ORIGINAL ARTICLE. R Graydon 1, SECM Gilchrist 1, IS Young 1, U Obermüller-Jevic 2, O Hasselwander 2 and JV Woodside 1.

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1 (2007) 61, & 2007 Nature Publishing Group All rights reserved /07 $ ORIGINAL ARTICLE Effect of lycopene supplementation on insulin-like growth factor-1 and insulin-like growth factor binding protein-3: a double-blind, placebo-controlled trial R Graydon 1, SECM Gilchrist 1, IS Young 1, U Obermüller-Jevic 2, O Hasselwander 2 and JV Woodside 1 1 Nutrition and Metabolism Group, Centre for Clinical and Population Science, Queen s University Belfast, Northern Ireland, Belfast, UK and 2 BASF Aktiengesellschaft, Ludwigshafen, Germany Objective: Studies have suggested a link between lycopene and insulin-like growth factor-1 (IGF-1). The aim of this study was to test the effect of lycopene supplementation on IGF-1 and binding protein-3 (IGFBP-3) status in healthy male volunteers. Design, setting, subjects and intervention: This was a 4 week randomized, double-blind, placebo-controlled study of lycopene supplementation (15 mg/day) in healthy male volunteers (n ¼ 20). Fasting blood samples were collected at baseline and after 4 weeks. Samples were analysed for lycopene by high-performance liquid chromatography (HPLC) and IGF-1 and IGFBP-3 by enzyme-linked immunosorbent assay (ELISA). Changes in end points from baseline were compared in those who received placebo versus those who received the lycopene supplement. Results: Median change in lycopene from baseline (post-supplement baseline) was higher in subjects in the intervention than those on placebo (lycopene group 0.29 (0.09, 0.46); placebo group 0.03 ( 0.11, 0.08) mmol/l; median (25th, 75th percentiles), Po0.01). There was no difference in median change in IGF-1 concentrations (lycopene group 0.6 ( 2.6, 1.9); placebo group 1.15 ( 2.88, 0.95) nmol/l, P ¼ 0.52), or median change in IGFBP-3 concentrations (lycopene group 245 ( 109, 484); placebo group 101 ( 34, 234) nmol/l, P ¼ 0.55) between intervention and control groups. Change in lycopene concentration was associated with the change in IGFBP-3 in the intervention group (r ¼ 0.78; P ¼ 0.008; n ¼ 10). Conclusions: Lycopene supplementation in healthy male subjects has no effect on IGF-1 or IGFBP-3 concentrations in a healthy male population. However, the association between change in lycopene concentration and change in IGFBP-3 in the intervention group suggests a potential effect of lycopene supplementation on IGFBP-3. Sponsorship: University-funded. (2007) 61, ; doi: /sj.ejcn ; published online 7 February 2007 Keywords: lycopene; insulin-like growth factor-1 (IGF-1); insulin-like growth factor binding protein-3 (IGFBP-3) Introduction Correspondence: Dr JV Woodside, Nutrition and Metabolism Group, Centre for Clinical and Population Science, Queen s University Belfast, Mulhouse Building, Grosvenor Road, Belfast BT12 6BJ, UK. j.woodside@qub.ac.uk Guarantor: JV Woodside. Contributors: RG completed the subject recruitment, laboratory analysis and drafted the manuscript, SECMG contributed to the laboratory analysis, developed the laboratory method for lycopene, and contributed to the current manuscript. ISY devised the original study design and contributed to current manuscript. UOJ and OH contributed to the study design and the current manuscript. JVW devised the original study design, carried out the statistical analyses, and contributed to the current manuscript. Received 27 April 2006; revised 24 November 2006; accepted 27 November 2006; published online 7 February 2007 It has been suggested that people who consume more tomatoes and tomato-based products are less likely to suffer from certain chronic diseases and cardiovascular disease (CVD) than those with low intakes (Rao and Agarwal, 1999). The mechanism by which lycopene may protect against chronic disease has yet to be established, but has been proposed to be through effects on insulin-like growth factor- 1 (IGF-1). A recent systematic review showed circulating IGF-1 to be associated with increased risk of prostate and pre-menopausal breast cancer (Renehan et al., 2004). A number of studies have suggested a link between lycopene

2 and IGF-1-stimulated cell proliferation in vitro (Levy et al., 1995; Karas et al., 2000), and an inverse association has been demonstrated between cooked tomato consumption and circulating IGF-1 concentrations in vivo (Mucci et al., 2001; Gunnell et al., 2003). Other studies have, however, failed to show an association between circulating IGF-1 and lycopene concentrations (Graydon et al., 2003; Vrieling et al., 2004). To date, no placebo-controlled intervention study has examined the effect of lycopene supplementation on IGF- 1, but the need for this research has been highlighted (Voskuil et al., 2005). The aim of this placebo-controlled study was to determine the effect of 4-weeks lycopene supplementation on IGF-1 or IGFBP-3 concentrations in healthy male volunteers. Materials and methods Subjects and study design The study was a 4-week randomized, double-blind, placebocontrolled study in healthy male volunteers, aged years, recruited from hospital staff and students. Volunteers gave a fasting blood sample at the outset of the study, and they were then randomized (using computer-generated random numbers) to receive either a lycopene supplement (15 mg) or placebo every day for 4 weeks. The lycopene supplement was synthetic in source, purity 496% and with an all-trans isomer content of 470%. The chosen dose was based on Hoppe et al. (2003), which, using the same does had resulted in a significant plasma increase in humans. Compliance was monitored by the return of the supplement or placebo packets at the end of the intervention period and counting of unused tablets. Subjects were asked not to change their diet during the course of the study, and did not take any other dietary supplements. At the end of the intervention another fasting blood sample was taken. Serum samples were kept in the dark for 1 h (plasma for 30 min at 41C) and separated by centrifugation. All samples were stored at 801C until analysis. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Queen s University, Belfast who follow the guidelines of the Royal College of Physicians, London. Lycopene assay and IGFBP-3 assay Lycopene was assessed using high performance liquid chromatography (HPLC) with diode array detection following extraction into heptane (Craft et al., 1992). The assay was reproducible (intra-batch CV ¼ 7.8%, inter-batch CV ¼ 12.8%), and was standardized against the appropriate NIST standard reference material. IGF-1 assay IGF-1 concentrations in serum were measured by enzymelinked immunosorbent assay (ELISA) (Immunodiagnostic Systems Ltd, UK). IGFBP-3 assay IGFBP-3 concentrations in serum were measured by ELISA (Immunodiagnostic Systems Ltd, UK). Statistical methods The change in lycopene, IGF-1 and IGFBP-3 was calculated (post-pre) and compared using the Mann Whitney U-test, owing to the small numbers of comparisons. Associations between continuous variables were tested by calculation of Spearmans rank correlation coefficients. A P-value of o0.05 for considered to be statistically significant. All statistical analyses were performed using SPSS for Windows, version Results A total of 20 subjects were recruited on to the study and all 20 recruited subjects completed the 4-week intervention. Table 1 shows the number of volunteers assigned to each intervention group, their median age and the percentage of whom smokers at baseline. None of these parameters differed between intervention groups. Table 2 shows the baseline median concentrations of lycopene, IGF-1, IGFBP-3 and IGF-1/IGFBP-3 ratio for the two intervention groups. There were no significant differences in these baseline parameters between intervention groups. Table 3 shows the median change in concentration of lycopene, IGF-1, IGFBP-3 and IGF-1/IGFBP-3 ratio for the two intervention groups. Subjects receiving lycopene supplementation had a significantly larger change in lycopene than those receiving placebo. There was no significant difference in the change in any other end point assessed between lycopene and placebo groups. Table 1 Baseline characteristics of study subjects by intervention group Lycopene Placebo N Age (years) 32 (26,41) 39 (28,48) % smokers Age shown as median (IQ range). Table 2 Median concentrations of lycopene, IGF-1, IGFBP-3 and IGF-1/ IGFBP-3 by intervention group at baseline Lycopene Placebo Lycopene (mmol/l) 0.38 (0.26,0.61) 0.48 (0.26,0.69) IGF-1 (ng/ml) (9.23,12.23) (11.75,16.68) IGFBP-3 (ng/ml) 4692 (4174,4966) 4907 (4234,5356) IGF-1/IGFBP (0.0019,0.0027) (0.0025,0.0032) Data presented as median (IQ range). 1197

3 1198 Table 3 Median change in lycopene, IGF-1, IGFBP-3 and IGF-1/IGFBP-3 during 4-week intervention period However, when the association between changes in lycopene, IGF-1 and IGFBP-3 were compared by intervention group, a significant association between change in IGFBP-3 and change in lycopene was demonstrated in the lycopene intervention group (see Figure 1). Discussion Lycopene Placebo P-value Lycopene (mmol/l) 0.29 (0.09,0.46) 0.03 ( 0.11,0.08) IGF-1 (ng/ml) 0.60 ( 2.6,1.9) 1.15 ( 2.88,0.95) 0.52 IGFBP-3 (ng/ml) 245 ( 109,484) 101 ( 34,234) 0.55 IGF-1/IGFBP-3 ( 10 3 ) 0.2 ( 0.5,0.3) 0.3 ( 0.6,0.2) 0.60 Lycopene and placebo groups were compared using a non-parametric Mann Whitney U-test. Data presented as median (IQ range). change in IGFBP-3 (ng/ml) change in lycopene (umol/l) r=0.78; p=0.008 Figure 1 Association between change in lycopene and change in IGFBP-3 in subjects receiving the lycopene supplement (15 mg/day). This randomized, double-blind intervention study has shown no significant effect of lycopene supplementation on either IGF-1 or IGFBP-3 in healthy male volunteers. However, the change in lycopene concentration in the lycopene intervention group was significantly positively associated with the change in IGFBP-3. In recent years, it has become widely accepted that the consumption of fruit and vegetables reduces the risk of a number of chronic diseases (Woodside et al., 2005). This is thought to be largely owing to their relatively high antioxidant potential, that is being able to protect tissue against free-radical-induced damage. Interest has focussed on lycopene (found mostly in tomatoes and tomato products), owing to its proposed antioxidant activity and the fact that lycopene has been shown to have both anticancer and anti-atherosclerotic properties, both in vitro, in vivo and in epidemiological studies. IGF-1 is thought to be a risk factor for breast and prostate cancer (Hankinson et al., 1998; Stattin et al., 2000), and this has been confirmed for premenopausal breast cancer and prostate cancer by metaanalysis (Renehan et al., 2004). A number of studies have suggested that lycopene s effect on chronic disease may be mediated through effects on IGF-1. One study (Levy et al., 1995) compared the effect of lycopene on cancer cell proliferation with a-carotene or b-carotene. Lycopene was much more effective than the other two compounds in inhibiting human breast, endometrial and lung cancer IGF- 1-stimulated cell growth. Another study (Karas et al., 2000) investigated the effect of lycopene on cell cycle progression and IGF-1 signalling in breast cancer cells. Lycopene inhibited IGF signalling, which was associated with IGFstimulated cell cycle progression. Recent work suggests that the inhibitory effect of lycopene on IGF-1 mitogenesis is mediated via attenuation of cyclin D1 levels (Nahum et al., 2006). No direct inference can be drawn from in vitro studies about circulating lycopene and IGF-1 concentrations. However, an epidemiological study (Mucci et al., 2001) examining the interaction of factors associated with prostate cancer risk found that there was a significant inverse association between cooked tomatoes and IGF-1. Similarly, another cross-sectional analysis in 344 disease-free men suggested a weak association between tomato consumption and circulating IGF-1 (Gunnell et al., 2003). However, a small trial of lycopene supplementation (over 3 weeks) found no effect on IGF-1 concentration, (which fell in both lycopene and control groups) in prostate cancer patients (Kucuk et al., 2001). This trial was, however, methodologically weak in that it was not placebo-controlled. The authors state that the fall in IGF-1 of control subjects could be owing to changes in their diet and lifestyle (Kucuk et al., 2001). All subjects had been told to increase their fruit and vegetable intake according to the National Cancer Institute s 5-a-day guidelines, and in fact lycopene concentrations increased significantly in both the intervention and control groups. Unfortunately, no data was collected on the subjects diet during the study period (Kucuk et al., 2001). Similarly, no data was collected on dietary intake of lycopene in the current study, although the inclusion of a placebo group in the current study reduces the likely impact of dietary changes during the study on findings. One other study has examined the effect of a tomato drink intervention on IGF-1 concentrations in healthy subjects. Although this was a food-based intervention, there was a placebo drink, and serum increases in lycopene were similar to those in the current study. There were no significant effects of the tomato drink intervention on IGF-1, but the change in lycopene was significantly associated with change in IGF-1. IGFBP-3 was not assessed in the study (Riso et al., 2006). This suggests some effect of lycopene intervention on

4 IGF-1, in contrast to the study reported here which only shows an effect on IGFBP-3. Given the findings of these previous studies, our aim was therefore to investigate whether placebo-controlled lycopene supplementation affected IGF-1 status in healthy male volunteers. However, when IGF-1 was measured pre- and post-supplement, there was no significant difference in change in IGF-1 between the intervention groups. The reason for this contrast with Riso et al. (2006) is unclear, as study designs in these studies were similar (apart from using a food-based rather than supplemental lycopene intervention). It has been proposed that the effect of lycopene on the IGF axis is mediated through IGFBP-3, as Gunnell et al. (2003) showed stronger associations between tomato consumption and the IGF-1/IGFBP-3 molar ratio, whereas Giovannucci et al. (2003) also showed an association between lycopene intake and circulating concentrations of IGFBP-3, but not IGF-1. In the current study, there was no significant difference in change in IGFBP-3 after lycopene supplementation, but the changes in IGFBP-3 in the intervention group were significantly associated with changes in lycopene concentrations, implying some effect of lycopene on IGFBP-3. A recent study (Liu et al., 2003) found that ferrets exposed to cigarette smoke and supplemented with lycopene had a significantly higher IGFBP-3 concentration and a lower IGF-1/IGFBP-3 ratio than ferrets exposed to smoke alone. They also found that lycopene supplementation inhibited smoke-induced squamous metaplasia in the lungs of ferrets. This suggests lycopene may have a role to play in cancer prevention by up-regulating IGFBP-3. IGFBP-3 has been proposed to have both IGF-dependent and IGF-independent anti-proliferative and pro-apoptotic effects which may relate to reduced cancer risk (Liu et al., 2003). We observed a heterogeneous response of serum lycopene concentrations to lycopene supplementation, and this is likely to account for our finding of no significant difference in IGFBP-3 between intervention and control groups. Percentage of baseline serum lycopene after the intervention period in the lycopene intervention group varied between 85 and 316% with a median value of 133%. The reason for this heterogeneous response is not known, as compliance, as assessed by return of empty supplement packets, was high. The change in lycopene concentration did not appear to depend on baseline lycopene concentrations, but was lower than previously reported in a study by Hoppe et al. (2003). In this study, supplementation with 15 mg lycopene resulted in a 0.58 mmol/l mean change after 4 weeks (Hoppe et al., 2003), whereas the current study observed a median increase in serum lycopene of 0.29 mmol/l. In summary, a 4-week double-blind, placebo-controlled intervention trial showed no significant effect of lycopene supplementation (15 mg/day) on IGF-1 or IGFBP-3. However, in subjects receiving the lycopene supplement, the change in IGFBP-3 was significantly associated with change in lycopene, suggesting a possibly beneficial effect of lycopene on IGFBP-3, and this should be tested in further well-designed intervention trials. Acknowledgements The lycopene and placebo capsules were kindly supplied by BASF Aktiengesellschaft, Ludwigshafen, Germany. We thank to Mr Alistair Magill for assistance with the IGF-1 assay. References Craft NE, Wise SA, Soares JH (1992). Optimisation of an isocratic high performance liquid chromatography separation of carotenoids. J Chromatogr 589, Giovannucci E, Pollak M, Liu Y, Platz EA, Majeed N, Rimm EB et al. (2003). Nutritional predictors of insulin-like growth factor I and their relationships to cancer in men. 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