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1.
Figure 3

Figure 3. Curcumin treatment enhances membrane β-catenin.. From: Curcumin Attenuates ?-catenin Signaling in Prostate Cancer Cells through Activation of Protein Kinase D1.

C4-2 cells were cultured on glass coverslips overnight in 12 well plates. The cells were treated with DMSO (upper panel) or curcumin (20 µM) (lower panel) for 1 h, washed, fixed and immunostained for β-catenin (red) and counter-stained with DAPI (blue). Higher β–catenin staining was observed on the cell surface at 1 h of curcumin treatment, compared to DMSO control treated cells. Original Magnifications 600×.

Vasudha Sundram, et al. PLoS One. 2012;7(4):e35368.
2.
Figure 1

Figure 1. Curcumin inhibits prostate cancer cell proliferation.. From: Curcumin Attenuates ?-catenin Signaling in Prostate Cancer Cells through Activation of Protein Kinase D1.

A). Chemical structure of curcumin. B). Effect of curcumin on proliferation of various prostate cancer cell lines. LNCaP, C4-2, DU145 and PC3 cell were treated with curcumin or vehicle control DMSO for 48 h and cell proliferation was determined using MTS assay. The percent cell proliferation was calculated by normalizing the proliferation of curcumin treated cells with proliferation of control treated cells. Concentration dependent inhibition in cell proliferation was observed with curcumin treatment. Mean ± SE; n = 3; *p<0.05.

Vasudha Sundram, et al. PLoS One. 2012;7(4):e35368.
3.
Figure 8

Figure 8. Curcumin inhibits prostate cancer growth in xenograft mouse model.. From: Curcumin Attenuates ?-catenin Signaling in Prostate Cancer Cells through Activation of Protein Kinase D1.

A) Effect of curcumin on prostate cancer growth. C4-2 prostate cancer cells were used to generate xenografts in male nude mice. Following tumor development, the mice were treated intra-tumorally with curcumin (n = 4) or DMSO (n = 3). The rate of tumor growth was measured after 7 day and the percent tumor growth following treatment was graphed. Curcumin effectively inhibits prostate cancer growth. B) Effect of curcumin on β-catenin localization. Tumor tissues from curcumin or control treated mice were processed for IHC staining using anti-β-catenin antibody. Enhanced staining of membranous β-catenin was observed in curcumin treated mice compared to control mice. Original Magnifications 400×.

Vasudha Sundram, et al. PLoS One. 2012;7(4):e35368.
4.
Figure 9

Figure 9. Schematic diagram showing possible signaling mechanisms modulated by curcumin mediated PKD1 activation.. From: Curcumin Attenuates ?-catenin Signaling in Prostate Cancer Cells through Activation of Protein Kinase D1.

Curcumin modulates a number of molecular pathways within the cancer cells including PKD1 signaling. Curcumin may suppress prostate cancer growth and metastasis by activating PKD1, which in turn may inhibit cell growth through the inhibition of β-catenin/TCF transcription activity, enhance cell-cell aggregation via enhanced translocation of β-catenin to the cell membrane and inhibit cell motility either directly by enhancing cell-cell aggregation and/or phosphorylating and inhibiting the function of sling shot 1 like (SSH1L) phosphatase or indirectly (dashed lines) by negatively regulating the expression of active cofilin via indirectly activating LIM kinase (LIMK).

Vasudha Sundram, et al. PLoS One. 2012;7(4):e35368.
5.
Figure 2

Figure 2. Curcumin activates PKD1.. From: Curcumin Attenuates ?-catenin Signaling in Prostate Cancer Cells through Activation of Protein Kinase D1.

A). Effect of curcumin on PKD1 levels. C4-2 cells were treated with 20 µM curcumin. At varying time points, the cells were harvested, and the lysates were resolved on SDS-PAGE, transferred onto a PVDF membrane and probed for total PKD1. β-actin was used as an internal loading control. The band intensities were densitometrically analyzed, normalized to β-actin levels and graphed. Curcumin treatment resulted in no marked change in PKD1 expression at 1, 3 and 6 h. B). Effect of curcumin on activation of PKD1. For determining the expression of activated/phosphorylated PKD1, blots were probed with phospho PKD1, total PKD1 and β-actin antibodies. The pPKD1 band intensity was normalized to total PKD1 levels and graphed. Curcumin treatment induced PKD1 activation/phosphorylation by 1 h, while no apparent changes were observed in the expression of total PKD1. Representative blots of three experiments are shown in the figure. AU- arbitrary units.

Vasudha Sundram, et al. PLoS One. 2012;7(4):e35368.
6.
Figure 4

Figure 4. PKD1 is required for curcumin induced enrichment of β-catenin on the membrane.. From: Curcumin Attenuates ?-catenin Signaling in Prostate Cancer Cells through Activation of Protein Kinase D1.

A). Silencing of PKD1 by PKD1 specific siRNA. C4-2 cells were transfected for 48 h with 25 nM control siRNA or PKD1 siRNA, lysed and immunoblotted for PKD1 and β-actin using specific antibodies. Quantitation of protein band intensities was performed by densitometric analysis. The PKD1 levels was normalized to β-actin levels and graphed. AU- arbitrary units. Immunoblotting shows over 95% suppression of PKD1 expression on transfection with PKD1 specific siRNA (lane 2) compared to control siRNA-transfected cells (lane 1). B). Suppression of PKD1 inhibits enrichment of membrane β-catenin levels. C4-2 cells were cultured on coverslips overnight. The cells were first transfected with either control siRNA (A1–D1; A2–D2) or PKD1 silencing siRNA (A3–D3; A4–D4) for 24 h, followed by treatment with vehicle control (DMSO) (A1–D1; A3–D3) or curcumin (20 µM) (A2–D2; A4–D4) for 1 h. The cells were immunostained for β-catenin (red) or PKD1 (green) and the nucleus was counter stained with DAPI (blue). Higher β–catenin staining was observed on the cell surface of control siRNA cells at 1 h of curcumin treatment (A2) compared to vehicle treatment (A1). However, siRNA mediated silencing of PKD1 (B3, B4) inhibited curcumin mediated enrichment of membrane β–catenin staining on the cell surface (A4 vs A3 and A2). Original Magnifications 600× with 2× zoom.

Vasudha Sundram, et al. PLoS One. 2012;7(4):e35368.
7.
Figure 6

Figure 6. Curcumin treatment attenuates colony formation and cell-cell aggregation.. From: Curcumin Attenuates ?-catenin Signaling in Prostate Cancer Cells through Activation of Protein Kinase D1.

A). Anchorage dependent colony formation assay. C4-2 cells (2000) were plated overnight, treated with indicated concentrations of curcumin for 14 days and examined for their colony forming ability. Representative photographs are shown. Curcumin showed a dose-dependent inhibition in anchorage dependent colony formation assay. Mean ± SE; n = 3; *p<0.05. B). Anchorage independent colony formation assay. C4-2 cells were seeded in 0.3% agarose and treated with varying concentrations of curcumin for 9 days. The number of colonies were counted and plotted. Curcumin treatment inhibited anchorage independent colony formation of C4-2 cells. Mean ± SE; n = 3; *p<0.01. C) Cell-cell aggregation assay. C4-2 cells treated with curcumin (15 µM) or DMSO for 1 h were harvested and assayed for cell-cell aggregation by incubating under gentle shaking conditions at 37°C in the presence of 5 mM CaCl2. After 6 h incubation, an aliquot of the reaction mixture was analyzed and photographed for cell-cell aggregation under phase contrast microscope. Larger cell-cell aggregates were observed in curcumin treated cells, compared to DMSO control cells. Original Magnifications 100×.

Vasudha Sundram, et al. PLoS One. 2012;7(4):e35368.
8.
Figure 7

Figure 7. Curcumin treatment inhibits cell motility through phosphorylation of cofilin.. From: Curcumin Attenuates ?-catenin Signaling in Prostate Cancer Cells through Activation of Protein Kinase D1.

A). Scratch assay. C4-2 cells were grown, until confluent, in plates containing IBIDI inserts. The inserts were removed from the plates to generate gaps (solid white lines show width of the gap; dashed lines border the gap) and phase contrast images of the same area of the gaps were taken at varying time intervals in the presence or absence of 20 µM curcumin. Curcumin treatment inhibited motility of C4-2 prostate cancer cells. B). Boyden's chamber assay. Equal numbers of C4-2 cells were seeded on the Boyden's chambers and incubated in the presence DMSO or curcumin (20 µM) for 24 h. Migrated cells were fixed, stained, counted and graphed. Curcumin inhibited motility of C4-2 cells. Mean ± SE; n = 3; *p<0.05. C). Effect of curcumin on the expression of actin remodeling proteins. Total cell lysates prepared from curcumin (20 µM) or DMSO treated C4-2 cells were processed for immunoblotting using specific antibodies. The densitometric quantitation of protein bands normalized to β-actin level is shown in graph. Curcumin treatment induced a marked increase in the expression of inactive phospho-cofilin compared to DMSO treated control cells. Minor change was also observed in the expression of Arp3. AU- arbitrary units.

Vasudha Sundram, et al. PLoS One. 2012;7(4):e35368.
9.
Figure 5

Figure 5. Curcumin inhibits β-catenin transcription activity in prostate cancer cells.. From: Curcumin Attenuates ?-catenin Signaling in Prostate Cancer Cells through Activation of Protein Kinase D1.

A) Effect of curcumin treatment on the cellular localization of β-catenin and PKD1. C4-2 cells treated with curcumin (20 µM) or DMSO for 24 h were immunostained for β-catenin (green) or PKD1 (red) and counter-stained with DAPI (blue). Curcumin treated cells showed lower cytoplasmic and higher membrane β-catenin staining compared to control cells. In addition, while PKD1 was predominantly localized in the cytoplasm in control cells, curcumin treated cells exhibited staining primarily on the cell membrane and in the nucleus (white arrows), with faint cytoplasmic staining. Original Magnifications 600× with 2× zoom. B) Effect of curcumin on nuclear β-catenin levels. Nuclear proteins isolated from C4-2 cells treated either with curcumin (20 µM) or DMSO were resolved on PVDF membrane and processed for immunoblotting using β-catenin antibody. Histone H1 protein was used as loading control. Densitometric quantitation of β-catenin band intensities, normalized to Histone H1 levels is shown in graph. Curcumin treatment markedly decreased the levels of nuclear β-catenin compared to vehicle treated cells. AU- arbitrary units. C) Effect of curcumin on β-catenin transcription activity in C4-2 prostate cancer cells. The β-catenin transcription activity was measured by transiently transfecting the cells with TCF luciferase reporter construct containing either TCF promoter binding sites (pTOP-FLASH) or mutant TCF promoter binding sites (pFOP-FLASH) along with internal control plasmid containing Renilla luciferase gene (pRL-TK). After 3 h, the cells were treated with curcumin (20 µM) or DMSO for 24 h. The β-catenin transcription activity was first normalized to Renilla luciferase activity, and expressed as a ratio of pTOP-FLASH/pFOP-FLASH activity. The activity of curcumin treated cells was normalized to activity of vehicle treated cells (considered 100%). Curcumin treatment significantly reduced β-catenin transcription activity in C4-2 cells compared to vehicle treated cells. Mean ± SE, n = 3, *p<0.01. D). Effect of curcumin on transcription of cyclin D1. The transcription of cyclin D1 was analyzed from cells treated with curcumin or vehicle control for 24 h. After reverse transcription of RNA to cDNA, PCR amplification of cyclin D1 or internal control GAPDH was carried out using gene specific primers. The amplified products were resolved on 1% agarose gel. The densitometric quantitation of cyclin D1 band intensities normalized to GAPDH levels is shown in graph. Curcumin treatment reduced the expression of cyclin D1. AU- arbitrary units. E). Immunoblot analyses. Cell lysates prepared from curcumin (20 µM) or DMSO treated C4-2 cells were resolved by SDS-PAGE and processed for immunoblotting using specific antibodies. Curcumin treatment markedly decreased cyclin D1 expression, whereas no effect was observed on the expression of total β-catenin, E-cadherin or Wnt 3a. Representative immunoblots from three experiments are shown.

Vasudha Sundram, et al. PLoS One. 2012;7(4):e35368.

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