Combined 5-FU and ChoKα Inhibitors as a New Alternative Therapy of Colorectal Cancer: Evidence in Human Tumor-Derived Cell Lines and Mouse Xenografts

<div><p>Background</p><p>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.</p><p>Methodology/Principal Findings</p><p>ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both <i>in vitro</i> and <i>in vivo</i> 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 <i>in vitro</i> in CRC-tumour derived cell lines, and <i>in vivo</i> 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 <i>in vitro</i> in three different human colon cancer cell lines, and <i>in vivo</i> 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.</p><p>Conclusion/Significance</p><p>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.</p></div

Cell cycle distribution in DLD-1 and SW620 from TCD-717 and 5-FU treatment, alone or in combination.

<p>2,5×10⁵ DLD-1 and SW620 cell lines were seeded in 6 well plates and incubated for 24 h with TCD-717 followed by 5-FU for 24 h alone or in combination. Combination of the two drugs increased cell death compared to the two drugs alone. Tables under the graphics indicate the percentage of the different phases of cell cycle when we treat with TCD-717 and 5-FU alone or in combination. *p<0.05 compared the different cell cycle phases vs. control.</p

Levels of TS and TK1 in SW620, HT29 and DLD-1 cell lines determined by RT-qPCR.

<p>2,5×10⁵ DLD-1, HT29 and SW620 cell lines were seeded in 6 well plates and incubated under optimal conditions for 24 h. Next, cells were exposed to different concentrations of ChoKα inhibitors for 24 h. Levels of TS and TK1 were analyzed by RT-qPCR as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064961#s4" target="_blank">Materials and Methods</a>. 18S was used as the endogenous control for normalisation.</p

Effect on cell viability of ChoKα inhibitors and 5-FU in DLD-1, HT29 and SW620 cell lines.

<p>6×10³ tumor cells were cultured in 96 well plates. After 24 h incubation, cells were exposed to TCD-717(left panels) for 24 h or MN58b (right panels) for 9 h. Thereafter the medium was changed for medium containing 5-FU for 60 h in plates previously treated with MN58b and for 24 h in plates treated with TCD-717. Cell viability was evaluated by MTT assay and represented as percentage of control, untreated cells. CI value in each case is the mean of three independent experiments, each performed in quadruplicates. CI<1 indicates a synergistic effect. A representative experiment of three independent experiments is shown.</p

<i>UPP1</i> mRNA in SW620, HT29 and DLD-1 cell lines.

<p>2,5×10⁵ DLD-1, HT29 and SW620 cell lines were seeded in 6 well plates and incubated 24 h under standard conditions. Thereafter, cells were treated with ChoKα inhibitors, TCD-717 (6 and 10 µM) and MN58b (10 and 15 µM) during 24 h. 18S was used as an endogenous control.</p

TS expression levels in tumor tissues from DLD-1 xenografts after treatment with MN58b plus 5-FU.

<p>Mice were inoculated with DLD-1 cells as indicated under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064961#s4" target="_blank">Materials and Methods</a> and either left untreated, or treated with MN58b or 5-FU alone or in combination. A) TS expression levels of each experiment group: Control, MN58b, 5-FU and combination group. Tubulin was used as loading control. B) Graph represents mean protein levels as TS/tubulin ratios for each group. Black bars represent total TS/tubulin values; Grey bars represent TS active values. (*) Statistically significant (p<0.05), compared to control group.</p

ChoKα expression levels in DLD-1, HT29 and SW620 cell lines by western blot.

<p>ChoKα protein levels of three colorectal tumor cell lines, DLD-1, HT29 and SW620 have been compared respect to the non tumoral colorectal cell line CCD-841. Below the western it is represented quantification levels (ChoKα/tubulin).</p

Induction of apoptosis in SW620, HT29 and DLD-1 cell lines after treatment with ChoKIs.

<p>(A–C) SW620, HT29 and DLD-1 cells were seeded as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064961#s4" target="_blank">Material and Methods</a> and treated with two concentrations of ChoKα inhibitors [TCD-717: 6 µM (left line) or 10 µM (right line); MN58b: 10 µM (left line) or 15 µM (right line)] for 24 h and, where indicated, with 5.5 µM 5-FU or vehicle for an additional 24 h. Protein expression was analyzed by Western blot using polyclonal antibody anti-PARP. Behind each graph, data shows protein levels (PARP/GAPDH) calculated by the mean ± SEM of three independent experiments. (A) SW620. (B) HT29. (C) DLD-1. (D) PARP and ChoKα expression levels in HT29 and SW620 cell lines transfected with a specific ChoKα siRNA determined by Western blot analysis or a control scramble siRNA (scr). Protein expression was analyzed by Western blot using ChoKα monoclonal antibody and polyclonal antibody anti-PARP. Below each Western the ratio PARP/Tubulin is represented. (E) Caspase 3 activity (upper panel) and its cleavage (lower panel) were determined in HT29 cells after treatment with MN58b for 24 h. (F) Caspase 3 enzymatic activity was also measured after transfection of HT29 cells with a ChoKα specific siRNA or a control siRNA (scr) for 48 h. Western blot represents the levels of ChoKα after transfection and its relative reduction normalized to GAPDH levels.</p

Effects of ChoK inhibition on p44/p42 MAPK levels and ceramide levels.

<p>(A) 2,5×10⁵ SW620 cells were seeded in 6 well plates and incubated for 24 h and exposed to two concentrations of ChoKα inhibitors [TCD-717: 6 µM (left line) or 10 µM (right line); MN58b: 10 µM (left line) or 15 µM (right line)]. Where indicated, medium was removed after treatment with ChoKα inhibitors and changed for fresh medium with 5.5 µM 5-FU for an additional 24 h. Protein expression was analyzed by western blot using monoclonal anti-phospho p44/p42 MAPK and anti-p44/p42 MAPK. The figure shows a representative blot of two independent experiments. (B) SW620 cells were grown and treated as indicated in (A) with 15 µM MN58b and 5.5 µM 5-FU alone or in combination for 24 hours. Protein expression was analyzed by western blot using anti-pAKT- Ser⁴⁷³, anti-AKT or anti-GAPDH. The figure shows on the top a representative blot, at the bottom protein levels (pAKT-Ser⁴⁷³/total AKT/GAPDH) from a representative experiment. (C) 2×10⁶ SW620 cells were seeded in p100 plates and incubated 24 h under standard conditions. Cells were then treated with 15 µM MN58b and 5.5 µM 5-FU alone or in combination for 24 hours. Total ceramide levels were analyzed by UPLC-TOF. Mean ± SD of two independent experiments performed in triplicate are shown.</p

Modulation of gene and miRNA expression by RE-2 in colon cancer cells.

<p>Microarray data of significantly modulated genes in SW620 colon cancer cells after treatment during 48 h with three different concentrations (30, 60 and 100 µg/mL) of RE-2.</p>a<p>FC: Fold change.</p