1 Institute of Nutrition and Food Technology, Department of Physiology, University of Granada, C/Ramón y Cajal 4, 18071, Granada, Spain, 2 Rowett Research Institute, Greenburn Road North, Bucksburn, Aberdeen AB21 9SB, Scotland, UK, 3 School of Life Sciences, Robert Gordon University, St Andrew Street, Aberdeen AB 25 1HG, Scotland, UK, 4 Department of Urology, Grampian Universities NHS Trust, Foresterhill, Aberdeen, Scotland, UK and 5 Department of Surgical and Nutritional Oncology, Aberdeen University Medical School, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK
6 To whom correspondence should be addressed Email: jjoh{at}ugr.es
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Abstract |
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Abbreviations: CLA, conjugated linoleic acids; COX, cyclooxygenase; c9,t11 CLA, cis-9, trans-11 CLA; LOX-5, 5-lipoxygenase; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; t10,c12 CLA, trans-10, cis-12 CLA
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Introduction |
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Diet is an identifiable risk factor associated with prostate cancer occurrence (1013). Some dietary constituents, particularly fatty acids, are implicated in cancer promotion and in contrast, others can suppress tumour development. So, identifying these last factors could be an effective non-invasive strategy for decreasing the incidence and severity of this disease or for an effective adjuvant treatment, especially in the hormone-refractory disease.
Conjugated linoleic acid (CLA) is a dietary fatty acid predominant in ruminant food products that has received considerable attention because of its anti-mutagenic and anticarcinogenic properties (1417). CLA is the generic term of a group of positional and geometric isomers of the omega-6 essential fatty acid linoleic acid (LA). Experimental studies in animal models and cells have shown that unlike the parent LA, which stimulates tumour growth, CLA is an effective inhibitor of many human cancers, including prostate cancer (17,18). However, the cellular mechanisms by which CLAs elicit these anticancer effects are not clear at present. Proposed mechanisms include modulation of eicosanoids synthesis and signal transduction, up-regulation of genes dependent on the transcription factors peroxisome proliferator-activated receptor (PPAR), inhibition of DNA adduct formation induced by exposure to carcinogens and induction of apoptosis (1417). In addition, recent findings suggest that not only does CLA affect many different pathways, but that individual isomers of CLA act differently and that some effects are induced and/or enhanced by these isomers apparently acting synergistically (15).
As a result of its inhibitory effects on tumorigenesis, CLA could be a novel dietary supplement for individuals with increased risk of prostate cancer or patients undergoing prostate cancer treatment. However, compared with other major cancers, such as breast or colon, the literature on CLA and prostate cancer is not extensive (1820). A better understanding of the anticarcinogenic properties of CLA preparations and their constituent isomers in prostate cancer could prove to be a novel nutritional management and an adjunct therapy in this disease.
Our aims are: (i) to evaluate the effects of a commercial preparation of a CLA mixture of isomers (cis-9, trans-11 and trans-10, cis-12 isomers in approximately a 50:50 ratio) and the individual isomers on the proliferation of prostate cancer cells in culture. (ii) To assess the effect of these fatty acids on gene expression (mRNA and protein levels) of different enzymes and oncoproteins involved in prostate cancer cell proliferation/progression. This includes enzymes of arachidonic acid metabolism/eicosanoid synthesis like cyclooxygenase 1 (COX-1), cyclooxygenase 2 (COX-2) and 5-lipoxygenase (5-LOX), and the oncoproteins involved in apoptosis (bcl-2) and cell cycle control (p21WAF1/Cip1), using the androgen-independent human prostate cancer cell line, PC-3. (iii) To determine whether individual isomers have the same regulatory effects on these enzymes and proteins as the mixture.
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Materials and methods |
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CLAs were obtained from Natural ASA (Hovdebygda, Norway). Purified CLAs [50:50 mixed isoforms and individual cis-9, trans-11 CLA (c9,t11 CLA) and trans-10, cis-12 CLA isoforms] in oil form were dissolved in 100% ethanol to give a 10 mM stock solution, which was further diluted in ethanol to produce the range of test concentrations. All fatty acids were stored under nitrogen at 80°C until used in the studies. A similar volume of ethanol, which did not contain any fatty acids, underwent similar preparation and storage and was used as a control, CLA-free medium. When the cells were sub-confluent, the respective media were replaced with new fresh medium containing CLA in concentrations of 25, 50, 100 and 150 µM and cells were treated with these concentrations for 24 h.
Cell proliferation/viability assessment
The modified colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to quantify cell viability/proliferation. It is a widely used in vitro assay for the measurement of cell proliferation that determines a reduction in cell viability (which equates with a reduction in cell numbers and therefore proliferation), based on the reduction of the tetrazolium salt by actively growing cells to produce a blue formazan product.
The MTT assay was purchased from Sigma (Sigma-Aldrich, Pool, Dorset) and standard manufacturer's protocol was followed for cell incubations and spectrophotometric determination of purple formazan dye. Briefly, cells were incubated in 96 well microplates in the appropriate complete medium with standardized densities/well for 24 h as a pre-incubation process. The medium was then removed and replaced by medium containing different CLA concentrations. After 24 h of treatment the medium was removed and cells were allowed to grow for an additional 48 h in new, fresh medium without CLA. After this period of time, MTT was added to each well (20 µl of 5 mg/ml in PBS solution) and plates were incubated for 4 h at 37°C. Absorbance was measured at 570 nm. Data were analysed by plotting cell numbers versus absorbance, allowing quantification of changes in cell viability/proliferation (Biolinx software, 2.2, Dynatech Lab., Billinghouse, West Sussex, UK). Ethanol and albumin containing media were used as controls. Three separate experiments were performed in triplicate (x9 observations) to obtain mean cell proliferation. The results are presented as mean ± standard error of the means (SEM).
Northern blot analysis (mRNA)
Total RNA was extracted from the cultured cells by the acid guanidinium thiocyanatephenolchloroform procedure of Chomczynski and Sacchi (21). RNA (10 µg) from each sample was subjected to electrophoresis on 1% agaroseformaldehyde gels to separate RNA species and transferred to a nylon membrane (Boehringer-Manheim, Manheim, Germany) by capillary blotting. RNA was fixed to the membrane by exposure to UV light and the membranes were stored dry until required. Membranes were pre-hybridized for at least 6 h at 42°C with 0.1 mg denatured salmon sperm DNA/ml in 50% (v/v) formamide, 10% (w/v) dextran sulfate, 0.2% (w/v) bovine serum albumin, 0.2% (w/v) polyvinylpyrrolidone, 0.2% (w/v) Ficoll, 0.1% (w/v) sodium pyrophosphate, 1% (w/v) SDS and 50 mM TrisHCl, pH 7.5. Finally, the membranes were hybridized for 24 h at 42°C with specific oligonucletide probes synthesized by Sigma Genosys (Cambs, UK) for cyclooxygenase-1 (5'-CCAGGGGCAGCCTGGATCCCTGCCCTTCTCAAGA-3'), cyclooxygenase-2 (5'-CTCGCCCGCG CCCTGCTGCTGTGCG CGGTCCTGG-3'), 5-lipoxygenase (5'-CAGTGGT TCGCCGGCACTGACGACTACATVTACC-3'), bcl-2 (5'-CACGCCCCATCCAGCATCCCGCGACCCGGTCG-3') and p21WAF1/Cip1 (5'-CAAAGGCCCGCTCTACATCTTCTGCCTTAGTCTCA-3') labelled with [32P]deoxycytidine triphosphate by 5-end labelling using T4 polynucleotide kinase from New England Biolabs (Hitchin, UK). Membranes were then washed to remove non-specifically bound probe and specific hybridization was then detected by electronic autoradiography using a Camberra Packard Instantimager (Packard, Pangbourne, Berks, UK) and densitometric analysis. After analysis, individual membranes were stripped by washing in 0.1% (w/v) SDS for 510 min at 95°C before re-hybridization to other specific probes.
The housekeeping 18 S probe was used last in order to correct any variation between loading of RNA on the gel or transfer to the nylon membrane.
Western blot analysis (protein)
After 24 h of treatment, cells were lysed in buffer [20 mM Tris pH 7.5, 0.25 M sucrose, 10 mM EGTA, 2 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium ß-glycerophosphate, 50 mM sodium fluoride, 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich, Poole, Dorset, UK) and 1% (v/v) Triton X-100]. The lysate was centrifuged at 14 000 r.p.m. and 4°C for 30 min, and the cleared supernatants were stored at 80°C until use. Protein concentrations were determined using the Bio-Rad (Hempstead, UK) assay.
Samples containing equal amounts of proteins (20 µg) were then subjected to electrophoretic fractionation on a 10% polyacrylamide gel under reducing conditions. Separated fractions were transferred onto a polyvinylidene difluoride (PVDF) membrane (Hybond-P, Amershan Pharmacia Biotech, Buckinghamshire, UK) by using a semi-dry transfer method (Transblot SD, Bio-Rad). Membranes were blocked with TBS-T (Tris-buffered saline0.1% Tween 20) containing 5% non-fat milk and incubated for 1 h with the human-specific antibodies (COX-2 and 5-LOX from Transduction Laboratories, Becton Dickinson, Oxford, UK) and COX-1, bcl-2 and p21WAF1/Cip1 from Santa Cruz Biotechnology (Santa Cruz, CA); they were then diluted according to the manufacturer's instructions. Following incubation with the antibodies, the membranes were washed three times for 5 min in TBS-T and incubated with the specific Horseradish peroxidase conjugated secondary antibodies (Santa Cruz Biotechnology) following the manufacturer's instructions. After washing two times for 5 min with TBS-T and one time with TBS, specific proteins were detected using an enhanced chemiluminescence system (Super Signal, Pierce Chemical, Rockford, IL).
Ponceau S and Coomasie brilliant blue (Sigma-Aldrich, Poole, Dorset, UK) staining were used for markers of the protein loading and protein transfer control, respectively.
Statistical analysis
For all groups, data are presented as the mean of the different experiments ± standard error of the mean (SEM). Comparison of mean values was assessed by one-way ANOVA followed by a post hoc Duncan's test. P values <0.05 were considered significant. Data were analysed using SPSS statistical software package (SPSS for Windows, 11.0.1, 2001; SPSS, Chicago, IL).
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Results |
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In contrast to the CLA-mix, c9,t11 CLA did not produce any effect on mRNA levels of p21WAF1/Cip1 at any concentration (Table II).
Treatment with trans-10, cis-12 CLA elicited a significant increase in mRNA levels of p21WAF1/Cip1 (Table II) (P < 0.05), with respect to the control values at 50, 100 and 150 µM (about a 23, 45 and 60% increase, respectively).
Effect of CLA on PC-3 proliferation (MTT assay)
Figure 5 summarizes the doseresponse effects of the CLA-mix and the individual isomers (c9,t11 CLA and t10,c12 CLA isomers) on the proliferation of PC-3 cancer cells in vitro. At 25 µM there were no effects on PC-3 proliferation with the three treatments. Mixed isomers of CLA (CLA-mix) were able to decrease prostate cancer cell proliferation at 100 and 150 µM (79.9 ± 3.3% of control and 59.3 ± 2.0% of control, respectively). Similar to the CLA-mix, the c9,t11 CLA isomer was only able to decrease PC-3 proliferation at 100 and 150 µM, although to a lesser degree than the mixture of isomers (12 and 20% inhibition, respectively). The only fatty acid that could decrease prostate cancer cell proliferation at 50 µM was the t10,c12 CLA isomer (about a 17% inhibition). This isomer was also the one that elicited the highest, significant decrease in PC-3 proliferation at 100 and 150 µM (66.0 ± 3.6% of control and 45.8 ± 4.3% of control, respectively) (P < 0.05).
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Discussion |
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The literature relating to CLA and its effects on prostate cancer is not extensive (1820) and although it has been shown that CLA has anti-proliferative effects in vitro in human prostate cancer cells and in vivo in animal models of human prostate cancer (1820), the underlying cell mechanisms are not well understood. Therefore, a better understanding of the molecular anticarcinogenic cell mechanisms affected by CLA mixtures and their constituent isomers in this type of cancer is important if we want to develop suitable dietary supplements for cancer prevention or for patients undergoing cancer treatment.
The colorimetric MTT assay was used to quantify cell viability and reduced proliferation, using a long-term incubation (1 day plus 2 days), which allows the determination of cells that remain viable and are capable of proliferating and those that remain viable but can not proliferate and/or the detection of delayed programmed cell death (21). Our results show a decrease in PC-3 proliferation elicited by CLA, although significant differences were observed between the isomers studied.
However, the MTT assay, although widely used to measure cell proliferation, will only give general information about the viability of cells and little about the possible mechanisms that could change the cell capacity for proliferation and viability. Therefore, in an effort to try to clarify the possible basic mechanisms of these different anti-proliferative properties of CLA, we have studied for the first time, their effects on gene expression of different enzymes and proteins involved in arachidonic acid metabolism (COX-1, COX-2 and 5-LOX), apoptosis (bcl-2) and cell cycle (p21WAF1/Cip1), all regarded as important pathways involved in the oncogenesis and progression of prostate cancer (49).
Increased eicosanoid biosynthesis in relation to enhanced prostate cancer development has been documented (4,6) making the enzymes involved in this metabolic pathway an attractive new target area for prostate cancer therapy. The pathways for eicosanoid synthesis have received a great deal of attention as possible targets for cancer inhibition, especially COX-2 (5,23,24), and lipoxygenase products, especially 5-LOX metabolites (6,25,26). Previous reports showed that inhibition or attenuation of COX-2 or 5-LOX expression or activities by various agents decreased prostate cancer proliferation by different pathways, such as apoptosis and cell cycle control (46,2326). Modulation of eicosanoids synthesis has been proposed as one of the major mechanisms by which CLA can elicit its anticancer effects (1417). However, information about CLA effects on gene expression of major enzymes involved in arachidonic acid metabolism to eicosanoids in cancer cells/tissues is extremely sparse.
In the experimental conditions reported here the mixture of CLA isomers used was only able to decrease the gene expression of 5-LOX at the highest concentration used and did not show any effect on cycloxygenase gene expression (COX-1 and COX-2) at this concentration. This effect appeared to be largely due to the cis-9, trans-12 CLA isomer in the mix which, when used alone, decreased 5-LOX mRNA at 100 and 150 µM and COX-2 protein levels but was without effect on COX-2 mRNA at the highest concentration. This could be due to instability of the COX-2 mRNA (27). In contrast, trans-10, cis-12 did not have any inhibitory effect on gene expression of these enzymes. Even at the highest concentrations used there was actually a non-significant increase in COX-2 protein. These results, in part, agree with those obtained by other authors who showed differences between these isomers in their effects on eicosanoid synthesis. They indicate that the main reason for reduced product formation is likely to be reduced gene expression of the important COX and LOX enzymes (28,29).
Another proposed mechanism by which CLA exerts its anticancer effects is through the induction of apoptosis (1417,30). Increased expression of Bcl-2, an important anti-apoptotic oncogene product, has been associated with the development of androgen-independent prostate cancer, and its down-regulation was associated with a decrease in prostate cancer proliferation (31). In our study, CLA was able to significantly decrease bcl-2 expression, which was regarded as an anticancer effect. This was apparently due largely to the trans-10, cis-12 isomer, which, when used individually, decreased bcl-2 mRNA and protein levels at 100 and 150 µM. Palombo et al. (19) showed recently that the trans-10, cis-12 isomer increased caspase 3 activity and induced apoptosis in PC-3 cells. However, our findings show an effect at a different level, specific oncogene expression, in the apoptotic cascade.
Finally, cell cycle checkpoints play a crucial role in maintaining tissue homeostasis. Loss of this control mechanism may contribute to the development of the malignant phenotype. Induction of p21WAF1/Cip1, a cyclin-dependent kinase inhibitor, which is capable of contributing to regulation of cell division (32), in prostate cancer cells, has been shown to reduce cell proliferation (33,34) and to induce apoptosis (33). This protein was shown by our group to be induced by CLA in breast cancer cells (30); however, there is no similar information for prostate cancer cells. In the present study we clearly show that CLA can also induce p21WAF1/CP1 in prostate cancer cells and that the trans-10, cis-12 CLA isomer is apparently responsible for this effect, even at 50 µM.
In conclusion, our results show an anti-proliferative, anti-viability effect of CLA on the androgen-independent human prostate cancer cell line PC-3. However, the anti-proliferative/anticancer effects of CLA mixtures and their constituent isomers on this cell line are not equivalent. These differences are apparently due to the different pathways modulated by the individual isomers. The trans-10, cis-12 CLA isomer appears to elicit the greatest effect and apparently works preferentially through modulation of genes involved in apoptosis and cell cycle control. The c9,t11 CLA isomer, in contrast, elicits its effects through regulation of genes involved in arachidonic acid metabolism and the subsequent attenuation of eicosanoid synthesis. These results are important because they highlight the fact that cancer is not a disease with a single aetiology for which there will be a single cure (7). CLA has clearly been implicated as a possible dietary factor for reducing both breast and prostate cancer through studies with animal models of the human disease and human cell studies in vitro (see above). If CLA is to be regarded as a suitable dietary adjuvant cancer therapy, it is important to understand the type of cancer cells targeted, the specific cell mechanisms affected and the type and concentration of CLA isomer to be used. In the case of androgen-independent prostate cancer cell it seems that the best CLA isomer for a possible adjuvant cancer therapy is the trans-10, cis-12 isomer, at least in our conditions.
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Acknowledgments |
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References |
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