黃連素抑制肺癌轉移與合併放射線療效之研究

The study of berberine in anti-metastasis and synergistic effect of radiotherapy on lung cancer

彭佩鈴(Pei-Ling Peng)

指導教授 ： 周芬碧

中山醫學大學/醫學院/生化暨生物科技研究所/博士(2008年)

https://doi.org/10.6834/CSMU.2008.00031

摘要

黃連素，是一種在許多中草藥都可以萃取出的生物鹼，目前的研究指出，它有許多抑菌及抗發炎的藥理作用。癌細胞的轉移往往是造成癌症病人復發死亡及不易根治的主要原因，其過程包括細胞外基質的降解及細胞轉移能力的改變。另一方面，放射線是目前臨床上肺癌治療的主要策略之一，然而受限於正常組織對放射線劑量的耐受性限制，以及癌細胞對放射線的敏感性程度不同，因而決定了治療是否成功的主要因素。在本篇論文中證實，黃連素在對正常肺臟細胞沒有毒性的濃度下，可顯著抑制具有高度轉移能力之人類肺癌細胞(A549)的侵襲及移動能力，利用gelatin-及casein-zymography分析方法，探討細胞外基質降解相關基因，matrix metalloproteinase-2 (MMP-2)及urokinase-type plasminogen activator (u-PA)的活性表現，結果發現berberine明顯地抑制MMP-2與u-PA的分泌量，並呈現濃度的效應關係；此外berberine也可以促進A549細胞tissue inhibitor matrix metalloproteinase-2 (TIMP-2)與plasminogen activator inhibitor-1 (PAI-1)之蛋白表現，進一步利用reverse transcription polymerase chain reaction (RT-PCR)，顯示其MMP-2、u-PA、TIMP-2與PAI-1的mRNA表現變化與蛋白表現量一致，顯示berberine對這些水解酶相關基因是調控是在轉錄的層次上；在作用機轉的探討上，結果發現berberine可顯著抑制p-Akt、c-Jun、c-fos與NF-κB的表達，進而抑制AP-1和NF-κB與DNA的結合能力，實驗中利用Akt的抑制劑LY294002處理時，發現其抑制A549細胞的MMP-2與u-PA的活性表現，同時間也抑制了細胞移動及侵襲的能力。另一部份，在合併放射線療效的實驗，結果顯示berberine會促進放射線毒殺肺癌細胞的能力；在動物實驗中，利用C57BL/6老鼠在皮下種植Lewis lung carcinoma，發現當老鼠給予berberine合併放射線時會明顯地抑制腫瘤大小；在細胞實驗中單獨處理放射線時對A549細胞的毒殺能力為SF2 = 0.423; D0 = 5.29 Gy，當合併berberine 5、10μM時則分別增加為SF2 =0.215: D0 = 2.70 Gy及SF2 = 0.099: D0 = 1.24 Gy。由電子顯微鏡觀察到合併處理的細胞出現大量的autophagosome，且acridine orange stain陽性的細胞數明顯地增加；經合併處理後細胞內autophagic protein: LC3-II及Beclin 1的表現量增加，細胞自噬的負調控者: p-mTOR， p-Akt及Bcl-2則表現量下降。在活體實驗中，腫瘤組織的免疫染色顯示Beclin 1及Bcl-2的表現也呈現相同於細胞實驗的結果，綜合以上結論，證實berberine增加放射線抑制肺癌癌瘤細胞的生長應是透過促進肺癌細胞自噬性機制死亡的結果。期望本論文結果可應用於肺癌的放射線臨床輔助治療。

關鍵字

黃連素 ； 轉移 ； 肺癌 ； 自噬性死亡 ； 放射線敏感性

並列摘要

Berberine is an isoquinoline derivative alkaloid isolated from many medicinal herbs. It is widely used in traditional Chinese medicine for the treatment of inflammation diseases and anti-microbial activities. Metastasis, a major cause of cancer death and the target of various treatment strategies, is a multi-step process involving cell proteolytic degradation of the extracellular matrix that is essential for achieving cell motility. Additional, ionizing radiation (IR) is one of the most commonly used and efficacious strategies for lung cancer therapies. However, there are some limitations in the clinical efficacy of radiotherapy, such as normal tissue tolerance and inherent tumor radioresistance, which could hinder successful outcome. In this study, we demonstrated that berberine exerted an inhibitory effect on the motility and invasion ability of a highly metastatic A549 cells under non-cytotoxic concentrations. A549 cells were treated with various concentrations of berberine for set periods, and then subjected to gelatin zymography, casein zymography, and Western blot to investigate the expression of matrix metalloproteinase-2 (MMP-2), urokinase-type plasminogen activator (u-PA), tissue inhibitor matrix metalloproteinase -2 (TIMP-2), and plasminogen activator inhibitor-1 (PAI-1). Berberine was found to decrease the expression of MMP-2 and u-PA in a concentration-dependent manner and enhance the expression of TIMP-2 and PAI-1. Further analysis with semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed that these alternations were all on the transcriptional levels. Berberine treatment was also resulted in an inhibition on the activation of p-Akt, and the DNA binding activities of c-Jun, c-Fos, and NF-κB. The treatment of p-Akt inhibitor, LY294002, decreased MMP-2 and u-PA activities and also invasion, migration ability of A549. In the radiation synergistic effect study, we demonstrated the radiosensitizing effect of berberine in both in vitro and in vivo lung cancer model. The possible therapeutic application of berberine to enhance radiotherapy was demonstrated by the LLC model in mice, showing that berberine combined with IR resulted in a substantial shrinkage of tumor. In in vitro experiments, compared with radiation alone (SF2 = 0.423; D0 = 5.29 Gy), berberine at 5.0 and 10μM concentrations in combination with radiation showed significant enhancement on radiation-induced clonogenic inhibition (SF2 =0.215: D0 = 2.70 Gy and SF2 = 0.099: D0 = 1.24 Gy) on A549 cells. The cellular ultrastructure showed the presence of autophagosome and an increased proportion of acridine orange stain–positive cells, demonstrating that berberine enhanced radiosensitivity via autophagy. The process involved LC3 modification, an increase in beclin-1 expression and down-regulation of p-mTOR, p-Akt, bcl-2. Furthermore, we performed immunohistochemistry to detect the level of beclin-1 and bcl-2 proteins in the xenograft. The results showed a remarkable increase in beclin-1 protein expression in the cotreatment groups. Besides, bcl-2 protein expression was reduced in berberine alone and cotreatment groups. Taken together, these observations demonstrated that autophagy was involved in the cotreatment-induced tumor elimination. Consequently, berberine was able to reinforce the killing effect of irradiation in local tumor growth. The results of this study provide evidence to support using natural compounds to reinforce the cell-killing effect of radiotherapy in human lung cancer cells via non-apoptotic cell death, and berberine could be used as adjuvant therapy to treat lung cancer.

並列關鍵字

berberine ； metastasis ； lung cancer ； autophagic cell death ； radiosensitivity

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