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PurposeThe naturally-occurring omega-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) is safe, well-tolerated and inexpensive, making it an attractive anti-cancer intervention. However, EPA has only modest anti-colorectal cancer (CRC) activity, when used alone. Both cyclooxygenase (COX) isoforms metabolise EPA and are over-expressed in CRC cells. We investigated whether COX inhibition increases the sensitivity of CRC cells to growth inhibition by EPA.MethodsA panel of 18 human and mouse CRC cell lines was used to characterize the differential sensitivity of CRC cells to the growth inhibitory effects of EPA. The effect of CRISPR-Cas9 genetic deletion and pharmacological inhibition of COX-1 and COX-2 on the anti-cancer activity of EPA was determined using in vitro and in vivo models.ResultsGenetic ablation of both COX isoforms increased sensitivity of CT26 mouse CRC cells to growth inhibition by EPA in vitro and in vivo. The non-selective COX inhibitor aspirin and the selective COX-2 inhibitor celecoxib increased sensitivity of several human and mouse CRC cell lines to EPA in vitro. However, in a MC38 mouse CRC cell tumour model, with dosing that mirrored low-dose aspirin use in humans, thereby producing significant platelet COX-1 inhibition, there was ineffective intra-tumoral COX-2 inhibition by aspirin and no effect on EPA sensitivity of MC38 cell tumours.ConclusionCyclooxygenase inhibition by non-steroidal anti-inflammatory drugs represents a therapeutic opportunity to augment the modest anti-CRC activity of EPA. However, intra-tumoral COX inhibition is likely to be critical for this drug-nutrient interaction and careful tissue pharmacodynamic profiling is required in subsequent pre-clinical and human studies.

The hydrophobicity of a drug can be a major challenge in its development and prevents the clinical translation of highly potent anti-cancer agents. We have used a lipid-based nanoemulsion termed Lipid-Oil-Nanodroplets (LONDs) for the encapsulation and in vivo delivery of the poorly bioavailable combretastatin A4 (CA4). Drug delivery with CA4 LONDs was assessed in a xenograft model of colorectal cancer. LC–MS/MS analysis revealed that CA4 LONDs, administered at a drug dose four times lower than drug control, achieved equivalent concentrations of CA4 intratumorally. We then attached CA4 LONDs to microbubbles (MBs) and targeted this construct to VEGFR2. A reduction in tumor perfusion was observed in CA4 LONDs-MBs treated tumors. A combination study with irinotecan demonstrated a greater reduction in tumor growth and perfusion (P = 0.01) compared to irinotecan alone. This study suggests that LONDs, either alone or attached to targeted MBs, have the potential to significantly enhance tumor-specific hydrophobic drug delivery.

Background Oral administration of the omega-3 fatty acid eicosapentaenoic acid (EPA), as the free fatty acid (FFA), leads to EPA incorporation into, and reduced growth of, experimental colorectal cancer liver metastases (CRCLM). Design: We performed a Phase II double-blind, randomised, placebo-controlled trial of EPA-FFA 2 g daily in patients undergoing liver resection surgery for CRCLM. The patients took EPA-FFA (n=43) or placebo (n=45) prior to surgery. The primary end-point was the CRCLM Ki67 proliferation index (PI). Secondary end-points included safety and tolerability of EPA-FFA, tumour fatty acid content and CD31-positive vascularity. We also analysed overall survival (OS) and disease-free survival (DFS). Results The median (range) duration of EPA-FFA treatment was 30 (12–65) days. Treatment groups were well matched with no significant difference in disease burden at surgery or preoperative chemotherapy. EPA-FFA treatment was well tolerated with no excess of postoperative complications. Tumour tissue from EPA-FFA-treated patients demonstrated a 40% increase in EPA content (p=0.0008), no difference in Ki67 PI, but reduced vascularity in ‘EPA-naive’ individuals (p=0.075). EPA-FFA also demonstrated antiangiogenic activity in vitro. In the first 18 months after CRCLM resection, EPA-FFA-treated individuals obtained OS benefit compared with placebo, although early CRC recurrence rates were similar. Conclusions EPA-FFA therapy is safe and well tolerated in patients with advanced CRC undergoing liver surgery. EPA-FFA may have antiangiogenic properties. Remarkably, limited preoperative treatment may provide postoperative OS benefit. Phase III clinical evaluation of prolonged EPA-FFA treatment in CRCLM patients is warranted. Trial Identifier: ClinicalTrials.gov NCT01070355.

As pre-clinical and clinical research interest in ω-3 polyunsaturated fatty acids (PUFA) increases, so does the need for a fast, accurate and reproducible analytical method to measure fatty acids (FA) in biological samples in order to validate potential prognostic and predictive biomarkers, as well as establishing compliance in ω-3 PUFA intervention trials. We developed a LC-ESI-MS/MS method suitable for high throughput development to measure FAs and validated it in the context of treatment with the ω-3 PUFA eicosapentaenoic acid (EPA). Uniquely we directly compared the LC-ESI-MS/MS method to a GC-MS protocol. We demonstrated the LC-ESI-MS/MS method is accurate and reproducible, with coefficients of variation consistently below 15% for each PUFA analysed. The relative FA content values correlated well with those obtained by GC-MS (r2 = 0.94, p < 0.001 for EPA) in vitro. The data obtained following analysis of FA content of liver tissues from mice fed an eicosapentaenoic acid enriched diet showed similar results to that of published studies in which GC-MS was used. The LC-ESI-MS/MS method allows concomitant analysis of unesterified (free, unbound) and esterified (bound) FAs in biological samples, allowing investigation of different PUFA pools in cells and tissues.

We investigated red blood cell (RBC) PUFA profiles, and the predictive value of RBC EPA content for tumour EPA exposure and clinical outcomes, in the EMT study, a randomised trial of EPA in patients awaiting colorectal cancer (CRC) liver metastasis surgery (Cockbain et al., 2014) [8]. There was a significant increase in RBC EPA in the EPA group (n=43; median intervention 30 days; mean absolute 1.26[±0.14]% increase; P

BACKGROUND AND PURPOSE The omega-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA) has antineoplastic activity at early stages of colorectal carcinogenesis, relevant to chemoprevention of colorectal cancer (CRC). We tested the hypothesis that EPA also has anti-CRC activity at later stages of colorectal carcinogenesis, relevant to treatment of metastatic CRC, via modulation of E-type PG synthesis. EXPERIMENTAL APPROACH A BALB/c mouse model, in which intrasplenic injection of syngeneic MC-26 mouse CRC cells leads to development of liver metastases, was used. Dietary EPA was administered in the free fatty acid (FFA) form for 2 weeks before and after ultrasound-guided intrasplenic injection of 1 × 106 MC-26 cells (n= 16 each group). KEY RESULTS Treatment with 5% (w w-1) EPA-FFA was associated with a reduced MC-26 mouse CRC cell liver tumour burden compared with control animals (median liver weight 1.03 g vs. 1.62 g; P < 0.034). Administration of 5% EPA-FFA was also linked to a significant increase in tumour EPA incorporation and lower intratumoural PGE2 levels (with concomitant increased production of PGE3). Liver tumours from 5% EPA-FFA- treated mice demonstrated decreased 5-bromo-2-deoxyuridine-positive CRC cell proliferation and reduced phosphorylated ERK 1/2 expression at the invasive edge of tumours. A concentration-dependent reduction in MC-26 CRC cell Transwell® migration following EPA-FFA treatment (50–200 µM) in vitro was rescued by exogenous PGE2 (10 µM) and PGE1-alcohol (1 µM). CONCLUSIONS AND IMPLICATIONS EPA-FFA inhibits MC-26 CRC cell liver metastasis. EPA incorporation is associated with a ‘PGE2 to PGE3 switch’ in liver tumours. Inhibition of PGE2-EP4 receptor-dependent CRC cell motility probably contributes to the antineoplastic activity of EPA.

NAD(P)H:quinone oxidoreductase-1 (NQO1) is a potential target for therapeutic intervention but attempts to exploit NQO1 using quinone-based bioreductive prodrugs have been largely compromised by toxicity to organs that inherently express high levels of NQO1. In an attempt to circumvent this problem, this study describes the development of a tripartite quinone-based drug delivery system, the ultimate objective of which is to release a targeted therapeutic agent following the reduction of a quinone “trigger” by NQO1. Molecular modeling of drug/NQO1 interactions were conducted prior to the synthesis of N-{4-[bis-(2-chloroethyl)-amino]-phenyl}-β,β,2,4,5-pentamethyl-3,6-dioxo-1,4-cyclohexadiene-1-propanamide (prodrug 1). Prodrug 1 is a good substrate for purified NQO1 (Vmax and Km values of 11.86 ± 3.09 μmol/min/mg and 2.70 ± 1.14 μmol/L, respectively) and liquid chromatography-mass spectrometry analysis of the metabolites generated showed that lactone 3 and aniline mustard 4 were generated in a time- and NQO1-dependent manner. Chemosensitivity studies showed that prodrug 1 is selectively toxic to cells that overexpress NQO1 under aerobic conditions, and comet assay analysis confirmed the presence of elevated interstrand cross-links in NQO1-rich compared with NQO1-deficient cells. Hypoxic sensitization (hypoxic cytotoxicity ratio = 15.8) was observed in T47D cells that overexpress cytochrome P450 reductase. In conclusion, the results of this study provide mechanistic proof of principle that a tripartite benzoquinone drug delivery system is enzymatically reduced to release an active therapeutic agent. Further development of this concept to fine-tune substrate specificity for specific reductases and/or the inclusion of alternative therapeutic agents is warranted. [Mol Cancer Ther 2007;6(12):3122–30]

Tumour response to Mitomycin C (MMC) is heterogenous and past attempts to predict clinical response based on enzyme activities have proven unsatisfactory. Using in vitro techniques, the aim of this study was to determine if the induction of DNA interstrand cross-links correlated with cellular response and to assess if DNA repair and induction of apoptosis influenced MMC chemosensitivity. Poor correlations were found between sensitivity and both DNA repair and induction of apoptosis suggesting that these processes do not play a major role in determining cellular response to MMC. In contrast, there was good correlation between the induction of DNA interstrand cross-links as determined by the alkaline comet assay and cellular response, suggesting that the biochemical events leading to DNA damage are the key factors that determine cellular response in vitro. Further studies are required to assess whether this approach as a mean of prediction has practical applications in vivo.

group): placebo; lurbinectedin at MTD (0.180mg/kg), 0.75MTD, 0.5MTD and 0.25MTD; gemcitabine at MTD (180mg/kg), 0.75MTD, 0.5MTD and 0.25MTD; and, lurbinectedin + gemcitabine at (1+1), (0.75+0.75), (0.50+0.50) and (0.25+0.25) MTDs. Treatments were given intravenously once per week during the placebo-treated survival time. The combination index (CI) was determined by the CI-isobol method. At 12 h post-treatment, placebo as well as lurbinectedin, gemcitabine and lurbinectedin + gemcitabine highly-dosed tumors were assayed for Hoescht and the number of stained cells in 5 high-power fields (×400) counted by 2 independent blinded counters. Results: The in vivo antitumor effect recorded in the experimental groups indicated synergism (CI

The mechanism of the anti-colorectal cancer (CRC) activity of the omega-3 fatty acid eicosapentaenoic acid (EPA) is not understood. We tested the hypothesis that EPA reduces expression of chemokine C-C motif ligand 2 (CCL2), a pro-inflammatory chemokine with known roles in metastasis. We measured CCL2 in clinical samples from a randomized trial of EPA in patients undergoing liver surgery for CRC liver metastasis (LM) and preclinical models. Genome-wide transcriptional profiling of tumors from EPA-treated patients was performed. EPA decreased CCL2 synthesis by CRC cells in a dose-dependent manner. CCL2 was localized to malignant epithelial cells in human CRCLM. EPA did not reduce CCL2 content in human or mouse tumors compare to control. However, EPA treatment was associated with decreased plasma CCL2 levels compared with controls (P=0.04). Reduction in plasma CCL2 following EPA treatment predicted improved disease-free survival (HR 0.32; P=0.003). Lack of ‘CCL2 response’ was associated with a specific CRCLM gene expression signature. In conclusion, reduction in plasma CCL2 in patients with CRCLM treated with EPA predicts better clinical outcome and a specific tumor gene expression profile. Further work is needed to validate CCL2 as a therapeutic response biomarker for omega-3 fatty acid treatment of CRC patients.