Lactate-induced MRP1 expression contributes to metabolism-based etoposide resistance in non-small cell lung cancer cells.

BACKGROUND: Metabolic reprogramming contributes significantly to tumor development and is tightly linked to drug resistance. The chemotherapeutic agent etoposide (VP-16) has been used clinically in the treatment of lung cancer but possess different sensitivity and efficacy towards SCLC and NSCLC. Here, we assessed the impact of etoposide on glycolytic metabolism in SCLC and NSCLC cell lines and investigated the role of metabolic rewiring in mediating etoposide resistance. METHODS: glycolytic differences of drug-treated cancer cells were determined by extracellular acidification rate (ECAR), glucose consumption, lactate production and western blot. DNA damage was evaluated by the comet assay and western blot. Chemoresistant cancer cells were analyzed by viability, apoptosis and western blot. Chromatin immunoprecipitation (ChIP) was used for analysis of DNA-protein interaction. RESULTS: Here we showed that exposure to chemotherapeutic drug etoposide induces an exacerbation of ROS production which activates HIF-1α-mediated the metabolic reprogramming toward increased glycolysis and lactate production in non-small cell lung cancer (NSCLC). We identified lactic acidosis as the key that confers multidrug resistance through upregulation of multidrug resistance-associated protein 1 (MRP1, encoded by ABCC1), a member of ATP-binding cassette (ABC) transporter family. Mechanistically, lactic acid coordinates TGF-ß1/Snail and TAZ/AP-1 pathway to induce formation of Snail/TAZ/AP-1 complex at the MRP1/ABCC1 promoter. Induction of MRP1 expression inhibits genotoxic and apoptotic effects of chemotherapeutic drugs by increasing drug efflux. Furthermore, titration of lactic acid with NaHCO3 was sufficient to overcome resistance. CONCLUSIONS: The chemotherapeutic drug etoposide induces the shift toward aerobic glycolysis in the NSCLC rather than SCLC cell lines. The increased lactic acid in extracellular environment plays important role in etoposide resistance through upregulation of MRP expression. These data provide first evidence for the increased lactate production, upon drug treatment, contributes to adaptive resistance in NSCLC and reveal potential vulnerabilities of lactate metabolism and/or pathway suitable for therapeutic targeting. Video Abstract The chemotherapeutic drug etoposide induces metabolic reprogramming towards glycolysis in the NSCLC cells. The secreted lactic acid coordinates TGF-ß1/Snail and TAZ/AP-1 pathway to activate the expression of MRP1/ABCC1 protein, thus contributing to chemoresistance in NSCLC.

[Atrazine changes meiosis and reduces spermatogenesis in male mice].

OBJECTIVE: To investigate the effects of exposure to atrazine on meiosis and spermatogenesis in adult male mice. METHODS: We divided 16 adult male Institute for Cancer Research (ICR) mice into a solvent control and an atrazine exposure group of an equal number and intraperitoneally injected with solvent dimethylsulfoxide (DMSO) and atrazine at 100 mg/kg/d, respectively. After 4 weeks of treatment, we obtained the body and testis weights of the mice, observed the changes in the testicular histomorphology, examined the cell apoptosis in the testis tissue, and determined the expressions of meiosis-related key genes in the spermatocytes by real-time fluorescence quantitative PCR. RESULTS: Compared with the controls, the mice treated with atrazine showed significantly less increase in the body weight (ï¼»11.2 ± 0.17ï¼½ vs ï¼»8.29 ± 0.51ï¼½ g, P < 0.05) and testis weight (ï¼»0.28 ± 0.01ï¼½ vs ï¼»0.24 ± 0.01ï¼½ g, P < 0.05), loosely arranged and thinned lumens of seminiferous tubules, disordered arrangement and reduced number of spermatogenic cells, decreased sperm concentration (ï¼»2.36 ± 0.14ï¼½ vs ï¼»0.90 ± 0.12ï¼½ ×106/ml, P < 0.01) and increased percentage of morphologically abnormal sperm in the epididymis tail (ï¼»8.60 ± 1.07ï¼½% vs ï¼»18.02 ± 1.71ï¼½%, P < 0.05), elevated apoptosis rate of spermatocytes, and down-regulated the expressions of SCP1, SCP3 and Rad51 mRNA in the spermatocytes (P < 0.05). CONCLUSIONS: Atrazine can reduce spermatogenesis in male mice by damaging testicular morphology, increasing the apoptosis of spermatocytes and down-regulating the expressions of meiosis-related genes in the spermatocytes.

[Effects of cigarette smoke on the oxidative damage of mice oocytes and developmental potential].

OBJECTIVE: To study the effects of cigarette smoke on the oxidative damage of mice oocytes and developmental potential. METHODS: A total of 96 SPF grade 6-week-old ICR female mice were randomly divided into control group, low-dose group, middle-dose group and high-dose group according to the principle of similar body weight. Twenty-four mice in each group were placed in a cigarette-lighting containers with the volume of 60, 32 and 23 L respectively. The control group was fed normally twice a day for 1 hour for 4 weeks. Weight and record daily, draw weight change curve; use kit to detect the activity of superoxide dismutase(SOD) and glutathione peroxidase(GPX) in ovarian tissue; use tissue section, indirect immunofluorescence and in vitro maturation technology to detect the activity of each group. The ovarian organ index, the level of reactive oxygen species(ROS) in oocyte and the first polar body rate were measured. The t test was used to compare the mean values of the two groups, and one-way ANOVA was used to compare the mean values of the multiple groups. RESULTS: Compared with the control group, the weight gain of mice in the treatment group was significantly slower(the weight gain in the high dose group was only 1. 05%±0. 41%). The ovarian organ index, SOD and GPX activity in the ovarian tissue and litter size(the high dose group was reduced to 5. 20%±1. 48%) were significantly decreased. The ROS level in oocyte was significantly increased, and oocyte maturation in vitro was the first. The polar body rate decreased(high dose group decreased to 49. 34%±5. 78%), and the difference was statistically significant(P<0. 05). CONCLUSION: Cigarette smoke exposure in mice can cause the decrease of ovarian organ index, SOD and GPX activity, the increase of ROS content in oocyte, the decrease of first polar body rate and litter size.

[Effects of cigarette smoke on chromatin configuration, DNA methylation and expression of related genes in mice oocytes].

OBJECTIVE: To study the effect of cigarette smoke on chromatin configuration, DNA methylation and expression of related genes in mice oocytes. METHODS: The clean grade ICR female mice were divided into 3 groups, control group, low dose group and high dose group. Animals were placed inside a perspex chamber( 18L) filled up with the smoke produced by 0, 1, 2 cigarettes, one hour a time, twice daily for 4weeks. Nuclear staining with Hoechst 33342, to observe the quality of oocyte and chromatin configuration. Analysis of the DNA methylation patterns by indirect immunofluorescence. The expressions of DNA methyltransferases( DNMT) 1, DNMT3a and DNMT3b mRNA were detected by quantitative real time PCR. RESULTS: With the increase of smoke concentration, the diameter of oocytes was significantly decreased( P <0. 05), the percentage of non surrounded nucleolus( NSN) chromatin configuration was significantly increased( P < 0. 05). In high dose group, the level of DNA methylation wassignificantly lower than that of control group( P < 0. 05). The expression of DNMT1 decreased with the increase of smoke concentration, but the expression level of DNMT3b was significantly decreased( P < 0. 05). CONCLUSION: Cigarette smoke exposure may alter oocyte chromatin configuration, decrease the expression level of DNMTs, resulted in oocytes DNA methylation decreased, so as to decrease the quality of oocytes.

Pubertal exposure to acrylamide disrupts spermatogenesis by interfering with meiotic progression in male mice.

Teenagers are a major group likely to love junk foods, such as potato chips and bread items, which contain high levels of acrylamide (AA). The increasing evidence suggests that AA exposure may be associated with decreased reproductive capacity in humans and animals. However, the reproductive toxicity of AA in pubertal males has not been fully elucidated. In this study, we evaluated the effects of pubertal AA exposure on adult spermatogenesis in male mice. Mice were exposed to AA at 0, 5, 10, 20, and 40 mg/kg/day by gavage from postnatal day 28 (PND28) to PND56. Our results showed that pubertal AA exposure increased apoptosis of germ cells in seminiferous tubules, decreased sperm concentration, and caused defects in sperm of adult mice. To explore the possible mechanisms of AA on spermatogenesis, the meiotic process was analyzed. The ratio of leptotene and zygotene spermatocytes increased, while the pachytene and diplotene spermatocytes decreased in AA-treated mice. Further analysis revealed that AA exposure disrupted the pattern of H2AX phosphorylation expansion, synapsis, and the crossover formation during meiotic prophase I (MPI). Taken together, these results indicate that pubertal AA exposure affects the spermatogenesis may be by disrupting the MPI progression of male mice.

The prognostic value of NLRP1/NLRP3 and its relationship with immune infiltration in human gastric cancer.

BACKGROUND: Inflammasomes are related to tumorigenesis and immune-regulation. Here, we investigated the prognostic value of the NLR family pyrin domain containing (NLRP) 1/NLRP3 inflammasome and its potential mechanisms in immune-regulation in gastric cancer (GC). METHODS: We analyzed the differential expression of NLRP1/NLRP3 between tumor and normal tissues using the Oncomine and Tumor Immune Estimate Resource (TIMER) databases. Immunohistochemistry and western blotting were used to detect NLRP1/NLRP3 protein expression in GC tissues. Correlations between NLRP1/NLRP3 expression levels and patient survival were analyzed using Kaplan-Meier survival curves. The relationships of NLRP1/NLRP3 expression and tumor-infiltrating immune cells/marker genes were assessed using the TIMER database. NLRP1/NLRP3 and immune checkpoint gene correlations were verified by single-gene co-expression analyses, and tumor immune-related pathways involving NLRP1/NLRP3 were analyzed using gene set enrichment analysis (GSEA). RESULTS: Elevated NLRP1/NLRP3 expression was significantly correlated with lymph node metastasis, poor survival, immune-infiltrating cell abundances, and immune cell markers. NLRP3 showed stronger correlations with immune infiltration and the prognosis of gastric cancer. NLRP1 and NLRP3 might be involved in the same tumor immune-related pathways. Thus, high NLRP1/NLRP3 expression promotes immune cell infiltration and poor prognosis in GC. NLRP1/NLRP3, particularly NLRP3, may have important roles in immune infiltration and may serve as a prognostic biomarker for GC. CONCLUSIONS: NLRP1/NLRP3, particularly NLRP3, may have important roles in immune infiltration and may serve as a prognostic biomarker for GC.

Mitochondrial genome characterization of Gryllodes sigillatus (Orthoptera: Gryllidae) and its phylogenetic implications.

Gryllodes sigillatus is a cricket widely distributed throughout the world. In this study, we reported the first complete mitogenome sequence of Genus Gryllodes and inferred its phylogeny. The mitogenome of G. sigillatus was 16,369 bp and consisted of a control region and a typical set of 37 genes. It was AT-rich with strong codon usage bias and possessed a gene arrangement of trnE-trnS1-trnN. Phylogenetic analysis indicated G. sigillatus was sister species to Velarifictorus hemelytrus, together belonging to the Family Gryllidae. Our findings would contribute to understanding mitogenomic evolution and phylogeny of Ensifera.

Acrylamide impairs the developmental potential of germinal vesicle oocytes by inducing mitochondrial dysfunction and autophagy/apoptosis in mice.

Background: Acrylamide (ACR), an important endogenous contaminant in carbohydrate-rich foods, has been involved in various negative effects on multiple organ networks, including the reproductive system. Previous studies have reported that ACR affects oocyte quality and fertility. Purpose: This study aimed to explore the toxic effects and regulatory mechanisms of ACR on mouse germinal vesicle (GV) oocytes. Research Design: In this study, adult female mice were exposed to ACR at 10 mg/kg/day/body weight through their drinking water continuously for 4 weeks. Study Sample and Data Analysis: The mitochondrial function, autophagy/apoptosis, and development potential of GV oocytes were investigated. Results: The results showed that ACR reduced the oocyte diameter, sperm-binding ability, parthenogenetic activation and in vitro fertilization (IVF) rate, and development potential of pre-implantation embryos. We also found that ACR exposure disrupted chromatin configuration, mitochondrial distribution, and membrane potential (Δφm) of oocytes. Actin filament expression was significantly reduced in both the membrane and cytoplasm of mouse oocytes. Moreover, ACR exposure increased LC3-positive signals, early apoptosis rate, aberrant ATG3, ATG5, LC3, Beclin1, and mTOR mRNA expression. Conclusions: These results suggest that ACR exposure can affect the developmental potential of GV oocytes by inducing mitochondrial dysfunction, actin filament assembly, and autophagy/apoptosis.

Molecular cloning and characterization of HSP60 gene in domestic pigeons (Columba livia) and differential expression patterns under temperature stress.

Heat shock protein 60 (HSP60) is a well-recognized multifunctional protein, playing a substantial role in protecting organisms from environmental stress. The domestic pigeon (Columba livia) is a promising model organism, with important economic and ecological value, and its health is susceptible to temperature stress. To explore the molecular characteristics, tissue expression profile, and response to temperature stress for HSP60 of Columba livia (ClHSP60), we firstly cloned and characterized the complete cDNA sequence and investigated its expression profile under optimal conditions and acute temperature stress. The cDNA of ClHSP60 contained 2257 nucleotides, consisting of 12 exons with length ranging from 65 to 590 bp. The open reading frame (ORF) encoded 573 amino acids with calculated molecular weight of 60.97 kDa that contained a number of structurally prominent domains or motifs. Under optimal temperature conditions, levels of ClHSP60 expression differed between all the tested tissues (the highest was noted in liver and the lowest in pectoralis major muscle). Under acute temperature stress, five patterns of change were detected in the tested tissues, suggesting that different tissues in domestic pigeons differentially responded to various temperature stress conditions. Upregulation of ClHSP60 expression was highest in the lung and pectoralis major muscle, reflecting the crucial role of these two tissues in temperature regulation. However, the crop, cerebrum, and heart showed little change or decreased ClHSP60 expression. The results indicate that ClHSP60 may be sensitive to and play pivotal roles in responding to acute temperature stress.

[miR-449a/b negatively regulates E2F1 to suppress proliferation of gastric cancer cells].

OBJECTIVE: To investigate the possible role of miR-449a/b in the occurrence of gastric cancer. METHODS: The expression of miR-449a/b and E2F1 mRNA in gastric cancer cells BGC-823 and gastric mucosal cells GES-1 were detected with qRT-PCR. miR-449a/b mimics or a negative control was transiently transfected into BGC-823 cells, and the changes in cell proliferation, apoptosis, and migration ability were assessed using CCK-8 assay, flow cytometry, and scratch wound healing assay, respectively. Western blotting was used to observe the effects of miR-449a/b upregulation on its target gene expression. The effects of transfection with an E2F1-over-expressing plasmid or an empty plasmid were analyzed on the expression level of miR-449a/b in BGC-823 cells using qRT-PCR and digital PCR. RESULTS: The gastric cancer cell line BGC-823 showed significantly a lowered expression of miR-449a/b compared with the normal gastric mucosal cell line GES-1 (P < 0.01). Overexpression of miR-449a/b obviously inhibited the proliferation and migration and promoted apoptosis of BGC-823 cells (P < 0.05). Overexpression E2F1 in the cells resulted in significantly up-regulated expression of miR-449a/b (P < 0.001). Upregulation of miR-449a/b caused significant down-regulation of its direct target genes CDK4 and CDK6 and also of the expression of E2F1 protein via the CDKs-pRb-E2F1 signaling pathway. CONCLUSIONS: The low expression of miR-449a/b and the high expression of E2F1 are both involved in the occurrence and progression of gastric cancer, and miR-449a/b negatively regulates E2F1 to inhibit the proliferation of gastric cancer cells.

A lactate-induced Snail/STAT3 pathway drives GPR81 expression in lung cancer cells.

Highly expressed G protein-coupled receptor 81 (GPR81), a receptor for lactate, is emerging as a critical regulator of tumor growth and metastasis. However, the mechanistic basis for its highly expression in cancer cells remains elusive. Here we report that tumor-derived lactate transcriptionally regulates GPR81 expression. We demonstrated that the transcriptional response of GPR81 to lactate is mediated by Signal transducer and activator of transcription 3 (STAT3). Mechanistically, lactate upregulates transcriptional factor Snail and induces the assembly of Snail/EZH2/STAT3 complex. Within this ternary complex, STAT3 activity is strongly enhanced. Consequently, the activated STAT3 by lactate directly binds GPR81promoter and activates its expression. These findings shed light on the transcriptional mechanism by which GPR81 expression is regulated in cancer cells, and provides mechanistic insight into how aberrant signaling and continually high lactate levels due to metabolic switch may yield a feed-forward/self-enabling loop to promote tumor progression.

[Prediction and evolution of B cell epitopes of hemagglutinin in human-infecting H6N1 avian influenza virus].

OBJECTIVE: To predict B cell epitopes of hemagglutinin (HA) of human-infecting H6N1 avian influenza virus and analyze their evolutionary characteristics. METHODS: The dataset was downloaded from GISAID and GenBank databases. And the linear and conformational B cell epitopes of HA were predicted separately by various bioinformatic software. Furthermore, the conservation, adaptation and other evolutionary characteristics were also analyzed by some bioinformatic means. RESULTS: Four linear epitopes (A, B, C and D) and two conformational epitopes (E and F) were obtained after consideration of multiple factors. And the C epitope and sites ( 41, 157, 186, 187) mutated easily, but the other epitopes were very conservative and the D epitope was the most conservative. Interestingly, the site 157 was identified under positive selection, suggesting that it may be a particularly important site to make the virus evade the attack from the host immune system. CONCLUSION: The HA of human-infecting H6N1 avian influenza virus has five conservative B cell epitopes (three linear and two conformational) and one site under positive selection. The findings would facilitate the vaccine development, virus control and pathogenesis understanding.

[Adaptive evolution of the hemagglutinin genes of the H6N1 avian influenza virus in Taiwan, China].

In Taiwan, the first human-infecting H6N1 avian influenza virus was isolated in 2013. To better understand the origin, evolutionary relationship and pathogenesis of the H6N1 virus, we studied the adaptive evolution and evolutionary dynamics of the hemagglutinin (HA) genes of the H6N1 virus in Taiwan. We felt that such studies woud contribute to the further study and control of the virus. Datasets were gained from the Flu and Global Initiative on Sharing All Influenza Data (GISAID) databases. Then, phylogenetic trees and evolutionary dynamics were reconstructed. The evolutionary rate and characterization of adaptive evolution were analyzed by bioinformatic methods. Results indicated that the HA genes of H6N1 in Taiwan were divided into at least five types, and that the new types that the infected human H6N1 belonged to could be local advantage type at present. Evolutionary dynamics revealed the viral population expanded first at the end of 1971, reduced sharply in 2008, and then increased slightly. Three sites were identified under positive selection, suggesting that various sites might increase the adaptive ability of the virus. Eighty-nine sites were under negative selection, revealing that these sites might play an important role in the replication and epidemiology of the virus. Interestingly, site 329 upstream from the cleavage site was also under negative selection, suggesting that this site might be associated with the virulence of H6N1. These data suggest that the HA genes of the Taiwanese H6N1 virus have been undergoing adaptive evolution, and that an outbreak may occur again. Hence, more attention should be paid to the identified sites, to enable timely monitoring and control of a future epidemic.