Green tea catechins reduce invasive potential of human melanoma cells by targeting COX-2, PGE2 receptors and epithelial-to-mesenchymal transition

Melanoma is the most serious type of skin disease and a leading cause of death from skin disease due to its highly metastatic ability. To develop more effective chemopreventive agents for the prevention of melanoma, we have determined the effect of green tea catechins on the invasive potential of human melanoma cells and the molecular mechanisms underlying these effects using A375 (BRAF-mutated) and Hs294t (Non-BRAF-mutated) melanoma cell lines as an in vitro model. Employing cell invasion assays, we found that the inhibitory effects of green tea catechins on the cell migration were in the order of (-)-epigallocatechin-3-gallate (EGCG)>(-)-epigallocatechin>(-)-epicatechin-3-gallate>(-)-gallocatechin>(-)-epicatechin. Treatment of A375 and Hs294t cells with EGCG resulted in a dose-dependent inhibition of cell migration or invasion of these cells, which was associated with a reduction in the levels of cyclooxygenase (COX)-2, prostaglandin (PG) E(2) and PGE(2) receptors (EP2 and EP4). Treatment of cells with celecoxib, a COX-2 inhibitor, also inhibited melanoma cell migration. EGCG inhibits 12-O-tetradecanoylphorbol-13-acetate-, an inducer of COX-2, and PGE(2)-induced cell migration of cells. EGCG decreased EP2 agonist (butaprost)- and EP4 agonist (Cay10580)-induced cell migration ability. Moreover, EGCG inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in A375 melanoma cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited cell migration. Inhibition of melanoma cell migration by EGCG was associated with transition of mesenchymal stage to epithelial stage, which resulted in an increase in the levels of epithelial biomarkers (E-cadherin, cytokeratin and desmoglein 2) and a reduction in the levels of mesenchymal biomarkers (vimentin, fibronectin and N-cadherin) in A375 melanoma cells. Together, these results indicate that EGCG, a major green tea catechin, has the ability to inhibit melanoma cell invasion/migration, an essential step of metastasis, by targeting the endogenous expression of COX-2, PGE(2) receptors and epithelial-to-mesenchymal transition.     

Grape seed proanthocyanidins inhibit the invasiveness of human HNSCC cells by targeting EGFR and reversing the epithelial-to-mesenchymal transition

Head and neck squamous cell carcinoma (HNSCC) is responsible for approximately 20,000 deaths per year in the United States. Most of the deaths are due to the metastases. To develop more effective strategies for the prevention of metastasis of HNSCC cells, we have determined the effect of grape seed proanthocyanidins (GSPs) on the invasive potential of HNSCC cell and the mechanisms underlying these effects using OSC19 cells as an in vitro model. Using cell invasion assays, we established that treatment of the OSC19 cells with GSPs resulted in a dose-dependent inhibition of cell invasion. EGFR is over-expressed in 90% of HNSCCs and the EGFR inhibitors, erlotinib and gefitinib, are being explored as therapies for this disease. We found that GSPs treatment reduced the levels of expression of EGFR in the OSC19 cells as well as reducing the activation of NF-κB/p65, a downstream target of EGFR, and the expression of NF-κB-responsive proteins. GSPs treatment also reduced the activity of ERK1/2, an upstream regulator of NF-κB and treatment of the cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, inhibited cell invasion. Overexpression of EGFR and high NF-κB activity play a key role in the epithelial-to-mesenchymal transition, which is of critical importance in the processes underlying metastasis, and we found treatment with GSPs enhanced the levels of epithelial (E-cadherin, cytokeratins and desmoglein-2) and reduced the levels of mesenchymal (vimentin, fibronectin, N-cadherin and Slug) biomarkers in the OSC19 cells. These results indicate that GSPs have the ability to inhibit HNSCC cell invasion, and do so by targeting the expression of EGFR and activation of NF-κB as well as inhibiting the epithelial-to-mesenchymal transition.     

Discovery of lesser known flavones as inhibitors of NF-κB signaling in MDA-MB-231 breast cancer cells—A SAR study

Seventeen flavonoids with different substitutions were evaluated for inhibition of nuclear factor-κB (NF-κB) signaling in the invasive breast cancer cell line MDA-MB-231. They were screened using an engineered MDA-MB-231 cell line reporting NF-κB activation. The modulation of expression of two NF-κB regulated genes involved in tumorigenesis, matrix metalloproteinase-9 (MMP-9), and cyclooxygenase-2 (COX-2) were also analyzed in these cells. Among the compounds tested, all except gossypetin and quercetagetin inhibited the activation of NF-κB, and the expression of MMP-9 and COX-2 to different degree. Methylated flavone, chrysoeriol (luteolin-3′-methylether), was found to be the most potent inhibitor of MMP-9 and COX-2 expressions. The effect of chrysoeriol on cell proliferation, cell cycle, apoptosis and metastasis was analyzed by established methods. Chrysoeriol caused cell cycle arrest at G2/M and inhibited migration and invasion of MDA-MB-231 cells. The structure–activity relations amongst the flavonoids as NF-κB signaling inhibitors was studied. The study indicates differences between the actions of various flavonoids on NF-κB activation and on the biological activities of breast cancer cells. Flavones in general, were more active than the corresponding flavonols.     

Interleukin-2 enhances the growth of human melanoma cells derived form primary but not from metastatic tumours

Previously, we demonstrated that in vitro treatment of B16F10 murine melanoma cells with interleukin-2 (IL-2) enhances proliferation and metastasis. To further investigate the role played by IL-2 in human melanomas, we studied the expression of IL-2/IL-2 receptor and the effect of IL-2 on the proliferation of melanoma cell lines derived from primary (A375 and RMS cell lines) and metastatic (Hs294T cell line) tumours. We found a constitutive expression of cytoplasmic IL-2 and alpha, beta and gamma-subunits of the IL-2R on the surface of the three melanoma cell lines. The presence of IL-2 in the culture increased the proliferation rate in A375 and RMS cell lines, but no effect was observed in Hs294T metastatic cells. Biologically active IL-2 could be found in the supernatant of the three melanoma cell lines, particularly in A375 and RMS cells, in which an inhibition of the proliferation rate was observed when IL-2 was blocked. Moreover, the combination of anti-IL-2R beta and anti-IL-2R gamma blocking antibodies induced a significant down-regulation of cell proliferation in the three melanoma cell lines, and the combination of anti-IL-2R alpha, anti-IL-2R beta and anti-IL-2R gamma blocking antibodies inhibited IL-2-mediated growth stimulation in A375 and Hs294T cell lines. In RMS cells, a more significant effect was observed when only IL-2R gamma was blocked. Finally, exogenous IL-2 modulated the IL-2 endogenously produced by melanoma cells. These data show that IL-2 may modulate the growth of melanoma cells through autocrine or/and paracrine mechanisms.     

Silymarin targets β-catenin signaling in blocking migration/invasion of human melanoma cells

Metastatic melanoma is a leading cause of death from skin diseases, and is often associated with activation of Wnt/β-catenin signaling pathway. We have examined the inhibitory effect of silymarin, a plant flavanoid from Silybum marianum, on cell migration of metastasis-specific human melanoma cell lines (A375 and Hs294t) and assessed whether Wnt/β-catenin signaling is the target of silymarin. Using an in vitro invasion assay, we found that treatment of human melanoma cell lines with silymarin resulted in concentration-dependent inhibition of cell migration, which was associated with accumulation of cytosolic β-catenin, while reducing the nuclear accumulation of β-catenin (i.e., β-catenin inactivation) and reducing the levels of matrix metalloproteinase (MMP) -2 and MMP-9 which are the down-stream targets of β-catenin. Silymarin enhanced: (i) the levels of casein kinase 1α, glycogen synthase kinase-3β and phosphorylated-β-catenin on critical residues Ser(45), Ser(33/37) and Thr(41), and (ii) the binding of β-transducin repeat-containing proteins (β-TrCP) with phospho forms of β-catenin in melanoma cells. These events play important roles in degradation or inactivation of β-catenin. To verify whether β-catenin is a potent molecular target of silymarin, the effect of silymarin was determined on β-catenin-activated (Mel 1241) and β-catenin-inactivated (Mel 1011) melanoma cells. Treatment of Mel 1241 cells with silymarin or FH535, an inhibitor of Wnt/β-catenin pathway, significantly inhibited cell migration of Mel 1241 cells, which was associated with the elevated levels of casein kinase 1α and glycogen synthase kinase-3β, and decreased accumulation of nuclear β-catenin and inhibition of MMP-2 and MMP-9 levels. However, this effect of silymarin and FH535 was not found in Mel 1011 melanoma cells. These results indicate for the first time that silymarin inhibits melanoma cell migration by targeting β-catenin signaling pathway.     

S100A8 and S100A9 promotes invasion and migration through p38 mitogen-activated protein kinase-dependent NF-κB activation in gastric cancer cells

S100A8 and S100A9 (S100A8/A9) are low-molecular weight members of the S100 family of calcium-binding proteins. Recent studies have reported S100A8/A9 promote tumorigenesis. We have previously reported that S100A8/A9 is mostly expressed in stromal cells and inflammatory cells between gastric tumor cells. However, the role of environmental S100A8/A9 in gastric cancer has not been defined. We observed in the present study the effect of S100A8/A9 on migration and invasion of gastric cancer cells. S100A8/ A9 treatment increased migration and invasionat lower concentrations that did not affect cell proliferation and cell viability. S100A8/A9 caused activation of p38 mitogenactivated protein kinase (MAPK) and nuclear factor-κB (NF-κB). The phosphorylation of p38 MAPK was not affected by the NF-κB inhibitor Bay whereas activation of NF-κB was blocked by p38 MAPK inhibitor SB203580, indicating that S100A8/A9-induced NF-κB activation is mediated by phosphorylation of p38 MAPK. S100A8/A9-induced cell migration and invasion was inhibited by SB203580 and Bay, suggesting that activation of p38 MAPK and NF-κB is involved in the S100A8/A9 induced cell migration and invasion. S100A8/A9 caused an increase in matrix metalloproteinase 2 (MMP2) and MMP12 expression, which were inhibited by SB203580 and Bay. S100A8/A9-induced cell migration and invasion was inhibited by MMP2 siRNA and MMP12 siRNA, indicating that MMP2 and MMP12 is related to the S100A8/A9 induced cell migration and invasion. Taken together, these results suggest that S100A8/A9 promotes cell migration and invasion through p38 MAPKdependent NF-κB activation leading to an increase of MMP2 and MMP12 in gastric cancer.     

Antitumor effects of the flavone chalcone: inhibition of invasion and migration through the FAK/JNK signaling pathway in human gastric adenocarcinoma AGS cells

Chalcones (benzylideneacetophenone) are cancer-preventive food components found in a human diet rich in fruits and vegetables. In this study, we first report the chemopreventive effect of chalcone in human gastric adenocarcinoma cell lines: AGS. The results showed that chalcone could inhibit the abilities of the adhesion, invasion, and migration by cell–matrix adhesion assay, Boyden chamber invasion/migration assay, and wound-healing assay. Molecular data showed that the effect of chalcone in AGS cells might be mediated via sustained inactivation of the phosphorylation of focal adhesion kinase (FAK) and c-Jun N-terminal kinase 1 and 2 (JNK1/2) signal involved in the downregulation of the expressions of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Next, chalcone-treated AGS cells showed tremendous decrease in the phosphorylation and degradation of inhibitor of kappaBα (IκBα), the nuclear level of NF-κB, and the binding ability of NF-κB to NF-κB response element. Furthermore, treating FAK small interfering RNA (FAK siRNA) and specific inhibitor for JNK (SP600125) to AGS cells could reduce the phosphorylation of JNK1/2 and the activity of MMP-2 and MMP-9. Our results revealed that chalcone significantly inhibited the metastatic ability of AGS cells by reducing MMP-2 and MMP-9 expressions concomitantly with a marked reduction on cell invasion and migration through suppressing and JNK signaling pathways. We suggest that chalcone may offer the application in clinical medicine.     

MHY884, a newly synthesized tyrosinase inhibitor,suppresses UVB-induced activation of NF-κB signaling pathway through the downregulation of oxidative stress

The skin is the primary target of prolonged and repeated ultraviolet (UVB) irradiation which induces cutaneous inflammation and pigmentation. Nuclear factor κB (NF-κB) is the major factor mediating UVB-induced inflammatory responses through the expression of various proinflammatory proteins such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). We have previously reported that the synthetic novel compound 4-(5-chloro-2,3-dihydrobenzo[d]thiazol-2-yl)-2,6-dimethoxyphenol (MHY884) strongly suppressed tyrosinase activity and melanin synthesis in B16F10 melanoma cells. In the present study, we investigated the effect of MHY884 on the inhibition of UVB-induced NF-κB activation and its proinflammatory downstream proteins through the suppression of oxidative stress in an in vivo model of photoaging. Generation of reactive oxygen species (ROS) and peroxynitrite was measured in vitro and in B16F10 melanoma cells to verify the scavenging activity of MHY884. MHY884 suppressed oxidative stress both in vitro and in the melanoma cells in a dose-dependent manner. Next, melanin-possessing hairless mice were pre-treated with MHY884 and then irradiated with UVB repeatedly. Topical application of MHY884 attenuated UVB-induced oxidative stress, resulting in reduced NF-κB activity. Pre-treatment with MHY884 inhibited Akt and IκB kinase α/β signaling pathways, leading to decreased translocation and phosphorylation of p65, a subunit of NF-κB. This result correlated with the expression levels of iNOS and COX-2 in the skin of MHY884-treated mice. Thus, the novel tyrosinase inhibitor MHY884 suppressed NF-κB activation signaling pathway by scavenging UVB-induced oxidative stress. The discovery of MHY884, a novel tyrosinase inhibitor that targets NF-κB signaling, is significant, because this compound is a promising protective agent against UVB-induced skin damage.     

Anti-cancer effects of celecoxib on nasopharyngeal carcinoma HNE-1 cells expressing COX-2 oncoprotein

Celecoxib is a selective cyclooxygenase-2 (COX-2) inhibitor with antitumor and antiangiogenic activities. To investigate the effects of celecoxib on nasopharyngeal carcinoma (NPC), HNE-1 cells were treated with celecoxib at various concentrations. MTT assay, migration assay and invasion assay were performed to observe the inhibitory activity of celecoxib on HNE-1 cells. Additionally, VEGF-A expression and radiation survival of NPC cell were also examined after treatment with celecoxib. Celecoxib treatment presented an anti-proliferation function in a time and dose-dependent manner on HNE-1 cells which highly express COX-2 protein. Celecoxib also displayed an obvious inhibitory activity on invasive capacity of NPC cells. Moreover, the celecoxib’s effects to suppress VEGF-A expression and enhance radiosensitivity were detected in HNE-1 cells. These findings implicate that application of celecoxib may be an effective strategy for NPC therapy.     

miR-145-5p inhibits tumor occurrence and metastasis through the NF-κB signaling pathway by targeting TLR4 in malignant melanoma

Compelling evidence shows that deregulated microRNAs (miRNAs) are important regulators in the progression of melanoma. miR-145-5p has been suggested to exhibit antitumorigenic activity in melanoma. However, the molecular mechanism underlying the biological activity of miR-145-5p in melanoma remains to be further understood. Herein, quantitative real-time polymerase chain reaction was used to examine the miR-145-5p expression in malignant melanoma tissues and cells. The interaction between miR-145-5p and toll-like receptor 4 (TLR4) was explored by bioinformatics analyses, luciferase reporter assay, and Western blot. The effects of miR-145-5p or combined with TLR4 on cell proliferation, colony formation, migration, and invasion abilities were investigated by (4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, colony formation, wound healing, and transwell assays, respectively. The melanoma xenograft tumor models were established to determine the biological activity of miR-145-5p in melanoma in vivo. In addition, the changes of the nuclear factor kappa B (NF-κB) pathway were analyzed by detecting the NF-κB activity and the NF-κB p65 protein level. We observed that the miR-145-5p expression was underexpressed in melanoma tissues and cells. miR-145-5p suppressed the TLR4 expression by binding to its 3′untranslated region in melanoma cells. Moreover, TLR4 overexpression abolished the inhibition of cell proliferation, colony formation, migration, and invasion abilities induced by miR-145-5p in melanoma cells. Meanwhile, miR-145-5p was confirmed to restrain melanoma tumor growth in vivo by targeting TLR4. Furthermore, miR-145-5p overexpression inactivated the NF-κB pathway in melanoma in vitro and in vivo, which was reversed by TLR4 overexpression. We concluded that miR-145-5p hindered the occurrence and metastasis of melanoma cells in vitro and in vivo by targeting TLR4 via inactivation of the NF-κB pathway.     

A novel derivative of decursin, CSL-32, blocks migration and production of inflammatory mediators and modulates PI3K and NF-κB activities in HT1080 cells

Decursin and related coumarin compounds in herbal extracts have a number of biological activities against inflammation, angiogenesis and cancer. We have analysed a derivative of decursin (CSL-32) for activity against inflammatory activation of cancer cells, such as migration, invasion and expression of pro-inflammatory mediators. The human fibrosarcoma cell line, HT1080, was treated with TNFα (tumour necrosis factor α) in the presence or absence of CSL-32. The cellular responses and modification of signalling adapters were analysed with respect to the production of pro-inflammatory mediators, as also migration, adhesion and invasion. Treatment of HT1080 cells with CSL-32 inhibited their proliferation, without affecting cell viability, and TNFα-induced expression of pro-inflammatory mediators, such as MMP-9 (matrix metalloproteinase-9) and IL-8 (interleukin-8). CSL-32 also suppressed phosphorylation and degradation of IκB (inhibitory κB), phosphorylation of p65 subunit of NF-κB (nuclear factor-κB) and nuclear translocation of NF-κB, which are required for the expression of pro-inflammatory mediators. In addition, CSL-32 inhibited invasion and migration of HT1080 cells, as also cellular adhesion to fibronectin, an ECM (extracellular matrix) protein. CSL-32 treatment resulted in a dose-dependent inhibition of PI3K (phosphoinositide 3-kinase) activity, required for the cellular migration. The analyses show that CSL-32 inhibits processes associated with inflammation, such as the production of pro-inflammatory mediators, as well as adhesion, migration and invasion in HT1080 cells.     

Numbl inhibits glioma cell migration and invasion by suppressing TRAF5-mediated NF-κB activation

The Notch signaling regulator Numblike (Numbl) is expressed in the brain, but little is known regarding its role in the pathophysiology of glial cells. In this paper, we report that Numbl expression was down-regulated in high-grade human glioma tissue samples and glioblastoma cell lines. To investigate the role of Numbl in glioma migration and invasion, we generated human glioma cell lines in which Numbl was either overexpressed or depleted. Overexpression of Numbl suppressed, while elimination of Numbl promoted, the migration and invasion of glioma cells. Numbl inhibited glioma migration and invasion by dampening NF-κB activity. Furthermore, Numbl interacted directly with tumor necrosis factor receptor-associated factor 5 (TRAF5), which signals upstream and is required for the activation of NF-κB, and committed it to proteasomal degradation by promoting K48-linked polyubiquitination of TRAF5. In conclusion, our data suggest that Numbl negative regulates glioma cell migration and invasion by abrogating TRAF5-induced activation of NF-κB.