探討人類骨橋蛋白在胰臟癌細胞對藥物轉運蛋白的調控作用

Abstract

胰臟癌多起源於胰管細胞的突變，而胰臟癌現今仍是最難治癒的癌症之一，比起其他癌症，現今科學對於它的了解也十分有限。在治療上，傳統化療藥物對於疾病進程的改善並無太大幫助，也難以抵擋其轉移至其他器官的能力。臨床統計，在患有胰臟癌的病人當中，只有10~15% 的病人可以進行病灶切除，但經過病灶切除的病人之中只有20% 可以有5年存活，故罹患胰臟癌的病人其預後極差。 由於胰臟癌初期難以察覺，所以發現得到胰臟癌通常為時已晚，若癌細胞已經轉移或評估難以手術切除，此時臨床上多半採用合併gemicitabine和其他化療藥物或只能以支持療法試圖使病人延長生命、減輕痛苦。但是，化療的成果有限，又因為胰臟癌的高度轉移性導致至今沒有一種化療藥物可以完全治癒胰臟癌，而支持療法所使用的藥物則必須審慎使用避免造成病人身體的負擔。即使合併多種療法，現今仍沒有出現可以明顯延長胰臟癌病患壽命的方式，所以人們尋求新的解答，將眼光放在探討胰臟癌的發生機轉，以及從分子生物學的角度切入進而開發新的標靶藥物試圖突破困境。 整體而言，癌症病人除了先天對化療藥物具有抗藥性外，若治療初期有成效，但最終仍產生抗藥性的原因大多是因為多重抗藥性的產生，而這跟ATP-binding cassette (ABC)藥物轉運蛋白有關。這些藥物轉運蛋白位於細胞膜，具有抗藥性的細胞會大量表現這些轉運蛋白，主動將藥物排出細胞，藉以降低化療藥物在胞內的累積濃度，影響治療效果。 人類骨橋蛋白，或稱骨調素 (osteopontin)，是一種磷酸化的醣蛋白，文獻指出有些癌症會過度表現人類骨橋蛋白，並且其表現可能和癌症進程與分期有關，包含膀胱癌、大腸癌、腎臟癌、咽喉癌、口腔癌及唾液腺癌。此外，至今研究認為人類骨橋蛋白雖難以作為癌症篩檢及確診的標記，但卻可作為癌症預後指標。 不過對於人類骨橋蛋白和藥物轉運蛋白間的關係令人好奇，但卻甚少研究可以幫助釐清它們之間調控的機轉。因此本篇研究著力於探討在胰臟癌中人類骨橋蛋白對於藥物轉運蛋白的調控，以及探討是否有新的治療方式可以有效降低癌細胞存活率，並探討其中可能的機轉。研究發現外給人類骨橋蛋白在人類胰臟癌細胞(MIA PaCa-2)確實可以使藥物轉運蛋白---ABCB1的mRNA和蛋白量表現增加。接著我們更加確認內生性人類骨橋蛋白在胰臟癌細胞中的地位，我們轉殖一小段具有專一性拮抗人類骨橋蛋白mRNA的shRNA後，發現隨著人類骨橋蛋白的減少，細胞ABCB1的表現量也隨之下降，並且細胞對於化療藥物doxorubicin的感受性增加，使得細胞死亡率顯著上升。此外，在本篇研究中亦發現合併使用αvβ3 integrin 的專一拮抗藥物HSA(C34S)-ARLDDL可以增強doxorubicin毒殺癌細胞的能力。另一方面，若是將轉殖人類骨橋蛋白shRNA的胰臟癌細胞重新給予人類骨橋蛋白，再以化療藥物毒殺，可以發現胰臟癌細胞的存活率提升。此外，為了模擬癌細胞長期接受化療藥物後產生的抗藥性現象，我們用doxorubicin治療癌細胞兩天後，發現藥物轉運蛋白在胰臟癌細胞中的表現確實明顯上升，而且人類骨橋蛋白在細胞中的表現量也顯著變多。已有研究指出ABCB1的增加與PI3K/ Akt調控有關。我們外給人類骨橋蛋白在胰臟癌細胞，確實發現可以增加FAK和Akt的磷酸化，因此，人類骨橋蛋白可能透過FAK/Akt路徑調控ABCB1的表現量。 總而言之，本篇研究證實人類骨橋蛋白可能藉由αvβ3 integrin在胰臟癌細胞降低對於化療藥物的感受性，並且合併使用disintegrin和doxorubicin可以為治療胰臟癌提供一個新的治療策略。

Pancreatic ductal adenocarcinoma remains a complicated and unsolved issue with conventional cancer treatments having little impact on its disease course. The fact that it is prone to metastasis makes the disease prognosis extremely poor. Survival is dependent on tumor burden and performance status at presentation. Only around 10~15% of patients may undergo resectable surgery, and 20% of them survive to 5 years. For locally advanced, unresectable, and metastatic lesion, treatment is palliative, or to combine gemcitabine-based chemotherapy. However, chemotherapy is never curative to date for metastatic disease due to its drug resistance to currently available treatments, and its potential palliative benefit must be carefully weighed against toxic effects. In addition, no randomized phase III trial has yet established a survival benefit for combination therapy. Therefore, new therapeutic strategies based on the molecular biology of pancreatic cancer seem to hold the greatest promise. Generally, in cancer patients who do not respond to chemotherapy, multidrug resistance (MDR) is usually mediated by the overexpression of various ATP-binding cassette (ABC) family of drug transporters located on the cytoplasmic side of the membrane, resulting in increased drug efflux from the resistant cell. Several studies using a broad range of tumor cell lines and chemotherapeutic agents have found that cellular transport proteins of the ABC family are involved in the penetration of drugs through the cell membrane and their intracellular accumulation to effective concentrations. Osteopontin (OPN), a phosphorylated glycoprotein, overexpresses in several cancers and is found to correlate significantly overall with tumor stage, including bladder, colon, kidney, larynx, mouth and salivary gland. In further studies to date, they support the hypothesis that osteopontin detected in tumor cells has a potential utility as a prognostic marker. Nevertheless, the relationship between osteopontin and drug transporters has not been well-understood. Therefore, in this study, we investigated the effect of osteopontin on the upregulation of drug transporters in pancreatic cancer, and found that treatment of osteopontin significantly upregulated the mRNA expression and protein level of drug transporter---ABCB1 in MIA PaCa-2 cells. We then further confirmed the important role of endogenous osteopontin in contribution to chemoresitance in pancreatic cancer. ABCB1 expression in pancreatic cancer cell was decreased by the knockdown of endogenous osteopontin. On the other hand, the cytotoxicity by doxorubicin was enhanced by the knockdown of osteopontin. HSA(C34S)-ARLDDL, an αvβ3 integrin antagonist, could potentiate cytotoxicity of doxorubicin, etiher at 0.5 μM or at 1 μM. In addition, we also found that osteopontin re-administration would attenuate the cytotoxicity caused by doxorubicin in MIA PaCa-2 cells. In addition, clinically long-term treatment of chemodrugs in patients with cancer would develop multidrug resistance eventually, and this was the common reason that treatment failed in vain. In our study, we treated the pancreatic cancer cells with doxorubicin for two days and verified this consequence. The data showed that long-term treatment of doxorubicin would upregulate the drug transporter and osteopontin expression. It is well-known that PI3K/ Akt pathway mediates ABCB1 expression. Further, we applied osteopontin to MIA PaCa-2 cells and found that osteopontin could increase the phosphorylation level of FAK and Akt. In conclusion, the result suggested that osteopontin played an important role in the regulation of drug sensitivity through αvβ3 integrin in pancreatic cancer cells, and chemotherapeutic drug in combination with disintegrin as a target therapy might be a promising treatment strategy in pancreatic cancer.