Department of Oncology, Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine, Shanghai 200062, China.
Chin J Integr Med. 2011 Jul;17(7):517-24. doi: 10.1007/s11655-011-0785-0. Epub 2011 Jul 3.
To investigate the in vitro effects and the primary mechanisms of Changweiqing (, CWQ) on antimetastasis and antiinvasion of hypoxic colon carcinoma cells. In addition, to provide experimental evidence for the Chinese medicinal theory of "strengthening the body's resistance to eliminate pathogenic factors" in the treatment of colorectal cancer, including its invasion and metastasis.
First, CWQ sera were prepared with serum-pharmacology methods. Then, the modified hypoxic chamber was designed and flushed with 5% CO(2) and 95% N(2) at 37 °C to induce a hypoxic environment. The effect of CWQ serum on the viability of LoVo cells was tested with MTT cytotoxicity assay. The wound model and chamber model were established to estimate the effects of CWQ serum on migration and invasion of LoVo cells. The model for cell adhesion was established to evaluate the effect of CWQ serum on LoVo cells' adhesion. The gelatin zymography model was performed to determine the effects of CWQ serum on the activities of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). The effects of CWQ serum on the hypoxia-inducible factor 1 α (HIF-1α) nuclear translocation and the mRNA level of vascular endothelial growth factor (VEGF) in LoVo cells were determined by Western blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses, respectively.
CWQ inhibited LoVo cells' migration based on wound healing assay. The inhibitive effect could reach about 68.00% under hypoxic culture and about 29.87% under normoxic culture when cells were treated with 10% CWQ serum for 24 h. The results from both cell invasion and adhesion assays showed that CWQ serum could dose-dependently repress the invasion of LoVo cells and inhibit cells from adhering to extra cellular matrix (ECM). Under the hypoxic culture condition, RT-PCR analysis showed that 10% CWQ serum had down-regulated the expression of VEGF by 45.87%, and the result of Western blot analysis provided further evidence. The HIF-1α amount in the nucleus of the LoVo cells was also diminished in a dose-dependent manner, as shown by the Western blot. Gel zymogram assay revealed that CWQ serum could suppress the activities of MMP-2 and MMP-9.
CWQ could effectively inhibit tumor metastasis in vitro The antimetastatic effects of CWQ were associated with the inhibition of cell motility, which was evidenced by inhibition of cell invasion and adhesion. The molecular mechanisms of the inhibition of tumor invasion by CWQ were due to the reduced expression of both HIF-1α and VEGF and the suppression of MMP-2 and MMP-9 expression.
研究肠外清（CWQ）对缺氧结肠癌细胞转移和侵袭的体外作用及初步机制。此外，为中医药理论“扶正祛邪”治疗大肠癌及其侵袭转移提供实验依据。
采用血清药理学方法制备 CWQ 血清。然后，设计改良缺氧室，在 37°C 下用 5%CO（2）和 95%N（2）冲洗以诱导缺氧环境。MTT 细胞毒性测定法检测 CWQ 血清对 LoVo 细胞活力的影响。建立划痕模型和腔室模型，估计 CWQ 血清对 LoVo 细胞迁移和侵袭的影响。建立细胞黏附模型，评价 CWQ 血清对 LoVo 细胞黏附的影响。明胶酶谱模型用于确定 CWQ 血清对基质金属蛋白酶-2（MMP-2）和基质金属蛋白酶-9（MMP-9）活性的影响。Western blot 和逆转录-聚合酶链反应（RT-PCR）分析分别检测 CWQ 血清对缺氧诱导因子 1α（HIF-1α）核转位和血管内皮生长因子（VEGF）mRNA 水平的影响。
CWQ 抑制 LoVo 细胞的迁移，基于划痕愈合试验。在缺氧培养下，当用 10%CWQ 血清处理 24 小时时，抑制率可达约 68.00%，在常氧培养下抑制率约为 29.87%。细胞侵袭和黏附试验的结果表明，CWQ 血清可剂量依赖性地抑制 LoVo 细胞的侵袭，并抑制细胞黏附到细胞外基质（ECM）上。在缺氧培养条件下，10%CWQ 血清通过 RT-PCR 分析下调 VEGF 的表达 45.87%，Western blot 分析提供了进一步的证据。Western blot 显示，LoVo 细胞核中 HIF-1α的量也呈剂量依赖性减少。凝胶酶谱分析表明，CWQ 血清可抑制 MMP-2 和 MMP-9 的活性。
CWQ 可有效抑制肿瘤的体外转移。CWQ 的抗转移作用与细胞运动的抑制有关，这通过抑制细胞侵袭和黏附得到证实。CWQ 抑制肿瘤侵袭的分子机制是由于 HIF-1α和 VEGF 的表达减少以及 MMP-2 和 MMP-9 表达的抑制。
