Bufalin inhibits migration and invasion in human hepatocellular carcinoma SK‐Hep1 cells through the inhibitions of NF‐kB and matrix metalloproteinase‐2/‐9‐signaling pathways

Metastasis plays an important role in mortality of cancer patients. Migration and invasion are the major characteristics of tumor metastasis. The induction of matrix metalloproteinases (MMPs) such as MMP‐2 and ‐9 are particularly important for the invasiveness of various cancer cells. Bufalin, a class of toxic steroids, was purified from the skin glands of Bufo gargarizans or Bufo melanostictus; it is known to inhibit proliferation of human cancer cells. In this study, we investigated the antiinvasive mechanisms of bufalin in the human hepatocellular cancer cell line SK‐Hep1. Bufalin significantly reduced serum‐induced cell invasion and migration. Furthermore, bufalin markedly inhibited MMP‐2 and ‐9 activity, mRNA expression and protein levels in SK‐Hep1 cells. Bufalin attenuated phosphoinisitide‐3‐kinase (PI3K) and phosphorylation of AKT which was associated with reduced levels of nuclear factor kappa B (NF‐κB). Bufalin also suppressed protein levels of FAK and Rho A, VEGF, MEKK3, MKK7, and uPA and it diminished NF‐κB translocation. Based on these observations, we propose that bufalin is acts as an antiinvasive agent by inhibiting MMP‐2 and ‐9 and involving PI3K/AKT and NF‐κB pathways. Bufalin is a potential therapeutic agent that may have efficacy in preventing the invasion and metastasis of malignant liver tumors. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 74–82, 2015.     

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.     

Tetrandrine inhibits human brain glioblastoma multiforme GBM 8401 cancer cell migration and invasion in vitro

Tetrandrine (TET) has been reported to induce anti‐cancer activity in many human cancer cells and also to inhibit cancer cell migration and invasion. However, there are no reports to show TET inhibits cell migration and invasion in human brain glioblastoma multiforme GBM 8401 cells. In this study, we investigated the anti‐metastasis effects of TET on GBM 8401 cells in vitro. Under sub‐lethal concentrations (from 1, 5 up to 10 μM), TET significantly inhibited cell mobility, migration and invasion of GBM 8401 cells that were assayed by wound healing and Transwell assays. Gelatin zymography assay showed that TET inhibited MMP‐2 activity in GBM 8401 cells. Western blotting results indicated that TET inhibited several key metastasis‐related proteins, such as p‐EGFR_(Tyr1068), SOS‐1, GRB2, Ras, p‐AKT_(Ser473) and p‐AKT_(Thr308), NF‐κB‐p65, Snail, E‐cadherin, N‐cadherin, NF‐κB, MMP‐2 and MMP‐9 that were significant reduction at 24 and 48 hours treatment by TET. TET reduced MAPK signaling associated proteins such as p‐JNK1/2 and p‐c‐Jun in GBM 8401 cells. The electrophoretic mobility shift (EMSA) assay was used to investigate NF‐κB and DNA binding was reduced by TET in a dose‐dependently. Based on these findings, we suggested that TET could be used in anti‐metastasis of human brain glioblastoma multiforme GBM 8401 cells in the future.     

Inhibitory action of naphtho[1,2‐b]furan‐4,5‐dione on hepatocyte growth factor‐induced migration and invasion of MDA‐MB‐231 cells: Mechanisms of action

Naphtho[1,2‐b]furan‐4,5‐dione (NFD), a bioactive component of Avicennia marina, has been shown to exhibit anticancer activity. The aim of the present study was to explore the effect of NFD on hepatocyte growth factor (HGF)‐induced cell migration and invasion of MDA‐MB‐231 human breast cancer cells, as well as the underlying mechanism of action. Cell viability was determined using the 3‐(4,5‐dimethyl‐2 thiazoyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide assay, western blot analysis was used to measure protein expression and cell migration and invasion were evaluated by the cell wound healing assay, Boyden chamber assay and gelatin zymography. When cells were treated with non‐toxic concentrations of NFD (1–3 μmol/L, 24 h), NFD concentration‐dependently inhibited HGF‐promoted cell migration and invasion. Simultaneously, NFD efficiently suppressed c‐Met phosphorylation and downstream activation of phosphatidylinositol 3‐kinase (PI3K) and Akt. In addition, NFD inhibited the phosphorylation of IκB kinases and IκBα and nuclear translocation of nuclear factor (NF)‐κB, as well as matrix metalloproteinase (MMP)‐9 activity. Furthermore, the c‐Met inhibitor PHA665752 (10 μmol/L) inhibited HGF‐induced MMP‐9 expression, cell migration and invasion, as well as the activation of PI3K/Akt, suggesting that PI3K/Akt activation occur downstream of c‐Met activation. In conclusion, the results of the present study suggest that NFD inhibits HGF‐induced invasion and migration of MDA‐MB‐231 cells via HGF‐ and/or c‐Met‐mediated PI3K/Akt and NF‐κB signalling pathways, leading to downregulation of MMP‐9 expression and cell migration.     

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.     

Chrysin inhibit human melanoma A375.S2 cell migration and invasion via affecting MAPK signaling and NF‐κB signaling pathway in vitro

Numerous evidences have shown that chrysin induced cytotoxic effects via induced cell cycle arrest and induction of cell apoptosis in human cancer cell lines, however, no information showed that chrysin inhibited skin cancer cell migration and invasion. In this study, we investigated anti‐metastasis mechanisms of chrysin in human melanoma cancer A375.S2 cells in vitro. Under sub‐lethal concentrations of chrysin (0, 5, 10, and 15 μM) which inhibits cell mobility, migration and invasion of A375.S2 cells that were assayed by wound healing and Transwell filter. That chrysin inhibited MMP‐2 activity in A375.S2 cells was investigated by gelatin zymography assay. Western blotting was used to examine protein expression and results indicated that chrysin inhibited the expression of GRB2, SOS‐1, PKC, p‐AKT (Thr308), NF‐κBp65, and NF‐κBp50 at 24 and 48 hours treatment, but only at 10‐15 μM of chrysin decreased Ras, PI3K, p‐c‐Jun, and Snail only at 48 hours treatment and only decrease p‐AKT(Ser473) at 24 hours treatment. Furthermore, chrysin (5‐15 μM) decreased the expression of uPA, N‐cadherin and MMP‐1 at 24 and 48 hours treatment but only decreased MMP‐2 and VEGF at 48 hours treatment at 10‐15 μM and 5‐15 μM of chrysin, respectively, however, increased E‐cadherin at 5‐15 μM treatment. Results of confocal laser microscopy systems indicated that chrysin inhibited expression of NF‐κBp65 in A375.S2 cells. Based on these observations, we suggest that chrysin can be used in anti‐metastasis of human melanoma cells in the future.     

AMPK‐dependent signaling modulates the suppression of invasion and migration by fenofibrate in CAL 27 oral cancer cells through NF‐κB pathway

Fenofibrate, a peroxisome proliferator‐activated receptor alpha (PPARα) agonist and lipid‐lowering agent, has been used worldwide for treatment of hyperlipidemia. The clinical trials demonstrate that fenofibrate possesses multiple pharmacological activities, including antitumor effects. However, the precise mechanisms in oral squamous cell carcinoma (OSCC) remain unclear. In this study, we investigated the anticancer effects of fenofibrate on the migration and invasion of human oral cancer CAL 27 cells. Fenofibrate inhibited the cell migration and invasion of CAL 27 cells by the wound healing and Boyden chamber transwell assays, respectively. In addition, fenofibrate reduced the protein expressions of MMP‐1, MMP‐2, MMP‐7, and MMP‐9 by Western blotting and inhibited enzyme activities of MMP‐2/‐9 using gelatin zymography assay. Results from immunoblotting analysis showed that the proteins of p‐LKB1 (Ser428), LKB1, p‐AMPKα (Thr172), p‐AMPKα1/α2 (Ser425/Ser491), p‐AMPKβ1 (Ser108), and AMPKγ1 were upregulated by fenofibrate; the levels of p‐IKKα/β (Ser176) and p‐IκBα were reduced in fenofibrate‐treated cells. Also, fenofibrate suppressed the expressions of nuclear NF‐κB p65 and p50 by immunoblotting and NF‐κB DNA binding activity by EMSA assay. The anti‐invasive effect of fenofibrate was attenuated by compound C [an adenosine 5′‐monophosphate‐activated protein kinase (AMPK) inhibitor] or dominant negative form of AMPK (DN‐AMPKα1). Thus, fenofibrate considerably inhibited metastatic behaviors of CAL 27 cells might be mediated through blocking NF‐κB signaling, resulting in the inhibition of MMPs; these effects were AMPK‐dependent rather than PPARα signaling. Our findings provide a molecular rationale, whereby fenofibrate exerts anticancer effects and additional beneficial effects for the treatment of cancer patients. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 866–876, 2016.     

Diallyl sulfide,diallyl disulfide,and diallyl trisulfide inhibit migration and invasion in human colon cancer colo 205 cells through the inhibition of matrix metalloproteinase‐2, ‐7, and ‐9 expressions

Diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS) are major organosulfur compounds exiting in garlic (Allium sativum). These compounds are reported to exhibit various pharmacological properties such as antibacteria, antiangiogenesis, anticancer, and anticoagulation, and they also induce cytotoxicity and induction of apoptosis in human cancer cells. Although these compounds show wide spectrum of biological activities, there are no reports to show that DAS, DADS, and DATS affected migration and invasion of human colon cancer cells, and their exact molecular mechanisms are not well investigated. Therefore, the purpose of this study was to determine whether DAS, DADS, and DATS affected the invasion and migration abilities of colo 205 human colon cancer cells. The results indicate that DAS, DADS, and DATS at 10 and 25 μM inhibited the migration and invasion of colo 205 cells in the order of DATS < DADS < DAS. DATS is the highest for inhibition of migration and invasion of colo 205 cells. DAS, DADS, and DATS induce downregulation expression of PI3K, Ras, MEKK3, MKK7, ERK1/2, JNK1/2, and p38 and then lead to the inhibition of MMP‐2, ‐7, and ‐9. DAS, DADS, and DATS inhibited NF‐κB and COX‐2 for leading to the inhibition of cell proliferation. Taken together, these results demonstrated that application of DAS, DADS, and DATS might serve as potential antimetastatic drugs. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 479–488, 2013.     

Regorefenib induces extrinsic/intrinsic apoptosis and inhibits MAPK/NF‐κB‐modulated tumor progression in bladder cancer in vitro and in vivo

The aim of the present study is to investigate anticancer effect and mechanism of regorafenib in bladder cancer in vitro and in vivo. Human bladder cancer TSGH 8301 cells were treated with regorafenib, NF‐κB, AKT, or mitogen‐activated protein kinase (MAPK) inhibitors for different time. The changes of cell viability, NF‐κB activation, apoptotic signaling transduction, and expression of tumor progression‐associated proteins were evaluated with MTT, NF‐κB reporter gene assay, flow cytometry, and Western blotting assay. TSGH 8301 tumor bearing mice were established and treated with vehicle (140 μL of 0.1% DMSO) or regorafenib (10 mg/kg/day by gavage) for 15 days. The changes of tumor volume, body weight, NF‐κB activation, MAPK activation, and tumor progression‐associated proteins (MMP‐9, XIAP, VEGF, and Cyclin‐D1) after regorafenib treatment were evaluated with digital caliper, digital weight, and ex vivo Western blotting assay. Our results demonstrated NF‐κB activation and protein levels of MMP‐9, XIAP, VEGF, and Cyclin‐D1 were significantly reduced by NF‐κB (QNZ), ERK (PD98059), and P38 (SB203580) inhibitors. Regorafenib also significantly induced extrinsic and intrinsic apoptotic signaling transduction in bladder cancer in vitro. In addition, regorafenib significantly inhibited tumor growth, NF‐κB, p38, ERK activation and expression of tumor progression‐associated proteins in bladder cancer in vitro and in vivo. Taken together, these results proved that regorafenib not only induced apoptosis through extrinsic and intrinsic pathways and but suppressed MAPK/ NF‐κB‐modulated tumor progression in bladder cancer.     

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.     

Inhibitory effect of Astragalus membranaceus root on matrix metalloproteinase‐1 collagenase expression and procollagen destruction in ultraviolet B‐irradiated human dermal fibroblasts by suppressing nuclear factor kappa‐B activity

Objectives The root of Astragalus membranaceus, regarded as a tonic in traditional Korean medicine, has been prescribed for long periods to treat chronic illness by boosting the immune system. Ultraviolet (UV) irradiation causes damage to skin connective tissue by degrading collagen, which is a major structural component of the extracellular matrix. Such damage is considered to be a cause of the wrinkling observed in premature ageing of the skin. This study has investigated the photo‐protective effect of A. membranaceus on UVB radiation‐induced activation of nuclear factor kappa‐B (NF‐κB) activity in human dermal fibroblasts. Methods Hs68 fibroblast cells cultured with various concentrations of A. membranaceus were exposed to UVB (40 mJ/cm²). Activation of NF‐κB P65 and expression of matrix metalloproteinase‐1 (MMP‐1) and type 1 procollagen were measured by Western blotting. Translocation of NF‐κB P65 and MMP‐1 regulation were also examined by immunocytochemistry. Key findings Western blotting and immunocytochemistry results showed that A. membranaceus inhibited UVB‐induced translocation of NF‐κB P65 and MMP‐1 expression. The data suggested that A. membranaceus restored type 1 procollagen synthesis by inhibiting NF‐κB P65 activity and MMP‐1 expression in UVB‐exposed human dermal fibroblasts. Conclusion A. membranaceus is a candidate for use in skin protection from UVB‐induced skin inflammation and photoageing.     

Puerarin Inhibits C‐Reactive Protein Expression via Suppression of Nuclear Factor κB Activation in Lipopolysaccharide‐Induced Peripheral Blood Mononuclear Cells of Patients with Stable Angina Pectoris

Abstract: Puerarin (4′‐7‐dihydroxy‐8‐β‐d ‐glucosylisoflavone), the most abundant isoflavone‐C‐glucoside extracted from the root of the plant Pueraria lobata, has demonstrated anti‐inflammatory activity in cellular models of inflammation. In this report, we examined the ability of puerarin to modulate C‐reactive protein (CRP) expression and key molecules in the nuclear factor kappa B (NF‐κB) pathway to determine its molecular target. The protein and mRNA levels of CRP were determined in lipopolysaccharide (LPS)‐induced peripheral blood mononuclear cells of patients with unstable angina pectoris. Also, we detected the I‐κBα phosphorylation and the p65NF‐κB expression in peripheral blood mononuclear cells under our experimental condition. The results indicated that puerarin inhibited the expression of the protein and mRNA levels of CRP in LPS‐induced peripheral blood mononuclear cells. Subsequently, we determined that the inhibition of CRP expression was because of a dose‐dependent inhibition of phosphorylation and degradation of inhibitor kappaB(I‐κB), which resulted in a reduction of p65NF‐κB nuclear translocation. We conclude that puerarin acts as an anti‐inflammatory agent by blocking NF‐κB signalling, and may possibly be developed as a useful agent for the chemoprevention of atherosclerosis.     

Apoptosis induction and ERK/NF‐κB inactivation are associated with magnolol‐inhibited tumor progression in hepatocellular carcinoma in vivo

Although hepatitis B and/or hepatitis C virus were recognized as major risk factor for the development of hepatocellular carcinoma (HCC), certain occupational, environmental, and lifestyle factors also play key roles in HCC tumorigenesis. Moreover, in molecular signaling route, extracellular signal‐regulated kinase (ERK)/nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) signaling was found to be overexpressed and linked to poor prognosis in HCC. Thus, to identify possible nature compound that can suppress ERK/NF‐κB may be benefit to HCC patient. Magnolol, a natural compound derived from herbal plant Magnolia officinalis, has been recognized as a liver protection and antitumor reagent. However, whether magnolol‐inhibited HCC progression correlates with disruption of ERK/NF‐κB signaling is remained unclear. In this studies, we performed SK‐Hep1/luc2 HCC bearing animal model to investigate the anticancer efficacy and mechanism of magnolol on tumor progression. Tumor size and tumor growth rate were dramatically suppressed after treatment of magnolol. In addition, expression of phospho‐ERK (p‐ERK), NF‐κB p65 (Ser536), and tumor progression‐associated proteins, such as matrix metallopeptidase 9 (MMP‐9), vascular endothelial growth factor (VEGF), X‐linked inhibitor of apoptosis protein (XIAP), and CyclinD1 were all significantly decreased by magnolol. Most important, major extrinsic and intrinsic apoptosis signaling factors, including active caspase‐8 and caspase‐9 were both enhanced by magnolol. This study indicated that apoptosis induction through extrinsic/intrinsic pathways and blockage of ERK/NF‐κB activation were associated with magnolol‐inhibited tumor progression in HCC in vivo.     

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.