sirolimus and Keloid

To investigate the effect of rapamycin on biological characteristics and autophagy of keloid fibroblasts, and the regulation of rapamycin in mTOR (mammalian target of rapamycin) signaling pathway and autophagy-related non-coding RNAs in keloid fibroblasts.. After Keloid fibroblasts were treated with rapamycin (10、50、100 nmol/L), and MTS assay was used to test the cell proliferation. The apoptosis of cells was tested by the flow cytometry analysis. The formation of autophagy was observed by TEM, and the Western Blot was used to detect the expression of autophagy-related protein LC3.Real-time PCR was used to detect the expression of genes of involued in mTOR pathway and autophagy-related non-coding RNAs. Statistical significance was determined using Paired-Samples t Test,P value less than 0.05 was considered statistically significant.. The ratio of 490nm was decreased significantly in rapamycin-treated keloid fibroblasts compared with that in untreated cells (P ＜ 0.05).Meanwhile the mRNA expressions of extracellular matrix (ECM) genes, including collagen-1 、α-SMA and Fibronectin, were inhibited by rapamycin (P ＜ 0.05).The flow cytometry analysis showed that the percent of apoptosis cells was not increased in rapamycin-induced cells (P ＞ O.05). The double-layer membrane structure of autophagosomes could be observed under the TEM in rapamycin-treated fibroblasts, accompanied by the increased expression of autophagy-related protein LC3.The mRNA expressions of downstream genes of mTOR pathway,4EBP1 and p70S6K,were down-regulated in rapamycin-treated fibroblasts, while the expressions of autophagy-related miRNAs, including miR-885-3p,miR-204,miR-101,miR-376b and lncRNA FLJ11812 were enhanced, and miR-30a,lncRNA HULC5 was decreased in rapamycin-treated fibroblasts (P ＜ 0.05).. Rapamycin could inhibit the proliferation of keloid fibroblasts, and could not affect the apoptosis of cells.However, rapamycin induced the autophagy of keloid fibroblasts through regulating the expression of autophagy-related non-coding RNAs and genes in the mTOR signaling pathway.

Topics: Apoptosis; Autophagy; Cell Line, Tumor; Cell Proliferation; Collagen Type I; Fibroblasts; Fibronectins; Humans; Immunosuppressive Agents; Keloid; MicroRNAs; Phosphorylation; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Excess scar formation after cutaneous injury can result in hypertrophic scar (HTS) or keloid formation. Modern strategies to treat pathologic scarring represent nontargeted approaches that produce suboptimal results. Mammalian target of rapamycin (mTOR), a central mediator of inflammation, has been proposed as a novel target to block fibroproliferation. To examine its mechanism of action, we performed genomewide microarray on human fibroblasts (from normal skin, HTS, and keloid scars) treated with the mTOR inhibitor, rapamycin. Hypertrophic scar and keloid fibroblasts demonstrated overexpression of collagen I and III that was effectively abrogated with rapamycin. Blockade of mTOR specifically impaired fibroblast expression of the collagen biosynthesis genes PLOD, PCOLCE, and P4HA, targets significantly overexpressed in HTS and keloid scars. These data suggest that pathologic scarring can be abrogated via modulation of mTOR pathways in procollagen and collagen processing.

Topics: Adult; Aged; Cicatrix, Hypertrophic; Collagen; Dose-Response Relationship, Drug; Down-Regulation; Female; Fibroblasts; Humans; Immunosuppressive Agents; Keloid; Male; Middle Aged; Phosphorylation; Protein Array Analysis; Sirolimus; TOR Serine-Threonine Kinases

Mammalian target of rapamycin (mTOR) is essential in controlling several cellular functions. This pathway is dysregulated in keloid disease (KD). KD is a common fibroproliferative dermal lesion with an ill-defined treatment strategy. KD demonstrates excessive matrix deposition, angiogenesis, and inflammatory cell infiltration. In KD, both total and phosphorylated forms of mTOR and p70(S6K)(Thr421/Ser424) are upregulated. Therefore, the aim of this study was to investigate adenosine triphosphate-competitive inhibitors of mTOR kinase previously unreported in keloid and their comparative efficacy with Rapamycin. Here, we present two mTOR kinase inhibitors, KU-0063794 and KU-0068650, that target both mTORC1 and mTORC2 signaling. Treatment with either KU-0063794 or KU-0068650 resulted in complete suppression of Akt, mTORC1, and mTORC2, and inhibition of keloid cell spreading, proliferation, migration, and invasive properties at a very low concentration (2.5 μmol  l(-1)). Both KU-0063794 and KU-0068650 significantly (P<0.05) inhibited cell cycle regulation and HIF1-α expression compared with that achieved with Rapamycin alone. In addition, both compounds induced shrinkage and growth arrest in KD, associated with the inhibition of angiogenesis, induction of apoptosis, and reduction in keloid phenotype-associated markers. In contrast, Rapamycin induced minimal antitumor activity. In conclusion, potent dual mTORC1 and mTORC2 inhibitors display therapeutic potential for the treatment of KD.

Topics: Adolescent; Adult; Aged; Cell Movement; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Inhibitors; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunosuppressive Agents; In Vitro Techniques; Keloid; Male; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Middle Aged; Morpholines; Multiprotein Complexes; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Pyrimidines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Young Adult

Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis during the wound healing process. As epithelial-mesenchymal interactions have been shown to regulate a plethora of genes in wound healing, we hypothesized that these interactions might have a role in modulating VEGF expression and angiogenesis. A two chamber co-culture model was used, wherein normal and keloid keratinocytes and fibroblasts were physically separated by membrane inserts while allowing cytokine diffusion. Cell lysates obtained from keratinocytes co-cultured with fibroblasts demonstrated increased expression of VEGF. An enzyme-linked immunosorbent assay (ELISA) showed significant increase in VEGF expression in co-culture conditioned media compared with controls. Additionally, the conditioned medium from keloid keratinocyte and fibroblast co-cultures increased proliferation and formation of complex three-dimensional capillary-like structures in human umbilical vein endothelial cells, emphasising the importance of epithelial-mesenchymal interactions in the angiogenic process. Immunostaining of keloid tissue localized VEGF in the basal layer of the epidermis and also demonstrated higher blood vessel density than normal skin. Keloid tissue extract also demonstrated increased expression of VEGF compared with normal skin. It is likely that epidermal VEGF exerts significant paracrine control over the dynamics and expression profile of underlying dermal fibroblasts. Addition of the inhibitors WP631, mitoxantrone, and Rapamycin to keloid keratinocyte and fibroblast co-cultures, downregulated secreted VEGF expression in a dose-dependent manner, suggesting therapeutic potential for these compounds in the treatment of keloid scars.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; Cell Differentiation; Cell Proliferation; Coculture Techniques; Culture Media, Conditioned; Daunorubicin; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Fibroblasts; Humans; Immunohistochemistry; Keloid; Keratinocytes; Mesoderm; Mitoxantrone; Neovascularization, Pathologic; Sirolimus; Statistics, Nonparametric; Up-Regulation; Vascular Endothelial Growth Factor A; Wound Healing

Keloid is a dermal fibroproliferative disorder characterized by excessive deposition of extracellular matrix (ECM) components such as collagen, glycoproteins and fibronectin. The mammalian target of rapamycin (mTOR) is a serine/theronine kinase which plays an important role in the regulation of metabolic processes and translation rates. Published reports have shown mTOR as regulator of collagen expression and its inhibition induces a decrease in ECM deposition. Our aim was to investigate the role of mTOR in keloid pathogenesis and investigate the effect of rapamycin on proliferating cell nuclear antigen (PCNA), cyclin D1, collagen, fibronectin and alpha-smooth muscle actin (alpha-SMA) expression in normal fibroblasts (NF) and keloid fibroblasts (KF). Tissue extracts obtained from keloid scar demonstrated elevated expression of mTOR, p70KDa S6 kinase (p70S6K) and their activated forms, suggesting an activated state in keloid scars. Serum stimulation highlighted the heightened responsiveness of KF to mitogens and the importance of mTOR and p70S6K during early phase of wound healing. Application of rapamycin to monoculture NF and KF, dose- and time-dependently downregulates the expression of cytoplasmic PCNA, cyclin D1, fibronectin, collagen and alpha-SMA, demonstrating the anti-proliferative effect and therapeutic potential of rapamycin in the treatment of keloid scars. The inhibitory effect of rapamycin was found to be reversible following recovery in the expression of proteins following the removal of rapamycin from the culture media. These results demonstrate the important role of mTOR in the regulation of cell cycle and the expression of ECM proteins: fibronectin, collagen and alpha-SMA.

Topics: Actins; Adolescent; Adult; Cells, Cultured; Child; Cicatrix, Hypertrophic; Collagen; Cyclin D; Cyclins; Dose-Response Relationship, Drug; Female; Fibroblasts; Fibronectins; Humans; In Vitro Techniques; Keloid; Male; Phosphorylation; Proliferating Cell Nuclear Antigen; Protein Kinases; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; TOR Serine-Threonine Kinases