Curcumin Attenuates β-catenin Signaling in Prostate Cancer Cells through Activation of Protein Kinase D1

Prostate cancer is the most commonly diagnosed cancer affecting 1 in 6 males in the US. Understanding the molecular basis of prostate cancer progression can serve as a tool for early diagnosis and development of novel treatment strategies for this disease. Protein Kinase D1 (PKD1) is a multifunctional kinase that is highly expressed in normal prostate. The decreased expression of PKD1 has been associated with the progression of prostate cancer. Therefore, synthetic or natural products that regulate this signaling pathway can serve as novel therapeutic modalities for prostate cancer prevention and treatment. Curcumin, the active ingredient of turmeric, has shown anti-cancer properties via modulation of a number of different molecular pathways. Herein, we have demonstrated that curcumin activates PKD1, resulting in changes in β-catenin signaling by inhibiting nuclear β-catenin transcription activity and enhancing the levels of membrane β-catenin in prostate cancer cells. Modulation of these cellular events by curcumin correlated with decreased cell proliferation, colony formation and cell motility and enhanced cell-cell aggregation in prostate cancer cells. In addition, we have also revealed that inhibition of cell motility by curcumin is mediated by decreasing the levels of active cofilin, a downstream target of PKD1. The potent anti-cancer effects of curcumin in vitro were also reflected in a prostate cancer xenograft mouse model. The in vivo inhibition of tumor growth also correlated with enhanced membrane localization of β-catenin. Overall, our findings herein have revealed a novel molecular mechanism of curcumin action via the activation of PKD1 in prostate cancer cells

Inflammasome Activation in Retinal Pigment Epithelium from Human Donors with Age-Related Macular Degeneration

Age-related macular degeneration (AMD), the leading cause of blindness in the elderly, is characterized by the death of retinal pigment epithelium (RPE) and photoreceptors. One of the risk factors associated with developing AMD is the single nucleotide polymorphism (SNP) found within the gene encoding complement factor H (CFH). Part of the innate immune system, CFH inhibits alternative complement pathway activation. Multi-protein complexes called inflammasomes also play a role in the innate immune response. Previous studies reported that inflammasome activation may contribute to AMD pathology. In this study, we used primary human adult RPE cell cultures from multiple donors, with and without AMD, that were genotyped for the Y402H CFH risk allele. We found complement and inflammasome-related genes and proteins at basal levels in RPE tissue and cell cultures. Additionally, treatment with rotenone, bafilomycin A, and ATP led to inflammasome activation. Overall, the response to priming and activation was similar, irrespective of disease state or CFH genotype. While these data show that the inflammasome is present and active in RPE, our results suggest that inflammasome activation may not contribute to early AMD pathology

PKD1 is required for curcumin induced enrichment of β-catenin on the membrane.

<p>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.</p

Curcumin treatment attenuates colony formation and cell-cell aggregation.

<p>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; *<i>p</i><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; <i>*p</i><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×.</p

Curcumin inhibits prostate cancer cell proliferation.

<p>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.</p

Curcumin inhibits β-catenin transcription activity in prostate cancer cells.

<p>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 <i>Renilla</i> 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 <i>Renilla</i> 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.</p

Schematic diagram showing possible signaling mechanisms modulated by curcumin mediated PKD1 activation.

<p>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 <i>via</i> 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 <i>via</i> indirectly activating LIM kinase (LIMK).</p

Curcumin treatment inhibits cell motility through phosphorylation of cofilin.

<p>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; *<i>p</i><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.</p

Curcumin inhibits prostate cancer growth in xenograft mouse model.

<p>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×.</p