Interaction of ω-3 polyunsaturated fatty acids with radiation therapy in two different colorectal cancer cell lines.

BACKGROUND & AIMS: This study aims at evaluating if docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) increases the efficacy of radiation therapy (RT) on two human colorectal cancer cell lines with different radio-sensitivity. METHODS: LS174T and HT-29 cells were treated with 20 or 50 µmol/L EPA or DHA followed by single X-ray RT of 0, 2 or 4 Gy, to evaluate cell survival, apoptosis, peroxide and malondialdehyde productions. Inflammation- and apoptosis-related proteins were analyzed by Western Blot. ANOVAs were used for statistical analysis. RESULTS: LS174T was more sensitive to RT than HT-29. DHA and to a lesser extent EPA increased cell death, apoptosis and peroxide production after RT in LS174T and to a lesser extent in HT-29 (p < 0.05). This was associated with increased expression of heat shock protein 70, decreased expression of NF-kB p65, COX-2 and Bcl-2 proteins. CONCLUSIONS: The effect of RT combination with DHA and to a lesser extent EPA was synergistic in the radio-sensitive LS174T cells, but additive in the radio-resistant HT-29 cells. This enhanced cytotoxicity was provoked at least partly by lipid peroxidation, which consequently modulated inflammatory response and induced apoptosis.

Colon cancer cell chemosensitisation by fish oil emulsion involves apoptotic mitochondria pathway.

Adjuvant use of safe compounds with anti-tumour properties has been proposed to improve cancer chemotherapy outcome. We aimed to investigate the effects of fish oil emulsion (FOE) rich in n-3 PUFA with the standard chemotherapeutic agents 5-fluorouracil (5-FU), oxaliplatin (OX) or irinotecan (IRI) on two human colorectal adenocarcinoma cells with different genetic backgrounds. The HT-29 (Bax+/+) and LS174T (Bax-/-) cells were co-treated for 24-72 h with 1 µm-5-FU, 1 µm-OX or 10 µm-IRI and/or FOE dilution corresponding to 24 µm-EPA and 20·5 µm-DHA. Soyabean oil emulsion (SOE) was used as isoenergetic and isolipid control. Cell viability, apoptosis and nuclear morphological changes were evaluated by cytotoxic colorimetric assay, flow cytometry analysis with annexin V and 4',6'-diamidino-2-phenylindole staining, respectively. A cationic fluorescent probe was used to evaluate mitochondrial dysfunction, and protein expression involved in mitochondrial apoptosis was determined by Western blot. In contrast to SOE, co-treatment with FOE enhanced significantly the pro-apoptotic and cytotoxic effects of 5-FU, OX or IRI in HT-29 but not in LS174T cells (two-way ANOVA, P <0.01). These results were confirmed by the formation of apoptotic bodies in HT-29 cells. A significant increase in mitochondrial membrane depolarisation was observed after the combination of 5-FU or IRI with FOE in HT-29 but not in LS174T cells (P <0.05). Co-administration of FOE with the standard agents, 5-FU, OX and IRI, could be a good alternative to increase the efficacy of chemotherapeutic protocols through a Bax-dependent mitochondrial pathway.

Vitamin E content in fish oil emulsion does not prevent lipoperoxidative effects on human colorectal tumors.

OBJECTIVE: The anticancer action exerted by polyunsaturated fatty acid peroxidation may not be reproduced by commercially available lipid emulsions rich in vitamin E. Therefore, we evaluated the effects of fish oil (FO) emulsion containing α-tocopherol 0.19 g/L on human colorectal adenocarcinoma cells and tumors. METHODS: HT-29 cell growth, survival, apoptosis, and lipid peroxidation were analyzed after a 24-h incubation with FO 18 to 80 mg/L. Soybean oil (SO) emulsion was used as an isocaloric and isolipidic control. In vivo, nude mice bearing HT-29 tumors were sacrificed 7 d after an 11-d treatment with intravenous injections of FO or SO 0.2 g ∙ kg(-1) ∙ d(-1) FO or SO to evaluate tumor growth, necrosis, and lipid peroxidation. RESULTS: The FO inhibited cell viability and clonogenicity in a dose-dependent manner, whereas SO showed no significant effect compared with untreated controls. Lipid peroxidation and cell apoptosis after treatment with FO 45 mg/L were increased 2.0-fold (P < 0.01) and 1.6-fold (P = 0.04), respectively. In vivo, FO treatment did not significantly affect tumor growth. However, immunohistochemical analyses of tumor tissue sections showed a decrease of 0.6-fold (P < 0.01) in the cell proliferation marker Ki-67 and an increase of 2.3-fold (P = 0.03) in the necrotic area, whereas malondialdehyde and total peroxides were increased by 1.9-fold (P = 0.09) and 7.0-fold (P < 0.01), respectively, in tumors of FO-treated compared with untreated mice. CONCLUSION: These results suggest that FO but not SO has an antitumor effect that can be correlated with lipid peroxidation, despite its vitamin E content.

Quercetin-mediated Mcl-1 and survivin downregulation restores TRAIL-induced apoptosis in non-Hodgkin's lymphoma B cells.

BACKGROUND: Non-Hodgkin's B-cell lymphomas account for approximately 70% of B-cell lymphomas. While its incidence is dramatically increasing worldwide, the disease is still associated with high morbidity due to ineffectiveness of conventional therapies, creating an urgent need for novel therapeutic approaches. Unconventional compounds, including polyphenols and the cytokine TRAIL, are being extensively studied for their capacity to restore apoptosis in a large number of tumors, including lymphomas. DESIGN AND METHODS: Molecular mechanisms of TRAIL-resistance and reactivation of the apoptotic machinery by quercetin in non-Hodgkin's lymphoma cell lines were determined by Hoescht, flow cytometry, Western blot, qPCR, by use of siRNA or pharmacological inhibitors of the mitochondrial pathway and by immunoprecipitation followed by post-translational modification analysis. RESULTS: Results demonstrate that quercetin, a natural flavonoid, restores TRAIL-induced cell death in resistant transformed follicular lymphoma B-cell lines, despite high Bcl-2 expression levels due to the chromosomal translocation t(14;18). Quercetin rescues mitochondrial activation by inducing the proteasomal degradation of Mcl-1 and by inhibiting survivin expression at the mRNA level, irrespective of p53. Restoration of the TRAIL pathway requires Bax and Bak but is independent of enhanced TRAIL DISC formation. CONCLUSIONS: We demonstrate that inactivation of survivin and Mcl-1 expression by quercetin is sufficient to restore TRAIL sensitivity in resistant non-Hodgkin's lymphoma B cells. Our results suggest, therefore, that combining quercetin with TRAIL treatments may be useful in the treatment of non-Hodgkin's lymphoma.

Angiogenesis as a potential target of pharmaconutrients in cancer therapy.

PURPOSE OF REVIEW: The aim of this review is to provide insight into tumor angiogenesis inhibition by pharmaconutrients through description of the most relevant and recent findings in cancer research. RECENT FINDINGS: Cancer growth needs oxygen and nutrients supplied through blood vessels to the tumor site. New vessel formation named angiogenesis can be prevented to avoid cancer invasion. Epidemiological studies suggested that specific food intakes could decrease incidence of many cancers. Recently, scientists were interested in the potential antitumor effects of nutrients because of their safety and general acceptance. Many excellent publications demonstrated that a large class of natural compounds including pharmaconutrients exhibits antitumoral activities in selected cancer types. This review focuses on the antiangiogenic role of natural products in cancer treatment, used alone or in combination with conventional chemotherapy. SUMMARY: There is strong evidence that natural diets influence cancer development by modulating signaling pathways. Our goal is to highlight the specific impact of specific nutrients in the modulation of vascular network leading to tumor angiogenesis inhibition.

Peroxynitrite-dependent killing of cancer cells and presentation of released tumor antigens by activated dendritic cells.

Dendritic cells (DCs), essential for the initiation and regulation of adaptive immune responses, have been used as anticancer vaccines. DCs may also directly trigger tumor cell death. In the current study, we have investigated the tumoricidal and immunostimulatory activities of mouse bone marrow-derived DCs. Our results indicate that these cells acquire killing capabilities toward tumor cells only when activated with LPS or Pam3Cys-SK4. Using different transgenic mouse models including inducible NO synthase or GP91 knockout mice, we have further established that LPS- or Pam3Cys-SK4-activated DC killing activity involves peroxynitrites. Importantly, after killing of cancer cells, DCs are capable of engulfing dead tumor cell fragments and of presenting tumor Ags to specific T lymphocytes. Thus, upon specific stimulation, mouse bone marrow-derived DCs can directly kill tumor cells through a novel peroxynitrite-dependent mechanism and participate at virtually all levels of antitumor immune responses, which reinforces their interest in immunotherapy.

Prognostic significance of TRAIL-R1 and TRAIL-R3 expression in metastatic colorectal carcinomas.

Drug resistance is believed to cause treatment failure in patients with metastatic colorectal carcinoma (CRC). Resistance to chemotherapy can involve different processes, including apoptosis, whose extrinsic pathway is regulated by expression of death-inducing TRAIL-R1 and -R2 and inhibitory TRAIL-R3 and -R4 cell surface receptors. Therefore, we investigated whether variations in their expression could influence the response to 5-Fluorouracil (5-FU) in metastatic CRC. We analysed TRAIL-R 1, -2, -3 and -4 expression by immuno-histochemistry in CRC, using tissue micro arrays, and found that concomitant low/medium TRAIL-R1 and high TRAIL-R3 expression in primary CRC is significantly associated with a poor response to 5-FU-based first-line chemotherapy and with shorter progression-free survival. Specifically, the median progression-free survival was 3.1 months (poor prognostic group) versus 10.1 in the good prognostic group. Thus, the combination of TRAIL-R1 and TRAIL-R3 expression might represent a predictive and prognostic factor of the response to 5-FU-based first-line chemotherapy in patients with metastatic CRC.

Gene expression signature in advanced colorectal cancer patients select drugs and response for the use of leucovorin, fluorouracil, and irinotecan.

PURPOSE: In patients with advanced colorectal cancer, leucovorin, fluorouracil, and irinotecan (FOLFIRI) is considered as one of the reference first-line treatments. However, only about half of treated patients respond to this regimen, and there is no clinically useful marker that predicts response. A major clinical challenge is to identify the subset of patients who could benefit from this chemotherapy. We aimed to identify a gene expression profile in primary colon cancer tissue that could predict chemotherapy response. PATIENTS AND METHODS: Tumor colon samples from 21 patients with advanced colorectal cancer were analyzed for gene expression profiling using Human Genome GeneChip arrays U133. At the end of the first-line treatment, the best observed response, according to WHO criteria, was used to define the responders and nonresponders. Discriminatory genes were first selected by the significance analysis of microarrays algorithm and the area under the receiver operating characteristic curve. A predictor classifier was then constructed using support vector machines. Finally, leave-one-out cross validation was used to estimate the performance and the accuracy of the output class prediction rule. RESULTS: We determined a set of 14 predictor genes of response to FOLFIRI. Nine of nine responders (100% specificity) and 11 of 12 nonresponders (92% sensitivity) were classified correctly, for an overall accuracy of 95%. CONCLUSION: After validation in an independent cohort of patients, our gene signature could be used as a decision tool to assist oncologists in selecting colorectal cancer patients who could benefit from FOLFIRI chemotherapy, both in the adjuvant and the first-line metastatic setting.

Impact of RNA degradation on gene expression profiles: assessment of different methods to reliably determine RNA quality.

DNA microarray technology enables investigators to measure the expression of several 1000 mRNA species simultaneously in a biological specimen. However, the reliability of the microarray technology to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. Thus, it is of critical importance to standardize sample-handling protocols and to perform a quality assessment of RNA preparations. In this report, 59 human tissue samples were used to evaluate the relationships between RNA quality and gene expression. From Affymetrix GeneChip array data analysis of these samples, we compared the performance of the 28S/18S ratio, two computer methods (RIN and degradometer) and our in-house RNA quality scale (RQS) in assessing RNA quality. The optimal RNA reliability threshold was determined for each method using statistical discrimination measures. We showed that RQS, RIN and degradometer have a similar capacity to detect reliable RNA samples whereas the 28S/18S ratio leads to a misleading categorization. Furthermore, we developed a new approach, based on clustering analyses of full chip expression, to control RNA quality after hybridization experiments. The combination of these methods, allowing monitoring of RNA quality prior to and after the hybridization experiments, ensured reliable and reproducible microarray data.

A lysophosphatidic acid analogue is revealed as a potent inhibitor of phosphatidylcholine synthesis, inducing apoptosis.

A previous study demonstrated that cross-desensitization experiments performed with the lysophosphatidic acid (LPA) analogues (R)- and (S)-N-palmitoyl-norleucinol 1-phosphate (PNPAs) inhibited LPA-induced platelet aggregation without any stereospecificity. Here we report opposite biological effects of the two enantiomers on mitogenesis of IMR-90 fibroblasts in relation to their respective metabolism. (R)PNPA was proliferative, while (S)PNPA induced apoptosis by specifically inhibiting phosphatidylcholine biosynthesis at the last step of the CDP-choline pathway controlled by cholinephosphotransferase. This effect was not direct but required dephosphorylation of PNPAs by ecto-lipid phosphate phosphatase before cellular uptake of the generated N-palmitoyl-norleucinols (PNOHs). Inhibition of cholinephosphotransferase by the derivative (S)PNOH was confirmed by an in vitro assay. (S)PNPA proapoptotic effects led us to clarify the mechanism linking cholinephosphotransferase inhibition to apoptosis. Three proapoptotic responses were observed: the activation of caspase-3, the production of ceramides from newly synthesized pools (as demonstrated by the inhibitor Fumonisin B1) and finally the activation of stress-activated protein kinase, p38 and c-Jun N-terminal kinases 1/2, as a result of ceramide increase. Thus our data demonstrate that synthetic analogues of LPA might display stereospecific effects leading to apoptosis independently of classical LPA-activated pathways.