Combined 5-FU and ChoKα inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts.

BACKGROUND: Colorectal cancer (CRC) is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα), an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy. METHODOLOGY/PRINCIPAL FINDINGS: ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS) and thymidine kinase (TK1) levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action. CONCLUSION/SIGNIFICANCE: Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

Choline kinase alpha depletion selectively kills tumoral cells.

Choline Kinase (ChoK) comprises a family of cytosolic enzymes involved in the synthesis of phosphatidylcholine (PC), the most abundant phospholipid in eukaryotic cell membranes. One of the ChoK isoforms, Choline Kinase alpha (ChoKalpha), is found over expressed in human tumours. Chemical inhibitors able to interfere with ChoK activity have proven to be effective antitumoral drugs in vitro and in vivo. To validate the use of selective ChoKalpha inhibitors in cancer therapy, we have developed a genetic strategy to interfere specifically with ChoKalpha activity based on the generation of a shRNA against the alpha isoform of ChoK. Here we demonstrate that specific inhibition of ChoKalpha by shRNA has antitumor activity. The specific depletion of ChoKalpha induces apoptosis in several tumor-derived cell lines from breast, bladder, lung and cervix carcinoma tumors, while the viability of normal primary cells is not affected. Furthermore, this selective antiproliferative effect is achieved both under in vitro and in vivo conditions, as demonstrated by an inducible ChoKalpha suppression system in human tumour xenografts. These results demonstrate that ChoKalpha inhibition is a useful antitumoral strategy per se, and provides definitive and non-ambiguous evidence that ChoKalpha can be used as an efficient and selective drug target for cancer therapy.