Andrographolide could inhibit human colorectal carcinoma Lovo cells migration and invasion via down-regulation of MMP-7 expression

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to have potent anti-cancer activity against human colorectal carcinoma Lovo cells by inhibiting cell-cycle progression. To further investigate the mechanism for the anti-cancer properties of Andro, it was used to examine the effect on migration and invasion of Lovo cells. The results of wound-healing assay and in vitro transwell assay revealed that Andro inhibited dose-dependently the migration and invasion of Lovo cells under non-cytotoxic concentrations. Using zymographic assay and RT-PCR, the results revealed that Andro diminished the activity and the mRNA and protein levels of MMP-7, but not MMP-2 or MMP-9. The down-regulation of MMP-7 appeared to be via the inactivation of activator protein-1 (AP-1) since the treatment with Andro suppressed the nuclear protein level of AP-1, which was accompanied by a decrease in DNA-binding level of the factor. Taken together, these results indicated that Andro reduces the MMP-7-mediated cellular events in Lovo cells, and provided a new mechanism for its anti-cancer activity.     

Celastrol inhibit the proliferation,invasion and migration of human cervical HeLa cancer cells through down-regulation of MMP-2 and MMP-9

The present study evaluated the anticancer potential of celastrol through down-regulation of matrix metalloproteinase-2 (MMP-2) and MMP-9. HeLa cells were incubated with different concentrations of celastrol (1, 10 and 100 µM) for 48h. Doxorubicin was used as a reference drug. Cancer cell migration, apoptosis, cell viability and mitochondrial fragmentation were evaluated following celastrol treatment. In addition, the expression level of MMP-2, MMP-9 and caspase-3 was evaluated following celastrol treatment. HeLa cell viability was 94.1 ± 7, 53.4 ± 4 and 36.3 ± 2% at 1-100 µM of celastrol, respectively. Apoptotic cell numbers were increased, and inhibition of larger wounds in cancer cells was observed following celastrol treatment. Celastrol-treated cells showed condensed nuclei and clumped mitochondria. Reduced expression of MMP-2 and MMP-9 and increased expression of caspase-3 were observed following celastrol treatment. Based on the experimental results, we are concluding that the celastrol was effective against HeLa cervical cancer cells.     

Cardiovascular drugs inhibit MMP-9 activity from human THP-1 macrophages

It is now recognized that atherosclerosis complications are related to the unstable character of the plaque rather than its volume. Vulnerable plaques often contain a large lipid core, a reduced content of smooth muscle cells, and accumulation of inflammatory cells. Colocalization of macrophages and active matrix metalloproteinases (MMPs) is likely relevant for atherosclerotic lesion disruption. Nevertheless, MMP activity and regulation by cardiovascular drugs remains poorly defined. In this study, we evaluated the effects of avasimibe, fluvastatin, and peroxisome proliferator-activated receptor (PPAR) ligands on 92-kDa gelatinase B (MMP-9) secretion by human THP-1 macrophages. THP-1 macrophages were treated with compounds for 48 h, and secreted MMP-9 protein was quantified by immunoassay. Avasimibe, fluvastatin, and PPARalpha agonists (fenofibric acid and Wy-14643) significantly reduced, in a concentration-dependent manner, MMP-9 protein (up to 67 +/- 5% for fenofibric acid). In these assays, the PPARgamma selective agonist rosiglitazone displayed a lower efficacy than other compounds. Enzymatic activity of MMP-9 was also decreased by all cardiovascular drugs tested. MMP-9 protein/activity inhibition by cardiovascular drugs was due, at least in part, to a decrease in MMP-9 mRNA. These results show that THP-1 macrophages could be an useful cellular model to investigate effects of compounds on plaque vulnerability through MMP-9 activity.     

Sulindac and its metabolites inhibit invasion of glioblastoma cells via down-regulation of Akt/PKB and MMP-2

Non-steroidal anti-inflammatory drug (NSAID), sulindac has chemopreventive and anti-tumorigenic properties, however, the molecular mechanism of this inhibitory action has not been clearly defined. The Akt/protein kinase B, serine/threonine kinase is well known as an important mediator of many cell survival signaling pathways. In the present study, we demonstrate that down-regulation of Akt is a major effect of anti-invasiveness property of sulindac and its metabolites in glioblastoma cells. Myristoylated Akt (MyrAkt) transfected U87MG glioblastoma cells showed increase invasiveness, whereas DN-Akt transfected cells showed decrease invasiveness indicating that Akt potently promoted glioblastoma cell invasion. MMP-2 promoter and enzyme activity were up-regulated in Akt kinase activity dependent manner. Sulindac and its metabolites down-regulated Akt phosphorylation, inhibited MMP-2 production, and significantly inhibited invasiveness of human glioblastoma cells. In addition, sulindac and LY294002, a selective inhibitor of phosphoinositide 3-kinase (PI3K), synergistically inhibited the invasion of glioblastoma cells. Furthermore, only celecoxib showed Akt phosphorylation reduction and an anti-invasivness in glioblastoma cells, whereas aspirin, ketoprofen, ketorolac, and naproxen did not. In conclusion, our results provide evidence that down-regulation of Akt pathway and MMP-2 may be one of the mechanisms by which sulindac and its metabolites inhibit glioblastoma cell invasion.     

Glucocorticoid receptor-induced down-regulation of MMP-9 by ginseng components, PD and PT contributes to inhibition of the invasive capacity of HT1080 human fibrosarcoma cells

We examined the effects of the purified ginseng components, panaxadiol (PD) and panaxatriol (PT), on the expression of matrix metalloproteinase-9 (MMP-9) in highly metastatic HT1080 human fibrosarcoma cell line. A significant down-regulation of MMP-9 by PD and PT was detected by Northern blot analysis. However, the expression of MMP-2 was not changed by treatment with PD and PT. Quantitative gelatin based zymography confirmed a markedly reduced expression of MMP-9, but not MMP-2 in the treatment of PD and PT. To investigate whether the reduced level of MMP-9 by PD and PT affects the invasive capacity of HT1080 cells, we conducted an in vitro invasion assay with PD and PT treated cells. The results of the in vitro invasion assay revealed that PD and PT reduced tumor cell invasion through a reconstituted basement membrane in the transwell chamber. Because of the similarity of chemical structure between PD, PT and dexamethasone (Dexa), a synthetic glucocorticoid, we investigated whether the down-regulation of MMP-9 by PD and PT were mediated by the nuclear translocation of glucocorticoid receptor (GR). Increased GR in the nucleus of HT1080 human fibrosarcoma cells treated by PD and PT was detected by immunocytochemistry. Western blot and gel retardation assays confirmed the increase of GR in the nucleus after treatment with PD and PT. These results suggest that GR-induced down-regulation of MMP-9 by PD and PT contributes to reduce the invasive capacity of HT1080 cells.     

Solvent-partitioned fractions from <Emphasis Type="Italic">Ishige okamurae</Emphasis> extract inhibit MMP-2 and MMP-9 activities in human fibrosarcoma cells in vitro

Matrix metalloproteinases (MMPs) are endopeptidases that take significant roles in extracellular matrix degradation and therefore linked with several complications such as metastasis of cancer progression, oxidative stress, and hepatic fibrosis. Ishige okamurae, a brown alga, has been reported to possess various bioactivities including but not limited to antiviral, antimicrobial, and anti-inflammatory. In this study, the potential of I. okamurae against cancer cell invasion was evaluated through MMP inhibitory effect in HT1080 fibrosarcoma cells in vitro. I. okamurae crude extract was fractionated with organic solvents, namely water (H₂O), n-butanol (n-BuOH), 85% aqueous methanol (85% aq. MeOH), and n-hexane (n-Hex). The non-toxicity of fractions was confirmed by MTT assay. All fractions inhibited the enzymatic activities of MMP-2 and MMP-9 according to gelatin zymography assay. Cell migration was also inhibited by 85% aq. MeOH fraction, significantly. In addition, both gene and protein expressions of MMP-2 and MMP-9, and tissue inhibitor of MMPs (TIMPs) were evaluated by RT-PCR and Western blotting, respectively. Fractions suppressed the mRNA and protein levels of MMP-2 and MMP-9 while elevating the TIMP-1 and TIMP-2, H₂O fraction being the least effective while 85% aq. MeOH fraction the most. Collectively, 85% aq. MeOH fraction from brown algae could be a potential inhibitor of MMPs, suggestively considering presence of poly-unsaturated fatty acids in high amounts.     

Disulfiram suppresses invasive ability of osteosarcoma cells via the inhibition of MMP-2 and MMP-9 expression

Cancer cells, characterized by local invasion and distant metastasis, are very much dependent on the extracellular matrix. The expression of matrix metalloproteinases (MMPs) has been implicated in the invasion and metastasis of cancer cells. In this study, we reported the effects of disulfiram, a clinically used anti-alcoholism drug, on tumor invasion suppression, as well as its effects on the activity of MMP-2 and MMP-9 in human osteosarcoma cells (U2OS). Disulfiram has been used for alcohol aversion therapy. However, recent reports have shown that disulfiram may have potential in the treatment of human cancers. Herewith, we showed that the anti-tumor effects of disulfiram, in an invasion assay using U2OS cells and that disulfiram has a type IV collagenase inhibitory activity that inhibits expression of genes and proteins responsible for both cell and non-cell mediated invasion on pathways. In conclusion, disulfiram inhibited expression of MMP-2 and MMP-9 and it regulated the invasion of human osteosarcoma cells. These observations raise the possibility of disulfiram being used clinical for the inhibition of cancer invasion.     

ATP potentiates interleukin-1 beta-induced MMP-9 expression in mesangial cells via recruitment of the ELAV protein HuR

Renal mesangial cells express high levels of matrix metalloproteinase 9 (MMP-9) in response to inflammatory cytokines such as interleukin (IL)-1 beta. We demonstrate here that the stable ATP analog adenosine 5'-O-(thiotriphosphate) (ATP gamma S) potently amplifies the cytokine-induced gelatinolytic content of mesangial cells mainly by an increase in the MMP-9 steady-state mRNA level. A Luciferase reporter gene containing 1.3 kb of the MMP-9 5'-promoter region showed weak responses to ATP gamma S but conferred a strong ATP-dependent increase in Luciferase activity when under the additional control of the 3'-untranslated region of MMP-9. By in vitro degradation assay and actinomycin D experiments we found that ATP gamma S potently delayed the decay of MMP-9 mRNA. Gel-shift and supershift assays demonstrated that three AU-rich elements (AREs) present in the 3'-untranslated region of MMP-9 are constitutively bound by complexes containing the mRNA stabilizing factor HuR. The RNA binding of these complexes was markedly increased by ATP gamma S. Mutation of each ARE element strongly impaired the RNA binding of the HuR containing complexes. Reporter gene assays revealed that mutation of one ARE did not affect the stimulatory effects by ATP gamma S, but mutation of all three ARE motifs caused a loss of ATP-dependent increase in luciferase activity without affecting IL-1 beta-inducibility. By confocal microscopy we demonstrate that ATP gamma S increased the nucleo cytoplasmic shuttling of HuR and caused an increase in the cytosolic HuR level as shown by cell fractionation experiments. Together, our results indicate that the amplification of MMP-9 expression by extracellular ATP is triggered through mechanisms that likely involve a HuR-dependent rise in MMP-9 mRNA stability.     

Cholesterol inhibits MMP-9 expression in human epidermal keratinocytes and HaCaT cells

Cholesterol is a major component of skin lipids and acts as a regulator of vesicular trafficking and signal transduction. However, the function of cholesterol on matrix metalloproteinases (MMPs) expression of human skin is not fully understood. Here, we investigated the effects of cholesterol on MMP-9 expression in normal human keratinocytes (NHK) and HaCaT cells. Basal level of MMP-9 expression was decreased by cholesterol in NHK. On the other hand, MMP-9 expression was increased by the cholesterol depletion agent, methyl-beta-cyclodextrin (MbetaCD), while it was inhibited by cholesterol repletion in HaCaT cells. MbetaCD induced ERK and JNK phosphorylation were prevented by cholesterol repletion. The inhibition of ERK and JNK decreased MbetaCD-induced MMP-9 expression. Therefore, our results suggest that cholesterol regulates MMP-9 expression through ERK and JNK-dependent pathways.