Glaucine inhibits breast cancer cell migration and invasion by inhibiting MMP-9 gene expression through the suppression of NF-κB activation.

Matrix metalloproteinase-9 (MMP-9) plays a central role in the invasion and metastasis of various types of cancer cells. Here, we demonstrate that glaucine, an alkaloid isolated from the plant Corydalis turtschaninovii tuber (Papaveraceae), can inhibit the migration and invasion of human breast cancer cells. We further show that glaucine significantly blocks phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 expression and activity in a dose-dependent manner. Results from reporter gene and electrophoretic mobility shift assays revealed that glaucine inhibits MMP-9 expression by suppressing activation of the nuclear transcription factor nuclear factor-κB (NF-κB). Moreover, glaucine attenuates PMA-induced IκBα degradation and nuclear translocation of NF-κB. Finally, we also found that glaucine inhibits invasion and MMP-9 expression in the highly metastatic MDA-MB-231 breast cancer cell line. Taken together, our findings indicate that the MMP-9 inhibitory activity of glaucine and its abilities to attenuate IκBα and NF-κB activities may be therapeutically useful as a novel means of controlling breast cancer growth and invasiveness.

The role of sLZIP in transcriptional regulation of c-Jun and involvement in migration and invasion of cervical cancer cells.

BACKGROUND/AIMS: Matrix metalloproteinase-9 (MMP-9) plays an important role in tumor invasion and metastasis through the breakdown of extracellular matrix. The c-Jun protein, a major component of the AP-1 transcription factor, is elevated in various cancers. Small leucine zipper protein (sLZIP) is a member of the leucine zipper transcription factor family. Although sLZIP is known to be involved in cancer cell migration and invasion, its biological roles in cancer development and the cellular target genes are not fully understood. In this study, we investigated the role of sLZIP in c-Jun expression, and their effects on expression of MMP-9 and migration of cervical cancer cells. METHODS AND RESULTS: sLZIP up-regulates transcription of c-Jun by binding directly to the CRE region in the c-Jun promoter. Elevated c-Jun due to sLZIP leads to activation of MMP-9 transcription by interaction with the AP-1 binding site in the MMP-9 promoter. c-Jun siRNA repressed migration and invasion of cervical cancer cells, whereas sLZIP recovered migration and invasion of cells transfected with c-Jun siRNA. Immunohistochemical analysis results revealed a significant correlation between the expressions of sLZIP and MMP-9 in clinical cervical specimens. CONCLUSION: These results indicate that sLZIP plays a role in expression of c-Jun, and migration and invasion of cervical cancer cells via regulation of MMP-9 transcription.

ER-to-lysosome Ca2+ refilling followed by K+ efflux-coupled store-operated Ca2+ entry in inflammasome activation and metabolic inflammation.

We studied lysosomal Ca2+ in inflammasome. Lipopolysaccharide (LPS) + palmitic acid (PA) decreased lysosomal Ca2+ ([Ca2+]Lys) and increased [Ca2+]i through mitochondrial ROS, which was suppressed in Trpm2-KO macrophages. Inflammasome activation and metabolic inflammation in adipose tissue of high-fat diet (HFD)-fed mice were ameliorated by Trpm2 KO. ER→lysosome Ca2+ refilling occurred after lysosomal Ca2+ release whose blockade attenuated LPS + PA-induced inflammasome. Subsequently, store-operated Ca2+entry (SOCE) was activated whose inhibition suppressed inflammasome. SOCE was coupled with K+ efflux whose inhibition reduced ER Ca2+ content ([Ca2+]ER) and impaired [Ca2+]Lys recovery. LPS + PA activated KCa3.1 channel, a Ca2+-activated K+ channel. Inhibitors of KCa3.1 channel or Kcnn4 KO reduced [Ca2+]ER, attenuated increase of [Ca2+]i or inflammasome activation by LPS + PA, and ameliorated HFD-induced inflammasome or metabolic inflammation. Lysosomal Ca2+ release induced delayed JNK and ASC phosphorylation through CAMKII-ASK1. These results suggest a novel role of lysosomal Ca2+ release sustained by ER→lysosome Ca2+ refilling and K+ efflux through KCa3.1 channel in inflammasome activation and metabolic inflammation.

Celastrol inhibits TGF-ß1-induced epithelial-mesenchymal transition by inhibiting Snail and regulating E-cadherin expression.

The epithelial-mesenchymal transition (EMT) is a pivotal event in the invasive and metastatic potentials of cancer progression. Celastrol inhibits the proliferation of a variety of tumor cells including leukemia, glioma, prostate, and breast cancer; however, the possible role of celastrol in the EMT is unclear. We investigated the effect of celastrol on the EMT. Transforming growth factor-beta 1 (TGF-ß1) induced EMT-like morphologic changes and upregulation of Snail expression. The downregulation of E-cadherin expression and upregulation of Snail in Madin-Darby Canine Kidney (MDCK) and A549 cell lines show that TGF-ß1-mediated the EMT in epithelial cells; however, celastrol markedly inhibited TGF-ß1-induced morphologic changes, Snail upregulation, and E-cadherin expression. Migration and invasion assays revealed that celastrol completely inhibited TGF-ß1-mediated cellular migration in both cell lines. These findings indicate that celastrol downregulates Snail expression, thereby inhibiting TGF-ß1-induced EMT in MDCK and A549 cells. Thus, our findings provide new evidence that celastrol suppresses lung cancer invasion and migration by inhibiting TGF-ß1-induced EMT.

A novel NLRP3 inhibitor as a therapeutic agent against monosodium urate-induced gout.

Background: Since NEK7 is critical for NLRP3 inflammasome activation, NEK7 inhibitors could be employed as therapeutic agents against gout, a representative disease caused by NLRP3 inflammasome. Methods: We designed NEK7 inhibitors based on biochemical kinome profiling of 2,7-substituted thieno[3,2-d]pyrimidine derivatives (SLC3031~3035 and SLC3037). Inflammasome activation was assessed by ELISA of IL-1b and immunoblotting of IL-1b maturation after treatment of bone marrow-derived macrophages with LPS+monosodium urate (MSU). NLPR3 binding to NEK7 and oligomerization were examined using immunoprecipitation and Blue Native gel electrophoresis, respectively. In vivo effect was investigated by studying gross and histopathological changes of food pad tissue of MSU-injected mice, together with assays of maturation of IL-1b and ASC speck in the tissue. Results: SLC3037 inhibited inflammasome by MSU and other inflammasome activators through blockade of NLRP3 binding to NEK7 or oligomerization, and subsequent ASC oligomerization/phosphorylation. SLC3037 significantly reduced foot pad thickness and inflammation by MSU, which was superior to the effects of colchicine. SLC3037 significantly reduced content or maturation of IL-1b and ASC speck in the food pad. The number and height of intestinal villi were decreased by colchicine but not by SLC3037. Conclusion: SLC3037, a NLRP3 inhibitor blocking NEK7 binding to NLRP3, could be a novel agent against diseases associated with NLRP3 inflammasome activation such as gout, cardiovascular diseases, metabolic syndrome or neurodegenerative diseases.

Human leucine zipper protein sLZIP induces migration and invasion of cervical cancer cells via expression of matrix metalloproteinase-9.

Extracellular proteolysis mediates tissue homeostasis. In cancer, altered proteolysis leads to abnormal tumor growth, inflammation, tissue invasion, and metastasis. Matrix metalloproteinase-9 (MMP-9) represents one of the most prominent proteinases associated with inflammation and tumorigenesis. The recently identified human transcription factor sLZIP is a member of the leucine zipper transcription factor family. Although sLZIP is known to function in ligand-induced transactivation of the glucocorticoid receptor, its exact functions and target genes are not known. In this study, we investigated the role of sLZIP in MMP-9 expression and its involvement in cervical cancer development. Our results show that sLZIP increased the expression of MMP-9 at both the mRNA and protein levels and the proteolytic activity of MMP-9 in HeLa and SiHa cells. sLZIP also increased the transcriptional activity of MMP-9 by binding directly to the cAMP-responsive element of the MMP-9 promoter region. Involvement of sLZIP in MMP-9 expression was further supported by the fact that ME-180 cells expressing sLZIP siRNA were refractory to MMP-9 expression. Results from wound healing and invasion assays showed that sLZIP enhanced both the migration and invasion of cervical cancer cells. The increased migration and invasion of HeLa and SiHa cells that were induced by sLZIP were abrogated by inhibition of the proteolytic activity of MMP-9. These results indicate that sLZIP plays a critical role in MMP-9 expression and is probably involved in invasion and metastasis of cervical cancer.

N-(4-hydroxyphenyl)retinamide inhibits breast cancer cell invasion through suppressing NF-KB activation and inhibiting matrix metalloproteinase-9 expression.

Synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) has been reported to exhibit anti-invasive and anti-metastatic activities by suppressing the enzymatic activity of matrix metalloproteinase (MMP)-9, but the underlying mechanism remains unclear. Here, we show that 4-HPR blocks the activity of MMP-9 in two ways: by reducing phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 secretion and by suppressing cell invasion through the downregulation of MMP-9 gene transcription in MCF-7 breast cancer cells. 4-HPR inhibits the transcriptional activity of MMP-9 by reducing the DNA-binding activity of NF-κB on the MMP-9 promoter as well as by inhibiting the degradation of IκBα, leading to cytoplasmic accumulation of NF-κB. We also found that 4-HPR inhibits invasion and MMP-9 expression in the highly metastatic breast cancer cell line MDA-MB-231. Thus, 4-HPR might be a potent anti-invasive agent that works by suppressing MMP-9 expression via the NF-κB signaling pathway.

The role of annexin A1 in expression of matrix metalloproteinase-9 and invasion of breast cancer cells.

Matrix metalloproteinase-9 (MMP-9) plays an important role in the invasion and metastasis of cancer cells. However, the regulatory mechanism of MMP-9 expression and its biological effects on breast cancer development remain obscure. In the current study, we examined the potential role of annexin A1 (ANXA1) in regulating migration and invasion in breast cancer cell lines. Both ANXA1 mRNA and protein are expressed in the highly invasive, hormone-insensitive human breast cancer cell lines MDA-MB-231 and SKBr3, but not in the hormone-responsive cell lines MCF-7 and T47D. Downregulation of ANXA1 expression with specific small interfering RNAs (ANXA1 siRNA) in MDA-MB-231 cells resulted in decreased cancer cell migration and invasion. Ablation of ANXA1 expression decreases the expression of MMP-9 at both the mRNA and protein levels and also reduces the proteolytic activity of MMP-9 in MDA-MB-231 cells. Moreover, silencing ANXA1 also decreases the transcriptional activity of MMP-9 by the suppression of nuclear factor kappa-B (NF-κB) activity. Collectively, these results indicate that ANXA1 functions as a positive regulator of MMP-9 expression and invasion of breast cancer cells through specific activation of the NF-κB signaling pathway.

Celastrol inhibits breast cancer cell invasion via suppression of NF-ĸB-mediated matrix metalloproteinase-9 expression.

BACKGROUND/AIMS: Metastasis is one of the main causes of death for patients with malignant tumors. Induction of matrix metalloproteinase (MMP)-9 is particularly important for the invasiveness of various cancer cells. Celastrol, a triterpenoid isolated from the traditional Chinese medicine, is known to inhibit the proliferation of a variety of tumor cells, including leukemia, glioma, prostate and breast cancer cells. In this study, we investigated the effect of celastrol on the migration and invasion of human breast carcinoma cells. RESULTS: We observed that celastrol suppressed phorbol 12-myristate 13-acetate (PMA)-induced invasion and migration of MCF-7 cells. We also found that celastrol inhibited PMA-induced MMP-9 expression at both the mRNA and the protein levels, and the proteolytic activity of MMP-9 in MCF-7 cells. Our results revealed that celastrol inhibited the transcriptional activity of MMP-9 by suppression of the DNA binding activity of NF-κB in the MMP-9 promoter, and inhibited degradation of IκBα and nuclear translocation of NF-κB. CONCLUSION: These results indicate that celastrol inhibits NF-κB-mediated MMP-9 expression, resulting in suppression of breast cancer cell invasion and migration that is induced by PMA. Celastrol is a potential agent for clinical use in preventing the invasion and metastasis of human malignant breast tumors.

TFEB-GDF15 axis protects against obesity and insulin resistance as a lysosomal stress response.

TFEB, a key regulator of lysosomal biogenesis and autophagy, is induced not only by nutritional deficiency but also by organelle stress. Here, we find that Tfeb and its downstream genes are upregulated together with lipofuscin accumulation in adipose tissue macrophages (ATMs) of obese mice or humans, suggestive of obesity-associated lysosomal dysfunction/stress in ATMs. Macrophage-specific TFEB-overexpressing mice display complete abrogation of diet-induced obesity, adipose tissue inflammation and insulin resistance, which is independent of autophagy, but dependent on TFEB-induced GDF15 expression. Palmitic acid induces Gdf15 expression through lysosomal Ca2+-mediated TFEB nuclear translocation in response to lysosomal stress. In contrast, mice fed a high-fat diet with macrophage-specific Tfeb deletion show aggravated adipose tissue inflammation and insulin resistance, accompanied by reduced GDF15 level. Finally, we observe activation of TFEB-GDF15 in ATMs of obese humans as a consequence of lysosomal stress. These findings highlight the importance of the TFEB-GDF15 axis as a lysosomal stress response in obesity or metabolic syndrome and as a promising therapeutic target for treatment of these conditions.

Human LZIP induces monocyte CC chemokine receptor 2 expression leading to enhancement of monocyte chemoattractant protein 1/CCL2-induced cell migration.

Chemokines and chemokine receptors play a role in migration of circulating leukocytes to the region of inflammation. Human LZIP is an uncharacterized transcription factor and is known to participate in leukotactin (Lkn)-1/CCL15-induced cell migration. We investigated the role of human LZIP in expression of CC chemokine receptors (CCRs) and its involvement in monocyte migration. RNase protection analysis showed that LZIP increased mRNA expression of CCR2 and CCR1 in THP-1 cells. Surface expressions of both CCR2 and CCR1 were also increased by LZIP. Results from an electrophoretic mobility shift assay showed that LZIP binds to the C/EBP element in the CCR2 promoter. LZIP also enhanced the chemotactic activities of monocyte chemoattractant protein-1/CCL2 and Lkn-1. These results suggest that LZIP regulates expression of chemokine receptors that are involved in monocyte migration.

CoCl2 induces PC12 cells apoptosis through p53 stability and regulating UNC5B.

The receptor uncoordinated 5B (UNC5B) induces apoptosis in the absence of its cognate ligand netrin-1. However, the role of UNC5B in hypoxia-induced apoptosis is not known. Here, we have demonstrated the biological functions of UNC5B in hypoxia-induced apoptosis and related regulatory pathways and examined the effects of UNC5B on p53-dependent apoptosis in PC12 cells under hypoxic conditions. First, we characterized p53-dependent PC12 cell death induced by CoCl2. Our data showed that CoCl2 increased p53 stabilization and transcriptional activity. The downregulation of p53 expression with specific small interfering RNA (p53 siRNA) in CoCl2-treated PC12 cells caused reduction in apoptosis, UNC5B expression, and p21 expression. Moreover, in PC12 cells, ectopic expression of UNC5B significantly enhanced apoptosis, while silencing of UNC5B with siRNA significantly inhibited apoptosis. In addition, netrin-1 significantly inhibited CoCl2-induced p53 stability and UNC5B expression and CoCl2-induced caspase-3 activity and cell death. Collectively, these results demonstrate a novel role for p53 in the control of CoCl2-induced apoptosis through the regulation of UNC5B.

Inhibitory effect of Trolox on the migration and invasion of human lung and cervical cancer cells.

The antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) is implicated in migration and invasion of metastatic tumors. However, the molecular mechanism underlying the effect of Trolox on metastatic cancer cells is not known. We found that a non-cytotoxic dose of Trolox decreased phorbol 12-myristate 13-acetate (PMA)-induced invasion and migration of both A549 and HeLa cancer cells. We also found that Trolox suppressed both the expression and the proteolytic activity of matrix metalloproteinase-9 (MMP-9), and that the promoter activity of PMA-induced MMP-9 was inhibited by Trolox. Our results show that Trolox inhibits the transcriptional activity of MMP-9 by suppression of NF-κB transactivation. These results indicate that Trolox inhibits NF-κB-mediated MMP-9 expression, leading to the suppression of migration and invasion in lung and cervical cancer cells. Trolox is a potential agent for clinical use in preventing the invasion and metastasis of human malignant lung and cervical cancers.

A novel isoform of human LZIP negatively regulates the transactivation of the glucocorticoid receptor.

The human leucine zipper protein (LZIP) is a basic leucine zipper transcription factor that is involved in leukocyte migration, tumor suppression, and endoplasmic reticulum stress-associated protein degradation. Although evidence suggests a diversity of roles for LZIP, its function is not fully understood, and the subcellular localization of LZIP is still controversial. We identified a novel isoform of LZIP and characterized its function in ligand-induced transactivation of the glucocorticoid receptor (GR) in COS-7 and HeLa cells. A novel isoform of human LZIP designated as "sLZIP" contains a deleted putative transmembrane domain (amino acids 229-245) of LZIP and consists of 345 amino acids. LZIP and sLZIP were ubiquitously expressed in a variety of cell lines and tissues, with LZIP being much more common. sLZIP was mainly localized in the nucleus, whereas LZIP was located in the cytoplasm. Unlike LZIP, sLZIP was not involved in the chemokine-mediated signal pathway. sLZIP recruited histone deacetylases (HDACs) to the promoter region of the mouse mammary tumor virus luciferase reporter gene and enhanced the activities of HDACs, resulting in suppression of expression of the GR target genes. Our findings suggest that sLZIP functions as a negative regulator in glucocorticoid-induced transcriptional activation of GR by recruitment and activation of HDACs.