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Carcinogenesis. 2009 May; 30(5): 808–817.
Published online 2009 February 20. doi:  10.1093/carcin/bgp044
PMCID: PMC2722146

Lupeol inhibits proliferation of human prostate cancer cells by targeting β-catenin signaling

Abstract

Lupeol, a dietary triterpene, was shown to decrease serum prostate-specific antigen levels and inhibit the tumorigenicity of prostate cancer (CaP) cells in vivo. Here, we show that Lupeol inhibits the proliferative potential of CaP cells and delineated its mechanism of action. Employing a focused microarray of human CaP-associated genes, we found that Lupeol significantly modulates the expression level of genes such as ERBB2, tissue inhibitor of metalloproteinases-3, cyclin D1 and matrix metalloproteinase (MMP)-2 that are known to be associated with proliferation and survival. A common feature of these genes is that all of them are known to either regulate or act as downstream target of β-catenin signaling that is highly aberrant in CaP patients. Lupeol treatment significantly (1) reduced levels of β-catenin in the cytoplasmic and nuclear fractions, (2) modulated expression levels of glycogen synthase kinase 3 beta (GSK3β)–axin complex (regulator of β-catenin stability), (3) decreased the expression level and enzymatic activity of MMP-2 (downstream target of β-catenin), (4) reduced the transcriptional activation of T Cell Factor (TCF) responsive element (marker for β-catenin signaling) in pTK-TCF-Luc-transfected cells and (5) decreased the transcriptional activation of MMP-2 gene in pGL2-MMP-2-Luc-transfected cells. Effects of Lupeol treatment on β-catenin degradation were significantly reduced in CaP cells where axin is knocked down through small interfering RNA transfection and GSK3β activity is blocked. Collectively, these data suggest the multitarget efficacy of Lupeol on β-catenin-signaling network thus resulting in the inhibition CaP cell proliferation. We suggest that Lupeol could be developed as an agent for chemoprevention as well as chemotherapy of human CaP.

Introduction

In recent years, there is an intense activity to identify novel therapeutic modalities and preventive approaches for prostate cancer (CaP). Epidemiological and laboratory studies suggest that diet-based naturally occurring agents, due to their ability to target multiple signaling pathways, their cost-effectiveness and most importantly their human acceptability could be ideal candidates for the treatment and prevention of human CaP (14). At the present time many such agents are being investigated in preclinical settings and emerging data with some of the agents in clinical settings is encouraging (3,4). We recently showed that Lupeol [Lup-20(29)-en-3β-ol], a diet-based triterpene found in fruits such as olive, mango, strawberry, grapes, figs and in several medicinal plants activates apoptotic machinery (Fas signaling that generally is impaired in CaP cells) and inhibits the tumorigenicity of human androgen-sensitive CaP cells with a concomitant decrease in serum prostate-specific antigen levels under in vivo conditions (5). We suggested that Lupeol could be developed as a potential agent for the treatment of human CaP (5). Recently, Lee et al. (6) showed that Lupeol treatment inhibits head and neck cancer in a mouse tumor xenograft model. In the current study, we provide evidence to show that Lupeol significantly reduces the proliferative and clonogenic potential of androgen-sensitive as well as androgen-insensitive CaP cells by modulating β-catenin-signaling pathway.

Materials and methods

Cell culture

Human CaP cells LNCaP and DU145 and fetal bovine serum were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in appropriate media containing 10% fetal bovine serum supplemented with 1% penicillin–streptomycin (Cellgro Mediatech, Herndon, VA).

[3H]-thymidine incorporation assay

Stock solution of Lupeol (Sigma, St Louis, MO) was prepared as described earlier (5). Cells grown in 24-well cluster plates were subjected to Lupeol treatment for 48 h, the last 16 h of which was in the presence of [3H]thymidine (0.5 μCi/ml). Cells were then washed twice with cold phosphate-buffered saline and then were incubated with trichloroacetic acid solution on ice for 30 min and subsequently, the acid-insoluble fraction was dissolved in 1 ml 1 M NaOH. Incorporated [3H]thymidine was quantified by liquid scintillation counting.

Colony formation studies

A total of 0.5% agar was prepared in RPMI containing 20% fetal calf serum (bottom layer). Cells (1 × 105 cell per 100 mm plate) in 20% fetal calf serum and 0.7% agarose (top layer) were plated and incubated at 37°C overnight before treatment with Lupeol. The medium was removed and replaced with fresh medium containing Lupeol every 3 days. After 21 days of incubation, the cells were stained with 0.05% Crystal Violet/methanol for 2 h and colonies were counted in two colony grids using a microscope.

Microarray analysis

LNCaP cells were treated with subtoxic dose (20 μM) of Lupeol. After 48 h of incubation, cells were harvested and RNA was isolated by using RNeasy kit (Qiagen, Valencia, CA). Next, 4 μg of RNA was enzymatically converted into complementary RNA, labeled and hybridized with the microarrays (imprinted with 288 well-characterized CaP-associated genes) as per vendor's protocol (Super Array, Frederick, MD) followed by detection with the chemiluminescent reagents and X-ray film. Data were acquired and analyzed by using GE super array software. A cutout point of 2-fold was selected for analysis. The microarray experiments were conducted three times independently.

Treatment of cells

For dose-dependent studies, the cells were treated with Lupeol (5–50 μM) for 48 h in complete cell medium. To investigate the phosphorylation of β-catenin by Lupeol, CaP cells were treated with Lupeol (20–40 μM) and cells were collected at 24 and 48 h later. After treatment with Lupeol, the cell lysates were prepared from harvested cells and stored at −80°C for later use.

Western blot analysis

Western blot analysis was performed as described earlier (5). Antibodies used in the immunoblotting were procured from Cell Signaling, Danvare, MA [anti-β-catenin, antiphospho-β-catenin, anti-Cdk2, anti-Cyclin D1, anti-cmyc, anti-matrix metalloproteinase (MMP)-2, anti-ERBB2, anti-insulin-like growth factor (IGF)-1R, anti-glycogen synthase kinase 3 beta (GSK3β), antiphospho-GSK3β and anti-axin], Santa Cruz Biotechnology, Santa Cruz, CA (anti-tissue inhibitor of metalloproteinases (TIMP)-3 and anti-Lamin,) and Sigma (anti-β-actin). Densitometry measurements of the scanned bands were performed using digitalized scientific software program UN-SCAN-IT (Silk Scientific Corporation, Orem, UT). Data were normalized to loading control.

GSK3β inhibition studies

CaP cells were pretreated with 0.1 μM of GSK3β inhibitor, (2′Z,3′E)-6-bromoindirubin-3′-oxime (BIO) (Calbiochem, San Diego, CA) for 4 h before Lupeol treatment (20 μM) for 24 h. Cell lysates were obtained for immunoblot and immunoprecipitation analysis.

Immunoprecipitation

Cell lysates were precleared by adding 1 μg of appropriate control immunoglobulin G (corresponding to the host species of primary antibody) together with 20 μl of resuspended volume of Protein A-Agarose (Santa Cruz Biotechnology) at 4°C for 30 min. Beads were pelleted and cell lysates were transferred to a fresh centrifuge tube. Equal amounts (200 μg) of cellular protein from the cell lysates were incubated overnight with 10 μl of specific primary antibodies (anti-human axin and anti-human GSK3β) at 4°C. After overnight incubation, 20 μl of protein A-Agarose was added and the mixture was incubated for 3 h. Immunoprecipitates were collected by centrifugation and were washed thrice with the cell lysis buffer [50 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (pH 7.55), 1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol and protease inhibitor cocktail). The final pellets were dissolved in 20 μl of 2× protein loading buffer. The expression levels of proteins (axin and GSK3β) were determined by immunoblot analysis. Further, to confirm the specificity of antibodies (axin and GSK3β immunoprecipitates) used for immunoprecipitation, a non-specific antibody (anti-cyclin E antibody) was used as a negative control. Cyclin E was immunoprecipitated in CaP cells and loaded in parallel on sodium dodecyl sulfate gels. The specificity of antibodies was confirmed by stripping immunoblots and reprobing them for Cyclin E (negative control). Finally, 10% of post-cleared protein was also loaded as input control on sodium dodecyl sulfate Gel. After detecting axin and GSK3β proteins, immunoblots were stripped and reprobed for β-actin to confirm the equal loading.

Small interfering RNA transfection

A total of 2 × 106 cells were transfected with 100 nM of small interfering RNA (siRNA) directed against axin (Santa Cruz Biotechnology). Control cells were transfected with scrambled siRNA (100 nM). After overnight incubation, transfected cells were treated with Lupeol (20–40 μM) and 24 h later were harvested and cell lysates were processed.

Transcriptional activity of TCF and MMP

The human MMP-2 promoter luciferase plasmid (pGL2-MMP-2-luc) was developed as described earlier (7). pTK-TCF-Luc (TopFlash and FopFlash) was procured from Upstate Laboratories (Lake Placid, NY). Cells were transfected with the plasmids (200 ng per well) for 24 h. Renilla luciferase (20 ng per well, pRL-TK; Promega, Madison, WI) was used as an internal control. In addition, for controls, the same amount of empty vectors were transfected in cells. After 12 h post-transfection, fresh media was added with Lupeol (5–10 μM) and incubated for 24 h. The cells were then harvested and transcriptional activity was measured in terms of luciferase activity in quadruplicates by using dual-luciferase reporter assay system (Promega). Relative luciferase activity was calculated with the values from vector alone group with or without Lupeol-treated group. To confirm the effect of Lupeol treatment on the general transcriptional machinery, CaP cells were transfected with 50 ng per well Renilla luciferase plasmid (pRL-TK). The transcriptional activity was measured in a dose- and time-dependent manner.

Gelatin zymography

Equal number of cells (1 × 106) were seeded in the plates and after 24 h were treated with Lupeol. After 48 h post-treatment, the conditioned media were harvested, concentrated and electrophoresed (10 μg protein) under non-reducing conditions. The gelatinolytic activity of MMP-2 was determined by employing zymography kit (Invitrogen, Carlsbad, CA) as per vendor's protocol.

Statistical analyses

Student's t-test for independent analysis was applied to evaluate differences between the treated and untreated cells with respect to the expression of various proteins using S-plus Software (Insightful, Seattle, WA). A P-value of <0.05 was considered to be statistically significant.

Results

Effect of Lupeol on androgen-sensitive CaP cell proliferation

Recently, we showed significant growth inhibitory effects of Lupeol (5–30 μM) on androgen-sensitive CaP cells without producing any effect on the viability of normal prostate epithelial cells (5). It is well known that proliferating cells exhibit increased thymidine incorporation into DNA that arises from increased growth factor expression and activity in cancer cells (8). We investigated the effect of Lupeol treatment on the rate of proliferation of androgen-sensitive LNCaP cells by measuring the rate of uptake of [3H]-thymidine by dividing cells. Lupeol treatment caused a significant decrease of [3H]-thymidine uptake by LNCaP cells suggesting the antiproliferative potential of Lupeol (Figure 1A).

Fig. 1.
Effect of Lupeol on proliferation, clonogenic potential, the expression level of proliferation-associated proteins and β-catenin signaling in LNCaP cells. (A) Histogram showing the rate of [3H]thymidine uptake in LNCaP cells treated with Lupeol. ...

Effect of Lupeol treatment on the clonogenic potential of androgen-sensitive CaP cells

Next, we asked whether treatment with Lupeol could exert greater activity on the formation of colonies, which allows an investigation over a longer period of time and that mimics cellular physiology in vivo. We observed that the colony-forming ability of LNCaP cells was inhibited by 20–50% (P < 0.05), after treatment with Lupeol (Figure 1B). Interestingly, even after 21 days, the colony formation was significantly decreased in Lupeol-treated cells as compared with control that exhibited numerous colonies (Figure 1B). These data suggest that the sustained effect of Lupeol on growth-promoting and proliferative property of androgen-sensitive CaP cells.

Effect of Lupeol treatment on expression of CaP-associated genes

To define the mechanism through which Lupeol inhibits or reduces the proliferation of CaP cells, we employed a focused microarray. The effect of subtoxic dose of Lupeol (20 μM) treatment on 288 well-characterized human CaP-associated genes was assessed. A cutout point of 2-fold was selected for analysis. Lupeol treatment was observed to modulate the messenger RNA expression level of several genes (Table I). Only those genes are represented in Table I that exhibit changes of 2- or >2-fold in their expression. We observed that Lupeol treatment significantly decreases the messenger RNA level of several genes associated with the growth, survival and proliferation of CaP cells. The prominent proliferation-associated genes whose expression was observed to be downregulated by Lupeol treatment are androgen receptor, IGF-1R, Cyclin D1, myc, MMP-2, Cdk2, Jun, nuclear factor kappa B1 and ERBB2 (Table I). In addition, Lupeol treatment was observed to upregulate the expression of several genes that are known to negatively regulate the survival and proliferation of human CaP cells (911). These include TIMP-3, KLK10, six-transmembrane epithelial antigen of the prostate (STEAP)-1 and insulin-like growth factor binding protein (IGFBP)-6 (Table I). Lupeol was also observed to induce the expression of TP53, RB1, RASSF1, MAPK12, PAR-4, PTEN, AGTR2 and CD82, genes that are known to negatively regulate the proliferation; however, the fold changes were <2-fold.

Table I.
List of selected genes modulated by Lupeol treatment of CaP cells

To further validate the microarray data at translational level, we treated LNCaP cells with Lupeol at the doses of 5–30 μM for 48 h. These doses were selected on the basis of our published data (5). To investigate whether the modulations induced by Lupeol occurs at translational level, we performed the immunoblot analysis of proliferation-associated proteins (Cdk2, c-myc, IGF-1R, ERBB2/HER2 and TIMP3) selected from the Table I. We observed that Lupeol treatment significantly decreased protein levels of in Cdk2, c-myc, IGF-1R and ERBB2 in LNCaP cells (Figure 1C). Lupeol was also observed to increase the level of TIMP-3 protein in cells in a dose-dependent manner (Figure 1D). It is well known that IGF-1R becomes activated after phosphorylation and the activated IGF-1R relays the tumorigenic signals in CaP cells (12,13). Next, we evaluated the phosphorylation of IGF-1R protein and observed that Lupeol treatment significantly decreased the phosphorylation of IGF-1R (Figure 1C). These data suggest that Lupeol-induced modulations observed at messenger RNA level corroborated with translational level changes in CaP cells.

Effect of Lupeol treatment on the expression level of β-catenin signaling in androgen-sensitive CaP cells

Lupeol was observed to induce modulation in the expression level of IGF-1R, ERBB2, TIMP3, cyclin D1 and MMP-2 (Table I) and it is noteworthy that the common feature shared by all of these genes is that these are directly or indirectly associated with β-catenin-signaling pathway (1416). These genes are known to either regulate or act as downstream targets of β-catenin signaling (1416). For example, IGF-1R is known to regulate the location, stability and transcriptional activity of β-catenin in cancer cells (17). Similarly, ERBB2/HER2 is reported to induce the expression of cyclin D1 whose expression is primarily known to be regulated by β-catenin (18). Further, β-catenin signaling is also reported to be increased in cells lacking TIMP-3 suggesting the regulatory action of TIMP-3 on β-catenin signaling (15). Compared with normal prostate epithelial cells, CaP cells have been reported to express high β-catenin protein levels (19). Stabilized β-catenin accumulates in the cytoplasm and translocates to the nucleus to induce transcriptional activation of proliferation-associated genes leading to increased proliferation of epithelial cells (20,21). Abnormal cytoplasmic/nuclear β-catenin expression has been reported to be associated with high Gleason scores in CaP patients (22). Since Lupeol was observed to modulate the expression of proliferation-associated genes (having direct or indirect association with β-catenin signaling), we next evaluated the effect of Lupeol on β-catenin signaling in CaP cells. Lupeol treatment was observed to significantly decrease the protein level of whole and cytoplasmic β-catenin protein in LNCaP cells in a dose-dependent manner (Figure 1E). We also observed that Lupeol treatment significantly decreases nuclear β-catenin level in LNCaP cells (Figure 1E). As evident from the densitometric analysis of immunoblots, Lupeol-induced β-catenin degradation was significant in LNCaP cells (Figure 1E).

Degradation of β-catenin in cells is known to be preceded by its phosphorylation. We next investigated the effect of Lupeol treatment on the phosphorylation of β-catenin in a time-dependent manner. As evident from the densitometric analysis, Lupeol treatment was observed to increase the phosphorylation of β-catenin at 24 h post-treatment (Figure 1F). At 48 h time point, Lupeol was observed to decrease the total pool of β-catenin (including phosphorylated, total, cytosolic and nuclear β-catenin) in CaP cells (Figure 1E). These data suggest that Lupeol treatment initiates the molecular events very early (24 h post-treatment) that ultimately result in the loss of β-catenin levels (Figure 1E).

GSK3β and axin are known to phosphorylate β-catenin protein leading to its degradation by proteasomes; thus, act as upstream regulators of β-catenin signaling (23,24). Increased phosphorylation of GSK3β is known to render β-catenin molecule defective and GSK3β and axin expressions are reported to be defective in CaP cells (2325). Next, we evaluated the effect of Lupeol treatment on GSK3β and axin protein levels. Lupeol treatment was observed to increase the GSK3β protein level in cells; however, marginal change in the phosphorylation of GSK3β was observed at the dose of 30 μM (Figure 2A). Further, to determine the role of GSK3β in Lupeol-induced β-catenin degradation, LNCaP cells were pretreated with the GSK3β inhibitor, BIO (0.1 μM), for 4 h prior to Lupeol treatment and cells were analyzed for β-catenin protein level. Inhibition of GSK3β by BIO prevented Lupeol-induced phosphorylation of β-catenin protein in LNCaP cells (Figure 2B). These findings further support the role of GSK3β activation in Lupeol-induced β-catenin degradation in CaP cells.

Fig. 2.
Effect of Lupeol treatment on the upstream regulators and downstream targets of β-catenin in CaP cells. (A) Immunoblots represent the effect of Lupeol treatment on the expression level of GSK3β, axin and the phosphorylation of GSK3β ...

The degradation of β-catenin depends on β-catenin phosphorylation, which occurs in a multiprotein complex containing axin, GSK3β and β-catenin. It is believed that in this complex, GSK3β phosphorylates the β-catenin primarily when it is bound to axin. Lupeol treatment was observed to increase the expression level of axin protein in cells (Figure 2A). To determine the effect of Lupeol treatment on the level of axin and GSK3β when in complex form, immunoprecipitation and immunoblot analyses were performed. As shown in Figure 2C, treatment of LNCaP cells with Lupeol induced the levels of axin and GSK3β in their complex thus increasing the possibility of β-catenin degradation in LNCaP cells.

To confirm that Lupeol-induced degradation of β-catenin is through the induction of axin protein, we investigated the effect of Lupeol treatment on the axin–GSK3β complex in LNCaP cells transfected with axin-targeted siRNA. As evident from immunoprecipitation analysis in axin–GSK3β complex in axin-suppressed cells, the level of axin protein was significantly reduced (Figure 2D). However, it is noteworthy that such cells when were treated with Lupeol exhibited increased axin protein levels in axin–GSK3β complex (Figure 2D). These data suggest that Lupeol destruction of β-catenin in human CaP cells is through the induction of axin protein level in axin–GSK3β complex.

β-Catenin acts to regulate the transcription of genes through the binding of a complex of β-catenin and T Cell Factor (Tcf) family of transcription factors to specific promoter elements (21,26). The decrease of nuclear β-catenin by Lupeol treatment suggested that β-catenin nuclear signaling might have been attenuated (Figure 1B). Next, we evaluated the effect of Lupeol treatment on the transcriptional activity of Tcf by transiently transfecting LNCaP cells with reporter plasmids bearing Tcf-4-binding sequences. At 24 h, Lupeol at 5 and 10 μM inhibited Tcf transcriptional activity by 50% (Figure 2E). The specificity of the Lupeol effect on Tcf reporters was confirmed by the fact that pFopflash (containing a ‘far from optimal’ Tcf binding site) was not influenced by Lupeol (Figure 2E).

β-Catenin is known to relieve the inhibition of TCF/lymphoid enhancer factor by repressors leading to transcriptional activation of target genes, such as c-myc, MMP-7, MMP-2 and cyclin D1 (21,26,27). Overexpression of β-catenin has been implicated in cell transformation and correlated with increased levels of Cyclin D1, MMP-2 and c-myc in human CaP cells (20,2830). Next, we determined the effect of Lupeol treatment to cells on the protein levels of Cyclin D1 and MMP-2. Lupeol treatment caused a significant decrease in the Cyclin D1 and MMP-2 protein levels in LNCaP cells (Figure 2F). We also determined the effect of Lupeol treatment on the transcriptional activation of MMP-2 gene. Lupeol (5 μM) significantly decreased MMP-2 transcriptional activation at 24 h post-treatment in cells (Figure 2G). To rule out the possibility that the effect of Lupeol is devoid of non-specific effect on general transcription, we transfected cells with 50 ng of pRLTK-luc plasmid and investigated its effect on Renilla in time-dependent manner in LNCaP cells. Lupeol exhibited no significant effect on Renilla luciferase activity (Figure 2H). MMP-2 is known to degrade the extracellular matrix when secreted by CaP cells in tissues. MMP-2 protein secreted by cells is known to exhibit gelatinolytic activity under in vitro conditions when analyzed by standard zymography. We observed that Lupeol treatment significantly decreases the gelatinolytic (enzymatic) activity of MMP-2 protein in LNCaP cells (Figure 2I).

Effect of Lupeol on β-catenin-signaling network in androgen-insensitive CaP cells

CaP disease in humans at the time of diagnosis is known to exhibit a heterogeneous system comprising of epithelial cells with different types of androgen status such as androgen-sensitive and androgen-insensitive cells. Since Lupeol was observed to inhibit growth and proliferation of androgen-sensitive CaP cells, we asked whether this effect of Lupeol is universal for all types of CaP cells. For this purpose, androgen-insensitive CaP cells DU145 were treated with Lupeol. These doses were selected on the basis of cell viability assay, where Lupeol (10–50 μM) was observed to inhibit the growth of androgen-insensitive cells (DU145 and PC-3) at 48 h without any adverse effect of normal prostate epithelial cells at these doses (data not shown). Lupeol treatment was observed to decrease the rate of proliferation of DU145 cells in a dose-dependent manner as was assessed by thymidine incorporation assay (Figure 3A). In addition, prolonged Lupeol treatment (for 21 days) was also observed to significantly inhibit the colony formation ability of DU145 (Figure 3B).

Fig. 3.
Effect of Lupeol on (A) proliferation of androgen-insensitive DU145 cells, (B) clonogenic potential of DU145 cells, (C) the expression level of prominent proliferation-associated proteins and (D) on β-catenin signaling in DU145 cells. (A) Histogram ...

Lupeol was observed to decrease the level of Cdk2, c-myc and ERBB2 proteins in DU145 cells (Figure 3C). Interestingly, Lupeol did not induce any modulation on total protein and phosphorylation level of IGF-1R in these cells (data not shown). Furthermore, Lupeol treatment caused a significant increase in the expression level of TIMP-3 protein in cells (Figure 3C). Next, we determined the effect of Lupeol treatment on β-catenin-signaling pathway in DU145 cells. Lupeol treatment was observed to cause a decrease in total, cytoplasmic and nuclear β-catenin levels in DU145 cells (Figure 3D). Lupeol was observed to significantly induce the phosphorylation of β-catenin in DU145 cells at 24 h time point in a dose-dependent manner (Figure 4A).

Fig. 4.
Effect of Lupeol on the phosphorylation of β-catenin and on GSK3β–axin complex in DU145 cells. (A) Immunoblots represent the effect of Lupeol on the phosphorylation of β-catenin in DU145 cells at 24 h post-treatment. Cells ...

Further, Lupeol treatment was observed to cause an increase in the expression level of axin protein with a peak effect at 10–20 μM (Figure 4B). Lupeol treatment caused a moderate decrease in the phosphorylation of GSK3β protein; however, no effect was observed on the total GSK3β protein levels in DU145 cells (Figure 4A). Effects of Lupeol treatment on the phosphorylation and degradation of β-catenin were minimal in BIO-pretreated DU145 cells suggesting the involvement of GSK3β in the Lupeol-induced effects (Figure 4C).

Next, we determined whether Lupeol has similar effect on GSK3β and axin levels in complex in DU145 cells as was observed in androgen-sensitive LNCaP cells. Treatment of DU145 cells with Lupeol induced the levels of axin–GSK3β complex formation (Figure 4D). In contrast, effect of Lupeol on GSK3β–axin complex in axin-silenced DU145 cells was only marginal (Figure 4E). Next, we determined effect of Lupeol treatment on the downstream targets of β-catenin. Lupeol treatment decreased the TCF promoter activity in DU145 cells (Figure 5A). Lupeol treatment also caused a decrease in the cyclin D1 and MMP-2 protein levels in cells (Figure 5B). Finally, Lupeol treatment resulted in decreased enzymatic activity of MMP-2 protein and transcriptional activation of MMP-2 gene in DU145 cells (Figure 5C and D).

Fig. 5.
Effect of Lupeol treatment on the (A) transcriptional activation TCF promoter, (B) expression level of Cyclin D1 and matrix mettaloproteinase-2 (MMP-2) proteins, (C) transcriptional activation MMP-2 gene and (D) on the enzyme activity of MMP-2 protein ...

Discussion

One difficulty in successfully treating CaP is that lesions respond differently to the treatment due to presence of a mixture of CaP cells comprising androgen-dependent and androgen-independent cells and the key to control of CaP seems to lie in the elimination of both types of CaP cells (without affecting the normal cells) (31). Recently, we showed that Lupeol induces apoptosis of LNCaP cells without any adverse effects on the viability of normal prostate epithelial cells (5). One major finding of this study is that Lupeol, a diet-based agent, caused inhibition in the growth and proliferation of human CaP cells irrespective of their androgen status. It is believed that the tumor cells bear the activation of proliferation-associated genes that directly or indirectly cross talk with β-catenin signaling forming a signaling network allowing CaP cells to survive, proliferate and acquire highly aggressive androgen-independent characteristics even after surgery and androgen ablation therapy (32). There exists a possibility that impediment of CaP may be addressed by targeting the β-catenin-associated signaling network that is impaired in CaP cells (33,34). Since lesions exhibit the activation of multiple signaling pathways, agents that target a single signaling pathway do not seem to be adequate in combating CaP. Based on our previous published data and the current study, we suggest that Lupeol could be such a multitarget agent. Lupeol is reported to inhibit the growth and proliferation of highly aggressive pancreatic cancer and melanoma cells and inhibit the skin tumorigenesis in a mouse model through the modulation of signaling pathways such as PI3K/Akt, nuclear factor kappa B1 and Ras/PKCα (3537). Lupeol is reported to exhibit various beneficial pharmacological activities under in vitro and in vivo conditions (5,3537). In the current study, we provide evidence that Lupeol adopts a strategy to target β-catenin-signaling network thus inhibiting the proliferation of human CaP cells of both androgen-dependent and androgen-independent nature.

Our data suggest that Lupeol modulates several signaling pathways in CaP cells. All the genes whose expression is modulated by Lupeol treatment (as listed in Table I) are known to be associated with the proliferation and/or survival of cancer cells. Our data is significant because Lupeol treatment decreases the expression of nuclear factor kappa B1 and tumor necrosis factor suggesting its potential against the inflammatory processes that is a common feature in CaP patients (38). ERBB2/HER2 is reported to be overexpressed in CaP patients and preclinical and clinical data show that the activation of the HER2-kinase axis is important for the progression of CaP to androgen-independent disease (39). Multiple pharmaceutical agents that block the ERBB2/HER2-kinase axis are currently being evaluated in patients with CaP (39). ERBB2 stimulation has been shown to activate androgen receptor in a ligand-independent fashion in CaP cells (40). It is noteworthy that Lupeol was observed to significantly decrease the expression level of ERBB2 in CaP cells (Table I and Figures 1 and and3).3). IGF-1R has been suggested to play an important role in the early androgen-dependent stages of CaP (12,13,41). Our data are significant as Lupeol was also observed to decrease the expression level and phosphorylation of IGF-1R in LNCaP cells that represents the androgen-dependent phase of CaP (Figure 1C). IGFBP6 has been reported to play an important role during the progression of CaP (11). IGFBP6 is an inhibitor of IGF-II and excess IGFBP6 has been reported to displace IGF-II from IGFBP2; thus, preventing it from potentiating the mitogenic action of IGF-II (42). Various studies have shown that increasing IGFBP6 level results in the decreased viability of human CaP cells; hence, IGFB6 has been suggested as a potential therapeutic target for human CaP treatment (43). Our data are significant because the transcriptional level of IGFBP6 was observed to be sharply increased by Lupeol treatment in CaP cells (Table I).

MMP-2 and myc proteins are highly expressed in CaP patients and are reported to be associated with survival, androgen independence, angiogenesis and invasion of CaP cells (22,29,30). A positive correlation has been shown to exist between MMP expression levels and Gleason score in CaP patients and c-myc was the first oncogene to be recognized as being overexpressed in human CaP (29,30,44). The proliferation pathway is known to be mediated by the ability of c-myc to activate several cyclins, including cyclin E and cyclin D2 (44). Interestingly, Lupeol treatment was observed to decrease the expression level of myc both at transcriptional as well as translational level (Figures 1 and and3).3). We also found that Lupeol significantly decreased the expression of MMP-2 both at transcriptional and translational level in CaP cells (Figures 2 and and5).5). MMP proteins possessing proteolytic properties when secreted in the extracellular vicinity are known to exert angiogenic and metastatic properties to the CaP cells, and Lupeol treatment was observed to decrease the proteolytic activity of secreted MMP-2 protein suggesting the efficacy of Lupeol against the spread of CaP cells (Figures 2 and and55).

The common feature shared by genes including MMP-2, myc, Cyclin D1, IGFBP6, IGF-1R, IGF2 and PI3K (Table I) is that these are directly or indirectly associated with β-catenin-signaling pathway (16). Various studies have shown that alterations in the β-catenin pathway may contribute to progression of CaP to androgen independence (32). Recent studies have shown that ~20–40% of hormone-refractory CaP samples exhibit nuclear localization of β-catenin (ref. 47 and references therein). Activation of the β-catenin pathway has been observed in CaP patients and a recent study showed that 32% of CaP patients with advanced disease carried mutations in β-catenin gene (45). Oncogenic activation of the β-catenin-signaling pathway has been reported to result in the abnormal accumulation of β-catenin. The translocation of β-catenin–TCF-4 complex to nucleus leads to transcriptional activation of target genes, such as c-myc, MMP-2 and cyclin D1 (29,30,33). Various reports suggest that β-catenin signaling cross talks with and is influenced by the alterations in the signaling pattern of growth factor receptors during the tumorigenesis process (36,37). It is noteworthy that in the current study, Lupeol was observed to restore the levels of active GSK3β–axin protein complex in the cytoplasm thus decreasing the stabilized β-catenin at upstream level of β-catenin signaling in both types of CaP cells (Figures 1 and and3).3). Lupeol treatment was also shown to decrease the level of β-catenin in nuclei of CaP cells thus inhibiting the transcription of proliferation-associated genes in CaP cells (Figures 1 and and3).3). These data further strengthen the suggestion that Lupeol is a potent inhibitor of β-catenin-signaling pathway in CaP cells.

To conclude, the multifaceted nature of the Lupeol against the β-catenin-signaling network that is involved in the proliferation and survival is probably to have numerous beneficial effects against the development, growth and progression of early (androgen dependent) as well as advanced stage (androgen independent) CaP in humans. Taken together, our present findings demonstrate the anticancer efficacy of Lupeol, with mechanistic rationale, against androgen-sensitive as well as androgen-insensitive human CaP cells. These observations warrant further in vivo efficacy studies in models that mimic progressive forms of human prostatic disease. The positive outcomes of such an in vivo study could form a strong basis for the development of Lupeol as a novel agent for human CaP prevention and/or intervention.

Funding

USA PHS (R03 CA130064 to Mohammad Saleem, Bhat, RO1 CA 78809, RO1 CA 101039, P50DK 65303 to H.M.). Department of Biotechnology, Ministry of Science and Technology, Government of India (BT/IN/BTON/Nich/09/2007 to I.M.).

Acknowledgments

We thankfully acknowledge Dr Etty N.Benveniste (University of Alabama at Birmingham, AL) for providing pGL2-MMP-2-luc reporter plasmid.

Conflict of Interest Statement: None declared.

Glossary

Abbreviations

BIO
(2′Z,3′E)-6-bromoindirubin-3′-oxime
CaP
prostate cancer
GSK3β
glycogen synthase kinase 3 beta
IGFBP
insulin-like growth factor binding protein
MMP
matrix metalloproteinase
siRNA
small interfering RNA
TCF
T Cell Factor
TIMP
tissue inhibitor of metalloproteinases

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