Diosgenin, a Steroidal Saponin, Inhibits Migration and Invasion of Human Prostate Cancer PC-3 Cells by Reducing Matrix Metalloproteinases Expression

BACKGROUND: Diosgenin, a steroidal saponin obtained from fenugreek (Trigonella foenum graecum), was found to exert anti-carcinogenic properties, such as inhibiting proliferation and inducing apoptosis in a variety of tumor cells. However, the effect of diosgenin on cancer metastasis remains unclear. The aim of the study is to examine the effect of diosgenin on migration and invasion in human prostate cancer PC-3 cells. METHODS AND PRINCIPAL FINDINGS: Diosgenin inhibited proliferation of PC-3 cells in a dose-dependent manner. When treated with non-toxic doses of diosgenin, cell migration and invasion were markedly suppressed by in vitro wound healing assay and Boyden chamber invasion assay, respectively. Furthermore, diosgenin reduced the activities of matrix metalloproteinase-2 (MMP-2) and MMP-9 by gelatin zymography assay. The mRNA level of MMP-2, -9, -7 and extracellular inducer of matrix metalloproteinase (EMMPRIN) were also suppressed while tissue inhibitor of metalloproteinase-2 (TIMP-2) was increased by diosgenin. In addition, diosgenin abolished the expression of vascular endothelial growth factor (VEGF) in PC-3 cells and tube formation of endothelial cells. Our immunoblotting assays indicated that diosgenin potently suppressed the phosphorylation of phosphatidylinositide-3 kinase (PI3K), Akt, extracellular signal regulating kinase (ERK) and c-Jun N-terminal kinase (JNK). In addition, diosgenin significantly decreased the nuclear level of nuclear factor kappa B (NF-κB), suggesting that diosgenin inhibited NF-κB activity. CONCLUSION/SIGNIFICANCE: The results suggested that diosgenin inhibited migration and invasion of PC-3 cells by reducing MMPs expression. It also inhibited ERK, JNK and PI3K/Akt signaling pathways as well as NF-κB activity. These findings reveal new therapeutic potential for diosgenin in anti-metastatic therapy

Primer pairs used in Quantitative Real-Time PCR.

<p>Primer pairs used in Quantitative Real-Time PCR.</p

Effect of diosgenin on migration of PC-3 cells.

<p>Cell monolayers were scraped by a sterile micropipette tip and the cells were treated with various concentrations of diosgenin for 24 h. (A) Cells migrated to the wounded region were photographed (100× magnification). (B) The wound area of the cell cultures were quantified in four fields in each treatment, and data were calculated from three independent experiments. Data are presented as as mean ± S.D. of three independent experiments. <i>*p</i><0.05, <i>**p</i><0.01, compared with the untreated control.</p

Effects of diosgenin on NF-κB activation.

<p>PC-3 cells were treated with various doses of diosgenin for 24 h. (A) The cytosolic and nuclear extracts were prepared and analyzed for IκBα degradation and NF-κB p65 translocation. β-actin and C23 were used as a cytosolic and nuclear protein loading control, respectively. (B) Determined levels of IκBα and NF-κB were quantified by densitometric analysis. The densitometric results were expressed as mean ± S.D. of three independent experiments. <i>*p</i><0.05, <i>**p</i><0.01, <i>***p</i><0.001, compared with the untreated control.</p

8-Hydroxydaidzein Downregulates JAK/STAT, MMP, Oxidative Phosphorylation, and PI3K/AKT Pathways in K562 Cells

A metabolite isolated from fermented soybean, 8-hydroxydaidzein (8-OHD, 7,8,4&prime;-trihydroxyisoflavone, NSC-678112), is widely used in ethnopharmacological research due to its anti-proliferative and anti-inflammatory effects. We reported previously that 8-OHD provoked reactive oxygen species (ROS) overproduction, and induced autophagy, apoptosis, breakpoint cluster region-Abelson murine leukemia viral oncogene (BCR-ABL) degradation, and differentiation in K562 human chronic myeloid leukemia (CML) cells. However, how 8-OHD regulates metabolism, the extracellular matrix during invasion and metastasis, and survival signaling pathways in CML remains largely unexplored. High-throughput technologies have been widely used to discover the therapeutic targets and pathways of drugs. Bioinformatics analysis of 8-OHD-downregulated differentially expressed genes (DEGs) revealed that Janus kinase/signal transducer and activator of transcription (JAK/STAT), matrix metalloproteinases (MMPs), c-Myc, phosphoinositide 3-kinase (PI3K)/AKT, and oxidative phosphorylation (OXPHOS) metabolic pathways were significantly altered by 8-OHD treatment. Western blot analyses validated that 8-OHD significantly downregulated cytosolic JAK2 and the expression and phosphorylation of STAT3 dose- and time-dependently in K562 cells. Zymography and transwell assays also confirmed that K562-secreted MMP9 and invasion activities were dose-dependently inhibited by 8-OHD after 24 h of treatment. RT-qPCR analyses verified that 8-OHD repressed metastasis and OXPHOS-related genes. In combination with DisGeNET, it was found that 8-OHD&rsquo;s downregulation of PI3K/AKT is crucial for controlling CML development. A STRING protein&ndash;protein interaction analysis further revealed that AKT and MYC are hub proteins for cancer progression. Western blotting revealed that AKT phosphorylation and nuclear MYC expression were significantly inhibited by 8-OHD. Collectively, this systematic investigation revealed that 8-OHD exerts anti-CML effects by downregulating JAK/STAT, PI3K/AKT, MMP, and OXPHOS pathways, and MYC expression. These results could shed new light on the development of 8-OHD for CML therapy

Effect of diosgenin on viabilities of PC-3 cell.

<p>Cells were treated with various concentrations of diosgenin for 24 h and 48 h. Cell viability is presented as mean ± S.D. of four independent experiments. <i>***p</i><0.001 compared with the untreated control.</p

Effect of diosgenin on invasion of PC-3 cells.

<p>Cells were treated with various concentrations of diosgenin for 24 h and cell invasion assay was performed. (A) The invaded cells were photographed (200× magnification). (B) The invaded PC-3 cells were counted in five random fields in each treatment, and data were calculated from three independent experiments. Data are presented as mean ± S.D. of three independent experiments. <i>*p</i><0.05, <i>**p</i><0.01, compared with the untreated control.</p

Effect of diosgenin on PC-3 induced tube formation of endothelial cell and VEGF expression.

<p>(A) HUVECs were seeded onto Matrigel and incubated with conditioned medium from PC-3 cells treated with diosgenin for 24 h. After 6 h, the enclosed networks of complete tubes were photographed (100× magnification). (B) The tubular lengths of the cells were measured using Image J software. (C) The mRNA expression of VEGF was determined and presented as mean ± S.D. of three independent experiments. <i>*p</i><0.05, <i>**p</i><0.01, compared with the untreated control.</p

Effect of diosgenin on the activities and expressions of MMP-2/9/7, EMMPRIN and TIMP-1/2 in PC-3 cells.

<p><b>(</b>A) PC-3 cells were treated with various concentrations of diosgenin for 24 h and the activities of MMP-9 and MMP-2 were determined by gelatin zymography. The expressions of MMP-9 and MMP-2 mRNA (B) and protein (C) were analyzed by RT-PCR and Western blotting, respectively. β-actin was used as an internal control. (D) The levels of MMP-2, -9, -7, EMMPRIN and TIMP-1,-2 mRNA were expressed as mean ± S.D. of three independent experiments. <i>*p</i><0.05, <i>**p</i><0.01, compared with the untreated control.</p