Beneficial effects of a new neuroprotective compound in neuronal cells and MPTP-administered mouse model of Parkinson's disease.

A new compound, a derivative of 3,4,5-trimethoxy-N-phenyl benzamide bearing an 8''-methylimidazopyridine moiety, is found to demonstrate neuroprotective effects by preventing cell death caused by oxidative stress. The compound possesses high solubility and metabolic stability, and inhibits MPTP-induced effects in vivo, indicating high potential as a therapeutic drug for Parkinson's disease.

Cost-Effectiveness Analysis of Cyclin-Dependent Kinase 4/6 Inhibitor Palbociclib for Inoperable or Recurrent Breast Cancer.

BACKGROUND: Palbociclib and endocrine therapy has been approved to treat hormone receptor-positive/human epidermal growth factor receptor 2-negative inoperable or recurrent breast cancer in Japan. However, this cotherapy imposes an economic burden on both patients and society because of its high cost. In this study, we assessed the cost-effectiveness of cotherapy with palbociclib and fulvestrant compared to fulvestrant monotherapy for inoperable or recurrent breast cancer. METHODS: The three-state Markov model was built by taking into count health stats in inoperable or recurrent breast cancer. The clinical outcomes of the therapies were drawn from published randomized controlled trials. Total regimen cost was calculated from medical receipts of patients at the Yamagata University Hospital. The cost-effectiveness was evaluated by the incremental cost-effectiveness ratio(ICER), in case that it was below 400,000 Yen per month. Markov chain Monte Carlo simulation was performed to assess probability. RESULTS: Acquisition cost of palbociclib and fulvestrant and fulvestrant monotherapy was 6,209,554 JPY and 780,870 JPY, and 25.7 and 22.8 months were achieved, respectively. ICER for the cotherapy was 1,847,721 JPY/quality adjusted life month(QALM)gained. CONCLUSIONS: The palbociclib and fulvestrant therapy provided better health outcomes than conventional fulvestrant monotherapy, but were costly and suggested to be less cost-effective.

Glutathione depletion results in S-nitrosylation of protein disulfide isomerase in neuroblastoma cells.

AIMS: Protein disulfide isomerase (PDI) is an essential enzyme involved in oxidative protein folding. PDI is S-nitrosylated in the brains of Alzheimer's disease patients, and S-nitrosylated PDI is considered one of main causes of Alzheimer's disease. However, the mechanisms underlying PDI S-nitrosylation have not yet been elucidated. Because glutathione (GSH) depletion is a pathological feature of Alzheimer's disease, we investigated the effect of GSH depletion on the S-nitrosylation level of PDI. MAIN METHODS: SH-SY5Y cells, which is a human derived neuroblastoma cells, were used in this study. Glutamate and buthionine sulfoximine (BSO) were used as GSH depletors. S-nitrosylated PDI was detected by biotin-switch assay. KEY FINDINGS: GSH depletion by glutamate, a cystine/glutamate antiporter xCT inhibitor, increased S-nitrosylated PDI at C343 in SH-SY5Y cells, and induced IRE1α phosphorylation. BSO, a γ-glutamylcysteine synthetase inhibitor, also increased S-nitrosylated PDI and phosphorylated IRE1α upon GSH depletion. Because S-nitrosylated PDI at C343 is stable in neuro cells, S-nitrosylated PDI by GSH depletion progresses to neurodegeneration by the induction of endoplasmic reticulum stress via phosphorylated IRE1α signaling from the early to late stage. Furthermore, treatment with neohesperidin, but not N-acetylcysteine (NAC), improved PDI S-nitrosylation level in GSH-depleted SH-SY5Y cells because nitrosylated compound of NAC induces PDI S-nitrosylation. SIGNIFICANCE: The results of our study provide a new insight into the mechanisms of neurodegeneration, and may be useful for the development of drugs for Alzheimer's diseases.

[Association of Abnormal Disulfide Bond Formation with Disease Development and Progression].

As intermolecular and intramolecular disulfide bridges in proteins play a vital role in the stability of the final protein structure, the disruption of disulfide bridges in proteins may lead to disease development and progression. Therefore, understanding the association of abnormal protein disulfide bond formation with disease development and progression can be useful for developing novel drugs for various diseases. Considering that disulfide-linked protein folding involves redox reactions in the endoplasmic reticulum, this process may be affected by oxidative stress. We hypothesized that oxidative stress-related diseases may be induced by abnormal protein disulfide bond formation. This review introduces the association of abnormal protein disulfide bond formation with disease development and progression.

Binding of Citrate-Fe3+ to Plastic Culture Dishes, an Artefact Useful as a Simple Technique to Screen for New Iron Chelators.

NaCT mediates citrate uptake in the liver cell line HepG2. When these cells were exposed to iron (Fe3+), citrate uptake/binding as monitored by the association of [14C]-citrate with cells increased. However, there was no change in NaCT expression and function, indicating that NaCT was not responsible for this Fe3+-induced citrate uptake/binding. Interestingly however, the process exhibited substrate selectivity and saturability as if the process was mediated by a transporter. Notwithstanding these features, subsequent studies demonstrated that the iron-induced citrate uptake/binding did not involve citrate entry into cells; instead, the increase was due to the formation of citrate-Fe3+ chelate that adsorbed to the cell surface. Surprisingly, the same phenomenon was observed in culture wells without HepG2 cells, indicating the adsorption of the citrate-Fe3+ chelate to the plastic surface of culture wells. We used this interesting phenomenon as a simple screening technique for new iron chelators with the logic that if another iron chelator is present in the assay system, it would compete with citrate for binding to Fe3+ and prevent the formation and adsorption of citrate-Fe3+ to the culture well. This technique was validated with the known iron chelators deferiprone and deferoxamine, and with the bacterial siderophore 2,3-dihydroxybenzoic acid and the catechol carbidopa.

The Effectiveness of Antidiabetic Drugs in Treating Dementia: A Peek into Pharmacological and Pharmacokinetic Properties.

Dementia dramatically affects the activities of daily living and quality of life; thus, many therapeutic approaches for overcoming dementia have been developed. However, an effective treatment regimen is yet to be developed. As diabetes is a well-known risk factor for dementia, drug repositioning and repurposing of antidiabetic drugs are expected to be effective dementia treatments. Several observational studies have been useful for understanding the effectiveness of antidiabetic drugs in treating dementia, but it is difficult to conclusively analyze the association between antidiabetic drug treatment and the risk of developing dementia after correcting for potential confounding factors. Mechanism-based approaches may provide a better understanding of the effectiveness of antidiabetic drugs for treating dementia. Since the peripheral circulation and the central nerve system are separated by the blood-brain barrier, it is important to understand the regulation of the central glucose metabolism. In this review, we discuss the pharmacological and pharmacokinetic properties of antidiabetic drugs in relation to treating dementia.

The safety of pranlukast and montelukast during the first trimester of pregnancy: A prospective, two-centered cohort study in Japan.

For leukotriene receptor antagonists (LTRAs), especially pranlukast, safety data during pregnancy is limited. Therefore, we conducted a prospective, two-centered cohort study using data from teratogen information services in Japan to clarify the effects of LTRA exposure during pregnancy on maternal and fetal outcomes. Pregnant women who being counseled on drug use during pregnancy at two facilities were enrolled. The primary outcome of this study was major congenital anomalies. The incidence of major congenital anomalies in women exposed to montelukast or pranlukast during the first trimester of pregnancy was compared with that of controls. Logistic regression analysis was performed to analyze the effects of maternal LTRA use during the first trimester of pregnancy on major congenital anomalies. The outcomes of 231 pregnant women exposed to LTRAs (montelukast n = 122; pranlukast n = 106; both n = 3) and 212 live births were compared with those of controls. The rate of major congenital anomalies in the LTRA group was 1.9%. Multivariable logistic regression analysis revealed that LTRA exposure was not a risk factor for major congenital anomalies (adjusted odds ratio, 0.78; 95% confidence interval, 0.23-2.05; p = 0.653). In addition, no significant difference was detected in stillbirth, spontaneous abortion, preterm birth, and low birth weight between the two groups. The present study revealed that montelukast and pranlukast were not associated with the risk of major congenital anomalies. Our findings suggest that LTRAs could be safely employed for asthma therapy during pregnancy.

Metabolic Alteration Analysis of Steroid Hormones in Niemann-Pick Disease Type C Model Cell Using Liquid Chromatography/Tandem Mass Spectrometry.

Niemann-Pick disease type C (NPC) is an autosomal recessive disease caused by a functional deficiency of cholesterol-transporting proteins in lysosomes, and exhibits various clinical symptoms. Since mitochondrial dysfunction in NPC has recently been reported, cholesterol catabolism to steroid hormones may consequently be impaired. In this study, we developed a comprehensive steroid hormone analysis method using liquid chromatography/tandem mass spectrometry (LC-MS/MS) and applied it to analyze changes in steroid hormone concentrations in NPC model cells. We investigated the analytical conditions for simultaneous LC-MS/MS analysis, which could be readily separated from each other and showed good reproducibility. The NPC phenotype was verified as an NPC model with mitochondrial abnormalities using filipin staining and organelle morphology observations. Steroid hormones in the cell suspension and cell culture medium were also analyzed. Steroid hormone analysis indicated that the levels of six steroid hormones were significantly decreased in the NPC model cell and culture medium compared to those in the wild-type cell and culture medium. These results indicate that some steroid hormones change during NPC pathophysiology and this change is accompanied by mitochondrial abnormalities.

Identification cholesterol metabolites altered before the onset of nonalcoholic steatohepatitis by targeted metabolomics.

Nonalcoholic steatohepatitis (NASH) is a disease with symptoms similar to those of alcoholic liver inflammation without alcohol intake. As an effective treatment strategy has not been established for this disease, a detailed understanding of the pathological progression mechanism is required. We focused on cholesterol metabolites, which are suspected to regulate NASH pathology, and investigated their relationship with the pathological progression in the early stages of NASH. First, the LC/MS/MS methods for bile acids and sterols were optimized and validated. Next, NASH model mice were established by feeding a choline-deficient, methionine-reduced high-fat diet, and the levels of hepatic cholesterol metabolites were measured. As a result, before the onset of NASH, desmosterol, 4ß-hydroxycholesterol, campesterol, sitosterol, secondary bile acids such as taurodeoxycholic acid significantly decreased by up to 1/38 of NASH model group. Autoxidation-generated sterols significantly increased 2- to 5-fold, and various primary bile acids such as conjugated ß-muricholic acids and cholic acids significantly increased 2- to 7-fold. In this study, the levels of cholesterol metabolites changed in the before the onset of NASH. These metabolic alterations involved in inflammation induction and detoxification for NASH may help the discovery of early diagnostic biomarkers in the future.

A Proton-Coupled Transport System for ß-Hydroxy-ß-Methylbutyrate (HMB) in Blood-Brain Barrier Endothelial Cell Line hCMEC/D3.

ß-Hydroxy-ß-methylbutyrate (HMB), a leucine metabolite, is used as a nutritional ingredient to improve skeletal muscle health. Preclinical studies indicate that this supplement also elicits significant benefits in the brain; it promotes neurite outgrowth and prevents age-related reductions in neuronal dendrites and cognitive performance. As orally administered HMB elicits these effects in the brain, we infer that HMB crosses the blood-brain barrier (BBB). However, there have been no reports detailing the transport mechanism for HMB in BBB. Here we show that HMB is taken up in the human BBB endothelial cell line hCMEC/D3 via H+-coupled monocarboxylate transporters that also transport lactate and ß-hydroxybutyrate. MCT1 (monocarboxylate transporter 1) and MCT4 (monocarboxylate transporter 4) belonging to the solute carrier gene family SLC16 (solute carrier, gene family 16) are involved, but additional transporters also contribute to the process. HMB uptake in BBB endothelial cells results in intracellular acidification, demonstrating cotransport with H+. Since HMB is known to activate mTOR with potential to elicit transcriptomic changes, we examined the influence of HMB on the expression of selective transporters. We found no change in MCT1 and MCT4 expression. Interestingly, the expression of LAT1 (system L amino acid transporter 1), a high-affinity transporter for branched-chain amino acids relevant to neurological disorders such as autism, is induced. This effect is dependent on mTOR (mechanistic target of rapamycine) activation by HMB with no involvement of histone deacetylases. These studies show that HMB in systemic circulation can cross the BBB via carrier-mediated processes, and that it also has a positive influence on the expression of LAT1, an important amino acid transporter in the BBB.

Glucose-induced oxidative stress leads to in S-nitrosylation of protein disulfide isomerase in neuroblastoma cells.

BACKGROUND: Dementia places a significant burden on both patients and caregivers. Since diabetes is a risk factor for dementia, it is imperative to identify the relationship between diabetes and cognitive disorders. Protein disulfide isomerase (PDI) is an enzyme for oxidative protein folding. PDI S-nitrosylation is observed in the brain tissues of Alzheimer's disease patients. The aim of this study is to clarify the relationship between PDI S-nitrosylation and diabetes. METHODS: We used SH-SY5Y cells cultured in high-glucose media. RESULTS: S-nitrosylated PDI level increased at 7 days and remained high till 28 days in SH-SY5Y cells cultured in high-glucose media. Using PDI wild-type- or PDI C343S-expressing SH-SY5Y cells, PDI C343 was identified as the site of glucose-induced S-nitrosylation. IRE1α and PERK were phosphorylated at day 14 in the SH-SY5Y cells cultured in high-glucose media, and the phosphorylated status was maintained to day 28. To determine the effect of S-nitrosylated PDI on endoplasmic reticulum stress signaling, SH-SY5Y cells were treated with S-nitrosocystein (SNOC) for 30 min, following which the medium was replaced with SNOC-free media and the cells were cultured for 24 h. Only phosphorylated IRE1α treated with SNOC was associated with PDI S-nitrosylation. Neohesperidin, a flavonoid in citrus fruits, is a natural antioxidant. The treatment with neohesperidin in the final 7 days of glucose loading reversed PDI S-nitrosylation and improved cell proliferation. CONCLUSION: Glucose loading leads to S-nitrosylation of PDI C343 and induces neurodegeneration via IRE1α phosphorylation. GENERAL SIGNIFICANCE: The results may be useful for designing curative treatment strategies for dementia.

Establishment of an analytical method for simultaneous quantitation of CDK4/6 inhibitors, aromatase inhibitors, and an estrogen receptor antagonist in human plasma using LC-ESI-MS/MS.

Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors (palbociclib, abemaciclib, and ribociclib) are used to treat human epithelial growth factor receptor (HER)-2 negative and hormone receptor (HR) positive advanced breast cancer in combination with aromatase inhibitors (letrozole, anastrozole) or an estrogen receptor antagonist (fulvestrant). Administration of these drugs frequently causes severe side effects, such as neutropenia and diarrhea. Therefore, therapeutic drug monitoring (TDM) of CDK4/6 inhibitors, aromatase inhibitors, and the estrogen receptor antagonist is considered important for ensuring the efficacy and safety of these drugs. In this study, we describe a simple, highly sensitive, and specific liquid chromatography/electrospray ionization tandem mass spectrometry method for simultaneous quantitation of the concentrations of palbociclib, abemaciclib, ribociclib, letrozole, anastrozole, and fulvestrant. In addition, we analyzed plasma samples from patients with HER2-negative and HR-positive advanced breast cancer treated with these compounds using this novel method. In our method, the intra-assay relative error (RE) values ranged from -12.8% to 12.9%, the inter-assay RE values ranged from -4.8% to 6.2%, and the coefficient of variation (CV) values for intra- and inter-assay were ≤8.6% and ≤13.3%, respectively. The analytes showed good stability with RE values ranging from -13.5% to 13.6% and CV values <10.4%. Moreover, all the samples from patients were successfully quantified, and were within the range of measurement. This method can be used for TDM of routine anticancer drugs in clinical practice and for pharmacokinetics/pharmacodynamics research in future studies.

High-throughput liquid chromatography/electrospray ionization-tandem mass spectrometry method using in-source collision-induced dissociation for simultaneous quantification of imatinib, dasatinib, bosutinib, nilotinib, and ibrutinib in human plasma.

Recent studies have shown that therapeutic drug monitoring of tyrosine kinase inhibitors (TKIs) could improve treatment efficacy and safety. A simple analytical method using high-performance LC/electrospray ionization-tandem mass spectrometry has been developed and validated for simultaneous quantification of BCR-ABL and Bruton's TKIs used for chronic leukemia (imatinib, dasatinib, bosutinib, nilotinib, and ibrutinib) in human plasma. Although these structures and physical properties are similar, owing to their different linear ranges, simultaneously determining the plasma levels of these five TKIs by applying optimal MS parameters remains difficult. A quantitative range exceeding 60,000-fold was required, and the linear dynamic ranges of imatinib, bosutinib, and nilotinib were limited because of the presence of a saturated detection signal. In this study, we applied the in-source collision-induced dissociation technique to control the ion amounts in mass spectrometry. This new method allowed rapid determination within 5 min with simple pretreatment. The method was validated according to the US Food and Drug Administration guidelines. Moreover, all samples of patients with chronic leukemia were successfully measured and their values were within the linear range of measurement. Therefore, our high-throughput analytical system is useful to measure the plasma concentrations of imatinib, dasatinib, bosutinib, nilotinib, and ibrutinib in clinical practice.

Development of a simultaneous analytical method for clozapine and its metabolites in human plasma using liquid chromatography/electrospray ionization tandem mass spectrometry with linear range adjusted by in-source collision-induced dissociation.

Clozapine (CLZ) is a key drug in treatment-resistant schizophrenia. Therapeutic drug monitoring (TDM) of CLZ and its metabolites, N-desmethylclozapine and clozapine N-oxide, is required to monitor and manage the risks of side effects. Although quantification methods for TDM have been developed for CLZ and its metabolites, they were not sufficiently accurate for the quantification of CLZ owing to the upper limits of the calibration curves. An analytical method using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry was developed and validated for the simultaneous measurement of CLZ and its metabolites in human plasma. To expand the concentration range of the calibration curves, we used a linear range shift technique using in-source collision-induced dissociation (CID). Using our approach, the linearity and quantitative range were improved compared to those reported by previous studies, and were sufficient for TDM in clinical practice. The intra- and inter-assay accuracy was 84.6%-114.8%, and the intra- and inter-assay precisions were ≤9.1% and ≤9.9%, respectively. Moreover, all samples from patients with treatment-resistant schizophrenia were successfully quantified. Therefore, our novel analytical method using in-source CID had the appropriate performance to measure the plasma concentrations of CLZ and its metabolites for TDM in clinical practice.

Simultaneous analysis of drugs administered to lung-transplanted patients using liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring.

Several drugs are administered to lung-transplanted patients, which are monitored using therapeutic drug monitoring (TDM). Therefore, we developed and validated a liquid chromatography-tandem mass spectrometry method to simultaneously analyze immunosuppressive drugs such as mycophenolic acid, antifungal drugs such as voriconazole and itraconazole, and its metabolite hydroxyitraconazole. Chromatographic separation was achieved using a C18 column and gradient flow of mobile phase comprising 20 mM aqueous ammonium formate and 20 mM ammonium formate-methanol solution. A simple protein precipitation treatment was performed using acetonitrile/methanol and mycophenolic acid-2 H3 , voriconazole-2 H3 , itraconazole-2 H4 , and hydroxyitraconazole-2 H4 as internal standards. The linearity ranges of mycophenolic acid, voriconazole, itraconazole, and hydroxyitraconazole were 100-20,000, 50-10,000, 5-1000, and 5-1000 ng/mL, respectively. The retention time of each target was less than 2 min. The relative errors in intra- and inter-day were within ±7.6%, the coefficient of variation was 8.9% or less for quality control low, medium, and high, and it was 15.8% or less for lower limit of quantitation. Moreover, the patient samples were successfully quantified, and they were within the linear range of measurements. Therefore, our new method may be useful for TDM in lung-transplanted patients.

Cysteine 343 in the substrate binding domain is the primary S-Nitrosylated site in protein disulfide isomerase.

Abnormal protein accumulations are typical pathological features for neurodegenerative diseases. Protein disulfide isomerase (PDI) is a critical enzyme in oxidative protein folding. S-nitrosylated PDI has been detected in the postmortem brain in neurodegenerative disease patients, but the effect of S-nitrosylation on PDI function and developing neurodegeneration was not clarified in detail. In this study, we identified that in vitro and in vivo S-nitrosylation of C343 in the b' domain of PDI occurs. Reduced recombinant human PDI (hPDI) reacted quickly with S-nitrosocompounds, with an observed increase in the expected S-nitrosylated species and the appearance of the disulfide state of the active sites. Both Mononitrosylated and dinitrosylated were observed from the S-nitrosylation of hPDI. Dinitrosylated species were S-nitrosylated both cysteines at active site. But, at least in part, mononitrosylated species were S-nitrosylated on cysteine 343 in the substrate binding b' domain. Although active site S-nitrosylation is reversible by reduced glutathione, S-nitrosylation of C343 is comparative stable. S-nitrosylation of PDI in SH-SY5Y cells was observed after the S-nitrosocysteine (SNOC) treatment and S-nitrosylated PDI was still detected 24 h after removing SNOC. While wild-type PDI was S-nitrosylated, the level of S-nitrosylation of the C343S mutant in over-expressed cells was substantially lower and only wild-type PDI of S-nitrosylation remained 24 h after removing SNOC in over-expressed cells. Both of in vitro and in vivo results suggested that S-nitrosylation of C343 in PDI may be the causative effect on physiological changes in neurodegerenative disease patients, and may be useful for the drug development for neurodegenerative diseases.

Development of a Diagnostic Screening Strategy for Niemann-Pick Diseases Based on Simultaneous Liquid Chromatography-Tandem Mass Spectrometry Analyses of N-Palmitoyl-O-phosphocholine-serine and Sphingosylphosphorylcholine.

Early diagnosis of Niemann-Pick diseases (NPDs) is important for better prognosis of such diseases. N-Palmitoyl-O-phosphocholine-serine (PPCS) is a new NPD biomarker possessing high sensitivity, and with its combination with sphingosylphosphocholine (SPC) it may be possible to distinguish NPD-C from NPD-A/B. In this study, a rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (method 1) and a validated LC-MS/MS analysis (method 2) of PPCS and SPC were developed, and we have proposed a diagnostic screening strategy for NPDs using a combination of serum PPCS and SPC concentrations. Nexera and API 5000 were used as LC-MS/MS systems. C18 columns with lengths of 10 and 50 mm were used for method 1 and 2, respectively. 2H3-Labeled PPCS and nor-SPC were used as internal standards. Selective reaction monitoring in positive-ion mode was used for MS/MS. Run times of 1.2 and 8 min were set for methods 1 and 2, respectively. In both methods 1 and 2, two analytes showed high linearity in the range of 1-4000 ng/mL. Method 2 provided high accuracy and precision in method validation. Serum concentrations of both analytes were significantly higher in NPD-C patients than those of healthy subjects in both methods. Serum PPCS correlated between methods 1 and 2; however, it was different in the case of SPC. The serum PPCS/SPC ratio was different in healthy subjects, NPD-C, and NPD-A/B. These results suggest that using a combination of the two LC-MS/MS analytical methods for PPCS and SPC is useful for diagnostic screening of NPDs.

Stimulatory effect on the transport mediated by organic anion transporting polypeptide 2B1.

Drug-drug interaction (DDI) is one of causes of adverse drug events and can result in life-threatening consequences. Organic anion-transporting polypeptide (OATP) 2B1 is a major uptake transporter in the intestine and contributes to transport various clinically used therapeutic agents. The intestine has a high risk of DDI, because it has a special propensity to be exposed to a high concentration of drugs. Thus, understanding drug interaction mediated by OATP2B1 in the absorption process is important for the prevention of adverse drug events, including decrease in the therapeutic effect of co-administered drugs. Acute drug interaction occurs through the direct inhibitory effect on transporters, including OATP2B1. Moreover, some compounds such as clinically used drugs and food components have an acute stimulatory effect on transport of co-administered drugs by OATP2B1. This review summarizes the acute stimulatory effect on the transport mediated by OATP2B1 and discusses the mechanisms of the acute stimulatory effects of compounds. There are two types of acute stimulatory effects, substrate-independent and -dependent interactions on OATP2B1 function. The facilitating translocation of OATP2B1 to the plasma membrane is one of causes for the substrate-independent acute stimulatory effect. On the contrary, the substrate-dependent effect is based on the direct binding to the substrate-binding site or allosteric progesterone-binding site of OATP2B1.

Chronic exposure to excess iron promotes EMT and cancer via p53 loss in pancreatic cancer.

Based on the evidence that hemochromatosis, an iron-overload disease, drives hepatocellular carcinoma, we hypothesized that chronic exposure to excess iron, either due to genetic or environmental causes, predisposes an individual to cancer. Using pancreatic cancer as our primary focus, we employed cell culture studies to interrogate the connection between excess iron and cancer, and combined in vitro and in vivo studies to explore the connection further. Ferric ammonium citrate was used as an exogenous iron source. Chronic exposure to excess iron induced epithelial-mesenchymal transition (EMT) in normal and cancer cell lines, loss of p53, and suppression of p53 transcriptional activity evidenced from decreased expression of p53 target genes (p21, cyclin D1, Bax, SLC7A11). To further extrapolate our cell culture data, we generated EL-KrasG12D (EL-Kras) mouse (pancreatic neoplastic mouse model) expressing Hfe+/+ and Hfe-/- genetic background. p53 target gene expression decreased in EL-Kras/Hfe-/- mouse pancreas compared to EL-Kras/Hfe+/+ mouse pancreas. Interestingly, the incidence of acinar-to-ductal metaplasia and cystic pancreatic neoplasms (CPN) decreased in EL-Kras/Hfe-/- mice, but the CPNs that did develop were larger in these mice than in EL-Kras/Hfe+/+ mice. In conclusion, these in vitro and in vivo studies support a potential role for chronic exposure to excess iron as a promoter of more aggressive disease via p53 loss and SLC7A11 upregulation within pancreatic epithelial cells.

Comprehensive and semi-quantitative analysis of carboxyl-containing metabolites related to gut microbiota on chronic kidney disease using 2-picolylamine isotopic labeling LC-MS/MS.

Carboxyl-containing metabolites, such as bile acids and fatty acids, have many important functions and microbiota is involved in the production of them. In the previous study, we found that the chronic kidney disease (CKD) model mice raised under germ-free conditions provided more severe renal damage than the mice with commensal microbiota. However, the precise influence by the microbiome and carboxyl-containing metabolites to the renal functions is unknown. In this study, we aimed to develop a novel chemical isotope labeling-LC-MS/MS method using the 2-picolylamine and its isotopologue and applied the analysis of effects of microbiome and CKD pathophysiology. The developed semi-quantitative method provided the high accuracy not inferior to the absolute quantification. By comparing of four groups of mice, we found that both microbiota and renal function can alter the composition and level of these metabolites in both plasma and intestine. In particular, the intestinal level of indole-3-acetic acid, short-chain fatty acids and n-3 type of polyunsaturated fatty acid, which play important roles in the endothelial barrier function, were significantly lower in germ-free conditions mice with renal failure. Accordingly, it is suggested these metabolites might have a renoprotective effect on CKD by suppressing epithelial barrier disruption.