統

造骨蛋白(osteopontin)是一種具有RGD序列的分泌型磷蛋白, 可以調節細胞內細胞骨架的功能還有基因的表現。細胞外的造骨蛋白可以透過與不同的組合蛋白(αvβ1, αvβ3, αvβ5, α4β1, and α9β1) 和細胞表面的接受體 CD44結合影響細胞黏附功能、細胞存活和細胞移行。許多癌症會大量表現造骨蛋白，而且在不同階段的癌症扮演著不同的重要角色。在不同的腫瘤上都發現, 造骨蛋白的表現與病人的存活率有相關性，造骨蛋白越多，病人的存活率越差。根據臨床研究指出，有腫瘤轉移現象的病人其血液中造骨蛋白的含量較一般正常人高許多。在本篇我們要探討造骨蛋白在癌細胞存活、癌症治療及抗藥性中所扮演的角色及發展藥物抑制其作用。
我們的研究發現，人類攝護腺癌細胞和人類惡性骨肉瘤細胞在缺氧的環境下會使造骨蛋白的表現增加。在惡性骨肉瘤細胞中還發現在缺氧的環境下會使葡萄糖轉移蛋白表現增加。在人類攝護腺癌細胞中發現，增加的造骨蛋白會促使藥物轉移蛋白-P-醣蛋白(P-glycoprotein)表現增加。P-醣蛋白是屬於ABC轉運蛋白(ABC-binding cassette transporter)家族之一。癌細胞會大量表現ABC轉運蛋白，使各種化合物被轉運出細胞外，降低化療藥物的治療效果。在實驗中，我們使用本身帶有自體螢光的化療藥物-唐黴素(daunomycin)來觀察ABC轉運蛋白將藥物排出細胞外的活性，並且發現造骨蛋白會促使ABC轉運蛋白將唐黴素排出細胞外的能力增加。此外，我們發現長時間給予低劑量唐黴素來刺激癌細胞產生抗藥性的同時，也造成造骨蛋白的大量表現。造骨蛋白會抑制唐黴素所誘發的細胞死亡現象，而此現象會被黏著分子(integrin) αvβ3抗體給拮抗。剔除細胞內造骨蛋白表現(osteopontin knockdown)會使唐黴素的毒性效果增加，誘發細胞死亡的現象。在此實驗中我們也使用其他屬於P-醣蛋白受質的化療藥物- paclitaxel, doxorubicin, actinomycin-D 和 rapamycin，都發現有相同的現象。在動物實驗中也證實，在造骨蛋白被剔除的腫瘤組中，唐黴素抑制腫瘤生長的現象明顯增加。這些實驗結果指出造骨蛋白在癌症治療產生抗藥性中是一個重要的治療標的，可以降低癌症細胞抗藥性產生。
在惡性骨肉瘤(osteosarcoma)的研究中，我們發現內生性造骨蛋白會與黏著分子αvβ3結合進而調節葡萄糖轉運蛋白(glucose transporter)第一型和第三型的表現，並且促進葡萄糖進入細胞內。將細胞內造骨蛋白剔除(osteopontin knockdown)會誘發百分之二十的惡性骨肉瘤細胞死亡。另外使用葡萄糖轉運蛋白抑制劑-皮根素(phloretin)也有誘發惡性骨肉瘤細胞死亡的現象。將皮根素作用在造骨蛋白剔除的細胞上，其誘發細胞死亡的現象有加成的效果。同時給予化療藥物(daunomycin, 5-Fu, etoposide, methotrexate)與低劑量的皮根素，其化療藥物所產生的毒性效果有加成現象。此現象指出，葡萄糖在惡性骨肉瘤中占有重要的促進生長角色，抑制造骨蛋白的產生而減少葡萄糖轉運蛋白的功能，或是直接給予葡萄糖轉運蛋白抑制劑都會造成惡性骨肉瘤的細胞死亡。在治療惡性骨肉瘤的病患時可以併用葡萄糖轉運蛋白的抑制劑與化療藥物，增加化療藥物對惡性骨肉瘤的治療效果。
根據以上的實驗我們知道造骨蛋白會透過與黏著分子αvβ3的結合而影響癌細胞，並且在癌症治療過程中所產生的抗藥性或是癌細胞存活狀態都扮演著重要的角色。在本實驗中，我們進一步研究組合蛋白拮抗劑用於治療癌症的效果。我們使用的黏著分子拮抗劑是一種Rhodostomin 變異體-RGD相關蛋白，HSA (C34S)-ARLDDL, PEG-ARLDDL, ARLDDL和KKKRT-ARGDNP。在腫瘤的實驗中我們發現這四種黏著分子拮抗劑可以有效地抑制攝護腺癌、黑色素癌和惡性骨肉瘤腫瘤的生長。此RGD蛋白也可以與化療藥物(daunomycin)合併使用，有加成抑制腫瘤生長的效果。這些RGD蛋白也具有抑制細胞黏附作用及抑制血管新生。
有些腫瘤細胞會表現較多的αvβ3黏著分子，而且黏著分子αvβ3會影響腫瘤發展。造骨蛋白和RGD相關蛋白都會透過αvβ3黏著分子來影響腫瘤的生長、細胞黏附及細胞存活。黏著分子αvβ3會是一個很好的癌症治療標的。我們的研究中更證明了αvβ3拮抗劑併用化療藥物在某些癌症可以增加治療效果，在未來腫瘤之治療可以提供另一種新的方向及策略。

Osteopontin, known as a secreted phosphoprotein, has a functional RGD domain and acts as a regulator of cytoskeleton dynamics and gene expression. Extracellular osteopontin functions through its interaction with cell surface receptors, including various integrins (αvβ1, αvβ3, αvβ5, α4β1, and α9β1) and CD44. Binding of osteopontin to these receptors can elicit a wild range of functions, such as cell adhesion, survival, and migration. Osteopontin is overexpressed in various cancers and has a crucial role in all stages of cancer. Elevated ostoepontin has been correlated with poor survival of patients with cancer in different tumor types. Accordingly, clinical studies have shown high osteopontin plasma concentration in patients with metastatic tumors compared with normal samples. Here we investigated the role of osteopontin in drug resistance, cancer cell survival and cancer therapy.
It was found that osteopontin was upregulated in hypoxic human prostate cancer cells and osteosarcoma cells. Glucose transporters were also regulated in the hypoxia condition. Osteopontin upregulated drug transporter-p-glycoprotein expression in prostate cancer cells. P-glycoprotein is a subfamily of ATP-binding cassette transporter (ABC transporter). Cancer cells overexpressing ABC transporters actively pump out a variety of compounds from cells and decrease the therapeutic effects of chemotherapeutic agents. Using daunomycin, a chemotherapeutic agent with autofluorescence, to evaluate the ABC transporter pump activity, and osteopontin was found to increase the drug pumping-out activity. Long-term treatment with low-dose of daunomyain contributed to a drug resistance condition and further enhanced the overexpression of osteopontin. Released osteopontin inhibited daunomycin-induced cell death, which was antagonized by αvβ3 antibody. Knockdown of endogenous osteopontin potentiated the daunomycin-induced apoptosis. Furthermore, knockdown of osteopontin also enhanced the cell death caused by other chemotherapeutic drugs, including paclitaxel, doxorubicin, actinomycin-D and rapamycin, which are also the p-glycoprotein substrate. The animal studies showed that osteopontin knockdown enhanced the cytotoxic action of daunomycin. These results indicate that osteopontin is a potential therapeutic target for cancer therapy to reduce the drug resistance in sensitive tumors.
On the other hand, endogenously released osteopontin regulated the expression of glucose transporter 1 and glucose transporter 3 in osteosarcoma and enhanced glucose uptake into cells via the αvβ3 integrin. Knockdown of osteopontin induced cell death in 20% of osteosarcoma cells. Phloretin, a glucose transporter inhibitor, also caused cell death by treatment alone. The phloretin-induced cell death was significantly enhanced in osteopontin knockdown osteosarcoma cells. Combination of a low dose of phloretin and chemotherapeutic drugs, such as daunomycin, 5-Fu, etoposide, and methotrexate, exhibited synergistic cytotoxic effects in three osteosarcoma cell lines. Inhibition of glucose transporters markedly potentiated the apoptotic sensitivity of chemotherapeutic drugs in osteosarcoma. These results indicate that the combination of a low dose of a glucose transporter inhibitor with cytotoxic drugs may be beneficial for treating osteosarcoma patients.
According to the results shown above studies, we know that osteopontin plays an important role in drug resistance and influences cancer cells survival during cancer therapy via integrin αvβ3. We thus investigated the effect of integrin antagonist, Rhodostomin mutant-RGD-related proteins, HSA (C34S)-ARLDDL, PEG-ARLDDL, ARLDDL and KKKRT-ARGDNP, in cancer therapy. During treatment of these four RGD-related proteins, they can inhibit prostate cancer, melanoma, osteosarcoma tumor growth, effectively. The inhibition of tumor growth effect is more significant when combination RGD-protein combined with chemotherapy drug of daunomycin. The RGD-proteins also can inhibit cancer cell adhesion and angiogenesis.
αvβ3 integrin is highly expressed in some tumor cells and involved in tumor progression. Osteopontin and RGD-related proteins influence tumor growth, cell adhesion, survival via αvβ3 integrins. αvβ3 integrin can be a good target for cancer therapy. Our studies demonstrate that inhibition of αvβ3 integrin with RGD-related proteins or in combination with chemotherapy improve the anticancer efficacy. This may be a new strategy for cancer therapy.

口試委員審定書
誌謝

Abbreviations…………………………………………………………………… 6
Abstract in Chinese……………………………………………………………... 9
Abstract in English……………………………………………………………... 12

Chapter 1. Introduction………………………………………………………. 16
1-1 Hypoxia regulates cancer environment…………………………………….. 17
1-2 Role of osteopontin in cancer………………………………………………. 26
1-3 Role of drug transporters in cancer………………………………………… 36
1-4 Role of glucose transporters in cancer……………………………………... 45
1-5 Role of integrins in cancer………………………………………………….. 52

Chapter 2. Materials and Methods…………………………………………... 59

Chapter 3. Upregulation of drug transporter expression by osteopontin in prostate cancer cells........ 71

Chapter 4. Osteopontin upregulates the expression of glucose transporters in osteosarcoma.... 98

Chapter 5. Inhibition of tumor growth by selective αvβ3 integrin antagonists... 118

Chapter 6. Conclusion and Perspective……………………………………… 136

Reference………………………………………………………………………. 140

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