Shimada Temiko, Nishimura Yoshihiro, Nishiuma Teruaki, Rikitake Yoshiyuki, Hirase Tetsuaki, Yokoyama Mitsuhiro
Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan.
Kobe J Med Sci. 2007;53(3):125-34.
Lung cancer is still a very severe disease which has a low survival rate due to local invasion and metastasis potentials in spite of many clinical challenges using anti-cancer drugs. Rho family small GTPases play pivotal roles in cell invasion and metastasis during carcinogenesis. In this study, we explored the inhibitory effect of adenoviral vector encoding dominant negative mutants of Rac, RhoA, and ROCK in human non-small cell lung carcinoma cell lines (A549 and SQ5) and mouse carcinoma cell line (Lewis lung carcinoma, LLC). These cells showed high expression of Rac, Rho, and ROCK, whereas only faint bands were detected in normal human lung epithelial cells, BET-1A. The efficiency of adenoviral vector transfer was stronger in A549 and SQ5 cells than LLC cells. Dominant negative forms of RhoA (Rho-DN) and Rac (Rac-DN) decreased cell proliferation in WST-8 assay and increased the number of apoptotic cells in both A549 and SQ5 cells by Hoechst 33258 and TUNEL staining. On the other hand, DN form of ROCK (ROCK-DN) did not show any apparent changes compared with the other proteins. Transwell chamber analysis showed that migration/invasion activity was significantly suppressed by gene transfection both in A549 and SQ5 cells and that ROCK-DN gene transfer required a higher multiplicity of infection to show effects similar to Rho and Rac. Although the effect of gene therapy is cell-dependent, these data suggest that adenoviral gene transfer with Rho family small GTPases is one good approach to lung cancer therapy.
肺癌仍然是一种非常严重的疾病，尽管使用抗癌药物面临许多临床挑战，但由于其具有局部侵袭和转移的潜力，生存率较低。Rho家族小GTP酶在致癌过程中的细胞侵袭和转移中起关键作用。在本研究中，我们探讨了编码Rac、RhoA和ROCK显性负突变体的腺病毒载体对人非小细胞肺癌细胞系（A549和SQ5）和小鼠癌细胞系（Lewis肺癌，LLC）的抑制作用。这些细胞中Rac、Rho和ROCK表达较高，而在正常人肺上皮细胞BET-1A中仅检测到微弱条带。腺病毒载体在A549和SQ5细胞中的转染效率强于LLC细胞。在WST-8实验中，RhoA（Rho-DN）和Rac（Rac-DN）的显性负形式降低了细胞增殖，通过Hoechst 33258和TUNEL染色发现，二者均增加了A549和SQ5细胞中的凋亡细胞数量。另一方面，与其他蛋白相比，ROCK的显性负形式（ROCK-DN）未显示任何明显变化。Transwell小室分析表明，基因转染显著抑制了A549和SQ5细胞的迁移/侵袭活性，并且ROCK-DN基因转移需要更高的感染复数才能显示出与Rho和Rac类似的效果。尽管基因治疗的效果因细胞而异，但这些数据表明，用Rho家族小GTP酶进行腺病毒基因转移是肺癌治疗的一种有效方法。
