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

• Main Authors: Pei-Ling, 彭佩鈴
• Other Authors: Fen-Pi Chou
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/29145743686167986707

博士 === 中山醫學大學 === 生化暨生物科技研究所 === 96 === 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.