Supplementary Table 1 from BRCA1 Induces Antioxidant Gene Expression and Resistance to Oxidative Stress

This study experimentally investigated the influence of the spatial layout of vegetation patches on the river hydro-morphodynamics and flow resistance. A unique large-scale outdoor experimental channel with artificial willows has been formed in the Korea Institute of Civil Engineering and Building Technology – River Experiment Center (KICT-REC). The primary data set is the flow velocity within a vegetation patch measured by a Nortek Acoustic Dopper Velocimeter (ADV), and the bed level changes recorded by 3D terrestrial laser scanning and manual survey before and after the experiment. The flow resistance evaluated through Manning's roughness coefficient was determined and compared for different vegetation layouts and densities. The experimental results suggest that flow characteristics in vegetation layouts strongly depend on the vertical structure and the density of the vegetation patch. Moreover, the relation between the flow resistance, the vegetation density, and the blockage area was investigated.

The purpose of this research is to design a simple and intuitive path tracker to minimise the distance error of a car-like delivery service robot using only a position sensor, i.e. global positioning system in outdoor environments. The authors proposed not only a single-point preview path tracker (SPP), but also a curvature-based preview control using a quadratic curve fitting technique. This method can provide flexibility in preview distance control even at a constant speed, and reduces the burden of an actuator for maintenance. Furthermore, the performance of the proposed adaptive SPP is considerably improved compared to that when using a fixed preview distance.

Pigmented skin equivalent models have been used in cosmetic, pharmaceutical and medical research to evaluate the skin pigmentation. Thus, reliable in vitro skin pigmentation models are highly demanded. Currently, the pigmented model is being developed by cultivating a constant ratio of keratinocytes and melanocytes but sometimes cells from different donors shown poor proliferation and differentiation in skin model. In this study, we isolated keratinocyte-adapted melanoblasts from keratinocyte culture and developed the pigmented reconstructed human epidermis models containing melanocytes and their progenitor cells derived from same donor of keratinocytes. We effectively isolated human melanoblasts using an easy and efficient method called 'calcium pulse'. The confluent cells were cultured with high-calcium medium (1.2 mM) for 3 days and changed back to the normal growth medium (0.06 mM calcium) for 7 days. The epidermal sheet was detached from the culture dish during PBS washing then we used remained cells on the dish as melanoblasts and reconstructed pigmented human epidermis models. The high calcium treatment increases the number of melanocytic cells in the keratinocyte culture. Moreover, pigmented reconstructed human epidermis models containing melanoblasts shown increased prolifereation and well-differentiated epidermis compare to the model consisting melanocytes. We successfully generated pigmented reconstructed human epidermis models including human melanoblasts reflecting in vivo human skin. Thus, the model used in this study represents a highly relevant tool to evaluate skin pigmentation and as an alternative to animal tests.

We previously designed a novel synthetic retinoid, seletinoid G, and demonstrated that seletinoid G as an anti-aging compound can increase the expression of extracellular matrix proteins (type I procollagen, tropoelastin, and fibrillin-1) in human skin in vivo. In addition to its anti-aging efficacy, however, other activities have not been studied. In this study, we investigated the wound healing effects of seletinoid G on human keratinocyte cell line (HaCaT) and skin equivalents. An automated time-lapse imaging technique was used to visualize real-time keratinocyte migration and in vitro wound healing effect of seletinoid G. Seletinoid G (12 μM) increased wound closure in a monolayer of line-scratched HaCaT cell line. The mRNA expression levels of keratin 1 and its heterodimer partner keratin 10 were significantly increased by seletinoid G treatment. In case of skin model containing both human epidermal keratinocytes in epidermis and human dermal fibroblasts in dermis, wounded skin equivalent model was prepared by the use of punch biopsy with 3 mm in diameter and topically treated with seletinoid G (12 and 25 μM) every other day for 3 – 6 days and then, their re-epithelialized areas were three-dimensionally measured by optical coherence tomography. Topical treatment with seletinoid G (12 and 25 μM) in phosphate-buffered saline significantly accelerated wound closure compared to vehicle-treated group. Our results suggest that seletinoid G could be helpful to improving wound re-epithelialization in skin.

Ganoderma lucidum has been used in China as a herbal medicine. Several studies suggested that Ganodermal lucidum polysaccharides have anti-tumor activities and anti-oxidation effects. This study was conducted to investigate the protective effect of G.lucidium in UVB-induced human fibroblasts and skin equivalent models. Fibroblasts exposed UVB increased MMP-1 protein expression and decreased procollagen protein expression. Treatment with G. lucidium inhibited UVB-induced MMP-1 expression and increased procollagen expression. Extracellular signal regulated kinase (ERK) activation was inhibited by treatment with G. lucidium. In addition, G. lucidium increased collagen expression in UVB-induced skin equivalent models analyzed by multiphoton microscopy. These results demonstrated that G. lucidium can inhibit UVB-induced MMP-1 expression by inhibiting the ERK pathways.

We recently developed the new depigmentation compound, AP736 (5-adamantan-1-yl-N-(2,4-dihydroxy-benzyl)-2,4-dimethoxy-benzamide), which strongly inhibits melanin synthesis in melanocytes. To show depigmentary effect, AP736 inhibits the expression of tyrosinase, tyrosinase-related protein (Tyrp)-1/2, and microphthalmia-associated transcription factor (MiTF). AP736 also suppresses the activation of cAMP-PKA-CREB upstream signaling pathway. As another possible mechanism, we have focused on microRNA expression in skin cells because MicroRNAs (miRNAs) are known to regulate skin biology, including the pigmentation process in melanocytes. Particularly, miR-125b is a potent inhibitor of steady-state melanogenesis, and also, down-regulates the expression of matrix metalloproteinase (MMPs) in several cells. Thus, we have tested whether AP736 induces miR-125b expression to exert the whitening effect and MMPs inhibition for anti-aging in epidermal skin cells. In the results, AP736 significantly increased the miR-125b expression in human epidermal melanocytes and keratinocytes. Notably, AP736 scavenged reactive oxygen species and inhibited the expression of MMP-1 and -3 expression in UVB-stimulated keratinocytes. These results suggest that AP736 not only has whitening ability but also anti-aging activity in UVB-stimulated human epidermal cells, effects that are attributable, at least in part, to the action of AP736 as a miR-125b inducer.

Extract of green tea leaf, Camellia sinensis, is one of the well known anti-oxidants and anti-inflammatory agents in nature. However the extract of green tea petiole (GTP), the stalk that attaches the leaf blade to the stem of green tea plant, has been less studied for its biological activities compared to other parts of green tea tree such as leaf, root and flower. In this study, we investigated the anti-inflammatory effect of GTP extract in human skin cells and 3D skin equivalent. Thus far it has been reported that abnormal TLRs and/or TLR-ligands signaling could lead to inflammation imbalance during cellular aging named inflamm-aging. Herein we used Poly(I:C), known as a specific ligand for TLR3, to mimic the inflammatory environment in the cell culture system. GTP extract reduced the induction of inflammatory cytokines mRNA level (TNF-alpha and IL-1beta) significantly in human keratinocytes via the inhibition of toll-like receptor 3 (TLR3) activation in presence of Poly(I:C). (p<0.001) It was also shown that GTP extract has the inhibitory effect on MMP-1 expression in Poly(I:C) stimulated human keratinocyte and 3D skin equivalent. Finally application of the skin care formulation containing GTP extract for 4 weeks significantly improved the skin elasticity and wrinkle formation around eyes in 26 Korean women (mean age 44.2 ± 6.0, p<0.05). Taken together, these results suggest that GTP extract can be a promising ingredient for cosmetic applications with the robust and dramatic anti-inflammatory and anti-ageing effect.

There are some evidences that human skin cell can sense mechanical stresses or temperature changes. Furthermore, it was suggested olfactory and optical receptor present in human skin cells recently. Considering evolutionary procedure, it is conceivable that there may be unusual sensing mechanism on human skin because sensory and neural systems originated from whole body. Likewise, it is not strange that skin has sound sensing mechanism. The hypersonic effect is known as a phenomenon that sounds containing ultrasonic frequencies above the human audible range affect cerebral blood flow and brain waves. It is interesting even when ultrasonic sounds were presented only to body surface, not to ears, subject's brain took hypersonic effect. The result suggests that human skin may have mechanisms sensing ultrasound directly. It was also reported that inaudible ultrasound at a frequency above 20 khz affected on the skin of rodents. Presenting of ultrasound on disrupted skin of rodents accelerated significant barrier recovery which was due to increased exocytosis of lamellar body from granular layer of epidermis. In this study, we hypothesized that human skin barrier function may altered by inaudible ultrasound treatment. To elucidate that, human skin equivalent (SE) barrier was disrupted with punch biopsy followed by ultrasound exposure. As expected, ultrasound promoted epidermal regeneration of SE. We used genomic analysis to get a clue how ultrasound accelerated epidermal regeneration. Gene set enrichment analysis revealed that fatty acid biosynthesis pathway had noteworthy enrichment score. Furthermore, we confirmed that the mRNA level of elongation of very long-chain fatty acid-4 (ELOVL4), which is a gene that may contribute skin barrier function, was elevated in ultrasound treated normal human keratinocytes (NHK). These data suggest that inaudible ultrasound may have beneficial effects on human skin regeneration.

Epigenetic regulation of gene expression is an emerging target to treat several human diseases including cancers. In cancers, expressions of many tumor suppressor genes are suppressed by hyper-methylation in their regulatory regions. Herein, we describe a novel carbazole SH-I-14 that decreased the level of the acetyl-STAT3 at the K685 residue. Mutation analysis revealed that SH-I-14 disrupted STAT3-DNMT1 interaction by removing acetyl group from K685 of STAT3. Finally, the inhibition of STAT3-DNMT1 interaction by SH-I-14 resulted in re-expression of tumor suppressor genes such as VHL and PDLIM4 through de-methylation of their promoter regions. In addition, SH-I-14 showed anti-proliferative effect in triple-negative breast cancer (TNBC) cell lines in vitro and anti-tumor effect in a mouse xenograft model of MDA-MB-231 tumor. Taken together, our results suggest that targeting acetyl-STAT3 (K685) provides potential therapeutic opportunity to treat a subset of human cancers.

Triple-negative breast cancer (TNBC) exhibits innate resistance to the EGFR inhibition despite high level expression of EGFR. Recently, we found that the proliferation of basal-like (BL) subtype TNBC cells is synergistically inhibited by combination of EGFR and PI3K/AKT inhibitors. On the contrary, TNBC cells of mesenchymal stem-like (MSL) subtype are resistant to these combinations. To identify potential synthetic lethal interaction of compounds for treatment of MSL subtype TNBC cells, we performed MTT screening of MDA-MB‑231 cells with a small library of receptor tyrosine kinase inhibitors (RTKIs) in the presence of gefitinib, an EGFR inhibitor. We identified MET inhibitors as potent RTKIs that caused synthetic lethality in combination with gefitinib in MDA-MB‑231 cells. We demonstrated that combination of a MET inhibitor SU11274 with various EGFR inhibitors resulted in synergistic suppression of cell viability (in MTT assay) and cell survival (in colony formation assay) of MSL subtype TNBC cells. We further demonstrated that SU11274 alone induced G2 arrest and gefitinib/SU11274 combination sustained the SU11274-induced G2 arrest in these cells. In addition, SU11274/gefitinib combination synergistically reduced the level of ribosomal protein S6 (RPS6) in MSL subtype TNBC cells. In addition, knockdown of RPS6 itself, in both HS578T and MDA-MB‑231, markedly reduced the proliferation of these cells. Taken together, our data suggest that dual targeting of EGFR and MET inhibits the proliferation of MSL subtype TNBC cells through downregulation of RPS6.

An F-box protein, β-TrCP recognizes substrate proteins and destabilizes them through ubiquitin-dependent proteolysis. It regulates the stability of diverse proteins and functions as either a tumor suppressor or an oncogene. Although the regulation by β-TrCP has been widely studied, the regulation of β-TrCP itself is not well understood yet. In this study, we found that the level of β-TrCP1 is downregulated by various protein kinase inhibitors in triple-negative breast cancer (TNBC) cells. A PI3K/mTOR inhibitor PI-103 reduced the level of β-TrCP1 in a wide range of TNBC cells in a proteasome-dependent manner. Concomitantly, the levels of c-Myc and cyclin E were also downregulated by PI-103. PI-103 reduced the phosphorylation of β-TrCP1 prior to its degradation. In addition, knockdown of β-TrCP1 inhibited the proliferation of TNBC cells. We further identified that pharmacological inhibition of mTORC2 was sufficient to reduce the β-TrCP1 and c-Myc levels. These results suggest that mTORC2 regulates the stability of β-TrCP1 in TNBC cells and targeting β-TrCP1 is a potential approach to treat human TNBC.

The aberrant activation of STAT3 occurs in many human cancers and promotes tumor progression. Phosphorylation of a tyrosine at amino acid Y705 is essential for the function of STAT3. Synthesized carbazole derived with fluorophore compound 12 was discovered to target STAT3 phosphorylation. Compound 12 was found to inhibit STAT3-mediated transcription as well as to reduce IL-6 induced STAT3 phosphorylation in cancer cell lines expressing both elevated and low levels of phospho-STAT3 (Y705). Compound 12 potently induced apoptosis in a broad number of TNBC cancer cell lines in vitro and was effective at inhibiting the in vivo growth of human TNBC xenograft tumors (SUM149) without any observed toxicity. Compound 12 also effectively inhibited the growth of human lung tumor xenografts (A549) harboring aberrantly active STAT3. In vitro and in vivo studies showed that the inhibitory effects of 12 on phospho-STAT3 were through up-regulation of the protein-tyrosine phosphatase PTPN6. Our present studies strongly support the continued preclinical evaluation of compound 12 as a potential chemotherapeutic agent for TNBC and cancers with constitutive STAT3 signaling.

Gemcitabine-based chemotherapy is the standard for treatment of pancreatic cancer; however, intrinsic and acquired resistance to gemcitabine commonly occurs. Aldehyde dehydrogenase 1A1 (ALDH1A1), one of the characteristic features of tumor-initiating and/or cancer stem cell (CSC) properties, is important in both intrinsic and acquired resistance to gemcitabine. In this study, we investigated the effectiveness of dasatinib, an SRC inhibitor, and gemcitabine combination to inhibit the survivals of parental (MIA PaCa-2/P) and gemcitabine-resistant (MIA PaCa-2/GR) cell lines. In MIA PaCa-2/GR cells, the levels of phospho-SRC and ALDH1A1 were increased compared to MIA PaCa-2/P cells. Inhibition of SRC by dasatinib or siRNA synergistically enhanced gemcitabine-induced anti-proliferative effects and induced apoptotic cell death in these cells. Furthermore, combination of SRC inhibition (either by dasatinib or siRNA) and gemcitabine significantly decreased the levels of ALDH1A1 expression. These results suggest that dasatinib and gemcitabine combination may be a potential therapeutic strategy to overcome gemcitabine resistance by decreasing the levels of ALDH1A1 expression.