Recombinant disintegrin domain of human ADAM9 inhibits migration and invasion of DU145 prostate tumor cells

One of the most important features of malignant cells is their capacity to invade adjacent tissues and metastasize to distant organs. This process involves the creation, by tumor and stroma cells, of a specific microenvironment, suitable for proliferation, migration and invasion of tumor cells. The ADAM family of proteins has been involved in these processes. This work aimed to investigate the role of the recombinant disintegrin domain of the human ADAM9 (rADAM9D) on the adhesive and mobility properties of DU145 prostate tumor cells. rADAM9D was able to support DU145 cell adhesion, inhibit the migration of DU145 cells, as well as the invasion of this cell line through matrigel in vitro. Overall this work demonstrates that rADAM9D induces specific cellular migratory properties when compared with different constructs having additional domains, specially those of metalloproteinase and cysteine-rich domains. Furthermore, we showed that rADAM9D was able to inhibit cell adhesion, migration and invasion mainly through interacting with α6β1 in DU145 tumor cell line. These results may contribute to the development of new therapeutic strategies for prostate cancer.     

Human Chorionic Gonadotropin β Induces Migration and Invasion via Activating ERK1/2 and MMP-2 in Human Prostate Cancer DU145 Cells

We previously demonstrated that human chorionic gonadotropin β (hCGβ) induced migration and invasion in human prostate cancer cells. However, the involved molecular mechanisms are unclear. Here, we established a stable prostate cancer cell line overexpressing hCGβ and tested hCGβ-triggered signaling pathways causing cell migration and invasion. ELISA showed that the hCGβ amount secreted into medium increased with culture time after the hCGβ-transfected cells were incubated for 3, 6, 9, 12 and 24 h. More, hCGβ standards promoted MAPK (ERK1/2) phosphorylation and increased MMP-2 expression and activity in both dose- and time-dependent manners in hCGβ non-transfected cells. In addition, hCGβ promoted ERK1/2 phosphorylation and increased MMP-2 expression and activity significantly in hCGβ transfected DU145 cells. Whereas ERK1/2 blocker PD98059 (25 µM) significantly downregulated phosphorylated ERK1/2 and MMP-2. Particularly, hCGβ promoted cell migration and invasion, yet the PD98059 diminished the hCGβ-induced cell motility under those conditions. These results indicated that hCGβ induced cell motility via promoting ERK1/2 phosphorylation and MMP-2 upregulation in human prostate cancer DU145 cells.     

Inhibition of platelets and tumor cell adhesion by the disintegrin domain of human ADAM9 to collagen I under dynamic flow conditions

This work aimed to investigate the role of the disintegrin domain of the human ADAM9 (ADAM9D) on the adhesion of breast tumor cells and platelets to collagen I, in a dynamic flow assay to simulate in vivo shear conditions. Recombinant ADAM9D was able to support tumor cell adhesion through binding to the β1 integrin subunit and also to inhibit the invasion through matrigel in vitro. In a dynamic flow assay ADAM9D inhibited about 75% and 65% of MDA-MB-231 tumor cells and platelet adhesion to collagen I, respectively. In addition, it was demonstrated that αVβ3 integrin is new interacting partner for ADAM9D. In conclusion, these results suggest a role for the disintegrin domain of ADAM9 in the metastatic process. Also, ADAM9D may be a tool for investigating the role of ADAMs in metastasis and cancer progression and for the design of selective inhibitors against the adhesion and extravasation of cancer cells.     

Dual roles of E-cadherin in prostate cancer invasion

The role(s) of E-cadherin in tumor progression, invasion, and metastasis remains somewhat enigmatic. In order to investigate various aspects of E-cadherin biological activity, particularly in prostate cancer progression, our laboratory cloned unique subpopulations of the heterogeneous DU145 human prostatic carcinoma cell line and characterized their distinct biological functions. The data revealed that the highly invasive, fibroblastic-like subpopulation of DU145 cells (designated DU145-F) expressed less than 0.1-fold of E-cadherin protein when compared to the parental DU145 or the poorly invasive DU145 cells (designated DU145-E). Experimental disruption of E-cadherin function stimulated migration and invasion of DU145-E and other E-cadherin-positive prostate cancer cell lines, but did not affect the fibroblastic-like DU145-F subpopulation. Within the medium of parental DU145 cells, the presence of an 80 kDa E-cadherin fragment was detected. Subsequent functional analyses revealed the stimulatory effect of this fragment on the migratory and invasive capability of E-cadherin-positive cells. These results suggest that E-cadherin plays an important role in regulating the invasive potential of prostate cancer cells through an unique paracrine mechanism.     

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.     

ADAM9 silencing inhibits breast tumor cell invasion in vitro

ADAM9 (A Disintegrin And Metalloproteinase 9) is a member of the ADAM protein family which contains a disintegrin domain. This protein family plays key roles in many physiological processes, including fertilization, migration, and cell survival. The ADAM proteins have also been implicated in various diseases, including cancer. Specifically, ADAM9 has been suggested to be involved in metastasis. To address this question, we generated ADAM9 knockdown clones of MDA-MB-231 breast tumor cells using silencing RNAs that were tested for cell adhesion, proliferation, migration and invasion assays. In RNAi-mediated ADAM9 silenced MDA-MB-231 cells, the expression of ADAM9 was lower from the third to the sixth day after silencing and inhibited tumor cell invasion in matrigel by approximately 72% when compared to control cells, without affecting cell adhesion, proliferation or migration. In conclusion, the generation of MDA-MB-231 knockdown clones lacking ADAM9 expression inhibited tumor cell invasion in vitro, suggesting that ADAM9 is an important molecule in the processes of invasion and metastasis.     

Protein kinase Cdelta signaling downstream of the EGF receptor mediates migration and invasiveness of prostate cancer cells

Tumor progression to the invasive phenotype occurs secondary to upregulated signaling from growth factor receptors that drive key cellular responses like proliferation, migration, and invasion. We hypothesized that Protein kinase Cdelta (PKCdelta)-mediated transcellular contractility is required for migration and invasion of prostate tumor cells. Two invasive human prostate cancer cell lines, DU145 cells overexpressing wildtype human EGFR (DU145WT) and PC3 cells, were studied. PKCdelta is overexpressed in these cells relative to normal prostate epithelial cells, and is activated downstream of EGFR leading to cell motility via modulation of myosin light chain activity. Abrogation of PKCdelta using Rottlerin and specific siRNA significantly decreased migration and invasion of both cell lines in vitro. Both PKCdelta and phosphorylated PKCdelta protein levels were higher in human prostate cancer tissue relative to normal donor prostate as assessed by Western blotting and immunohistochemistry. Thus, we conclude that PKCdelta inhibition can limit migration and invasion of prostate cancer cells.     

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.     

p120ctn and P-Cadherin but Not E-Cadherin Regulate Cell Motility and Invasion of DU145 Prostate Cancer Cells

Background

Adherens junctions consist of transmembrane cadherins, which interact intracellularly with p120ctn, ß-catenin and α-catenin. p120ctn is known to regulate cell-cell adhesion by increasing cadherin stability, but the effects of other adherens junction components on cell-cell adhesion have not been compared with that of p120ctn.

Methodology/Principal Findings

We show that depletion of p120ctn by small interfering RNA (siRNA) in DU145 prostate cancer and MCF10A breast epithelial cells reduces the expression levels of the adherens junction proteins, E-cadherin, P-cadherin, ß-catenin and α-catenin, and induces loss of cell-cell adhesion. p120ctn-depleted cells also have increased migration speed and invasion, which correlates with increased Rap1 but not Rac1 or RhoA activity. Downregulation of P-cadherin, β-catenin and α-catenin but not E-cadherin induces a loss of cell-cell adhesion, increased migration and enhanced invasion similar to p120ctn depletion. However, only p120ctn depletion leads to a decrease in the levels of other adherens junction proteins.

Conclusions/Significance

Our data indicate that P-cadherin but not E-cadherin is important for maintaining adherens junctions in DU145 and MCF10A cells, and that depletion of any of the cadherin-associated proteins, p120ctn, ß-catenin or α-catenin, is sufficient to disrupt adherens junctions in DU145 cells and increase migration and cancer cell invasion.     

AaHIV a sodium channel scorpion toxin inhibits the proliferation of DU145 prostate cancer cells

Prostate cancer is the most highly diagnosed cancer in men worldwide. It is characterized by high proliferation, great invasion and metastatic potential. Sodium channel subtypes have been identified as highly expressed in different prostate cancer cell lines. In this study, we have screened the negatively charged fractions of Androctonus australis (Aa) scorpion venom to identify active peptides on DU145 prostate cancer cells proliferation. The most active compound was identified to be the sodium channel peptide AaHIV with an IC₅₀ value of 15 μM. At this concentration, AaHIV had low effect on the adhesion of DU145 cells to fibronectin. When compared to other Na⁺ channel Aa toxins, AaHIV was found to be 2 times more active than AaHI and AaHII on DU145 cells proliferation and slightly less active than AaHII on their adhesion. The three peptides are inactive on DU145 cells migration. AaHIV was found to be 16 times more active than veratridine, asteroidal alkaloid from plants of the lily family widely used as a sodium channel activator. Electrophysiological experiments showed that the AaHIV toxin activates Nav1.6 channel, suggesting that this sodium channel subtype is implicated in the proliferation of DU145 prostate cancer cells.     

Distinct Phenotypes of Human Prostate Cancer Cells Associate with Different Adaptation to Hypoxia and Pro-Inflammatory Gene Expression

Hypoxia and inflammation are strictly interconnected both concurring to prostate cancer progression. Numerous reports highlight the role of tumor cells in the synthesis of pro-inflammatory molecules and show that hypoxia can modulate a number of these genes contributing substantially to the increase of cancer aggressiveness. However, little is known about the importance of the tumor phenotype in this process. The present study explores how different features, including differentiation and aggressiveness, of prostate tumor cell lines impact on the hypoxic remodeling of pro-inflammatory gene expression and malignancy. We performed our studies on three cell lines with increasing metastatic potential: the well differentiated androgen-dependent LNCaP and the less differentiated and androgen-independent DU145 and PC3. We analyzed the effect that hypoxic treatment has on modulating pro-inflammatory gene expression and evaluated the role HIF isoforms and NF-kB play in sustaining this process. DU145 and PC3 cells evidenced a higher normoxic expression and a more complete hypoxic induction of pro-inflammatory molecules compared to the well differentiated LNCaP cell line. The role of HIF1α and NF-kB, the master regulators of hypoxia and inflammation respectively, in sustaining the hypoxic pro-inflammatory phenotype was different according to cell type. NF-kB was observed to play a main role in DU145 and PC3 cells in which treatment with the NF-kB inhibitor parthenolide was able to counteract both the hypoxic pro-inflammatory shift and HIF1α activation but not in LNCaP cells. Our data highlight that tumor prostate cell phenotype contributes at a different degree and with different mechanisms to the hypoxic pro-inflammatory gene expression related to tumor progression.     

ADAM17通过EGFR-PI3K/Akt/p27通路调控前列腺癌细胞增殖

ADAM17是金属蛋白酶家族(ADAMs)成员之一,研究发现ADAM17可以通过水解细胞表面蛋白的胞外结构域导致肿瘤细胞的增殖和转移.本课题前期研究结果显示,与LNCap细胞相比,ADAM17在DU145细胞中高表达,且与细胞增殖相关.为了研究ADAM17与前列腺癌细胞增殖相关基因p27表达的关系及调控机制,我们采用RNAi技术下调ADAM17的表达,加入PMA(一种ADAM17的激活剂)上调ADAM17的表达,通过细胞计数和CCK-8方法检测细胞增殖,RT-PCR检测p27mRNA的表达,Western印迹检测ADAM17的表达;进一步阻断EGFR和PI3K/Akt信号转导,RT-PCR方法检测p27mRNA的表达,Western印迹检测ADAM17、EGFR、pEGFR、Akt和pAkt的表达.结果显示ADAM17的表达与前列腺癌细胞的增殖呈正相关(P0.05);p27mRNA的表达与ADAM17的表达呈负相关(P0.05);分别阻断EGFR和PI3K/Akt信号转导通路,同时使ADAM17表达增加,与对照组(单独PMA处理组)相比,p27mRNA的表达均增加(P0.05).提示ADAM17调控前列腺癌细胞增殖相关基因p27表达是通过EGFR-PI3K/Akt信号通路实现的.     

Somatostatin Derivate (smsDX) Attenuates the TAM-Stimulated Proliferation,Migration and Invasion of Prostate Cancer via NF-κB Regulation

Tumor development and progression are influenced by macrophages of the surrounding microenvironment. To investigate the influences of an inflammatory tumor microenvironment on the growth and metastasis of prostate cancer, the present study used a co-culture model of prostate cancer (PCa) cells with tumor-associated macrophage (TAM)-conditioned medium (MCM). MCM promoted PCa cell (LNCaP, DU145 and PC-3) growth, and a xenograft model in nude mice consistently demonstrated that MCM could promote tumor growth. MCM also stimulated migration and invasion in vitro. Somatostatin derivate (smsDX) significantly attenuated the TAM-stimulated proliferation, migration and invasion of prostate cancer. Immunohistochemistry revealed that NF-κB was over-expressed in PCa and BPH with chronic inflammatory tissue specimens and was positively correlated with macrophage infiltration. Further investigation into the underlying mechanism revealed that NF-κB played an important role in macrophage infiltration. SmsDX inhibited the paracrine loop between TAM and PCa cells and may represent a potential therapeutic agent for PCa.     

Hypoxia stabilizes GAS6/Axl signaling in metastatic prostate cancer

The receptor tyrosine kinase Axl is overexpressed in a variety of cancers and is known to play a role in proliferation and invasion. Previous data from our laboratory indicate that Axl and its ligand growth arrest-specific 6 (GAS6) may play a role in establishing metastatic dormancy in the bone marrow microenvironment. In the current study, we found that Axl is highly expressed in metastatic prostate cancer cell lines PC3 and DU145 and has negligible levels of expression in a nonmetastatic cancer cell line LNCaP. Knockdown of Axl in PC3 and DU145 cells resulted in decreased expression of several mesenchymal markers including Snail, Slug, and N-cadherin, and enhanced expression of the epithelial marker E-cadherin, suggesting that Axl is involved in the epithelial-mesenchymal transition in prostate cancer cells. The Axl-knockdown PC3 and DU145 cells also displayed decreased in vitro migration and invasion. Interestingly, when PC3 and DU145 cells were treated with GAS6, Axl protein levels were downregulated. Moreover, CoCl(2), a hypoxia mimicking agent, prevented GAS6-mediated downregulation of Axl in these cell lines. Immunochemical staining of human prostate cancer tissue microarrays showed that Axl, GAS6, and hypoxia-inducible factor-1α (Hif-1α; indicator of hypoxia) were all coexpressed in prostate cancer and in bone metastases compared with normal tissues. Together, our studies indicate that Axl plays a crucial role in prostate cancer metastasis and that GAS6 regulates the expression of Axl. Importantly, in a hypoxic tumor microenvironment Axl expression is maintained leading to enhanced signaling.     

蝎毒多肽提取物抑制DU- 145 细胞 COX- 2 和MMP- 9 表达的研究

目的：研究蝎毒多肽提取物（peptide extract from scorpion venom,PESV）对雄激素非依赖性人前列腺癌细胞株DU-145COX-2和MMP-9表达的影响,进一步探讨其抗血管生成的分子机制,为抗前列腺癌骨转移提供有效的治疗手段。方法：采用免疫组只化学方法检测PESV对COX-2、MMP-9蛋白表达的影响,应用RT-PCR检测PESV对MMP-9在mRNA水平表达的影响。结果：蝎毒多肽提取物（40μg／mL）作用于前列腺癌细胞后,COX-2、MMP-9蛋白表达水平明显下调（P〈0．05）,进一步检测发现MMP-9在mRNA水平亦明显下降（P〈0．05）。结论：蝎毒多肽提取物（PESV）通过抑制前列腺癌细胞血管生成因子COX-2的表达而发挥其抗血管生成作用,具有临床应用价值。     

Small Molecular Inhibitors Block TRPM4 Currents in Prostate Cancer Cells,with Limited Impact on Cancer Hallmark Functions

Transient receptor potential melastatin 4 (TRPM4) is a broadly expressed Ca²⁺ activated monovalent cation channel that contributes to the pathophysiology of several diseases.For this study, we generated stable CRISPR/Cas9 TRPM4 knockout (K.O.) cells from the human prostate cancer cell line DU145 and analyzed the cells for changes in cancer hallmark functions. Both TRPM4-K.O. clones demonstrated lower proliferation and viability compared to the parental cells. Migration was also impaired in the TRPM4-K.O. cells. Additionally, analysis of 210 prostate cancer patient tissues demonstrates a positive association between TRPM4 protein expression and local/metastatic progression. Moreover, a decreased adhesion rate was detected in the two K.O. clones compared to DU145 cells.Next, we tested three novel TRPM4 inhibitors with whole-cell patch clamp technique for their potential to block TRPM4 currents. CBA, NBA and LBA partially inhibited TRPM4 currents in DU145 cells. However, none of these inhibitors demonstrated any TRPM4-specific effect in the cellular assays.To evaluate if the observed effect of TRPM4 K.O. on migration, viability, and cell cycle is linked to TRPM4 ion conductivity, we transfected TRPM4-K.O. cells with either TRPM4 wild-type or a dominant-negative mutant, non-permeable to Na⁺. Our data showed a partial rescue of the viability of cells expressing functional TRPM4, while the pore mutant was not able to rescue this phenotype. For cell cycle distribution, TRPM4 ion conductivity was not essential since TRPM4 wild-type and the pore mutant rescued the phenotype.In conclusion, TRPM4 contributes to viability, migration, cell cycle shift, and adhesion; however, blocking TRPM4 ion conductivity is insufficient to prevent its role in cancer hallmark functions in prostate cancer cells.