Abstract 980: Downregulation of choline kinase does not affect endothelial cell proliferation
Authors: Mori Noriko , Gadiya Mayur , Wildes Flonne , Krishnamachary Balaji , Bhujwalla Zaver M. 
Published: June 2012

Elevated phosphocholine (PC) and high choline kinase (Chk) expression are typically observed in cancer. Chk, the enzyme that converts choline (Cho) to PC, is being evaluated as a novel target in cancer treatment using pharmacological and molecular inhibition. We have previously shown that both transient transfection and stable expression of siRNA (siRNA-chk) and shRNA against choline kinase-α (Chk) significantly reduced proliferation in breast cancer cells [1] and tumors [2]. The downregulation of Chk in nonmalignant MCF-12A cells resulted in an almost negligible effect on PC and proliferation [3]. Since endothelial cells are a key component of vasculature and are exposed to agents that are delivered systemically, it is important to determine the effect of Chk on endothelial cells in normal and tumor tissue. We have examined the proliferation and PC levels of human umbilical vein endothelial cells (HUVEC) after transient siRNA-chk transfection and compared the results with human breast cancer cells (MDA-MB-231).

MDA-MB-231 and HUVEC were used in this study. Cells were transiently transfected with 100 nM siRNA-chk for 48 hours using DhamaFECT. Cells were harvested to determine protein and mRNA levels at 48 hour post-transfection. Quantitative real-time PCR (q-RT-PCR) was performed to determine mRNA level using iQ SYBR Green Supermix and gene-specific primers in the iCycler real-time PCR detection system. Fully relaxed 1H MR spectra of water-soluble cell extracts were acquired on a Bruker Avance 500 MR spectrometer. PC level was quantified as mM using integrals of around 3.225 ppm signal in the 1H NMR spectra relative to cell number, cell volume (MDA-MB-231: 2050 µm3 and HUVEC: 4530 µm3), and an internal concentration standard. To exam the prolifelation/viability, cells were transfected with siRNA for 48 hours, changed to culture medium and cultured another 3 days, following which an MTS assay was performed.

After siRNA-chk transfection, Chk mRNA levels of MDA-MB-231 and HUVEC were comparable. Basal levels of Chk mRNA and Chk protein in HUVEC were low to start with, and it was difficult to downregulate Chk in HUVEC further. Immunoblot analysis showed significant downregulation of Chk protein in MDA-MB-231, and downregulation to a lesser extent in HUVEC after transfection of siRNA-chk. MTS assay result showed no significant reduction of proliferation in HUVEC after siRNA-chk transfection, while MDA-MB-231 showed a significant reduction of proliferation. The level of PC in HUVEC was about one tenth compared to MDA-MB-231. PC level was significantly reduced in MDA-MB-231 after siRNA-chk transfection but there was about 10% reduction in HUVEC. These data suggest that Chk inhibition will not affect endothelial cells during systemic administration, nor will it affect tumor vasculature.

[1] Glunde K et al, Cancer Res, 65, (2005); [2] Krishnamachary B et al, Cancer Res, 69, (2009); [3] Mori N et al, Cancer Res, 67, (2007).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 980. doi:10.1158/1538-7445.AM2011-980