Seunghee Bae

Resveratrol alters microRNA expression profiles in A549 human non-small cell lung cancer cells

Resveratrol is a plant phenolic phytoalexin that has been reported to have antitumor properties in several types of cancers. In particular, several studies have suggested that resveratrol exerts antiproliferative effects against A549 human non-small cell lung cancer cells; however, its mechanism of action remains incompletely understood. Deregulation of microRNAs (miRNAs), a class of small, noncoding, regulatory RNA molecules involved in gene expression, is strongly correlated with lung cancer. In this study, we demonstrated that resveratrol treatment altered miRNA expression in A549 cells. Using microarray analysis, we identified 71 miRNAs exhibiting greater than 2-fold expression changes in resveratrol-treated cells relative to their expression levels in untreated cells. Furthermore, we identified target genes related to apoptosis, cell cycle regulation, cell proliferation, and differentiation using a miRNA target-prediction program. In conclusion, our data demonstrate that resveratrol induces considerable changes in the miRNA expression profiles of A549 cells, suggesting a novel approach for studying the anticancer mechanisms of resveratrol.

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.

Cooperative actions of p21WAF1 and p53 induce Slug protein degradation and suppress cell invasion

How the p53 transcription factor/tumor suppressor inhibits cell invasion is poorly understood. We demonstrate that this function of p53 requires its direct interaction with p21WAF1, a transcriptional target of p53, and that both p21 and p53 bind to Slug, which promotes cell invasion. Functional studies reveal that p21 and p53 cooperate to facilitate Mdm2‐dependent Slug degradation and that this p53 function is mimicked by p53R273H, a mutant lacking trans‐activating activity. These actions of p21 and p53 are induced by γ‐irradiation of cells and also operate in vivo. This is the first study to elucidate a mechanism involving p53 and p21 cooperation.

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.

Analysis of the microRNA expression profile of normal human dermal papilla cells treated with 5α-dihydrotestosterone.

Clinical evidence has demonstrated that the accumulation of 5α-dihydrotestosterone (DHT) in dermal papilla cells (DPCs) is implicated in androgenetic alopecia. Whether this accumulation in DHT may have direct cellular effects leading to androgenetic alopecia remains to be elucidated. The present study aimed to determine whether DHT affects cell growth, cell cycle arrest, cell death, senescence and the induction of reactive oxygen species (ROS), and whether these effects are mediated by microRNA (miRNA)-dependent mechanisms. The cell viability and cell cycle were determined, levels of ROS were examined and senescence-associated β-galactosidase assays were performed in normal human DPCs (nHDPCs). Furthermore, miRNA expression profiling was performed using an miRNA microarray to determine whether changes in the expression levels of miRNA were associated with the cellular effects of DHT. The results revealed that DHT decreased cell growth by inducing cell death and G2 cell cycle arrest, and by increasing the production of ROS and senescence in the nHDPCs. In addition, 55 miRNAs were upregulated and 6 miRNAs were downregulated in the DHT-treated nHDPCs. Bioinformatic analysis demonstrated that the putative target genes of these upregulated and downregulated miRNAs were involved in cell growth, cell cycle arrest, cell death, senescence and the production of ROS. Specifically, the target genes of five highly upregulated and downregulated miRNAs were identified and were associated with the aforementioned effects of DHT. These results demonstrated that the expression of miRNA was altered in the DHT-treated nHDPCs and suggest the potential mechanisms of DHT-induced cell growth repression, cell cycle arrest, cell death, senescence and induction of ROS.

Identification of ultraviolet B radiation-induced microRNAs in normal human dermal papilla cells

Ultraviolet (UV) radiation impairs intracellular functions by directly damaging DNA and by indirectly generating reactive oxygen species (ROS), which induce cell cycle arrest and apoptosis. UV radiation can also alter gene expression profiles, including those of mRNA and microRNA (miRNA). The effects of UV radiation on cellular functions and gene expression have been widely documented in human skin cells such as keratinocytes, melanocytes and dermal fibroblasts, but the effect it has on other types of skin cell such as dermal papilla cells, which are crucial in the induction of hair follicle growth, remains unknown. In the current study, the effect of UV radiation on physiological changes and miRNA-based expression profiles in normal human dermal papilla cells (nHDPs) was investigated. UVB radiation of ≥50 mJ/cm2 displayed high cytotoxicity and apoptosis in a dose-dependent manner. In addition, ROS generation was exhibited in UVB-irradiated nHDPs. Furthermore, using miRNA microarray analysis, it was demonstrated that the expression profiles of 42 miRNAs in UVB-irradiated nHDPs were significantly altered compared with those in the controls (35 upregulated and 7 downregulated). The biological functions of the differentially expressed miRNAs were studied with gene ontology analysis to identify their putative target mRNAs, and were demonstrated to be involved in cell survival- and death-related functions. Overall, the results of the present study provide evidence that miRNA-based cellular mechanisms may be involved in the UVB-induced cellular response in nHDPs.

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.