Krishnamachary Balaji, Glunde Kristine, Wildes Flonne, Mori Noriko, Takagi Tomoyo, Raman Venu, Bhujwalla Zaver M
Johns Hopkins University In Vivo Cellular Molecular Imaging Center Program, The Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Cancer Res. 2009 Apr 15;69(8):3464-71. doi: 10.1158/0008-5472.CAN-08-4120. Epub 2009 Mar 31.
Elevated phosphocholine (PC) and total choline (tCho) metabolites are widely established characteristics of most cancer cells, including breast cancer. Effective silencing of choline kinase (chk), the enzyme that converts choline to PC, is associated with reduced tumor growth. The functional importance and down-regulation of chk using RNA interference has been previously established. Here, we report on the preclinical evaluation of lentiviral vector-mediated down-regulation of chk using short hairpin RNA (shRNA) in established tumors derived from human breast cancer cells. Concentrated lentivirus expressing shRNA against chk was injected i.v. in the tail vein of MDA-MB-231 tumor-bearing female severe combined immunodeficient mice. Transduction efficiency in cells and tumors in vivo was assessed optically by enhanced green fluorescent protein expression and additionally from chk mRNA and protein levels. An 80% reduction in chk mRNA and protein was achieved following approximately 90% transduction efficiency in cells. After transduction with chk-shRNA, (1)H magnetic resonance spectroscopy (MRS) of cell and tumor extracts showed decreases in PC and tCho levels (P < 0.01 and 0.05, respectively) in comparison with controls. PC levels were monitored noninvasively by (31)P MRS in tumors and by (1)H MRS in cell and tumor tissue extracts. Noninvasive (31)P MR spectra of chk-shRNA-transduced tumors in vivo showed lower PC and phosphomonoester levels that were associated with reduced tumor growth and proliferation. This study shows the use of lentiviral vectors to target chk in a human breast cancer xenograft and noninvasive MRS detection of this targeting.
升高的磷酸胆碱（PC）和总胆碱（tCho）代谢物是包括乳腺癌在内的大多数癌细胞广泛确立的特征。胆碱激酶（chk）可将胆碱转化为PC，有效沉默该酶与肿瘤生长减缓相关。此前已证实使用RNA干扰对chk进行功能抑制和下调。在此，我们报告了在源自人乳腺癌细胞的已建立肿瘤中，利用短发夹RNA（shRNA）通过慢病毒载体介导下调chk的临床前评估。将表达针对chk的shRNA的浓缩慢病毒经尾静脉注射到携带MDA - MB - 231肿瘤的雌性严重联合免疫缺陷小鼠体内。通过增强绿色荧光蛋白表达以及chk mRNA和蛋白水平，以光学方式评估体内细胞和肿瘤中的转导效率。在细胞转导效率约为90%后，chk mRNA和蛋白水平降低了80%。用chk - shRNA转导后，细胞和肿瘤提取物的氢磁共振波谱（MRS）显示，与对照组相比，PC和tCho水平降低（分别为P < 0.01和0.05）。通过磷磁共振波谱（31P MRS）在肿瘤中以及通过氢磁共振波谱（1H MRS）在细胞和肿瘤组织提取物中对PC水平进行无创监测。体内经chk - shRNA转导的肿瘤的无创31P磁共振波谱显示PC和磷酸单酯水平较低，这与肿瘤生长和增殖减少相关。本研究展示了利用慢病毒载体在人乳腺癌异种移植模型中靶向chk以及对这种靶向作用进行无创MRS检测。
