ABSTRACT
Ubiquitin C-terminal hydrolase-L1 (UCH-L1), which catalyzes the hydrolysis of ubiquitin esters and amides, is highly expressed in brain. Recently, UCH-L1 has been found to increase cancer cell migration and invasion by modulating hydrogen peroxide generated by NADPH oxidase 4 (NOX4). Because angiogenesis is also mediated by hydrogen peroxide, we explored the role of UCH-L1 in angiogenesis in human umbilical vein endothelial cells (HUVECs). Silencing UCH-L1 suppressed tubule formation in HUVECs, indicating that UCH-L1 promotes angiogenesis in vitro. This was confirmed using in vivo Matrigel plug studies of HUVECs, after overexpressing or silencing UCH-L1. Silencing UCH-L1 significantly suppressed VEGF-induced ROS levels as well as activation of VEGFR, both of which are required for angiogenesis. This study also showed that UCH-L1 promotes angiogenesis of HUVECs, as well as invasion in cancer cells, by up-regulating ROS by deubiquitination of NOX4, suggesting that UCH-L1 plays a key role in angiogenesis of HUVECS by regulating ROS levels by deubiquitination of NOX4.
Subject(s)
Endothelial Cells/physiology , Hydrogen Peroxide/metabolism , NADPH Oxidase 4/metabolism , Neovascularization, Physiologic/physiology , Ubiquitin Thiolesterase/metabolism , Vascular Endothelial Growth Factor A/metabolism , Cells, Cultured , Endothelial Cells/cytology , Gene Expression Regulation, Enzymologic/physiology , Humans , Reactive Oxygen Species/metabolism , Receptors, Vascular Endothelial Growth Factor/metabolism , Ubiquitination/physiologyABSTRACT
This study explored the role of ubiquitin C-terminal hydrolase-L1 (UCH-L1) in the production of ROS and tumor invasion. UCH-L1 was found to increase cellular ROS levels and promote cell invasion. Silencing UCH-L1, as well as inhibition of H2O2 generation by catalase or by DPI, a NOX inhibitor, suppressed the migration potential of B16F10 cells, indicating that UCH-L1 promotes cell migration by up-regulating H2O2 generation. Silencing NOX4, which generates H2O2, with siRNA eliminated the effect of UCH-L1 on cell migration. On the other hand, NOX4 overexpressed in HeLa cells happens to be ubiquitinated, and NOX4 following deubiquitination by UCH-L1, restored H2O2-generating activity. These in vitro findings are consistent with the results obtained in vivo with catalase (-/-) C57BL/6J mice. When H2O2 and UCH-L1 levels were independently varied in these animals, the former by infecting with H2O2-scavenging adenovirus-catalase, and the latter by overexpressing or silencing UCH-L1, pulmonary metastasis of B16F10 cells overexpressing UCH-L1 increased significantly in catalase (-/-) mice. In contrast, invasion did not increase when UCH-L1 was silenced in the B16F10 cells. These findings indicate that H2O2 levels regulated by UCH-L1 are necessary for cell invasion to occur and demonstrate that UCH-L1 promotes cell invasion by up-regulating H2O2 via deubiquitination of NOX4.
Subject(s)
Catalase/physiology , Cell Movement , Hydrogen Peroxide/metabolism , Lung Neoplasms/secondary , Melanoma, Experimental/pathology , NADPH Oxidases/metabolism , Ubiquitin Thiolesterase/metabolism , Animals , Apoptosis , Blotting, Western , Cell Proliferation , HeLa Cells , Humans , Immunoenzyme Techniques , Immunoprecipitation , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Male , Melanoma, Experimental/genetics , Melanoma, Experimental/metabolism , Mice , Mice, Inbred C57BL , Mice, Nude , NADPH Oxidases/genetics , Neoplasm Invasiveness , Protein Processing, Post-Translational , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Reactive Oxygen Species/metabolism , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Tumor Cells, Cultured , Ubiquitin/metabolism , Ubiquitin Thiolesterase/antagonists & inhibitors , Ubiquitin Thiolesterase/genetics , Xenograft Model Antitumor AssaysABSTRACT
Ocimum sanctum has been known to possess various beneficial properties including anti-oxidative, anti-inflammatory and anti-cancer activities. In the present study, we investigated that ethanol extracts of O. sanctum (EEOS) had anti-metastatic activity through activation of anti-oxidative enzymes. EEOS exerted cytotoxicity against Lewis lung carcinoma (LLC) cells. Also, EEOS significantly inhibited cell adhesion and invasion as well as activities of matrix metalloproteinase-9 (MMP-9), but not MMP-2, indicating the important role of MMP-9 in anti-metastatic regulation of EEOS. In addition, EEOS significantly reduced the tumor nodule formation and lung weight in LLC-injected mice. Inhibitory effect of EEOS on metastasis was further confirmed by using hematoxylin and eosin (H&E) staining. Notably, we also found that EEOS enhanced activities of anti-oxidative enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) in a concentration-dependent manner. Taken together, our findings support that EEOS can be a potent anti-metastatic candidate through inactivation of MMP-9 and enhancement of anti-oxidant enzymes.
Subject(s)
Antioxidants/metabolism , Ethanol/chemistry , Matrix Metalloproteinase Inhibitors , Neoplasm Metastasis/prevention & control , Ocimum/chemistry , Plant Extracts/pharmacology , Animals , Carcinoma, Lewis Lung/pathology , Catalase/metabolism , Cell Line, Tumor , Glutathione Peroxidase/metabolism , Mice , Superoxide Dismutase/metabolismABSTRACT
Although Ocimum sanctum has been used extensively for its medicinal values in India and China, its antitumor activity against human nonsmall cell lung carcinoma (NSCLC) A549 cells has not been investigated until now. Therefore, the antitumor mechanism of ethanol extracts of Ocimum sanctum (EEOS) was elucidated in A549 cells in vitro and the Lewis lung carcinoma (LLC) animal model. EEOS exerted cytotoxicity against A549 cells, increased the sub-G1 population and exhibited apoptotic bodies in A549 cells. Furthermore, EEOS cleaved poly(ADP-ribose)polymerase (PARP), released cytochrome C into cytosol and simultaneously activated caspase-9 and -3 proteins. Also, EEOS increased the ratio of proapoptotic protein Bax/antiapoptotic protein Bcl-2 and inhibited the phosphorylation of Akt and extracellular signal regulated kinase (ERK) in A549 cancer cells. In addition, it was found that EEOS can suppress the growth of LLC inoculated onto C57BL/6 mice in a dose-dependent manner. Overall, these results demonstrate that EEOS induces apoptosis in A549 cells via a mitochondria caspase dependent pathway and inhibits the in vivo growth of LLC, suggesting that EEOS can be applied to lung carcinoma as a chemopreventive candidate.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Apoptosis/drug effects , Carcinoma, Lewis Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Ocimum , Plant Extracts/therapeutic use , Animals , Antineoplastic Agents, Phytogenic/pharmacology , Carcinoma, Lewis Lung/metabolism , Carcinoma, Non-Small-Cell Lung/metabolism , Caspase 3/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Cytochromes c/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Lung Neoplasms/metabolism , Mice , Mice, Inbred C57BL , Phosphorylation , Phytotherapy , Plant Extracts/pharmacology , Plant Leaves , Poly(ADP-ribose) Polymerases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , bcl-2-Associated X Protein/metabolismABSTRACT
Carcinoma of the stomach is reportedly the second most common cancerous condition affecting the general population. Administration of antioxidants is reported to effectively alleviate the risk of gastric carcinoma. Therefore, we assessed the protective role of naringenin, an antioxidant and naturally occurring citrus flavanone, on gastric carcinogenesis induced by MNNG (200 mg/kg body weight) and S-NaCl (1 mL per rat) in Wistar rats (obtained from the Central Animal House Facility, University of Madras, Taramani Campus, Chennai, India). The animals were divided into 5 groups, and the effects of naringenin on simultaneous and posttreated stages of MNNG were tested. Cancer risk was analyzed along with their antioxidant status. The LPO levels in the experimental groups were assessed as an index of oxidative milieu. Altered redox status was subsequently investigated by assaying the superoxide and hydroxyl radicals, the enzymatic antioxidants (SOD, CAT, GPx), and the nonenzymatic antioxidants viz reduced GSH, vitamin C, and vitamin E. In the presence of MNNG, cancer incidence and LPO levels were significantly increased, whereas enzymatic (SOD, CAT, and GPx) and nonenzymatic antioxidant activities (GSH, Vitamins C, and E) were decreased in the treated rats compared with control rats. Administration of naringenin to gastric carcinoma-induced rats largely up-regulated the redox status to decrease the risk of cancer. We conclude that up-regulation of antioxidants by naringenin treatment might be responsible for the anticancer effect in gastric carcinoma.
Subject(s)
Antineoplastic Agents/pharmacology , Antioxidants/metabolism , Carcinoma/drug therapy , Flavanones/pharmacology , Stomach Neoplasms/drug therapy , Weight Loss/drug effects , Animals , Carcinoma/chemically induced , Carcinoma/pathology , Cysteine/analogs & derivatives , Disease Models, Animal , Gastric Mucosa/metabolism , Lipid Peroxidation/drug effects , Male , Methylnitronitrosoguanidine/toxicity , Oxidation-Reduction , Random Allocation , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Stomach Neoplasms/chemically induced , Stomach Neoplasms/pathology , Superoxide Dismutase/metabolism , Thiobarbituric Acid Reactive Substances/metabolismABSTRACT
Lithospermum erythrorhizon has been used for treatment of inflammatory diseases and cancer as a folk remedy. Based on the evidences that anti-inflammatory agents frequently exert antiangiogenic activity, thus we examined comparatively the antiangiogenic activities of three naphthoquinone derivatives (shikonin, acetylshikonin, and isobutyroylshikonin) isolated from the plant. Three derivatives exhibited weak cytotoxicity against human umbilical vein endothelial cells (HUVECs) with IC50 of over 20 microM. Shikonin had more specific inhibitory effects on proliferation and vascular endothelial growth factor (VEGF) production by VEGF compared with different derivatives. All of derivatives significantly suppressed the migration of VEGF treated HUVECs at different optimal concentrations. Also, shikonin and acetylshikonin significantly disrupted VEGF-induced tube formation. Furthermore, three derivatives effectively downregulated the expression of urokinase-type plasminogen activator (uPA), but not its receptor uPAR. Additionally, shikonin significantly inhibited tumor growth in LLC-bearing mice, whereas its derivatives had relatively mild effects. Taken together, our findings suggest that shikonin and its derivatives exhibit the antiangiogenic and antitumorigenic effects by suppressing proliferation and angiogenic factors.
