Aim: Protein L-isoaspartyl O-methyltransferase (PIMT) regulates cell adhesion in various cancer cell lines through activation of integrin αv and the PI3K pathway. The epithelial mesenchymal transition (EMT) enables epithelial cells to acquire the characteristics of mesenchymal cells, and to allow them to migrate for metastasis. Here, we examined the relationship between PIMT and EMT with attached or detached MDA-MB 231 cells. Methods: Human breast cancer cell line MDA-MB-231 cells were maintained in a suspension on poly-HEMA in the presence or absence of PIMT siRNA or ERK inhibitor PD98059. The mRNAs and proteins were analyzed using RT-PCR and immunoblotting, respectively. Results: During cellular incubation under detached conditions, PIMT, integrin αv and EMT proteins, such as Snail, Slug and matrix metalloproteinase 2 (MMP-2), were significantly increased in correlation with the phosphorylation of ERK1/2. The ERK inhibitor PD98059 (25 µmol/L) strongly suppressed the expression of the proteins and PIMT. Interestingly, PIMT siRNA blocked the phosphorylation of ERK and the expression of the EMT proteins. Additionally, PIMT and ERK phosphorylation were both co-activated by treatment with TGF-β (10 ng/mL) and TNF-α (10 ng/mL). Conclusion: A tight cross-regulation exists between ERK and PIMT in regards to their activation and expression during the EMT. Because the role of PIMT in the EMT and metastatic processes remains unclear, in this study, we explored the involvement of PIMT in the regulation of the detachment and attachment of the anoikis-resistant cell line MDA-MB-231, an aggressive breast cancer cell line with a highly invasive, migrative, and metastatic characters [10] , by culturing the cells in poly-HEMA (2-hydroxyethylmethacrylate)-coated dishes and introducing siRNA specific for PIMT. Materials and methods MaterialsLiCl and poly-HEMA were purchased from Sigma-Aldrich (St Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin solution (10 000 unit/mL and 10 mg/mL, respectively), fetal bovine serum (FBS) and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Gibco BRL (Gaithersburg, MD, USA). MDA-MB 231 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Moloney murine leukemia virus (M-MLV) reverse transcriptase, polymerase chain reaction (PCR) premix, and Sapphire Super Taq were purchased from Rexgene Biotech Co, Ltd (Ochang, Korea). All primers used for PCR were purchased from Bioneer (Taejeon, Korea). A mixture of Stealth TM /siRNA duplex oligoribonucleotides against PIMT and Lipofectamine TM RNAiMAX was purchased from Invitrogen (Carlsbad, CA, USA). Rabbit anti-PIMT antiserum was produced against recombinant PIMT proteins of porcine brain as previously described (Koh and Hong, unpublished data). Antibodies against MAPK, MMP-2, MMP-9, N-cadherin, integrin αv, phospho-GSK3 (Tyr 279/216 ), phospho-ERK1/2 (Thr 202 /Tyr 204 ), phospho-MEK1 (Ser 218/222 )/MEK2 (Ser 222/226 ), phospho-Akt1/PkBα (Ser 473 ) and phospho-p90RSK (Ser 380 ) were...
Edelweiss (Leontopodium Alpinum) in the family Asteraceae is a wildflower that grows in rocky limestone places. Here, we investigated the efficacy of edelweiss callus culture extract (Leontopodium Alpinum callus culture extract; LACCE) using multiple assays from in vitro to in vivo as well as transcriptome profiling. Several in vitro assay results showed the strong antioxidant activity of LACCE in response to UVB treatment. Moreover, LACCE suppressed inflammation and wrinkling; however, moisturizing activity was increased by LACCE. The clinical test in vivo demonstrated that constant application of LACCE on the face and skin tissues improved anti-periorbital wrinkles, skin elasticity, dermal density, and skin thickness compared with the placebo. The RNA-Sequencing results showed at least 16.56% of human genes were expressed in keratinocyte cells. LACCE up-regulated genes encoding several KRT proteins; DDIT4, BNIP3, and IGFBP3 were involved in the positive regulation of the developmental process, programmed cell death, keratinization, and cornification forming skin barriers, which provide many advantages in the human skin. By contrast, down-regulated genes were stress-responsive genes, including metal, oxidation, wounding, hypoxia, and virus infection, suggesting LACCE did not cause any harmful stress on the skin. Our comprehensive study demonstrated LACCE is a promising agent for anti-aging cosmetics.
Camellia japonica L. is a flowering tree with several medicinal and cosmetic applications. Here, we investigated the efficacy of C. japonica placenta extract (CJPE) as a potential therapeutic agent for promotion of hair growth and scalp health by using various in vitro and in vivo assays. Moreover, we performed transcriptome analysis to examine the relative expression of human follicle dermal papilla cells (HFDPC) in response to CJPE by RNA‐sequencing (RNA‐seq). In vitro assays revealed upregulation of the expression of hair growth marker genes in HFDPC after CJPE treatment. Moreover, in vivo clinical tests with 42 adult female participants showed that a solution containing 0.5% CJPE increased the moisture content of the scalp and decreased the scalp's sebum content, dead scalp keratin, and erythema. Furthermore, RNA‐seq analysis revealed key genes in HFDPC which are associated with CJPE. Interestingly, genes associated with lipid metabolism and cholesterol efflux were upregulated. Genes upregulated by CJPE are associated with several hormones, including parathyroid, adrenocorticotropic hormone, α‐melanocyte‐stimulating hormone (alpha‐MSH), and norepinephrine, which are involved in hair follicle biology. Furthermore, some upregulated genes are associated with the regulation of axon guidance. In contrast, many genes downregulated by CJPE are associated with structural components of the cytoskeleton. In addition, CJPE suppressed genes associated with muscle structure and development. Taken together, this study provides extensive evidence that CJPE may have potential as a therapeutic agent for scalp treatment and hair growth promotion.
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