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 were treated with vehicle (dimethyl sulfoxide + alcohol) only or specified concentrations of Lupeol and the expression of phosphorylated β-catenin was determined by western blot analysis in whole-cell lysates. (B) Immunoblots represent the effect of Lupeol treatment on the expression level of GSK3β, axin and on the phosphorylation of GSK3β protein in DU145 cells. Cells were treated with vehicle only or specified concentrations of Lupeol and the expression of GSK3β, axin and phosphorylated GSK3β were determined by western blot analysis. (C) Immunoblots represent the effect of Lupeol on the phosphorylation of β-catenin in DU145 cells at 24 h post-treatment in presence of GSK3β inhibitor (BIO). Cells treated with BIO and Lupeol as described under Materials and Methods and the expression of phosphorylated β-catenin was determined by western blot analysis. The immunoblots (Figure 5A–C) are representative of three independent experiments with similar results. Equal loading of proteins was confirmed by stripping immunoblots and reprobing them for β-actin. ‘V’ represents vehicle-treated cells. The values above the immunoblots represent relative densities (in terms of fold units) of the bands normalized to β-actin. (D) Immunoblots represent the effect of Lupeol on GSK3β and axin complex in DU145 cells. DU145 cells were treated with the specified concentrations of Lupeol for 48 h and were processed for the immunoprecipitation analysis as described under Materials and Methods. (E) Immunoblots represent the effect of Lupeol on the GSK3β and axin complex in axin-silenced DU145 cells. Axin-silenced DU145 cells were treated with Lupeol and immunoprecipitation analysis was performed as described under Materials and Methods. Input control consisted of 10% of post-cleared protein. After detecting axin protein in input samples, immunoblots were stripped and reprobed for β-actin to confirm the equal loading. The immunoblots shown here are representative of three independent experiments with similar results. (D and E) V represents vehicle-treated cells, ‘WB’ represents western blot and ‘IP’ represents immunoprecipitation.

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 showing the rate of [³H]thymidine uptake in DU145 cells treated with Lupeol. DU145 cells were treated with Lupeol in the presence of [³H]thymidine and [³H]thymidine incorporation was quantified as described under Materials and Methods. Each bar in the histogram represents mean ± standard error, ‘*’ indicates P < 0.05. All experiments were repeated three times with similar results. (B) Histogram showing number of colonies formed by DU145 cells treated with Lupeol. Cells seeded in agarose and incubated at 37°C were treated with Lupeol as described under Materials and Methods. After 21 days of incubation, the cells were stained with crystal violet–methanol and colonies were counted. Each bar in the histogram represents mean ± standard error, * indicates P < 0.05. All experiments were repeated three times with similar results. (C and D) Immunoblots represent the effect of Lupeol treatment on the expression level of Cdk2, c-myc, ERBB2, TIMP3, β-catenin (total, cytosolic and nuclear), GSK3β and axin proteins in DU145 cells. Cells were treated with vehicle (dimethyl sulfoxide + alcohol) only or specified concentrations of Lupeol and the expression levels of proteins were determined by western blot analysis. Equal loading was confirmed by probing the immunoblots for β-actin and Lamin. The immunoblots shown here are representative of three independent experiments with similar results. ‘C’ represents untreated control and V represents vehicle. The values above the immunoblots represent relative densities (in terms of fold units) of the bands normalized to β-actin.

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 in DU145 cells. (A) Histogram represents the effect of Lupeol treatment on the transcriptional activation of TCF (marker of β-catenin activity) in DU145 cells. pTK-TCF-Luc (pTopFlash)-transfected DU145 cells were treated with Lupeol as described under Materials and Methods. pFopFlash and Renilla luciferase were used as negative and internal control, respectively. For controls, the same amount of empty vectors were transfected in cells. (B) Immunoblots represents the effect of Lupeol treatment on the expression level of Cyclin D1 and MMP-2 proteins. DU145 cells were treated with vehicle (dimethyl sulfoxide + alcohol) only or specified concentrations of Lupeol and expression of Cyclin D1 and MMP-2 proteins were determined by western blot analysis. Equal loading was confirmed by stripping immunoblots and reprobing them for β-actin. The immunoblots shown here are representative of three independent experiments with similar results. ‘C’ represents untreated control and ‘V’ represents vehicle-treated cells. The values above the immunoblots represent relative densities (in terms of fold units) of the bands normalized to β-actin. (C) Image represents the gelatinolytic effect of CaP cells treated with Lupeol. The gelatinolytic activity of MMP-2 in the conditioned media harvested from treated cells was determined by employing zymography kit as described under Materials and Methods (D) Histogram represents the effect of Lupeol treatment on the transcriptional activation of MMP-2 in CaP cells. pGL2-MMP-2-luc-transfected cells were treated with Lupeol and the transcriptional activity was measured in terms of luciferase activity as described under Materials and Methods. Relative luciferase activity was calculated with the values from vector alone group with or without Lupeol-treated group.

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. Cells were subjected to Lupeol treatment for 48 h, the last 16 h of which in the presence of [³H]thymidine (0.5 μCi/ml). Incorporated [³H]thymidine was quantified by liquid scintillation counting as described under Materials and Methods. Each bar in the histogram represents mean ± standard error, ‘*’ indicates P < 0.05. All experiments were repeated three times with similar results. (B) Histogram showing number of colonies formed by LNCaP cells treated with Lupeol. Cells seeded in agarose and incubated at 37°C were treated with Lupeol as described under Materials and Methods. After 21 days of incubation, the cells were stained with crystal violet–methanol and colonies were counted. Each bar in the histogram represents mean ± standard error, * indicates P < 0.05. All experiments were repeated three times with similar results. (C–E) Immunoblots represent the effect of Lupeol treatment on the protein level of Cdk2, c-myc, ERBB2, IGF-1R, TIMP3, β-catenin (total, cytosolic and nuclear), phosphorylated-IGF-1R, GSK3β and axin in LNCaP cells. Cells were treated with vehicle (dimethyl sulfoxide + alcohol) only or specified concentrations of Lupeol for 48 h. (F) Immunoblots represent the effect of Lupeol on the phosphorylation of β-catenin in LNCaP cells at 24 h post-treatment. Cells were treated with vehicle (dimethyl sulfoxide + alcohol) only or specified concentrations of Lupeol for 24 h and harvested (Figure 1C–F). Proteins levels in these experiments were determined by western blot analysis by using specific antibody. The immunoblots shown here are representative of three independent experiments with similar results. The details are described under Material and Methods. ‘V’ represents vehicle. Values above the immunoblots represent relative densities (in terms of fold units) of the bands normalized to β-actin. Equal loading was confirmed by stripping immunoblots and reprobing them for β-actin (for total and cytosolic fractions) and Lamin (for nuclear fraction).

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 protein in LNCaP cells. Cells were treated with vehicle (dimethyl sulfoxide + alcohol) only or specified concentrations of Lupeol for 48 h. The expression level of GSK3β, axin and phosphorylated GSK3β were determined by western blot analysis. Equal loading was confirmed by stripping immunoblots and reprobing them for β-actin. (B) Immunoblots represent the effect of Lupeol on the phosphorylation of β-catenin in LNCaP cells at 24 h post-treatment in presence of GSK3β inhibitor (BIO). CaP cells were pretreated with 0.1 μM of BIO for 4 h before Lupeol treatment (20 μM) for 24 h. The expression of phosphorylated β-catenin was determined by western blot analysis in total cell lysates. Equal loading was confirmed by stripping immunoblots and reprobing them for β-actin. The immunoblots shown here (Figure 2A and B) are representative of three independent experiments with similar results. The details are described under Materials and Methods. ‘V’ represents vehicle. Values above the immunoblots represent relative densities (in terms of fold units) of the bands normalized to β-actin. (C) Immunoblots represent the effect of Lupeol on the GSK3β and axin complex in LNCaP cells. LNCaP cells were treated with the specified concentrations of Lupeol (20 μM) for 48 h. Cells were harvested and total cell lysates were processed for the immunoprecipitation analysis as described under Materials and Methods. V represents vehicle. (D) Immunoblots represent the effect of Lupeol on the GSK3β and axin complex in axin-silenced LNCaP cells. Cells transfected with 100 nM of siRNA directed against axin and scrambled siRNA (100 nM) were treated with Lupeol and vehicle alone. Cells were harvested after 24 h post-Lupeol treatment and cell lysates were processed for immunoprecipitation analysis as described under Materials and Methods. The expression of GSK3β and axin proteins in the immunocomplex was determined by western blot analysis. Input control consisted of 10% of post-cleared protein. After detecting axin protein in input samples, immunoblots were stripped and reprobed for β-actin to confirm the equal loading. The immunoblots shown here are representative of three independent experiments with similar results. V represents vehicle-treated cells. (E) Histogram represents the effect of Lupeol treatment on the transcriptional activation of TCF (marker of β-catenin activity) in LNCaP cells. pTK-TCF-Luc (pTopFlash)-transfected LNCaP cells were treated with Lupeol and the transcriptional activity was measured as described under Materials and Methods. pFopFlash was used as a negative control and Renilla luciferase was used as an internal control. Relative luciferase activity was calculated with the values from vector alone group with or without Lupeol-treated group. (F) Immunoblots represent the effect of Lupeol treatment on the expression level of Cyclin D1 and MMP-2 proteins. Cells were treated with vehicle (dimethyl sulfoxide + alcohol) only or specified concentrations of Lupeol and the expression of Cyclin D1 and MMP-2 were determined in whole-cell lysates. Equal loading was confirmed by stripping immunoblots and reprobing them for β-actin. The immunoblots shown here are representative of three independent experiments with similar results. ‘C’ represents untreated cells and V represents vehicle-treated cells. The values above the immunoblots represent relative densities (in terms of fold units) of the bands normalized to β-actin. (G) Histogram represents the effect of Lupeol treatment on the transcriptional activation of MMP-2 in LNCaP cells. pGL2-MMP-2-luc-transfected LNCaP cells were treated with Lupeol and the transcriptional activity was measured in terms of luciferase activity as described under Materials and Methods. Relative luciferase activity was calculated with the values from vector alone group with or without Lupeol-treated group. (H) Histogram represents the effect of Lupeol treatment on Renilla luciferase activity in LNCaP cells. LNCaP cells were transfected with pRLTK-luc plasmid (50 ng) and treated with Lupeol. After 24 h of Lupeol treatment, the transcriptional activity was measured as described under Materials and Methods. Data are presented as percent luciferase activity. Each bar in the histogram represents mean ± standard error. (I) Image represents the gelatinolytic effect of LNCaP cells treated with Lupeol. The gelatinolytic activity of MMP-2 was determined in conditioned media harvested from treated cells by employing zymography as described under Materials and Methods.