Self-assembled adipose-derived mesenchymal stem cells as an extracellular matrix component- and growth factor-enriched filler.

The clinical application of mesenchymal stem cells (MSCs) is attracting attention due to their excellent safety, convenient acquisition, multipotency, and trophic activity. The clinical effectiveness of transplanted MSCs is well-known in regenerative and immunomodulatory medicine, but there is a demand for their improved viability and regenerative function after transplantation. In this study, we isolated MSCs from adipose tissue from three human donors and generated uniformly sized MSC spheroids (∼100 µm in diameter) called microblocks (MiBs) for dermal reconstitution. The viability and MSC marker expression of MSCs in MiBs were similar to those of monolayer MSCs. Compared with monolayer MSCs, MiBs produced more extracellular matrix (ECM) components, including type I collagen, fibronectin, and hyaluronic acid, and growth factors such as vascular endothelial growth factor and hepatocyte growth factor. Subcutaneously injected MiBs showed skin volume retaining capacity in mice. These results indicate that MiBs could be applied as regenerative medicine for skin conditions such as atrophic scar by having high ECM and bioactive factor expression.

Changes in handwashing and hygiene product usage patterns in Korea before and after the outbreak of COVID-19.

BACKGROUND: The World Health Organization declared COVID-19, the disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a global pandemic on March 11, 2020. Non-pharmaceutical interventions such as social distancing, handwashing, using hand sanitizer, and wearing facial masks are recommended as the first line of protection against COVID-19. Encouraging hand hygiene may be one of the most cost-effective means of reducing the global burden of disease. METHODS: This study uses a web-based questionnaire to evaluate the usage patterns and consumer perceptions of the effectiveness and health safety of bar soap, liquid hand soap, and hand sanitizer products before and after the spread of COVID-19. RESULTS: The results show that since the outbreak of COVID-19, the number of consumers who primarily use bar soap has decreased from 71.8 to 51.4%, the number of those who primarily use liquid hand soap has increased from 23.5 to 41.3%, and the number of those who use and carry hand sanitizer has increased. The frequency of use, duration of use, and amount used of all three products have increased significantly since the COVID-19 outbreak. Finally, consumer perception of the products' preventive effect against COVID-19 is higher for liquid hand soap and hand sanitizer than it is for bar soap. CONCLUSIONS: Because use of hand sanitizers has increased, public health guidelines must address the potential risks associated them. Our data also show that the public is abiding by the recommendations of the regulatory authorities. As handwashing has become important in preventing COVID-19 infections, the results of our study will support the development of better handwashing guidelines and a public health campaign. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12302-021-00517-8.

The mixture effects of bisphenol derivatives on estrogen receptor and androgen receptor.

Bisphenol A (BPA) is a well-known for endocrine-disrupting chemical (EDC) and is one of the highest amounts of chemicals produced worldwide. Some countries restrict the use of BPA, which is widely used in the production of a variety products. Considering the toxicity and limitations on use of BPA, efforts are needed to find safer alternatives. Increasingly, bisphenol F (BPF) and bisphenol S (BPS) are alternatives of BPA, which is increasing their exposure levels in various environments. There are many ways to assess whether a chemical is an EDC. Here, we evaluated the endocrine-disrupting risks of the bisphenols by investigating their agonist and antagonist activities with the estrogen (ER), androgen (AR), and aryl hydrocarbon (AhR) receptors. Our results showed that BPA, BPS, and BPF (BPs) have estrogen agonist and androgen antagonist activities and decrease the ERα protein level. Interestingly, a mixture of the BPs had ER and anti-AR activity at lower concentrations than BPs alone. The activation of AhR was not a concentration-dependent effect of BPs, although it was increased significantly. In conclusion, BPs have estrogen agonist and androgen antagonist activities, and the effect of exposure to a BPs mixture differs from that of BPs alone.

Overexpression and Selective Anticancer Efficacy of ENO3 in STK11 Mutant Lung Cancers.

Oncogenic gain-of-function mutations are clinical biomarkers for most targeted therapies, as well as represent direct targets for drug treatment. Although loss-of-function mutations involving the tumor suppressor gene, STK11 (LKB1) are important in lung cancer progression, STK11 is not the direct target for anticancer agents. We attempted to identify cancer transcriptome signatures associated with STK11 loss-offunction mutations. Several new sensitive and specific gene expression markers (ENO3, TTC39C, LGALS3, and MAML2) were identified using two orthogonal measures, i.e., fold change and odds ratio analyses of transcriptome data from cell lines and tissue samples. Among the markers identified, the ENO3 gene over-expression was found to be the direct consequence of STK11 loss-of-function. Furthermore, the knockdown of ENO3 expression exhibited selective anticancer effect in STK11 mutant cells compared with STK11 wild type (or recovered) cells. These findings suggest that ENO3 -based targeted therapy might be promising for patients with lung cancer harboring STK11 mutations.

Dissecting phenotypic responses of the druggable targetome in cancers.

Although a large amount of screening data comprising target genes and/or drugs tested against cancer cell line panels are available, different assay conditions and readouts limit the integrated analysis and batch-to-batch comparison of these data. Here, we systematically produced and analyzed the anticancer effect of the druggable targetome to understand the varied phenotypic outcomes of diverse functional classes of target genes. A library of siRNAs targeting ~4,800 druggable genes was screened against cancer cell lines under 2D and/or 3D assay conditions. The anticancer effect was simultaneously measured by quantifying cell proliferation and/or viability. Hit rates varied significantly depending on assay conditions and/or phenotypic readouts. Functional classes of hit genes were correlated with the microenvironment difference between the 2D monolayer cell proliferation and 3D sphere formation assays. Furthermore, multiplexing of cell proliferation and viability measures enabled us to compare the sensitivity and resistance responses to the gene knockdown. Many target genes that inhibited cell proliferation increased the single-cell-level viability of surviving cells, leading to an increase in self-renewal potential. In this study, combinations of parallel 2D/3D assays and multiplexing of cell proliferation and viability measures provided functional insights into the varied phenotypic outcomes of the cancer targetome.

Indazole-Cl inhibits hypoxia-induced cyclooxygenase-2 expression in vascular smooth muscle cells.

Atherosclerosis is the most common root cause of arterial disease, such as coronary artery disease and carotid artery disease. Hypoxia is associated with the formation of macrophages and increased inflammation and is known to be present in lesions of atherosclerotic. Vascular smooth muscle cells (VSMCs) are one of the major components of blood vessels, and hypoxic conditions affect VSMC inflammation, proliferation and migration, which contribute to vascular stenosis and play a major role in the atherosclerotic process. Estrogen receptor (ER)-ß is thought to play an important role in preventing the inflammatory response in VSMCs. In this report, we studied the anti-inflammatory effect of indazole (In)-Cl, an ERß-specific agonist, under conditions of hypoxia. Expression of cyclooxygenase-2 reduced by hypoxia was inhibited by In-Cl treatment in VSMCs, and this effect was antagonized by an anti-estrogen compound. Additionally, the production of reactive oxygen species induced under conditions of hypoxia was reduced by treatment with In-Cl. Increased cell migration and invasion by hypoxia were also dramatically decreased following treatment with In-Cl. The increase in cell proliferation following treatment with platelet-derived growth factor was attenuated by In-Cl in VSMCs. RNA sequencing analysis was performed to identify changes in inflammation-related genes following In-Cl treatment in the hypoxic state. Our results suggest that ERß is a potential therapeutic target for the suppression of hypoxia-induced inflammation in VSMCs.

The effects of bisphenol A, benzyl butyl phthalate, and di(2-ethylhexyl) phthalate on estrogen receptor alpha in estrogen receptor-positive cells under hypoxia.

Endocrine-disrupting chemicals (EDCs) are widely used in various consumer goods. Consequently, humans are constantly exposed to EDCs, which is associated with a variety of endocrine-related diseases. In this study, we demonstrated the effects of bisphenol A (BPA), benzyl butyl phthalate (BBP), and di(2-ethylhexyl) phthalate (DEHP) on estrogen receptor alpha (ERα) expression under normoxia and hypoxia. First, we confirmed the effects of EDCs on ER activity using OECD Test Guideline 455. Compared to the 100% activity induced by 1 nM 17-ß-estradiol (positive control), BPA and BBP exhibited 50% ERα activation at concentrations of 1.31 µM and 4.8 µM, respectively. In contrast, and consistent with previous reports, DEHP did not activate ERα. ERα is activated and degraded by hypoxia in breast cancer cells. BPA, BBP, and DEHP enhanced ERα-mediated transcriptional activity under hypoxia. All three EDCs decreased ERα protein levels under hypoxia in MCF-7 cells. The transcriptional activity of hypoxia-inducible factor-1 was decreased and secretion of vascular endothelial growth factor (VEGF) was increased by BPA and BBP under hypoxia in MCF-7 cells, but not by DEHP. All three EDCs decreased the ERα protein expression level in Ishikawa human endometrial adenocarcinoma cells, and DEHP caused a weak decrease in VEGF secretion under hypoxia. These results demonstrate down-regulation of ERα by EDCs may influence the pathological state associated with hypoxia.

Assessment of endocrine-disrupting activities of alternative chemicals for bis(2-ethylhexyl)phthalate.

Plastic products are closely intertwined with modern life. Some plasticizers used in making plastics, such as phthalates, are reported to be endocrine-disrupting chemicals. Plasticizers can be released into the environment, and health risks related to plasticizer exposure have been reported. In addition, due to plastic waste that flows into the ocean, microplastics have been found in marine products, including non-biological seawater products such as sea salt. Plastics can affect the body via a variety of pathways, and therefore safer alternative chemicals are needed. Three chemicals were evaluated: acetyl tributyl citrate (ATBC), triethyl 2-acetylcitrate (ATEC), and trihexyl O-acetylacitrate (ATHC), replacing bis(2-ethylhexyl)phthalate (DEHP), a typical plasticizer. The endocrine-disrupting activities of each chemical, including estrogenic or anti-estrogenic activity (test guideline (TG) No. 455), androgenic or anti-androgenic activity (TG No. 458), steroidogenesis (TG No. 456), and estrogenic properties via a short-term screening test using the uterotrophic assay (TG No. 440), were assessed in accordance with the Organisation for Economic Co-operation and Development guidelines for chemical testing. Our results showed that DEHP, ATBC, ATEC, ATHC possess no estrogenic activity, whereas DEHP, ATBC and ATHC demonstrate anti-estrogenic activity and ATBC anti-androgenic activity. DEHP and ATHC exhibited a disruption in steroidogenesis activities. Additional tests are necessary, but our results suggest that ATEC is a good candidate plasticizer providing a suitable alternative to DEHP.

QSurface: fast identification of surface expression markers in cancers.

BACKGROUND: Cell surface proteins have provided useful targets and biomarkers for advanced cancer therapies. The recent clinical success of antibody-drug conjugates (ADCs) highlights the importance of finding selective surface antigens for given cancer subtypes. We thus attempted to develop stand-alone software for the analysis of the cell surface transcriptome of patient cancer samples and to prioritize lineage- and/or mutation-specific over-expression markers in cancer cells. RESULTS: A total of 519 genes were selected as surface proteins, and their expression was profiled in 14 cancer subtypes using patient sample transcriptome data. Lineage/mutation-oriented analysis was used to identify subtype-specific surface markers with statistical confidence. Experimental validation confirmed the unique over-expression of predicted surface markers (MUC4, MSLN, and SLC7A11) in lung cancer cells at the protein level. The differential cell surface gene expression of cell lines may differ from that of tissue samples due to the absence of the tumor microenvironment. CONCLUSIONS: In the present study, advanced 3D models of lung cell lines successfully reproduced the predicted patterns, demonstrating the physiological relevance of cell line-based 3D models in validating surface markers from patient tumor data. Also QSurface software is freely available at http://compbio.sookmyung.ac.kr/~qsurface .

Integrated analysis of transcriptomes of cancer cell lines and patient samples reveals STK11/LKB1-driven regulation of cAMP phosphodiesterase-4D.

The recent proliferation of data on large collections of well-characterized cancer cell lines linked to therapeutic drug responses has made it possible to identify lineage- and mutation-specific transcriptional markers that can help optimize implementation of anticancer agents. Here, we leverage these resources to systematically investigate the presence of mutation-specific transcription markers in a wide variety of cancer lineages and genotypes. Sensitivity and specificity of potential transcriptional biomarkers were simultaneously analyzed in 19 cell lineages grouped into 228 categories based on the mutational genotypes of 12 cancer-related genes. Among a total of 1,455 category-specific expression patterns, the expression of cAMP phosphodiesterase-4D (PDE4D) with 11 isoforms, one of the PDE4(A-D) subfamilies, was predicted to be regulated by a mutant form of serine/threonine kinase 11 (STK11)/liver kinase B1 (LKB1) present in lung cancer. STK11/LKB1 is the primary upstream kinase of adenine monophosphate-activated protein kinase (AMPK). Subsequently, we found that the knockdown of PDE4D gene expression inhibited proliferation of STK11-mutated lung cancer lines. Furthermore, challenge with a panel of PDE4-specific inhibitors was shown to selectively reduce the growth of STK11-mutated lung cancer lines. Thus, we show that multidimensional analysis of a well-characterized large-scale panel of cancer cell lines provides unprecedented opportunities for the identification of unexpected oncogenic mechanisms and mutation-specific drug targets.

Overexpression of ERß is sufficient to inhibit hypoxia-inducible factor-1 transactivation.

Estrogen receptor (ER) ß is predicted to play an important role in the prevention of breast cancer development and progression. We have previously shown that ERß suppresses hypoxia inducible factor (HIF)-1-mediated transcription through aryl hydrocarbon receptor nuclear translocator (ARNT) degradation via ubiquitination processes. In this study, we attempted to examine the effect of ERß specific ligand on HIF-1 inhibition in ERß positive PC3 cells and ERß transfected MCF-7 cells. ERß specific agonist diarylpropionitrile (DPN) stimulated estrogen response element (ERE)-luciferase activity in a similar fashion to estradiol in PC3 cells. We observed that DPN down-regulates the ARNT protein levels leading to an attenuation of hypoxia-induced hypoxia response element (HRE)-driven luciferase reporter gene activation in PC3 cells. Treatment of DPN reduced vascular endothelial growth factor (VEGF) expression and co-treatment with ERß specific antagonist PHTPP abrogated the effect in PC3 cells. We then examined the effect of DPN in ERß transfected MCF-7 cells. HIF-1 transcriptional activity repression by ERß was not further reduced by DPN, as examined by HRE-driven luciferase assays. Expression of ERß significantly decreased VEGF secretion and ARNT expression under hypoxic conditions. However, DPN did not additionally affect this suppression in MCF-7 cells transfected with ERß. This result shows that unliganded ERß is sufficient to inhibit HIF-1 in systems of overexpression.

Glucocorticoids suppress hypoxia-induced COX-2 and hypoxia inducible factor-1α expression through the induction of glucocorticoid-induced leucine zipper.

BACKGROUND AND PURPOSE: The COX-2/PGE2 pathway in hypoxic cancer cells has important implications for stimulation of inflammation and tumourigenesis. However, the mechanism by which glucocorticoid receptors (GRs) inhibit COX-2 during hypoxia has not been elucidated. Hence, we explored the mechanisms underlying glucocorticoid-mediated inhibition of hypoxia-induced COX-2 in human distal lung epithelial A549 cells. EXPERIMENTAL APPROACH: The expressions of COX-2 and glucocorticoid-induced leucine zipper (GILZ) in A549 cells were determined by Western blot and/or quantitative real time-PCR respectively. The anti-invasive effect of GILZ on A549 cells was evaluated using the matrigel invasion assay. KEY RESULTS: The hypoxia-induced increase in COX-2 protein and mRNA levels and promoter activity were suppressed by dexamethasone, and this effect of dexamethasone was antagonized by the GR antagonist RU486. Overexpression of GILZ in A549 cells also inhibited hypoxia-induced COX-2 expression levels and knockdown of GILZ reduced the glucocorticoid-mediated inhibition of hypoxia-induced COX-2 expression, indicating that the inhibitory effects of dexamethasone on hypoxia-induced COX-2 are mediated by GILZ. GILZ suppressed the expression of hypoxia inducible factor (HIF)-1α at the protein level and affected its signalling pathway. Hypoxia-induced cell invasion was also dramatically reduced by GILZ expression. CONCLUSION AND IMPLICATIONS: Dexamethasone-induced upregulation of GILZ not only inhibits the hypoxic-evoked induction of COX-2 expression and cell invasion but further blocks the HIF-1 pathway by destabilizing HIF-1α expression. Taken together, these findings suggest that the suppression of hypoxia-induced COX-2 by glucocorticoids is mediated by GILZ. Hence, GILZ is a potential key therapeutic target for suppression of inflammation under hypoxia.

Hypoxia enhances ligand-occupied androgen receptor activity.

Hypoxia and the androgen receptor (AR) play important roles in the development and progression of prostate cancer. In this study, the combined effects of dihydrotestosterone (DHT) and hypoxia on AR-mediated transactivation were investigated. Hypoxia alone did not induce a detectable ARE-mediated response in the absence of DHT. DHT-induced AR transcriptional activity was dramatically increased by hypoxia or ectopic expression of HIF-1α, as determined by introducing ARE-responsive reporter plasmids into LNCaP prostate cancer cells. The secretion of VEGF was enhanced by the combination of hypoxia and DHT as compared to each treatment alone. These effects were not due to increased expression of the AR or HIF-1α as a result of hypoxia and DHT treatment. These results provide evidence that hypoxia may stimulate as yet unknown factors, which further stimulate AR signal transduction pathways.

Hypoxia-inducible factor 1 alpha represses the transcription of the estrogen receptor alpha gene in human breast cancer cells.

The estrogen receptor is one of the most important transcription factors in breast tumor growth and development. We, and others, have previously shown that hypoxia induces rapid ERα protein degradation by a proteasome-mediated pathway in breast cancer cells, which is linked with ERα activation. However, no report has shown the effect of hypoxia on ESR1 gene regulation at the transcriptional level. In this report, we show that hypoxia repressed the expression of ERα mRNA in MCF-7 and T47D human breast cancer cells, but not in human endometrial Ishikawa cells, although ERα degradation under hypoxia was also observed in Ishikawa cells. This indicates that ESR1 transcriptional repression and ERα protein downregulation by hypoxia are regulated by distinct mechanisms. Repression of ESR1 gene transcription occurred at the transcriptional level as a result of decreased recruitment of RNA polymerase II at the proximal promoter of the ESR1 locus in response to stabilization of the HIF-1α protein under hypoxia. Our data show that hypoxia induces repression of the ESR1 gene, which may facilitate hormone insensitivity in the tumor microenvironment.

Estrogen receptor beta inhibits transcriptional activity of hypoxia inducible factor-1 through the downregulation of arylhydrocarbon receptor nuclear translocator.

INTRODUCTION: Estrogen receptor (ER) ß is predicted to play an important role in prevention of breast cancer development and metastasis. We have shown previously that ERß inhibits hypoxia inducible factor (HIF)-1α mediated transcription, but the mechanism by which ERß works to exert this effect is not understood. METHODS: Vascular endothelial growth factor (VEGF) was measured in conditioned medium by enzyme-linked immunosorbent assays. Reverse transcription polymerase chain reaction (RT-PCR), Western blotting, immunoprecipitation, luciferase assays and chromatin immunoprecipitation (ChIP) assays were used to ascertain the implication of ERß on HIF-1 function. RESULTS: In this study, we found that the inhibition of HIF-1 activity by ERß expression was correlated with ERß's ability to degrade aryl hydrocarbon receptor nuclear translocator (ARNT) via ubiquitination processes leading to the reduction of active HIF-1α/ARNT complexes. HIF-1 repression by ERß was rescued by overexpression of ARNT as examined by hypoxia-responsive element (HRE)-driven luciferase assays. We show further that ERß attenuated the hypoxic induction of VEGF mRNA by directly decreasing HIF-1α binding to the VEGF gene promoter. CONCLUSIONS: These results show that ERß suppresses HIF-1α-mediated transcription via ARNT down-regulation, which may account for the tumour suppressive function of ERß.