Sucrose nonfermenting AMPK-related kinase (SNARK) regulates exercise-stimulated and ischemia-stimulated glucose transport in the heart.

The signaling mechanisms mediating myocardial glucose transport are not fully understood. Sucrose nonfermenting AMP-activated protein kinase (AMPK)-related kinase (SNARK) is an AMPK-related protein kinase that is expressed in the heart and has been implicated in contraction-stimulated glucose transport in mouse skeletal muscle. We first determined if SNARK is phosphorylated on Thr208 , a site critical for SNARK activity. Mice were treated with exercise, ischemia, submaximal insulin, or maximal insulin. Treadmill exercise slightly, but significantly increased SNARK Thr208 phosphorylation. Ischemia also increased SNARK Thr208 phosphorylation, but there was no effect of submaximal or maximal insulin. HL1 cardiomyocytes were used to overexpress wild-type (WT) SNARK and to knockdown endogenous SNARK. Overexpression of WT SNARK had no effect on ischemia-stimulated glucose transport; however, SNARK knockdown significantly decreased ischemia-stimulated glucose transport. SNARK overexpression or knockdown did not alter insulin-stimulated glucose transport or glycogen concentrations. To study SNARK function in vivo, SNARK heterozygous knockout mice (SNARK+/- ) and WT littermates performed treadmill exercise. Exercise-stimulated glucose transport was decreased by ~50% in hearts from SNARK+/- mice. In summary, exercise and ischemia increase SNARK Thr208 phosphorylation in the heart and SNARK regulates exercise-stimulated and ischemia-stimulated glucose transport. SNARK is a novel mediator of insulin-independent glucose transport in the heart.

Clinical significance of BRAF non-V600E mutations on the therapeutic effects of anti-EGFR monoclonal antibody treatment in patients with pretreated metastatic colorectal cancer: the Biomarker Research for anti-EGFR monoclonal Antibodies by Comprehensive Cancer genomics (BREAC) study.

BACKGROUND: Patients with BRAFV600E-mutated metastatic colorectal cancer (mCRC) have a poorer prognosis as well as resistance to anti-EGFR antibodies. However, it is unclear whether BRAF mutations other than BRAFV600E (BRAFnon-V600E mutations) contribute to anti-EGFR antibody resistance. METHODS: This study was composed of exploratory and inference cohorts. Candidate biomarkers identified by whole exome sequencing from super-responders and nonresponders in the exploratory cohort were validated by targeted resequencing for patients who received anti-EGFR antibody in the inference cohort. RESULTS: In the exploratory cohort, 31 candidate biomarkers, including KRAS/NRAS/BRAF mutations, were identified. Targeted resequencing of 150 patients in the inference cohort revealed 40 patients with RAS (26.7%), 9 patients with BRAFV600E (6.0%), and 7 patients with BRAFnon-V600E mutations (4.7%), respectively. The response rates in RAS, BRAFV600E, and BRAFnon-V600E were lower than those in RAS/BRAF wild-type (2.5%, 0%, and 0% vs 31.9%). The median PFS in BRAFnon-V600E mutations was 2.4 months, similar to that in RAS or BRAFV600E mutations (2.1 and 1.6 months) but significantly worse than that in wild-type RAS/BRAF (5.9 months). CONCLUSIONS: Although BRAFnon-V600E mutations identified were a rare and unestablished molecular subtype, certain BRAFnon-V600E mutations might contribute to a lesser benefit of anti-EGFR monoclonal antibody treatment.

Development of Highly Selective Fluorescent Probe Enabling Flow-Cytometric Isolation of ALDH3A1-Positive Viable Cells.

Aldehyde dehydrogenase (ALDH) is overexpressed in some subpopulations of stem cells and cancer cells. We have designed and synthesized the first selective fluorescent probe for class 3 ALDH (ALDH3A1). This probe enabled the visualization of ALDH3A1-positive cells by fluorescence microscopy as well as flow-cytometric isolation of ALDH3A1-positive viable cells from a human Caucasian esophageal squamous cell line (OE21) that heterogeneously expresses ALDH3A1.

Highly oxygenated antiausterity agents from the leaves of Uvaria dac.

From the chloroform extract of the leaves of Uvaria dac, four new highly-oxygenated cyclohexene derivatives named uvaridacols I-L (1-4) were isolated together with nine previously reported compounds (5-13). Their structures were determined based on the extensive NMR spectroscopic data and circular dichroism spectroscopic analysis. Among the new compounds, uvaridacol L (4) displayed strong preferential cytotoxicity in the nutrient deprived medium against five different tested pancreatic cancer cell lines, PANC-1 (PC50, 20.1µM), PSN-1 (PC50, 9.7µM), MIA PaCa-2 (PC50, 29.1µM), Capan-1 (73.0µM) and KLM-1 (25.9µM).

Constituents of the Rhizomes of Boesenbergia pandurata and Their Antiausterity Activities against the PANC-1 Human Pancreatic Cancer Line.

Human pancreatic cancer cell lines have a remarkable tolerance to nutrition starvation, which enables them to survive under a tumor microenvironment. The search for agents that preferentially inhibit the survival of cancer cells under low nutrient conditions represents a novel antiausterity strategy in anticancer drug discovery. In this investigation, a methanol extract of the rhizomes of Boesenbergia pandurata showed potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrient-deprived conditions, with a PC50 value of 6.6 µg/mL. Phytochemical investigation of this extract led to the isolation of 15 compounds, including eight new cyclohexene chalcones (1-8). The structures of the new compounds were elucidated by NMR spectroscopic data analysis. Among the isolated compounds obtained, isopanduratin A1 (14) and nicolaioidesin C (15) exhibited potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under nutrition-deprived conditions, with PC50 values of 1.0 and 0.84 µM, respectively.

Hypermutation and unique mutational signatures of occupational cholangiocarcinoma in printing workers exposed to haloalkanes.

Cholangiocarcinoma is a relatively rare cancer, but its incidence is increasing worldwide. Although several risk factors have been suggested, the etiology and pathogenesis of the majority of cholangiocarcinomas remain unclear. Recently, a high incidence of early-onset cholangiocarcinoma was reported among the workers of a printing company in Osaka, Japan. These workers underwent high exposure to organic solvents, mainly haloalkanes such as 1,2-dichloropropane (1,2-DCP) and/or dichloromethane. We performed whole-exome analysis on four cases of cholangiocarcinoma among the printing workers. An average of 44.8 somatic mutations was detected per Mb in the genome of the printing workers' cholangiocarcinoma tissues, approximately 30-fold higher than that found in control common cholangiocarcinoma tissues. Furthermore, C:G-to-T:A transitions with substantial strand bias as well as unique trinucleotide mutational changes of GpCpY to GpTpY and NpCpY to NpTpY or NpApY were predominant in all of the printing workers' cholangiocarcinoma genomes. These results were consistent with the epidemiological observation that they had been exposed to high concentrations of chemical compounds. Whole-genome analysis of Salmonella typhimurium strain TA100 exposed to 1,2-DCP revealed a partial recapitulation of the mutational signature in the printing workers' cholangiocarcinoma. Although our results provide mutational signatures unique to occupational cholangiocarcinoma, the underlying mechanisms of the disease should be further investigated by using appropriate model systems and by comparison with genomic data from other cancers.

Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma.

Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells.

Phase I trial of GBS-01 for advanced pancreatic cancer refractory to gemcitabine.

GBS-01, an extract from the fruit of Arctium lappa L. is an orally administered drug rich in arctigenin, which has been reported to exert antitumor activity by attenuating the tolerance of cancer cells to glucose deprivation. We investigated the maximum tolerated dose of GBS-01 based on the frequency of the dose-limiting toxicities (DLTs) and pharmacokinetics in patients with advanced pancreatic cancer refractory to gemcitabine. GBS-01 was given orally at escalating doses from 3.0 g (containing 1.0 g burdock fruit extract) to 12.0 g q.d. A DLT was defined as a grade 4 hematological toxicity and grade 3 or 4 non-hematological toxicity appearing during the first 28 days of treatment. Fifteen patients (GBS-01 dose level 1 [3.0 g], three patients; dose level 2 [7.5 g], three patients; and dose level 3 [12.0 g], nine patients) were enrolled. None of the patients at any of the three dose levels showed any sign of DLTs. The main adverse events were increased serum γ-glutamyl transpeptidase, hyperglycemia, and increased serum total bilirubin; however, all the toxicities were mild. Of the 15 patients, 1 showed confirmed partial response and 4 patients had stable disease. The median progression-free and overall survival of the patients were 1.1 and 5.7 months, respectively. The pharmacokinetic study revealed a high bioavailability of arctigenin and rapid conjugation of the drug with glucuronic acid. The recommended dose of GBS-01 was 12.0 g q.d, and favorable clinical responses were obtained. This trial was registered at UMIN-CTR (http://www.umin.ac.jp/ctr/index-j.htm), identification number UMIN000005787.

Synthesis of new tricyclic thiolactams as potent antitumor agent for pancreatic cancer.

We synthesized the novel tricyclic thiolactams 2a-d, 3d-k, having a benzyl or substituted benzyl substituent on the nitrogen of indole subunit, and their preferential cytotoxicity under both nutrient-deprived medium (NDM) and Dulbecco's modified Eagle's medium (DMEM) was evaluated against a human pancreatic cancer cell line PANC-1. Among the tested compounds, the 4'-hydroxy derivative 3d showed the most potent cytotoxicity in NDM (PC50 1.68µM) although the moderate preferential cytotoxicity (PC50 1.68µM in NDM vs PC50 20µM in DMEM). The 3'-hydroxy derivative 3e exhibited the most preferential cytotoxicity (PC50 1.96µM in NDM vs less than 50% inhibition at 30µM in DMEM). The benzyl 2a and halogenated benzyl derivatives 2b,c showed no cytotoxicity in NDM. In addition, the indole (10, PC50 173.7µM), lactone (11, PC50 131.7µM), and lactam (12, PC50 44.8µM) derivatives showed week or moderate cytotoxicity in NDM. These results indicated that the hydroxy group on the benzyl substituent and tricyclic thiolactam ring were essential for the cytotoxicity in NDM against PANC-1 cell line. Moreover, 3'-hydroxy derivative 3e compound exhibited antitumor activity against the pancreatic ductal adenocarcinoma (PDAC) xenograft model in vivo.

Chemical Constituents of Mangifera indica and Their Antiausterity Activity against the PANC-1 Human Pancreatic Cancer Cell Line.

Human pancreatic cancer cell lines such as PANC-1 have an altered metabolism, enabiling them to tolerate and survive under extreme conditions of nutrient starvation. The search for candidates that inhibit their viability during nutrition starvation represents a novel antiausterity strategy in anticancer drug discovery. A methanol extract of the bark of Mangifera indica was found to inhibit the survival of PANC-1 human pancreatic cancer cells preferentially under nutrient-deprived conditions with a PC50 value of 15.5 µg/mL, without apparent toxicity, in normal nutrient-rich conditions. Chemical investigation on this bioactive extract led to the isolation of 19 compounds (1-19), including two new cycloartane-type triterpenes, mangiferolate A (1) and mangiferolate B (2). The structures of 1 and 2 were determined by NMR spectroscopic analysis. Among the isolated compounds, mangiferolate B (2) and isoambolic acid (12) exhibited potent preferential cytotoxicity against PANC-1 human pancreatic cancer cells under the nutrition-deprived condition with PC50 values of 11.0 and 4.8 µM, respectively.

Aberrant transcriptional regulations in cancers: genome, transcriptome and epigenome analysis of lung adenocarcinoma cell lines.

Here we conducted an integrative multi-omics analysis to understand how cancers harbor various types of aberrations at the genomic, epigenomic and transcriptional levels. In order to elucidate biological relevance of the aberrations and their mutual relations, we performed whole-genome sequencing, RNA-Seq, bisulfite sequencing and ChIP-Seq of 26 lung adenocarcinoma cell lines. The collected multi-omics data allowed us to associate an average of 536 coding mutations and 13,573 mutations in promoter or enhancer regions with aberrant transcriptional regulations. We detected the 385 splice site mutations and 552 chromosomal rearrangements, representative cases of which were validated to cause aberrant transcripts. Averages of 61, 217, 3687 and 3112 mutations are located in the regulatory regions which showed differential DNA methylation, H3K4me3, H3K4me1 and H3K27ac marks, respectively. We detected distinct patterns of aberrations in transcriptional regulations depending on genes. We found that the irregular histone marks were characteristic to EGFR and CDKN1A, while a large genomic deletion and hyper-DNA methylation were most frequent for CDKN2A. We also used the multi-omics data to classify the cell lines regarding their hallmarks of carcinogenesis. Our datasets should provide a valuable foundation for biological interpretations of interlaced genomic and epigenomic aberrations.

Epidermal growth factor receptor (EGFR) signaling regulates global metabolic pathways in EGFR-mutated lung adenocarcinoma.

Genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancer cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer cell metabolism has not yet been clearly elucidated. Here we show that EGFR signaling plays an important role in aerobic glycolysis in EGFR-mutated LAD cells. EGFR-tyrosine kinase inhibitors (TKIs) decreased lactate production, glucose consumption, and the glucose-induced extracellular acidification rate (ECAR), indicating that EGFR signaling maintained aerobic glycolysis in LAD cells. Metabolomic analysis revealed that metabolites in the glycolysis, pentose phosphate pathway (PPP), pyrimidine biosynthesis, and redox metabolism were significantly decreased after treatment of LAD cells with EGFRTKI. On a molecular basis, the glucose transport carried out by glucose transporter 3 (GLUT3) was downregulated in TKI-sensitive LAD cells. Moreover, EGFR signaling activated carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the first step in de novo pyrimidine synthesis. We conclude that EGFR signaling regulates the global metabolic pathway in EGFR-mutated LAD cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, which is an attractive target for the development of more effective targeted therapies to treat patients with EGFR-mutated LAD.

[Therapeutic strategies targeting cancer--specific metabolism].

Basic, clinical and translational metabolic researches in cancer area have been extensively tried to discover and develop novel cancer metabolism drugs. Since tumor cells have metabolic dependencies that distinguish them from their normal counterparts, targeted inhibition of these metabolic dependencies is considered a promising therapeutic strategy against cancer. For example the representative cancer metabolism is that cancer cells exhibit profound metabolic alterations by choosing aerobic glycolysis to metabolize glucose to lactate regardless of the presence of adequate oxygen, although normal cells which mainly utilize glucose by using mitochondrial oxidative phosphorylation to generate ATP. In this review, we focus on several important oncogenes and enzymes, whose alterations have contributed extensively to the metabolic phenotype of cancer cells, with an emphasis on the therapeutic targets.

Effect of a poly(ADP-ribose) polymerase-1 inhibitor against esophageal squamous cell carcinoma cell lines.

Effective molecular target drugs that improve therapeutic efficacy with fewer adverse effects for esophageal cancer are highly anticipated. Poly(ADP-ribose) polymerase (PARP) inhibitors have been proposed as low-toxicity agents to treat double strand break (DSB)-repair defective tumors. Several findings imply the potential relevance of DSB repair defects in the tumorigenesis of esophageal squamous cell carcinoma (ESCC). We evaluated the effect of a PARP Inhibitor (AZD2281) on the TE-series ESCC cell lines. Of these eight cell lines, the clonogenic survival of one (TE-6) was reduced by AZD2281 to the level of DSB repair-defective Capan-1 and HCC1937 cells. AZD2281-induced DNA damage was implied by increases in γ-H2AX and cell cycle arrest at G2/M phase. The impairment of DSB repair in TE-6 cells was suggested by a sustained increase in γ-H2AX levels and the tail moment calculated from a neutral comet assay after X-ray irradiation. Because the formation of nuclear DSB repair protein foci was impaired in TE-6 cells, whole-exome sequencing of these cells was performed to explore the gene mutations that might be responsible. A novel mutation in RNF8, an E3 ligase targeting γ-H2AX was identified. Consistent with this, polyubiquitination of γ-H2AX after irradiation was impaired in TE-6 cells. Thus, AZD2281 induced growth retardation of the DSB repair-impaired TE-6 cells. Interestingly, a strong correlation between basal expression levels of γ-H2AX and sensitivity to AZD2281was observed in the TE-series cells (R(2)  = 0.5345). Because the assessment of basal DSB status could serve as a biomarker for selecting PARP inhibitor-tractable tumors, further investigation is warranted.

Muscle-specific knock-out of NUAK family SNF1-like kinase 1 (NUAK1) prevents high fat diet-induced glucose intolerance.

NUAK1 is a member of the AMP-activated protein kinase-related kinase family. Recent studies have shown that NUAK1 is involved in cellular senescence and motility in epithelial cells and fibroblasts. However, the physiological roles of NUAK1 are poorly understood because of embryonic lethality in NUAK1 null mice. The purpose of this study was to elucidate the roles of NUAK1 in adult tissues. We determined the tissue distribution of NUAK1 and generated muscle-specific NUAK1 knock-out (MNUAK1KO) mice. For phenotypic analysis, whole body glucose homeostasis and muscle glucose metabolism were examined. Quantitative phosphoproteome analysis of soleus muscle was performed to understand the molecular mechanisms underlying the knock-out phenotype. Nuak1 mRNA was preferentially expressed in highly oxidative tissues such as brain, heart, and soleus muscle. On a high fat diet, MNUAK1KO mice had a lower fasting blood glucose level, greater glucose tolerance, higher insulin sensitivity, and higher concentration of muscle glycogen than control mice. Phosphoproteome analysis revealed that phosphorylation of IRS1 Ser-1097 was markedly decreased in NUAK1-deficient muscle. Consistent with this, insulin signaling was enhanced in the soleus muscle of MNUAK1KO mice, as evidenced by increased phosphorylation of IRS1 Tyr-608, AKT Thr-308, and TBC1D4 Thr-649. These observations suggest that a physiological role of NUAK1 is to suppress glucose uptake through negative regulation of insulin signaling in oxidative muscle.

Mitochondrial fumarate reductase as a target of chemotherapy: from parasites to cancer cells.

Recent research on respiratory chain of the parasitic helminth, Ascaris suum has shown that the mitochondrial NADH-fumarate reductase system (fumarate respiration), which is composed of complex I (NADH-rhodoquinone reductase), rhodoquinone and complex II (rhodoquinol-fumarate reductase) plays an important role in the anaerobic energy metabolism of adult parasites inhabiting hosts. The enzymes in these parasite-specific pathways are potential target for chemotherapy. We isolated a novel compound, nafuredin, from Aspergillus niger, which inhibits NADH-fumarate reductase in helminth mitochondria at nM order. It competes for the quinone-binding site in complex I and shows high selective toxicity to the helminth enzyme. Moreover, nafuredin exerts anthelmintic activity against Haemonchus contortus in in vivo trials with sheep indicating that mitochondrial complex I is a promising target for chemotherapy. In addition to complex I, complex II is a good target because its catalytic direction is reverse of succinate-ubiquionone reductase in the host complex II. Furthermore, we found atpenin and flutolanil strongly and specifically inhibit mitochondrial complex II. Interestingly, fumarate respiration was found not only in the parasites but also in some types of human cancer cells. Analysis of the mitochondria from the cancer cells identified an anthelminthic as a specific inhibitor of the fumarate respiration. Role of isoforms of human complex II in the hypoxic condition of cancer cells and fetal tissues is a challenge. This article is part of a Special Issue entitled Biochemistry of Mitochondria, Life and Intervention 2010.

Identification of a lung adenocarcinoma cell line with CCDC6-RET fusion gene and the effect of RET inhibitors in vitro and in vivo.

Rearrangements of the proto-oncogene RET are newly identified potential driver mutations in lung adenocarcinoma (LAD). However, the absence of cell lines harboring RET fusion genes has hampered the investigation of the biological relevance of RET and the development of RET-targeted therapy. Thus, we aimed to identify a RET fusion positive LAD cell line. Eleven LAD cell lines were screened for RET fusion transcripts by reverse transcription-polymerase chain reaction. The biological relevance of the CCDC6-RET gene products was assessed by cell growth, survival and phosphorylation of ERK1/2 and AKT with or without the suppression of RET expression using RNA interference. The efficacy of RET inhibitors was evaluated in vitro using a culture system and in an in vivo xenograft model. Expression of the CCDC6-RET fusion gene in LC-2/ad cells was demonstrated by the mRNA and protein levels, and the genomic break-point was confirmed by genomic DNA sequencing. Mutations in KRAS and EGFR were not observed in the LC-2/ad cells. CCDC6-RET was constitutively active, and the introduction of a siRNA targeting the RET 3' region decreased cell proliferation by downregulating RET and ERK1/2 phosphorylation. Moreover, treatment with RET-inhibitors, including vandetanib, reduced cell viability, which was accompanied by the downregulation of the AKT and ERK1/2 signaling pathways. Vandetanib exhibited anti-tumor effects in the xenograft model. Endogenously expressing CCDC6-RET contributed to cell growth. The inhibition of kinase activity could be an effective treatment strategy for LAD. LC-2/ad is a useful model for developing fusion RET-targeted therapy.

Sucrose nonfermenting AMPK-related kinase (SNARK) mediates contraction-stimulated glucose transport in mouse skeletal muscle.

The signaling mechanisms that mediate the important effects of contraction to increase glucose transport in skeletal muscle are not well understood, but are known to occur through an insulin-independent mechanism. Muscle-specific knockout of LKB1, an upstream kinase for AMPK and AMPK-related protein kinases, significantly inhibited contraction-stimulated glucose transport. This finding, in conjunction with previous studies of ablated AMPKalpha2 activity showing no effect on contraction-stimulated glucose transport, suggests that one or more AMPK-related protein kinases are important for this process. Muscle contraction increased sucrose nonfermenting AMPK-related kinase (SNARK) activity, an effect blunted in the muscle-specific LKB1 knockout mice. Expression of a mutant SNARK in mouse tibialis anterior muscle impaired contraction-stimulated, but not insulin-stimulated, glucose transport. Whole-body SNARK heterozygotic knockout mice also had impaired contraction-stimulated glucose transport in skeletal muscle, and knockdown of SNARK in C2C12 muscle cells impaired sorbitol-stimulated glucose transport. SNARK is activated by muscle contraction and is a unique mediator of contraction-stimulated glucose transport in skeletal muscle.

Loss of HGF activator inhibits foveolar hyperplasia induced by oxyntic atrophy without altering gastrin levels.

Spasmolytic polypeptide/trefoil family factor 2 expressing metaplasia (SPEM) is induced by oxyntic atrophy and is known as a precancerous or paracancerous lesion. We now have sought to determine whether hepatocyte growth factor (HGF) influences the development of SPEM and oxyntic atrophy. DMP-777, a parietal cell ablating reagent, was administered to HGF activator (HGFA)-deficient mice and wild-type mice. Gastric mucosal lineage changes were analyzed in the DMP-777 treatment phase and recovery phase. Both wild-type and HGFA knockout mice showed SPEM, and there was no difference in SPEM development. However, after cessation of DMP-777, HGFA-deficient mice showed delayed recovery from SPEM compared with wild-type mice. Foveolar cell hyperplasia and the increase in proliferating cells after parietal cell loss were reduced in HGFA-deficient mice. The HGFA does not affect emergence of SPEM. However, the absence of HGFA signaling causes a delay in the recovery from SPEM to normal glandular composition. HGFA also promotes foveolar cell hyperplasia and mucosal cell proliferation in acute oxyntic injury.

Hypoglycemic/hypoxic condition in vitro mimicking the tumor microenvironment markedly reduced the efficacy of anticancer drugs.

Tumor tissues are often hypoxic because of defective vasculature. We previously showed that tumor tissues are also often deprived of glucose. The efficacy of anticancer drugs is affected by the tumor microenvironment, partly because of the drug delivery and cellular drug resistance; however, the precise mechanisms remain to be clarified. In the present study, we attempted to clarify whether hypoglycemic/hypoxic condition, which mimics the tumor microenvironment, might induce drug resistance, and if it did, to elucidate the underlying mechanisms. Pancreatic cancer-derived PANC-1 cells were treated with serial dilutions of anticancer drugs and incubated in either normoglycemic (1.0 g/L glucose) or hypoglycemic (0 g/L glucose) and normoxic (21% O(2)) or hypoxic (1% O(2) ) conditions. The 50% inhibitory concentration of gemcitabine was 1000 times higher for PANC-1 cells incubated under the hypoglycemic/hypoxic condition than for those incubated under the normoglycemic/normoxic condition. Conventional anticancer drugs target rapidly growing cells, so that non-proliferating or slowly proliferating cells usually show resistance to drugs. Though the cell cycle was delayed, sufficient cellular uptake and DNA incorporation of gemcitabine occurred under the hypoglycemic/hypoxic condition to cause DNA lesions and S-phase arrest. To overcome hypoglycemic/hypoxia-induced drug resistance, we examined kinase inhibitors targeting Chk1 or cell-survival signaling pathways. Among the compounds examined, the combination of UCN-01 and LY294002 partially sensitized the cells to gemcitabine under the hypoglycemic/hypoxic condition. These findings suggested that the adoption of suitable strategies may enhance the cytotoxicities of clinically used anticancer drugs against cancer cells.