Rubus idaeus extract suppresses migration and invasion of human oral cancer by inhibiting MMP‐2 through modulation of the Erk1/2 signaling pathway

Raspberries (Rubus idaeus L.) have been extensively studies worldwide because of their beneficial effects on health. Recently reports indicate that crude extracts of Rubus idaeus (RIE) have antioxidant and anticancer ability. The aim of this study was to evaluate the mechanism of its antimetastatic ability in oral cancer cells. In this study, SCC‐9 and SAS oral cancer cells were subjected to a treatment with RIE and then analyzed the effect of RIE on migration and invasion. The addition of RIE inhibited the migration and invasion ability of oral cancer cells. Real time PCR, western blot and zymography analysis demonstrated that mRNA, protein expression and enzyme activity of matrix metalloproteinases‐2 (MMP‐2) were down‐regulated by RIE. Moreover, the phosphorylation of Focal adhesion kinase (FAK), src, and extracellular signal‐regulated kinase (ERK) were inhibited after RIE treatment. In conclusion, these results demonstrated that RIE exerted an inhibitory effect of migration and invasion in oral cancer cells and alter metastasis by suppression of MMP‐2 expression through FAK/Scr/ERK signaling pathway. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1037–1046, 2017.     

Antimetastatic potentials of salvianolic acid A on oral squamous cell carcinoma by targeting MMP‐2 and the c‐Raf/MEK/ERK pathway

The metastasis of oral squamous cell carcinoma (OSCC) is one of the most important causes of cancer‐related deaths. Thus, various therapeutic strategies have been developed to prevent the metastasis of OSCC. Salvianolic acid A (SAA), a traditional Chinese medicine, has antithrombosis, antiplatelet, anti‐inflammation, and antitumor activities. Here, we provide molecular evidence indicating that SAA exerts its antimetastatic effects by markedly inhibiting the invasion and migration of oral squamous SCC‐9 and SCC‐25 cells. SCC‐9 and SCC‐25 cells were treated with various concentrations of SAA to further investigate the precise involvement of SAA in cancer metastasis. The results of zymography, and Western blotting indicated that SAA treatment may decrease matrix metallopoteinase‐2 (MMP‐2) expression. SAA also inhibited p‐c‐Raf, p‐MEK1/2, and p‐ERK1/2 protein expression. In addition, treating SCC‐9 cells with U0126, a MEK‐specific inhibitor, decreased MMP‐2 expression and concomitantly inhibited cell migration. Our findings suggested that SAA inhibits the invasion and migration of OSCC by inhibiting the c‐Raf/MEK/ERK pathways that control MMP‐2 expression. Our findings provide new insights into the molecular mechanisms that underlie the antimetastatic effect of SAA and are thus valuable for the development of treatment strategies for metastatic OSCC.     

Inhibitory effects of Leucaena leucocephala on the metastasis and invasion of human oral cancer cells

Oral cancer is one of the most common cancers worldwide, and metastasis is recognized as a major factor causing its low survival rate. The inhibition of metastasis progress and the improvement of the survival rate for oral cancer are critical research objectives. Leucaena leucocephala from the mimosa branch Leucaena genus is native to Central and South America and has been used as a traditional remedy for treating various disorders. Previous studies have demonstrated antioxidant, anti‐inflammatory as well as anticancer properties of L. leucocephala plant materials. However, the molecular mechanism underlying the anticancer effect induced by L. leucocephala remains unclear. In this study, we investigated the effect of L. leucocephala extract (LLE) on SCC‐9 and SAS oral cancer cells and examined the potential inhibitory mechanisms involved. The results indicated that LLE attenuated the migration and invasion abilities of both SCC‐9 and SAS cells by reducing the activity and protein expression of matrix metalloproteinases‐2 (MMP‐2). Regarding mitogen‐activated protein kinase (MAPK) pathways, the phosphorylation of ERK1/2 and p38 exhibited a significant inhibitory effect in the presence of LLE. The application of ERK inhibitor and p38 inhibitor confirmed that both signalling transduction pathways were involved in the inhibition of cell metastasis. These data indicate that L. leucocephala could be a potent therapeutic agent for the prevention and treatment of oral cancer and a prominent plant source for anticancer research in the future.     

Daphnetin inhibits TNF‐α and VEGF‐induced angiogenesis through inhibition of the IKKs/IκBα/NF‐κB,Src/FAK/ERK1/2 and Akt signalling pathways

Coumarins, identified as plant secondary metabolites possess diverse biological activities including anti‐angiogenic properties. Daphnetin (DAP), a plant derived dihydroxylated derivative of coumarin has shown significant pharmacological properties such as anticancer, anti‐arthritic and anti‐inflammatory. The present study was performed to investigate the anti‐angiogenic potential of DAP, focusing on the mechanism of action. The in vivo anti‐angiogenic potential of DAP was evaluated by vascular endothelial growth factor (VEGF)‐induced rat aortic ring (RAR) assay and chick chorioallantoic membrane (CAM) assay. For in vitro evaluation, wounding migration, transwell invasion, tube formation and apoptosis assays were performed on VEGF (8 ng/mL)‐induced human umbilical vein endothelial cells (HUVECs). The cellular mechanism of DAP was examined on TNFα (10 ng/mL) and VEGF‐induced HUVECs by extracting the mRNA and protein levels using RT‐qPCR and western blotting. Our data demonstrated that DAP inhibited the in vivo angiogenesis in the RAR and CAM assay. DAP also inhibited the different steps of angiogenesis, such as migration, invasion, and tube formation in HUVECs. DAP inhibited nuclear factor‐κB signalling together including TNF‐α induced IκBα degradation; phosphorylation of IκB kinase (IKKα/β) and translocation of the NF‐κB‐p65 protein. Furthermore, western blotting revealed that DAP significantly down‐regulated the VEGF‐induced signalling such as c‐Src, FAK, ERK1/2 and the related phosphorylation of protein kinase B (Akt) and VEGFR2 expressions. DAP reduced the elevated mRNA expression of iNOS, MMP2 and also, induced apoptosis in VEGF‐stimulated HUVECs by the caspase‐3 dependent pathway. Taken together, this study reveals that DAP may have novel prospective as a new multi‐targeted medication for the anti‐angiogenesis and cancer therapy.     

Pomegranate extract inhibits migration and invasion of oral cancer cells by downregulating matrix metalloproteinase‐2/9 and epithelial‐mesenchymal transition

Discovering drug candidates for the modulation of metastasis is of great importance in inhibiting oral cancer malignancy. Although most pomegranate extract applications aim at the antiproliferation of cancer cells, its antimetastatic effects remain unclear, especially for oral cancer cells. The aim of this study is to evaluate the change of two main metastasis characters, migration and invasion of oral cancer cells. Further, we want to explore the molecular mechanisms of action of pomegranate extract (POMx) at low cytotoxic concentration. We found that POMx ranged from 0 to 50 μg/mL showing low cytotoxicity to oral cancer cells. In the case of oral cancer HSC‐3 and Ca9‐22 cells, POMx inhibits wound healing migration, transwell migration, and matrix gel invasion. Mechanistically, POMx downregulates matrix metalloproteinase (MMP)‐2 and MMP‐9 activities and expressions as well as epithelial‐mesenchymal transition (EMT) signaling. POMx upregulates extracellular signal‐regulated kinases 1/2 (ERK1/2), but not c‐Jun N‐terminal kinase (JNK) and p38 expression. Addition of ERK1/2 inhibitor (PD98059) significantly recovered the POMx‐suppressed transwell migration and MMP‐2/?9 activities in HSC‐3 cells. Taken together, these findings suggest to further test low cytotoxic concentrations of POMx as a potential antimetastatic therapy against oral cancer cells.     

Antimetastatic effects of Eclipta prostrata extract on oral cancer cells

Eclipta prostrata, a traditional Chinese medication, has been used for the treatment of several diseases. However, the molecular mechanism underlying the effects of Eclipta prostrata extracts (EPE) on human oral cancer cell metastasis remains unclear. We thus examined the effects of EPE on metastasis promoting proteins in oral cancer. Our results revealed that the EPE attenuated SCC‐9, HSC‐3, and TW2.6 cell migration and invasiveness by reducing matrix metalloproteinase (MMP)‐2 enzyme activities. In addition, Western blot analysis revealed that EPE significantly reduced the levels of phosphorylated extracellular signal‐regulated kinase 1/2 (ERK 1/2) but not those of c‐Jun N‐terminal kinase (JNK) 1/2 and p38. In conclusion, we found that EPE could inhibit oral cancer metastasis through the inhibition of MMP‐2 expression. Therefore, EPE may be used to prevent the metastasis of oral cancer, and has the potential to be applied to cancer treatment.     

Bufalin‐inhibited migration and invasion in human osteosarcoma U‐2 OS cells is carried out by suppression of the matrix metalloproteinase‐2, ERK,and JNK signaling pathways

Bufalin has been shown to exhibit multiple pharmacological activities, including induction of apoptosis in many types of cancer cell lines. Osteosarcoma is a type of cancer which is difficult to treat and the purpose of this study was to investigate the effects of bufalin on the migration and invasion of human osteosarcoma U‐2 OS cells. The wound healing assay and Boyden chamber transwell assay were used for examining the migration of U‐2 OS cells. Western blotting and gelatin zymography assays were used for theexpression and activities of metalloproteinase (MMP)‐2, MMP‐7 or MMP‐9 levels. Western blotting analysis also was used for measuring the levels of growth factor receptor‐bound protein 2 (GRB2), son of sevenless homolog 1 (SOS1), c‐Jun N‐terminal kinases 1/2 (JNK1/2), extracellular signal‐regulated kinase 1/2 (ERK1/2), and p38 in bufalin‐treated U‐2 OS cells. Bufalin inhibited the cell migration and invasion of U‐2 OS cells in vitro. Moreover, bufalin reduced MMP‐2 and MMP‐9 enzyme activities of U‐2 OS cells. Bufalin also suppressed the protein level of MMP‐2 and reduced the levels of mitogen‐activated protein kinases (MAPKs) such as JNK1/2 and ERK1/2 signals in U‐2 OS cells. Our results suggest that signaling pathways for bufalin‐inhibited migration and invasion of U‐2 OS cells might be mediated through blocking MAPK signaling and resulting in the inhibition of MMP‐2. Bufalin could be a useful agent to develop as a novel antitumor agent by virtue of its ability to inhibit tumor cell migration and invasion. © 2011 Wiley Periodicals, Inc. Environ Toxicol 29: 21–29, 2014.     

p38 mitogen-activated protein kinase contributes to angiotensin II-stimulated migration of rat aortic smooth muscle cells

In this study, we clarified the intracellular mechanism of angiotensin II (Ang II) in promoting migration in rat aortic smooth muscle cells (RASMCs). RASMC migration was measured with the Boyden chamber assay, and the result was confirmed with an aortic sprout assay. The activities of kinases were investigated by western blot analysis. Ang II enhanced RASMC migration, which was chemotaxis directed, and induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2 (ERK1/2), and heat shock protein 27 (Hsp27). Ang II-enhanced cell migration was inhibited by SB203580 (a p38 MAPK inhibitor) and piceatannol (a spleen tyrosine kinase inhibitor), but only partially by PD98059 (an ERK inhibitor) and PP2 (a Src inhibitor). The Ang II-stimulated phosphorylation of p38 MAPK and Hsp27 in RASMCs was inhibited by piceatannol and SB203580. The phosphorylation of ERK1/2 stimulated by Ang II was suppressed by PD98059, piceatannol, and PP2. Ang II increased the sprout outgrowth from aortic rings and this response was attenuated by pretreatment with SB203580, PD98059, PP2, or piceatannol. These results suggest that p38 MAPK contributes to the regulation of the Ang II-induced chemotactic migration of vascular smooth muscle cells, which is mediated by Hsp27 phosphorylation.     

Mechanisms for prostaglandin E2 formation caused by proteinase-activated receptor-1 activation in rat gastric mucosal epithelial cells

Proteinase-activated receptor-1 (PAR1), a thrombin receptor, plays a protective role in gastric mucosa via prostanoid formation. Thus, we studied effects of PAR1 stimulation on prostaglandin E(2) (PGE(2)) formation in rat normal gastric mucosal epithelial RGM1 cells and analyzed the underlying signal transduction mechanisms. The PAR1-activating peptide (PAR1-AP) and thrombin increased PGE(2) release from RGM1 cells for 18h, an effect being suppressed by inhibitors of COX-1, COX-2, MEK, p38 MAP kinase (p38 MAPK), protein kinase C (PKC), Src and EGF receptor-tyrosine kinase (EGFR-TK), but not JNK and matrix metalloproteinase (MMP)/a disintegrin and metalloproteinases (ADAMs). PAR1-AP caused persistent (6h or more) and transient (5min) phosphorylation of ERK and p38 MAPK, respectively, followed by delayed reinforcement at 18h. PAR1-AP up-regulated COX-2 in a manner dependent on MEK and EGFR-TK, but not p38 MAPK. The PAR1-mediated persistent ERK phosphorylation was reduced by inhibitors of Src and EGFR-TK. PAR1-AP actually phosphorylated EGF receptors and up-regulated mRNA for heparin-binding-EGF (HB-EGF), the latter effect being blocked by inhibitors of Src, EGFR-TK and MEK. Heparin, an inhibitor for HB-EGF, suppressed PAR1-mediated PGE(2) formation and persistent ERK phosphorylation. These results suggest that PAR1 up-regulates COX-2 via persistent activation of MEK/ERK that is dependent on EGFR-TK activation following induction of HB-EGF, leading to PGE(2) formation. In addition, our data also indicate involvement of COX-1, PKC and p38 MAPK in PAR1-triggered PGE(2) formation. PAR1, thus stimulates complex multiple signaling pathways responsible for PGE(2) formation in RGM1 cells.     

Spleen tyrosine kinase participates in src-mediated migration and proliferation by PDGF-BB in rat aortic smooth muscle cells

Tyrosine kinases, Src and spleen tyrosine kinase (Syk), play crucial roles in cell responses to platelet-derived growth factor (PDGF) and may have their functional interactions. In this study, we focused on investigating the roles of Syk in the regulation of Src signaling in PDGF-mediated vascular cell responses. Migration, proliferation, and activity of kinases were determined in rat aortic smooth muscle cells (RASMCs). PDGF-BB (10 ng/mL) induced the migration and proliferation of RASMCs, which were significantly inhibited by PP2 (10 microM) and piceatannol (30 microM), inhibitors of Src and Syk, respectively. The phosphorylation of Syk induced by PDGF-BB was abolished by PP2. PDGF-BB increased the co-association of the PDGFbeta-receptor and the kinases, Src or Syk, and its maximal binding to Src was achieved in a shorter time than that to Syk. PDGF-BB stimulated the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) 1/2, which was inhibited by PP2 and piceatannol. PDGF-BB-induced proliferation and migration were inhibited by SB203580 (30 microM) and PD98059 (30 microM), inhibitors of p38 MAPK and ERK1/2, respectively. These results imply that Syk is regulated by Src kinase, which participates in migration and proliferation in response to PDGF-BB in RASMCs.     

Crude extract of Rheum palmatum L inhibits migration and invasion of LS1034 human colon cancer cells acts through the inhibition of matrix metalloproteinase‐2/‐9 by MAPK signaling

Crude extract of Rheum palmatum L. (CERP) has been used to treat different diseases in the Chinese population for decades. In this study, we investigated the anti‐metastasis effects of CERP on LS1034 human colorectal cancer cells in vitro and examined potential mechanisms of its effects. CERP significantly inhibited cell migration and invasion of LS1034 cells. We also found that CERP inhibited protein levels of matrix metalloproteinases‐2 (MMP‐2) and matrix metalloproteinases‐9 (MMP‐9), and cytosolic NF‐kB p65, RHO A, ROCK 1. Furthermore, we found CERP inhibited protein levels of GRB2, SOS1, MKK7, FAK, Rho A, ROCK 1, VEGF, PKC, AKT, phosphor‐AKT (Thr308), Cyclin D, iNOS, COX2, NF‐kB p65, p‐ERK1/2, p‐JNK1/2, p‐p38, p‐c‐jun, MMP‐2, MMP‐9, MMP‐1, MMP‐7, MMP‐10, UPA and increased the protein level of Ras in LS1034 cells. In conclusion, our results suggest that CERP may be used as a novel anti‐metastasis agent for the treatment of human colon cancer cells. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 852–863, 2015.     

Glucose suppresses IL‐1β‐induced MMP‐1 expression through the FAK,MEK, ERK,and AP‐1 signaling pathways

Osteoarthritis (OA) commonly affects the synovial joint and is characterized by degradation of articular cartilage. Increased matrix metalloproteinase (MMP) activity plays a major role in this degradation. Dextrose (D‐glucose) prolotherapy has shown promising activity in the treatment of different musculoskeletal disorders, including OA. However, little is known about the role of glucose on MMP inhibition in OA therapy. We found that stimulating chondrocytes with the proinflammatory cytokine interleukin‐1β (IL‐1β) increased the expression of MMP‐1, MMP‐3, and MMP‐13. Glucose reduced this increase in MMP‐1 expression, but had no effect upon MMP‐3 or MMP‐13 expression. Analyses using a focal adhesion kinase (FAK) inhibitor, MEK inhibitors (U0126 and PD98059), an ERK inhibitor, AP‐1 inhibitors (curcumin and tanshinone), or siRNAs demonstrated that the FAK, MEK, ERK, and AP‐1 pathways mediate IL‐1β‐induced increases in MMP‐1 expression. Glucose antagonized IL‐1β‐promoted phosphorylation of FAK, MEK, ERK, and c‐Jun. Thus, glucose decreased IL‐1β‐induced MMP‐1 expression through the FAK, MEK, ERK, and AP‐1 signaling cascades. These findings may provide a better understanding of the mechanisms of prolotherapy on inhibiting MMP expression.     

WISP-1 increases MMP-2 expression and cell motility in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. WISP-1 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, Nov) family of matricellular proteins. However, the effect of WISP-1 on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that WISP-1 increased the migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells (JJ012 cells). We also found that human chondrosarcoma tissues had significant expression of the WISP-1 which was higher than that in normal cartilage. α5β1 monoclonal antibody and MAPK kinase (MEK) inhibitors (PD98059 and U0126) inhibited the WISP-1-induced increase of the migration and MMP-2 up-regulation of chondrosarcoma cells. WISP-1 stimulation increased the phosphorylation of focal adhesion kinase (FAK), MEK and extracellular signal-regulated kinase (ERK). In addition, NF-κB inhibitors also suppressed the cell migration and MMP-2 expression enhanced by WISP-1. Moreover, WISP-1 increased NF-κB luciferase activity and binding of p65 to the NF-κB element on the MMP-2 promoter. Taken together, our results indicated that WISP-1 enhances the migration of chondrosarcoma cells by increasing MMP-2 expression through the α5β1 integrin receptor, FAK, MEK, ERK, p65 and NF-κB signal transduction pathway.     

The crude extract of Corni Fructus inhibits the migration and invasion of U‐2 OS human osteosarcoma cells through the inhibition of matrix metalloproteinase‐2/‐9 by MAPK signaling

Osteosarcoma is the most common primary malignancy of the bone cancers. In the Chinese population, the crude extract of Corni Fructus (CECF) has been used as Traditional Chinese medicine to treat several different diseases for hundreds of years. In the present study, effects of CECF on inhibition of migration and invasion in U‐2 OS human osteosarcoma cells were examined. CECF significantly inhibited migration and invasion of U‐2 OS human osteosarcoma cells. We also found that CECF inhibited activities of matrix metalloproteinases‐2 (MMP‐2) and matrix metalloproteinases‐9 (MMP‐9). CECF decreased protein levels of FAK, PKC, SOS1, MKK7, MEKK3, GRB2, NF‐κB p65, COX‐2, HIF‐1α, PI3K, Rho A, ROCK‐1, IRE‐1α, p‐JNK1/2, p‐ERK1/2, p‐p38, Ras, p‐PERK, MMP‐2, MMP‐9, and VEGF in U‐2 OS cells. Results of this study indicate that CECF may have potential as a novel anticancer agent for the treatment of osteosarcoma by inhibiting migration and invasion of cancer cells © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 53–63, 2015.     

Kaempferol suppresses cell migration through the activation of the ERK signaling pathways in ARPE‐19 cells

Kaempferol is a flavonoid with anticancer and anti‐metastasis activity in different cancer‐cell lines. However, the underlying mechanisms by which kaempferol acts on human retinal pigment epithelial (ARPE‐19) cells remain unclear. In this study, we demonstrated that kaempferol inhibited migration and invasion in ARPE‐19 cells at non‐toxic dosages. We discovered that kaempferol obviously reduced the enzyme activity and protein expression of matrix metalloproteinase‐2 by increasing the phosphorylated levels of extracellular signal‐regulated kinases 1/2 (ERK1/2) signaling pathways. Additionally, ERK1/2‐specific inhibitor PD98059 significantly reversed kaempferol's inhibitory effects on migration and expression of MMP‐2 in ARPE‐19 cells. Overall, our results are the first to demonstrate that kaempferol is capable of inhibiting cell migration by targeting ERK1/2 signaling in human retinal pigment epithelial cells.     

Anti‐angiogenic potential of scopoletin is associated with the inhibition of ERK1/2 activation

Angiogenesis plays an important role in many diseases, such as cancer, rheumatoid arthritis, and diabetic retinopathy. Specific inhibitors of angiogenesis are therefore expected to be potential candidate therapeutics for these disorders. Recently, several naturally occurring coumarins and synthetic analogues have proved to hinder new vessel formation. The present study was undertaken to investigate the effects of scopoletin, a phenolic coumarin compound with various biological activities on endothelial cell activation and resultant angiogenesis. Scopoletin had no cytotoxic effect on endothelial cells at the concentrations tested but suppressed the endothelial cell migration and disrupted rat tail collagen tube formation at concentrations of 62.5, 125, 250, and 500 µM, whereas it only moderately inhibited the proliferation and adhesion of endothelial cells. Notably, scopoletin (500 µM) selectively downregulated serum‐induced ERK1/2 phosphorylation, without affecting endothelial cell p38 MAPK or JNK phosphorylation. These findings demonstrate that scopoletin has anti‐angiogenic properties that are manifest mainly through inhibiting migration and tube formation of endothelial cells via downregulating ERK1/2 activation. Scopoletin may potentially be useful for the treatment of angiogenesis‐mediated diseases and could serve as a structural basis for screening for more potent synthetic analogues. Drug Dev Res 2009. © 2009 Wiley‐Liss, Inc.