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Carcinogenesis Advance Access originally published online on June 13, 2006
Carcinogenesis 2007 28(1):28-37; doi:10.1093/carcin/bgl085
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Reactive oxygen species regulate insulin-induced VEGF and HIF-1{alpha} expression through the activation of p70S6K1 in human prostate cancer cells

Qiong Zhou{dagger}, Ling-Zhi Liu{dagger}, Beibei Fu, Xiaowen Hu, Xianglin Shi, Jing Fang* and Bing-Hua Jiang*

The Institute for Nutritional Sciences, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences Shanghai 200031, China

*To whom correspondence should be addressed. Email: bhjiang{at}sibs.ac.cn or jfang{at}sibs.ac.cn


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF-1) are important regulators of angiogenesis. HIF-1 is composed of HIF-1{alpha} and HIF-1ß subunits, and regulates VEGF expression at transcriptional level. In this study, we demonstrated that insulin induced H2O2 production and p70S6K1 activation in PC-3 prostate cancer cells. The inhibition of H2O2 production by catalase abolished insulin-induced p70S6K1 activation. H2O2 production is also required for insulin-induced VEGF and HIF-1{alpha} expression in the cells. Over-expression of p70S6K1 or HIF-1{alpha} reversed catalase- and rapamycin-inhibited VEGF transcriptional activation. These results suggest that insulin induced HIF-1{alpha} and VEGF expression through H2O2 production and p70S6K1 activation in prostate cancer cells. In addition, we found that inhibition of p70S6K1 by rapamycin decreased prostate tumor angiogenesis, suggesting that p70S6K1 plays an important role in tumor angiogenesis. These results provide some useful information for prostate cancer therapy in the future.

Abbreviations: HIF-1, hypoxia-inducible factor 1; MVD, micro-vessel density; PBS, phosphate-buffered saline; PI3K, phosphatidylinositol 3-kinase; ROS, reactive oxygen species; VEGF, Vascular endothelial growth factor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Reactive oxygen species (ROS) play an important role in certain cellular functions (1). ROS include hydrogen peroxide (H2O2), superoxide anion and hydroxyl radicals. High levels of ROS production are observed in cancer cells (2,3). Recent reports suggest that ROS play an important role in angiogenesis (4–7). ROS, especially H2O2, are also induced by a variety of external stimulators including growth factors (8–12). However, the role of ROS production in cancer cells in response to growth factors remains to be elucidated.

Vascular endothelial growth factor (VEGF) plays a critical role in tumor angiogenesis (13,14). Many stimuli including hypoxia, growth factors and oxidative stress can increase VEGF expression in cancer cells in vitro (15,16). Increased level of VEGF is correlated with increased microvessel counts and poor prognosis in many human cancers including prostate cancer (17–21). The correlation is mainly contributed to the ability of VEGF to stimulate endothelial cell proliferation, cell migration, protease expression and neoangiogenesis (18,22–24). VEGF expression is mainly regulated at transcriptional level by hypoxia-inducible factor 1 (HIF-1) in response to hypoxia (25). HIF-1 is composed of HIF-1{alpha} and HIF-1ß subunits. HIF-1 activates the transcription of many genes that are involved in multiple aspects of tumor growth including angiogenesis, cell survival, invasion and glucose metabolism (26). High levels of HIF-1 expression are observed in many human cancers, and are correlated with tumorigenesis (26). HIF-1{alpha} protein expression is also regulated by phosphatidylinositol 3-kinase (PI3K)/AKT pathway in response to growth factors and cytokines (22,27). Our recent studies showed that PI3K/AKT signaling may regulate VEGF and HIF-1 expression for tumor growth and angiogenesis (28–30). MEK/ERK signaling is also involved in mediating VEGF and HIF-1 expression in cancer cells (31–33). P70S6K1, a downstream target of AKT, is implicated in regulating HIF-1{alpha} expression (34–37).

Prostate cancer is the second leading cause of cancer death among men (38). Recent studies have showed that elevated ROS production is associated with prostate tumor growth and angiogenesis (39–41). Recent studies show that insulin plays an important role in prostate cancer cells, and that hyperinsulinaemia is one of the risk factors for clinical prostate cancer (42–44). Insulin can stimulate the proliferation of prostate cancer cell line in vitro (45), and elevated level of insulin is a risk factor for the development of prostate cancer and for recurrence of prostate cancer in patients following radiation treatment (46,47). Insulin cross-talks with insulin-like growth factor (IGF) axis to mediate several important cellular functions, and levels of circulating insulin concentration is important in determining IGF-I bioactivity (46,48–50). IGF-I pathway is involved in cancers of endometrium, breast and prostate (46,51–53). High levels of insulin also increase free IGF-I levels by reducing the level of IGF-binding proteins including IGFBP-3 (46,54). However, the mechanism of insulin in regulating prostate cancer cells still remains to be elucidated.

In the present study, we plan to study whether (i) insulin induces VEGF and HIF-1{alpha} expression through ROS generation in PC-3 cells; (ii) insulin increases VEGF transcriptional activity via ROS production, p70S6K1 activation and HIF-1{alpha} expression; (iii) insulin activates VEGF and HIF-1{alpha} expression mediated by PI3K/AKT and MEK/ERK signaling pathways through ROS production; and (iv) p70S6K1 activation is required for prostate cancer cell-induced tumor angiogenesis. These studies would determine the role of ROS production in mediating insulin effects in prostate cancer cells, and help to understand the mechanism of insulin in regulating prostate cancer cells.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cell culture and reagents
PC-3 prostate cancer cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin and 5% CO2 at 37°C. Catalase, insulin, rapamycin PD98059 and U0126 were purchased from Sigma (St Louis, MO). Rapamycin was dissolved in dimethyl sulphoxide, and stored at –20°C. Antibodies against HIF-1{alpha} and HIF-1ß were from BD Biosciences (Franklin Lakes, NJ, USA). Antibodies against phospho-AKT, AKT and phospho-p70S6K1 were from Cell Signaling (Beverly, MA, USA). The antibodies against p70S6K1, p-ERK and ERK were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The growth-factor-reduced phenol red-free Matrigel is from BD Biosciences (Bedford, MA, USA).

Construction of plasmids
VEGF promoter reporter pGL-Stu1 containing a 2.65 Kb KpnI–BssHII fragment of the human VEGF gene promoter was cloned into the pGL2 basic luciferase vector as described previously (25). Plasmid encoding human HIF-1{alpha} was inserted into pCEP4 vector (25,55). Plasmid encoding an active form of p70S6K1 has been described previously (56).

DCFH-DA staining
PC-3 cells were plated onto a glass slip in the 12-well plate at 8 x 104 cells/well, and incubated at 37°C for 24 h. The cells were then cultured in serum-free medium for 24 h, followed by the pretreatment with catalase (750 U/ml) for 0.5 h. DCFH-DA (5 µM) was added and incubated with the cells for 10 min. Cells were then stimulated with insulin (200 nM) for 5 min. The cells were washed twice with phosphate-buffered saline (PBS), and fixed with 10% buffered formalin. The images were captured with a fluorescence microscope.

Immunoblotting
Immunoblotting was performed as described previously (34). In brief, PC-3 cells were seeded in 60 mm dishes and cultured to 70–80% confluence. The cells were washed once with PBS, and cultured in serum-free medium for 24 h. After treatment with insulin and rapamycin or catalase, the cells were harvested and lysed. The supernatant was collected. Aliquots of proteins were resolved on SDS–PAGE, and transferred to nitro-cellulose membrane. Proteins of interest were detected by immunoblotting using specific antibodies.

Reverse transcription–PCR (RT–PCR)
PC-3 cells were cultured to 70% confluence in 60 mm dishes. The cells were washed once with PBS, and then cultured in serum-free medium for 24 h. The cells were pretreated with various doses of rapamycin (0, 5 and 10 nM) for 30 min, followed by the incubation for 8 h in the absence or presence of 200 nM insulin. Total RNAs were extracted and used for cDNA synthesis by reverse transcription. The primers used for PCR are as follows: VEGF upstream primer, 5'-TCGGGCCTCCGAAACCATGA-3'; VEGF downstream primer, 5'-CCTGGTGAGAGATCTGGTTC-3'; GAPDH upstream primer, 5'-CCACCCATGGCAAATTCCATGGCA-3'; and GAPDH downstream primer, 5'-TCTAGACGGCAGGTCAGGTCCACC-3'. The PCR procedure is 95°C for 3 min, followed by 28 cycles of 95°C for 1 min, 59°C for 30 s, 72°C for 1 min.

Enzyme-linked immunosorbent assay
The levels of VEGF protein secreted by the cells in the medium were determined by a VEGF ELISA kit (R&D Systems). In brief, PC-3 cells were cultured to 70–80% confluence, then switched into serum-free medium and cultured for 24 h. The cells were pretreated with rapamycin (0, 5, and 10 nM) for 30 min, followed by the incubation for 20 h in the absence or presence of 200 nM insulin. The medium was collected, and VEGF protein concentrations were measured by ELISA according to the manufacturer's instructions. The results were normalized to the number of cells per plate.

Transient transfection and luciferase assay
PC-3 cells were plated in 12-well plates and cultured to 50% confluence. Cells were transiently transfected with VEGF promoter reporter and pCMV-galactosidase plasmids using Lipofectamine from Invitrogen according to the manufacturer's instructions. The transfected cells were cultured for 20 h, followed by the incubation in serum-free medium for 24 h. The cells were pretreated by rapamycin or catalase for 30 min, followed by the incubation with insulin for 15 h. Cells were washed with PBS, and lysed with reporter lysis buffer from Promega (Madison, WI, USA). Luciferase (Luc) activities of the cell extracts were determined using the luciferase assay system (Promega). ß-Galactosidase (ß-gal) activity was measured in assay buffer (100 mM phosphate, 2 mM MgCl2, 100 mM mercaptoethanol and 1.33 mg/ml o-nitrophenyl ß-D-galactopyranoside, pH 7.5). Relative Luc activity (defined as VEGF reporter activity) was calculated as the ratio of Luc/ß-gal activity, and normalized to that of the control.

In vivo angiogenesis assay
Male BALB/cA-nu nude mice (4-weeks-old) were purchased from Shanghai Experimental Animal Center (Chinese Academy of Sciences, China), and maintained in pathogen-free conditions. PC-3 cells at 90% confluence were trypsinized, washed once with PBS and re-suspended in serum-free medium (3 x 107 cells/ml). Aliquots of cells (3 x 106 cells) were mixed with 0.2 ml growth factor-reduced phenol red-free Matrigel in the presence of rapamycin (20 nM). For control group, the cells were mixed with Matrigel and solvent. The mixture was injected subcutaneously into both flanks of nude mice. The Matrigel mixed with the serum-free medium alone was used as a negative control. The Matrigel plugs were removed from the mice 11 days after the implantation, and trimmed of the surrounding tissues. The plugs were weighed and immersed immediately in lysis buffer (1 mM EDTA and 5 mM phosphate, pH 8), and incubated at 4°C for 24 h. Hemoglobin content was measured using a Drabkin's reagent kit (Sigma) as per the manufacturer's instruction. The data represented the mean ± SD (n = 8) from two independent experiments.

Immunohistochemical staining for CD31
Tumor samples were fixed in Zinc Fixative (BD Biosciences) according to the manufacture's instruction and stored at 4°C. The fixed samples were treated in 30% sucrose for 12 h, and serial 5 µm frozen sections were prepared and mounted on slides coated with 3-amino propyltriethoxy silane. Monoclonal CD31 antibody from BD Bioscience was used for the staining, and detected through streptavidin–biotin–horseradish peroxidase complex (SABC) formation. In brief, the sections were washed three times in 1x PBS and incubated for 20 min in 0.3% hydrogen peroxide/methanol. The slides were washed three times with 1x PBS, then incubated with 10% normal goat serum or 0.3% Triton X-100 at 37°C for 20 min. The sections were incubated with the CD31 primary antibody (1:100). The samples were incubated in a humid chamber at 4°C for 16 h. After three washes in 1x PBS, the slides were incubated for 1 h with rat anti-mouse secondary IgG, and detected by incubation with SABC compound for 0.5 h at 37°C. Sections were rinsed several times in 1x PBS. Sections incubated with preimmune IgG instead of the primary antibodies were used as a negative control. The relative angiogenesis level was estimated by micro-vessel density (MVD) as described previously (57,58). Briefly, slides were first scanned under low power (x40) in order to determine three ‘hot-spots’ or areas with the maximum number of microvessels, which were then evaluated at x200 magnification. The number of microvessels in each field was determined and their average number was expressed as MVD per field.

Statistical analysis
The data represent mean ± SD from three independent experiments except where indicated. Statistical analysis was performed by Student's t-test at a significance level of P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Insulin stimulated H2O2 production in PC-3 cells
There is accumulating evidence for the importance of ROS as secondary messengers in a variety of cellular functions (59). ROS, especially H2O2, can be induced by growth factors in several types of cells (60,61). To determine whether insulin could induce H2O2 formation in PC-3 cells, the cells were cultured in serum-free medium, followed by the treatment of insulin. As shown in Figure 1, the H2O2 level in serum-starved PC-3 cells was low, and insulin increased the H2O2 production. Pretreatment of the cells with catalase, a scavenger of H2O2, abrogated the insulin-induced H2O2 generation. These results suggest that insulin induces H2O2 production in PC-3 cells.


Figure 1
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  		Fig. 1 Insulin induced generation of H2O2 in PC-3 cells. PC-3 cells were plated onto a glass slip in the 12-well plate at 8 x 104 cells/well, and incubated at 37°C for 24 h. The cells were then cultured in serum-free medium for 24 h, followed by pretreatment without or with catalase (750 U/ml) for 30 min. DCFH-DA (5 µM) was added and incubated with the cells for 10 min. The cells were stimulated with insulin (200 nM) as indicated for 5 min. Then the cells were washed and fixed. The images were captured with a fluorescence microscope.

 
Insulin induced p70S6K1 activation, which required H2O2 production in PC-3 cells
As shown in Figure 2A, p70S6K1 was activated by insulin in PC-3 cells. Because insulin induced generation of H2O2 (Figure 1), we tested whether insulin activated p70S6K1 through H2O2 production. Pretreatment of the cells with catalase, the scavenger of H2O2, greatly suppressed the phosphorylation of p70S6K1 induced by insulin (Figure 2B). This result suggests that insulin-induced activation of p70S6K1 requires H2O2 production. To exclude the possibility that catalase affects p70S6K1 phosphorylation independently of its H2O2 scavenging activity, the heat-killed catalase was used as a control. The heat-inactivated catalase had no effect on insulin-induced p70S6K1 activation (Figure 2C). The catalase treatment did not affect the cell viability under the same experimental conditions (data not shown). To further confirm that H2O2 is able to activate p70S6K1, we treated PC-3 cells directly with H2O2. As shown in Figure 2D, H2O2 induced phosphorylation of p70S6K1 in a dose-dependent manner. Addition of catalase blocked the effect of H2O2 in inducing p70S6K1 phosphorylation (Figure 2E). These results suggest that insulin-induced H2O2 production plays an important role in p70S6K1 activation.


Figure 2
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  		Fig. 2 H2O2 production is required for insulin-induced p70S6K1 activation. (A) PC-3 cells were cultured to 70% confluence, then starved in serum-free medium for 24 h. The cells were treated by different concentrations of insulin (0, 50, 100 and 200 nM) for 2 h. (B) PC-3 cells were cultured and starved as above. Then the cells were pretreated by catalase (0, 750, 1500 and 3000 U/ml) for 30 min, followed by stimulation with insulin (200 nM) for 2 h. (C) The cells were pretreated by heat-killed catalase (3000 U/ml), followed by stimulation with insulin (200 nM) for 2 h. (D) PC-3 cells were cultured and starved as above, then treated with different concentrations of H2O2 (0, 50, 100, 150 and 200 µM) for 2 h. (E) PC-3 cells were cultured and starved, pretreated by catalase (0, 750, 1500 and 3000 U/ml) for 30 min, followed by treated with H2O2 (150 µM) for 2 h. Total protein extracts were subjected to immunoblotting analysis using specific antibodies against p-p70S6K1 and total p70S6K1, respectively.

 
P70S6K1 is a downstream target of PI3K/AKT, and H2O2 has been recognized as a positive regulator of PI3K pathway (1). We therefore wanted to know whether the effect of H2O2 on p70S6K1 was through PI3K/AKT pathway. We tested the effects of catalase on AKT phosphorylation in response to insulin in PC-3 cells, and found that catalase did not significantly affect insulin-stimulated AKT phosphorylation (Figure 3A).


Figure 3
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  		Fig. 3 Insulin-induced ERK activation was inhibited by catalase. PC-3 cells were cultured to 70% confluence, then starved in serum-free medium for 24 h. The cells were pretreated with catalase (0, 750, 1500 and 3000 U/ml) for 30 min, followed by the treatment of insulin (200 nM) for 2 h. Aliquots of proteins were analyzed by immunoblotting using the specific antibodies as indicated. (A) Effects of catalase on insulin-induced phospho-AKT levels. (B) Effects of catalase on insulin-induced phospho-ERK levels. (C) The serum-starved PC-3 cells were pretreated with ERK inhibitor PD98059 for 30 min, followed by stimulation with insulin (200 nM) for 15 min. (D) The PC-3 cells were pretreated with ERK inhibitor U0126, then treated by insulin as above. The cell lysates were subjected to immunoblotting analysis.

 
Since MEK/ERK signaling is also known to regulate p70S6K1 activation (62), we next determined whether insulin induced p70S6K1 activation via ERK. We first examined the effects of catalase on phosphorylation of ERK1/2 induced by insulin. Addition of catalase inhibited insulin-induced ERK phosphorylation (Figure 3B), indicating that H2O2 is required for insulin-induced activation of ERK. Inhibition of ERK activation by MEK1/ERK inhibitors, PD98059 and U0126 blocked insulin-stimulated p70S6K1 phosphorylation (Figures 3C and 3D). These results suggest that insulin induced p70S6K1 activation through H2O2 production and ERK1/2 signaling in PC-3 cells.

Insulin-induced HIF-1{alpha} expression requires p70S6K1 and H2O2 production in PC-3 cells
It is reported that p70S6K1 is involved in HIF-1{alpha} expression (34–37). In PC-3 cells, insulin induced HIF-1{alpha} expression, which was inhibited by rapamycin, an inhibitor of mTOR/p70S6K1 (Figures 4A and 4B). Since we showed that H2O2 production is required for insulin-induced p70S6K1 phosphorylation (Figure 2B), we expected that insulin-induced H2O2 could play a role in HIF-1{alpha} expression in PC-3 cells. To test this, PC-3 cells were pretreated with catalase, then stimulated with insulin. Catalase treatment inhibited insulin-induced expression of HIF-1{alpha} (Figure 4C), suggesting that H2O2 production is required for insulin-induced HIF-1{alpha} expression.


Figure 4
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  		Fig. 4 Rapamycin and H2O2 are required for insulin-induced HIF-1{alpha} expression. (A) PC-3 cells were cultured to 70% confluence, then starved in serum-free medium for 24 h. The cells were treated by different concentrations of insulin (0, 50, 100 and 200 nM) as indicated for 6 h. HIF-1{alpha} expression was determined by immunoblotting. (B) PC-3 cells were cultured and starved as above. Then, the cells were pretreated by rapamycin (0, 5, 10 and 20 nM) for 30 min, followed by treatment with insulin (200 nM) for 6 h. HIF-1{alpha} and phopho-p70S6K1 levels were determined by immunoblotting. (C) The starved PC-3 cells were pretreated with catalase (0, 750, 1500 and 3000 U/ml) for 30 min, followed by stimulation with insulin (200 nM) for 6 h. Protein expression was determined by immunoblotting.

 
Insulin induced VEGF expression through H2O2 production and p70S6K1 activation
VEGF, the critical regulator of angiogenesis, is mainly regulated by HIF-1 through the binding of HIF-1 to the hypoxia-responsive element of VEGF promoter region. In our subsequent experiments, we determined whether insulin-induced H2O2 is involved in regulation of VEGF expression in response to insulin in PC-3 cells. We first determined the transcriptional activation of VEGF by employing the VEGF promoter reporter. As shown in Figure 5A, the transcriptional activity of VEGF was induced by insulin and suppressed by catalase. The catalase treatment did not affect the cell viability (Figure 5B). Next, we determined VEGF expression by performing semi-quantitative RT–PCR. Similarly, the VEGF mRNA levels were enhanced in response to insulin treatment and decreased by catalase treatment (Figure 5C). In addition, we showed that rapamycin inhibited insulin-induced VEGF transcriptional activation and its mRNA levels (Figures 5D and 5E). We also detected VEGF protein levels in the medium by ELISA as described previously (63), and showed that insulin induced VEGF production and rapamycin suppressed insulin-induced VEGF production (Figure 5F). Cell viability was not affected by rapamycin treatment in the same experiment (Figure 5G). These results implied that insulin-induced VEGF expression is H2O2-dependent.


Figure 5
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  		Fig. 5 (A) H2O2 production and p70S6K1 activation were required for insulin-induced VEGF expression. PC-3 cells were transfected with VEGF reporter plasmid (0.4 µg) and pCMV-ß-galactoside plasmids (0.2 µg) as described in Materials and methods. After the transfection, the cells were pretreated with different concentration of catalase (0, 750 and 1500 U/ml) for 30 min. Insulin (200 nM) was added to the cells with incubation for 15 h. Luc assay was performed as described in Materials and methods. (B) Low dose of catalase had no significant toxic effect on cell viability. PC-3 cells were seeded into 12-well plate and cultured as above. Various doses of catalase (0, 750 and 1500 U/ml) were added to the cells and cultured for 20 h. Cell viability was determined by the trypan blue dye exclusion method. (C) PC-3 cells were cultured to 70% confluence, then cultured in serum-free medium for 24 h. The cells were treated by catalase (0, 750 and 1500 U/ml) for 30 min, followed by an 8 h incubation with 200 nM insulin. Total RNAs were extracted and used for RT–PCR for detecting VEGF and GAPDH mRNA levels. (D) PC-3 cells were transfected and starved as in (A). After starvation, the cells were pretreated with rapamycin (0, 5 and 10 nM) for 30 min. Insulin (200 nM) was added to the cells and incubated for 15 h. Luc assay was performed as described above. (E) PC-3 cells were cultured and starved. Then the cells were pretreated by rapamycin (0, 5 and 10 nM) for 30 min, followed by treatment with insulin (200 nM) for 8 h. Total RNAs were extracted and used for RT–PCR. Asterisk indicates significant difference when compared to the value of the control (P < 0.05). ‘#’ indicates significant difference when compared to that treated with insulin alone (P < 0.05). (F) Effect of rapamycin on VEGF protein level. PC-3 cells were cultured to 70% confluence, then starved in serum-free medium for 24 h. The cells were pretreated by solvent or rapamycin (5 and 10 nM) for 30 min, followed by a 24 h incubation with 200 nM insulin. VEGF protein concentrations in the medium were measured by ELISA assays. The results were normalized to the number of cells per plate. Asterisk indicates significant difference when compared to the value of control (P < 0.05). ‘#’ indicates significant difference when compared to that treated with insulin alone (P < 0.05). (G) Cell viability with rapamycin treatment. PC-3 cells were cultured in serum-free medium for 24 h. The cells were pretreated by solvent or rapamycin (5 and 10 nM) for 30 min, followed by a 24 h incubation with 200 nM insulin. Cell viability was determined by the trypan blue exclusion method.

 
Insulin induced VEGF transcriptional activation through HIF-1{alpha} expression
To know whether the induction of VEGF by insulin is via HIF-1{alpha}, we determined the effects of forced expression of wild-type HIF-1{alpha} on VEGF transcriptional activation. Rapamycin inhibited insulin-induced VEGF transcriptional activity, which was restored by over-expression of HIF-1{alpha} (Figure 6A). Over-expression of HIF-1{alpha} also reversed catalase-inhibited VEGF transcriptional activation induced by insulin (Figure 6B). The result suggests that insulin induces VEGF-transcriptional activation through HIF-1{alpha} expression.


Figure 6
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  		Fig. 6 Insulin induces VEGF transcriptional activation through HIF-1{alpha} expression. (A) PC-3 cells (50–70% confluence) in 12-well plates were transfected with VEGF reporter plasmid (0.4 µg), pCMV-ß-galactoside plasmids (0.2 µg), with or without HIF-1{alpha} plasmid (0.25 µg). Empty vector was used to make total transfected DNA to the equal amount. After transfection, the cells were cultured overnight, and switched to serum-free medium for 20 h. The starved cells were pretreated with rapamycin (10 nM) for 30 min. Insulin (200 nM) was added and the cells were incubated for 15 h. Luc assay was determined as above. Cellular protein extracts were used to analyze expression of HIF-1{alpha} and HIF-1ß by immunoblotting. (B) PC-3 cells were treated as described above except the pretreatment of catalase (750 U/ml). Asterisk indicates significant difference when compared to the control (P < 0.05). ‘#’ indicates significant difference when compared to that treated with insulin alone (P < 0.05). ‘{Delta}’ indicates a significant difference when compared to that treated with insulin and rapamycin or catalase (P < 0.05).

 
Over-expression of p70S6K1 reversed the inhibitory effects of rapamycin and catalase in VEGF transcriptional activation
We next determined whether over-expression of active p70S6K1 (rapamycin resistant) (56) could reverse the rapamycin- and catalase-inhibited VEGF expression induced by insulin. Rapamycin inhibited VEGF transcriptional activation, and the inhibition was reversed by forced expression of active p70S6K1 in PC-3 cells, which correlated with p70S6K1 protein expression (Figure 7A). Similarly, catalase inhibited VEGF transcriptional activation by insulin, which was reversed by over-expression of p70S6K1 (Figure 7B). These results suggest that insulin induces VEGF transcription via p70S6K1 activation.


Figure 7
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  		Fig. 7 Overexpression of p70S6K1 reversed the VEGF transcriptional activation inhibited by rapamycin or catalase in PC-3 cells. (A) PC-3 cells (50–70% confluence) were transfected with VEGF reporter plasmid (0.4 µg), pCMV-ß-galactoside plasmids (0.2 µg) with or without active form of p70S6K1 plasmid (0.3 µg). Empty vector was used to make total transfected DNA to the equal amount. After transfection, the cells were cultured overnight, and switched to serum-free medium for 20 h. The starved cells were pretreated with rapamycin (10 nM) for 30 min. Insulin (200 nM) was added to the cells and incubated for 15 h. Luc assay was determined as above. Cellular protein extracts were used to analyze expression of p-p70S6K1 and total p70S6K1 by immunoblotting. (B) PC-3 cells were treated as described above except the treatment of catalase (750 U/ml). Asterisk indicates a significant difference when compared to the value of the control (P < 0.05). ‘#’ indicates a significant difference when compared to that treated with insulin alone (P < 0.05). ‘{Delta}’ indicates a significant difference when compared to that of insulin and rapamycin or catalase (P < 0.05).

 
Forced expression of ERK1/2 activator, MEK1 reversed the inhibitory effect of catalase in VEGF transcriptional activation
To determine whether ERK1/2 is downstream of insulin and H2O2 for regulating VEGF transcriptional activation, the cells were co-transfected with VEGF promoter reporter and MEK1. Forced expression of MEK1 completely restored catalase-inhibited VEGF transcriptional activation (Figure 8), suggesting that ERK1/2 is a downstream signaling molecule of H2O2 in the cells.


Figure 8
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  		Fig. 8 Overexpression of MEK1 reversed the catalase-inhibited VEGF transcriptional activation. PC-3 cells were transfected with VEGF reporter plasmid (0.4 µg) and pCMV-ß-galactoside plasmids (0.2 µg) with or without active form of MEK1 plasmid (0.2 µg). Empty vector (pcDNA3) was used to make total transfected DNA to the equal amount. After transfection, the cells were cultured overnight, and switched to serum-free medium for 20 h. The starved cells were pretreated with catalase (750 U/ml) for 30 min, then incubated with insulin (200 nM) for 15 h. Asterisk indicates a significant difference when compared to the value of the control (P < 0.05). ‘#’ indicates a significant difference when compared to that treated by insulin alone (P < 0.05). ‘{Delta}’ indicates a significant difference when compared to that treated with insulin and catalase (P < 0.05).

 
p70S6K1 is important for prostate tumor angiogenesis
We and others showed that p70S6K1 plays a role in regulating HIF-1{alpha} and VEGF expression in cancer cells (34–37). However, the role of p70S6K1 in angiogenesis in vivo is not known yet. In the present work, we determined the inhibitory effects of p70S6K1 on prostate tumor angiogenesis in nude mice by Matrigel plug assay. Compared with the Matrigel alone, PC-3 cells greatly induced angiogenesis, which was inhibited by rapamycin treatment (Figure 9A). The hemoglobin contents in the plug were used as relative angiogenesis index. Little hemoglobin was produced in the Matrigel plug without the cells. The presence of PC-3 cells greatly increased the hemoglobin content up to 7 mg/g plug (Figure 9B). Addition of rapamycin decreased hemoglobin level by 50%, indicating that rapamycin inhibited angiogenesis (Figure 9B). CD31 staining of the tumor tissue was performed to determine MVD. Treatment of rapamycin decreased blood vessel formation in the tumor tissues (Figure 9C). Quantitative analysis showed that rapamycin treatment decreased MVD to 30% of the solvent group (Figure 9D). The phosphorylation of p70S6K1 was inhibited in the xenograft tissue (Figure 9E). Taken together, these results suggest that p70S6K1 plays an important role in tumor cell-induced angiogenesis.


Figure 9
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  		Fig. 9 Rapamycin inhibits prostate tumor angiogenesis in vivo. PC-3 cells were cultured to 90% confluence. The cells were trypsinized and resuspended in serum-free medium at 3 x 107 cells/ml. Aliquots of the cells (0.1 ml) were mixed with 0.2 ml of phenol red-free Matrigel in the absence or presence of rapamycin (20 nM), and injected subcutaneously into the nude mice as described in Materials and methods. The Matrigel alone was used as a negative control. The mice were euthanized 11 days after the injection, and the Matrigel plugs were removed from nude mice and photographed immediately. (A) The representative Matrigel plugs (scale bar, 3 mm) are shown. (B) The hemoglobin content levels. The Matrigel plugs were liquefied in lysis buffer at 4°C for 24 h. Hemoglobin levels were determined using Drabkin's solution (Sigma) according to the manufacturer's instructions. Asterisk indicates a significant difference when compared to the value of the control (P < 0.05). ‘#’ indicates a significant difference when compared to that of PC-3 cells alone (P < 0.05). (C) The representative CD31-stained sections. Sections of tumors were stained with monoclonal antibody against CD31. The result showed that rapamyicn decreased microvessel density (MVD) in PC-3 xenografts. (D) Quantative analysis of MVD in the tumor sections was performed. The graph represents the mean ± SD from five different tumor sections. (E) Analysis of p70S6K1 phosphorylation. The PC-3 xenografts were snap-frozen in liquid nitrogen and crushed in a mortar rapidly. Total protein extracts were obtained and analyzed by immunoblotting.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
HIF-1 and VEGF play an important role in angiogenesis. Upregulation of HIF-1 expression is observed in many common cancers, and induced by genetic and environmental factors (64). The PI3K/AKT signaling plays an important role in regulating HIF-1{alpha} expression (22,27,65,66). It has been reported that activation of PI3K/AKT by growth factors, cytokines and other stimulators induced expression of HIF-1{alpha} and VEGF in human cancer cells (22,67–69). Inhibition of PI3K/AKT signaling suppressed expression of HIF-1 and VEGF in vitro and tumor angiogenesis in vivo (22,70,71). P70S6K1 was also observed to play a role in regulating HIF-1{alpha} and VEGF expression (34–37). Recent evidence has demonstrated the importance of ROS as secondary messengers in a variety of cellular functions (1,59). Redox signaling of HIF-1{alpha} is an emotive subject and the argument for ROS signaling to this protein is not entirely clear. In a few types of cells, some growth factors, cytokines and/or other stimulators induce ROS production such as H2O2 (8–12). H2O2 can stimulate cascades of cell signals including PI3K/AKT and MEK/ERK pathways (1). In the present work, we found that insulin induced H2O2 generation (Figure 1), and insulin-induced H2O2 is important for inducing HIF-1 and VEGF expression (Figures 4C, 5A and 5C). Interestingly, catalase treatment did not significantly affect the insulin-induced phosphorylation of AKT (Figure 3A), suggesting that insulin-induced H2O2 activates p70S6K1 through other signaling pathway in PC-3 cells. The reason that AKT is not sensitive to insulin-induced H2O2 signaling in PC-3 cells could be interesting, which requires further study (72).

Insulin-induced H2O2 is required for p70S6K1 activation (Figures 2A and 2B), and catalase did not affect AKT phosphorylation induced by insulin (Figure 3A), suggesting that H2O2 may regulate p70S6K1 through other pathways in PC-3 cells. We found that depletion of H2O2 by catalase in PC-3 cells suppressed the ERK activation (Figure 3B), and MAP kinase inhibitors PD98059 and U0126 decreased insulin-induced p70S6K1 phosphorylation (Figures 3C and 3D). These results suggest that H2O2/ERK signaling is involved in p70S6K1 activation in response to insulin in PC-3 cells. This result is consistent with other study that ERK activation is required for p70S6K1 activation (62).

Both PI3K/AKT and MEK/ERK signaling pathways are implicated in HIF-1{alpha} and VEGF expression (33,35). p70S6K1 may act as a converging point of the two signaling pathways, and plays a crucial role in regulating HIF-1{alpha} and VEGF expression. In this study, we found that MEK/ERK pathway, but not PI3K/AKT pathway is required for inducing p70S6K1 activation through H2O2 production in response to insulin treatment. This result indicates the specificity of ROS signaling induced by insulin, which would be a very interesting subject for the future study. In this work, we showed that (i) insulin-induced H2O2 production is required for HIF-1 and VEGF expression; (ii) insulin induced H2O2 production to activate p70S6K1, which in turn regulates HIF-1 and VEGF expression; and (iii) p70S6K1 plays an important role in tumor angiogenesis.


   Footnotes
 
{dagger}These authors contributed equally to this work. Back


   Acknowledgments
 
This work was supported by research grants 30570962 and 30470361 from National Natural Science Foundation of China; and by research grants from the Science and Technology Commission of Shanghai Municipality (04DZ14007 and 05DJ14009).

Conflict of Interest Statement: None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received January 8, 2006; revised May 8, 2006; accepted May 16, 2006.


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