Retinoic acid receptor α activity in proximal tubules prevents kidney injury and fibrosis.

Chronic kidney disease (CKD) is characterized by a gradual loss of kidney function and affects ~13.4% of the global population. Progressive tubulointerstitial fibrosis, driven in part by proximal tubule (PT) damage, is a hallmark of late stages of CKD and contributes to the development of kidney failure, for which there are limited treatment options. Normal kidney development requires signaling by vitamin A (retinol), which is metabolized to retinoic acid (RA), an endogenous agonist for the RA receptors (RARα, ß, γ). RARα levels are decreased in a mouse model of diabetic nephropathy and restored with RA administration; additionally, RA treatment reduced fibrosis. We developed a mouse model in which a spatiotemporal (tamoxifen-inducible) deletion of RARα in kidney PT cells of adult mice causes mitochondrial dysfunction, massive PT injury, and apoptosis without the use of additional nephrotoxic substances. Long-term effects (3 to 4.5 mo) of RARα deletion include increased PT secretion of transforming growth factor ß1, inflammation, interstitial fibrosis, and decreased kidney function, all of which are major features of human CKD. Therefore, RARα's actions in PTs are crucial for PT homeostasis, and loss of RARα causes injury and a key CKD phenotype.

PASTICCINO2 interacting with Golgi Anti-Apoptotic Proteins resists endoplasmic reticulum stress dependent on very long-chain fatty acids.

The endoplasmic reticulum (ER) is crucial for maintaining cell homeostasis because it is the primary site for synthesizing secreted and transmembrane proteins and lipids. The unfolded protein response (UPR) is activated to restore ER homeostasis under ER stress. However, the relationship between lipids and the ER stress response in plants is not well understood. Arabidopsis Golgi anti-apoptotic proteins (GAAPs) are involved in resisting ER stress. To elucidate the function of GAAPs, PASTICCINO2 (PAS2), involved in very long-chain fatty acid (VLCFA) synthesis, was found to interact with GAAPs and IRE1. Single pas2 and gaap1/gaap2pas2 double mutants exhibited increased seedling damage and impaired UPR response under chronic ER stress. Site mutation combined with genetic analysis revealed that the role of PAS2 in resisting ER stress depended on its VLCFA synthesis domain. VLCFA contents were upregulated under ER stress, which required GAAPs. Exogenous VLCFAs partially restored the defect in UPR upregulation caused by PAS2 or GAAP mutations under chronic ER stress. These findings demonstrate that the association of PAS2 with GAAPs confers plant resistance to ER stress by regulating VLCFA synthesis and the UPR. This provides a basis for further studies on the connection between lipids and cell fate decisions under stress.

Retinoic Acid Receptor ß Loss in Hepatocytes Increases Steatosis and Elevates the Integrated Stress Response in Alcohol-Associated Liver Disease.

In alcohol-associated liver disease (ALD), hepatic reductions in vitamin A and perturbations in vitamin A metabolism are common. However, the roles that the vitamin A receptors, termed retinoic acid receptors (RARs), may have in preventing the pathophysiology of ALD remains unclear. Our prior data indicate that a RARß agonist limits the pathology of alcohol-related liver disease. Thus, we generated liver-specific AlbCre-RARß knockout (BKO) mice and compared them to wild type (WT) mice in an early ALD model. Both strains showed similar blood ethanol concentrations and ETOH-metabolizing enzymes. However, the livers of pair-fed-BKO and ETOH-BKO mice developed higher levels of steatosis and triglycerides than pair-fed-WT and ETOH-WT mice. The increased hepatic steatosis observed in the pair-fed-BKO and ETOH-BKO mice was associated with higher lipid synthesis/trafficking transcripts and lower beta-oxidation transcripts. ETOH-BKO mice also exhibited a higher integrated stress response (ISR) signature, including higher transcript and protein levels of ATF4 and its target, 4-EBP1. In human hepatocytes (HepG2) that lack RARß (RARß-KO), ETOH treatments resulted in greater reactive oxygen species compared to their parental cells. Notably, even without ETOH, ATF4 and 4-EBP1 protein levels were higher in the RARß-KO cells than in their parental cells. These 4-EBP1 increases were greatly attenuated in cultured ATF4-deficient and RARß/ATF4-deficient HepG2, suggesting that RARß is a crucial negative regulator of 4-EBP1 through ATF4 in cultured hepatocytes. Here, we identify RARß as a negative regulator of lipid metabolism and cellular stress in ALD.

Quassinoids from Twigs of Harrisonia perforata (Blanco) Merr and Their Anti-Parkinson's Disease Effect.

Six new C-20 and one new C-19 quassinoids, named perforalactones F-L (1-7), were isolated from twigs of Harrisonia perforata. Spectroscopic and X-ray crystallographic experiments were conducted to identify their structures. Through oxidative degradation of perforalactone B to perforaqussin A, the biogenetic process from C-25 quassinoid to C-20 via Baeyer-Villiger oxidation was proposed. Furthermore, the study evaluated the anti-Parkinson's disease potential of these C-20 quassinoids for the first time on 6-OHDA-induced PC12 cells and a Drosophila Parkinson's disease model of PINK1B9. Perforalactones G and I (2 and 4) showed a 10-15% increase in cell viability of the model cells at 50 µM, while compounds 2 and 4 (100 µM) significantly improved the climbing ability of PINK1B9 flies and increased the dopamine level in the brains and ATP content in the thoraces of the flies.

[Separation, characterization and anti-psoriasis effect of self-assembled nanoparticles from Shaoyao Gancao Decoction].

This study aims to separate and characterize self-assembled nanoparticles(SAN) from Shaoyao Gancao Decoction(SGD) and determine the content of active compounds. Further, we aimed to observe the therapeutic effect of SGD-SAN on imiquimod-induced psoriasis in mice. The separation of SGD was performed by dialysis, and the separation process was optimized by single factor experiment. The SGD-SAN isolated under the optimal process was characterized, and the content of gallic acid, albiflorin, paeoniflorin, liquiritin, isoliquiritin apioside, isoliquiritin, and glycyrrhizic acid in each part of SGD was determined by HPLC. In the animal experiment, mice were assigned into a normal group, a model group, a methotrexate group(0.001 g·kg~(-1)), and SGD, SGD sediment, SGD dialysate, and SGD-SAN groups of different doses(1, 2, and 4 g·kg~(-1)) respectively. The psoriasis grade of mice was evaluated based on the pathological changes of skin lesions, the content of inflammatory cytokines, organ index and other indicators. The results showed that SAN obtained by centrifugation at 13 000 r·min~(-1) for 30 min was stable after dialysis for 4 times, which were uniform spherical nanoparticles with the particle size of(164.43±1.34) nm, the polydispersity index of(0.28±0.05), and the Zeta potential of(-12.35±0.80) mV. The active compound content accounted for more than 70% of SGD. Compared with the model group, SAN and SGD decreased the skin lesion score, spleen index, and inflammatory cytokine levels(P<0.05 or P<0.01) and alleviated the skin thickening and infiltration of inflammatory cells. However, the sediment group and the dialysate group had no obvious effect. SGD showed a good therapeutic effect on imiquimod-induced psoriasis in mice, and SAN demonstrated the effect equivalent to SGD in a dose-dependent manner. Therefore, we conclude that the SAN formed during decocting is the main active form of SGD, which can lower the levels of inflammatory cytokines, promote the normal differentiation of keratinocytes, and reduce the infiltration of inflammatory cells in the treatment of psoriasis lesions in mice.

A retinoic acid receptor ß2 agonist attenuates transcriptome and metabolome changes underlying nonalcohol-associated fatty liver disease.

Nonalcohol-associated fatty liver disease (NAFLD) is characterized by excessive hepatic accumulation of fat that can progress to steatohepatitis, and currently, therapeutic options are limited. Using a high-fat diet (HFD) mouse model of NAFLD, we determined the effects of the synthetic retinoid, AC261066, a selective retinoic acid receptor ß2 (RARß2) agonist, on the global liver transcriptomes and metabolomes of mice with dietary-induced obesity (DIO) using genome-wide RNA-seq and untargeted metabolomics. We found that AC261066 limits mRNA increases in several presumptive NAFLD driver genes, including Pklr, Fasn, Thrsp, and Chchd6. Importantly, AC261066 limits the increases in the transcript and protein levels of KHK, a key enzyme for fructose metabolism, and causes multiple changes in liver metabolites involved in fructose metabolism. In addition, in cultured murine hepatocytes, where exposure to fructose and palmitate results in a profound increase in lipid accumulation, AC261066 limits this lipid accumulation. Importantly, we demonstrate that in a human hepatocyte cell line, RARß is required for the inhibitory effects of AC261066 on palmitate-induced lipid accumulation. Finally, our data indicate that AC261066 inhibits molecular events underpinning fibrosis and exhibits anti-inflammatory effects. In conclusion, changes in the transcriptome and metabolome indicate that AC261066 affects molecular changes underlying multiple aspects of NAFLD, including steatosis and fibrosis. Therefore, we suggest that AC261066 may have potential as an effective therapy for NAFLD.

Golgi anti-apoptotic proteins redundantly counteract cell death by inhibiting production of reactive oxygen species under endoplasmic reticulum stress.

Maintaining proteostasis in the endoplasmic reticulum (ER) is critical for cell viability and plant survival under adverse conditions. The unfolded protein response (UPR) pathways interact with reactive oxygen species (ROS) to precisely trigger adaptive outputs or cell death under ER stress with varying degrees. However, little information is known about the relationship between UPR signalling and ROS regulation. Here, Arabidopsis GOLGI ANTI-APOPTOTIC PROTEIN1 (GAAP1)-GAAP4 were found to play redundant positive roles under ER stress. Genetic analysis showed that GAAP4 played a role in INOSITOL-REQUIRING ENZYME (IRE1)-dependent and -independent pathways. In addition, GAAPs played negative roles to activate the adaptive UPR under conditions of stress. Quantitative biochemical analysis showed that mutations in GAAP genes decreased the oxidised glutathione content and altered the pattern of ROS and glutathione in early ER stress. When plants were challenged with unmitigated ER stress, mutations in GAAP advanced ROS accumulation, which was associated with a decline in adaptive UPR. These data indicated that GAAPs resist cell death by regulating glutathione content to inhibit ROS accumulation and maintain UPR during ER stress. They provide a basis for further analysis of the regulation of cell fate decision under ER stress.

Fenretinide Reduces Intestinal Mucin-2-Positive Goblet Cells in Chronic Alcohol Abuse.

INTRODUCTION: Alcohol-induced thickening of the gut mucosal layer and increased expression of goblet cell gel-forming mucins, such as mucin-2 (MUC2) are associated with disruptions to the gut barrier in alcoholic liver disease (ALD). Interest in drugs that can target gut mucins in ALD has grown; however to date, no studies have examined the properties of drugs on expression of gut mucins in models of ALD. We previously demonstrated that at 10 mg/kg/day, the drug fenretinide (N-[4-hydroxyphenyl] retinamide [Fen]), a synthetic retinoid, mitigates alcohol-associated damage to the gut barrier and liver injury in a murine model of ALD. METHODS: In this study, we specifically sought to examine the effects of Fen on gut goblet cells, and expression of mucins, including MUC2 using a 25-day Lieber-DeCarli model of chronic alcohol intake. RESULTS: Our results show that chronic alcohol intake increased gut-mucosal thickening, goblet cell numbers, and mRNA and protein expression of MUC2 in both the ileum and colon. Alcohol intake was associated with marked decreases in ileal and colonic Notch signaling, levels of Notch ligands Dll1 and Dll4, and increases in the expression of Notch-associated genes indispensable for goblet cell specification, including Math1 and Spdef. Interestingly, ileal and colonic expression of KLF4, which is involved in terminal differentiation of goblet cells, was reduced in mice chronically fed alcohol. Coadministration of alcohol with Fen at 10 mg/kg/day significantly reduced alcohol-associated increases in ileal and colonic mucosal thickening, ileal Muc2, colonic Muc2, Muc5ac and Muc6 mRNAs, and goblet cell numbers. We also found that Fen strongly prevented alcohol-mediated suppression of the Notch ligand Dll1, Notch signaling, and alcohol-induced increases in expression of Notch-associated goblet cell specification genes in both the ileum and colon. In the absence of alcohol, Fen treatments alone at 10 mg/kg/day had no effects on any of the goblet cell-related endpoints. CONCLUSION: These data show for the first time that the drug Fen possesses mucosal layer-modulating properties in response to chronic alcohol abuse. These data warrant further preclinical examination of Fen given the need for anti-ALD drugs and emerging evidence of a role for intestinal goblet cell mucins in the progression of ALD.

EZH2 knockout in oral cavity basal epithelia causes more invasive squamous cell carcinomas.

Oral squamous cell carcinoma (oral SCC) is an aggressive disease and despite intensive treatments, 5-year survival rates for patients have remained low in the last 20 years. Enhancer of zeste homolog 2 (EZH2), part of polycomb repressive complex 2 (PRC2), is highly expressed in human oral SCC samples and cell lines and has been associated with greater epithelia-to-mesenchymal transition (EMT), invasion and metastasis. Here, we developed a tamoxifen-regulated, transgenic mouse line (KcEZH2) in which EZH2 is selectively knocked out (KO) in some tongue epithelial basal stem cells (SCs) in adult mice. EZH2 KO SCs do not show the H3K27me3 mark, as assessed by double-label immunofluorescence. We used this mouse line to assess EZH2 actions during oral tumorigenesis with our immunocompetent 4-nitroquinoline 1-oxide model of oral SCC. We report that higher percentages of mice with invasive SCCs and high-grade neoplastic lesions are observed in mice containing EZH2 KO SCs (KcEZH2-2TΔ and KcEZH2-5TΔ mice). Moreover, EZH2 expression does not correlate with the expression of markers of invasive SCCs. Finally, EZH2 KO cells that are E-cadherin+ are present at invasion fronts infiltrating underlying muscle tissue. Our findings indicate that the knockout of EZH2 in basal SCs of tongue epithelia results in more aggressive carcinomas, and this should be considered when targeting EZH2 as a therapeutic strategy.

A Retinoic Acid Receptor ß 2 Agonist Improves Cardiac Function in a Heart Failure Model.

We previously demonstrated that the selective retinoic acid receptor (RAR) ß 2 agonist AC261066 reduces oxidative stress in an ex vivo murine model of ischemia/reperfusion. We hypothesized that by decreasing oxidative stress and consequent fibrogenesis, AC261066 could attenuate the development of contractile dysfunction in post-ischemic heart failure (HF). We tested this hypothesis in vivo using an established murine model of myocardial infarction (MI), obtained by permanent occlusion of the left anterior descending coronary artery. Treating mice with AC261066 in drinking water significantly attenuated the post-MI deterioration of echocardiographic indices of cardiac function, diminished remodeling, and reduced oxidative stress, as evidenced by a decrease in malondialdehyde level and p38 mitogen-activated protein kinase expression in cardiomyocytes. The effects of AC261066 were also associated with a decrease in interstitial fibrosis, as shown by a marked reduction in collagen deposition and α-smooth muscle actin expression. In cardiac murine fibroblasts subjected to hypoxia, AC261066 reversed hypoxia-induced decreases in superoxide dismutase 2 and angiopoietin-like 4 transcriptional levels as well as the increase in NADPH oxidase 2 mRNA, demonstrating that the post-MI cardioprotective effects of AC261066 are associated with an action at the fibroblast level. Thus, AC261066 alleviates post-MI cardiac dysfunction by modulating a set of genes involved in the oxidant/antioxidant balance. These AC261066 responsive genes diminish interstitial fibrogenesis and remodeling. Since MI is a recognized major cause of HF, our data identify RARß 2 as a potential pharmacological target in the treatment of HF. SIGNIFICANCE STATEMENT: A previous report showed that the selective retinoic acid receptor (RAR) ß 2 agonist AC261066 reduces oxidative stress in an ex vivo murine model of ischemia/reperfusion. This study shows that AC261066 attenuates the development of contractile dysfunction and maladaptive remodeling in post-ischemic heart failure (HF) by modulating a set of genes involved in oxidant/antioxidant balance. Since myocardial infarction is a recognized major cause of HF, these data identify RARß 2 as a potential pharmacological target in the treatment of HF.

Controllable Strategy for Metal-Organic Framework Light-Driven [2 + 2] Cycloaddition Reactions via Solvent-Assisted Linker Exchange.

Flexible olefinic trans-1,2-bis(4-pyridyl)ethene linkers were postsynthetically introduced into the metal-organic frameworks (MOFs) containing parallel rigid 4,4'-bipyridine linkers with a spacing of less than 4.2 Å by the linker exchange strategy, and then, the MOF satisfied Schmidt criteria could be obtained. Eventually, MOF products connected by cyclobutane derivatives were formed by the photochemical [2 + 2] cycloaddition reaction under UV irradiation.

Perforalactones D and E, two new C-20 quassinoids with potential activity to induce lysosomal biogenesis from the twigs of Harrisonia perforata (Blanco) Merr.

Two new quassinoids (1 and 2) were isolated from the twigs of Harrisonia perforata (Blanco) Merr. Perforalactone E (2) possesses an uncommon hexacyclic 1α,12α:5α,13α-dicyclo-9ßH-picrasane skeleton. Its structure was determined based on spectroscopic data and X-ray crystallography. Compounds 1 and 2 could significantly induce lysosomal biogenesis through transcriptional activation of lysosomal genes.

[An integrative metabolomics and network pharmacology method for exploring bioactive components and preliminary pharmacodynamics in medicinal parts of Harrisonia perforata].

In this paper,metabolomics and network pharmacology were used to investigate the bioactive components of Harrisonia perforata and their possible mechanisms of action. Metabolites in the flowers,fruits,branches,leaves and stalks of H. perforata were analyzed by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Meanwhile,multiple statistical analysis methods including principal component analysis( PCA) and orthogonal partial least squares discriminant analysis( OPLS-DA)were applied to screen and identify differential compounds. With metabolomics method,9 differential compounds were preliminarily identified from leaves and other non-traditional medicinal parts. Subsequently,these compounds were explored by using network pharmacology. With gastrointestinal absorption and drug-likeness as limiting conditions,they were imported into the Swiss ADME,from which 7 compounds with potential medicinal activity were obtained. Then,their targets were predicted by PharmMapper,with Human Protein Targets Only and Normalized Fit Score>0. 9 set as limiting conditions,and 60 standardized potential targets were identified with Uniprot. KEGG( Kyoto encyclopedia of genes and genomes) pathway data was obtained using metascape and the " potential active ingredients-target-pathway" network was constructed with Cytoscape 3. 7. 2. The enrichment analysis of KEGG demonstrated that the 60 targets were enriched in 78 signaling pathways( min overlap: 3,P value cutoff: 0. 01,min enrichment: 1. 5),many of which are related to anti-bacteria,anti-inflammation and anti-virus,such as IL-17 signaling pathway,RIG-I-like receptor signaling pathway and NOD-like receptor signaling pathway. Finally,depending on the clinical activity of H. perforata,the relevant signaling pathways were analyzed through experimental data and literature. Dehydroconiferyl alcohol was reported to have the anti-inflammatory effect and perforamone D to possess the antimycobacterial activity. The KEGG pathway enrichment analysis showed that dehydroconiferyl alcohol could act on the Alzheimer's disease( AD) signaling pathway by targeting CDK5 R1 and BACE1. ACh E inhibitor is the most promising drug to treat AD,while dehydroconiferyl alcohol has been proved to inhibit ACh E according to literature. The experimental results revealed that the extract of leaves of H. perforata can effectively inhibit the growth of Staphylococcus aureus. These are consistent with the enrichment analysis results of KEGG. This study explored the bioactive components and pharmacodynamics of the leaves of the H. perforata,laying a theoretical foundation for its in-depth development and rational application.

Amelioration of Diabetic Nephropathy Using a Retinoic Acid Receptor ß2 Agonist.

Vitamin A (VA) and its derivatives, known as retinoids, play critical roles in renal development through retinoic acid receptor ß2 (RARß2). Disruptions in VA signaling pathways are associated with the onset of diabetic nephropathy (DN). Despite the known role of RARß2 in renal development, the effects of selective agonists for RARß2 in a high-fat diet (HFD) model of DN are unknown. Here we examined whether AC261066 (AC261), a highly selective agonist for RARß2, exhibited therapeutic effects in a HFD model of DN in C57BL/6 mice. Twelve weeks of AC261 administration to HFD-fed mice was well tolerated with no observable side effects. Compared with HFD-fed mice, HFD + AC261-treated mice had improved glycemic control and reductions in proteinuria and urine albumin-to-creatinine ratio. Several cellular hallmarks of DN were mitigated in HFD + AC261-treated mice, including reductions in tubule lipid droplets, podocyte (POD) effacement, endothelial cell collapse, mesangial expansion, and glomerular basement membrane thickening. Mesangial and tubule interstitial expression of the myofibroblast markers α-smooth muscle actin (α-SMA) and type IV collagen (Col-IV) was lower in HFD + AC261-treated mice compared with HFD alone. Ultrastructural and immunohistochemistry analyses showed that, compared with HFD-fed mice, HFD + AC261-treated mice showed preservation of POD foot process and slit-diaphragm morphology, an increase in the levels of slit-diagram protein podocin, and the transcription factor Wilms tumor-suppressor gene 1 in PODs. Given the need for novel DN therapies, our results warrant further studies of the therapeutic properties of AC261 in DN.

A Retinoic Acid ß2-Receptor Agonist Exerts Cardioprotective Effects.

We previously discovered that oral treatment with AC261066, a synthetic selective agonist for the retinoic acid ß2-receptor, decreases oxidative stress in the liver, pancreas, and kidney of mice fed a high-fat diet (HFD). Since hyperlipidemic states are causally associated with myocardial ischemia and oxidative stress, we have now investigated the effects of AC261066 in an ex vivo ischemia/reperfusion (I/R) injury model in hearts of two prototypic dysmetabolic mice. We found that a 6-week oral treatment with AC261066 in both genetically hypercholesterolemic (ApoE-/-) and obese (HFD-fed) wild-type mice exerts protective effects when their hearts are subsequently subjected to I/R ex vivo in the absence of added drug. In ApoE-/- mice this cardioprotection ensued without hyperlipidemic changes. Cardioprotection consisted of attenuation of infarct size, diminution of norepinephrine (NE) spillover, and alleviation of reperfusion arrhythmias. This cardioprotection was associated with a reduction in oxidative stress and mast cell (MC) degranulation. We suggest that the reduction in myocardial injury and adrenergic activation, and the antiarrhythmic effects, result from decreased formation of oxygen radicals and toxic aldehydes known to elicit the release of MC-derived renin, promoting the activation of the local renin-angiotensin system leading to enhanced NE release and reperfusion arrhythmias. Because these beneficial effects of AC261066 occurred at the ex vivo level following oral drug treatment, our data suggest that AC261066 could be viewed as a therapeutic means to reduce I/R injury of the heart, and potentially also be considered in the treatment of other cardiovascular ailments such as chronic arrhythmias and cardiac failure.

Combination of bexarotene and the retinoid CD1530 reduces murine oral-cavity carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide.

We investigated the effects of bexarotene (a retinoid X receptor agonist), CD1530 (a retinoic acid receptor γ selective agonist), and the combination of these two drugs for the prevention of oral carcinogenesis induced by the carcinogen 4-nitroquinoline 1-oxide (4-NQO) in a mouse model of human oral-cavity and esophageal squamous-cell carcinoma previously generated in our laboratory. We observed decreased numbers of neoplastic tongue lesions and reduced lesion severity in the 4-NQO plus CD1530 (4N+C) and 4-NQO plus bexarotene plus CD1530 (4N+B+C) groups compared with the 4-NQO group. RNA-Seq analyses showed increases in transcripts in cell proliferation/cell cycle progression pathways in the 4-NQO vs. the untreated group. In addition, ß-catenin and matrix metallopeptidase 9 (MMP9) protein levels and reactive oxygen species (ROS), as assessed by 4-hydroxynonenal (4-HNE) staining, were elevated in tongue tissues 17 wk after the termination of the 4-NQO treatment. The 4N+B, 4N+C, and 4N+B+C groups showed dramatically lower levels of ß-catenin, MMP9, and 4-HNE staining compared with the 4-NQO group. The major reduction in 4-HNE staining in the retinoid treatment groups suggests a novel mechanism of action, reduction of ROS, by which bexarotene and CD1530 inhibit carcinogenesis.

Cross-reacting material 197 reverses the resistance to paclitaxel in paclitaxel-resistant human ovarian cancer.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been proven to be a promising chemotherapeutic target for ovarian cancer. Our previous studies have demonstrated that inhibition of HB-EGF by the special inhibitor, cross-reacting material 197 (CRM197), potently inhibits the anti-tumor activity in paclitaxel-resistant ovarian cancer. Here, we found that inhibition of HB-EGF by CRM197 significantly reverses the resistance to paclitaxel in paclitaxel-resistant ovarian carcinoma cell line (A2780/Taxol). A2780/Taxol cells over-expressed HB-EGF and epidermal growth factor receptor (EGFR) and CRM197 notably suppressed the expression of HB-EGF and EGFR. Experiments performed in vitro and in vivo further suggested that CRM197 markedly down-regulated the ATP-binding cassette sub-family B member 1 (ABCB1/MDR1) messenger RNA (mRNA) expression (P = 0.01), plasma membrane glycoprotein (P-gp) protein (P = 0.009), and P-gp-mediated efflux (P = 0.007) through inhibition of nuclear factor-κB (NF-κB) expression, which were classical chemoresistance-related targets with respect to paclitaxel therapy. Meanwhile, inhibition of HB-EGF enhanced caspase-3 activity to induce apoptosis via MDR1 inhibition in A2780/Taxol cells (P = 0.038). Collectively, HB-EGF is a molecular target for the resistance of ovarian cancer to paclitaxel and CRM197 as a HB-EGF-targeted agent might be a chemosensitizing agent for paclitaxel-resistant ovarian carcinoma. Our findings provide novel possible mechanisms for HB-EGF to be a target to restore the chemosensitivity to paclitaxel.

Changes of Regulatory T Cells in the Early Stage of Obesity Mice and Their Modulation on Macrophage Subtypes in Visceral Adipose Tissue.

Objective To investigate the changes of regulatory T cells (Tregs) and whether Tregs can modulate the distribution of macrophage subtypes in visceral adipose tissue in the early stage of obesity.Methods After C57BL/6 mice obesity models were successfully established,metabolic parameters and numbers of Tregs and M1/M2 macrophage were measured at 4,10,and 20 weeks.The changes of metabolic parameters and adipose tissue inflammation in obesity mice after rapamycin intervention were evaluated. Results The early-stage obesity models were successfully established.Compared with normal diet mice,high fat diet mice had significantly higher epididymal adipose tissue mass and serum leptin levels(P<0.05).However,there was no statistical difference in blood glucose and insulin levels between these two groups(All P>0.05). Macrophages infiltration in adipose tissue in high fat diet mice gradually increased with time,coincident with decrease in Treg numbers. Increased numbers of Treg,improved metabolic parameters,and decreased ratio of M1/M2 can be seen after rapamycin intervention in mice.Conclusion The decrease of Tregs in the early stage of obesity may contribute to abnormal distribution of macrophage subtypes in visceral adipose.

Identification of Ethanol and 4-Nitroquinoline-1-Oxide Induced Epigenetic and Oxidative Stress Markers During Oral Cavity Carcinogenesis.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a cancer that is characterized by its high morbidity and mortality rates. While tobacco use and alcohol consumption are 2 major contributing factors for HNSCC carcinogenesis, how the combination of tobacco and alcohol increases HNSCC risk is not understood. METHODS: We combined the 4-nitroquinoline-1-oxide (4-NQO) oral carcinogenesis and Meadows-Cook alcohol mouse models to elucidate the molecular events and to identify the novel biomarkers associated with oral cancer development. RESULTS: By genome-wide RNA-seq of tongue samples (3 mice per group), we identified changes in transcripts that mediate alcohol metabolism and oxidative stress (Aldh2, Aldh1a3, Adh1, Adh7, and Cyp2a5) in mice treated with 4-NQO followed by ethanol (4-NQO/EtOH) as compared to the vehicle control/untreated (V.C./Untr.) samples. We measured major, global increases in specific histone acetylation and methylation epigenetic marks (H3K27ac, H3K9/14ac, H3K27me3, and H3K9me3) in the oral cavities of V.C./EtOH, 4-NQO/Untr., and 4-NQO/EtOH treatment groups compared to the V.C./Untr. group. We detected changes in histone epigenetic marks near regulatory regions of genes involved in ethanol metabolism by chromatin immunoprecipitation. For instance, the Aldh2 promoter showed increased H3K27me3 marks, and Aldh2 mRNA levels were reduced by 10-fold in 4NQO/EtOH versus V.C./Untr. tongue samples. 4-NQO/EtOH treatment also caused increases in markers of oxidative stress, including 4-HNE, MCT4/SLC16a3, and TOM20, as measured by immunohistochemistry. CONCLUSIONS: We delineate a mechanism by which 4-NQO and ethanol can regulate gene expression during the development of HNSCC and suggest that histone epigenetic marks and oxidative stress markers could be the novel biomarkers and targets for the prevention of HNSCC.

Cross-reacting material 197, a heparin-binding EGF-like growth factor inhibitor, reverses the chemoresistance in human cisplatin-resistant ovarian cancer.

Cross-reacting material 197 (CRM197), a specific HB-EGF inhibitor, has been proven to be a promising antitumor agent for ovarian cancer therapy. Our previous studies have shown that CRM197 has potent antitumor activity in human cisplatin-resistant ovarian cancer. However, the relationship between CRM197 and the resistance to cisplatin remains unclear. Here, we report that CRM197 significantly reverses the resistance to cisplatin in cisplatin-resistant ovarian carcinoma cell line (A2780/CDDP). We established xenograft nude mice models with A2780 and A2780/CDDP cells. Notably, we observed that CRM197 suppresses the expression of HB-EGF and epidermal growth factor receptor in A2780/CDDP cells and xenografts harboring the overexpression of HB-EGF and epidermal growth factor receptor. Experiments conducted in vitro and in vivo suggest that CRM197 markedly downregulates the expression of excision repair cross-complementing group 1 (P = 0.002) and DNA repair capacity in A2780/CDDP tumor (P < 0.001) by inactivation of extracellular signal-regulated kinase signaling, providing novel possible mechanisms for the ability of CRM197 to restore drug sensitivity. These results suggest that CRM197 as an HB-EGF inhibitor might be a cisplatin-chemosensitizing agent for the treatment of ovarian carcinoma with resistance to cisplatin.