In-Sook An

Akt is negatively regulated by the MULAN E3 ligase

The serine/threonine kinase Akt functions in multiple cellular processes, including cell survival and tumor development. Studies of the mechanisms that negatively regulate Akt have focused on dephosphorylation-mediated inactivation. In this study, we identified a negative regulator of Akt, MULAN, which possesses both a RING finger domain and E3 ubiquitin ligase activity. Akt was found to directly interact with MULAN and to be ubiquitinated by MULAN in vitro and in vivo. Other molecular assays demonstrated that phosphorylated Akt is a substantive target for both interaction with MULAN and ubiquitination by MULAN. The results of the functional studies suggest that the degradation of Akt by MULAN suppresses cell proliferation and viability. These data provide insight into the Akt ubiquitination signaling network.

Ginsenoside Rh2 mediates changes in the microRNA expression profile of human non-small cell lung cancer A549 cells

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer insensitive to chemotherapy. Efforts are, therefore, directed toward understanding the molecular mechanisms of chemotherapy insensitivity and the development of new anticancer drugs. Ginsenoside Rh2, one of the components in ginseng saponin, has been shown to have anti-proliferative effect on human NSCLC cells and is being studied as a therapeutic drug for NSCLC. microRNAs (miRNAs) are small, non-coding RNA molecules that play a key role in cancer progression and prevention. However, the miRNA portrait of ginsenoside Rh2-treated NSCLC cells has not yet been studied. In this study, we identified a unique set of changes in the miRNA expression profile in response to Rh2 treatment in the human NSCLC cell line A549. Using miRNA microarray analysis, we identified 44 and 24 miRNAs displaying changes in expression greater than 2-fold in Rh2-treated A549 cells. In addition, using an miRNA target prediction program, we discovered that these miRNAs are predicted to have several target genes related to angiogenesis, apoptosis, chromatic modification, cell proliferation and differentiation. Thus, these results may assist in the better understanding of the anticancer mechanism of Rh2 in NSCLC.

Titrated extract of Centella asiatica provides a UVB protective effect by altering microRNA expression profiles in human dermal fibroblasts

The titrated extract of Centella asiatica (TECA) is a reconstituted mixture comprising of asiatic acid, madecassic acid, asiaticoside and madecassoside, and is used as a therapeutic agent in wound healing and also as an anti-microbial, anticancer and anti-aging agent. Although these properties and the associated cell signaling pathways have been elucidated, the cellular mechanism of anti-photoaging upon ultraviolet (UV) exposure in normal human dermal fibroblasts (NHDFs) remains unknown. In this study, we investigated the photoprotective role of TECA via microRNA (miRNA) expression profiling analysis. Low dose of TECA did not exhibit toxicity and showed a protective effect against UVB irradiation in NDHFs. miRNA microarray experiments revealed that specific miRNAs were altered by TECA stimulation in UVB-irradiated NHDFs. Functional bioinformatic analysis showed that the putative target genes of the altered miRNAs were associated with the positive regulation of cell proliferation, anti-apoptosis, small GTPase- and Ras-mediated signal transduction and activation of MAPKK. Therefore, these results suggest that TECA may serve as a potential natural chemoprotective agent against UVB-mediated damage in NHDFs through changes in the expression of specific miRNAs.

Oridonin protects HaCaT keratinocytes against hydrogen peroxide-induced oxidative stress by altering microRNA expression

microRNAs (miRNAs) have been shown to function as primary regulators of a variety of biological processes, including proliferation, differentiation and apoptosis in human keratinocytes. However, the biological significance of miRNAs in the defense against oxidative stress in keratinocytes remains to be elucidated. In this study, we demonstrate that oridonin, a diterpenoid compound isolated from Rabdosia rubescens with established antioxidant properties, protects HaCaT human keratinocytes from oxidative stress induced by exposure to hydrogen peroxide (H2O2). Our data demonstrate that low doses of oridonin (1-5 µM) protect keratinocytes against H2O2-induced apoptosis in a concentration- and time-dependent manner. Moreover, as shown by our results, oridonin markedly decreased H2O2-induced reactive oxygen species production in HaCaT cells. Oridonin mediated these effects by altering miRNA expression. Bioinformatics analysis identified several putative target genes of the differentially expressed miRNAs. Assessment of their gene ontology annotation revealed that these target genes are likely involved in cell growth and inhibition of apoptosis. Thus, the data from this study establish a role for miRNAs in mediating oridonin-induced protective effects against oxidative stress in human keratinocytes.

The hypoxia-mimetic agent cobalt chloride induces cell cycle arrest and alters gene expression in U266 multiple myeloma cells

Hypoxia is a common feature of tumors that occurs across a wide variety of malignancies. Multiple myeloma is an incurable malignant disorder of plasma cells in the bone marrow. Although bone marrow hypoxia is crucial for normal hematopoiesis, the effect of hypoxia on multiple myeloma is poorly understood. In this study, we demonstrated that cobalt chloride (CoCl2)-mediated hypoxia decreased cell viability and altered gene expression in U266 human multiple myeloma cells. CoCl2 induced the loss of cell viability in a concentration-dependent manner. In addition, FACS analysis revealed that the loss of cell viability was related to apoptosis. Using microarray analysis, we identified mRNA expression profile changes in response to CoCl2 treatment in U266 cells. Four hundred and fifty-two mRNAs exhibited >2-fold changes in expression in CoCl2-treated U266 cells compared to their expression in control cells. A follow-up bioinformatics study revealed that a great number of genes with altered expression were involved in apoptosis, cell cycle, transcription and development. In conclusion, these results provide novel evidence that CoCl2-mediated hypoxia affects the expression profiles of genes that are functionally related to apoptosis and angiogenesis in U266 multiple myeloma cells.

Troxerutin induces protective effects against ultraviolet B radiation through the alteration of microRNA expression in human HaCaT keratinocyte cells.

Ultraviolet light B (UVB), contained in sunlight, induces damaging effects on skin by impairing cells in the epidermis and dermis. In particular, keratinocytes in the epidermis are those cells which are mainly affected by UVB light. UVB radiation induces cell death, growth arrest, DNA damage and restricts cell migration. Various phytochemicals have been shown to alleviate UVB-induced cellular damage. Troxerutin is a natural flavonoid rutin mainly found in extracts of Sophora japonica, and is a well-known antioxidant and anti-inflammatory compound used in experimental mouse models. In this study, we examined the effects of troxerutin on UVB-induced damage in HaCaT cells. HaCaT cells were pre-treated with troxerutin (0-10 µM) and then exposed to UVB radiation (50 mJ/cm2). Cell viability, cell cycle and migration assays were performed to determine the protective effects of troxerutin on the cells. DNA repair activity was also measured. Troxerutin protected the cells against UVB-induced damage, such as cell death, growth arrest, restriction of cell migration and decreased DNA repair activity in HaCaT cells. Analyses of microRNA (miRNA) expression demonstrated that the protective effects of troxerutin correlated with alterations in miRNA expression, as indicated by Gene Ontology analyses of putative target genes. Overall, our data demonstrate that troxerutin exerts protective effects against UVB-induced damage by regulating miRNA expression.

Arctiin blocks hydrogen peroxide-induced senescence and cell death though microRNA expression changes in human dermal papilla cells

BackgroundAccumulating evidence indicates that reactive oxygen species (ROS) are an important etiological factor for the induction of dermal papilla cell senescence and hair loss, which is also known alopecia. Arctiin is an active lignin isolated from Arctium lappa and has anti-inflammation, anti-microbial, and anti-carcinogenic effects. In the present study, we found that arctiin exerts anti-oxidative effects on human hair dermal papilla cells (HHDPCs).ResultsTo better understand the mechanism, we analyzed the level of hydrogen peroxide (H2O2)-induced cytotoxicity, cell death, ROS production and senescence after arctiin pretreatment of HHDPCs. The results showed that arctiin pretreatment significantly inhibited the H2O2-induced reduction in cell viability. Moreover, H2O2-induced sub-G1 phase accumulation and G2 cell cycle arrest were also downregulated by arctiin pretreatment. Interestingly, the increase in intracellular ROS mediated by H2O2 was drastically decreased in HHDPCs cultured in the presence of arctiin. This effect was confirmed by senescence associated-beta galactosidase (SA-β-gal) assay results; we found that arctiin pretreatment impaired H2O2-induced senescence in HHDPCs. Using microRNA (miRNA) microarray and bioinformatic analysis, we showed that this anti-oxidative effect of arctiin in HHDPCs was related with mitogen-activated protein kinase (MAPK) and Wnt signaling pathways.ConclusionsTaken together, our data suggest that arctiin has a protective effect on ROS-induced cell dysfunction in HHDPCs and may therefore be useful for alopecia prevention and treatment strategies.

MicroRNA-1290 promotes asiatic acid‑induced apoptosis by decreasing BCL2 protein level in A549 non‑small cell lung carcinoma cells

Asiatic acid, a triterpenoid derived from Centella asiatica, is a putative anticancer agent in several types of cancer cells. Investigations of its biological role in negative regulation of cell growth have focused on the extent of induction of apoptosis in a cell-type-specific manner. In this study, we identified an important regulator of asiatic acid-induced cell death, microRNA (miR)-1290, which sensitizes cells to asiatic acid-induced cytotoxicity and negatively regulates BCL2 expression. Asiatic acid significantly upregulated miR-1290, and asiatic acid-induced cell death was shown to be dependent on miR-1290 activity. Molecular assays demonstrated that BCL2 mRNA is a direct target of miR-1290-mediated RNA interference. The results of functional studies suggest that miR-1290 suppresses cell viability and cell cycle progression. These data provide insight into miR-1290-mediated cellular mechanisms in asiatic acid-treated A549 non-small cell lung carcinoma cells.

Identification of microRNAs involved in growth arrest and cell death in hydrogen peroxide-treated human dermal papilla cells

microRNAs (miRNAs) are small non‑coding RNAs that regulate various biological processes by interfering with the translation of target genes. Several studies have suggested that miRNAs are involved in cellular responses to hydrogen peroxide (H2O2). Reactive oxygen species (ROS) are involved in hair malignancies, however, the H2O2‑induced, miRNA‑dependent regulatory mechanisms of human dermal papilla (HDP) cells are not fully understood. Our previous study demonstrated that changes in miRNA expression function to regulate growth arrest and apoptosis in UVB‑irradiated HDPs. In the present study, miRNA expression was profiled in HDPs treated with H2O2. The transcriptome analysis of H2O2‑treated HDPs enabled the identification of 68 differentially expressed miRNAs (62 were upregulated and 6 were downregulated) and 14,316 putative target genes of the miRNAs. Gene ontology (GO) analysis was utilized to verify that the putative target genes of the altered miRNAs were associated with H2O2‑induced cell growth arrest and apoptosis. This bioinformatics analysis indicated that H2O2‑response pathways involved in growth arrest and apoptosis were significantly affected. The identification of miRNAs and their putative targets may offer new therapeutic strategies for H2O2‑induced hair follicle disorders.

Arctiin induces an UVB protective effect in human dermal fibroblast cells through microRNA expression changes

Ultraviolet (UV) radiation induces severe alterations in the molecular and cellular components of normal human dermal fibroblast (NHDF) cells by disrupting many intracellular transduction cascades. Although UV responses have been well documented at the genome and proteome levels, UV protective effects have not been elucidated at these levels. The aim of the present study was to demonstrate that arctiin, a phytochemical isolated from the plant Arctium lappa, induced a protective effect against UVB radiation by changing microRNA (miRNA) expression profiles. Using flow cytometry, and water-soluble tetrazolium salt (WST-1)-based cell viability, wound healing, and DNA repair assays we showed that pretreatment with arctiin prior to UVB irradiation reduced UVB-induced apoptosis, cell migration defects, and DNA damage in NHDF cells. It was also found that arctiin‑induced UVB protection is associated with altered miRNA expression profiles. Bioinformatic analysis revealed that the deregulated miRNAs were functionally involved in mitogen-activated protein kinase (MAPK) signaling and cancer signaling pathways. The results suggest that arctiin acts as a UVB protective agent by altering specific miRNA expression in NHDF cells.