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Departments of Physiological Science (T.H.N., R.J.B., P.-S.L.), Pediatrics (P.C.), and Urology (W.J.A.), University of California, Los Angeles, California 90095
Address all correspondence and requests for reprints to: R. James Barnard, Ph.D., Department of Physiological Science, University of California, 4303 Life Science Building, 621 Charles Young Drive South, Los Angeles, California 90095. E-mail: jbarnard{at}physci.ucla.edu.
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Introduction |
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Not only is the prostate an androgen-dependent gland, it also depends on several circulating and local growth factors for normal development (5, 6). We previously reported that placing men on a low fat diet along with daily aerobic exercise reduced serum levels of free testosterone, estradiol, and insulin (7). These changes in serum hormones in vivo were associated with a significant reduction in serum-stimulated LNCaP cell growth in vitro (8). When the hormones were added back to the post-diet and exercise (post-DE) serum, individually or in combination, and LNCaP cells were grown in medium containing patient serum, there was still a significant reduction in LNCaP cell growth compared with the preintervention serum (7). These results suggested the involvement of other mitogenic factors. Epidemiological data have implicated IGF-I as a risk factor for prostate cancer (9, 10, 11). Thus, we studied the effects of diet and exercise on the IGF axis. Serum IGF-I was significantly reduced, whereas IGF binding protein-1 (IGFBP-1) was significantly increased (12). Serum IGFBP-3, which normally binds 95% of IGF-I in the circulation, was unchanged (12). The observed changes in IGF-I and IGFBP-1 were correlated with the reduction in serum-stimulated LNCaP cell growth and associated with the induction of apoptosis. These results suggest that serum IGF-I and IGFBP-1 levels may be important biomarkers for prostate cancer risk. Therefore, an understanding of the mechanisms of action of IGF-I and IGFBP-1 would be crucial to determine whether they may be specific mediators for the effects of diet and exercise on prostate cancer cell growth and apoptosis. The current study extends our work on IGF-I and IGFBP-1 and specifically investigates the mechanism of IGFBP-1 action on LNCaP cell growth and apoptosis using IGF-I receptor (IGF-IR)-neutralizing antibodies (IGF-IR-Ab) and IGF-I analogs with significantly reduced affinity for IGFBPs (13, 14) or IGF-IR (15). It is important to determine how IGFBP-1 works, because our previous findings (8) indicate that IGF-I and IGFBP-1 are two important factors whose serum levels are greatly affected by diet and exercise and exhibit opposite effects on prostate cancer cell growth as well as apoptosis.
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Materials and Methods |
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10% kcal), high fiber (35–40 g/1000 kcal) diet and performed 30–60 min of aerobic exercise daily at the Pritikin Longevity Center residential program, as previously described (8). Twelve-hour fasting morning serum was obtained from each subject before and after the 11-d intervention. The study subjects did not have prostate cancer, and their anthropometric and lipid profiles have previously been reported (12).
Cell culture/bioassay
LNCaP cells from American Type Culture Collection (Manassas, VA) were grown in 75-cm2 flasks (Falcon, BD Biosciences, Franklin Lakes, NJ) in RPMI 1640 medium without phenol red, supplemented with 10% fetal bovine serum (FBS), 200 IU penicillin, 200 mg/ml streptomycin, and 4 nM L-glutamine (Omega Scientific, Inc., Tarzana, CA). The cultures were maintained in a humidified incubator (37 C, 5% CO2) and passaged routinely at 80% confluence. Cells were collected using 0.25% trypsin-EDTA (Sigma-Aldrich Corp., St. Louis, MO), and a limited number of passages (not >10) were used for experiments performed in 5 replicates. LNCaP cells (5 x 103) were plated in each well of a 96-well tissue culture plate (Falcon) containing 100 µl 10% FBS medium and allowed to attach for 24 h. After 24 h, medium was replaced with serum-free medium (SFM), and treatments [FBS, subject serum, IGF-I, IGFBP-1, long-R3-IGF-I (LR3), AL31Leu60-IGF-I (Leu60), 
-IR3, and/or antihuman IGF-IR antibody] were added to constitute a total of 100 µl in each well. The doses of IGF-I, IGFBP-1, LR3, and/or Leu60 had been adjusted to the 10% of serum in the medium. Doses of IGF-I and IGFBP-1 added reflect the serum changes resulting from diet and exercise. After treatment, the cells were incubated (37 C, 5% CO2) for 48 h. Cell growth was assessed by the MTS assay (Promega Corp., Madison, WI). Cell growth after diet and exercise was also studied using LAPC-4, androgen-dependent prostate cancer cells.
IGF-I was a gift from Dr. Barbara Lippe (Pharmacia Biotech, Stockholm, Sweden), and IGFBP-1 was a gift from Dr. Paul Fielder (Genentech, Inc., South San Francisco, CA). Additional recombinant human IGF-I (I-3769) and phosphorylated IGFBP-1 (I-0524) were purchased from Sigma-Aldrich Corp. Long-R3-IGF-I and Leu60 were purchased from GroPep (Adelaide, Australia). Two IGF-IR-Ab (clones -IR3 and 33255.111) were purchased from Oncogene (Boston, MA).
Apoptosis study
Apoptosis was determined after the 48-h incubation period by annexin V and terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling assays (Roche Molecular Biochemicals). Medium was aspirated, and 50 µl staining solution (annexin V-fluorescein, propidium iodide, and binding buffer) was added to each well and incubated for 15 min at 25 C. Annexin V-fluorescein binds to negatively charged membrane phosphatidylserine and stains apoptotic as well as necrotic cells. Propidium iodide stains DNA of leaky necrotic cells only. For the TUNEL assay, cells were first fixed with 50 µl paraformaldehyde solution (4% in PBS, pH 7.4) for 1 h at 25 C. Each well was then washed with 200 µl PBS and permeabilized with 50 µl permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice (4 C). Each well was washed again with PBS and aspirated completely. Twenty microliters (20 µl) of TUNEL reaction mixture containing fluorescein-deoxy-UTP were added to each well and incubated in a humidified chamber for 60 min at 37 C in the dark. Fluorescein-deoxy-UTP labels DNA strand breaks and allows the direct detection of DNA fragmentation by fluorescent microscopy. Cells were detected at x20 using an inverted fluorescent microscope (Axiovert 135M, Carl Zeiss, New York, NY). Images were digitized with an attached SPOT Digital Camera (Diagnostic Instruments, Inc., Sterling Heights, MI). Apoptotic and necrotic staining were quantified by pixel histogram (Adobe Systems, Mountain View, CA) and confirmed by manual counting (r = 0.98).
Statistical analysis
Statistical analyses (InStat statistical software, GraphPad Software, Inc., San Diego, CA) were performed by ANOVA, followed by Newman-Keuls post hoc analyses. P < 0.05 was considered significant. Data are expressed as the mean ± SE.
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Results |
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). Post-DE subject serum had lower IGF-I and higher IGFBP-1 levels compared with baseline levels, as reported previously (12). Adding IGFBP-1 to baseline serum at concentrations of 20 ng/ml (the reduction of serum IGFBP-1 over 11 d of diet and exercise) and 50 ng/ml (reduction with 14 yr DE) reduced LNCaP cell growth by 23% and 29%, respectively (Fig. 1). The addition of IGFBP-1 to baseline serum also induced apoptosis of LNCaP cells (Fig. 2). Adding the IGF-IR blocker to baseline serum reduced LNCaP cell growth from 100 ± 8% to 62 ± 5% FBS at 10 nmol/ml and down to 33 ± 2% FBS at a 100 nmol/ml serum concentration. Addition of IGF-I to post-DE serum at concentrations of 60 ng/ml (11-d diet and exercise reduction) and 200 ng/ml (14-yr diet and exercise reduction) stimulated LNCaP cell growth significantly and was dose dependent (Fig. 1). Tests conducted using LAPC-4 cells showed that cell growth was reduced from 92 ± 4% FBS at baseline to 62 ± 2% FBS after 11 d of DE. These results all point to an important role for the IGF axis in prostate cancer development.
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). IGFBP-1 significantly reduced the stimulatory effect of IGF-I when the two factors were added together to LNCaP cell cultures. Addition of IGF-I resulted in a minimal level of apoptosis (Fig. 4), whereas substantially elevated levels of apoptosis were seen when IGFBP-1 was added alone or in combination with IGF-I.
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-IR3, was used as a negative control. LR3 has significantly reduced affinity for IGFBPs, but still binds the IGF-IR. Leu60 has significantly reduced affinity for the IGF-IR, but still binds IGFBPs. IGF-I, LR3, and Leu60 (60 ng/ml) stimulated LNCaP cell growth beyond FBS-stimulated levels (Fig. 5). When added in combination, IGFBP-1 significantly attenuated the stimulatory effects of IGF-I, LR3, and Leu60 on FBS-stimulated LNCaP cell cultures. However, the effect of IGFBP-1 on LR3-stimulated growth was significantly less than that on IGF-I and Leu60 (Fig. 5), and the level of growth due to LR3/IGFBP-1 was significantly higher than those due to IGF-I/ IGFBP-1 and Leu60/IGFBP-1. The level of growth with LR3 plus IGFBP-1 was not significantly different from that with IGF-I alone. -IR3 effectively blocked IGF-stimulated growth signaling.
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). When added in combination, IGFBP-1 induced a substantial amount of apoptosis in the presence of IGF-I and Leu60, but not LR3. The level of apoptosis due to the LR3/IGFBP-1 combination was not statistically different from the level due to LR3, IGF-I, or Leu60 alone, but was significantly different from the levels due to IGF-I/IGFBP-1 and Leu60/IGFBP-1. The -IR3 induced significant apoptosis of LNCaP cells.
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-IR3 was also used as a negative control. At a physiological serum concentration (100 ng/ml), IGF-I stimulated the growth of LNCaP cell cultures in 10% FBS to a greater extent than in SFM (Fig. 7). IGFBP-1 inhibited growth in FBS-stimulated cultures and to a lesser extent in SFM cultures. IGF-IR-Ab effectively inhibited FBS-stimulated growth and growth in SFM. IGF-I did not stimulate FBS or SFM LNCaP cell cultures in the presence of IGF-IR-Ab. IGFBP-1 did not induce further growth reduction in the presence of IGF-IR-Ab. Mouse IgG (10 µg/ml) and BSA (200 ng/ml) were also added to FBS and SFM LNCaP cultures as controls and were shown to have no inhibitory effect on LNCaP cell growth (data not shown).
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Discussion |
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IGF-I, however, did not stimulate LNCaP cell growth in SFM to the extent it did when added to FBS. This suggests that IGF-I is an important growth factor for serum-stimulated growth, and IGF-I may enhance the mitogenic effects of other serum factors. In SFM, LNCaP cells still slowly proliferate due to autocrine/paracrine IGF-I production and growth stimulation (16, 17). However, for optimal growth, other factors are required along with IGF-I (18, 19). Iwamura et al. (5) reported that IGF-I could not stimulate the growth of androgen-dependent LNCaP cells in steroid- and growth factor-free medium, which also supports the fact that IGF-I interacts with other serum factors to promote cell growth. It is, however, important to consider the enhancement of growth due to IGF-I addition in the presence of serum, which may more closely represent the role of IGF-I in the development of prostate cancer.
A number of IGFBPs have recently emerged as growth modulators themselves, possessing properties that are beyond their traditional roles as carrier proteins (20, 21, 22). In the present study IGFBP-1 antagonized the IGF-I growth stimulatory effects on LNCaP cell growth and induced apoptosis. We hypothesized that IGFBP-1 may have IGF-independent effects on LNCaP cell growth and apoptosis, and studied this by using IGF-IR-Ab and IGF-I analogs with altered binding affinities for IGFBPs (13, 14) or IGF-IR (15).
We demonstrated that IGF-I, LR3 (an IGF-I analog with reduced affinity for IGFBPs), and Leu60 (an IGF-I analog with reduced affinity for IGF-IR) all significantly stimulated LNCaP cells grown in 10% FBS. This is in accordance with the findings of Bryant et al. (23), who showed that LR3 or its muteins retained growth-promoting activity equivalent to or higher than that of IGF-I. Similarly, Clemmons et al. (24) found that in the absence of exogenous IGFBP-1, IGF-I analogs with reduced affinity for IGFBP-1 were more potent than IGF-I in stimulating smooth muscle cell DNA synthesis. Leu60 does not have a direct effect on the IGF-IR due to its loss of affinity for the receptor (15), but was found to enhance FBS-stimulated LNCaP cell growth. There are two possible mechanisms explaining the observed effects of Leu60. First, Leu60 may compete with IGF-I in the medium for IGFBPs, thus displacing more free IGF-I to act on LNCaP cells, as suggested by Luddick et al. (25) and Lowman et al. (26). Second, Leu60 may have an allosteric affinity for IGFBPs, especially IGFBP-1, resulting in the inactivation of these binding proteins. This has been reported for IGFBP-3 by Wu et al. (27).
As both IGF-I and Leu60 still bind IGFBPs with high affinity (15), the addition of exogenous IGFBP-1 to cultures containing these growth factors significantly reduced their growth stimulatory effects and induced apoptosis, compared with their individual effects without IGFBP-1. Thus, the IGF-dependent effect of IGFBP-1 was present, and IGFBP-1 appeared to be acting through sequestering circulating IGF-I and Leu60. The percentage of IGFBP-1 inhibition through this IGF-dependent mechanism, however, was unclear, and the presence and extent of IGF-independent activity of IGFBP-1 were also uncertain.
Interestingly, exogenous addition of LR3 alone resulted in the greatest level of LNCaP growth stimulation compared with that of IGF-I and Leu60. This may be due to the fact that LR3 has significantly reduced affinity for IGFBPs, but still binds IGF-IR (15). Thus, LR3 interaction with the IGF-IR was not influenced by IGFBPs in the medium. Adding IGFBP-1 together with LR3 to LNCaP cultures resulted in a significant growth reduction, but not a significant increase in apoptosis, compared with LR3 alone. The level of growth due to combined LR3 and IGFBP-1, however, was not statistically different from that with IGF-I alone. The level of growth due to LR3/IGFBP-1 was significantly higher compared with that with IGF-I/IGFBP-1 and Leu60/IGFBP-1, whereas the level of apoptosis was significantly lower.
The results using these analogs suggested that IGFBP-1 may have IGF-independent effects. As LR3 does not bind IGFBP-1, we expected that the addition of IGFBP-1 should have no effect on LNCaP cell growth if there was no IGF-independent effect and an inhibitory effect if there was an IGF-independent effect. The level of cell growth observed when IGFBP-1 was added along with LR3 was in the presence of 10% FBS. Although IGFBP-1 had no effect on LR3 activity, it may have sequestered some of the circulating IGF-I present in FBS. Reducing available growth factors would reduce cell growth, but only to a lesser extent, as when LR3 was present. Furthermore, the level of growth due to LR3/IGFBP-1 was not significantly differently from that with 60 ng/ml IGF-I alone, implying that the level of growth due to LR3/IGFBP-1 was still high and that the IGFBP-1 effect was minor. This is supported by the fact that IGFBP-1 addition to LR3 did not result in a significant increase in apoptosis compared with LR3, IGF-I, or Leu60 alone.
To investigate whether the effects of IGFBP-1 were mediated through the type I IGF receptor, an anti-IGF-IR-Ab was used. The experiment was performed in the presence or absence of FBS to confirm observations. The results showed that IGF-IR-Ab inhibited the growth of both FBS-stimulated and SFM LNCaP cell cultures. Adding IGF-I together with IGF-IR-Ab did not stimulate cell growth, confirming that IGF signaling was abolished. If IGFBP-1 had an IGF-independent effect on LNCaP cell growth, we would expect to see a further reduction of growth in the presence of IGF-IR-Ab. In contrast, we did not detect any further growth inhibition due to IGFBP-1 in the presence of IGF-IR-Ab. Thus, these results suggest that IGFBP-1 did not inhibit LNCaP prostate cancer cell growth via an IGF-independent mechanism, but acted mainly through sequestering bioactive IGF-I in the system (IGF-dependent effect).
Our findings are in contrast with the report by Perks et al. (28), who showed that IGFBP-1 could interact with integrin receptors to induce focal adhesion kinase (FAK) dephosphorylation and induce detachment and apoptosis of breast cancer cells. After demonstrating the similar effects of IGFBP-1, which contains RGD sequences, and a synthetic RGD-containing peptide on breast cancer cells, the researchers concluded that IGFBP-1 induced apoptosis of breast cancer cells via integrin-mediated IGF-independent effects. The main difference between our study and that by Perks et al. (28) is the amount of IGFBP-1 used. Perks et al. (28) used much higher concentrations (400 and 800 ng/ml) of IGFBP-1 in their study, whereas the highest amount of IGFBP-1 used in our study was 200 ng/ml. Furthermore, at 400 ng/ml IGFBP-1, Perks et al. (28) could not detect a significant level of FAK dephosphorylation compared with that without IGFBP-1. Significant FAK dephosphorylation was only detected with 800 ng/ml added IGFBP-1. Therefore, with a lower physiological amount of IGFBP-1 used in our study, we could not attribute the effects of IGFBP-1 on LNCaP prostate cancer cell growth and apoptosis to an integrin-mediated IGF-independent mechanism.
In conclusion, we demonstrate that IGF-I is an important factor in the serum for LNCaP prostate cancer cell growth and survival and that IGFBP-1 antagonizes its effect in an IGF-dependent fashion to induce apoptosis. The interaction of IGF-I with the IGF-IR is critical for LNCaP survival and proliferation, and IGFBP-1 functions to modulate IGF-I bioavailability. Because these two factors are regulated by nutrition and physical activity (12), IGF-I and IGFBP-1 may emerge as primary factors in determining prostate cancer risk and/or promotion. Our previous (7, 12) and present studies are the only ones to our knowledge to characterize the effects of a lifestyle modification, including a low fat, high fiber diet and daily aerobic exercise, on serum IGF-I and IGFBP-1 with special implications for prostate cancer prevention. The decrease in serum IGF-I and increase in circulating IGFBP-1 resulting from diet and exercise substantially reduce bioavailable IGF and prostate cancer cell growth. These experimental results support prospective studies where IGF-I had been reported to be a biomarker for predicting prostate cancer (29, 30, 31). IGF-I has also been reported to be a risk factor in case-control studies (32, 33, 34). In addition, one of the case-control studies (33) found that elevated IGFBP-1 reduced the risk of cancer. The elucidated mechanisms of IGF-I and IGFBP-1 actions on prostate cancer growth and apoptosis in vitro may serve as the basis for further animal and human trials on prostate cancer prevention via diet and exercise.
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Footnotes |
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Abbreviations: DE, Low fat diet and exercise intervention; FAK, focal adhesion kinase; FBS, fetal bovine serum; IGFBP-1, IGF binding protein-1; IGF-IR, IGF-I receptor; IGF-IR-Ab, IGF-IR-neutralizing antibody; Leu60, AL31Leu60-IGF-I; LR3, long-R3-IGF-I; SFM, serum-free medium.
Received October 3, 2002.
Accepted for publication February 12, 2003.
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