Negative regulation of thyroid adenoma-associated protein (THADA) in the cardiac glycoside-induced anti-cancer effect.

Cardiac glycosides, known as inhibitors of Na+,K+-ATPase, have anti-cancer effects such as suppression of cancer cell proliferation and induction of cancer cell death. Here, we examined the signaling pathway elicited by cardiac glycosides in the human hepatocellular carcinoma HepG2 cells and human epidermoid carcinoma KB cells. Three kinds of cardiac glycosides (ouabain, oleandrin, and digoxin) inhibited the cancer cell proliferation and decreased the expression level of thyroid adenoma-associated protein (THADA). Interestingly, the knockdown of THADA inhibited cancer cell proliferation, and the proliferation was significantly rescued by re-expression of THADA in the THADA-knockdown cells. In addition, the THADA-knockdown markedly decreased the expression level of L-type amino acid transporter LAT1. Cardiac glycosides also reduced the LAT1 expression. The LAT1 inhibitor, JPH203, significantly weakened the cancer cell proliferation. These results suggest that the binding of cardiac glycosides to Na+,K+-ATPase negatively regulates the THADA-LAT1 pathway, exerting the anti-proliferative effect in cancer cells.

An adverse outcome pathway-based approach to assess the neurotoxicity by combined exposure to current-use pesticides.

Exposure to multiple pesticides in daily life has become an important public health concern. However, the combined effects of pesticide mixtures have not been fully elucidated by the conventional toxicological testing used for individual chemicals. Grouping of chemicals by mode of action using common key events (KEs) in the adverse outcome pathway (AOP) as endpoints could be applied for efficient risk assessment of combined exposure to multiple chemicals. The purpose of this study was to investigate whether exposure to multiple pesticides has synergistic neurotoxic effects on mammalian nervous systems. According to the AOP-based approach, we evaluated the effects of 10 current-use pesticides (4 neonicotinoids, 4 pyrethroids and 2 phenylpyrazoles) on the common KEs in AOPs for neurotoxicity, such as KEs involving mitochondrial and proteolytic functions, in a mammalian neuronal cell model. Our data showed that several pyrethroids and phenylpyrazoles partly shared the effects on several common KEs, including decreases in mitochondrial membrane potential and proteasome activity and increases in autophagy activity. Furthermore, we also found that combined exposure to a type-I pyrethroid permethrin or a type-II pyrethroid deltamethrin and the phenylpyrazole fipronil decreased the cell viability and the benchmark doses much more than either single exposure, indicating that the pair exhibited synergistic effects, since the combination indexes were less than 1. These findings revealed that novel pairs of different classes of pesticides with similar effects on common KEs exhibited synergistic neurotoxicity and provide new insights into the risk assessment of combined exposure to multiple chemicals.

Parkinson's disease-associated ATP13A2/PARK9 functions as a lysosomal H+,K+-ATPase.

Mutations in the human ATP13A2 (PARK9), a lysosomal ATPase, cause Kufor-Rakeb Syndrome, an early-onset form of Parkinson's disease (PD). Here, we demonstrate that ATP13A2 functions as a lysosomal H+,K+-ATPase. The K+-dependent ATPase activity and the lysosomal K+-transport activity of ATP13A2 are inhibited by an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase, thapsigargin, and K+-competitive inhibitors of gastric H+,K+-ATPase, such as vonoprazan and SCH28080. Interestingly, these H+,K+-ATPase inhibitors cause lysosomal alkalinization and α-synuclein accumulation, which are pathological hallmarks of PD. Furthermore, PD-associated mutants of ATP13A2 show abnormal expression and function. Our results suggest that the H+/K+-transporting function of ATP13A2 contributes to acidification and α-synuclein degradation in lysosomes.

Regulation of Paracellular Fluxes of Amino Acids by Claudin-8 in Normal Mouse Intestinal MCE301 Cells.

The ingested proteins are catabolized to di/tri-peptides and amino acids (AAs), which are absorbed through various transporters in the small intestinal and colonic epithelial cells. Tight junctions (TJs) are formed between neighboring cells and restrict paracellular fluxes to mineral ions and aqueous molecules. However, it is unknown whether the TJs are implicated in the control of paracellular fluxes to AAs. The paracellular permeability is controlled by claudins (CLDNs), which comprise a family of over 20 members. Here, we found that CLDN8 expression is decreased by AAs deprivation in normal mouse colon-derived MCE301 cells. The reporter activity of CLDN8 was not significantly changed by AAs deprivation, whereas the stability of CLDN8 protein was decreased. MicroRNA analysis showed that AAs deprivation increases the expression of miR-153-5p which targets CLDN8. The AAs deprivation-induced decline of CLDN8 expression was reversed by a miR-153-5p inhibitor. The CLDN8 silencing enhanced the paracellular fluxes to AAs, especially middle molecular size AAs. The expression levels of colonic CLDN8 and miR-153-5p in aged mice were lower and higher than those in young mice, respectively. We suggest that AAs deprivation downregulates CLDN8-dependent barrier function, mediated by the elevation of miR-153-5p expression in the colon, in order to enhance the AAs absorption.

Physiological consequences of space flight, including abnormal bone metabolism, space radiation injury, and circadian clock dysregulation: Implications of melatonin use and regulation as a countermeasure.

Exposure to the space environment induces a number of pathophysiological outcomes in astronauts, including bone demineralization, sleep disorders, circadian clock dysregulation, cardiovascular and metabolic dysfunction, and reduced immune system function. A recent report describing experiments aboard the Space Shuttle mission, STS-132, showed that the level of melatonin, a hormone that provides the biochemical signal of darkness, was decreased during microgravity in an in vitro culture model. Additionally, abnormal lighting conditions in outer space, such as low light intensity in orbital spacecraft and the altered 24-h light-dark cycles, may result in the dysregulation of melatonin rhythms and the misalignment of the circadian clock from sleep and work schedules in astronauts. Studies on Earth have demonstrated that melatonin regulates various physiological functions including bone metabolism. These data suggest that the abnormal regulation of melatonin in outer space may contribute to pathophysiological conditions of astronauts. In addition, experiments with high-linear energy transfer radiation, a ground-based model of space radiation, showed that melatonin may serve as a protectant against space radiation. Gene expression profiling using an in vitro culture model exposed to space flight during the STS-132 mission, showed that space radiation alters the expression of DNA repair and oxidative stress response genes, indicating that melatonin counteracts the expression of these genes responsive to space radiation to promote cell survival. These findings implicate the use of exogenous melatonin and the regulation of endogenous melatonin as countermeasures for the physiological consequences of space flight.

ZBTB2 links p53 deficiency to HIF-1-mediated hypoxia signaling to promote cancer aggressiveness.

Aberrant activation of the hypoxia-inducible transcription factor HIF-1 and dysfunction of the tumor suppressor p53 have been reported to induce malignant phenotypes and therapy resistance of cancers. However, their mechanistic and functional relationship remains largely unknown. Here, we reveal a mechanism by which p53 deficiency triggers the activation of HIF-1-dependent hypoxia signaling and identify zinc finger and BTB domain-containing protein 2 (ZBTB2) as an important mediator. ZBTB2 forms homodimers via its N-terminus region and increases the transactivation activity of HIF-1 only when functional p53 is absent. The ZBTB2 homodimer facilitates invasion, distant metastasis, and growth of p53-deficient, but not p53-proficient, cancers. The intratumoral expression levels of ZBTB2 are associated with poor prognosis in lung cancer patients. ZBTB2 N-terminus-mimetic polypeptides competitively inhibit ZBTB2 homodimerization and significantly suppress the ZBTB2-HIF-1 axis, leading to antitumor effects. Our data reveal an important link between aberrant activation of hypoxia signaling and loss of a tumor suppressor and provide a rationale for targeting a key mediator, ZBTB2, to suppress cancer aggressiveness.

Roles of Macrophages in Advanced Liver Fibrosis, Identified Using a Newly Established Mouse Model of Diet-Induced Non-Alcoholic Steatohepatitis.

Macrophages play critical roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). However, it is unclear which macrophage subsets are critically involved in the development of inflammation and fibrosis in NASH. In TSNO mice fed a high-fat/cholesterol/cholate-based diet, which exhibit advanced liver fibrosis that mimics human NASH, we found that Kupffer cells (KCs) were less abundant and recruited macrophages were more abundant, forming hepatic crown-like structures (hCLS) in the liver. The recruited macrophages comprised two subsets: CD11c+/Ly6C- and CD11c-/Ly6C+ cells. CD11c+ cells were present in a mesh-like pattern around the lipid droplets, constituting the hCLS. In addition, CD11c+ cells colocalized with collagen fibers, suggesting that this subset of recruited macrophages might promote advanced liver fibrosis. In contrast, Ly6C+ cells were present in doughnut-like inflammatory lesions, with a lipid droplet in the center. Finally, RNA sequence analysis indicates that CD11c+/Ly6C- cells promote liver fibrosis and hepatic stellate cell (HSC) activation, whereas CD11c-/Ly6C+ cells are a macrophage subset that play an anti-inflammatory role and promote tissue repair in NASH. Taken together, our data revealed changes in liver macrophage subsets during the development of NASH and shed light on the roles of the recruited macrophages in the pathogenesis of advanced fibrosis in NASH.

Cardiac glycosides stimulate endocytosis of GLUT1 via intracellular Na+ ,K+ -ATPase α3-isoform in human cancer cells.

Glucose transporter GLUT1 plays a primary role in the glucose metabolism of cancer cells. Here, we found that cardiac glycosides (CGs) such as ouabain, oleandrin, and digoxin, which are Na+ ,K+ -ATPase inhibitors, decreased the GLUT1 expression in the plasma membrane of human cancer cells (liver cancer HepG2, colon cancer HT-29, gastric cancer MKN45, and oral cancer KB cells). The effective concentration of ouabain was lower than that for inhibiting the activity of Na+ ,K+ -ATPase α1-isoform (α1NaK) in the plasma membrane. The CGs also inhibited [3 H]2-deoxy- d-glucose uptake, lactate secretion, and proliferation of the cancer cells. In intracellular vesicles of human cancer cells, Na+ ,K+ -ATPase α3-isoform (α3NaK) is abnormally expressed. Here, a low concentration of ouabain inhibited the activity of α3NaK. Knockdown of α3NaK significantly inhibited the ouabain-decreased GLUT1 expression in HepG2 cells, while the α1NaK knockdown did not. Consistent with the results in human cancer cells, CGs had no effect on GLUT1 expression in rat liver cancer dRLh-84 cells where α3NaK was not endogenously expressed. Interestingly, CGs decreased GLUT expression in the dRLh-84 cells exogenously expressing α3NaK. In HepG2 cells, α3NaK was found to be colocalized with TPC1, a Ca2+ -releasing channel activated by nicotinic acid adenine dinucleotide phosphate (NAADP). The CGs-decreased GLUT1 expression was significantly inhibited by a Ca2+ chelator, a Ca2+ -ATPase inhibitor, and a NAADP antagonist. The GLUT1 decrease was also attenuated by inhibitors of dynamin and phosphatidylinositol-3 kinases (PI3Ks). In conclusion, the binding of CGs to intracellular α3NaK elicits the NAADP-mediated Ca2+ mobilization followed by the dynamin-dependent GLUT1 endocytosis in human cancer cells.

Effect of steam-processing of the Panax ginseng root on its inducible activity on granulocyte-colony stimulating factor secretion in intestinal epithelial cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng root has been used as tonic in traditional Chinese medicine (TCM) and traditional Japanese Kampo medicine. Steam processing of Panax ginseng root is carried out to enhance its nourishing effects on qi. AIM OF THE STUDY: In order to explore the mechanism of these beneficial effects behind the steam processing of the P. ginseng root, we evaluated effectiveness of processing on the granulocyte-colony stimulating factor (G-CSF) secretion in intestinal epithelial cell-like MCE301 cells. MATERIALS AND METHODS: We collected P. ginseng root samples in the markets of China and Japan. Fresh or dried samples were steamed for different time lengths and subsequently dried and extracted. MCE301 cells were incubated with the medium containing various P. ginseng root extracts, while the concentration of G-CSF in the medium was measured. We also investigated the active ingredients by size exclusion HPLC. RESULTS: The extracts of fresh P. ginseng hairy root samples steamed for more than 6 h significantly induced G-CSF secretion, and the maximum activity was recorded at a 9-h steaming. The same activity was noted when already dried P. ginseng hairy root samples were steamed. The extracts of fresh P. ginseng hairy root without steam processing and those of fresh P. ginseng root body samples with steam processing exhibited no activities. The active ingredients of steamed P. ginseng hairy root samples were high-molecular-weight compounds with an average molecular weight of 758 kDa, and the activity was mediated by the toll-like receptor (TLR) 9. CONCLUSIONS: Our results shed on more light on the mechanism underlying the appearance of immunostimulatory activity of the P. ginseng hairy root induced by steam processing.

SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

Hypoxia is associated with tumor radioresistance; therefore, a predictive marker for tumor hypoxia and a rational target to overcome it have been sought to realize personalized radiotherapy. Here, we show that serine protease inhibitor Kazal type I (SPINK1) meets these 2 criteria. SPINK1 expression was induced upon hypoxia (O2 < 0.1%) at the transcription initiation level in a HIF-dependent manner, causing an increase in secreted SPINK1 levels. SPINK1 proteins were detected both within and around hypoxic regions of xenografted and clinical tumor tissues, and their plasma levels increased in response to decreased oxygen supply to xenografts. Secreted SPINK1 proteins enhanced radioresistance of cancer cells even under normoxic conditions in EGFR-dependent and nuclear factor erythroid 2-related factor 2-dependent (Nrf2-dependent) manners and accelerated tumor growth after radiotherapy. An anti-SPINK1 neutralizing antibody exhibited a radiosensitizing effect. These results suggest that SPINK1 secreted from hypoxic cells protects the surrounding and relatively oxygenated cancer cells from radiation in a paracrine manner, justifying the use of SPINK1 as a target for radiosensitization and a plasma marker for predicting tumor hypoxia.

Neurotoxicity of a pyrethroid pesticide deltamethrin is associated with the imbalance in proteolytic systems caused by mitophagy activation and proteasome inhibition.

Pyrethroids are one of the most commonly used classes of synthetic pesticides in the world. Recent laboratory and epidemiological evidence suggested that pyrethroids have potential adverse effects in the mammalian brain; however, the underlying mechanisms of the neurotoxic effects of pyrethroids have not been fully elucidated. In the present study, we investigated the mechanisms of effects of a type II pyrethroid deltamethrin (DM) in a neuronal cell model focusing on the proteolytic function, including autophagy and the ubiquitin-proteasome system. We confirmed that a micromolar concentration of DM dose-dependently decreased the cell viability and induced apoptotic cell death. Our results showed that DM enhanced autophagy in association with an accumulation of autophagosomes and increase in the levels of autophagy markers LC3-II/LC3-I ratio and p62 which were much elevated in the presence of lysosomal inhibitors bafilomycin A1 and chloroquine. We also found that DM caused a dysfunction of mitochondria with a decrease of mitochondrial membrane potential and mitochondrial DNA copy number as well as colocalization with autophagosomes. Moreover, a decrease in the activities of three major proteasomal enzymes and an accumulation of ubiquitinated proteins were observed by the exposure to DM. Transcriptome analysis revealed that up-regulated genes supported the activation of autophagy with induction of cellular stress responses including oxidative stress and endoplasmic reticulum stress, while down-regulated genes related to the cell cycle and DNA replication. These findings provide novel insights into the neurotoxicity of DM which underlie the imbalance in proteolytic function caused by mitophagy activation and proteasome inhibition.

Low-intensity ultrasound inhibits melanoma cell proliferation in vitro and tumor growth in vivo.

PURPOSE: To determine the effect of low-intensity ultrasound on cancer cell proliferation in vitro and tumor growth in vivo. METHODS: In vitro, several cancer cell lines were exposed to low-intensity ultrasound at 0.11 W/cm2 for 2 min. Of the cell lines screened, melanoma C32 is one of the cell lines that showed sensitivity to growth inhibition by ultrasound and was therefore used in succeeding experiments. In vivo, under the same ultrasound conditions used in vitro, C32 tumors in mice were exposed to ultrasound daily for 2 weeks, and the tumor volumes were monitored weekly using sonography. RESULTS: In vitro, C32 cell growth was inhibited, attaining 43.2% inhibition on the 3rd day. In vivo, tumor growth was significantly inhibited, with the treated tumors exhibiting 2.7-fold slowed tumor growth vs. untreated tumors at week 2. Such inhibition was not associated with increased cell death. Several genes related to the cell cycle and proliferation were among those significantly regulated. CONCLUSION: These findings highlight the potential of low-intensity ultrasound to inhibit tumor growth in a noninvasive, safe, and easy-to-administer way. In addition, this may suggest that the mechanical stress induced by ultrasound on C32 cells may have affected the intrinsic biomolecular mechanism related to the cell growth of this particular cell line. Further research is needed to identify which of the regulated genes played key roles in growth inhibition.

Overexpression of D-dopachrome tautomerase increases ultraviolet B irradiation-induced skin tumorigenesis in mice.

Ultraviolet irradiation (UV) exposure is the leading factor underlying the development of skin malignancies. D-dopachrome tautomerase (D-DT), a functional homolog of macrophage migration inhibitory factor (MIF), has functional similarities to MIF. However, its role, unlike the role of MIF in photocarcinogenesis, is unknown. We therefore explored the role of D-DT in photocarcinogenesis by developing D-DT transgenic (D-DT Tg) mice and provided a research model for future studies targeting D-DT. Chronic UVB exposure accelerated tumor development in D-DT Tg mice compared with wild-type (WT) mice, with a higher incidence of tumors observed in D-DT Tg mice than in WT mice. In D-DT Tg irradiated mouse keratinocytes, the p53, PUMA, and Bax expression was lower than that in WT mice. These results indicate that D-DT Tg overexpression confers prevention against UVB-induced apoptosis in keratinocytes. Taken together, these findings support D-DT as a functionally important cytokine in photocarcinogenesis and potential therapeutic target for the prevention of photocarcinogenesis.

Survival of detached cancer cells is regulated by movement of intracellular Na+,K+-ATPase.

Beginning of metastasis, cancer cells detach from the primary tumor and they can survive even under loss of anchorage; however, the detachment-elicited mechanisms have remained unknown. Here, we found that Na+,K+-ATPase α3-isoform (α3NaK) in human cancer cells is dynamically translocated from intracellular vesicles to the plasma membrane when the attached cells are detached and that this mechanism contributes to the survival of the detached (floating) cancer cells. α3NaK was detected in the plasma membrane of floating cancer cells in peritoneal fluids of patients, while it was in the cytoplasm of the cells in primary tumor tissues. On cancer cell detachment, we also found the focal-adhesion-kinase-dependent Ca2+ response that induces the α3NaK translocation via nicotinic acid adenine dinucleotide phosphate pathway. Activation of AMP-activated protein kinase was associated with the translocated α3NaK in the plasma membrane. Collectively, our study identifies a unique mechanism for survival of detached cancer cells, opening up new opportunities for development of cancer medicines.

Berberine induces anti-atopic dermatitis effects through the downregulation of cutaneous EIF3F and MALT1 in NC/Nga mice with atopy-like dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with severe pruritus. Berberine, a naturally occurring isoquinoline alkaloid, has anti-inflammatory effects. This study investigated the effects and molecular mechanisms of berberine on AD-like symptoms in mice. In this study, NC/Nga mice with atopy-like dermatitis (dermatitis mice), fibroblast and mast cells were used. In dermatitis mice, intermittent oral administrations of berberine 3 times a week for 12 days inhibited skin symptom, itching, cutaneous infiltration of eosinophils and mast cells, and the expression of cutaneous eotaxin, macrophage migration inhibitory factor (MIF) and IL-4. Berberine also attenuated IL-4/MIF-induced eotaxin in fibroblasts and allergen-induced MIF and IL-4 in mast cells. In mast cells, the GeneChip® microarray showed that antigen increased the expression of EIF3F and MALT1, inhibited by berberine. The siRNAs for them inhibited the expression of MIF and IL-4 in antigen-stimulated mast cells. These results suggest that berberine improves AD-like symptoms through the inhibition of the eotaxin and pro-inflammatory cytokine expression and the related inflammatory cell recruitment. It is also suggested that the downregulation of EIF3F and MALT1 by berberine is involved in suppressing the cytokine expression. Taken together, berberine or berberine-containing crude drugs are expected to contribute to the improvement of AD symptoms.

Genetic response to low­intensity ultrasound on mouse ST2 bone marrow stromal cells.

Although low­intensity ultrasound (LIUS) is a clinically established procedure, the early cellular effect of LIUS on a genetic level has not yet been studied. The current study investigated the early response genes elicited by LIUS in bone marrow stromal cells (BMSCs) using global­scale microarrays and computational gene expression analysis tools. Mouse ST2 BMSCs were treated with LIUS [ISATA, 25 mW/cm2 for 20 min with a frequency of 1.11 MHz in a pulsed­wave mode (0.2­s burst sine waves repeated at 1 kHz)], then cultured for 0.5, 1 and 3 h at 37˚C. The time course of changes in gene expression was evaluated using GeneChip® high­density oligonucleotide microarrays and Ingenuity® Pathway Analysis tools. The results were verified by reverse transcription­quantitative polymerase chain reaction (RT­qPCR). A single exposure of LIUS did not affect cell morphology, cell growth or alkaline phosphatase activity. However, 61 upregulated and 103 downregulated genes were identified from 0.5 to 3 h after LIUS treatment. Two significant gene networks, labeled E and H, were identified from the upregulated genes, while a third network, labeled T, was identified from the downregulated genes. Gene network E or H containing the immediate­early genes FBJ osteosarcoma oncogene and early growth response 1 or the heat shock proteins heat shock protein 1a/b was associated mainly with the biological functions of bone physiology and protein folding or apoptosis, respectively. Gene network T containing transcription factors fos­like antigen 1 and serum response factor was also associated with the biological functions of the gene expression. RT­qPCR indicated that the expression of several genes in the gene networks E and H were elevated in LIUS­treated cells. LIUS was demonstrated to induce gene expression after short application in mouse ST2 BMSCs. The results of the present study provide a basis for the elucidation of the detailed molecular mechanisms underlying the cellular effects of LIUS.

Inverse regulation of claudin-2 and -7 expression by p53 and hepatocyte nuclear factor 4α in colonic MCE301 cells.

Colonic epithelial cells move up along the crypt villus axis and are differentiated into absorptive or secretory cells. Claudin-7 (CLDN7), a tight junctional protein, is mainly located at the surface of crypt, whereas CLDN2 is located at the bottom. However, the expression mechanism and function of these CLDNs are not fully understood. The expression levels of CLDN2 and CLDN7 were altered depending on the culture days in MCE301 cells derived from mouse colon. The nuclear levels of transcriptional factors p53 and hepatocyte nuclear factor 4α (HNF4α) at day 21 were higher than those at day 7. Tenovin-1 (TEN), a p53 activator, increased the nuclear levels of p53 and HNF4α. The mRNA level and promoter activity of CLDN7 were increased by TEN, whereas those of CLDN2 were decreased. The changes of CLDNs expression were inhibited by p53 and HNF4α siRNAs. The association between p53 and HNF4α was elevated by TEN. In addition, the binding of p53 and HNF4α to the promoter region of CLDN2 and CLDN7 was enhanced by TEN. Transepithelial electrical resistance was decreased by TEN, but paracellular fluxes of lucifer yellow and dextran were not. In the Ussing chamber assay, TEN increased dilution potential and the ratio of permeability of Cl- to Na+. Both p53 and HNF4α were highly expressed at the surface of mouse colon crypt. We suggest that p53 and HNF4α alter the paracellular permeability of Cl- to Na+ mediated by the inverse regulation of CLDN2 and CLDN7 expression in the colon.

Bioinformatics analysis of the microRNA-mRNA network in sebaceous gland carcinoma of the eyelid.

Sebaceous gland carcinoma (SGC) of the eyelid is an uncommon aggressive tumor with a relatively high rate of local recurrence and a poor prognosis following metastasis. However, the molecular mechanisms underlying the pathogenesis of SGC remain unclear. The purpose of the present study was to clarify microRNA (miRNA) expression profiles in SGC and to explore novel miRNA­mRNA networks of SGC. A small RNA­sequencing analysis was performed to identify miRNAs differentially expressed between SGC and sebaceous adenoma control samples. Bioinformatics analyses were conducted to reveal biological functions, canonical pathways and molecular interaction networks using integrated miRNA­mRNA datasets, including mRNA expression profiles of SGC from our previous study. The present results demonstrated that 16 upregulated miRNAs and 516 downregulated mRNAs were associated with loss of lipid metabolism function and enriched in cholesterol biosynthesis pathways. By contrast, 29 downregulated miRNAs and 194 upregulated mRNAs were mainly associated with the promotion of cell survival and proliferation in addition to enrichment of DNA damage­induced cell cycle­regulation pathways. Furthermore, network analyses revealed that the upregulated miRNAs, miR­130a­3p and miR­939­5p, and the downregulated miRNAs, miR­146a­5p, miR­149­3p, miR­193a­3p, miR­195­5p and miR­4671­3p, could be upstream regulators related to these functional changes of SGC. These results improved the understanding of molecular mechanisms of SGC and may help to improve the diagnosis of SGC.

Trichohyalin-like 1 protein plays a crucial role in proliferation and anti-apoptosis of normal human keratinocytes and squamous cell carcinoma cells.

Epidermal differentiation is a complex process that requires the regulated and sequential expression of various genes. Most fused-type S100 proteins are expressed in the granular layer and it is hypothesized that these proteins may be associated with cornification and barrier formation. We previously identified a member of the fused-type S100 proteins, Trichohyalin-like 1 (TCHHL1) protein. TCHHL1 is distributed in the basal layer of the normal epidermis. Furthermore, the expression is markedly increased in cancerous/non-cancerous skin samples with the hyperproliferation of keratinocytes. We herein examined the role of TCHHL1 in normal human keratinocytes (NHKs) and squamous cell carcinoma (SCC). The knockdown of TCHHL1 by transfection with TCHHL1 siRNA significantly inhibited proliferation and induced the early apoptosis of NHKs. In TCHHL1-knockdown NHKs, the level of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation was markedly decreased. In addition, the slight inhibition of v-akt murine thymoma viral oncogene homolog (AKT) phosphorylation and upregulation of forkhead box-containing protein O1(FOXO1), B-cell lymphoma2 (BCL2) and Bcl2-like protein 11 (BCL2L11) was observed. Skin-equivalent models built by TCHHL1-knockdown NHKs showed a markedly hypoplastic epidermis. These findings highlight that TCHHL1 plays an important role in homeostasis of the normal epidermis. TCHHL1 was expressed in the growing cells of cutaneous SCC; therefore, we next examined an association with the cell growth in HSC-1 cells (a human SCC line). In HSC-1 cells, the knockdown of TCHHL1 also suppressed cell proliferation and induced apoptosis. These cells showed an inhibition of phosphorylation of ERK1/2, AKT and signal transducers and activator of transcription 3, and the significant upregulation of FOXO1, BCL2, and BCL2L11. Accordingly, TCHHL1 is associated with survival of cutaneous SCC. In addition, we hypothesize that TCHHL1 may be a novel therapeutic target in cutaneous SCC.

Honey isomaltose contributes to the induction of granulocyte-colony stimulating factor (G-CSF) secretion in the intestinal epithelial cells following honey heating.

We have previously discovered that heated honey but not unheated honey could induce the secretion of granulocyte-colony stimulating factor (G-CSF) in the MCE301 intestinal epithelial cells. The objective of this study was to identify compounds in honey that could contribute to this activity. We bought several kinds of commercial honey samples derived from different flowers, as well as corn syrup samples, in the markets of China and Japan, and heated them at 180 °C for 30 min. MCE301 cells were treated with the medium containing the samples, and G-CSF levels in the medium were measured by ELISA. By comparing their activities and sugar contents, we discovered that isomaltose was primarily implicated. The optimum heating conditions for isomaltose were at 180 °C for 60 min or at 200 °C for 15-30 min, and these time- and temperature-dependencies were similar to those of honey in our previous study. When heated isomaltose was partitioned by dialysis, the active ingredients were transferred into a high-molecular-weight fraction. By size-exclusion HPLC analysis, the average molecular weight of heated isomaltose was 790 kDa. When heated isomaltose was hydrolyzed by acids, glucose was subsequently produced. Maltose, sucrose, turanose, and trehalose did not exhibited any activity when heated at 180 °C for 60 min, indicating that the glucose groups with α(1 → 6)-binding in the isomaltose molecule play important roles in its activity when oxidatively polymerized by heat. The stimulating activity of heated isomaltose was inhibited by toll-like receptor 4 (TLR4) inhibitor, suggesting that heated isomaltose activates TLR4 to induce G-CSF. Since G-CSF is clinically used for cancer patients to accelerate their recovery from neutropenia following chemotherapy or accompanied with aplastic anemia, these findings indicate that honey which contains high level of isomaltose could improve immunosuppressive conditions when honey is heated, and that heated isomaltose might be of potential therapeutic use in patients with compromised immunity caused by chemotherapeutic agents.