Prostaglandin E2 receptor EP1 transactivates EGFR/MET receptor tyrosine kinases and enhances invasiveness in human hepatocellular carcinoma cells

Recent evidence indicates that cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) are involved in hepatocarcinogenesis. This study was designed to evaluate the possible interaction between the COX-2 and EGFR signaling pathways in human hepatocellular carcinoma (HCC) cells. Immunohistochemical analysis using serial sections of human HCC tissues revealed positive correlation between COX-2 and EGFR in HCC cells (P < 0.01). Overexpression of COX-2 in cultured HCC cells (Hep3B) or treatment with PGE(2) or the selective EP(1) receptor agonist, ONO-DI-004, increased EGFR phosphorylation and tumor cell invasion. The PGE(2)-induced EGFR phosphorylation and cell invasiveness were blocked by the EP(1) receptor siRNA or antagonist ONO-8711 and by two EGFR tyrosine kinase inhibitors, AG1478 and PD153035. The EP(1)-induced EGFR transactivation and cell invasion involves c-Src, in light of the presence of native binding complex of EP(1)/Src/EGFR and the inhibition of PGE(2)-induced EGFR phosphorylation and cell invasion by the Src siRNA and the Src inhibitor, PP2. Further, overexpression of COX-2 or treatment with PGE(2) also induced phosphorylation of c-Met, another receptor tyrosine kinase critical for HCC cell invasion. Moreover, activation of EGFR by EGF increased COX-2 promoter activity and protein expression in Hep3B and Huh-7 cells, whereas blocking PGE(2) synthesis or EP(1) attenuated EGFR phosphorylation induced by EGF, suggesting that the COX-2/PGE(2)/EP(1) pathway also modulate the activation of EGFR by its cognate ligand. These findings disclose a cross-talk between the COX-2/PGE(2)/EP(1) and EGFR/c-Met signaling pathways that coordinately regulate human HCC cell invasion.     

Lectin BS‐I inhibits cell migration and invasion via AKT/GSK‐3β/β‐catenin pathway in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is most common malignant cancer worldwide; however, the mortality rate of HCC remains high due to the invasion and metastasis of HCC. Thus, exploring novel treatments to prevent the invasion of HCC is needed for improving clinical outcome of this fatal disease. In this study, we identified lectin from Bandeiraea simplicifolia seeds (BS‐I) binds to metastasis‐associated HCC cell surface glycans by a lectin microarray and inhibits HCC cell migration and invasion through downregulating the matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9 (MMP9) and urokinase‐type plasminogen activator (uPA) production. These effects of BS‐I were mediated by inhibiting the activation of AKT/GSK‐3β/β‐catenin pathway and depended on specificity of lectin BS‐I binding to GalNAc. GSK3β inhibitors rescued BS‐I‐mediated inhibition of migration and invasion of HCC cell. Further, we identified that lectin BS‐I interacts with sGrp78, affects membrane localization of sGrp78 and attenuates the binding of sGrp78 and p85 to inhibit the activation of AKT/GSK‐3β/β‐catenin pathway. Overexpression of Grp78 or P85 rescues BS‐I‐mediated inhibition of migration and invasion of HCC cell. These findings demonstrated for the first time that BS‐I can act as a novel potential drug to prevent the invasion of HCC.     

Fluvastatin, a lipophilic statin, induces apoptosis in human hepatocellular carcinoma cells through mitochondria-operated pathway

Fluvastatin, a lipophilic statin, was known to inhibit proliferation and induce apoptosis in many cancer cells. Its potential anticancer was evaluated in three hepatocellular carcinoma (HCC) cell lines (HepG2, SMMC-7721 and MHCC-97H). Cells were treated with fluvastatin in vitro and its effect on cell proliferation, cell cycle, invasion and apoptosis was determined. Mechanism of apoptosis induced by fluvastatin on HCC cell lines was also investigated through western blotting and mitochondrial membrane potential (MMP) analysis. It was observed that fluvastatin inhibited proliferation of HCC cells by inducing apoptosis and G2/M phase arrest in a dose-dependent manner. The results of cell invasion assay revealed that fluvastatin significantly decreased the invasion potency of HCC cells. A mitochondria-operated mechanism for fluvastatin induced apoptosis might be involved and was supported by Western blotting and MMP analysis. After fluvastatin treatment, expression of Bcl-2 and procaspase-9 were downregulated, cytochrome c (cytosolic extract), Bax and cleaved-caspase-3 protein expression were increased. Furthermore, a breakdown of MMP in HCC cells was observed. To conclude, these results have provided a rationale for clinical investigations of fluvastatin in future as a potential anticancer reagent for growth control of HCC.     

Down‐regulation of Notch‐1 and Jagged‐1 inhibits prostate cancer cell growth,migration and invasion,and induces apoptosis via inactivation of Akt,mTOR, and NF‐κB signaling pathways

Notch signaling is involved in a variety of cellular processes, such as cell fate specification, differentiation, proliferation, and survival. Notch‐1 over‐expression has been reported in prostate cancer metastases. Likewise, Notch ligand Jagged‐1 was found to be over‐expressed in metastatic prostate cancer compared to localized prostate cancer or benign prostatic tissues, suggesting the biological significance of Notch signaling in prostate cancer progression. However, the mechanistic role of Notch signaling and the consequence of its down‐regulation in prostate cancer have not been fully elucidated. Using multiple cellular and molecular approaches such as MTT assay, apoptosis assay, gene transfection, real‐time RT‐PCR, Western blotting, migration, invasion assay and ELISA, we found that down‐regulation of Notch‐1 or Jagged‐1 was mechanistically associated with inhibition of cell growth, migration, invasion and induction of apoptosis in prostate cancer cells, which was mediated via inactivation of Akt, mTOR, and NF‐κB signaling. Consistent with these results, we found that the down‐regulation of Notch‐1 or Jagged‐1 led to decreased expression and the activity of NF‐κB downstream genes such as MMP‐9, VEGF, and uPA, contributing to the inhibition of cell migration and invasion. Taken together, we conclude that the down‐regulation of Notch‐1 or Jagged‐1 mediated inhibition of cell growth, migration and invasion, and the induction of apoptosis was in part due to inactivation of Akt, mTOR, and NF‐κB signaling pathways. Our results further suggest that inactivation of Notch signaling pathways by innovative strategies could be a potential targeted approach for the treatment of metastatic prostate cancer. J. Cell. Biochem. 109: 726–736, 2010. © 2010 Wiley‐Liss, Inc.     

Long non-coding RNA PAARH promotes hepatocellular carcinoma progression and angiogenesis via upregulating HOTTIP and activating HIF-1α/VEGF signaling

Hepatocellular carcinoma (HCC) is one of the leading lethal malignancies and a hypervascular tumor. Although some long non-coding RNAs (lncRNAs) have been revealed to be involved in HCC. The contributions of lncRNAs to HCC progression and angiogenesis are still largely unknown. In this study, we identified a HCC-related lncRNA, CMB9-22P13.1, which was highly expressed and correlated with advanced stage, vascular invasion, and poor survival in HCC. We named this lncRNA Progression and Angiogenesis Associated RNA in HCC (PAARH). Gain- and loss-of function assays revealed that PAARH facilitated HCC cellular growth, migration, and invasion, repressed HCC cellular apoptosis, and promoted HCC tumor growth and angiogenesis in vivo. PAARH functioned as a competing endogenous RNA to upregulate HOTTIP via sponging miR-6760-5p, miR-6512-3p, miR-1298-5p, miR-6720-5p, miR-4516, and miR-6782-5p. The expression of PAARH was significantly positively associated with HOTTIP in HCC tissues. Functional rescue assays verified that HOTTIP was a critical mediator of the roles of PAARH in modulating HCC cellular growth, apoptosis, migration, and invasion. Furthermore, PAARH was found to physically bind hypoxia inducible factor-1 subunit alpha (HIF-1α), facilitate the recruitment of HIF-1α to VEGF promoter, and activate VEGF expression under hypoxia, which was responsible for the roles of PAARH in promoting angiogenesis. The expression of PAARH was positively associated with VEGF expression and microvessel density in HCC tissues. In conclusion, these findings demonstrated that PAARH promoted HCC progression and angiogenesis via upregulating HOTTIP and activating HIF-1α/VEGF signaling. PAARH represents a potential prognostic biomarker and therapeutic target for HCC.Subject terms: Cancer microenvironment, Oncogenes, Translational research     

A Feedback Inhibition between miRNA-127 and TGFβ/c-Jun Cascade in HCC Cell Migration via MMP13

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is increasing in frequency in the U.S. The major reason for the low postoperative survival rate of HCC is widespread intrahepatic metastasis or invasion, and activation of TGFβ signaling is associated with the invasive phenotype. This study aims at determining the novel function of miR-127 in modulating HCC migration. Overexpression of miR-127 inhibits HCC cell migration, invasion and tumor growth in nude mice. MiR-127 directly represses matrix metalloproteinase 13 (MMP13) 3′UTR activity and protein expression, and diminishes MMP13/TGFβ-induced HCC migration. In turn, TGFβ decreases miR-127 expression by enhancing c-Jun-mediated inhibition of miR-127 promoter activity. In contrast, p53 transactivates miR-127 promoter and induces miR-127 expression, which is antagonized by c-Jun. The inhibition of miR-127 by c-Jun is through TGFβ-mediated ERK and JNK pathways. The lower miR-127 expression shows a negative correlation with the higher MMP13 expression in a subset of human HCC specimens. This is the first report elucidating a feedback regulation between miR-127 and the TGFβ/c-Jun cascade in HCC migration via MMP13 that involves a crosstalk between the oncogene c-Jun and tumor suppressor p53.     

Low level of low-density lipoprotein receptor-related protein 1 predicts an unfavorable prognosis of hepatocellular carcinoma after curative resection

Background

Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor involved in receptor-mediated endocytosis and cell signaling. The aim of this study was to elucidate the expression and mechanism of LRP1 in hepatocellular carcinoma (HCC).

Methods

LRP1 expression in 4 HCC cell lines and 40 HCC samples was detected. After interruption of LRP1 expression in a HCC cell line either with specific lentiviral-mediated shRNA LRP1 or in the presence of the LRP1-specific chaperone, receptor-associated protein (RAP), the role of LRP1 in the migration and invasion of HCC cells was assessed in vivo and in vitro, and the expression of matrix metalloproteinase (MMP) 9 in cells and the bioactivity of MMP9 in the supernatant were assayed. The expression and prognostic value of LRP1 were investigated in 327 HCC specimens.

Results

Low LRP1 expression was associated with poor HCC prognosis, with low expression independently related to shortened overall survival and increased tumor recurrence rate. Expression of LRP1 in non-recurrent HCC samples was significantly higher than that in early recurrent samples. LRP1 expression in HCC cell lines was inversely correlated with their metastatic potential. After inhibition of LRP1, low-metastatic SMCC-7721 cells showed enhanced migration and invasion and increased expression and bioactivity of MMP9. Correlation analysis showed a negative correlation between LRP1 and MMP9 expression in HCC patients. The prognostic value of LRP1 expression was validated in the independent data set.

Conclusions

LRP1 modulated the level of MMP9 and low level of LRP1 expression was associated with aggressiveness and invasiveness in HCCs. LRP1 offered a possible strategy for tumor molecular therapy.     

Oridonin effectively reverses the drug resistance of cisplatin involving induction of cell apoptosis and inhibition of MMP expression in human acute myeloid leukemia cells

Cisplatin is the first generation platinum-based chemotherapy agent. However, the extensive application of cisplatin inevitably causes drug resistance, which is a major obstacle to cancer chemotherapy. Oridonin is a diterpenoid isolated from Rabdosia rubescens with potent anticancer activity. The aim of our study is to investigate the role of oridonin to reverse the cisplatin-resistance in human acute myeloid leukemia (AML) cells. The effect of oridonin on human AML cell proliferation was evaluated by MTT assay, cell migration and invasion were evaluated by transwell migration and invasion assays in cisplatin-resistant human AML cells. Furthermore, cell apoptosis was examined by flow cytometry. The inhibitive effect of oridonin in vivo was determined using xenografted nude mice. In addition, the expressions of MMP2 and MMP9 were detected by Western blot. There was a synergistic antitumor effect between cisplatin and oridonin on cisplatin-resistant human AML cells in vitro and in vivo. In addition, the combination of cisplatin and oridonin synergistically induced cell apoptosis. Furthermore, the combination treatment not only inhibited AML cell migration and invasion, but more significantly, decreased the expressions of MMP2 and MMP9 proteins. Our results suggest that the synergistic effect between both agents is likely to be driven by the inhibition of MMP expression and the resulting increased apoptosis.     

Overexpression of Ras Homologous C (RhoC) Induces Malignant Transformation of Hepatocytes In Vitro and in Nude Mouse Xenografts

Ras homologous C (RhoC) is expressed in various cancers, including hepatocellular carcinoma (HCC). In this study, we first analyzed RhoC expression in 46 HCC tissue specimens and found that RhoC expression was significantly increased in HCC tissues compared to the adjacent normal liver tissues. Next, we investigated the role of RhoC in malignant transformation of normal hepatocytes. The HL7702 cell line was stably transfected with a RhoC expression vector and then subjected to cell proliferation, differentiation, colony formation, migration and invasion assays, as well as nude mouse xenograft assays. Gene expressions in these cells were determined using RT-PCR and Western blot. Overexpression of RhoC significantly promoted proliferation and anchorage-independent growth of HL7702 cells, but suppressed cell differentiation, as compared with the parental cells and the empty vector-transfected control cells. Moreover, RhoC overexpression induced migration and invasion of HL7702 cells in vitro. Molecularly, RhoC increased the expression of cell cycle-related genes, matrix metalloprotease 2 (MMP2), MMP9 and vascular endothelial growth factor (VEGF). In addition, RhoC-transfected cells formed tumors in nude mice, whereas vector-transfected HL7702 cells did not form any tumors in nude mice. This study demonstrated the role of RhoC overexpression in malignant transformation of normal human hepatocytes, suggesting that RhoC may function as an oncogene in hepatocytes.     

miR-150-5p Inhibits Hepatoma Cell Migration and Invasion by Targeting MMP14

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. Despite progress in diagnostics and treatment of HCC, its prognosis remains poor because the molecular mechanisms underlying hepatocarcinogenesis are not well understood. In the study, we focused on identifying the role of miRNAs in HCC progression. miRNA microarray was used to analyze the differentially expressed miRNAs, and the results were validated by qPCR. We found that the miR-150-5p expression is down-regulated in HCC tissues compared with pair non-tumor tissues. miR-150-5p expression is also decreased in metastatic cancer tissues compared with pair primary tissues, indicating that miR-150-5p may be involved in HCC metastasis. Functionally, miR-150-5p inhibition significantly promotes hepatoma cell migration and invasion, whereas miR-150-5p overexpression suppresses cancer cell migration and invasion in vitro. The matrix metalloproteinase 14 (MMP14) is identified as a new target gene of miR-150-5p. miR-150-5p markedly inhibits MMP14 expression in hepatoma cells, and miR-150-5p expression is negative correlation with MMP14 expression in vivo. More important, re-expression of MMP14 in hepatoma cells partially reverses the effect of miR-150-5p in inhibiting cell invasion.     

microRNA-489 Plays an Anti-Metastatic Role in Human Hepatocellular Carcinoma by Targeting Matrix Metalloproteinase-7

Dysregulation of microRNAs (miRNAs) is actively involved in the pathogenesis and tumorigenicity of hepatocellular carcinoma (HCC). miR-489 was found to play either oncogenic or tumor suppressive roles in human cancers. Recent study reported that the levels of miR-489 in late recurrent HCC patients were evidently higher than that in early recurrent cases, suggesting that miR-489 may function as a tumor suppressive miRNA in HCC. Yet, the clinical value and biological function of miR-489 remain rarely known in HCC. Here, we presented that miR-489 level in HCC tissues was notably reduced compared to matched non-cancerous specimens. Its decreased level was evidently correlated with adverse clinical parameters and poor prognosis of HCC patients. Accordingly, the levels of miR-489 were obviously down-regulated in HCC cells. Ectopic expression of miR-489 in HCCLM3 and MHCC97H cells prominently inhibits the migration and invasion of tumor cells and reduced lung metastases in vivo, while miR-489 knockdown increased these behaviors of HepG2 and MHCC97L cells. Mechanically, miR-489 negatively regulated matrix metalloproteinase-7 (MMP7) abundance in HCC cells. Herein, MMP7 was found to be a downstream molecule of miR-489 in HCC. An inversely correlation between miR-489 and MMP7 was confirmed in HCC specimens. MMP7 knockdown prohibited cell migration and invasion while MMP7 overexpression showed opposite effects on HCC cells. Furthermore, restoration of MMP7 expression could abrogate the anti-metastatic effects of miR-489 on HCCLM3 cells with enhanced cell migration and invasion. Altogether, miR-489 potentially acts as a prognostic predictor and a drug-target for HCC patients.     

RGD肽对肺癌A549 细胞增殖侵袭能力的影响及其机制研究

目的:研究RGD肽对肺癌A549细胞增殖凋亡及侵袭迁移的影响,并探讨其作用机制。方法:不同浓度RGD肽处理肺癌A549细胞后,MTT检测肺癌细胞的增殖能力,流式细胞仪检测肺癌细胞凋亡及周期分布,Transwell检测其迁移及侵袭能力的变化,Western blot检测RGD肽对肺癌A549细胞MMP2、MMP9的表达水平影响。结果:当RGD肽浓度增加至50 mg/L时,肺癌A549细胞增殖明显受到抑制,且这种抑制作用呈剂量依赖关系;RGD肽组A549细胞G0/G1期细胞比例增高,细胞凋亡率由(6.1±0.1)%增至(15.2±0.5)%;在迁移和侵袭试验中,RGD肽组A549细胞的穿膜细胞数分别由123±10和43±10降至45±5和18±5;RGD肽组A549细胞MMP2、MMP9表达水平显著降低。结论:RGD肽对肺癌A549细胞的增殖有明显抑制作用,并促进其凋亡,可能与RGD肽改变其周期分布有关,RGD肽可明显抑制A549细胞的迁移及侵袭,可能与其下调MMP2、MMP9的表达相关。     

Raf kinase inhibitor protein mediated signaling inhibits invasion and metastasis of hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is the most common type of liver cancer with high mortality and poor prognosis. Mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways have been implicated in promoting tumor cell proliferation and invasion of HCC cells.MethodsAs a potential inhibitor of tumor metastasis, the role of Raf kinase inhibitor protein (RKIP) in HCC development and the functional relevance with MAPK and NF-κB signaling pathways were investigated. The levels of RKIP expression were examined in human HCC tissues and correlated with tumor stages and metastatic status. Function of RKIP in cellular proliferation, migration, invasion and apoptosis was investigated in HCC cell lines by either overexpressing or knocking down RKIP expression. Mouse xenograft model was established to assess the effect of RKIP expression on tumor growth.ResultsOur results demonstrated decreased RKIP expression in HCC tissues and a strong correlation with tumor grade and distant metastasis. Manipulation of RKIP expression in HCCLM3 and HepG2 cells indicated that RKIP functioned to inhibit HCC cell motility and invasiveness, and contributed to tumor growth inhibition in vivo. Mechanistic studies showed that the function of RKIP was mediated through MAPK and NF-κB signaling pathways. However, cell type-dependent RKIP regulation on these two pathways was also suggested, indicating the complex nature of signaling network.ConclusionOur study provides a better understanding on the molecular mechanisms of HCC metastasis and sets the foundation for the development of targeted therapeutics for HCC.     

Inactivation of uPA and its receptor uPAR by 3,3′‐diindolylmethane (DIM) leads to the inhibition of prostate cancer cell growth and migration

3,3′‐Diindolylmethane (DIM) has been studied for its putative anti‐cancer properties, especially against prostate cancer; however, its exact mechanism of action remains unclear. We recently provided preliminary data suggesting down‐regulation of uPA during B‐DIM (a clinically active DIM)‐induced inhibition of invasion and angiogenesis in prostate cancer cells. Since the expression and activation of uPA plays important role in tumorigenicity, and high endogenous levels of uPA and uPAR are found in advanced metastatic cancers, we investigated their role in B‐DIM‐mediated inhibition of prostate cancer cell growth and motility. Using PC3 cells, we found that B‐DIM treatment as well as the silencing of uPA and uPAR by siRNAs led to the inhibition of cell growth and motility. Conversely, over‐expression of uPA/uPAR in LNCaP and C4‐2B cells resulted in increased cell growth and motility, which was effectively inhibited by B‐DIM. Moreover, we found that uPA as well as uPAR induced the production of VEGF and MMP‐9, and that the down‐regulation of uPA/uPAR by siRNAs or B‐DIM treatment resulted in the inhibition of VEGF and MMP‐9 secretion which could be responsible for the observed inhibition of cell migration. Interestingly, silencing of uPA/uPAR led to decreased sensitivity to B‐DIM indicating important role of uPA/uPAR in B‐DIM‐mediated regulation of prostate cancer cell growth and migration. Our data suggest that chemopreventive and/or therapeutic activity of B‐DIM is in part due to down‐regulation of uPA–uPAR leading to reduced production of VEGF/MMP‐9 which ultimately leads to the inhibition of cell growth and migration of aggressive prostate cancer cells. J. Cell. Biochem. 107: 516–527, 2009. © 2009 Wiley‐Liss, Inc.     

c‐Cbl regulates glioma invasion through matrix metalloproteinase 2

c‐Cbl, a multifunctional adaptor and an E3 ubiquitin ligase, plays a role in such cytoskeleton‐mediated events as cell adhesion and migration. Invasiveness of human glioma is dependent on cell adhesion, migration, and degradation of extracellular matrix (ECM). However, the function of c‐Cbl in glioma invasion has never been investigated. We report here, for the first time, that c‐Cbl plays a positive role in the invasion of ECM by SNB19 glioma cells. RNAi‐mediated depletion of c‐Cbl decreases SNB19 cell invasion and expression of matrix metalloproteinase 2 (MMP2). Consistent with these findings, SNB19 cells expressing wild‐type, but not mutant c‐Cbl show increased invasion and MMP2 expression. We demonstrate that the observed role of c‐Cbl in invasion of SNB19 cells is not mediated by the previously shown effects of c‐Cbl on cell adhesion and migration or on EGFR signaling. Together, our results suggest that c‐Cbl promotes glioma invasion through up‐regulation of MMP2. J. Cell. Biochem. 111: 1169–1178, 2010. © 2010 Wiley‐Liss, Inc.