CD24 gene inhibition and TIMP-4 gene upregulation by Imperata cylindrica's root extract prevents metastasis of CaSki cells via inhibiting PI3K/Akt/snail signaling pathway and blocking EMT.

ETHNOPHARMACOLOGICAL RELEVANCE: Imperata cylindrica (L.) Raeusch (Gramineae) is a medicinal spice traditionally used in the treatment of hypertension and cancer. AIM OF THE STUDY: To assess the anti-metastatic potential of the methanol extract of I. cylindrica roots and determined its mechanisms of action. MATERIAL AND METHODS: The growth inhibition activity of I. cylindrica root extract in vitro and in vivo in human cervical cancer. The scratch assay and Boyden Chamber assay were used to determine the anti-migrative and anti-invasion actions of the plant extract. The whole-genome gene expression profiling using RNA-Seq was performed to determine the differentially expressed genes in CaSki cells after exposure to I. cylindrica to identify its targeted genes related to metastasis. Using protein analysis (western blotting) and gene expression analysis (RTqPCR), the targeted pathways of the key genes that were initially identified with RNA-Seq, were evaluated. RESULTS: I. cylindrica extract showed dose-dependent cytotoxicity in vitro and in vivo in mice bearing tumors. Furthermore, I. cylindrica root extract significantly inhibited cell migration and cell invasion. After the genome-wide transcriptome analysis, we found that important genes involved in cancer progression and metastasis of cervical cancer, that is, CD24 and TIMP-4 were significantly downregulated and upregulated, respectively. Moreover, I. cylindrica root extract significantly inhibited the PI3/AKT/Snail signaling pathway and blocked the EMT of CaSki cells. CONCLUSION: These findings provide an anti-metastatic mechanism of action of I. cylindrica root extract toward the human cervical cancer suggesting that this plant maybe developed into selective chemotherapy.

Inhibition of Invasion by Polyphenols from Citrus Fruit and Berries in Human Malignant Glioma Cells In Vitro.

BACKGROUND/AIM: The outlook for patients with high grade glioma (HGG) remains dismal. Hence, attention has focused on numerous innovative treatments. Our group has proposed a strategy on the use of a combination of polyphenols, as anti-invasive agents for the management of these neoplasms. MATERIALS AND METHODS: The aim of this study was to evaluate the in vitro effects of citrus flavonoids (tangeretin, nobiletin, naringin and limonin) and berry extracts (chokeberry, elderberry and bilberry) on selected mediators of invasion in 2 HGG cell cultures. RESULTS: The IC50 values could only be determined for tangeretin and chokeberry extract. The rest were non-functional in this context. Immunocytochemistry and flow cytometry results showed that chokeberry extract was most effective in down-regulating the expression of CD44. Similarly, RT-PCR data supported its ability to reduce gene expression of MMP-14 and EGFR. 2D invasion assays confirmed that inhibition is greater with chokeberry extract. CONCLUSION: Both polyphenols have anti-invasive potential but chokeberry extract is a stronger agent for glioma management.

Carotenoids in Cancer Metastasis-Status Quo and Outlook.

Metastasis represents a major obstacle in cancer treatment and the leading cause of cancer-related deaths. Therefore, the identification of compounds targeting the multi-step and complex process of metastasis could improve outcomes in the management of cancer patients. Carotenoids are naturally occurring pigments with a plethora of biological activities. Carotenoids exert a potent anti-cancer capacity in various cancer models in vitro and in vivo, mediated by the modulation of signaling pathways involved in the migration and invasion of cancer cells and metastatic progression, including key regulators of the epithelial-mesenchymal transition and regulatory molecules, such as matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), urokinase plasminogen activator (uPA) and its receptor (uPAR), hypoxia-inducible factor-1α (HIF-1α), and others. Moreover, carotenoids modulate the expression of genes associated with cancer progression and inflammatory processes as key mediators of the complex process involved in metastasis. Nevertheless, due to the predominantly preclinical nature of the known anti-tumor effects of carotenoids, and unclear results from certain carotenoids in specific cancer types and/or specific parts of the population, a precise analysis of the anti-cancer effects of carotenoids is essential. The identification of carotenoids as effective compounds targeting the complex process of cancer progression could improve the outcomes of advanced cancer patients.

Photobiomodulation of extracellular matrix enzymes in human nucleus pulposus cells as a potential treatment for intervertebral disk degeneration.

Intervertebral disc (IVD) degeneration is associated with imbalances between catabolic and anabolic responses, regulated by extracellular matrix (ECM)-modifying enzymes such as matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors of metalloproteinases (TIMPs). Potential contributing factors, such as interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, derived from infiltrated, activated macrophages within IVD tissues, can trigger abnormal production of ECM-modifying enzymes and progression of IVD degeneration. Novel therapies for regulating ECM-modifying enzymes can prevent or ameliorate IVD degeneration. Photobiomodulation (PBM), known to regulate wound repair, exhibits regenerative potential by modulating biological molecules. This study examined the effects of PBM, administered at various wavelengths (630, 525, and 465 nm) and energy densities (16, 32, and 64 J/cm2), on the production of ECM-modifying enzymes in replicated degenerative IVD. Our results showed that PBM selectively inhibited the production of ECM-modifying enzymes in a dose- and wavelength-dependent manner, suggesting that it could be a novel tool for treating symptomatic IVD degeneration.

Xinmailong mitigated epirubicin-induced cardiotoxicity via inhibiting autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Using insects, such as the cockroach, for the treatment of disease has a long history in traditional Chinese medicine. Xinmailong (XML) Injection, a bioactive composite extracted from Periplaneta americana (a species of cockroach), shows reasonable protective effects against cardiovascular injury and was approved for the use in the treatment of cardiac dysfunction in 2006, yet its cardio protective mechanisms remain unclear. AIM: The present study aims to examine the protective effects of XML against epirubicin-induced cardiotoxicity in vivo and determine its underlying mechanisms. MATERIALS AND METHODS: The chemical characteristics of XML were identified using high performance liquid chromatography (HPLC). Rats were intraperitoneally injected with epirubicin and then treated with XML for 14 days. Survival rate, echocardiography, electrocardiographic recordings and Masson staining were used to evaluate the cardioprotective activity of XML. Western blot and quantitative real time reverse transcriptase polymerase chain reaction (RT-PCR) analyses were used to investigate the molecular mechanisms underlying the actions of XML. RESULTS: XML treatment significantly enhanced the survival rate of rats from epirubicin-induced heart failure. XML prevented left ventricle dilatation, improved cardiac function. Furthermore, treatment with XML also significantly inhibited the accumulation of collagen, reduced the levels of mRNA for matrix metalloproteinases-9 (Mmp9) and transforming growth factor-ß 1(Tgfb1). This action of XML therefore might be responsible, at least in part, for the attenuation of cardiac fibrotic remodeling. XML inhibited autophagy as evidenced by the decreased accumulation of Beclin1 and autophagy related 7 (Atg7), which are necessary to form autophagosome structures. Protein kinase B (PKB/Akt), phosphatidylinositol 3 kinase (PI3K) and B cell lymphoma2 (Bcl2) levels were up-regulated, while significantly decreased protein levels for phosphorylated P38 and extracellular regulated protein kinases 1/2 (Erk1/2) were observed in the XML treated rats. The autophagy related results suggested that the increase in PI3K/Akt levels and inhibition of the phosphorylation of P38 MAPK and Erk1/2 contributed to the anti-autophagic activity of XML. CONCLUSIONS: Our data suggest that XML may be effective for mitigating epirubicin-induced cardiomyopathy and inhibits autophagy via activating the PI3K/Akt signaling pathway and inhibiting the Erk1/2 and P38 MAPK signaling pathways.

Unripe Rubus coreanus Miquel suppresses migration and invasion of human prostate cancer cells by reducing matrix metalloproteinase expression.

Rubus coreanus Miquel (RCM) is used to promote prostate health and has been shown to have anti-oxidant and anti-carcinogenic activities. However, the effects and mechanisms of RCM on prostate cancer metastasis remain unclear. PC-3 and DU 145 cells were treated with ethanol or water extract of unripe or ripe RCM and examined for cell invasion, migration, and matrix metalloproteinases (MMPs) activity and expression. Phosphoinositide 3-kinase (PI3K) and Akt activities were examined. Unripe RCM extracts exerted significant inhibitory effects on cell migration, invasion, and MMPs activities. A significant reduction in MMPs activities by unripe RCM ethanol extract treatment (UE) was associated with reduction of MMPs expression and induction of tissue inhibitors of metalloproteinases (TIMPs) expression. Furthermore, PI3K/Akt activity was diminished by UE treatment. In this study, we demonstrated that UE decreased metastatic potential of prostate cancer cells by reducing MMPs expression through the suppression of PI3K/Akt phosphorylation, thereby decreasing MMP activity and enhancing TIMPs expression.

ß-Carotene inhibits neuroblastoma cell invasion and metastasis in vitro and in vivo by decreasing level of hypoxia-inducible factor-1α.

Neuroblastoma is the most prevalent extracranial solid tumor in childhood and has poor clinical outcome due to its high potential for metastasis. Consequently, an understanding of the mechanisms that modulate cancer cell invasion, migration and metastasis is important for the development of more effective chemotherapeutic agents. While ß-carotene is a vitamin A precursor that has been shown to exert antioxidant and anticancer effects, the anti-metastatic effects of ß-carotene on neuroblastoma cells remain poorly understood. The aim of the present study was to investigate the anti-metastatic effects of ß-carotene on highly malignant SK-N-BE(2)C neuroblastoma cells in vitro and in vivo. Treatment of SK-N-BE(2)C cells with ß-carotene was found to attenuate the migratory and invasive capabilities of the cells. In addition, the enzymatic activity and expression of matrix metalloproteinase (MMP)-2 was suppressed following ß-carotene treatment under both normoxia and hypoxia. To induce metastasis, immunodeficient nude mice were injected with SK-N-BE(2)C cells via the tail vein in vivo. The incidence of liver metastasis and mean tumor volume in mice that were administered ß-carotene was decreased compared to controls. Furthermore, mRNA levels of MMPs, membrane-type (MT) 2 MMP and tissue inhibitors of metalloproteinases in liver tumor tissues were also lower following ß-carotene treatment. Level of hypoxia-inducible factor-1α (HIF-1α) and its downstream targets, vascular endothelial growth factor and glucose transporter 1 (GLUT1), were lower both in vitro and in vivo following ß-carotene treatment. In conclusion, the present study provides the first evidence that ß-carotene may represent an effective chemotherapeutic agent by regulating the invasion and metastasis of neuroblastoma via HIF-1α.

Adipose tissue-derived stem cell therapy for prevention and treatment of erectile dysfunction in a rat model of Peyronie's disease.

Peyronie's disease (PD) is a localized connective tissue disorder that involves the tunica albuginea (TA) of the penis. While surgical correction remains the gold standard, the search for an effective and less invasive therapy continues. The objective of this study was to evaluate the effects of intratunical injection of adipose tissue-derived stem cells (ADSCs) for the prevention and treatment of erectile dysfunction in a rat model of PD. Twenty-four male Sprague-Dawley rats (300-350 g) were randomly divided into four groups: sham, PD, PD + ADSC (prevention) and PD + ADSC (treatment). All rats underwent penile injections into the TA with 50 µL vehicle (sham) or 0.5 µg transforming growth factor (TGF)-ß1 (remaining groups). The ADSC groups received intratunical injections with 0.5 million rat-labelled ADSCs on day 0 (prevention) or day 30 (treatment). Forty-five days following TGF-ß1 injection, rats underwent cavernous nerve stimulation (CNS) with total intracavernous-to-mean arterial pressure ratio (ICP/MAP) and total ICP recorded to measure response to therapy. Tissues were evaluated histologically and for mRNA expression of tissue inhibitors of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs) and zymographic activity of MMPs. Statistical analysis was performed by analysis of variance followed by the Tukey test for post hoc comparisons. In both prevention and treatment groups, intratunical injection of ADSCs resulted in significantly higher ICP/MAP and total ICP in response to CNS compared with the PD group. Local injection of ADSCs prevented and/or reduced Peyronie's-like changes by decreasing the expression of TIMPs, and stimulating expression and activity of MMPs. This study documents the preventive and therapeutic benefits of ADSC on penile fibrosis and erectile function in an animal model of PD.

Melatonin inhibits IL1ß-induced MMP9 expression and activity in human umbilical vein endothelial cells by suppressing NF-κB activation.

Matrix metalloproteinases (MMPs) have been involved in inflammatory and degradative processes in pathologic conditions. The purpose of this study was to investigate the protective effect of melatonin in human umbilical vein endothelial cell (HUVEC) monolayer permeability and the regulation of MMP9 induced by interleukin 1ß (IL1ß (IL1B)) in HUVECs. Protection studies were carried out with melatonin, a well-known antioxidant and antiinflammatory molecule. MMP9 expression was increased with IL1ß induction in HUVECs. Melatonin showed a barrier-protective role by downregulation of MMP9 and upregulation of tissue inhibitor of metalloproteinase-1 expression in HUVECs. Meanwhile, melatonin also decreased sodium fluorescein permeability and counteracted the downregulation of vascular endothelial cadherin and occludin expression in HUVECs. During inflammatory stimulus, nuclear factor-κB (NF-κB) plays a significant role in regulating MMP genes expression, thus the function of NF-κB in HUVECs' barrier disruption was investigated. IL1ß induced nuclear translocation of NF-κB in HUVECs and regulated MMP9 expression. However, NF-κB translocation into the nucleus was inhibited significantly by melatonin. Our results show that melatonin decreases the permeability of monolayer endothelial cell induced by IL1ß. At the same time, melatonin decreased the expression and activity of MMP9 by a NF-κB-dependent pathway in HUVECs induced by IL1ß.

Protective effect of (2'S)-columbianetin from Corydalis heterocarpa on UVB-induced keratinocyte damage.

A salt tolerant plant, Corydalis heterocarpa has been used as a folk medicine to treat travail and spasm. Recent studies have also reported antioxidant and antiinflammatory activities of compounds isolated from C. heterocarpa. In this study, the protective effect of (2'S)-columbianetin isolated from C. heterocarpa on UVB-induced human keratinocyte (HaCaT) damage was investigated. First, the appropriate energy level of UVB irradiation was determined using MTT and LDH assays. And then the protective effect of (2'S)-columbianetin on UVB induced HaCaT damage was evaluated by measuring; the changes in cell viability, LDH release level, ROS generation, cell cycle arrest and MMP expression levels. Finally, the effect of compound on MAPK and AP-1 signaling pathways were studied to understand the underlying signaling mechanisms. Result demonstrated that the presence of (2'S)-columbianetin suppressed the reactive oxygen species (ROS) generation, cell cycle arrest at sub-G1 phase and down regulation of MMP expression in UVB treated HaCaT cells. Furthermore, stress activated signaling pathways (ASK1-MAPK) and AP-1 signaling pathway were regulated by (2'S)-columbianetin treatment. These results suggest that (2'S)-columbianetin could be effectively used to protect human keratinocytes from UVB induced damage.

Diverse patterns of cyclooxygenase-independent metalloproteinase gene regulation in human monocytes.

BACKGROUND AND PURPOSE Matrix metalloproteinase (MMP) production from monocyte/macrophages is implicated in matrix remodelling and modulation of inflammation. However, knowledge of the patterns and mechanisms of gene regulation of MMPs and their endogenous tissue inhibitors (TIMPs) is fragmentary. MMP up-regulation may be a target for cyclooxygenase (COX) and prostaglandin (PG) receptor inhibition, but the extent and mechanisms of COX-independent MMP up-regulation are unclear. EXPERIMENTAL APPROACH We studied MMP mRNA expression and selected protein levels in human peripheral blood monocytes before and after adhesion, upon stimulation with bacterial lipopolysaccharide (LPS), PGE(2) or forskolin and after culturing with monocyte colony-stimulating factor on plastic or human fibronectin for up to 7 days. KEY RESULTS Monocyte adherence for 2 h transiently up-regulated COX-2, MMP-1, MMP-7 and MMP-10 mRNAs, and persistently up-regulated MMP-2, MMP-9, MMP-14 and MMP-19 mRNAs. LPS, PGE(2) or forskolin selectively increased MMP-1, MMP-9, MMP-10, MMP-12 and MMP-14 mRNAs. LPS increased PGE(2) production through COX but up-regulated MMP levels independently of COX. Differential dependence on inhibition of p42/44 and p38 mitogen-activated protein kinases, c-jun N-terminal kinase and inhibitor of κB kinase2 paralleled the diverse patterns of MMP stimulation by LPS. Differentiation on plastic increased mRNA levels of MMP-7, MMP-9, MMP-12 and MMP-14 and TIMP-2 and TIMP-3 independently of COX; fibronectin accelerated MMP but not TIMP up-regulation. CONCLUSIONS AND IMPLICATIONS Adhesion, LPS stimulation and maturation of human monocytes lead to selective, COX-independent MMP and TIMP gene regulation, which is a potential target for selective inhibition by signalling kinase inhibitors.

Possible roles for folic acid in the regulation of trophoblast invasion and placental development in normal early human pregnancy.

In addition to its role in the prevention of neural tube defects, folic acid has many other physiological functions, including cell proliferation, DNA replication, and antioxidant protection. The aim of this study was to determine the role that folic acid has in regulating placental trophoblast development. Placental explants from placentae at gestational age 7 wk (n = 3) were cultured in folic acid at concentrations of 10(-6) M, 10(-8) M, and 10(-10) M. Extravillous trophoblast (EVT) invasion was assessed following 6-day culture, and explants were used for immunohistochemical evaluation of proliferation (MKI67) and apoptosis (active caspase 3). In addition, an array was performed on cell culture supernatants to examine a range of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs). Folic acid increased the invasion of EVT cells in this explant model by between 83% and 19% (P = 0.005), and this was associated with increased MKI67 positivity and decreased active caspase 3 positivity; this effect was concentration dependent and showed a biphasic response. In addition, culture in folic acid increased vascular density, as determined by anti-CD31 immunostaining (P = 0.05). The increase in EVT invasion correlated with increased placental explant secretion of MMP2 (P = 0.01), MMP3 (P = 0.01), and MMP9 (P = 0.02). This study demonstrates that folic acid is potentially important in a number of crucial early stages of placental development, including EVT invasion, angiogenesis, and secretion of MMPs, and highlights the need for further studies to address the benefit of longer-term folic acid supplementation throughout pregnancy to prevent pregnancy disorders associated with deficient placental development, including preeclampsia.

Effects of the 532-nm and 1,064-nm Q-switched Nd:YAG lasers on collagen turnover of cultured human skin fibroblasts: a comparative study.

Cultured human skin fibroblasts were irradiated twice successively with the 1.5 J/cm(2) of 532-nm and 1,064-nm lasers, respectively. The mRNA of procollagen, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), heat-shock protein 70 (Hsp70), interleukin-6 (IL-6) and transforming growth factor beta (TGF-beta) were analyzed at 24 and 48 h post-irradiation by using RT-PCR. Both lasers significantly increased the expression of type I and III procollagen, TIMP1, and TIMP2, but decreased MMP1 and MMP2 expression. The 1,064-nm laser initiated TGF-beta expression while the 532-nm laser elicited the increase of Hsp70 and IL-6. The increase/decrease rates of procollagen, TIMPs and MMPs for the 1,064-nm laser were higher than that of the 532-nm laser. Thus, both lasers effectively accelerated collagen synthesis and inhibited collagen degradation. Collagen synthesis induced by the 1,064-nm laser might be partly due to the upregulation of TGF-beta expression, while the increase of Hsp70 and IL-6 might be partly responsible for collagen synthesis stimulated by the 532-nm laser. With the parameters used in this study, the 1,064-nm infrared laser is more effective in promoting the beneficial molecular activities than the 532-nm visible laser.

Pro-oxidant-mediated hepatic fibrosis and effects of antioxidant intervention in murine dietary steatohepatitis.

The mechanistic significance of oxidative stress to fibrogenesis in the methionine and choline-deficient (MCD) diet-induced model of steatohepatitis was evaluated by antioxidant intervention, using either vitamin E or L-2-oxothiazolidine-4-carboxylate (OTC), a cysteine precursor that promotes glutathione synthesis. Significant depletion of hepatic reduced glutathione (GSH) and elevation of thiobarbituric acid reactive substances (TBARS) occurred from week 3 in association with hepatic injury in mice fed the MCD diet. Hepatic stellate cell (HSC) activation and increased collagen alpha1(I) mRNA expression, together with morphologic fibrosis were evident from week 5. Vitamin E repleted GSH, reduced TBARS, steatosis, inflammation, HSC activation and collagen alpha1(I) mRNA expression, and ameliorated fibrosis. Vitamin E did not effect the expression of either profibrogenic cytokines (transforming growth factor-beta 1, connective tissue growth factor) or matrix remodeling enzymes (tissue inhibitor of metalloproteinase-1 and -2, matrix metalloproteinase-2 and -13). Despite repletion of hepatic GSH in OTC-supplemented mice, the initial benefit in the reduction of hepatic TBARS and inhibition of collagen alpha1(I) mRNA expression at week 5, failed to protect these mice from hepatic injury or fibrosis at later time points. Oxidative stress or products of lipid peroxidation mediate HSC activation and collagen gene expression directly in the MCD model of steatohepatitis. Vitamin E but not glutathione augmentation can interrupt this pathogenic process.

[Triptolide evaluates DNA methylation level of matrix metalloproteinase 9 gene in human fibrosarcoma HT-1080 cells].

OBJECTIVE: The effect of triptolide on the DNA methylation level of MMP-9 gene and the mRNA expression of tissue inhibitors of met-alloproteinases (TIMPs) were examined in human fibrosarcoma HT-1080 cells to explore the molecular mechanisms involved in the anticancer activity of triptolide. METHOD: HT-1080 cells were cultured in MEM containing 10% newborn calf serum and 1% penicillin-streptomycin. Triptolide was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 goL-1 and stored at -20 degrees C. Triptolide was freshly diluted with culture medium perior to use and directly added to cell cultures at the indicated concentration, and incubated for 72 hours at 37 degrees C in a humidified atmosphere with 5% CO2, with changes of reagents every 24 hours. Methylation specific PCR (MSP)was applied to assess the methylation status of MMP-9 gene promoter, and semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was employed to measure the mRNA expression of tissue inhibitors of metalloproteinases (TIMPs) in human fibrosarcoma HT-1080 cells after 72 hours of treatment with 6 nmol x L(-1), 12 nmol x L(-1) or 18 nmol x L(-1) triptolide, respectively. RESULTS: The methylation index of MMP-9 gene promoter was statistically elevated in HT-1080 cells after 72 hours of treatment with 18 nmol L(-1) triptolide, compared with those in controls (0.61 +/- 0.10 vs 0.39 +/- 0.10, P < 0.05), while no significant difference was noted between 6 nmol x L(-1) or 12 nmol x L(-1) triptolide treated HT-1080 cells and controls (0.40 +/- 0.15 vs 0.39 +/- 0.10, 0.46 +/- 0.20 vs 0.39 +/- 0.10, respectively, both P > 0.05). The mRNA expression of TIMP-1, -2, -3 or -4 was not significantly changed in HT-1080 cells after 72 hours of treatment with the indicated concentrations of triptolide, respectively compared with those in controls (all P > 0.05). CONCLUSION: The results demonstrated that triptolide upregulates the methylation level of MMP-9 gene in HT-1080 cells in vitro.

Beneficial regulation of matrixmetalloproteinases and their inhibitors, fibrillar collagens and transforming growth factor-beta by Polypodium leucotomos, directly or in dermal fibroblasts, ultraviolet radiated fibroblasts, and melanoma cells.

The extracellular matrix (ECM) that gives tissue its structural integrity is remodeled in skin aging/photoaging and cancer via the increased expression/activities of matrixmetalloproteinases (MMP), inhibition of the tissue inhibitors of matrix metalloproteinases (TIMP), or inhibition of collagen synthesis. Transforming growth factor-beta (TGF-beta), a predominant regulator of the ECM, is inhibited in aging/photoaging and stimulated in carcinogenesis. P. leucotomos (fern) extract has potential to counteract these alterations via its antioxidant, anti-inflammatory and photoprotective properties. The goal of this research was to determine the efficacy of P. leucotomos to (a) directly inhibit MMP-1, 2, 3, and 9 activities, (b) inhibit MMP-2, and stimulate TIMPs, fibrillar collagens and TGF-beta in non-irradiated or ultraviolet (UV) radiated fibroblasts, and (c) inhibit MMPs and TGF-beta, and stimulate TIMPs in melanoma cells. To this purpose, we examined the direct effect of P. leucotomos (0-1%) on MMPs' activities, and its effects on the expression (protein and/or transcription levels) of (1) MMPs and TIMPs in dermal fibroblasts, and melanoma cells, (2) TGF-beta in non-irradiated, UVA (2.5 J/cm2) or UVB (2.5 mJ/cm2) irradiated fibroblasts, and melanoma cells, and (3) types I, III, and V collagen in non-irradiated or UV irradiated fibroblasts. P. leucotomos directly inhibited the activities of MMPs as well as the expression of MMPs in fibroblasts, and melanoma cells while stimulating the expression of TIMPs in these cells. P. leucotomos stimulated types I, III, and V collagen in non-irradiated fibroblasts, and types I and V collagen in UV radiated fibroblasts. P. leucotomos had predominant stimulatory effects on TGF-beta expression in non-irradiated or UV radiated fibroblasts, and inhibited TGF-beta expression in melanoma cells. The effects of P. leucotomos were largely similar to that of ascorbic acid. P. leucotomos demonstrated dual protective effects on the ECM via its inhibition of the ECM proteolytic enzymes and the stimulation of the structural ECM collagens. The effects of P. leucotomos on fibroblasts and melanoma cells may be partly via its cell-specific regulation of TGF-beta expression and partly via its antioxidant property. The intake or topical application of P. leucotomos may be beneficial to skin health, in aging and cancer prevention or treatment.

alpha-Lipoic acid modulates extracellular matrix and angiogenesis gene expression in non-healing wounds treated with hyperbaric oxygen therapy.

alpha-Lipoic acid (LA) has been found previously to accelerate wound repair in patients affected by chronic wounds who underwent hyperbaric oxygen (HBO) therapy. Because proteinases are important in wound repair, we hypothesized that LA may regulate matrix metalloproteinase (MMP) expression in cells that are involved in wound repair. Patients undergoing HBO therapy were double-blind randomized into two groups: the LA group and the placebo group. Gene expression profiles for MMPs and for angiogenesis mediators were evaluated in biopsies collected at the first HBO session, at the seventh HBO session, and after 14 days of HBO treatment. ELISA tests were used to validate microarray expression of selected genes. LA supplementation in combination with HBO therapy downregulated the inflammatory cytokines and the growth factors which, in turn, affect MMPs expression. The disruption of the positive autocrine feedback loops that maintain the chronic wound state promotes progression of the healing process.

All-trans retinoic acid enhances murine dendritic cell migration to draining lymph nodes via the balance of matrix metalloproteinases and their inhibitors.

Cancers escape immune surveillance through the manipulation of the host's immune system. Sequestration of dendritic cells (DCs) within tumor tissues and the subsequent inhibition of their migration is one of the several mechanisms by which tumors induce immunosuppression. In view of recent findings depicting the improvement of tumor immune responses in cancer patients following all-trans retinoic acid (ATRA) treatment, we sought to identify the effects of ATRA on DC mobility in the context of tumor immunotherapy. Our results demonstrate that ATRA, added to differentiating murine bone marrow progenitor cells, enhances the invasive capacity of the resulting DCs. Immature DCs injected intratumorally in mice show increased accumulation in draining lymph nodes, but not in nondraining lymph nodes and spleens, when differentiated in the presence of ATRA. The in vitro migration of mature DCs through the basement membrane matrix toward the lymphoid chemokines CCL19 and CCL21 is enhanced in these cells, albeit not in the presence of a matrix metalloproteinase (MMP) inhibitor. An increase in MMP production with a simultaneous decrease in the production of their inhibitors (tissue inhibitors of matrix metalloproteinase or TIMPs) is provoked by ATRA. This affects the MMP/TIMP balance in DCs, in particular that of MMP-9 and TIMP-1, favoring protease activity and thus allowing for enhanced DC mobilization. In conclusion, this study demonstrates that ATRA is capable of improving DC trafficking in a tumor milieu and, in view of the encouraging results obtained in the clinic, further supports the notion that ATRA might be a valuable chemical adjuvant to current immunotherapeutic strategies for cancer.

Effects of hyperbaric oxygen on gene expressions of procollagen, matrix metalloproteinase and tissue inhibitor of metalloproteinase in injured medial collateral ligament and anterior cruciate ligament.

Animal experiments were performed to investigate whether and how the administration of hyperbaric oxygen (HBO) affects gene expressions of procollagens, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in injured medial collateral ligament (MCL) and anterior cruciate ligament (ACL). In 64 Sprague-Dawley rats, the MCL of the left knee was lacerated at the midsubstance, and the ACL of the left knee was lacerated adjacent to the tibial insertion in another 64 rats. Of these, 32 rats with lacerated MCL and 32 rats with lacerated ACL were housed in individual cages at normal atmospheric pressure (Groups MC and AC, respectively), while the remaining 64 rats were exposed to 100% oxygen at 2.5 atmospheres absolute for 2 h for 5 days a week (Groups MH and AH, respectively). Rats were sacrificed at 3, 7, 14 and 28 days postoperatively. After macroscopic examination, bilateral MCLs were harvested from Groups MC and MH, and bilateral ACLs from Groups AC and AH. Total RNA was extracted from each specimen and gene expressions of type I and type III procollagens, MMP-2, -9 and -3, and TIMP-1 and -2 were estimated using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Macroscopically, lacerated MCL healed by scar tissue formation, the amount of which appeared to be greater in Group MH than in Group MC. In contrast, no lacerated ACLs united, and little, if any, differences were apparent in macroscopic findings between Groups AH and AC. Gene expression of type I procollagen was significantly greater in Group MH than in Group MC at 7 days postoperatively and was also significantly greater in Group AH than in Group AC at 28 days (P<0.05). No significant differences in type III procollagen gene expression were noted between Groups MH and MC or between Groups AH and AC. In addition, no significant differences in gene expressions of MMPs were seen in either ligament, except that gene expression of MMP-13 was significantly lower at 7 days in Group MH than in Group MC (P<0.05). Gene expressions of TIMPs did not differ significantly between Groups MH and MC in each time interval, whereas gene expressions of TIMPs were significantly greater in Group AH than in Group AC at 7, 14 and 28 days for TIMP-1 and at 3, 7 and 14 days for TIMP-2 (P<0.05). RT-PCR results suggested that HBO enhances structural protein synthesis and inhibits degradative processes by enhancing TIMP activities in the lacerated ACL. However, none of the lacerated ACLs united macroscopically despite administration of HBO, indicating that the effect of HBO is insufficient for healing of the injured ACL. If HBO therapy is used as an adjunctive therapy after primary repair of the injured ACL, the success rate of surgery seems likely to be increased.

Prevention of high-fat diet-induced adipose tissue remodeling in obese diabetic mice by n-3 polyunsaturated fatty acids.

OBJECTIVE: Obesity is associated with reduced insulin sensitivity and extensive reorganization of adipose tissue. As polyunsaturated fatty acids (PUFA) appear to inhibit diabetes development, we investigated PUFA effects on markers of matrix remodeling in white adipose tissue. METHODS AND PROCEDURE: Male obese diabetic (db/db) mice were treated with either a low-fat standard diet (LF), or high-fat diets rich in saturated and monounsaturated fatty acids (HF/S), n-6 PUFA (HF/6) or the latter including marine n-3 PUFA (HF/3). White adipose tissue was analyzed for gene expression, fatty acid composition and by immunofluorescence. RESULTS: HF/S treatment increased adipose tissue expression of a number of genes involved in matrix degradation including matrix metalloproteinase (MMP)-12, -14 and cathepsin K, L and S compared with LF. MMP-12 gene was expressed in macrophages and adipocytes, and MMP-12 protein colocalized with both cell types. In addition, mean adipocyte area increased by 1.6-fold in HF/S-treated mice. Genes essential for collagen production, such as procollagen I, III, VI, tenascin C and biglycan were upregulated in HF/S-treated animals as well. N-3 PUFA supplementation resulted in enrichment of these fatty acids in adipose tissue. Moreover, n-3 PUFA inhibited the HF/S-induced upregulation of genes involved in matrix degradation and production I restored mean adipocyte area and prevented MMP-12 expression in macrophages and adipocytes. CONCLUSION: N-3 PUFA prevent high-fat diet-induced matrix remodeling and adipocyte enlargement in adipose tissue of obese diabetic mice. Such changes could contribute to diabetes prevention by n-3 PUFA in obese patients.