Transforming growth factor‐β1 modulates metalloproteinase‐2 and ‐9, nitric oxide,RhoA and α‐smooth muscle actin expression in colon adenocarcinoma cells

Colon carcinoma invasiveness is a process involving cell–cell and cell–matrix alterations, local proteolysis of the ECM (extracellular matrix) or changes in cytokine and growth factor levels. In order to evaluate the role of TGF‐β1 (transforming growth factor‐β1) and small G protein RhoA in tumour progression, the influence of TGF‐β1 treatment or RhoA‐associated kinase inhibitor on the production of NO (nitric oxide) and MMP‐2 and MMP‐9 (metalloproteinases‐2 and ‐9) was analysed in three human colon adenocarcinoma cell lines (HT29, LS180, SW948) representing different stages of tumour development. All the tested cell lines produced low amounts of MMP‐2 and MMP‐9. rhTGF‐β1 and the synthetic Rho kinase inhibitor (Y‐27632) decreased MMP‐2 secretion by colon cancer cells, especially in the most advanced stage of colon cancer. rhTGF‐β1 decreased NO secretion by cells, while Y‐27632 had no effect on it. Immunoblotting with anti‐RhoA antibodies followed by densitometry revealed that RhoA levels were slightly increased after incubation of colon carcinoma cells (SW948) with rhTGF‐β1. rhTGF‐β1 induced α‐smooth muscle actin (α‐SMA) expression, especially in high Duke's grade of colon cancer, while Y‐27632 blocked it. Summing up, in colon carcinoma cells, TGF‐β1 and RhoA protein may regulate tumour invasiveness measured as MMP, NO and α‐SMA expression or assayed using motility data and may be a good target for cancer therapy.     

PAX3 silencing inhibits prostate cancer progression through the suppression of the TGF‐β/Smad signaling axis

Multiple studies have confirmed the pro‐oncogenic effects of PAX3 in an array of cancers, but its role in prostate cancer (PCa) remains largely undefined. The aim of this study is to investigate the role of PAX3 in PCa. PAX3 expression was compared between PCa tumor tissue and nontumor tissues and PCa cell lines and normal prostate epithelial cells (PNT2) by western blot analysis and immunohistochemistry staining. MTT and immunofluorescence assays were used to detect PCa cell proliferation. Flow cytometry was used to evaluate cell apoptosis in PCa. Transwell assays were used for the determination of cell migration and PCa cell invasion. PAX3 expression was higher in PCa tissues and human PCa cell lines. Moreover, PAX3 silencing inhibited the proliferation, metastasis, and epithelial–mesenchymal transition (EMT) of PCa cells, and increased the rates of apoptosis. PAX3 silencing inhibited transforming growth factor‐β (TGF‐β)/Smad signaling in PCa cells. The effects of si‐PAX3 on the proliferation, apoptosis, metastasis, and EMT of PCa cells were alleviated by TGF‐β1 treatment. PAX3 silencing inhibits PCa progression through the inhibition of TGF‐β/Smad signaling. This reveals PAX3 as a novel biomarker and therapeutic target for future PCa treatments.     

Crosstalk between Smad2/3 and specific isoforms of ERK in TGF‐β1‐induced TIMP‐3 expression in rat chondrocytes

This study investigated the roles of ERK1 and ERK2 in transforming growth factor‐β1 (TGF‐β1)‐induced tissue inhibitor of metalloproteinases‐3 (TIMP‐3) expression in rat chondrocytes, and the specific roles of ERK1 and ERK2 in crosstalk with Smad2/3 were investigated to demonstrate the molecular mechanism of ERK1/2 regulation of TGF‐β1 signalling. To examine the interaction of specific isoforms of ERK and the Smad2/3 signalling pathway, chondrocytes were infected with LV expressing either ERK1 or ERK2 siRNA and stimulated with or without TGF‐β1. At indicated time‐points, TIMP‐3 expression was determined by real‐time PCR and Western blotting; p‐Smad3, nuclear p‐Smad3, Smad2/3, p‐ERK1/2 and ERK1/2 levels were assessed. And then, aggrecan, type II collagen and the intensity of matrix were examined. TGF‐β1‐induced TIMP‐3 expression was significantly inhibited by ERK1 knock‐down, and the decrease in TIMP‐3 expression was accompanied by a reduction of p‐Smad3 in ERK1 knock‐down cells. Knock‐down of ERK2 had no effect on neither TGF‐β1‐induced TIMP‐3 expression nor the quantity of p‐Smad3. Moreover, aggrecan, type II collagen expression and the intensity of matrix were significantly suppressed by ERK1 knock‐down instead of ERK2 knock‐down. Taken together, ERK1 and ERK2 have different roles in TGF‐β1‐induced TIMP‐3 expression in rat chondrocytes. ERK1 instead of ERK2 can regulate TGF‐β/Smad signalling, which may be the mechanism through which ERK1 regulates TGF‐β1‐induced TIMP‐3 expression.     

MicroRNA‐144‐3p suppressed TGF‐β1‐induced lung cancer cell invasion and adhesion by regulating the Src–Akt–Erk pathway

Lung cancer remains a leading cause to cancer‐related death worldwide. The anti‐cancer ability of microRNA‐144‐3p has been reported in many cancer types. This study focused on the mechanisms underlying miR‐144‐3p in inhibiting lung cancer. The expression levels of miR‐144‐3p and steroid receptor coactivator (Src) in different lung cancer cell lines and those in bronchial epithelial cells (16HBE) were compared. miR‐144‐3p mimic and siSrc were transfected into A549 cells. Under the conditions of transforming growth factor‐β1 (TGF‐β1). Small interfering transfection or TGF‐β1 treatment, cell invasive and adhesive abilities were analyzed by Transwell and cell adhesion assays. miR‐144‐3p inhibitor and siSrc were co‐transfected into A549 cells and the changes in cell invasion and adhesion were detected. The activation of Src–protein kinase B–extracellular‐regulated protein kinases (Src–Akt–Erk) pathway was determined using Western blot. The downregulated miR‐144‐3p and upregulated Src were generally detected in lung cancer cell lines and were the most significant genes in A549 cells. Both miR‐144‐3p overexpression and Src inhibition could obviously inhibit the invasion and adhesion abilities of A549 cells in the presence or absence of the effects of TGF‐β1. The inhibition of Src could block the promotive effects of miR‐144‐3p inhibitor and TGF‐β1 on cell invasion and adhesion. Furthermore, we found that miR‐144‐3p could negatively regulate the phosphorylation levels of Akt and Erk. Our data indicated the essential role of Src in the mechanisms underlying TGF‐β1‐induced cell invasion and adhesion of lung cancer, and that miR‐144‐3p could effectively suppress TGF‐β1‐induced aggressive lung cancer cells by regulating Src expression.     

Transforming growth factor-beta1 regulates the fate of cultured spinal cord-derived neural progenitor cells

Abstract. Objectives: We have evaluated the physiological roles of transforming growth factor‐β1 (TGF‐β1) on differentiation, migration, proliferation and anti‐apoptosis characteristics of cultured spinal cord‐derived neural progenitor cells. Methods: We have used neural progenitor cells that had been isolated and cultured from mouse spinal cord tissue, and we also assessed the relevant reaction mechanisms using an activin‐like kinase (ALK)‐specific inhibitory system including an inhibitory RNA, and found that it involved potential signalling molecules such as phosphatidylinositol‐3‐OH kinase (PI3K)/Akt and mitogen‐activated protein kinase (MAPK)/extracellular signal‐regulated kinase (ERK1/2). Results and Conclusions: Transforming growth factor‐β1‐mediated cell population growth was activated after treatment and was also effectively blocked by an ALK41517‐synthetic inhibitor (4‐(5‐benzo(1,3) dioxol‐5‐yl‐4‐pyridine‐2‐yl‐1H‐imidazole‐2‐yl) benzamide (SB431542) and ALK siRNA, thereby indicating the involvement of SMAD2 in the TGF‐β1‐mediated growth and migration of these neural progenitors cells (NPC). In the present study, TGF‐β1 actively induced NPC migration in vitro. Furthermore, TGF‐β1 demonstrated extreme anti‐apoptotic behaviour against hydrogen peroxide‐mediated apoptotic cell death. At low dosages, TGF‐β1 enhanced (by approximately 76%) cell survival against hydrogen peroxide treatment via inactivation of caspase‐3 and ‐9. TGF‐β1‐treated NPCs down‐regulated Bax expression and cytochrome c release; in addition, the cells showed up‐regulated Bcl‐2 and thioredoxin reductase 1. They also had increased p38, Akt and ERK1/2 phosphorylation, showing the involvement of both the PI3K/Akt and MAPK/ERK1/2 pathways in the neuroprotective effects of TGF‐β1. Interestingly, these effects operate on specific subtypes of cells, including neurones, neural progenitor cells and astrocytes in cultured spinal cord tissue‐derived cells. Lesion sites of spinal cord‐overexpressing TGF‐β1‐mediated prevention of cell death, cell growth and migration enhancement activity have been introduced as a possible new basis for therapeutic strategy in treatment of neurodegenerative disorders, including spinal cord injuries.     

The response of extracellular signal-regulated kinase (ERK) to androgen-induced proliferation in the androgen-sensitive prostate cancer cell line, LNCaP.

The mechanisms by which androgens stimulate proliferation of prostate cancer cells are poorly understood. It has been proposed that androgen stimulation may induce the mitogen-activated protein (MAP) kinase system in prostate cancer cells and lead to cellular proliferation. We attempted to evaluate the role of the extracellular signal-regulated kinase (ERK) pathway in the stimulation by androgens of prostate cancer cell proliferation. Androgen-sensitive prostate cancer cell line (LNCaP) cells plated on sterile glass coverslips were treated with 10(-8) M dihydrotestosterone (DHT) or epidermal growth factor (EGF) (10 ng/ml) for periods ranging from 1 min to 96 h. The proliferative index of the cells, evaluated by immunoperoxidase staining of cells with an antibody to Ki-67, was increased at least two-fold at all time points from 5 min to 48 h following exposure to either DHT or EGF. Immunohistochemical evaluation of ERK1/2 and pERK (activated ERK) demonstrated high levels of ERK1/2 in untreated LNCaP cells, while pERK was expressed at much lower levels. Following treatment with DHT, no change in staining intensity for either ERK1/2 or pERK was observed, while treatment with EGF resulted in no change in ERK1/2, but significantly increased cytoplasmic staining for pERK at all time points beyond 2 min. These results were confirmed by Western blot analysis of ERK1/2 and pERK expression in these cell lines following treatment with DHT or EGF. Our findings suggest that the proliferative response of prostate cancer cells to androgens, unlike the proliferative response to EGF, is not mediated by the activation of ERK1/2, and that currently undefined pathways other than those involving ERK1/2 are involved.     

Issue information

Galectin‐3 (Gal‐3) plays a critical role in vascular inflammation and fibrosis. The role of TGF‐β1 in mediating pulmonary vascular fibrosis is well documented; thus, we suspected that Gal‐3 could be an important factor in TGF‐β1‐induced fibrosis in pulmonary adventitial fibroblasts (PAFs). We treated rats with monocrotaline (MCT) and cultured PAFs with TGF‐β1 to stimulate fibrosis. We found that MCT injection induced vessel thickening and extracellular matrix deposition in vivo. TGF‐β1 stimulated the production of collagen and fibronectin (Fn) protein in vitro. TGF‐β1 promoted the expression of Gal‐3 and its translocation, while silencing Gal‐3 reduced Col‐1a deposition. Blockage of STAT3 decreased the expression of Gal‐3 induced by TGF‐β1. Gal‐3 increased Col‐1a accumulation and downregulated matrix metallopeptidase 9 (MMP‐9) expression in PAFs, but it did not affect Fn expression. These findings demonstrate that Gal‐3 is required for TGF‐β1‐stimulated vascular fibrosis via a STAT3 signaling cascade and that MMP‐9 is also involved in TGF‐β1/Gal‐3‐induced vascular fibrosis.     

Epidermal growth factor and ionizing radiation up-regulate the DNA repair genes XRCC1 and ERCC1 in DU145 and LNCaP prostate carcinoma through MAPK signaling

This work examined the importance of radiation-induced and ligand-induced EGFR-ERK signaling for the regulation of DNA repair proteins XRCC1 and ERCC1 in prostate carcinoma cells, DU145 (TP53(mut)), displaying EGFR-TGFA-dependent autocrine growth and high MAPK (ERK1/2) activity, and LNCaP (TP53(wt)) cells expressing low constitutive levels of ERK1/2 activity. Using quantitative RT-PCR and Western analyses, we determined that ionizing radiation activated the DNA repair genes XRCC1 and ERCC1 in an ERK1/2-dependent fashion for each cell line. After irradiation, a rapid increase followed by a decrease in ERK1/2 activity preceded the increase in XRCC1/ERCC1 expression in DU145 cells, while only the rapid decrease in ERK1/2 preceded the increase in XRCC1/ERCC1 expression in LNCaP cells. Administration of EGF, however, markedly increased the up-regulation of phospho-ERK, ERCC1 and XRCC1 in both cell lines. Although the EGFR inhibitor tyrphostin (AG-1478) and the MEK inhibitor PD90859 both attenuated EGF-induced levels of the ERCC1 and XRCC1 protein, PD98059 blocked the induction of ERCC1 and XRCC1 by radiation more effectively in both cell lines. Inhibition of ERK at a level that reduced the up-regulation of DNA repair led to the persistence of apurinic/apyrimidinic (AP) sites of DNA damage and increased cell killing. Taken together, these data imply a complex control of DNA repair activation that may be more generally dependent on MAPK (ERK1/2) signaling than was previously noted. These data provide novel insights into the capacity of the EGFR-ERK signaling to modulate DNA repair in cancer cells and into the functional significance of this signaling.     

The response of extracellular signal-regulated kinase (ERK) to androgen-induced proliferation in the androgen-sensitive prostate cancer cell line,LNCaP

The mechanisms by which androgens stimulate proliferation of prostate cancer cells are poorly understood. It has been proposed that androgen stimulation may induce the mitogen-activated protein (MAP) kinase system in prostate cancer cells and lead to cellular proliferation. We attempted to evaluate the role of the extracellular signal-regulated kinase (ERK) pathway in the stimulation by androgens of prostate cancer cell proliferation. Androgen-sensitive prostate cancer cell line (LNCaP) cells plated on sterile glass coverslips were treated with 10^?8 M dihydrotestosterone (DHT) or epidermal growth factor (EGF) (10 ng/ml) for periods ranging from 1 min to 96 h. The proliferative index of the cells, evaluated by immunoperoxidase staining of cells with an antibody to Ki-67, was increased at least two-fold at all time points from 5 min to 48 h following exposure to either DHT or EGF. Immunohistochemical evaluation of ERK1/2 and pERK (activated ERK) demonstrated high levels of ERK1/2 in untreated LNCaP cells, while pERK was expressed at much lower levels. Following treatment with DHT, no change in staining intensity for either ERK1/2 or pERK was observed, while treatment with EGF resulted in no change in ERK1/2, but significantly increased cytoplasmic staining for pERK at all time points beyond 2 min. These results were confirmed by Western blot analysis of ERK1/2 and pERK expression in these cell lines following treatment with DHT or EGF. Our findings suggest that the proliferative response of prostate cancer cells to androgens, unlike the proliferative response to EGF, is not mediated by the activation of ERK1/2, and that currently undefined pathways other than those involving ERK1/2 are involved.     

The response of extracellular signal-regulated kinase (ERK) to androgen-induced proliferation in the androgen-sensitive prostate cancer cell line, LNCaP

The mechanisms by which androgens stimulate proliferation of prostate cancer cells are poorly understood. It has been proposed that androgen stimulation may induce the mitogen-activated protein (MAP) kinase system in prostate cancer cells and lead to cellular proliferation. We attempted to evaluate the role of the extracellular signal-regulated kinase (ERK) pathway in the stimulation by androgens of prostate cancer cell proliferation. Androgen-sensitive prostate cancer cell line (LNCaP) cells plated on sterile glass coverslips were treated with 10^-8 M dihydrotestosterone (DHT) or epidermal growth factor (EGF) (10 ng/ml) for periods ranging from 1 min to 96 h. The proliferative index of the cells, evaluated by immunoperoxidase staining of cells with an antibody to Ki-67, was increased at least two-fold at all time points from 5 min to 48 h following exposure to either DHT or EGF. Immunohistochemical evaluation of ERK1/2 and pERK (activated ERK) demonstrated high levels of ERK1/2 in untreated LNCaP cells, while pERK was expressed at much lower levels. Following treatment with DHT, no change in staining intensity for either ERK1/2 or pERK was observed, while treatment with EGF resulted in no change in ERK1/2, but significantly increased cytoplasmic staining for pERK at all time points beyond 2 min. These results were confirmed by Western blot analysis of ERK1/2 and pERK expression in these cell lines following treatment with DHT or EGF. Our findings suggest that the proliferative response of prostate cancer cells to androgens, unlike the proliferative response to EGF, is not mediated by the activation of ERK1/2, and that currently undefined pathways other than those involving ERK1/2 are involved.     

Correlation between TGF‐β1 expression and proteomic profiling induced by severe acute respiratory syndrome coronavirus papain‐like protease

Severe acute respiratory syndrome (SARS) coronavirus (SARS‐CoV) papain‐like protease (PLpro), a deubiquitinating enzyme, demonstrates inactivation of interferon (IFN) regulatory factor 3 and NF‐κB, reduction of IFN induction, and suppression of type I IFN signaling pathway. This study investigates cytokine expression and proteomic change induced by SARS‐CoV PLpro in human promonocyte cells. PLpro significantly increased TGF‐β1 mRNA expression (greater than fourfold) and protein production (greater than threefold). Proteomic analysis, Western blot, and quantitative real‐time PCR assays indicated PLpro upregulating TGF‐β1‐associated genes: HSP27, protein disulfide isomerase A3 precursor, glial fibrillary acidic protein, vimentin, retinal dehydrogenase 2, and glutathione transferase omega‐1. PLpro‐activated ubiquitin proteasome pathway via upregulation of ubiquitin‐conjugating enzyme E2–25k and proteasome subunit alpha type 5. Proteasome inhibitor MG‐132 significantly reduced expression of TGF‐β1 and vimentin. PLpro upregulated HSP27, linking with activation of p38 MAPK and ERK1/2 signaling. Treatment with SB203580 and U0126 reduced PLpro‐induced expression of TGF‐β1, vimentin, and type I collagen. Results point to SARS‐CoV PLpro triggering TGF‐β1 production via ubiquitin proteasome, p38 MAPK, and ERK1/2‐mediated signaling.     

RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo

The invasive ability of tumor cells plays a key role in prostate cancer metastasis and is a major cause of treatment failure. Urokinase plasminogen activator-(uPA) and its receptor (uPAR)-mediated signaling have been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study was undertaken to investigate the biological roles of uPA and uPAR in prostate cancer cell invasion and survival, and the potential of uPA and uPAR as targets for prostate cancer therapy. uPA and uPAR expression correlates with the metastatic potential of prostate cancer cells. Thus, therapies designed to inhibit uPA and uPAR expression would be beneficial. LNCaP, DU145, and PC3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as demonstrated by their capacity to invade the extracellular matrix. In this study we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human uPA and uPAR. These small interfering RNA constructs significantly inhibited uPA and uPAR expression at both the mRNA and protein levels in the highly metastatic prostate cancer cell line PC3. Our data demonstrated that uPA-uPAR knockdown in PC3 cells resulted in a dramatic reduction of tumor cell invasion as indicated by a Matrigel invasion assay. Furthermore, simultaneous silencing of the genes for uPA and uPAR using a single plasmid construct expressing shRNAs for both uPA and uPAR significantly reduced cell viability and ultimately resulted in the induction of apoptotic cell death. RNA interference for uPA and uPAR also abrogated uPA-uPAR signaling to downstream target molecules such as ERK1/2 and Stat 3. In addition, our results demonstrated that intratumoral injection with the plasmid construct expressing shRNAs for uPA and uPAR almost completely inhibited established tumor growth and survival in an orthotopic mouse prostate cancer model. These findings uncovered evidence of a complex signaling network operating downstream of uPA-uPAR that actively advances tumor cell invasion, proliferation, and survival of prostate cancer cells. Thus, RNA interference-directed targeting of uPA and uPAR is a convenient and novel tool for studying the biological role of the uPA-uPAR system and raises the potential of its application for prostate cancer therapy.