Isoliquiritigenin inhibits migration and invasion of prostate cancer cells: possible mediation by decreased JNK/AP-1 signaling

Isoliquiritigenin (ISL, 4,2′,4′-trihydroxychalcone), which is found in licorice, shallot and bean sprouts, is a potent antioxidant with anti-inflammatory and anti-carcinogenic effects. The purpose of this study was to investigate the effects of ISL treatment on the migration, invasion and adhesion characteristics of DU145 human prostate cancer cells. DU145 cells were cultured in the presence of 0–20 μmol/L ISL with or without 10 μg/L epidermal growth factor (EGF). ISL inhibited basal and EGF-induced cell migration, invasion and adhesion dose dependently. ISL decreased EGF-induced secretion of urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP)-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and vascular endothelial growth factor (VEGF), but increased TIMP-2 secretion in a concentration-dependent manner. In addition, ISL decreased the protein levels of integrin-α2, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), and mRNA levels of uPA, MMP-9, VEGF, ICAM and integrin-α2. Furthermore, basal and EGF-induced activator protein (AP)-1 binding activity and phosphorylation of Jun N-terminal kinase (JNK), c-Jun and Akt were decreased after ISL treatment. However, phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase was not altered. The JNK inhibitor SP600125 inhibited basal and EGF-induced secretion of uPA, VEGF, MMP-9 and TIMP-1, as well as AP-1 DNA binding activity and cell migration. These results provide evidence for the role of ISL as a potent antimetastatic agent, which can markedly inhibit the metastatic and invasive capacity of prostate cancer cells. The inhibition of JNK/AP-1 signaling may be one of the mechanisms by which ISL inhibits cancer cell invasion and migration.     

Isoliquiritigenin (ISL) inhibits ErbB3 signaling in prostate cancer cells

Isoliquiritigenin (ISL), a flavonoid found in licorice, shallot, and bean sprouts, has been identified as a potent anti-tumor promoting agent. We previously demonstrated that ISL reduces cell proliferation and induces apoptosis in DU145 human prostate cancer cells and MAT-LyLu (MLL) rat prostate cancer cells. Overexpression of members of the ErbB receptor family is a frequently observed event in several human cancers, and ErbB receptors currently constitute the primary targets of anticancer strategies. In order to elucidate the mechanisms underlying the ISL regulation of prostate cancer cell proliferation, the present study attempted to determine whether ISL inhibits heregulin (HRG)-beta-induced ErbB3 signaling. DU145 and MLL cells were cultured in serum-free medium with ISL and/or HRG-beta. Exogenous HRG-beta alone was shown to effect an increase in the numbers of viable cells, whereas HRG-beta did not counteract the ISL-induced growth inhibition. ISL reduced the protein and mRNA levels of ErbB3 in a dose-dependent manner, but exerted no effect on HRG protein levels. Immunoprecipitation/Western blot studies indicated that ISL inhibited the HRG-beta-induced tyrosine phosphorylation of ErbB3, the recruitment of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) to ErbB3, and Akt phosphorylation in DU145 cells. These results indicate that ISL inhibits the proliferation of prostate cancer cells, at least in part, via the inhibition of ErbB3 signaling and the PI3K/Akt pathway.     

Piceatannol prevents lipopolysaccharide (LPS)-induced nitric oxide (NO) production and nuclear factor (NF)-kappaB activation by inhibiting IkappaB kinase (IKK)

The effect of piceatannol on lipopolysaccharide (LPS)-induced nitric oxide (NO) production was examined. Piceatannol significantly inhibited NO production in LPS-stimulated RAW 264.7 cells. The inhibition was due to the reduced expression of an inducible isoform of NO synthase (iNOS). The inhibitory effect of piceatannol was mediated by down-regulation of LPS-induced nuclear factor (NF)-kappaB activation, but not by its cytotoxic action. Piceatannol inhibited IkappaB kinase (IKK)-alpha and beta phosphorylation, and subsequently IkappaB-alpha phosphorylation in LPS-stimulated RAW 264.7 cells. On the other hand, piceatannol did not affect activation of mitogen-activated protein (MAP) kinases including extracellular signal regulated kinase 1/2 (Erk1/2), p38 and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Piceatannol inhibited the phosphorylation of Akt and Raf-1 molecules, which regulated the activation of IKK-alpha and beta phosphorylation. The detailed mechanism of the inhibition of LPS-induced NO production by piceatannol is discussed.     

Transforming growth factor beta1 stimulates urokinase plasminogen activator system on prostate cancer cells

The effect of TGFbeta1 on the proliferation and plasminogen activator system (PA) of two prostate carcinoma cell lines, PC3 and DU145, was investigated. PA, particularly urokinase plasminogen activator (uPA), has been implicated in extracellular proteolysis, local invasiveness, metastatic spread and angiogenesis. High levels of uPA and plasminogen activator inhibitor-1 (PAI-1) correlate with poor prognosis in several cancers. TGFbeta1 had no significant effect on the proliferation of either cell line. TGFbeta1 increased the production of uPA in PC3 and DU145 cells. Despite the very low PAI-1 protein levels in both cell lines, TGFbeta1 treatment resulted in a remarkable increase in PAI-1 secretion. PAI-2 protein was also increased by 59% in the PC3 cells. A divergent effect of TGFbeta1 on the uPA enzyme activity was observed (28% decrease in PC3 and 131% increase in DU145 cells). Overall, TGFbeta1 treatment did not affect the invasion of reconstituted basement membrane of PC3 cells. In addition to the uPA:PAI-1 ratio, the presence of PAI-2 may be an important factor in the determination of metastatic sites for prostate cancer cells. In conclusion, the potential contribution of TGFbeta1 to tumor invasion may be considered as positive, based on both loss of growth inhibition and stimulation of components of the invasive system of prostate carcinoma.     

Growth factor/growth factor receptor loops in autocrine growth regulation of human prostate cancer DU145 cells

Autocrine growth factors produced by epithelial cells mediate the development and proliferation of neoplastic human prostate tissue. Various approaches have been used to down-regulate neoplastic growth of prostate cancer using natural flavonoids, soluble receptors, pseudo-ligands, monoclonal antibodies and tyrosine kinase inhibitors (tyrphostins). Selected growth factor/growth factor receptor loops (mainly TGFα/EGFR and IGFs/IGFIR) have been proposed as regulators of prostate cancer cell growth. We have previously determined that blockade of IGFIR or VEGF2R signaling pathways by tyrphostin AG1024 and SU1498 inhibits autocrine growth and viability of DU145 cells in vitro. Recently, we compared the activity of AG1024 and SU1498 with the inhibiting effect of tyrphostin A23 (a selective inhibitor of EGFR). The results described in this paper confirm that DU145 cells do not produce IGFI or EGF. In contrast, DU145 cells produce a great amount of VEGF, much more than TGFα (about 60-fold), and VEGF may be the real autocrine growth factor of the investigated cells. The results indicate that the growth of DU145 may be regulated by at least three autocrine loops: TGFα/EGFR, IGFII/IGFIR and VEGF/VEGFR2. Neither AG1024 nor SU1498 affected the production of TGFα substantially, which excludes the possibility that IGFRs or VEGFR2 inhibitors arrest the growth of these cells by inhibition of synthesis and/or secretion of TGFα. The obtained data indicate that all tree investigated tyrphostins (AG1024, SU1498 and A23) inhibit signal transmission by Akt (PKB), ERK(1/2), Src and STAT in a similar manner. A comparison of the effects of the investigated tyrphostins indicates that TGFα, IGFII and VEGF stimulate cell growth by affecting the same signaling pathway. The hypothesis was confirmed by the effect of the investigated tyrphostins on activation of EGFR. All these inhibitors decreased phosphorylation of EGFR to the same extent, and after the same time of incubation with cell culture. These results strongly suggest that stimulation of EGFR kinase is the main step in the initiation of mitogen signaling in DU145 cells, regardless of the type of ligand (TGFα, IGFs or VEGF) and their specific receptors.     

The inhibitory effects of capillarisin on cell proliferation and invasion of prostate carcinoma cells

Objectives

Capillarisin (Cap), an active component of Artemisia capillaris root extracts, is characterized by its anti‐inflammatory, anti‐oxidant and anti‐cancer properties. Nevertheless, the functions of Cap in prostate cancer have not been fully explored. We evaluated the potential actions of Cap on the cell proliferation, migration and invasion of prostate carcinoma cells.

Materials and methods

Cell proliferation and cell cycle distribution were measured by water‐soluble tetrazolium‐1 and flow cytometry assays. The expression of cyclins, p21, p27, survivin, matrix metallopeptidase (MMP2 and MMP9) were assessed by immunoblotting assays. Effects of Cap on invasion and migration were determined by wound closure and matrigel transmigration assays. The constitutive and interlukin‐6 (IL‐6)‐inducible STAT3 activation of prostate carcinoma cells were determined by immunoblotting and reporter assays.

Results

Capillarisin inhibited androgen‐independent DU145 and androgen‐dependent LNCaP cell growth through the induction of cell cycle arrest at the G0/G1 phase by upregulating p21 and p27 while downregulating expression of cyclin D1, cyclin A and cyclin B. Cap decreased protein expression of survivin, MMP‐2, and MMP‐9 and therefore blocked the migration and invasion of DU145 cells. Cap suppressed constitutive and IL‐6‐inducible STAT3 activation in DU145 and LNCaP cells.

Conclusions

Our data indicate that Cap blocked cell growth by modulation of p21, p27 and cyclins. The inhibitory effects of Cap on survivin, MMP‐2, MMP‐9 and STAT3 activation may account for the suppression of invasion in prostate carcinoma cells. Our data suggest that Cap might be a therapeutic agent in treating advanced prostate cancer with constitutive STAT3 or IL‐6‐inducible STAT3 activation.

     

Differential expression and effects of gp130 cytokines and receptors in prostate cancer cells

High levels of circulating interleukin-6 (IL6), and possibly neuroendocrine (NE) differentiation, correlate with advanced prostate cancer (PCa). IL6 has many overlapping biological effects with the related gp130 cytokines LIF and OSM that can be explained by the shared usage of the signalling receptor, gp130. We set out to determine whether LIF and OSM can substitute for IL6 in PCa, particularly in relation to neuroendocrine differentiation. Expression analysis of the gp130 cytokines and receptors by RT-PCR, Southern blotting and immunohistochemistry showed that they are widely expressed in LNCaP, DU145 and PC3 cells, but not in normal prostate epithelial PZ-HPV-7 cells. IL6, but not LIF or OSM inhibited proliferation, induced NE differentiation and tyrosine phosphorylation of STAT3 in LNCaP cells. The data suggests that IL6 has a unique role in the progression of PCa.     

Complement C1q Activates Tumor Suppressor WWOX to Induce Apoptosis in Prostate Cancer Cells

Background

Tissue exudates contain low levels of serum complement proteins, and their regulatory effects on prostate cancer progression are largely unknown. We examined specific serum complement components in coordinating the activation of tumor suppressors p53 and WWOX (also named FOR or WOX1) and kinases ERK, JNK1 and STAT3 in human prostate DU145 cells.

Methodology/Principal Findings

DU145 cells were cultured overnight in 1% normal human serum, or in human serum depleted of an indicated complement protein. Under complement C1q- or C6-free conditions, WOX1 and ERK were mainly present in the cytoplasm without phosphorylation, whereas phosphorylated JNK1 was greatly accumulated in the nuclei. Exogenous C1q rapidly restored the WOX1 activation (with Tyr33 phosphorylation) in less than 2 hr. Without serum complement C9, p53 became activated, and hyaluronan (HA) reversed the effect. Under C6-free conditions, HA induced activation of STAT3, an enhancer of metastasis. Notably, exogenous C1q significantly induced apoptosis of WOX1-overexpressing DU145 cells, but not vehicle-expressing cells. A dominant negative and Y33R mutant of WOX1 blocked the apoptotic effect. C1q did not enhance p53-mediated apoptosis. By total internal reflection fluorescence (TIRF) microscopy, it was determined that C1q destabilized adherence of WOX1-expressing DU145 cells by partial detaching and inducing formation of clustered microvilli for focal adhesion particularly in between cells. These cells then underwent shrinkage, membrane blebbing and death. Remarkably, as determined by immunostaining, benign prostatic hyperplasia and prostate cancer were shown to have a significantly reduced expression of tissue C1q, compared to age-matched normal prostate tissues.

Conclusions/Significance

We conclude that complement C1q may induce apoptosis of prostate cancer cells by activating WOX1 and destabilizing cell adhesion. Downregulation of C1q enhances prostate hyperplasia and cancerous formation due to failure of WOX1 activation.     

Acacetin, a flavonoid, inhibits the invasion and migration of human prostate cancer DU145 cells via inactivation of the p38 MAPK signaling pathway

Acacetin (5,7-dihydroxy-4′-methoxyflavone), a flavonoid compound, has anti-peroxidative and anti-inflammatory effects. The effect of acacetin on antimetastasis in human prostate cancer DU-145 cells was investigated. First, the result demonstrated acacetin could exhibit an inhibitory effect on the abilities of the adhesion, invasion, and migration by cell–matrix adhesion assay, wound-healing assay, and Boyden chamber assay. Data also showed acacetin could inhibit the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) involved in the downregulation of the expressions of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and urokinase-type plasminogen activator (u-PA) at both the protein and mRNA levels. Next, acacetin significantly decreased the nuclear levels of nuclear factor kappa B (NF-κB), c-Fos, and c-Jun. Also, the treatment with acacetin to DU145 cells also leads to a dose-dependent inhibition on the binding ability of NF-κB and activator protein-1 (AP-1). Furthermore, the treatment of inhibitors specific for p38 MAPK (SB203580) to DU145 cells could cause reduced expressions of MMP-2, MMP-9, and u-PA. These results showed acacetin could inhibit the invasion and migration abilities of DU145 cells by reducing MMP-2, MMP-9, and u-PA expressions through suppressing p38 MAPK signaling pathway and inhibiting NF-κB- or AP-1-binding activity. These findings proved acacetin might be offered further application as an antimetastatic agent.