研究肺癌多重抗藥性細胞株的抗藥性與轉移的機制

第一部分研究對微管束化療藥物產生抗藥性後，其抗性機制和如何反轉抗藥性在癌症的化學治療中，多重抗藥性是導致治療失敗很大的原因。因此，我們利用A549和H1299肺癌細胞株篩選出對docetaxel和vincristine具有抗藥性的細胞株。它們均具有多重抗藥性的表現型，同時我們也利用它們來研究如何反轉抗藥性的方法。所有的抗藥株都對docetaxel、vincristine和doxorubicin具有交叉抗藥性，但是只有A549/D16有ATP-binding cassette (ABC) transporter B1 (ABCB1) 的高度表達，其他抗藥細胞株則沒有，不論在RNA或蛋白層面都可觀察到。我們也進一步使用Rhodamin-123偵測ABCB1的活性，也得到同樣的結果。表示多重抗藥性有ABC transporter和非ABC transporter兩種類型的存在。此外，我們發現同時具有抑制ABCB1和阻斷L型鈣離子通道的藥物-verapamil，可以反轉抗藥細胞株的抗藥性，不論是否具有ABCB1的表現。另外兩種L型鈣離子通道阻斷劑-diltiazem和nifedipine也同樣具有反轉抗藥性的效用，而且diltiazem和nifedipine的藥效比verapamil差。我們的結果顯示除了ABCB1之外，鈣離子通道的活性在docetaxel和vincristine所引起的多重抗藥性也扮演很重要的角色。因此，抑制鈣離子通道可能是一個解決多重抗藥性的方法。我們也利用小孢子靈芝免疫調節蛋白-GMI，來提高抗藥細胞株中自由態鈣離子的濃度，且在免疫缺陷小鼠的背部種植A549/D16抗藥株，胃管餵食GMI，發現GMI會透過細胞自噬和細胞凋亡，抑制腫瘤的生長。此外，我們在細胞實驗中，利用GMI、thapsigargin和tunicamycin來研究細胞自噬所扮演的角色。Thapsigargin會因為細胞大量表現ABCB1而失去毒殺的效用，而tunicamycin的效用不會因為ABCB1表達與否，均可毒殺多重抗藥性的細胞株。我們利用Annexin V、西方點墨法和caspase的抑制劑 (Z-VAD-FMK) 來證明thapsigargin或tunicamycin所引起的細胞凋亡現象。同時也利用偵測細胞內酸性囊狀胞器、LC3-II蛋白的增加、p62蛋白的減少和使用ATG5的shRNA，來證明thapsigargin或tunicamycin所導致多重抗藥性細胞株的細胞自噬的現象。在thapsigargin和tunicamycin中，shATG5可以減少細胞自噬作用，但不會減少細胞凋亡的現象。我們發現，抗藥細胞株在處理GMI後，磷酸化Akt和磷酸化p70s6k的表現會被抑制，但與磷酸化ERK是無關的。我們的結果顯示，GMI會透過抑制Akt/mTOR的路境造成細胞自噬作用的產生，這個細胞自噬作用會導致細胞死亡，暗示GMI可能是用來解決多重抗藥性的方法。第二部分研究對愛寧達產生抗藥性後，其轉移能力的機制癌症能夠威脅生命除了因為抗藥性的產生外，另一個很大的原因，是因為癌細胞會轉移擴散所導致。在實驗室之前的研究中，我們發現Pemetrexed的抗藥株– CL1/A200相較於親代細胞株有較高的移行與侵入能力，所以我們利用Pemetrexed的抗藥株來研究轉移的機制。在我們的研究中發現Pemetrexed的抗藥株，不論是A549/A400或CL1/A200相較於親代細胞株都有較高的移行和和侵入能力，我們進一步使用動物模式，也可以發現Pemetrexed的抗藥株有較高的轉移能力。這個較佳的轉移能力是透過上皮-間質型態轉換 (The epithelial-mesenchymal transition, EMT)的方式，上皮細胞的標記基因：E-cadherin 表達降低；間質細胞的標記基因：Fibronectin表達增加所造成的。我們進一步利用ERK的抑制劑或siRNA抑制ERK的表現，發現EMT的相關轉錄因子ZEB1也會隨之減少，導致E-cadherin和Fibronectin也會跟著變動，同時Pemetrexed抗藥株的移行和侵入能力也會降低。由此可知Pemetrexed的抗藥株會透過磷酸化ERK來調控EMT的轉錄相關因子- ZEB1，進一步調控E-cadherin和Fibronectin的表現，達到促進細胞轉移的能力。此外我們也發現長春花鹼類的化療藥物可以反轉其抗藥性，在IC50的實驗中，Pemetrexed的抗藥株對Vincristine比上親代細胞有較佳的敏感性，且在動物模式的實驗中也發現Vinblastine會抑制A549/A400腫瘤的生長。我們的研究提供未來可能可以使用ERK的抑制劑或長春花鹼類的化療藥物，來治療對Pemetrexed產生抗性或有轉移現象的患者。

關鍵字

肺癌；多重抗藥性；轉移

並列摘要

part1 Multidrug resistance (MDR) of cancer cells to cytotoxic drugs significantly impedes chemotherapeutic treatment. The purpose of this study is to characterize docetaxel (DOC) or vincristine (VCR) selected A549 and H1299 non-small cell lung cancer (NSCLC) sublines that exhibit MDR phenotypes and followed by re-sensitization study. Although all drug resistant sublines showed cross-resistance to DOC, VCR, and doxorubicin (DXR), the expression of ATP-binding cassette (ABC) transporter B1 (ABCB1) gene was found to be strongly induced in DOC but not in VCR resistant A549 sublines by quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR). In DOC and VCR resistant H1299 sublines, moderate expression of ABCB1 was detected. The levels of ABCB1 protein and efflux activities were further examined by immunoblotting and rhodamin-123 staining assay. The results showed that both ABC and non-ABC mediated MDR are existed. Furthermore, verapamil (VER), an inhibitor of ABCB1 and an L-type calcium channel blocker, is capable of reversing the resistance in all drug-resistant sublines independent of ABCB1 expression. Importantly, VER only sensitizes resistant sublines but has no effect on parental cancer cells. Other L-type calcium channel blockers, such as diltiazem (DIL) and nifedipine (NIF), also sensitize MDR sublines without interfering with ABCB1 activity but with lower efficacy than VER. Our data showed that in addition to ABCB1, calcium channel activity may play a crucial role in DOC- and VCR-acquired MDR. Therefore, inhibition of calcium influx may provide a new target to modulate MDR in chemotherapy. We demonstrated that a fungal protein from Ganoderma microsporum, GMI, elevates the intracellular calcium level and reduces the growth of MDR subline via autophagy and apoptosis, regardless of p-glycoprotein (P-gp) overexpression, in mice xenograft tumors. In addition, we examined the roles of autophagy in the death of MDR A549 lung cancer sublines by GMI, thapsigargin (TG) and tunicamycin (TM) in vitro. Cytotoxicity of TG was inhibited by overexpressed P-gp. However, TM-induced death of MDR sublines was independent of P-gp level. Combinations of TG and TM with either docetaxel or vincristine showed no additional cytotoxic effects on MDR sublines. TG- and TM-mediated apoptosis of MDR sublines was demonstrated on AnnexinV assay and Western blot and repressed by pan-caspase inhibitor (Z-VAD-FMK). Treatment of MDR sublines with TG and TM also augmented autophagy with accumulation of LC3-II proteins, breakdown of p62 and formation of acidic vesicular organelles (AVOs). Inhibition of ATG5 by shRNA silencing significantly reduced autophagy and cell death but not apoptosis following TG or TM treatment. GMI treatment inhibited the phosphorylation of Akt/ S473 and p70S6K/T389. Interestingly, the phosphorylation of ERK was not associated with GMI-induced autophagy. We conclude that autophagy plays a pro-death role in acquired MDR and upregulation of autophagy by GMI via Akt/mTOR inhibition provides a potential strategy for overcoming MDR in the treatment of lung cancers. part 2 Beyond the drug resistance, matastasis is another life-threatening factor of cancer. In our previous study, the migration and invasion capacity of Pemetrexed resistant CL1 sublines are higher than their parental cells. Therefore, we investigated the metastatic mechanisms of Pemetrexed-resistant lung cancer cells. In this study, we found that A549/A400 and CL1/A200, two Pemetrexed-resistant lung cancer cells, exhibited higher migration and invasion abilities than the parental cells. Using animal model, Pemetrexed-resistant lung cancer cells have high metastatic capacity. We demonstrated that epithelial-mesenchymal transition (EMT) was associated with downregulation of E-cadherin and upregulation of fibronectin, which leaded to a higher metastatic ability in Pemetrexed-resistant lung cancer cells. The EMT phenomenon and metastatic ability of Pemetrexed-resistant cell lines were abolished by ERK inhibitor and siRNA, suggesting that ERK activation play an important role in Pemetrexed-resistant cell metastasis. Furthermore, compared with the parental cells, Pemetrexed-resistant A549 cells were more sensitive to Vincristine. Using xenograft model, Vinblastine significantly inhibited the A549/A400 tumor growth. Our results demonstrated that ERK/EMT pathway activation leads to the high metastatic ability in Pemetrexed-resistant lung cancer cells. Furthermore, Vinblastine, a clinically used anti-cancer drug, reversed the drug-resistance in Pemetrexed-resistant cells. Our finding provides a new insight into treating the Pemetrexed-resistant lung cancer cells.

並列關鍵字

lung cancer ； multidrug resistant ； metastasis

參考文獻

Baehrecke EH (2005) Autophagy: dual roles in life and death? Nature reviews 6: 505-510

Barker JM, Silvestri GA (2002) Lung cancer staging. Curr Opin Pulm Med 8: 287-293

Barr RK, Bogoyevitch MA (2001) The c-Jun N-terminal protein kinase family of mitogen-activated protein kinases (JNK MAPKs). Int J Biochem Cell Biol 33: 1047-1063

Barrallo-Gimeno A, Nieto MA (2005) The Snail genes as inducers of cell movement and survival: implications in development and cancer. Development 132: 3151-3161

Bernales S, Papa FR, Walter P (2006) Intracellular signaling by the unfolded protein response. Annual review of cell and developmental biology 22: 487-508

國際替代計量

研究肺癌多重抗藥性細胞株的抗藥性與轉移的機制

全文下載

主題瀏覽