ROS-mediated Different Homeostasis of Murine Corneal Epithelial Progenitor Cell Line under Oxidative Stress.

The role of ROS in stem cell biology has not been fully illustrated and understood. Here we compared the different responses and investigated the mechanism underlying oxidative stress induced by hydrogen peroxide (H2O2) between murine corneal epithelial progenitor cell line (TKE2) and mature murine corneal epithelial cells (MCE). TKE2 showed a different homeostasis and strong resistance to H2O2. TKE2 reduced the production of ROS, inhibited ROS generation enzyme NADPH oxidase 4 (NOX4), and increased dual specificity phosphatase 6 (DUSP6). Furthermore, TKE2 activated nuclear factor (erythroid-derived 2)-like 2 (NRF2) signaling pathway, regulated miR-125B1 and miR-29B1, and elevated levels of antioxidants glutathione S-transferase P (GSTP) and superoxide dismutases (SOD). The association with ROS of the cells was also verified by RNA interference approach and pharmacological antagonization. In addition, TKE2 enhanced the autophagy after exposure to H2O2. The novel evidence suggests that TKE2 cells have different homeostasis and strong antioxidant properties against oxidative stress via the regulation of ROS formation and pathway.

The oxidant role of 4-hydroxynonenal in corneal epithelium.

4-Hydroxynonenal (4-HNE or HNE) is a main endogenous product of cellular lipid peroxidation in tissues and is reported to play pathogenic roles in eye diseases. Here we investigated the association between 4-HNE and oxidative stress in the corneal epithelium. 4-HNE suppressed the cell viability of human corneal epithelial cells (HCE) in a concentration dependent manner. 4-HNE significantly increased the level of 3-Nitrotyrosine (3-NT), a marker of oxidative stress, in HCE cells and corneal epithelium of rats by immunofluorescent staining and Western blot analysis. To its underlying mechanistic on ROS system, 4-HNE elevated the ROS generation enzyme NADPH oxidase 4 (NOX4) and induced the activation of NF-E2-related factor-2 (NRF2) and its downstream effectors: NAD(P)H dehydrogenase (quinone 1) (NQO1) and glutathione S-transferase P (GSTP). Furthermore, N-acetylcysteine (NAC), an antioxidant and ROS scavenger, antagonized the inhibitory and oxidant effects of 4-HNE on the corneal epithelial cells. In conclusion, 4-HNE plays an oxidant role in the corneal epithelium and this work provides a new strategy for the pathogenesis and treatment of corneal diseases.

Synthesis and evaluation of trehalose-based compounds as novel inhibitors of cancer cell migration and invasion.

As a continuous research for the discovery of trehalose-based anti-invasive agents, we developed a convenient synthetic approach for the preparation of 6,6'-dideoxy-6,6'-bis(acylamino)-α,α-D-trehaloses. A series of trehalose-based amides were prepared through the trityl protection of the two primary hydroxyls of α,α-D-trehalose, benzoylation, the removal of the trityl protective group, mesylation, azidation, catalytic hydrogenation in the presence of hydrochloride, coupling reaction with a variety of acids, and subsequent debenzoylation and deacetylation in some cases. Compound 8b, 6,6'-dideoxy-6,6'-bis(2-hydroxybenzamide)-α,α-D-trehalose, was just as potent as the natural brartemicin against the invasion of murine colon 26-L5 cells. It exhibited no cytotoxicity on human breast adenocarcinoma MDA-MB-231 and murine colon 26-L5 cells. It can significantly inhibit the migration and invasion of the MDA-MB-231 cells. The anti-invasive effect of 8b was possibly related to its inhibitory activity on MMP-9, its suppression on the expression of MMP-9 and VEGF, and its deactivation of Akt.

SERPINA3K plays antioxidant roles in cultured pterygial epithelial cells through regulating ROS system.

We recently demonstrated that SERPINA3K, a serine proteinase inhibitor, has antioxidant activity in the cornea. Here we investigated the antioxidant effects of SERPINA3K on the pterygial, which is partially caused by oxidative stress in pathogenesis. The head part of primary pterygial tissue was dissected and then cultured in keratinocyte serum-free defined medium (KSFM). The cultured pterygial epithelial cells (PECs) were treated with SERPINA3K. The cell proliferation and migration of PECs were measured and analyzed. Western blot and quantitative real-time polymerase chain reaction (PCR) assay were performed. It showed that SERPINA3K significantly suppressed the cell proliferation of PECs in a concentration-dependent manner, compared with cultured human conjunctival epithelial cells. SERPINA3K also inhibited the cell migration of PECs. Towards its underlying mechanism, SERPINA3K had antioxidant activities on the PECs by significantly inhibiting NADPH oxidase 4 (NOX4), which is an important enzyme of ROS generation, and by elevating the levels of key antioxidant factors of ROS: such as NAD(P)H dehydrogenase (quinone 1) (NQO1), NF-E2-related factor-2 (NRF2) and superoxide dismutases (SOD2). Meanwhile, SERPINA3K down-regulated the key effectors of Wnt signaling pathway: ß-catenin, nonphospho-ß-catenin, and low-density lipoprotein receptor-related protein 6 (LRP6). We provided novel evidence that SERPINA3K had inhibitory effects on pterygium and SERPINA3K played antioxidant role via regulating the ROS system and antioxidants.

Inhibition of invasion and metastasis by DMBT, a novel trehalose derivative, through Akt/GSK-3ß/ß-catenin pathway in B16BL6 cells.

Invasion, either directly or via metastasis formation, is the main cause of death in cancer patients. Development of efficient anti-invasive agents is an important research challenge. 6,6'-bis (2,3-dimethoxybenzoyl)-a, a-d-trehalose (DMBT), one of brartemicin analogs, was found to be the most potent anti-invasive agent, but the underlying mechanisms are poorly understood. Our current study was to explore the effects of DMBT on invasion and metastasis in B16BL6 cells. Antiproliferation assay and trypan blue exclusion assay showed that no obvious inhibitory or cytotoxic effect of DMBT was found in B16BL6 cells. Wound healing demonstrated that DMBT could inhibit cell migration compared with the normal group. Transwell experiments showed that DMBT could significantly inhibit invasion to the reconstituted basement membrane (P<0.01). We examined the effects of lung metastasis produced by highly metastatic B16BL6 melanoma cells by using experimental metastasis models and BLI analysis. DMBT could significantly suppress lung metastasis in mice. Results from immunohistochemical staining, Western blotting and real-time PCR indicated that the chemopreventive effect of DMBT was attributed to the inhibition of the VEGF and MMP-9 through Akt/GSK-3ß/ß-catenin and Akt/mTOR signaling pathways. These results suggested that DMBT could be a promising lead molecule for the anti-metastasis and serve as a therapeutic agent to inhibit cancer cell invasion and metastasis.