Anticancer effects of alpelisib on PIK3CA-mutated canine mammary tumor cell lines.

Canine mammary tumors (CMTs) are commonly observed in old and unspayed female dogs. Recently, dogs have been increasingly spaying at a young age to prevent mammary tumors. These CMTs require extensive local excision and exhibit a high probability of metastasis to the regional lymph nodes and lungs during malignancy. However, the molecular and biological mechanisms underlying CMT development have not been fully elucidated, and research in this area is limited. Therefore, in this study, we established new CMT cell lines by isolating cells from tumor tissues and investigated phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), a target for human breast cancer. PIK3CA mutations were observed at a similar loci as in the human PIK3CA gene in half of all canine samples. Furthermore, we investigated whether alpelisib, a PIK3CA inhibitor approved by the U.S. Food and Drug Administration for human breast cancer treatment, along with fulvestrant, is effective for CMT treatment. Alpelisib exerted stronger anticancer effects on cell lines with PIK3CA mutations than on the wild-type cell lines. In conclusion, we established new CMT cell lines with PIK3CA mutations and confirmed the efficacy of alpelisib for CMT treatment in vitro.

Surface proteins of Propionibacterium freudenreichii MJ2 inhibit RANKL-induced osteoclast differentiation by lipocalin-2 upregulation and lipocalin-2-mediated NFATc1 inhibition.

Osteoclasts degrade bone and osteoclast differentiation has been implicated in bone destruction in rheumatoid arthritis. The dairy bacterium Propionibacterium freudenreichii MJ2 (MJ2) isolated from raw milk inhibits osteoclast differentiation and ameliorates collagen-induced arthritis. This study aimed to investigate the inhibitory effect of the surface proteins of MJ2 on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and explain the underlying mechanism. The murine macrophage cell line RAW 264.7 was used to study the inhibition of osteoclast differentiation. The surface proteins significantly inhibited RANKL-induced osteoclast differentiation in a protein concentration-dependent manner by inhibiting the expression of genes and proteins related to osteoclast differentiation. RNA microarray analysis showed that the surface proteins significantly upregulated lipocalin-2 (lcn2) expression. In addition, they downregulated c-fos and NFATc1 and inhibited the expression of NFATc1-downstream genes Atp6v0d2, Calcr, and Ctsk. siRNA silencing of lcn2 decreased the extent of surface protein inhibition on osteoclast differentiation, suggesting that lcn2 plays an important role in the inhibition of RANKL-induced osteoclast differentiation. In conclusion, surface proteins of MJ2 show inhibitory effects on RANKL-induced osteoclast differentiation by upregulating lcn2 expression, in turn downregulating NFATc1, leading to the inhibition of NFATc1-downstream osteoclastogenesis-related gene expression.

Specific activation of hypoxia-inducible factor-2α by propionate metabolism via a ß-oxidation-like pathway stimulates MUC2 production in intestinal goblet cells.

Microbiota-derived short-chain fatty acids (SCFAs) are known to stimulate mucin expression in the intestine, which contributes to the gut mucosal immune responses, and the gut mucosal immune system extends to the brain and other organs through several axes. Hypoxia-inducible factors (HIFs), especially HIF-1α, are known to act as the master regulator of mucin expression, however, underlying mechanism of mucin expression during hypoxia by SCFAs remains unclear. In this study, we investigated the mechanism of MUC2 expression by propionate, an SCFA, in intestinal goblet cells. The real time oxygen consumption rate (OCR) and ATPase activity were measured to investigate the induction of hypoxia by propionate. Using 2-dimensional electrophoresis (2-DE), microarray analysis, and siRNA-induced gene silencing, we found that propionate is metabolized via a ß-oxidation-like pathway instead of the vitamin B12-dependent carboxylation pathway (also known as the methylmalonyl pathway). We verified the results by analyzing several intermediates in the pathway using LC-MS and GC-MS. Propionate metabolism via the ß-oxidation-like pathway leads to the depletion of oxygen and thereby induces hypoxia. Analysis of HIFs revealed that HIF-2α is the primary HIF whose activation is induced by propionate metabolism in a hypoxic environment and that HIF-2α regulates the expression of MUC2. Thus, hypoxia induced during propionate metabolism via a ß-oxidation-like pathway specifically activates HIF-2α, stimulating MUC2 production in LS 174 T goblet cells. Our findings show that propionate-induced selective HIF-2α stimulation contributes to intestinal mucosal defense.

Remofuscin induces xenobiotic detoxification via a lysosome-to-nucleus signaling pathway to extend the Caenorhabditis elegans lifespan.

Lipofuscin is a representative biomarker of aging that is generated naturally over time. Remofuscin (soraprazan) improves age-related eye diseases by removing lipofuscin from retinal pigment epithelium (RPE) cells. In this study, the effect of remofuscin on longevity in Caenorhabditis elegans and the underlying mechanism were investigated. The results showed that remofuscin significantly (p < 0.05) extended the lifespan of C. elegans (N2) compared with the negative control. Aging biomarkers were improved in remofuscin-treated worms. The expression levels of genes related to lysosomes (lipl-1 and lbp-8), a nuclear hormone receptor (nhr-234), fatty acid beta-oxidation (ech-9), and xenobiotic detoxification (cyp-34A1, cyp-35A1, cyp-35A2, cyp-35A3, cyp-35A4, cyp-35A5, cyp-35C1, gst-28, and gst-5) were increased in remofuscin-treated worms. Moreover, remofuscin failed to extend the lives of C. elegans with loss-of-function mutations (lipl-1, lbp-8, nhr-234, nhr-49, nhr-8, cyp-35A1, cyp-35A2, cyp-35A3, cyp-35A5, and gst-5), suggesting that these genes are associated with lifespan extension in remofuscin-treated C. elegans. In conclusion, remofuscin activates the lysosome-to-nucleus pathway in C. elegans, thereby increasing the expression levels of xenobiotic detoxification genes resulted in extending their lifespan.

Oxyresveratrol Ameliorates Dextran Sulfate Sodium-Induced Colitis in Rats by Suppressing Inflammation.

Colitis causes destruction of the intestinal mucus layer and increases intestinal inflammation. The use of antioxidants and anti-inflammatory agents derived from natural sources has been recently highlighted as a new approach for the treatment of colitis. Oxyresveratrol (OXY) is an antioxidant known to have various beneficial effects on human health, such as anti-inflammatory, antibacterial activity, and antiviral activity. The aim of this study was to investigate the therapeutic effect of OXY in rats with dextran sulfate sodium (DSS)-induced acute colitis. OXY ameliorated DSS-induced colitis and repaired damaged intestinal mucosa. OXY downregulated the expression of pro-inflammatory cytokine genes (TNF-α, IL-6, and IL-1ß) and chemokine gene MCP-1, while promoting the production of anti-inflammatory cytokine IL-10. OXY treatment also suppressed inflammation via inhibiting cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in the colon, as well as the activity of myeloperoxidase (MPO). OXY exhibited anti-apoptotic effects, shifting the Bax/Bcl-2 balance. In conclusion, OXY might improve DSS-induced colitis by restoring the intestinal mucus layer and reducing inflammation within the intestine.

Probiotic Propionibacterium freudenreichii MJ2 Enhances Osteoblast Differentiation and Mineralization by Increasing the OPG/RANKL Ratio.

Osteoblast differentiation is important for the development of bone and the maintenance of bone density. Propionibacterium freudenreichii is a probiotic with an anti-inflammatory property. The aim of this study was to investigate the enhancement effect of P. freudenreichii MJ2 (MJ2) isolated from raw milk on osteoblast differentiation, mineralization, and its signaling pathway. For in vitro and in vivo experiments, human fetal osteoblastic cell line hFOB 1.19 and an ovariectomized rat model were used, respectively. Expression levels of genes and proteins related to osteoblast differentiation and mineralization were measured by real-time polymerase chain reaction (qPCR) and Western blotting, respectively. Alizarin red S staining was performed to measure osteoblast mineralization. Heat-killed MJ2 (hkMJ2)-treated cells showed significantly increased osteoblast differentiation via an increase in the osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL) ratio and significantly increased osteoblast mineralization by stimulating the expression of bone morphogenetic protein 2 and runt-related transcription factor 2. Additionally, oral administration of live or heat-killed MJ2 to ovariectomized rats inhibited osteoporosis-induced bone loss. Specifically, surface proteins isolated from MJ2 promoted osteoblast differentiation and mineralization. In conclusion, MJ2 enhanced osteoblast differentiation and mineralization through the OPG/RANKL signaling pathway and the effective component of MJ2 might be its surface proteins.

Propionibacterium freudenreichii Inhibits RANKL-Induced Osteoclast Differentiation and Ameliorates Rheumatoid Arthritis in Collagen-Induced Arthritis Mice.

Osteoclast differentiation is crucial for bone absorption, and osteoclasts are involved in bone destruction in rheumatoid arthritis (RA). Dairy Propionibacterium freudenreichii is used as a cheese starter and possesses prebiotic and postbiotic properties. It is known to stimulate the growth of bifidobacteria and produces valuable metabolites, such as vitamin B12 and propionic acid. However, limited information is available on the beneficial effects of P. freudenreichii on human disease. Herein, we aimed to investigate the inhibitory effect of P. freudenreichii MJ2 (MJ2) isolated from raw milk on osteoclast differentiation and evaluate the improvement in RA. The murine macrophage cell line, RAW 264.7, and a collagen-induced arthritis (CIA) mouse model were used to perform in vitro and in vivo studies, respectively. Heat-killed P. freudenreichii MJ2 (hkMJ2)-treated cells significantly inhibited RANKL-induced osteoclast differentiation and TRAP activity. HkMJ2-treated cells exhibited significantly decreased expression of genes and proteins related to RANKL-induced osteoclast differentiation. MJ2 administration decreased the arthritic score in the CIA mouse model. Live and dead MJ2 inhibited bone loss and afforded protection against bone erosion and joint damage in CIA mice. MJ2 decreased the levels of collagen-specific antibodies and inflammatory cytokines and the expression of osteoclast differentiation-related genes and proteins in CIA mice. Interestingly, live and dead MJ2 showed similar RA improvement effects in CIA mice. In conclusion, P. freudenreichii MJ2 inhibited osteoclast differentiation by inhibiting the NF-κB signaling pathway and ameliorated CIA.

Exogenous NAD+ Stimulates MUC2 Expression in LS 174T Goblet Cells via the PLC-Delta/PTGES/PKC-Delta/ERK/CREB Signaling Pathway.

BACKGROUND: MUC2, a major component of the mucus layer in the intestine, is associated with antimicrobial activity and gut immune system function. Currently, mucin is mainly known for its critical function in defense against toxic molecules and pathogens. In this study, we investigated the stimulatory effects of exogenous nicotinamide adenine dinucleotide (NAD+) on the expression of MUC2 in LS 174T goblet cells. METHODS: Genes related to MUC2 synthesis were measured by quantitative real-time PCR (qPCR). To analyze the gene expression profiles of NAD+-treated LS 174T goblet cells, RNA sequencing was performed. MUC2 expression in the cells and secreted MUC2 were measured by immunocytochemistry (ICC) and ELISA, respectively. RESULTS: NAD+ significantly stimulated MUC2 expression at mRNA and protein levels and increased the secretion of MUC2. Through RNA sequencing, we found that the expression of genes involved in arachidonic acid metabolism increased in NAD+-treated cells compared with the negative control cells. NAD+ treatment increased phospholipase C (PLC)-δ and prostaglandin E synthase (PTGES) expression, which was inhibited by the appropriate inhibitors. Among the protein kinase C (PKC) isozymes, PKC-δ was involved in the increase in MUC2 expression. In addition, extracellular signal-regulated kinase (ERK)1/2 and cyclic AMP (cAMP) response element-binding protein (CREB) transcript levels were higher in NAD+-treated cells than in the negative control cells, and the enhanced levels of phosphorylated CREB augmented MUC2 expression. CONCLUSIONS: Exogenous NAD+ increases MUC2 expression by stimulating the PLC-δ/PTGES/PKC-δ/ERK/CREB signaling pathway.

Dairy Propionibacterium freudenreichii ameliorates acute colitis by stimulating MUC2 expression in intestinal goblet cell in a DSS-induced colitis rat model.

An intact mucus layer is important in managing inflammatory bowel disease (IBD). Dairy Propionibacterium freudenreichii has probiotic potential, produces propionic acid and is known to promote health. The aim of this study was to evaluate the effects of P. freudenreichii on the improvement of colitis. LS 174T goblet cells and a dextran sodium sulfate (DSS)-induced colitis rat model were used to investigate the P. freudenreichii-induced stimulation of mucin production in vitro and in vivo, respectively. The mRNA and protein expression levels of MUC2, a main component of intestinal mucus, increased in the supernatant of P. freudenreichii culture (SPFC)-treated LS 174 cells. The SPFC and live P. freudenreichii (LPF) reduced the disease activity index (DAI) in the rats with DSS-induced colitis. After treatment with SPFC or LPF, the mRNA levels of typical pro-inflammatory cytokines decreased and the inflammatory state was histologically improved in the rats with DSS-induced colitis. The SPFC and LPF treatments increased the gene and protein expression levels of MUC2 in the rats with DSS-induced colitis compared with the expression levels in the negative control rats, and immunohistochemistry (IHC) showed an increase of the intestinal MUC2 level. In addition, SPFC and LPF augmented the level of propionate in the faeces of the rats with DSS-induced colitis. In conclusion, P. freudenreichii might improve acute colitis by restoring goblet cell number and stimulating the expression of MUC2 in intestinal goblet cells.

Oxyresveratrol Induces Autophagy via the ER Stress Signaling Pathway, and Oxyresveratrol-Induced Autophagy Stimulates MUC2 Synthesis in Human Goblet Cells.

BACKGROUND: Autophagy is a cell protection system invoked to eliminate the damaged organelles and misfolded proteins that induce various stresses, including endoplasmic reticulum (ER) stress. Autophagy can control mucin secretion in goblet cells. Oxyresveratrol (OXY), an antioxidant, stimulates expression of MUC2. Thus, we investigated the effect of OXY on autophagy and found that OXY-induced autophagy stimulates MUC2 expression in human intestinal goblet cells. METHODS: Autophagy-related genes and proteins were examined by quantitative real-time PCR (qPCR) and Western blotting, respectively. Autophagy was assessed by immunocytochemistry (ICC). To analyze the protein expression profiles of OXY-treated LS 174T goblet cells, two-dimensional electrophoresis (2DE) and peptide mass fingerprinting (PMF) were performed. MUC2 expression in cells was evaluated by ICC. RESULTS: OXY significantly increased the expression levels of genes related to autophagy induction, and activated phagosome elongation resulted in the formation of autophagosomes. OXY also activated the ER stress signaling pathway and promoted MUC2 synthesis, which was inhibited by treatment with an autophagy inhibitor. CONCLUSION: OXY induces autophagy via the ER stress signaling pathway, and OXY-induced autophagy increases MUC2 production in intestinal goblet cells.