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.     

Lysophosphatidic acid inhibits serum deprivation-induced autophagy in human prostate cancer PC-3 cells

Lysophosphatidic acid (LPA) is a platelet-enriched bioactive lysophospholipid. By binding to its cognitive G protein-coupled receptors, which are encoded by endothelial differentiation genes (edgs), LPA regulates various cellular activities including proliferation, survival, and migration. Currently, little is known about the influences of LPA on autophagy, a pivotal mechanism for cell survival during conditions of starvation. Herein we present data indicating that LPA attenuates starvation-induced autophagy, by monitoring the percentage of LC3-II, an autophagy indicator, in human prostate PC-3 cells. In addition, by using cells stably expressing EGFP-LC3, LPA is shown to inhibit the formation of autophagosomes in serum-starved conditions. Our results suggest that in these conditions, LPA inhibits autophagy, which might facilitate early cancer development.     

Transport of methotrexate into PC-3 human prostate cancer cells

Recombinant BmRad51 and BmDmc1, silkworm homologs of the Escherichia coli RecA proteins catalyzing the homologous DNA pairing, were purified from E. coli cells carrying expression vectors. These possessed different enzymatic properties in the joint molecule formation between single-stranded circular DNA and homologous linear double-stranded DNA. The requirement of single-stranded circular DNA for the efficient reaction was twofold higher in BmRad51 than in BmDmc1. Although able to mediate the joint molecule formation independently, a complex of the two enzymes formed prior to single-stranded DNA binding was found to have augmented efficiency of the pairing reaction.     

Crk II silencing down-regulates IGF-IR and inhibits migration and invasion of prostate cancer cells

Crk (C10 regulator of kinase) adaptor proteins are highly expressed in many types of human cancers and often contribute to aggressive cancer phenotypes. Crk II, a member of CRK family, has been reported to regulate cell migration and metastasis in breast cancer cells. However, its role in other cancer types has not been reported. In this study, we investigated the molecular function of Crk II in prostate cancer (PCa) cells (CWR-22rv1) in vitro and using a mouse tumor model. Results showed that Crk II knockdown by shRNA-mediated silencing (Crk II-shRNA) in the PCa cells significantly inhibited both cancer cell migration and invasion in cell culture study. Crk II-shRNA cancer cells also significantly decreased colony formation in vitro, but had no significant reduction of tumor volume after 4 weeks of cancer cell xenografting in vivo when compared to the scramble control. Interestingly, Crk II-shRNA cancer cells showed a greatly reduced level of insulin-like growth factor 1 receptor (IGF-1R) and decreased signaling of the IGF-1R/PI3K/Akt axis upon IGF-1 ligand stimulation. A close interaction between Crk II and IGF-1R was demonstrated upon co-immunoprecipitation of IGF-1R with Crk II protein. Further, treatment of cells with either proteosomal degradation or protein synthesis inhibitor showed higher proportion of ubiquitin-associated IGF-1R and faster degradation of IGF-1R in Crk II-shRNA cells in comparison with that in the control cancer cells. Taken together, these data suggest that Crk II plays an important role in the regulation of IGF-1R protein stability and affects downstream of IGF-1R signaling pathways. Therefore, targeting Crk-II can block IGF-1R growth signaling and suppress cancer cell invasion and progression.     

Extracellular matrix protein fibronectin induces matrix metalloproteinases in human prostate adenocarcinoma cells PC-3

Abstract

Studies on interaction of tumor cells with ECM components showed increased extracellular protease activity mediated by the family of matrix metalloproteinases (MMPs). Here we studied the effect of human prostate adenocarcinoma PC-3 cells–fibronectin (FN) interaction on MMPs and the underlying signaling pathways. Culturing of PC-3 cells on FN-coated surface upregulated MMP-9 and MMP-1. This response is abrogated by the blockade of α₅ integrin. siRNA and inhibitor studies indicate possible involvement of phosphatidyl-inositol-3-kinase (PI-3K), focal adhesion kinase (FAK) and nuclear factor-kappaB (NF-κB) in FN-induced upregulation of MMPs. FN treatment also enhanced phosphorylation of FAK, PI3K, protein kinase B (PKB or Akt), nuclear translocation of NF-κB, surface expression of CD-44, and cell migration. Our findings indicate that, binding of PC-3 cells to FN, possibly via α_5β1 integrin, induces signaling involving FAK, PI-3K, Akt, NF-κB followed by upregulation of MMP-9 and MMP-1. CD-44 may have role in modulating MMP-9 activity.     

Quercetin inhibits invasion, migration and signalling molecules involved in cell survival and proliferation of prostate cancer cell line (PC-3)

Urokinase-type plasminogen activator (uPA) is a serine protease that is involved in cancer progression, especially invasion and metastasis including prostate cancer. uPA activation is mediated by transactivation of uPAR and epidermal growth factor receptor (EGF-R) in prostate cancer progression. Prostate cancer (PC-3) cells have highly invasive capacity and they express uPA and uPAR gene. PC-3 cells are treated with quercetin, which inhibits invasion and migration of PC-3 cells. Quercetin downregulates uPA, uPAR and EGF, EGF-R mRNA expressions. Quercetin inhibits cell survival factor β-catenin, NF-κB and also proliferative signalling molecules such as p-EGF-R, N-Ras, Raf-1, c.Fos c.Jun and p-c.Jun protein expressions. But quercetin increased p38 mitogen-activated protein kinase protein expression. Our results suggest that quercetin inhibit migration and invasion of prostate cancer cells. It shows the value for treatment of invasive and metastasis type of prostate cancer.     

TGF-beta inhibits human cutaneous melanoma cell migration and invasion through regulation of the plasminogen activator system

Over the past decades, the incidence of cutaneous melanoma in developed countries has increased faster than any other cancer. Although most patients have localized disease at the time of diagnosis and are cured by surgical excision of the primary tumor, melanoma can be highly malignant and the survival dramatically decreases for advanced stage melanomas. It is thus necessary to understand the progression of this disease. Cell migration and invasion promote tumor metastasis, the major cause of melanoma cancer morbidity and death. In this study, we investigated the role of the TGFβ/Smad signaling pathway in melanoma tumor progression and found TGFβ to potently inhibit both cell migration and invasion in human melanoma cell lines, established from different patients. Furthermore, we elucidated the molecular mechanisms by which TGFβ exerts its effects and found the plasminogen activation system (PAS) to play a central role in the regulation of these effects. We found TGFβ to strongly up-regulate the Plasminogen Activator Inhibitor-1 (PAI-1) in melanoma cells, leading to reduced plasmin generation and activity and, in turn to inhibition of cell migration and invasion. Together, our results define TGFβ as a potent suppressor of tumor progression in cutaneous melanoma, inhibiting both cell migration and invasion.     

Unripe Rubus coreanus Miquel suppresses migration and invasion of human prostate cancer cells by reducing matrix metalloproteinase expression

Rubus coreanus Miquel (RCM) is used to promote prostate health and has been shown to have anti-oxidant and anti-carcinogenic activities. However, the effects and mechanisms of RCM on prostate cancer metastasis remain unclear. PC-3 and DU 145 cells were treated with ethanol or water extract of unripe or ripe RCM and examined for cell invasion, migration, and matrix metalloproteinases (MMPs) activity and expression. Phosphoinositide 3-kinase (PI3K) and Akt activities were examined. Unripe RCM extracts exerted significant inhibitory effects on cell migration, invasion, and MMPs activities. A significant reduction in MMPs activities by unripe RCM ethanol extract treatment (UE) was associated with reduction of MMPs expression and induction of tissue inhibitors of metalloproteinases (TIMPs) expression. Furthermore, PI3K/Akt activity was diminished by UE treatment. In this study, we demonstrated that UE decreased metastatic potential of prostate cancer cells by reducing MMPs expression through the suppression of PI3K/Akt phosphorylation, thereby decreasing MMP activity and enhancing TIMPs expression.     

Delphinidin inhibits BDNF-induced migration and invasion in SKOV3 ovarian cancer cells

Brain-derived neurotrophic factor (BDNF), the TrkB ligand, is associated with aggressive malignant behavior, including migration and invasion, in tumor cells and a poor prognosis in patients with various types of cancer. Delphinidin is a diphenylpropane-based polyphenolic ring structure-harboring compound, which exhibits a wide range of pharmacological activities, anti-tumor, anti-oxidant, anti-inflammatory, anti-angiogenic and anti-mutagenic activity. However, the possible role of delphinidin in the cancer migration and invasion is unclear. We investigated the suppressive effect of delphinidin on the cancer migration and invasion. Thus, we found that BDNF enhanced cancer migration and invasion in SKOV3 ovarian cancer cell. To exam the inhibitory role of delphinidin in SKOV3 ovarian cancer migration and invasion, we investigated the use of delphinidin as inhibitors of BDNF-induced motility and invasiveness in SKOV3 ovarian cancer cells in vitro. Here, we found that delphinidin prominently inhibited the BDNF-induced increase in cell migration and invasion of SKOV3 ovarian cancer cells. Furthermore, delphinidin remarkably inhibited BDNF-stimulated expression of MMP-2 and MMP-9. Also, delphinidin antagonized the phosphorylation of Akt and nuclear translocation of NF-κB permitted by the BDNF in SKOV3 ovarian cancer cells. Taken together, our findings provide new evidence that delphinidin suppressed the BDNF-induced ovarian cancer migration and invasion through decreasing of Akt activation.     

Genistein inhibits the contact-stimulated migration of prostate cancer cells

The results of several epidemiological studies have suggested that a soybean-based diet is associated with a lower risk of prostate cancer. We investigated the effect of the soy isoflavone genistein on the proliferation and contact-stimulated migration of rat prostatic carcinoma MAT-LyLu and AT-2 cell lines. Genistein almost completely inhibited the growth of both MAT-LyLu and AT-2 cells in the concentration range from 25 to 100 μM, but the addition of 1 μM genistein to the medium significantly stimulated the proliferation of both cell lines. Additionally, at concentrations above 25 μM, genistein showed a potent cytotoxic effect. However, the central finding of this study is that at physiologically relevant concentrations (1 μM and 10 μM), genistein inhibits the motility of prostate cancer cells stimulated by homo-and heterotypic contacts. These results show that at physiological concentrations, genistein exerts an inhibitory effect on the migration of prostate cancer cells and suggest that it may be one of the factors responsible for the anti-metastatic activity of plant isoflavonoids     

Simvastatin inhibits the proliferation of human prostate cancer PC-3 cells via down-regulation of the insulin-like growth factor 1 receptor

Recently, statins have been being studied for their proapoptic and antimetastatic effects. However, the exact mechanisms of their anticancer action are still unclear. Dolichyl phosphate is a nonsterol isoprenoid derivative in the mevalonate pathway that affects the expression of the Insulin-like growth factor 1 receptor (IGF-1R). IGF-1R activation is required for prostate cell proliferation; therefore, IGF-1R inhibitory agents may be of preventive and/or therapeutic value. In this study, the effects of simvastatin on IGF-1R signaling in prostate cancer PC-3 cells were examined. Simvastatin suppressed proliferation and induced apoptosis of PC-3, and the expression of IGF-1R was suppressed by simvastatin. Knockdown of IGF-1R by siRNA led to inhibition of proliferation of PC-3. Simvastatin also inhibited IGF-1-induced activation of both ERK and Akt signaling and IGF-1-induced PC-3 cell proliferation. Our results suggest statins are potent inhibitors of the IGF-1/IGF-1R system in prostate cancer cells and may be beneficial in prostate cancer treatment.     

Invadopodia and matrix degradation, a new property of prostate cancer cells during migration and invasion

The present study demonstrated that invadopodia are associated with invasion by degradation of matrix in prostate cancer cells PC3. To find out the presence of invadopodia in PC3 cells, we performed a few comparative analyses with osteoclasts, which utilize podosomes for migration. Our investigations indeed demonstrated that invadopodia are comparable to podosomes in the localization of Wiskott-Aldrich syndrome protein (WASP)/matrix metalloproteinase-9 and the degradation of matrix. Invadopodia are different from podosomes in the localization of actin/vinculin, distribution during migration, and the mode of degradation of extracellular matrix. Invadopodia enable polarized invasion of PC3 cells into the gelatin matrix in a time-dependent manner. Gelatin degradation was confined within the periphery of the cell. Osteoclasts demonstrated directional migration with extensive degradation of matrix underneath and around the osteoclasts. A pathway of degradation of matrix representing a migratory track was observed due to the rearrangement of podosomes as rosettes or clusters at the leading edge. Reducing the matrix metalloproteinase-9 levels by RNA interference inhibited the degradation of matrix but not the formation of podosomes or invadopodia. Competition experiments with TAT-fused WASP peptides suggest that actin polymerization and formation of invadopodia involve the WASP-Arp2/3 complex pathway. Moreover, PC3 cells overexpressing osteopontin (OPN) displayed an increase in the number of invadopodia and gelatinolytic activity as compared with PC3 cells and PC3 cells expressing mutant OPN in integrin-binding domain and null for OPN. Thus, we conclude that OPN/integrin alphavbeta3 signaling participates in the process of migration and invasion of PC3 cells through regulating processes essential for the formation and function of invadopodia.     

Galectin-1-matured human monocyte-derived dendritic cells have enhanced migration through extracellular matrix

Dendritic cells (DCs) are potent mediators of the immune response, and can be activated by exogenous pathogen components. Galectin-1 is a member of the conserved beta-galactoside-binding lectin family that binds galactoside residues on cell surface glycoconjugates. Galectin-1 is known to play a role in immune regulation via action on multiple immune cells. However, its effects on human DCs are unknown. In this study, we show that galectin-1 induces a phenotypic and functional maturation in human monocyte-derived DCs (MDDCs) similar to but distinct from the activity of the exogenous pathogen stimuli, LPS. Immature human MDDCs exposed to galectin-1 up-regulated cell surface markers characteristic of DC maturation (CD40, CD83, CD86, and HLA-DR), secreted high levels of IL-6 and TNF-alpha, stimulated T cell proliferation, and showed reduced endocytic capacity, similar to LPS-matured MDDCs. However, unlike LPS-matured DCs, galectin-1-treated MDDCs did not produce the Th1-polarizing cytokine IL-12. Microarray analysis revealed that in addition to modulating many of the same DC maturation genes as LPS, galectin-1 also uniquely up-regulated a significant subset of genes related to cell migration through the extracellular matrix (ECM). Indeed, compared with LPS, galectin-1-treated human MDDCs exhibited significantly better chemotactic migration through Matrigel, an in vitro ECM model. Our findings show that galectin-1 is a novel endogenous activator of human MDDCs that up-regulates a significant subset of genes distinct from those regulated by a model exogenous stimulus (LPS). One unique effect of galectin-1 is to increase DC migration through the ECM, suggesting that galectin-1 may be an important component in initiating an immune response.     

Depletion of the histone chaperone tNASP inhibits proliferation and induces apoptosis in prostate cancer PC-3 cells

Background  

NASP (Nuclear Autoantigenic Sperm Protein) is a histone chaperone that is present in all dividing cells. NASP has two splice variants: tNASP and sNASP. Only cancer, germ, transformed, and embryonic cells have a high level of expression of the tNASP splice variant. We examined the consequences of tNASP depletion for prostate cancer PC-3 cells.     

Laminar shear stress inhibits high glucose-induced migration and invasion in human bladder cancer cells

High glucose has been known to play a pathogenic role in the development and progression of bladder cancer in diabetics, whereas the leading cause of death in such patients is mainly attributed to hyperglycemia-enhanced metastasis. In addition to the impact of glucose, cancer cells may be affected by laminar shear stress (LSS) generated from interstitial, blood, and/or lymphatic fluid flows during metastasis. Although the effect of flow-induced mechanical force on cancer pathophysiology has been extensively investigated, very little is understood regarding the cells that are simultaneously stimulated by LSS and hyperglycemia. To address this issue, the influence of LSS on bladder cancer cell motility in a hyperglycemic environment was examined. Based on the results of cell movement and protein expression analyses, we found that both cell migration and invasion were up- and downregulated by 25 mM glucose and 12 dynes/cm² LSS, respectively. Furthermore, the motility of the cells with simultaneous hyperglycemic and LSS stimulations was significantly reduced compared with that of the cells stimulated by high glucose alone (P?<?0.05), demonstrating that the LSS rather than hyperglycemia played the dominant role in regulation of cell motility. These results implied that LSS with an intensity ≥?12 dynes/cm² may serve as a feasible tool to reduce bladder cancer motility in diabetics.     

Uptake of 5-methyltetrahydrofolate into PC-3 human prostate cancer cells is carrier-mediated

Uptake of 5-methyltetrahydrofolate into the PC-3 human prostate cancer cells was linear for the first 60 min. There was no difference in the initial rate of uptake in cells incubated in folate-free medium for 24 or 48 hr compared to control cells grown in folate-containing medium. The initial rate of 5-methyltetrahydrofolate uptake showed little dependence on extracellular pH and it was independent of extracellular sodium ions. Transport of 5-methyltetrahydrofolate into PC-3 cells was saturable - K(m) = 0.74 micro M and V(max) = 7.78 nmol/10(9)cells/min and these kinetic constants were not different in cells incubated for 24 hr in folate-free medium (K(m) = 0.80 +/- 0.22, V(max) = 8.52 +/- 0.50; P = 0.09, N = 3). Uptake of 5-methyltetrahydrofolate was inhibited by structural analogs with the K(i) values being 0.50, 1.79, and 31.8 micro M for 5-formyltetrahydrofolate, methotrexate, and folic acid, respectively. Uptake of 5-methyltetrahydrofolate was inhibited by the energy poisons, sodium cyanide, sodium arsenate, p-chloromercuriphenylsulfonate, and sodium azide. Uptake was inhibited by increasing concentrations of sulfate and phosphate ions, suggesting that 5-methyltetrahydrofolate may be transported by an anion-exchange mechanism. These results show that 5-methyltetrahydrofolate is transported into PC-3 prostate cancer cells by a carrier-mediated process.     

Knockdown of SRPX2 inhibits the proliferation,migration, and invasion of prostate cancer cells through the PI3K/Akt/mTOR signaling pathway

Sushi repeat‐containing protein X‐linked 2 (SRPX2), a novel chondroitin sulfate proteoglycan, is reported to play a critical role in tumorigenesis. However, the expression and functional role of SRPX2 in prostate cancer have not been defined. Thus, the aim of this study was to investigate the expression and functional role of SRPX2 in human prostate cancer. Our results showed that the expression of SRPX2 was obviously increased in human prostate cancer tissues and cell lines. In addition, knockdown of SRPX2 inhibited the proliferation, migration, and invasion of prostate cancer cells, as well as prevented the epithelial‐mesenchymal transition process in prostate cancer cells. Mechanically, knockdown of SRPX2 efficiently inhibited the activation of PI3K/Akt/mTOR pathway in prostate cancer cells. Taken together, these data demonstrated that knockdown of SRPX2 inhibits the proliferation and metastasis in human prostate cancer cells, partly through the PI3K/Akt/mTOR signaling pathway. Thus, SRPX2 may be a novel therapeutic target for the treatment of prostate cancer.