Identification of disease-specific genes related to immune infiltration in nonalcoholic steatohepatitis using machine learning algorithms.

To identify disease signature genes associated with immune infiltration in nonalcoholic steatohepatitis (NASH), we downloaded 2 publicly available gene expression profiles, GSE164760 and GSE37031, from the gene expression omnibus database. These profiles represent human NASH and control samples and were used for differential genes (DEGs) expression screening. Two machine learning methods, the Least Absolute Shrinkage and Selection Operator regression model and Support Vector Machine Recursive Feature Elimination, were used to identify candidate disease signature genes. The CIBERSORT deconvolution algorithm was employed to analyze the infiltration of 22 immune cell types in NASH. Additionally, we constructed a NASH cell model using HepG2 cells treated with oleic acid and free fatty acids. The construction of the cell model was verified using oil red O staining, and Western blotting was used to detect the protein expression of the disease signature genes in both control and model groups. As a result, a total of 262 DEGs were identified. These DEGs were primarily associated with metal ion transmembrane transporter activity, sodium ion transmembrane transporter protein activity, calcium ion, and neuroactive ligand-receptor interactions. FOS, IGFBP2, dual-specificity phosphatase 1 (DUSP1), and IKZF3 were identified as disease signature genes of NASH by the least absolute shrinkage and selection operator and Support Vector Machine Recursive Feature Elimination algorithms for DEGs analysis. The receiver operating characteristic curves showed that FOS, IGFBP2, DUSP1, and IKZF3 had good diagnostic value (area under receiver operating characteristic curve > 0.8). These findings were validated in the GSE89632 dataset and through cellular assays. Immunocyte infiltration analysis revealed that NASH was associated with CD8 T cells, CD4 T cells, follicular helper T cells, resting NK cells, eosinophils, regulatory T cells, and γδ T cells. The FOS, IGFBP2, DUSP1, and IKZF3 genes were specifically associated with follicular helper T cells. Lipid droplet aggregation significantly increased in HepG2 cells treated with oleic acid and free fatty acids, indicating successful construction of the cell model. In this model, the expression of FOS, IGFBP2, and DUSP1 was significantly decreased, while that of IKZF3 was significantly elevated (P < .01, P < .001) compared with the control group. Therefore, FOS, IGFBP2, DUSP1, and IKZF3 can be considered as disease signature genes associated with immune infiltration in NASH.

Benzo(a)pyrene regulates chaperone-mediated autophagy via heat shock protein 90.

AIMS: Some studies have shown that the Benzo(a)pyrene (BaP) exposure induced oxidative damage, DNA damage and autophagy, but the molecular mechanism is not clear. Heat shock protein 90 (HSP90) is regarded as an important target in cancer therapy and a key factor in autophagy. Therefore, this study aims to clarify the new mechanism of BaP regulating CMA through HSP90. MAIN METHODS: C57BL mice were fed with BaP at a dose of 25.3 mg/kg. A549 cells were treated with different concerntrations of BaP, and MTT assay was used to observe the effect of BaP on the proliferation of A549 cells. DNA damage was detected by alkaline comet assay. Focus experiment for detection of γ-H2AX by immunofluorescence. The mRNA expression of HSP90, HSC70 and Lamp-2a was detected by qPCR. The protein expressions of HSP90, HSC70 and Lamp-2a were detected by Western blot. Next, we knocked down HSP90 expression by the HSP90 Inhibitor, NVP-AUY 922, exposed or HSP90α shRNA lentivirus transduction in A549 cells. KEY FINDINGS: In these studies, we first found that heat shock protein 90 (HSP90), heat shock cognate 70 (HSC70) and lysosomal-associated membrane protein type 2 receptor (Lamp-2a) expressions of C57BL mice lung tissue and A549 cells exposed to BaP were significant increase, as well as BaP induced DNA double-strand breaks (DSBs) and activated DNA damage responses, as evidenced by comet assay and γ-H2AX foci analysis in A549 cells. Our results demonstrated BaP induced CMA and caused DNA damage. Next, we knocked down HSP90 expression by the HSP90 Inhibitor, NVP-AUY 922, exposed or HSP90α shRNA lentivirus transduction in A549 cells. HSC70 and Lamp-2a expressions of these cells exposed to BaP were not significant increase, which showed that BaP inducted CMA was mediated by HSP90. Further, HSP90α shRNA prevented BaP induced of BaP which suggested BaP regulated CMA and caused DNA damage by HSP90. Our results elucidated a new mechanism of BaP regulated CMA through HSP90. SIGNIFICANCE: BaP regulated CMA through HSP90. HSP90 is involved in the regulation of gene instability induced by DNA damage by BaP, which promotes CMA. Our study also revealed that BaP regulates CMA through HSP90. This study fills the gap of the effect of BaP on autophagy and its mechanism, which will lead to a more comprehensive understanding of the action mechanism of BaP.

Role and mechanism of benzo[a]pyrene in the transformation of chronic obstructive pulmonary disease into lung adenocarcinoma.

OBJECTIVE: This experiment is explores the genes that play a key role, their expression changes and the biological processes in the transformation of chronic obstructive pulmonary disease (COPD) into lung adenocarcinoma (LAC). Meanwhile, identify the effects of Benzo[a]pyrene (BaP) in the conversion of COPD into LAC. METHODS: 1. Differential expression genes of COPD and LAC were screened and analyzed by high-throughput microarray data between the two diseases and their respective control groups. 2. The screened genes were used for routine bioinformatics analysis such as functional analysis, expression verification, protein interaction analysis and functional enrichment. 3. Cigarette smoke extract (CSE) combined with lipopolysaccharide (LPS) was used to establish an in vitro COPD model. 4. MTT assay was used to detect the influence of B(a)P in effect on A549 cell proliferation. CCK-8, Transwell invasion test and scratch test were used to detect the cell proliferation, invasion and migration ability, while qPCR and Western Blot tests were used to observe the cell proliferation, apoptosis and changes in related indicators such as EMT. 5. Experimental method of separately adding agonists (tBHQ) and inhibitors (DIC) of NQO1 was used to confirm the effect of NQO1 on A549 cell proliferation, apoptosis, migration and invasion. 6. To further clarify whether BaP exerted effect on cell proliferation, apoptosis, migration and invasion through NQO1, we knocked down NQO1 gene and then infecting cells with BaP. RESULTS: 1. We screened genes of COPD and LAC using datasets from GSE151052, GSE118370, and GSE140797. After screening, the genes upregulated in COPD and downregulated in LAC were RTKN2, SLC6A4, and HBB, the gene downregulated in COPD and upregulated in LAC was NQO1, the genes downregulated in both COPD and LAC were FPR1, LYVE1 and PKHD1L1. 2. The main signaling pathways in which the target genes were enriched are cell cycle, EMT, PI3K/AKT, and apoptosis. In the data included GEPIA, PKHD1L1, FPR1, LYVE1, RTKN2, HBB, and SLC6A4 were significantly downregulated and NQO1 was upregulated in LAC relative to controls. In addition, there were 46 interaction proteins in the target genes, and the functions they enriched included hydrogen peroxide catabolism, etc. 3. When A549 cell was stimulated with 100 ng/mL LPS+ 10% CSE, the COX-2 expression indicated that COPD model in vitro was successfully established. 4. The optimal dose and action time were screened which were 1 µM and 24 h. Compared to the control group, COPD and BaP group increased cell proliferation and invasion capabilities. On the basis of COPD, adding BaP could further increase the proliferation and migration capabilities. Interestingly, the levels of NQO1 decreased in COPD models, while increased by BaP. 5. tBHQ can increase the proliferation and migration capacity of A549 cells, which is inhibited by the addition of DIC. 6. The enhanced proliferation, migration and invasion of A549 cells by BaP were attenuated after knockdown of NQO1. CONCLUSION: Our study reveals that PKHD1L1, FPR1, LYVE1, RTKN2, HBB, SLC6A4 and NQO1 may play an important role in the conversion of COPD to LAC. High NQO1 expression may increase the proliferation and migration ability of A549 cells, and BaP may promote the EMT state by increasing the expression of NQO1, thereby making the COPD model in vitro expose the tumor characteristics.

Study on molecular mechanism of benzo (ɑ) pyrene on CMA by HSP90ɑ and HIF-1ɑ.

OBJECTIVE: The effects of benzo (α) pyrene (BaP) on chaperone mediated autophagy (CMA) through heat shock protein 90 (HSP90) and hypoxia- inducible factor-1 (HIF-1) are studied by RNA interference and subcutaneous tumor formation technique in nude mice. METHODS: 40 nude mice that were inoculated with the silenced HSP90ɑ A549 cells line under the armpits of the forelimbs were divided into 4 groups, and were intragastrically administered with 1.80 mg/kg/d BaP-corn oil solutionfor for 60d (except the Control group), and the growth curves of nude mice and transplanted tumors were recorded. The size and morphological changes of tumors were observed by small animal imaging technique. qPCR, Western blot and Immunohistochemistry were used to detect the expression of HSP90ɑ, HSC70 and Lamp-2A. A549 cells were treated with 0.1 µmol/L, 1 µmol/L and 10 µmol/L BaP for 24 h, EPO and HIF-1ɑ concentration and HIF-1ɑ protein expression were detected by Elisa and Western blot; A549 cells were treated with 10 µmol/L BaP and HIF-1ɑ inhibitor for 24 h, qPCR, Western blot and Immunofluorescence methods were used to detect the expression of HSP90ɑ, HSC70 and Lamp-2A. RESULTS: The weight of nude mice and transplanted tumors silenced HSP90ɑ was reduced by BaP; the expression of HSP90ɑ, HSC70, Lamp-2A mRNA and proteins in transplanted tumor tissues silenced HSP90ɑ were reduced by BaP; the total number of bioluminescence photons of transplanted tumors silenced HSP90ɑ were reduced by BaP. The concentration of EPO and HIF-1ɑ and the expression of HIF-1ɑ protein in A549 cells was increased by 10 µmol/L BaP; with HIF-1ɑ inhibitors treated, HSP90ɑ, HSC70, Lamp-2A mRNA and proteins expression and the fluorescence intensity of HSP90ɑ were decreased of A549 cells. CONCLUSIONS: The growth of transplanted tumor in nude mice is promoted by BaP, and is inhibited when HSP90ɑ was silenced. BaP promotes the occurrence of CMA by promoting the expression of HSP90ɑ and HIF-1ɑ, which are vital regulatory genes of BaP activation of CMA.

Interaction between AhR and HIF-1 signaling pathways mediated by ARNT/HIF-1ß.

BACKGROUND: The main causes of lung cancer are smoking, environmental pollution and genetic susceptibility. It is an indisputable fact that PAHs are related to lung cancer, and benzo(a) pyrene is a representative of PAHs. The purpose of the current investigation was to investigate the interaction between AhR and HIF-1 signaling pathways in A549 cells, which provide some experimental basis for scientists to find drugs that block AhR and HIF-1 signaling pathway to prevent and treat cancer. METHODS: This project adopts the CYP1A1 signaling pathways and the expression of CYP1B1 is expressed as a measure of AhR strength index. The expression of VEGF and CAIX volume as a measure of the strength of the signal path HIF-1 indicators. Through the construction of plasmid vector, fluorescence resonance energy transfer, real-time quantitative PCR, western blotting and immunoprecipitation, the interaction between AhR signaling pathway and HIF-1 signaling pathway was observed. RESULTS: BaP can enhance the binding ability of HIF-1α protein to HIF-1ß/ARNT in a dose-dependent manner without CoCl2. However, the binding ability of AhR protein to HIF-1ß/ARNT is inhibited by HIF-1α signaling pathway in a dose-dependent manner with CoCl2. CONCLUSION: It is shown that activation of the AhR signaling pathway does not inhibit the HIF-1α signaling pathway, but activation of the HIF-1α signaling pathway inhibits the AhR signaling pathway.

Mechanism of Synsepalum dulcificum Daniell. Inhibiting Lung Adenocarcinoma.

Objective: Synsepalum dulcificum Daniell. (SD) is a natural plant fruit and is famous for containing miraculin. It has been reported that SD can be used as an adjuvant treatment to correct patients' loss of taste during the antitumor process, but the effect of SD itself as an antitumor is not clear. In this study, we investigated the mechanism of action of SD on lung adenocarcinoma using network pharmacology. Materials and Methods. The components of SD were identified by liquid chromatography-mass spectrometry, and then the compounds that affect tumor immunity of SD were screened and the related targets were predicted by TCMIO database. At the same time, the results were associated with lung adenocarcinoma targets included in the MalaCards and CTD databases, so as to construct a compound-target action network diagram and explore the mechanism of SD in the treatment of lung adenocarcinoma. In in vitro experiments, cell viability was determined and western blotting was used to detect the related expression of action targets to determine the therapeutic effect of SD. Results. In this experiment, 335 chemical components were identified in SD, and 107 components were related to tumor immunity. After screening by ADME, it was found that 11 compounds might be inhaled into the human body and affect the growth of lung adenocarcinoma. In vitro experiments showed that SD could inhibit the growth of lung adenocarcinoma A549 cells. SD could reduce the expression of PCNA (P < 0.05) and significantly increase the expression of Caspase-3 (P < 0.05). The results of further experiments showed that SD could significantly reduce the phosphorylation of EGFR (P < 0.05), and SD could also effectively inhibit the expression of JAK and STAT3 phosphorylation (P < 0.01) and inhibit the expression of PI3K and AKT phosphorylation (P < 0.01). Conclusion. SD can inhibit the growth of lung adenocarcinoma A549 cells and the potential mechanism was found to be the inhibition of EGFR/JAK/STAT3 and EGFR/PI3K/AKT signaling pathway, and the substance basis for SD to exert antitumor effect may be catechin, taxifolin, betaine, epigallocatechin gallate, erucamide, guanosine, kaempferol, lanosterol, morin, oleanolic acid, and quercetin.

Molecular mechanism of benzo [a] pyrene regulating lipid metabolism via aryl hydrocarbon receptor.

BACKGROUND: Benzo [a] pyrene (BaP), a potent carcinogen, has been proved that it has toxicological effects via activation the aryl hydrocarbon receptor (AhR) pathway. AhR can participate in regulating lipogenesis and lipolysis. This topic will verify whether BaP regulates lipid metabolism via AhR. METHODS: (1) C57BL/6 mice were gavaged with BaP for 12 weeks to detect serum lipids, glucose tolerance, and insulin resistance. Morphological changes in white adipose tissue (WAT) were detected by Hematoxylin and Eosin staining. The mRNA expression levels of adipogenesis-related factors included recombinant human CCAAT/enhancer binding protein alpha (C/EBPα), peroxisome proliferator-activated receptor gamma (PPARγ), and fatty acid binding protein 4 (FABP4) and inflammatory factors included nuclear factor kappa-B (NF-κB), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor alpha (TNF-α) were detected using PCR. (2) Neutral lipid content changes in differentiated 3 T3-L1 adipocytes treated with BaP with and w/o AhR inhibitor were detected by Oil red staining. The protein expression levels of adipogenesis- and decomposition-related factors included PPARγ coactivator-1 alpha (PGC-1α), and peroxisome proliferation-activated receptor alpha (PPARα) were detected using western blotting. The mRNA expression levels of inflammatory factors were detected using PCR. RESULTS: (1) BaP inhibited body weight gain, decreased lipid content, increased lipid levels, and decreased glucose tolerance and insulin tolerance in mice; (2) BaP reduced the expressions of C/EBPα, PPARγ, FABP4, PGC-1α, and PPARα and increased the expressions of NF-κB, MCP-1, and TNF-α by activating AhR. CONCLUSION: BaP inhibit fat synthesis and oxidation while inducing inflammation by activating AhR, leading to WAT dysfunction and causing metabolic complications.

Lysophosphatidylcholine promotes intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression in human umbilical vein endothelial cells via an orphan G protein receptor 2-mediated signaling pathway.

The oxLDL-based bioactive lipid lysophosphatidylcholine (LPC) is a key regulator of physiological processes including endothelial cell adhesion marker expression. This study explored the relationship between LPC and the human umbilical vein endothelial cell expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) with a particular focus on the regulation of the LPC-G2A-ICAM-1/VCAM-1 pathway in this context. We explored the LPC-inducible role of orphan G protein receptor 2 (G2A) in associated regulatory processes by using human kidney epithelial (HEK293) cells that had been transfected with pET-G2A, human umbilical vein endothelial cells (HUVECs) in which an shRNA was used to knock down G2A, and western blotting and qPCR assays that were used to confirm changes in gene expression. For in vivo studies, a rabbit model of atherosclerosis was established, with serum biochemistry and histological staining approaches being used to assess pathological outcomes in these animals. The treatment of both HEK293 cells and HUVECs with LPC promoted ICAM-1 and VCAM-1 upregulation, while incubation at a pH of 6.8 suppressed such LPC-induced adhesion marker expression. Knocking down G2A by shRNA and inhibiting NF-κB activity yielded opposite outcomes. The application of a Gi protein inhibitor had no impact on LPC-induced ICAM-1/VCAM-1 expression. Atherosclerotic model exhibited high circulating LDL and LPC levels as well as high aortic wall ICAM-1/VCAM-1 expression. Overall, these results suggested that the LPC-G2A-ICAM-1/VCAM-1 pathway may contribute to the atherogenic activity of oxLDL, with NF-κB antagonists representing potentially viable therapeutic tools for the treatment of cardiovascular disease.

The Impact of the SARS-COV-2 Pandemic on the Mental Health and Employment Decisions of Medical Students in North China.

Background: The outbreak of severe respiratory syndrome coronavirus 2 (SARS-COV-2) has led to long periods of social isolation for individuals across the world. Although medical students generally have a high prevalence of mental health problems, they have received less attention than other groups concerning the impact of SARS-COV-2. Therefore, the present study investigated the mental health status, risk factors, and protective factors for mental health problems in medical students in North China during the SARS-COV-2 pandemic. Methods: A WeChat-based survey, which included the Depression Anxiety Stress Scale-21 and measures of social demographics, was performed twice. Risk and protective factors were identified by binary logistic regression analysis. Results: A total of 702 effective questionnaires were collected in two separate surveys. In total, 24.55% of medical students were suffering anxiety to different degrees of severity, 13.18% were suffering depression in the first survey, and 3.71% wanted to give up working in primary medical care during the SARS-COV-2 pandemic in the second survey. In contrast, during the SARS-COV-2 pandemic, a risk factor for anxiety and depression was gender which is male, while being knowledgeable about the SARS-COV-2 pandemic and having a lower academic burden were both protective factors. Conclusions: Measures are required to prevent increases in mental health problems in medical students. Our findings suggest that increasing knowledge about the SARS-COV-2 pandemic and reducing academic burden in medical students is extremely important during the SARS-COV-2 pandemic.

Relationship between Methylation of FHIT and CDH13 Gene Promoter Region and Liver Cancer.

In order to demonstrate the relationship between methylation of fragile histidine triad (FHIT) and T-cadherin/H-cadherin (CDH13) genes and liver cancer, the methylation status of FHIT and CDH13 was detected in healthy individuals and in Mongolian and Han patients with liver cancer. The phenol-chloroform method was used to extract genomic DNA. The methylation specific polymerase chain reaction method was applied to detect the methylation status of FHIT and CDH13. The relationship between smoking and alcohol consumption and gene (FHIT and CDH13) methylation was analyzed. There was significant difference in methylation rate of FHIT (72.67%, 34.67%) and CDH13 (72.0%, 28.0%) between liver cancer patients and healthy individuals of Mongolian descent (P<0.05), as well as that of FHIT (68%, 30.67%) and CDH13 (64%, 26%) between liver cancer patients and healthy individuals of Han individuals (P<0.05). There was also a relationship between smoking and drinking and the methylation of FHIT and CDH13 (P<0.05). Thus, the methylation of FHIT and CDH13 had a relationship with liver cancer incidence. Smoking and alcohol ingestion may promote the methylation of FHIT and CDH13.

High RAD18 Expression is Associated with Disease Progression and Poor Prognosis in Patients with Gastric Cancer.

BACKGROUND: RAD18 plays an important role in DNA damage repair by inducing monoubiquitinated PCNA (mUB-PCNA) in both cancer and normal tissues. Previous studies have not determined the significance of RAD18 expression in clinical gastric cancer (GC) samples. Thus, this study aimed to clarify the expression and functional significance of RAD18 in GC. METHODS: Overall, 96 resected GC samples were subjected to an immunohistochemical analysis of RAD18. GC cell lines were also subjected to functional RNA interference analyses of RAD18. RESULTS: RAD18 expression was predominantly nuclear and was observed at higher levels in GC tissues than in normal tissues. In GC tissues, strong RAD18 expression was associated with progression of lymph node metastasis (p = 0.0001), lymphatic invasion (p = 0.0255), venous invasion (p < 0.0001), recurrence (p = 0.028), and disease stage (p = 0.0253). Moreover, GC patients with high tumor RAD18 expression had shorter overall survival (p = 0.0061) and recurrence-free survival durations (p = 0.035) than those with low tumor RAD18 expression. RAD18 knockdown inhibited GC proliferation and invasiveness and increased chemosensitivity by suppressing mUB-PCNA. CONCLUSIONS: RAD18 expression may be a useful marker of progression and poor prognosis of GC. Moreover, therapeutic strategies that target RAD18 might be a novel chemosensitizer to eradicate the refractory GC.

Molecular mechanism of the dual regulatory roles of ATP on the αγ heterodimer of human NAD-dependent isocitrate dehydrogenase.

Human NAD-dependent isocitrate dehydrogenase (NAD-IDH) is responsible for the catalytic conversion of isocitrate into α-ketoglutarate in the Krebs cycle. This enzyme exists as the α2ßγ heterotetramer composed of the αß and αγ heterodimers. Our previous biochemical data showed that the αγ heterodimer and the holoenzyme can be activated by low concentrations of ATP but inhibited by high concentrations of ATP; however, the molecular mechanism was unknown. Here, we report the crystal structures of the αγ heterodimer with ATP binding only to the allosteric site (αMgγMg+CIT+ATP) and to both the allosteric site and the active site (αMg+ATPγMg+CIT+ATP). Structural data show that ATP at low concentrations can mimic ADP to bind to the allosteric site, which stabilizes CIT binding and leads the enzyme to adopt an active conformation, revealing why the enzyme can be activated by low concentrations of ATP. On the other hand, at high concentrations ATP is competitive with NAD for binding to the catalytic site. In addition, our biochemical data show that high concentrations of ATP promote the formation of metal ion-ATP chelates. This reduces the concentration of free metal ion available for the catalytic reaction, and thus further inhibits the enzymatic activity. The combination of these two effects accounts for the inhibition of the enzyme at high concentrations of ATP. Taken together, our structural and biochemical data reveal the molecular mechanism for the dual regulatory roles of ATP on the αγ heterodimer of human NAD-IDH.

High expression of carcinoembryonic antigen and telomerase reverse transcriptase in circulating tumor cells is associated with poor clinical response to the immune checkpoint inhibitor nivolumab.

The present study aimed to enrich circulating tumor cells (CTCs) from blood samples using a new size-sorting CTC chip. The present study also set out to identify a blood sensitivity marker for the immune checkpoint inhibitor nivolumab in patients with advanced, pre-treatment lung cancer. The CTC sorting efficacy of the chip was investigated and the large cell fraction of blood samples from 15 patients with pre-treatment lung cancer who were later administered nivolumab were purified. The expression levels of carcinoembryonic antigen (CEA), human Telomerase Reverse Transcriptase (hTERT), cytokeratin19 (CK19), and programmed death ligand-1 (PD-L1) were investigated to clarify the association between these CTC markers and the clinical response to nivolumab. The CTC chip effectively enriched cells from lung cancer cell line PC-9. The large cell fraction had a high expression of CEA and hTERT, with the former being significantly associated with the clinical response to nivolumab. The expression of CEA and hTERT in CTCs derived from the blood of a patient with lung cancer were also validated. The evaluation of CEA and possibly hTERT in CTCs collected by the CTC chip may represent a promising predictive blood marker for sensitivity to nivolumab. To the best of our knowledge this is the first report to describe the predictive CTC marker for nivolumab in pre-treatment patients.

Nuclear heat shock protein 110 expression is associated with poor prognosis and hyperthermo-chemotherapy resistance in gastric cancer patients with peritoneal metastasis.

AIM: To investigate the significance of heat shock protein 110 (HSP110) in gastric cancer (GC) patients with peritoneal metastasis undergoing hyperthermo-chemotherapy. METHODS: Primary GC patients (n = 14) with peritoneal metastasis or positive peritoneal lavage cytology who underwent distal or total gastrectomy between April 2000 and December 2011 were enrolled in this study. The patients underwent postoperative intraperitoneal hyperthermo-chemotherapy using a Thermotron RF-8 heating device two weeks after surgery. We analyzed nuclear HSP110 expression in surgically resected tumors using immunohistochemistry. Additionally, the effect of HSP110 suppression on hyptherthermo-chemosensitivity was assessed in vitro in the MKN45 GC cell line using the HSP inhibitor KNK437. RESULTS: HSP110 immnohistochemical staining in 14 GC patients showed that five (35.7%) samples belonged to the low expression group, and nine (64.3%) samples belonged to the high expression group. Progression-free survival was significantly shorter in the HSP110 high-expression group than in the low-expression group (P = 0.0313). However, no significant relationships were identified between HSP110 expression and the clinicopathological characteristics of patients. Furthermore, high HSP110 expression was not an independent prognostic factor in GC patients with peritoneal metastasis (P = 0.0625). HSP110 expression in MKN45 cells was suppressed by KNK437 at the hyperthermic temperature of 43 °C in vitro. Comparison of MKN45 cell proliferation in the presence and absence of KNK437 at 43 °C, revealed that proliferation was significantly decreased when HSP110 was inhibited by KNK437. Additionally, HSP110 suppression via HSP inhibitor treatment increased cellular sensitivity to hyperthermo-chemotherapy in vitro. CONCLUSION: The expression of nuclear HSP110 in GC patients might be a new marker of chemosensitivity and a therapeutic target for patients who are tolerant to existing hyperthermo-chemotherapies.

Structural basis for the functional role of the Shu complex in homologous recombination.

The Shu complex, a conserved regulator consisting of Csm2, Psy3, Shu1 and Shu2 in budding yeast, plays an important role in the assembly of the Rad51-ssDNA filament in homologous recombination. However, the molecular basis for the assembly of the Shu complex and its functional role in DNA repair is still elusive. Here, we report the crystal structure of the yeast Shu complex, revealing that Csm2, Psy3, Shu1 and Shu2 interact with each other in sequence to form a V-shape overall structure. Shu1 adopts a structure resembling the ATPase core domain of Rad51 and represents a new Rad51 paralog. Shu2 assumes a novel structural fold consisting of a conserved zinc-finger containing SWIM domain and a small insertion domain. The functional roles of the key residues are validated using mutagenesis and in vitro pull-down and in vivo yeast growth studies. Structural analysis together with available biological data identifies two potential DNA-binding sites, one of which might be responsible for binding the ssDNA region of the 3'-overhang DNA and the other for the dsDNA region. Collectively, these findings reveal the molecular basis for the assembly of the Shu complex and shed new insight on its functional role in homologous recombination.

High STMN1 Expression is Associated with Cancer Progression and Chemo-Resistance in Lung Squamous Cell Carcinoma.

BACKGROUND: Known as a microtubule-destabilizing protein, STMN1 (gene symbol: STMN1) regulates the dynamics of microtubules, cell cycle progress, and chemo-resistance against taxane agents. It is highly expressed in various human cancers and involved in cancer progression as well as poor prognosis. METHODS: Expression of STMN1 was examined by immunohistochemistry using FFPE tissue sections from 186 patients with lung squamous cell carcinoma (LSCC). Analysis of STMN1 suppression was performed for STMN1 small interfering RNA (siRNA)-transfected LSCC cell lines to determine the change in proliferation, invasive and apoptosis abilities, and paclitaxel sensitivity. RESULTS: The cytoplasmic STMN1 expression in LSCC was higher than in normal tissues. The high expression was significantly associated with vascular invasion (P = 0.0477) and poor prognosis. In addition, the proliferating and invasive abilities were decreased, and the apoptosis ability and paclitaxel sensitivity were increased in STMN1-suppressed LSCC cells compared with control cells. CONCLUSION: The results suggest that STMN1 is a prognostic factor that also is associated with caner progression and chemo-resistance. Therefore, STMN1 could be a predictor for poor prognosis and a potential therapeutic target in LSCC.

Stathmin1 expression is associated with aggressive phenotypes and cancer stem cell marker expression in breast cancer patients.

Stathmin1 (STMN1) regulates progression in various cancers. The present study aimed to determine the relationship between STMN1 expression and several cancer-related markers in breast cancer. Using immunohistochemistry, we evaluated STMN1, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, Ki-67, epidermal growth factor receptor (EGFR), CK5/6, CD44, CD24, aldehyde dehydrogenase 1, E-cadherin, epithelial cell adhesion molecule, and vimentin in 237 breast cancer patients and the clinical significance of STMN1. STMN1 expression was evaluated in 51 breast cancer cell lines, and the prognostic value of STMN1 was calculated. Higher STMN1 expression was detected in cancer tissues and was predominantly localized in the cytoplasm. High STMN1 expression was associated with the triple negative subtype, nuclear grade progression, high expression of Ki-67, EGFR, CK5/6, E-cadherin and high CD44/low CD24. According to gene expression-based outcome for breast cancer online and the Kaplan-Meier plotter, STMN1 expression was higher in basal-type cell lines than in luminal-type cell lines, and overall survival and post-progression survival in the high STMN1 expression breast cancer patients were shorter than in low STMN1 expression patients. High STMN1 expression is a possible marker of breast cancer aggressiveness in association with proliferation, phenotype and cancer stem cell type.

Heat shock-induced HIKESHI protects cell viability via nuclear translocation of heat shock protein 70.

Heat shock proteins (HSPs), particularly HSP70, help restore normal cellular function following damage caused by stressors. HSP expression in tumor tissues indicates cancer progression, and while the development of HSP inhibitors is progressing, these substances are not widely used to treat cancer. HIKESHI (C11orf73) does not control the intracellular movement of HSP70 at normal temperatures; however, it does regulate the function and movements of HSP70 during heat shock. In this study, we examined the intracellular movement of HSP70 during heat shock to investigate the significance of HIKESHI expression in gastric cancer (GC) and determine if HIKESHI inhibition has cytotoxic effects. We examined HIKESHI using GC cell lines and immunostaining in 207 GC tissue samples. HIKESHI expression in GC tissues was associated with the progression of lymphatic invasion. Suppressing HIKESHI using siRNA did not affect cell viability at normal temperatures. However, suppressing HIKESHI during heat shock inhibited HSP70 nuclear transport and suppressed cell viability. Our results suggest that HIKESHI is a marker of cancer progression and that the combination of HIKESHI inhibition and hyperthermia is a therapeutic tool for refractory GC.

High STMN1 level is associated with chemo-resistance and poor prognosis in gastric cancer patients.

BACKGROUND: Stathmin1 (STMN1) is a cytosolic phosphoprotein that regulates cellular microtubule dynamics and is known to have oncogenic activity. Despite several reports, its roles in gastric cancer (GC) remain unclear owing to a lack of analyses of highly metastatic cases. This study aimed to investigate STMN1 as a prognostic and predictive indicator of response to paclitaxel therapy in patients with GC, including inoperable cases. METHODS: Immunohistochemical analysis of STMN1 was performed on both operable (n=95) and inoperable GC (n=61) samples. The roles of STMN1 in cancer cell proliferation and sensitivity to a microtubule-targeting drug, paclitaxel, were confirmed by knockdown experiments using GC cell lines. RESULTS: Multivariate and Kaplan-Meier analyses demonstrated that high STMN1 was predictive of poor prognosis in both the groups. In the operable cohort, STMN1 expression correlated with cancer curability, recurrence, and resistance to adjuvant therapy. A correlation with paclitaxel resistance was observed in inoperable cases. Knockdown of STMN1 in GC cell lines inhibited proliferation and sensitised the cells to paclitaxel by enhancing apoptosis. CONCLUSIONS: STMN1 is a possible biomarker for paclitaxel sensitivity and poor prognosis in GC and could be a novel therapeutic target in metastatic GC.

Low Expression of FBXO45 Is Associated with Gastric Cancer Progression and Poor Prognosis.

BACKGROUND: F-Box protein 45 (FBXO45) is reported to be associated with cancer aggressiveness. We investigated the relationship between FBXO45 and clinicopathological factors in gastric cancer (GC). MATERIALS AND METHODS: We used immunohistochemistry to investigate FBXO45 expression in 104 GC samples; the prognostic value of FBXO45 was also calculated. RESULTS: FBXO45 levels in GC were higher than in normal tissues. Patients with relatively low FBXO45 expression (n=58) had increased cancer progression and poorer prognosis than those with high expression (n=46). Low FBXO45 expression was an independent negative prognostic factor in patients with GC. Using the public Kaplan-Meier plotter database, we showed that survival in patients with low expression of FBXO45 in GC was shorter than that in those with high FBXO45 expression, regardless of lymph node metastasis. CONCLUSION: A low FBXO45 expression level in GC tissues may be a powerful predictor of poor prognosis. FBXO45 might, therefore, be a promising candidate for a molecular targeted therapy in GC.