RBC and WBC fatty acid composition following consumption of an omega 3 supplement: Lessons for future clinical trials

<p>Abstract</p> <p>Background</p> <p>Results from increasing numbers of <it>in vitro </it>and <it>in vivo </it>studies have demonstrated that omega 3 fatty acids incorporated in cell culture media or in the diet of the animals can suppress the growth of cancers. When human clinical trials are initiated to determine the ability of omega 3 fatty acids to alter growth or response to chemotherapeutic interventions of cancers, it will be essential to determine the omega 3 intake of individuals in the trial to determine compliance with consumption of the supplement and to correlate with endpoints of efficacy. We wondered if the fatty acid composition of RBCs might accurately indicate incorporation of omega 3 fatty acids in the WBCs. In this report we determine and compare the changes in fatty acid compositions of red blood cells and white blood cells in response to consumption of three doses of an omega 3 fatty acid supplement.</p> <p>Results</p> <p>We found that the fraction of omega 3 fatty acids in both red blood cells and white blood cells increased following consumption of the supplement. There was a linear, dose responsive increase in the fraction of omega 3 fatty acids in red blood cells but the increase in omega 3 in white blood cells was not linear. The magnitude of increase in omega 3 fatty acids was different between the two cell types.</p> <p>Conclusions</p> <p>Fatty acid analysis of red blood cells is a good measure of compliance with supplement consumption. However, fatty acid analysis of white blood cells is needed to correlate changes in fatty acid composition of white blood cells with other biochemical changes in the white blood cells.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov NCT00899353.</p

Inhibition of Cholinergic Signaling Causes Apoptosis in Human Bronchioalveolar Carcinoma

Recent case-controlled clinical studies show that bronchioalveolar carcinomas (BAC) are correlated with smoking. Nicotine, the addictive component of cigarettes, accelerates cell proliferation through nicotinic acetylcholine receptors (nAChR). In this study, we show that human BACs produce acetylcholine (ACh) and contain several cholinergic factors including acetylcholinesterase (AChE), choline acetyltransferase (ChAT), choline transporter 1 (CHT1, SLC5A7), vesicular acetylcholine transporter (VAChT, SLC18A3), and nACh receptors (AChRs, CHRNAs). Nicotine increased the production of ACh in human BACs, and ACh acts as a growth factor for these cells. Nicotine-induced ACh production was mediated by α7-, α3β2-, and β3-nAChRs, ChAT and VAChT pathways. We observed that nicotine upregulated ChAT and VAChT. Therefore, we conjectured that VAChT antagonists, such as vesamicol, may suppress the growth of human BACs. Vesamicol induced potent apoptosis of human BACs in cell culture and nude mice models. Vesamicol did not have any effect on EGF or insulin-like growth factor-II–induced growth of human BACs. siRNA-mediated attenuation of VAChT reversed the apoptotic activity of vesamicol. We also observed that vesamicol inhibited Akt phosphorylation during cell death and that overexpression of constitutively active Akt reversed the apoptotic activity of vesamicol. Taken together, our results suggested that disruption of nicotine-induced cholinergic signaling by agents such as vesamicol may have applications in BAC therapy

Consumption of high ω-3 fatty acid diet suppressed prostate tumorigenesis in C3(1) Tag mice

Prostate cancer incidence and mortality are high in the Western world and high ω-6/ω-3 PUFA in the Western diet may be a contributing factor. We investigated whether changing from a diet that approximates ω-6 fat content of the Western diet to a high ω-3 fat diet at adulthood might reduce prostate cancer risk. Female SV 129 mice that had consumed a high ω-6 diet containing corn oil for 2 weeks were bred with homozygous C3(1)Tag transgenic male mice. All male offspring were weaned to the corn oil diet (CO) until postpuberty when half of the male offspring were transferred to a high ω-3 diet containing canola oil and fish oil concentrate (FS). High ω-3 diet increased ω-3 and decreased ω-6 fat content of mice tissues. Average weights of prostate and genitourinary bloc were significantly lower in mice consuming high ω-3 diet at adulthood (CO-FS) than mice fed a lifetime high ω-6 diet (CO–CO). There was slower progression of tumorigenesis in dorsalateral prostate of CO-FS than in CO–CO mice. CO-FS mice had slightly lower plasma testosterone level at 24 and 40 weeks, significantly lower estradiol level at 40 weeks and significantly less expressed androgen receptor (AR) in the dorsalateral prostate at 40 weeks than CO–CO mice. Consumption of high ω-3 diet lowered the expression of genes expected to increase proliferation and decrease apoptosis in dorsalateral prostate. Our results suggest that consumption of high ω-3 diet slows down prostate tumorigenesis by lowering estradiol, testosterone and AR levels, promoting apoptosis and suppressing cell proliferation in C3(1)Tag mice

A phase II study of vinflunine in bladder cancer patients progressing after first-line platinum-containing regimen

A multicentre phase II trial to determine the efficacy of vinflunine as second-line therapy in patients with advanced transitional cell carcinoma (TCC) of the bladder; secondary objectives were to assess duration of response, progression-free survival (PFS) and overall survival (OS), and to evaluate the toxicity associated with this treatment. Patients had tumours that failed or progressed after first-line platinum-containing regimens for advanced or metastatic disease, or had progressive disease after platinum-containing chemotherapy given with adjuvant or neoadjuvant intent. Response and adverse events were assessed according to WHO criteria and NCI-CTC (version 2), respectively. Out of 51 patients treated with 320 mg m−2 of vinflunine, nine patients responded to the therapy yielding an overall response rate of 18% (95% CI: 8.4–30.9%), and 67% (95%CI: 52.1–79.3%) achieved disease control (PR+SD). Of note, responses were seen in patients with relatively poor prognostic factors such as a short (<12 months) interval from prior platinum therapy (19%, including an 11% response rate in those progressing <3 months after platinum treatment), prior treatment for metastatic disease (24%), prior treatment with vinca alkaloids (14%) and visceral involvement (20%). The median duration of response was 9.1 months (95% CI: 4.2–15.0) and the median PFS was 3.0 months (95% CI: 2.4–3.8). The median OS was 6.6 months (95% CI: 4.8–7.6). The main haematological toxicity was grade 3–4 neutropenia, observed in 67% of patients (42% of cycles). Febrile neutropenia was observed in five patients (10%) and among them two were fatal. Constipation was frequently observed (but was manageable and noncumulative) and was grade 3–4 in only 8% of patients. The incidence of grade 3 nausea and vomiting was very low (4 and 6% of patients, respectively). Neither grade 3–4 sensory neuropathy nor severe venous irritation was observed. Moreover, and of importance in this particular study population, no grade 3–4 renal function impairment was observed. Vinflunine is an active agent for the treatment of platinum-pretreated bladder cancer, and these results warrant further investigation in phase III trials, either as monotherapy or in combination with other agents as treatment of advanced/metastatic TCC of the bladder

Omega-3 eicosapentaenoic acid decreases CD133 colon cancer stem-like cell marker expression while increasing sensitivity to chemotherapy.

Colorectal cancer is the third leading cause of cancer-related death in the western world. In vitro and in vivo experiments showed that omega-3 polyunsaturated fatty acids (n-3 PUFAs) can attenuate the proliferation of cancer cells, including colon cancer, and increase the efficacy of various anticancer drugs. However, these studies address the effects of n-3 PUFAs on the bulk of the tumor cells and not on the undifferentiated colon cancer stem-like cells (CSLCs) that are responsible for tumor formation and maintenance. CSLCs have also been linked to the acquisition of chemotherapy resistance and to tumor relapse. Colon CSLCs have been immunophenotyped using several antibodies against cellular markers including CD133, CD44, EpCAM, and ALDH. Anti-CD133 has been used to isolate a population of colon cancer cells that retains stem cells properties (CSLCs) from both established cell lines and primary cell cultures. We demonstrated that the n-3 PUFA, eicosapentaenoic acid (EPA), was actively incorporated into the membrane lipids of COLO 320 DM cells. 25 uM EPA decreased the cell number of the overall population of cancer cells, but not of the CD133 (+) CSLCs. Also, we observed that EPA induced down-regulation of CD133 expression and up-regulation of colonic epithelium differentiation markers, Cytokeratin 20 (CK20) and Mucin 2 (MUC2). Finally, we demonstrated that EPA increased the sensitivity of COLO 320 DM cells (total population) to both standard-of-care chemotherapies (5-Fluorouracil and oxaliplatin), whereas EPA increased the sensitivity of the CD133 (+) CSLCs to only 5-Fluorouracil

Analysis of fatty acids incorporation by gas chromatography.

<p>Total lipids were extracted from COLO 320 DM cells treated for 96 hours with control vehicle (CTRL VH) or a range of concentrations of (<b>A</b>) EPA or (<b>B</b>) SA, (6.25–12.5–25 uM). Gas chromatography was performed to study the fatty acids incorporation and their metabolites by COLO 320 DM cells. Results are presented as relative percentage with respect to the CTRL VH. Results represent the mean ± SD of at least three experiments. <i>Black bars</i>: compound used for the treatment detected with GC; <i>Light Grey bars</i>: DPA (<i>docosapentaenoic Acid</i>, C22:5, n-3); <i>Dark Grey bars: DHA (docosahexaenoic Acid, C22:6, n-3)</i>. (SA: stearic acid C18:0).</p

Differential effects of fatty acids on COLO 320 DM CD133 (+) and CD133 (−) cell number.

<p>COLO 320 DM cells were treated for 0–96 hours with vehicle control (CTRL VH) or 6.25–12.5–25 uM of (<b>A</b>) EPA and (<b>B</b>) SA. MTT assay of EPA and SA treated cells vs. vehicle control (CTRL VH) treated cells. <i>p</i> values were calculated with one-way ANOVA test with a Tukey's multiple comparison post-test on the various treatments within each time point. *p≤0.05; **p≤0.01; ***p≤0.001; n = 5. COLO 320 DM cells were also treated for 96 hours with vehicle control (CTRL VH) or a range of concentrations of fatty acids (6.25–12.5–25 uM), (<b>C</b>) EPA and (<b>D</b>) SA. Viable cells were counted and labeled with a monoclonal antibody anti-CD133 to determine the percentage of CD133 (+) cells in the treated groups. Results represent CD133 (+) and CD133 (−) cell numbers of ± SD of at least three experiments. <i>p</i> values were calculated with Student's t-test on treated samples vs. CTRL VH (* p≤0.05).</p

Sensitivity of COLO 320 DM total population and CSLCs cells to Oxaliplatin and 5-Fluorouracil following treatment with 25 uM EPA.

<p>(<b>A</b>) COLO 320 DM cells were treated with a range of Oxaliplatin (0.005–0.1 mM) or 5-Fluorouracil (0.05–2 mM) concentrations to determine the inhibitory concentration of 25% (IC25) and 50% (IC50). (<b>B</b>) Cells from COLO 320 DM total population were pre-treated for 48 hours with 25 uM EPA or SA. Afterwards cells were exposed for 24 hours with Oxaliplatin (<i>IC25, 2.5 </i><i>uM; IC50, 10 </i><i>uM</i>) and 5 Fluorouracil (<i>IC25, 100 </i><i>uM; IC50, 1.5 </i><i>mM</i>). (<b>C</b>) CD133 (+) cells were magnetically sorted from the total population of COLO 320 DM and were pre-treated for 48 hours with 25 uM EPA or SA and then exposed for 24 hours to IC25 and IC50 of Oxaliplatin and 5-Fluorouracil. Results represent the mean ± SD of at least three experiments. <i>p</i> values were calculated with Student's t-test on treated samples vs. CTRL VH (* p≤0.05, ** p≤0.01, *** p≤0.001).</p