RNA interference-mediated silencing of laminin receptor 1 (LR1) suppresses migration and invasion and down-regulates matrix metalloproteinase (MMP)-2 and MMP-9 in trophoblast cells: implication in the pathogenesis of preeclampsia

Shallow trophoblast invasion is a common pathological feature of preeclampsia. The 67 kDa laminin receptor 1 (LR1) is a laminin-binding protein that has been reported to be down-regulated in preeclamptic placentas. The aim of the present study was to determine the functional role of LR1 in the migration and invasion of the trophoblast cell line, JEG3 cells. RNA interference mediated by plasmid expressing LR1 short hairpin RNA (shRNA) was utilized to knockdown LR1 expression in JEG3 cells. We found that the mRNA and protein expression levels of LR1 were significantly reduced in LR1-specific shRNA transfected cells compared with the untransfected and control shRNA transfected cells. The wound healing and Transwell invasion assays demonstrated that LR1 knockdown remarkably suppressed the migration and invasion potential of JEG3 cells. The gelatin zymography assay showed that LR1 knockdown greatly reduced matrix metalloproteinase (MMP)-2 and MMP-9 activities in the culture supernatants. Western blot analysis showed that LR1 shRNA significantly decreased expression levels of MMP-2, MMP-9 and phospho-extracellular signal-regulated kinase, but increased expression levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in comparison to the control vector-transfected cells. In conclusion, our data support an important role for LR1 in regulating trophoblast invasion and migration, and suggest a possible pathological mechanism of preeclampsia.     

Liver X receptor α (LXRα) promoted invasion and EMT of gastric cancer cells by regulation of NF-κB activity

Aberrant expression of Liver X receptor α (LXRα) has been frequently reported in various types of cancers excluding gastric cancer (GC). Moreover, the role of LXRα in human GC has not been previously reported. In this study, we investigated the effect of LXRα down-regulation on invasion and EMT of GC. The expression of LXRα in GC cell lines was detected by real-time PCR. The LXRα siRNA was transiently transfected into GC cells using Lipofectamine? 2000 reagent. Subsequently, cell invasive ability was evaluated by Transwell assays. Western blot and real-time PCR were used to determined the expressions of matrix metalloproteinase-2 and -9 (MMP-2 and -9), E-cadherin, N-cadherin, Vimentin, Snail, Slug, and Twist in GC cells. In addition, the effect of LXRα down-regulation on the phosphoinositide 3-kinase (PI3K)/Akt/nuclear factor (NF)-κB signaling pathway was explored by Western blot. From our results, we found that the expression of LXRα was significantly increased in GC tissues and cell lines. Knockdown of LXRα suppressed the invasive ability of GC cells. The levels of MMP-2 and -9 were dramatically decreased by down-regulating LXRα. In addition, we found a decrease of N-cadherin, Twist, and Slug expressions and an increase of E-cadherin expression, but no influence on the expression levels of Vimentin and Snail. We also found that LXRα down-regulation might suppress the phosphorylation of Akt, NF-κB, and IκB. Collectively, our results indicated that down-regulation of LXRα was shown to suppress invasion and EMT of GC cells by decreasing the expressions of related proteins through inhibiting the PI3K/Akt/NF-κB signaling pathway.     

Curcumin suppresses the TPA-induced invasion through inhibition of PKCα-dependent MMP-expression in MCF-7 human breast cancer cells

Curcumin (diferuloylmethane) is a polyphenol derived from the plant turmeric (Curcuma longa), which is commonly used as a spice. Although anti-carcinogenic, anti-oxidant, anti-inflammation, and anti-angiogenic properties have been reported, the effect of curcumin on breast cancer metastasis is unknown. Matrix metalloproteinase-9 (MMP-9) is a major component in cancer cell invasion. In this study, we investigated the inhibitory effect of curcumin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion and the molecular mechanisms involved in MCF-7 cells. Our results showed that curcumin inhibits TPA-induced MMP-9 expression and cell invasion through suppressing NF-κB and AP-1 activation. Also, curcumin strongly repressed the TPA-induced phosphorylation of p38 and JNK and inhibited TPA-induced translocation of PKCα from the cytosol to the membrane, but did not affect the translocation of PKCδ. These results indicate that curcumin-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of the PKCα, MAPK and NF-κB/AP-1 pathway in MCF-7 cells. Curcumin may have potential value in restricting breast cancer metastasis.     

Focal adhesion kinase mediates TGF-β1-induced renal tubular epithelial-to-mesenchymal transition in vitro

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which participates in many important cellular processes such as cell adhesion and migration. However, the role of FAK in renal tubular epithelial-to-mesenchymal transition (EMT) is still unknown. FAK was knocked down by transfection of specific small interfering RNA (siRNA) in cultured HK-2 cells, then the cells were stimulated with transforming growth factor-beta 1 (TGF-β1). The expression of FAK, α-smooth muscle actin (α-SMA),E-cadherin, Akt, matrix metallopeptidase-9 (MMP-9),tissue inhibitor of metalloproteinase-1 (TIMP-1), and collagen IV were detected by RT-PCR, Western blot and immunofluorescence methods, respectively. Cell migration was determined by transwell assay. The results suggest that the expression of FAK was up-regulated in HK-2 cells when incubated with TGF-β1(10 μg/l), which was accompanied by reduced expression of E-cadherin and increased expression of α-SMA. All these changes were restored by FAK siRNA. Akt phosphorylation was induced by the treatment with TGF-β1, which was blocked by FAK siRNA. TGF-β1-induced down-regulation of E-cadherin was recovered by a PI3K/Akt inhibitor, LY294002, without affecting the expression of FAK. Functionally, TGF-β1 induced an increase in MMP-9 expression, as well as decreased expression of TIMP-1 and collagen IV, which were all restored by the FAK siRNA transfection. In addition, FAK siRNA significantly reduced TGF-β1-induced cells migration. In conclusion, FAK may play a crucial role in mediating TGF-β1-induced EMT through the activation of Akt pathway.     

Downregulation of monocarboxylate transporter 1 inhibits the invasion and migration through suppression of the PI3K/Akt signaling pathway in human nasopharyngeal carcinoma cells

Monocarboxylate transporter 1 (MCT1) has been reported to be correlated wtih decreased survival and advanced stage of progression in a series of human tumor cells and primary cancers. Specifically, MCT1 has been documented to be involved in tumor progression, including invasion and migration. Here, we investigated the mechanism and effect of regulation of MCT1 on invasion and migration of nasopharyngeal carcinoma (NPC) cells. In the study, we firstly demonstrated that the expression of MCT1 in CNE2Z cells was obviously higher than that in HNE1 cells. Downregulation of MCT1 inhibited the invasion and migration in CNE2Z cells, upregulated the expression of E-cadherin, TIMP (tissue inhibitor of metalloproteinase)-2 and TIMP-1, and suppressed the expression of matrix metalloproteinases (MMP)-9 and MMP-2. Correspondingly, upregulation of MCT1 enhanced the invasive and migratory potential in HNE1 cells, increased the expression of MMP-9 and MMP-2, and downregulated the expression of E-cadherin, TIMP-2 and TIMP-1. The mechanistic study demonstrated that the effect of MCT1 might be correlated with PI3K/Akt signaling pathway. LY294002, a PI3K inhibitor, increased the inhibition of invasion and migration mediated by downregulation of MCT1 in CNE2Z cells. These findings collectively suggested that MCT1 might act as a new regulator to improve invasion and migration of NPC cells and be correlated with activating the PI3K/Akt pathway.     

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.     

AMP-activated protein kinase suppresses matrix metalloproteinase-9 expression in mouse embryonic fibroblasts

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tissue remodeling under both physiological and pathological conditions. Although MMP-9 expression is low in most cells and is tightly controlled, the mechanism of its regulation is poorly understood. We utilized mouse embryonic fibroblasts (MEFs) that were nullizygous for the catalytic α subunit of AMP-activated protein kinase (AMPK), which is a key regulator of energy homeostasis, to identify AMPK as a suppressor of MMP-9 expression. Total AMPKα deletion significantly elevated MMP-9 expression compared with wild-type (WT) MEFs, whereas single knock-out of the isoforms AMPKα1 and AMPKα2 caused minimal change in the level of MMP-9 expression. The suppressive role of AMPK on MMP-9 expression was mediated through both its activity and presence. The AMPK activators 5-amino-4-imidazole carboxamide riboside and A769662 suppressed MMP-9 expression in WT MEFs, and AMPK inhibition by the overexpression of dominant negative (DN) AMPKα elevated MMP-9 expression. However, in AMPKα(-/-) MEFs transduced with DN AMPKα, MMP-9 expression was suppressed. AMPKα(-/-) MEFs showed increased phosphorylation of IκBα, expression of IκBα mRNA, nuclear localization of nuclear factor-κB (NF-κB), and DNA-binding activity of NF-κB compared with WT. Consistently, selective NF-κB inhibitors BMS345541 and SM7368 decreased MMP-9 expression in AMPKα(-/-) MEFs. Overall, our results suggest that both AMPKα isoforms suppress MMP-9 expression and that both the activity and presence of AMPKα contribute to its function as a regulator of MMP-9 expression by inhibiting the NF-κB pathway.     

MMP-15 is upregulated in preeclampsia, but does not cleave endoglin to produce soluble endoglin

Preeclampsia is a major pregnancy complication, characterized by severe endothelial dysfunction, hypertension and maternal end-organ damage. Soluble endoglin is an anti-angiogenic protein released from placenta and thought to play a central role in causing the endothelial dysfunction and maternal organ injury seen in severe preeclampsia. We recently reported MMP-14 was the protease producing placentally-derived soluble endoglin by cleaving full-length endoglin present on the syncytiotrophoblast surface. This find identifies a specific drug target for severe preeclampsia; interfering with MMP-14 mediated cleavage of endoglin could decrease soluble endoglin production, ameliorating clinical disease. However, experimental MMP-14 inhibition alone only partially repressed soluble endoglin production, implying other proteases might have a role in producing soluble endoglin. Here we investigated whether MMP-15--phylogenetically the closest MMP relative to MMP-14 with 66% sequence similarity--also cleaves endoglin to produce soluble endoglin. MMP-15 was localized to the syncytiotrophoblast layer of the placenta, the same site where endoglin was localized. Interestingly, it was significantly (p?=?0.03) up-regulated in placentas from severe early-onset preeclamptic pregnancies (n?=?8) compared to gestationally matched preterm controls (n?=?8). However, siRNA knockdown of MMP-15 yielded no significant decrease of soluble endoglin production from either HUVECs or syncytialised BeWo cells in vitro. Importantly, concurrent siRNA knockdown of both MMP-14 and MMP-15 in HUVECS did not yield further decrease in soluble endoglin production compared to MMP-14 siRNA alone. We conclude MMP-15 is up-regulated in preeclampsia, but does not cleave endoglin to produce soluble endoglin.     

Suppression of 12-O-Tetradecanoylphorbol-13-Acetate-Induced MCF-7 Breast Adenocarcinoma Cells Invasion/Migration by α-Tomatine Through Activating PKCα/ERK/NF-κB-Dependent MMP-2/MMP-9 Expressions

α-Tomatine, isolated from Lycopersicon esculentum Linn., is a naturally occurring glycoalkaloids in immature green tomatoes. Some reports demonstrated that α-tomatine had various anti-carcinogenic properties. First, the result demonstrated α-tomatine could inhibit TPA-induced the abilities of the adhesion, morphology/actin cytoskeleton arrangement, invasion, and migration by cell–matrix adhesion assay, immunofluorescence stain assay, Boyden chamber invasion assay, and wound-healing assay. Data also showed α-tomatine could inhibit the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and protein kinase C-α (PKCα) involved in the downregulation of the enzyme activities and messenger RNA levels of matrix metalloproteinase-2/9 (MMP-2/MMP-9) induced by TPA. Next, α-tomatine also strongly inhibited TPA-induced the activation of nuclear factor kappa B (NF-κB) and phospho-inhibitor of kappa Bα (phospho-IκBα). In addition, TPA-induced translocation of PKC-α from cytosol to membranes, and suppression of TPA elicited the expression of PKC-α by adding the PKC-α inhibitors, GF-109203X and Gö-6983. The treatment of specific inhibitor for ERK (U0126) to MCF-7 cells could inhibit TPA-induced MMP-2/MMP-9 and phospho-ERK along with an inhibition on cell invasion and migration. Application of α-tomatine to prevent the invasion/migration of MCF-7 cells through blocking PKCα/ERK/NF-κB activation is first demonstrated herein.     

Sulforaphane controls TPA-induced MMP-9 expression through the NF-κB signaling pathway,but not AP-1, in MCF-7 breast cancer cells

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)-butane] is an isothiocyanate found in some cruciferous vegetables, especially broccoli. Sulforaphane has been shown to display anti-cancer properties against various cancer cell lines. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix (ECM), plays an important role in cancer cell invasion. In this study, we investigated the effect of sulforaphane on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion in MCF-7 cells. TPA-induced MMP-9 expression and cell invasion were decreased by sulforaphane treatment. TPA substantially increased NF-κB and AP-1 DNA binding activity. Pre-treatment with sulforaphane inhibited TPA-stimulated NF-κB binding activity, but not AP-1 binding activity. In addition, we found that sulforaphane suppressed NF-κB activation, by inhibiting phosphorylation of IκB in TPA-treated MCF-7 cells. In this study, we demonstrated that the inhibition of TPA-induced MMP-9 expression and cell invasion by sulforaphane was mediated by the suppression of the NF-κB pathway in MCF-7 cells. [BMB Reports 2013; 46(4): 201-206]