Grape Seed Proanthocyanidins Inhibit Melanoma Cell Invasiveness by Reduction of PGE2 Synthesis and Reversal of Epithelial-to-Mesenchymal Transition

Melanoma is the leading cause of death from skin disease due, in large part, to its propensity to metastasize. We have examined the effect of grape seed proanthocyanidins (GSPs) on melanoma cancer cell migration and the molecular mechanisms underlying these effects using highly metastasis-specific human melanoma cell lines, A375 and Hs294t. Using in vitro cell invasion assays, we observed that treatment of A375 and Hs294t cells with GSPs resulted in a concentration-dependent inhibition of invasion or cell migration of these cells, which was associated with a reduction in the levels of cyclooxygenase (COX)-2 expression and prostaglandin (PG) E2 production. Treatment of cells with celecoxib, a COX-2 inhibitor, or transient transfection of melanoma cells with COX-2 small interfering RNA, also inhibited melanoma cell migration. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate, an inducer of COX-2, enhanced the phosphorylation of ERK1/2, a protein of mitogen-activated protein kinase family, and subsequently cell migration whereas both GSPs and celecoxib significantly inhibited 12-O-tetradecanoylphorbol-13-acetate -promoted cell migration as well as phosphorylation of ERK1/2. Treatment of cells with UO126, an inhibitor of MEK, also inhibited the migration of melanoma cells. Further, GSPs inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in melanoma cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited cell migration. Additionally, inhibition of melanoma cell migration by GSPs was associated with reversal of epithelial-mesenchymal transition process, which resulted in an increase in the levels of epithelial biomarkers (E-cadherin and cytokeratins) while loss of mesenchymal biomarkers (vimentin, fibronectin and N-cadherin) in melanoma cells. Together, these results indicate that GSPs have the ability to inhibit melanoma cell invasion/migration by targeting the endogenous expression of COX-2 and reversing the process of epithelial-to-mesenchymal transition

Silymarin Targets β-Catenin Signaling in Blocking Migration/Invasion of Human Melanoma Cells

Metastatic melanoma is a leading cause of death from skin diseases, and is often associated with activation of Wnt/β-catenin signaling pathway. We have examined the inhibitory effect of silymarin, a plant flavanoid from Silybum marianum, on cell migration of metastasis-specific human melanoma cell lines (A375 and Hs294t) and assessed whether Wnt/β-catenin signaling is the target of silymarin. Using an in vitro invasion assay, we found that treatment of human melanoma cell lines with silymarin resulted in concentration-dependent inhibition of cell migration, which was associated with accumulation of cytosolic β-catenin, while reducing the nuclear accumulation of β-catenin (i.e., β-catenin inactivation) and reducing the levels of matrix metalloproteinase (MMP) -2 and MMP-9 which are the down-stream targets of β-catenin. Silymarin enhanced: (i) the levels of casein kinase 1α, glycogen synthase kinase-3β and phosphorylated-β-catenin on critical residues Ser45, Ser33/37 and Thr41, and (ii) the binding of β-transducin repeat-containing proteins (β-TrCP) with phospho forms of β-catenin in melanoma cells. These events play important roles in degradation or inactivation of β-catenin. To verify whether β-catenin is a potent molecular target of silymarin, the effect of silymarin was determined on β-catenin-activated (Mel 1241) and β-catenin-inactivated (Mel 1011) melanoma cells. Treatment of Mel 1241 cells with silymarin or FH535, an inhibitor of Wnt/β-catenin pathway, significantly inhibited cell migration of Mel 1241 cells, which was associated with the elevated levels of casein kinase 1α and glycogen synthase kinase-3β, and decreased accumulation of nuclear β-catenin and inhibition of MMP-2 and MMP-9 levels. However, this effect of silymarin and FH535 was not found in Mel 1011 melanoma cells. These results indicate for the first time that silymarin inhibits melanoma cell migration by targeting β-catenin signaling pathway

Association of SP-D, MBL and I-NOS genetic variants with pulmonary tuberculosis

Background : Pulmonary tuberculosis is caused by Mycobacterium tuberculosis . It is a multifactorial disease with both host as well as pathogen factors contributing to susceptibility and protection from the disease. Various reports have highlighted important roles of lung surfactant protein D (SP-D), mannan-binding lectin (MBL) and I-NOS in innate immune defense against M. tuberculosis Aims : The present study investigated the role of polymorphisms in three candidate genes encoding Lung surfactant protein D, Mannan binding lectin and Inducible Nitric oxide synthase, in susceptibility and protection to pulmonary tuberculosis. Settings and Design : A case-control association study of SNP&apos;s in lung surfactant protein D (SP-D), mannan-binding lectin (MBL) and I-NOS with pulmonary tuberculosis in Indian population was carried out. This involved sequencing of all the coding exons of lung surfactant protein D (SP-D) , while, exon 1 (collagen region) and exon 4 (carbohydrate recognition domain) of mannan-binding lectin (MBL) and exons 2, 8 and 16 of I-NOS and their flanking intronic regions for single nucleotide polymorphisms in DNA samples isolated from 30 pulmonary tuberculosis patients and 30 controls of Indian population. Statistical analysis: Various allele frequencies were calculated using online two by two table (home.clara.net/sisa/). Odds ratio and P values were calculated at 95% confidence interval (CI). Results : A total of fourteen single nucleotide polymorphisms (5 in SP-D , 5 in MBL and 4 in I-NOS ) were observed of which four (G459A SP-D , G274T I-NOS , G1011A and T357G MBL ) have not been reported earlier. Four single nucleotide polymorphisms viz. G459A of exon 7 of SP-D ( P =0.00, odds ratio (OR) = 4.96, 2.18 OR< 11.31), G875A of exon 1 of MBL ( P = 0.00 or= 3.85 1.66 OR< 8.88), G300A of intron 8 ( P =0.00 or=4.04, 2.20< OR< 7.42) and G274T of intron 16 of I-NOS ( P =0.00 or=4.46, 2.40 OR< 8.28) showed significant association with TB in Indian population. Conclusion: The present study has led to identification of 4 SNP&apos;s in SP-D , MBL and I-NOS associated with pulmonary tuberculosis in Indian population

Grape proanthocyanidin inhibit pancreatic cancer cell growth in vitro and in vivo through induction of apoptosis and by targeting the PI3K/Akt pathway.

Pancreatic cancer is an aggressive malignancy that is frequently diagnosed at an advanced stage with poor prognosis. Here, we report the chemotherapeutic effects of bioactive proanthocyanidins from grape seeds (GSPs) as assessed using In Vitro and In Vivo models. Treatment of human pancreatic cancer cells (Miapaca-2, PANC-1 and AsPC-1) with GSPs In Vitro reduced cell viability and increased G2/M phase arrest of the cell cycle leading to induction of apoptosis in a dose- and time-dependent manner. The GSPs-induced apoptosis of pancreatic cancer cells was associated with a decrease in the levels of Bcl-2 and Bcl-xl and an increase in the levels of Bax and activated caspase-3. Treatment of Miapaca-2 and PANC-1 cells with GSPs also decreased the levels of phosphatidylinositol-3-kinase (PI3K) and phosphorylation of Akt at ser(473). siRNA knockdown of PI3K from pancreatic cancer cells also reduced the phosphorylation of Akt. Further, dietary administration of GSPs (0.5%, w/w) as a supplemented AIN76A control diet significantly inhibited the growth of Miapaca-2 pancreatic tumor xenografts grown subcutaneously in athymic nude mice, which was associated with: (i) inhibition of cell proliferation, (ii) induction of apoptosis of tumor cells, (iii) increased expression of Bax, reduced expression of anti-apoptotic proteins and activation of caspase-3-positive cells, and (iv) decreased expression of PI3K and p-Akt in tumor xenograft tissues. Together, these results suggest that GSPs may have a potential chemotherapeutic effect on pancreatic cancer cell growth

Dietary GSPs reduce the proliferation potential and inhibit PI3K signaling pathway in xenograft tissues.

<p>(<b>A</b>) Immunohistochemical analysis of tumor samples obtained from mice at the termination of the experiment described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043064#pone-0043064-g005" target="_blank">Figure 5</a> was used to detect Ki-67-positive cells as an estimate of the proliferation index. Ki-67-positive cells were counted in four to five different fields of a section, and data are summarized in terms of percent positive cells from 5 tumor xenograft samples. (<b>B</b>) Dietary GSPs reduce the levels of PI3K (p110, p85) and p-Akt in tumor xenograft tissues. Western blot analysis was performed to analyze the levels of these proteins. (<b>C</b>) Dietary GSPs decrease the levels of p-Akt in tumor samples. Immunohistochemical data show a reduction in the immunostaining intensity of p-Akt in tumor xenograft samples from GSPs-treated mice. Representative micrographs are shown from 4 tumor samples/group. Phospho-Akt-positive cells were counted in four to five different fields of a section, and data are summarized in terms of percent positive cells from 5 tumor xenograft samples (right panel). Statistical significance <i>vs</i>. tumor samples from mice receiving the control diet only, ^*<i>P</i><0.001.</p

GSPs induce apoptosis in human pancreatic cancer cells in a dose-dependent manner.

<p>(<b>A</b>) Miapaca-2 and PANC-1 cells were treated with different concentrations of GSPs (0, 20, 40 and 60 µg/mL) in complete medium for 48 h, then harvested for the analysis of apoptotic cells by FACS analysis using the Annexin V-Alexa Fluor488 Apoptosis Vybrant Assay Kit (Alexa488) following the manufacturer's protocol. The lower right (LR) quadrant of the FACS histograms indicates the percentage of early apoptotic cells (Alexa488-stained cells) and the upper right (UR) quadrant indicates the percentage of late apoptotic cells (Alexa488+ propidium iodide-stained cells). (<b>B</b>) Total percentages of apoptotic cells in each treatment group after 48 h are summarized with data presented as the mean ± SD of two experiments. Significant difference <i>vs.</i> control group (non-GSPs-treated), *<i>P</i><0.001; **<i>P</i><0.05. (<b>C</b>) Treatment of Miapaca-2 and PANC-1 cells with varying concentrations of GSPs for 48 h results in a dose-dependent reduction in the expression levels of the anti-apoptotic proteins (Bcl-2 and Bcl-xl) while increasing the expression of the pro-apoptotic protein Bax. GSPs also increase the activation level of caspase-3 in cells.</p

Effect of GSPs on melanoma cell migration.

<p>(<b>A</b>) Migration ability of human melanoma cells and comparison with normal human epidermal melanocytes (HEMa). Equal numbers of human melanoma cells (A375 and Hs294t) and HEMa were subjected to cell migration using standard Boyden chamber assay. Twenty four h later, migratory cells were detected on the membrane after staining with crystal violet. The migratory cells were counted and the results expressed as the mean number of migratory cells ± SD per microscopic field (n = 3). Significantly lower <i>versus</i> melanoma cells, ^*<i>P</i><0.001. (<b>B</b>) Treatment of human melanoma cancer cells (A375 and Hs294t) with GSPs for 24 h inhibit migration of cells in a concentration-dependent manner compared to non-GSPs-treated control cells. (<b>C</b>) The migratory cells were counted and the results expressed as the mean number of migratory cells ± SD/microscopic field. Significant inhibition <i>versus</i> non-GSPs-treated control, ^*<i>P</i><0.001.</p

Effect of GSPs and celecoxib on TPA-induced activation of ERK1/2 and melanoma cell migration.

<p>(<b>A</b>) Effect of TPA or its combination with GSPs on melanoma cell migration. Treatment of A375 cells with TPA, a stimulator of COX-2, significantly enhances cell migration (<b>^†</b><i>P</i><0.001) compared with non-TPA-treated control cells. (<b>B & C</b>) Treatment of A375 cells with TPA results in activation of ERK1/2. Treatment of cells with GSPs (40 µg/mL) or celecoxib (40 µM) inhibits TPA-induced activation of ERK1/2 protein, and simultaneously inhibits TPA-enhanced migration of melanoma cells. The data on cell migration capacity are summarized in Panel <b>C</b>. Significant inhibition versus TPA-treatment alone, ^*<i>P</i><0.001. (<b>D</b>) Treatment of melanoma cells with MEK inhibitor (UO126, 80 µM) resulted in inhibition of the activation of ERK1/2 as well as inhibition of cell migration compared to non-MEK inhibitor-treated control cells. The data are expressed as the mean number of migratory cells± SD/microscopic field, n = 3. Significant difference versus controls ^*<i>P</i><0.001.</p