Nuclear Expression Levels Research Articles

ARID1A expression in hepatocellular carcinoma and relation to tumor recurrence after microwave ablation.

Aim of the studyAT-rich interactive domain 1A (ARID1A) is a subunit of the switch/sucrose non-fermentable chromatin remodeling complex, which is commonly mutated in human cancers. The clinical and pathological significance of ARID1A alteration in hepatocellular carcinoma (HCC) has not yet been clarified. The present study aimed to evaluate the clinical significance of the ARID1A gene signature in HCC and its relation to the likelihood of tumor recurrence after microwave ablation (MWA).Material and methodsThis study included 50 patients with cirrhotic HCC of Barcelona Clinic Liver Cancer stages 0/A eligible for MWA. Tumor and peri-tumor biopsies were obtained just prior to MWA and assessed for tumor pathological grade and ARID1A expression by immunohistochemistry. Patients were followed for one year after complete tumor ablation to detect any recurrence.ResultsTumor size (MCp = 0.010) and α-fetoprotein level (p = 0.013) can effectively predict the response to MWA. Nuclear expression of ARID1A was significantly lower in HCC compared to the corresponding peri-tumor cirrhotic liver tissues (p = 0.002), but no significant difference in ARID1A cytoplasmic expression was found. Nuclear ARID1A expression level in HCC showed a significantly negative relation to tumor size (MCp = 0.006), pathological grade (MCp = 0.046) and post-MWA tumor recurrence (FEp = 0.041).ConclusionsARID1A loss may enhance HCC aggressiveness and post-MWA tumor recurrence. ARID1A could be a potential target to select HCC patients for future therapies.

Anlotinib inhibits the proliferation, migration and invasion, and induces apoptosis of breast cancer cells by downregulating TFAP2C.

The vascular endothelial growth factor receptor (VEGFR) network contributes to breast cancer pathogenesis and progression. Anlotinib is a highly potent multi-target tyrosine kinase inhibitor that has been previously shown to exert antitumor effects in various types of cancer. The aim of the present study is to investigate the effect of Anlotinib against breast cancer cells in vitro and uncover the possible underlying mechanisms. The human breast cancer cell line MCF-7 was treated with different concentrations of Anlotinib, before cell proliferation, migration, invasion and apoptosis were assessed using colony formation, wound healing, Transwell and TUNEL staining assays. In addition, the expression of transcription factor AP-2γ (TFAP2C) following Anlotinib stimulation was measured using reverse transcription-quantitative PCR and western blot analysis. TFAP2C was overexpressed in MCF-7 using transfection with a pcDNA3.1 vector, before the aforementioned experiments were repeated. The results revealed that Anlotinib impaired cell viability and colony formation, reduced proliferating cell nuclear antigen, Ki-67, MMP2, MMP9 and Bcl-2 expression levels, and inhibited cell migration and invasion. By contrast, the expression levels of tissue inhibitor of metalloproteinase 1, the frequency of apoptotic cells, the expression of Bax and the cleaved caspase-3/caspase-3 ratio increased in a concentration-dependent manner. Additionally, the expression of TFAP2C decreased after Anlotinib treatment. However, TFAP2C overexpression partially blocked the effects of Anlotinib on the proliferation, migration, invasion and apoptosis of MCF-7 cells. To conclude, Anlotinib suppressed proliferation, migration and invasion, whilst inducing apoptosis of MCF-7 cells, which may be partially dependent on the inhibition of TFAP2C expression.

Overexpression of melanoma-associated antigen A2 has a clinical significance in embryonal carcinoma and is associated with tumor progression.

Melanoma-associated antigen A2 (MAGE-A2) is a member of the cancer-testis antigen family differentially overexpressed in a variety of malignancies and is associated with tumor development. However, clinical significance and prognostic value of MAGE-A2 in different histological subtypes of testicular germ cell tumors (TGCTs) have not been explored. Here, we aimed to investigate the clinical significance and prognostic impact of MAGE-A2 expression in TGCTs compared to benign tumors as well as adjacent normal tissues and then between seminomas and non-seminomas groups using immunohistochemistry on tissue microarrays. The results indicated a statistically significantdifference between overexpression of MAGE-A2 and histological subtypes of TGCTs. A statistically significant association was found between a high level of nuclear expression of MAGE-A2 protein and advanced pT stage (P = 0.022), vascular invasion (P = 0.037), as well as involvement of rete testis (P = 0.022) in embryonal carcinomas. Increased nuclear expression of MAGE-A2 was observed to be associated with more aggressive behaviors and tumor progression rather than cytoplasmic expression in these cases. Further, high level nuclear expression of MAGE-A2 had shorter disease-specific survival (DSS) or progression-free survival (PFS) compared to patients with moderate and low expression of MAGE-A2, however, without a statistically significant association. Our results confirm that increased nuclear expression of MAGE-A2 has a clinical significance in embryonal carcinomas and is associated with progression of disease. Moreover, MAGE-A2 may act as a potential predictive biomarker for the prognosis in embryonal carcinomas if follow-up period becomes longer. Further investigations for the biological function of MAGE-A2 are required in future studies.

Targeting Non-Canonical NFκB2 to Determine the Effects on Radiation Sensitization

<h3>Purpose/Objective(s)</h3> Homologous recombination (HR) provides repair of DNA double stranded breaks and lesion that block DNA replication. While non-homologous end joining (NHEJ) proficiency is commonly attributed to tumor cell resistance to radiation, HR also plays a role. This is supported by the observation that simultaneous disruption of both pathways makes cells more sensitive to radiation than either pathway alone. Thus, HR inhibition may improve radiation sensitization. Since NF-κB is known to influence HR gene expression, we hypothesize that targeting NF-κB signaling pathways, specifically non-canonical signaling, may cause radiation sensitization. <h3>Materials/Methods</h3> The Cancer Cell Line Encyclopedia (CCLE) was used to evaluated <b>NFKB2</b> gene expression levels in various cell lines. Immunofluorescence, using an NFKB2 antibody (Santa Cruz sc-7386), was performed in several cell lines (including NHI-H650, A-172, MCF-7, and SK-N-BE2) to compare total NFKB2 protein expression level and nuclear protein expression level to CCLE data. Cells were irradiated using an irradiator at radiation doses between 0.0 Gy and 8.0 Gy. Cells were fixed 24 hours after irradiation and immunofluorescence was performed using NFKB2 antibody. We performed CRISPR-Cas mutagenesis to generate U2OS clones, which carry null mutations in either <b>NFKB2</b> (encodes p100/p52) or <b>Relb</b>. We used U2OS cells with a safe harbor control locus for a control cell line. Surviving cell fraction was determined after a single fraction of radiation at 2.0 Gy by using a fixable viability staining kit. <h3>Results</h3> <b>NFKB2</b> gene expression corresponds with total non-canonical NF-κB factor p52 protein expression and nuclear p52 protein expression at baseline when results from the Cancer Cell Line Encyclopedia (CCLE) are compared to p52 immunofluorescent protein expression levels. We find nuclear protein expression of p52 increases with increasing doses of radiation from 0.0 Gy-8.0 Gy. We compared a CRISPR knockout of <b>NFKB2</b> and <b>Relb</b> in a U2OS cell line to a U2OS cell line with a safe harbor control. We found that the surviving fraction of cells is decreased in the <b>NFKB2</b> CRISPR knockout compared to the safe harbor control when given 2.0 Gy of radiation and examined 24 hours later (<i>P</i> = 0.034), but is not decreased in the <b>Relb</b> CRISPR knockout (<i>P</i> = 0.558). <h3>Conclusion</h3> NFKB2 total and nuclear protein expression increases as radiation dose increases. Knocking out <b>NFKB2</b> causes radiation sensitization, suggesting that NFKB2 may be a useful target for future cancer therapies.

Knockdown of LMTK3 in the Endometrioid Adenocarcinoma Cell Line Ishikawa: Inhibition of Growth and Estrogen Receptor α.

ObjectiveThe curative effect of high-efficiency progesterone and other therapeutic drugs for endometrioid adenocarcinoma patients with preservation of reproductive capacity has not been satisfactory so far. Novel therapeutic drugs need to be explored.MethodsWe investigated the cytoplastic and nuclear expression levels of LMTK3 between endometrioid adenocarcinoma tissues and adjacent endometrial tissues by immunohistochemistry. We detected the effects of LMTK3 on cell viability of Ishikawa cells by CCK-8. We detected the effects of LMTK3 on cell cycle and apoptosis of Ishikawa cells by flow cytometry. We also detected the effects of LMTK3 knockdown on mRNA and protein levels of ERα by qRT-PCR and western blotting, respectively. We also used the cBioPortal online database to analyze the coexpression of LMTK3 and ESR1 in 1647 UCEC samples.ResultsWe used TMAs to identify that LMTK3 was mainly detected in the cytoplasm of endometrioid tissues, and cytoplasmic LMTK3 expression in endometrioid tissues was higher than that in adjacent endometrial tissues (P < 0.05). LMTK3 knockdown decreased the proliferation of Ishikawa cells through decreasing cell viability (P < 0.01), increasing G1 (P < 0.001) arrest, and promoting apoptosis (P < 0.01). There was a positive correlation between the mRNA expression levels of LMTK3 and ESR1 (Spearman: P=2.011e-5, R=0.13; Pearson: P=7.18e-8, R=0.17). Knockdown of LMTK3 also reduced the mRNA (P < 0.001) and protein (P < 0.001) levels of ERα.ConclusionsInhibitors of LMTK3 may be a possible future treatment for ERα and LMTK3 highly expressed endometrioid adenocarcinoma following appropriate studies.

MicroRNA-216a-5p in lipopolysaccharide-induced endothelial injury.

MicroRNAs (miRNAs/miRs) are a type of non-coding RNA that are closely associated with disease development and treatment. The present study aimed to investigate the role of miR-216a-5p in lipopolysaccharide (LPS)-induced endothelial injury in vitro. The EdU assay was performed to detect EdU-positive cells, while flow cytometric analysis was performed to detect apoptotic cells. Reverse transcription-quantitative PCR and western blot analyses were performed to detect the expression levels of miR-216a-5p, Toll-like receptor 4 (TLR4), MyD88 and nuclear factor (NF)-κB(p65) and phosphorylated (p)-NF-κB(p65). Furthermore, p-NF-κB(p65) nuclear expression level was detected via cellular immunofluorescence. The dual-luciferase reporter assay was performed to verify the association between miR-216a-5p and TLR4. The results demonstrated that the number of EdU-positive cells significantly decreased, the apoptotic rate significantly increased, and TLR4, MyD88 and NF-κB(p65) mRNA expression levels were significantly upregulated.TLR4, MyD88 and p-NF-κB(p65) protein expression levels were significantly upregulated and p-NF-κB(p65) nuclear concentration was significantly enhanced in the small interfering RNA-miR-216a-5p and LPS groups (P<0.001, respectively) compared with the negative control group. However, the addition of miR-216a-5p significantly increased the number of EdU-positive cells, significantly decreased the apoptotic rate and significantly downregulated the mRNA expression levels of TLR4, MyD88 and NF-κB(p65), as well as the protein expression levels of TLR4, MyD88 and p-NF-κB(p65). In addition, the p-NF-κB(p65) nuclear concentration was significantly decreased in the miR-216a-5p group (P<0.001, respectively) compared with the LPS group. Taken together, the results suggest that overexpression of miR-216a-5p suppresses the effects of LPS induced endothelial injury.

The Keap1/Nrf2-ARE signaling pathway is involved in atrazine induced dopaminergic neurons degeneration via microglia activation

ObjectiveTo investigate the mechanisms of ATR-induced dopaminergic toxicity by microglia activation and the response of the Keap1/ Nrf2- ARE signaling pathway. MethodsWistar rats were treated with 50, 100 and 200 mg/kg ATR and BV-2 microglia cells were treated with 50, 100 μM ATR or 100 ng/mL LPS, respectively. Rats behavioral responses and histopathological changes were monitored. Immunohistochemical and immunofluorescence analysis detected Iba-1 and TH+ cells in rats. Keap1/Nrf2-ARE signaling-related proteins and inflammatory factors from BV-2 cells and rats were detected using ELISA, Western blot and Real-time PCR. ResultsAfter ATR treatment, the grip strength of Wistar rats was significantly decreased, and anxiety were clearly observed. TH+ neurons were reduced, however, the number of microglia cells and Iba-1 levels were increased clearly in SN. The release of ROS, TNF-α and IL-Iβ were increased, and levels of SOD and GSH-Px were significantly decreased. Keap1 mRNA expression and protein levels were decreased, while nuclear Nrf2 mRNA expression and protein levels were both increased in vivo and in vitro. ConclusionATR could significantly activate microglia and exacerbate neurotoxicity and neuroinflammation, leading to accelerate dopaminergic neuron cell death by inhibiting Keap1/Nrf2-ARE signaling pathway.

Ginkgo biloba extract (EGb 761) mitigates methotrexate-induced testicular insult in rats: Targeting oxidative stress, energy deficit and spermatogenesis

Methotrexate (MTX) is commonly used as a therapeutic agent in the treatment of malignancies and autoimmune disorders. Risk of subsequent infertility following MTX administration has been reported as a significant side effect due to testicular toxicity. The aim of the study was to evaluate the modulatory effects of Ginkgo biloba (standardized extract, EGb 761) on MTX-induced testicular oxidative stress, energy deficits and spermatogenic status in rats. All groups received intraperitoneal injection of MTX (0.5 mg/kg) twice weekly for four weeks except the control group that received the corresponding vehicles. Other groups received oral EGb761, at doses 25, 50 or 100 mg/kg/day, for four weeks, concurrently with MTX. Blood and semen sampling followed by testis dissection were performed 24 h after last EGb 761 treatment. Semen was examined for sperm progressive motility, percent of normal spermatozoa and sperm cell concentration as well as seminal plasma essential and non-essential amino acids. Serum LH, FSH and testosterone were detected as well as testicular MDA, GSH, GSSG, TNF-α, IL-1β, IL-6, NF-κB and the nuclear, cytoplasmic and mRNA expression levels of Nrf-2 besides the testicular cell energy; AMP, ADP and ATP. Histopathological studies of interstitial fibrosis and the severity of germ cell degeneration were also conducted. MTX induced significant decline in sperm quality along with decreased essential and non-essential amino acids in seminal plasma. MTX reduced serum FSH, LH and testosterone as well as testicular ATP, GSH and Nrf2, while increased levels of testicular ADP, AMP, MDA, GSSG and TNF-α. Results were confirmed by prominent interstitial fibrosis and severe germ cell degeneration in MTX group. Concurrent treatment with EGb 761 alleviated MTX-induced testicular insult evidenced by amelioration of oxidative stress biomarkers, energy functions, seminal sperms abnormalities and spermatogenesis status. The present study suggests a beneficial role of EGb 761 in MTX-induced testicular injury and subsequent distortion of spermatogenesis.

SPIB acts as a tumor suppressor by activating the NFkB and JNK signaling pathways through MAP4K1 in colorectal cancer cells

Spi-B transcription factor (SPIB) is a member of the E-twenty-six (ETS) transcription factor family. Previous studies have shown that the expression of SPIB is downregulated in human colorectal cancer tissues. The purpose of our study was to explore the biological function and related mechanism of SPIB in colorectal cancer cells. Our study found that SPIB could inhibit the proliferation, migration and invasion of CRC cells; inhibit angiogenesis; and induce CRC cells cycle arrest in G2/M phase and promote the apoptosis of CRC cells. We also found that compared with the control group, the 50% inhibitory concentration (IC50) values of oxaliplatin and 5-FU in the SPIB overexpression group were significantly reduced. Western blot results showed that the overexpression of SPIB upregulated cleaved-PARP(c-PARP), nuclear factor kB p65 (NFkB p65), phospho-NFkB p65 (p-NFkB P65), JNK1, and C-Jun protein expression levels compared with the control group. The silence of SPIB downregulated c-PARP, NFκB p65, p-NFκB p65, JNK1, and C-Jun protein expression levels. A dual-luciferase reporter assay showed that SPIB could activate the promoter of MAP4K1 and enhance the expression of MAP4K1. After silencing MAP4K1, the protein expression levels of c-PARP, NFkB P65, p-NFkB P65, JNK1, and C-Jun were downregulated. In summary, we found that SPIB is a tumor suppressor in colorectal cancer cells and that SPIB sensitizes colorectal cancer cells to oxaliplatin and 5-FU, SPIB exerts its anti-colorectal cancer effect by activating the NFkB and JNK signaling pathways through MAP4K1. The above findings may provide a reference for new molecular markers and therapeutic targets for CRC.

Oestrogen-regulated protein SLC39A6: a biomarker of good prognosis in luminal breast cancer

PurposeThe outcome of the luminal oestrogen receptor-positive (ER +) subtype of breast cancer (BC) is highly variable and patient stratification needs to be refined. We assessed the prognostic significance of oestrogen-regulated solute carrier family 39 member 6 (SLC39A6) in BC, with emphasis on ER + tumours.Materials and methodsSLC39A6 mRNA expression and copy number alterations were assessed using the METABRIC cohort (n = 1980). SLC39A6 protein expression was evaluated in a large (n = 670) and annotated series of early-stage (I–III) operable BC using tissue microarrays and immunohistochemistry. The associations between SLC39A6 expression and clinicopathological parameters, patient outcomes and other ER-related markers were evaluated using Chi-square tests and Kaplan–Meier curves.ResultsHigh SLC39A6 mRNA and protein expression was associated with features characteristic of less aggressive tumours in the entire BC cohort and ER + subgroup. SLC39A6 protein expression was detected in the cytoplasm and nuclei of the tumour cells. High SLC39A6 nuclear expression and mRNA levels were positively associated with ER + tumours and expression of ER-related markers, including the progesterone receptor, forkhead box protein A1 and GATA binding protein 3. In the ER + luminal BC, high SLC39A6 expression was independently associated with longer BC-specific survival (BCSS) (P = 0.015, HR 0.678, 95% CI 0.472‒0.972) even in those who did not receive endocrine therapy (P = 0.001, HR 0.701, 95% CI 0.463‒1.062).ConclusionSLC39A6 may be prognostic for a better outcome in ER + luminal BC. Further functional studies to investigate the role of SLC39A6 in ER + luminal BC are warranted.

MiR‑4306 inhibits the malignant behaviors of colorectal cancer by regulating lncRNA FoxD2‑AS1.

MicroRNA (miR)-4306 and FoxD2-adjacent opposite strand RNA 1 (FOXD2-AS1) are cancer-related genes involved in tumor progression. However, the potential functional roles of miR-4306 and FoxD2-AS1 in colorectal cancer (CRC) development remain unknown. The present study aimed to investigate the biological functions and the molecular mechanisms of miR-4306 and FoxD2-AS1 in CRC. Reverse transcription-quantitative PCR analysis was performed to determine the expression levels of FoxD2-AS1 and miR-4306 in CRC tissues and cell lines. Functional experiments, including Cell Counting Kit-8, colony formation, cell cycle assays and western blotting, were conducted to examine the effects of FoxD2-AS1 and miR-4306 on the malignant behaviors of CRC cells. In addition, the relationship between FoxD2-AS1 and miR-4306 was assessed using a dual-luciferase reporter assay and Pearson's correlation analysis. Compared with normal samples and cells, FoxD2-AS1 expression was increased and miR-4306 expression was decreased in CRC tissues and cells. Functional experiments demonstrated that silencing FoxD2-AS1 inhibited proliferation and induced cell arrest at G0/G1 phase in CRC cells, while the overexpression of FoxD2-AS1 showed opposite results. Ki-67 and proliferating cell nuclear antigen expression levels were decreased after transfection with small interfering RNA FoxD2-AS1, but were increased after transfection with FoxD2-AS1 overexpression plasmid. Furthermore, investigations into the underling mechanism revealed that FoxD2-AS1 functioned as a molecular sponge of miR-4306. The inhibitory effects of FoxD2-AS1 silencing on CRC progression were reversed by miR-4306 knockdown. Collectively, the present study demonstrated that FoxD2-AS1 functioned as an oncogene in CRC progression, and that miR-4306 could inhibit the malignant behaviors of CRC by regulating FoxD2-AS1. Thus, the current study provided a promising therapeutic target for CRC treatment.

Evaluation of nuclear PGAM2 value in hepatocellular carcinoma prognosis.

Phosphoglycerate mutase (PGAM) is a critical enzyme in glycolysis. PGAM2 is abundant in several types of tissues and malignant tumours. However, there is limited information regarding their clinicopathological significance in dysplastic nodules and hepatocellular carcinoma (HCC). This study aims to investigate the prognostic value of PGAM2 as a new biomarker for HCC. The PGAM2 expression level was evaluated by immunohistochemistry in liver cirrhosis (n = 10), low-grade dysplastic nodules (n = 15), high-grade dysplastic nodules (n = 15) and HCCs (n = 20) and 178 pairs of HCC and adjacent peritumoral liver tissues. We selected X-tile software for counting cut-point based on the outcomes for prognosis analysis, and used Kaplan-Meier analysis and Cox regression analysis can assess the prognosis of clinicopathologic parameters. Nuclear PGAM2 was significantly overexpressed in peritumoral liver tissues compared with HCC tissues (P = 0.0010). Kaplan-Meier analyses of 178 HCC samples revealed that nuclear PGAM2's high expression level, but not cytoplasmic PGAM2, was significantly related to good overall survival rate (OS). In addition, univariate and multivariate Cox analyses indicated nuclear PGAM2 expression could be regarded as valuable predictors for OS in HCC. PGAM2 was highly expressed in HCC tissues than liver cirrhosis tissues, and nuclear PGAM2's high expression might demonstrate HCC patients have poor postoperative results. Thus, nuclear PGAM2 can be regarded as valuable predictors for OS in HCC patients after surgery.

Anti-Aging Effect of Urolithin A on Bovine Oocytes In Vitro.

Simple SummaryPost-ovulatory and maternal oocyte aging impair female reproductive capacity through several mechanisms that are not fully understood. Urolithin A (UA) is a natural compound previously identified to exert an anti-aging effects in several cells, which has never been used in bovine germinal cells. Our goal was to study UA effect on the developmental potential of the female gamete and the surround cumulus cells obtained from young and adult cows. A model for in vitro aging of female gametes was implemented to study different problems associated with reproductive aging and fertility impairment. Results confirmed that aging exerts a harmful effect on oocyte quality measured by using different parameters and gene expression levels of cumulus cells. Moreover, UA supplementation was an effective way to prevent oocyte aging, improving the subsequent bovine embryonic development.Oxidative stress and mitochondrial dysfunction have been associated with the age-related decline of oocyte quality and strategies for their prevention are currently quested. Urolithin A (UA) is a natural metabolite with pro-apoptotic and antioxidant effects, capable of preventing the accumulation of dysfunctional mitochondria in different aged cells. UA has never been tested in bovine oocytes. Our aim was to study the effect of UA on the developmental potential of cumulus-oocyte-complexes (COCs) and granulosa cells’ (GCs) expression of important genes related to reproductive competence. Nuclear maturation progression, mitochondrial membrane potential (MMP) and developmental competence of physiologically mature (22 h) and in vitro aged oocytes (30 h of IVM) obtained from prepubertal and adult females, either supplemented with UA or not were assessed. Additionally, the amount of mRNA of several genes (NFE2L2, NQO1, and mt-DN5) and the number of mt-ND5 DNA copies were quantified in cultured GCs from prepubertal and adult females, either supplemented with UA or not. Our study confirmed the harmful effect of oocyte aging on the nuclear maturation progression, MMP, developmental competence and gene expression levels. UA treatment during in vitro maturation enhanced (p < 0.05) the maturation rate and subsequent developmental capacity of aged oocytes. A positive effect (p < 0.05) of UA on physiological maturation, MMP and embryonic development was also identified. UA also interfered on the expression profile of NFE2L2 and NQO1 genes in GCs cultures. Our findings demonstrate that UA supplementation is an effective way to prevent oocyte aging and improves the subsequent bovine embryonic development.

Validation of FOXM1 As a Therapeutic Target in Acute Myeloid Leukemia

Objectives:Forkhead box M1 (FOXM1) is a transcription factor of the Forkhead family that induces genes critical in executing the mitotic program. The FOXM1 regulatory network is a major predictor of adverse survival outcomes across 18000 human tumor expression signatures (Gentles. Nat Med 2015). FOXM1 interacts with nucleophosmin (NPM) in cancer cells and NPM determines the cellular localization of FOXM1. In the OCI-AML3 cell line and in AML primary samples with mutant alleles of NPM1, we have shown that FOXM1 and mutant NPM are re-localized to the cytoplasm (Khan. Leukemia 2017). We postulated that nuclear export of FOXM1 may underlie the chemotherapy responsiveness in NPM1mut patients. We conducted a multi-institution study to investigate the nuclear expression level of FOXM1 as a predictor of chemotherapy resistance in AML and validate it as a therapeutic target.Methods:Adult patients diagnosed with intermediate-risk AML were identified at Northwestern Memorial and the University of Illinois Hospitals. A total of 102 patients were included in the study with equivalent representation from each institution. Clinical data were collected by chart review, and corresponding bone marrow biopsy samples were retrieved under an IRB approved protocol. Tissue sections were stained with a FOXM1 antibody and scanned images were analyzed using HALO 2.0 software. All biopsy specimens were reviewed by a hematopathologist for adequacy. The percentage of total nuclei expressing FOXM1, nuclear FOXM1 intensity (optical density), and the FOXM1 nuclear:cytoplasmic ratio were quantified. Statistical analysis was performed by the Design and Analysis Core, UIC.Results:Patient characteristics are summarized in Table 1. Out of 102 patients, 77 (75%) achieved a complete remission with or without count recovery (CR/CRi). We used lines of induction therapy needed to achieve remission as a measure of chemotherapy resistance. Patients were stratified as needing 1 or >1 line of induction therapy. Induction therapy included cytarabine and anthracycline in 82 cases and hypomethylator in 19 cases.We found that patients needing >1 line of induction therapy had substantially higher levels of nuclear FOXM1 in their diagnostic bone marrow biopsy compared to patients who responded to first-line therapy (20.4% vs. 11.4% positive nuclei; p= 0.066). In logistic regression analysis, the percentage of FOXM1 positive nuclei (OR 1.85 for 10% increase in positive nuclei, p=0.02) and average nuclear intensity (OR 2.18 for 0.1 U increase in OD, p=0.05) were able to predict resistance to first-line chemotherapy. Due to variation in post-induction therapies, the institutions were considered separately for survival analysis. Nuc:Cyto FOXM1 ratio was able to predict inferior overall survival in a single institution cohort analyzed (n=45) in a model incorporating standard clinicopathologic prognostic factors. Multivariate regression analysis is shown in Table 2.We then proceeded to validate nuclear FOXM1 as a therapeutic target in AML. Short hairpin knockdown of FOXM1 in AML cell lines results in decreased colony size and number (KG-1 shFOXM1: 0.35 normalized to control, p=0.01; MV4-11 shFOXM1: 0.61, p=0.03). We have shown that proteasome inhibitors stabilize HSP70 which is a negative regulator of FOXM1 (Halasi. JBC 2016). The novel proteasome inhibitor ixazomib was tested for in vitro effect on FOXM1 in AML cells. Ixazomib inhibits the transcriptional activity of FOXM1 using a luciferase reporter cell line with inducible FOXM1. Treatment with ixazomib significantly decreases FOXM1 mRNA and inhibits FOXM1 protein expression in AML cell lines and patient samples (n=12) resulting in increased apoptosis by caspase cleavage. This is consistent with the FOXM1 auto-regulation loop (Halasi. Cell Cycle 2009). The therapeutic efficacy of ixazomib in AML was then tested in vivo. Single agent treatment with ixazomib compared to vehicle in an murine xenograft model of AML showed significant reduction in tumor volume after 3 weeks of treatment (0.89cm3 vs 1.62cm3, n=8 per group) and downregulation of FOXM1 expression.Conclusion:Our work shows for the first time that nuclear FOXM1 confers chemotherapy resistance in a clinically homogenous group of intermediate risk AML patients. Moreover we show that ixazomib, a well-tolerated novel proteasome inhibitors has therapeutic efficacy in AML models that correlates with suppression of FOXM1. [Display omitted] DisclosuresKhan:Teva: Speakers Bureau; Takeda: Research Funding; Gilead: Consultancy.

Clinicopathological significance of the EMT-related proteins and their interrelationships in prostate cancer. An immunohistochemical study.

The chronic inflammation influences a microenvironment, where as a result of losing control over tissue homeostatic mechanisms, the carcinogenesis process may be induced. Inflammatory response cells can secrete a number of factors that support both initiation and progression of cancer and also they may consequently induct an epithelial-mesenchymal transition (EMT), the process responsible for development of distant metastasis. Macrophage migration inhibitory factor (MIF) acts as a pro-inflammatory cytokine that is considered as a link between chronic inflammation and tumor development. MIF can function as a modulator of important cancer-related genes expression, as well as an activator of signaling pathways that promotes the development of prostate cancer. The study was performed on FFPE tissues resected from patients who underwent radical prostatectomy. To investigate the relationship of studied proteins with involvement in tumor progression and initiation of epithelial-to-mesenchymal transition (EMT) process, we selected clinicopathological parameters related to tumor progression. Immunohistochemical analyses of MIF, SOX-4, β-catenin and E-cadherin were performed on TMA slides. We found a statistically significant correlation of overall β-catenin expression with the both lymph node metastasis (p<0.001) and presence of angioinvasion (p = 0.012). Membrane β-catenin expression was associated with distant metastasis (p = 0.021). In turn, nuclear MIF was correlated with lymph node metastasis (p = 0.003). The positive protein-protein correlations have been shown between the total β-catenin protein expression level with level of nuclear SOX-4 protein expression (r = 0.27; p<0.05) as well as negative correlation of β-catenin expression with level of nuclear MIF protein expression (r = -0.23; p<0.05). Our results seem promising and strongly highlight the potential role of MIF in development of nodal metastases as well as may confirm an involvement of β-catenin in disease spread in case of prostate cancer.

Expression profiling of RTL1 in human breast cancer tissues and cell lines

Breast cancer (BC) is the most common cancer in females. In this regard, the identification of molecular alterations driving BC is an immediate need for developing effective immunotherapeutic tools. Here we investigated the expression of a placenta-specific protein, Retrotransposon-like 1 (RTL1) in a series of BC tissues and cell lines. RTL1-specific polyclonal antibody was generated and characterized. Using tissue microarray immunohistochemistry, expression of RTL1 in a total of 147 BC and 36 non-malignant breast tissues was investigated and the association of patient's clinicopathological parameters with RTL1 expression was then examined. Expression of RTL1 in four BC cells was assessed by flow cytometry, immunofluorescent staining and Western blotting. We observed a mixture pattern of nuclear and cytoplasmic RTL1 expression in most tissues examined, however nuclear expression was found to be dominant pattern of expression. The level of nuclear RTL1 expression was significantly higher in BC tissues (P < 0.001). A statistically significant association between nuclear RTL1 expression and histological grade and vascular invasion was found (P < 0.001 and P < 0.05). All cell lines expressed RTL1 with varying degrees at their surface. The most invasive BC cell line MDA-MB-231, compared to T-47D, SKBR3 and MCF7 expressed higher levels of RTL1 at their surface. Cells with a low level of surface expression, expressed high levels of intracellular RTL1 expression. Our antibody reacted with a specific band of about 125 KD in normal human placenta and all cell lines examined. In contrast to placenta, two additional bands were also observed in cancer cell lines. Our results showed for the first time that RTL1 is differentially expressed in BC compared to non-malignant breast tissues and is associated with a higher grade and vascular invasion. In BC cells with high metastatic and invasive potential, this antigen is mostly confined to cell surface compartment indicating the possibility of using antibody-based immunotherapy for advanced metastatic BC patients.

The aggravation of allergic airway inflammation with dibutyl phthalate involved in Nrf2-mediated activation of the mast cells

Dibutyl phthalate (DBP)-an organic pollutant-is ubiquitous in the environment. DBP as an immune adjuvant is related to the development of multiple allergic diseases. However, the current research involving DBP-induced pulmonary toxicity remains poorly understood. Therefore, this research aimed to explore the adverse effect and potential mechanism of DBP exposure on the lungs in rats. In our study, ovalbumin was used to build a rat model of allergic airway inflammation to study any harmful effect of DBP exposure on lung tissues. Rats were treated by intragastric administration of DBP (500 mg kg−1 or 750 mg kg−1) and/or subcutaneous injection of SFN (4 mg kg−1). The results of histopathological analysis, cell count, and myeloperoxidase showed that DBP promoted the inflammatory damage of lungs. In the lung tissues, the detection of terminal deoxynucleotidyl transferase dUNT nick end labeling and oxidative stress indices showed that DBP significantly increased the level of apoptosis and oxidative stress. Western blot analysis indicated that DBP raised the expression level of thymic stromal lymphopoietin and reduced the nuclear expression level of nuclear factor-erythroid-2-related factor 2 (Nrf2), which was further verified by quantitative real-time PCR. Meanwhile, DBP treatment markedly up-regulated the inflammatory cytokines such as IL-4 and IL-13, and rat mast cell protease-2, a marker secreted by mast cells (MCs). Conversely, sulforaphane, a Nrf2 inducer, ameliorated the pulmonary damage induced by DBP in the above. Altogether, our data provides a new insight into the impacts of the activation of MCs on the DBP-induced pulmonary toxicity as well as the safety evaluation of DBP.

Overexpression of the receptor for advanced glycation end-products in the auditory cortex of rats with noise-induced hearing loss

BackgroundThe receptor for advanced glycation end-products (RAGE) is involved in neuroinflammation. This study investigated the changes in RAGE expression following noise-induced hearing loss.MethodsThree-week-old female Sprague–Dawley rats were exposed to 115 dB SPL white noise for 4 h daily for 3 d (noise group, n = 16). In parallel, age and sex-matched control rats were raised under standard conditions without noise exposure (control group, n = 16). After 2 h (noise immediate, n = 8) and 4 wk (noise 4-week, n = 8) of noise exposure, the auditory cortex was harvested and cytoplasmic and nuclear fractions were isolated. The gene expression levels of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL6), interleukin 1 beta (IL1β), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and RAGE were evaluated using real-time reverse transcription polymerase chain reaction. The protein expression levels of nuclear RAGE and cytosolic RAGE were evaluated using western blotting. Additionally, matrix metalloproteinase 9 (MMP9) was pharmacologically inhibited in the noise immediate group, and then nuclear and cytosolic RAGE expression levels were evaluated.ResultsThe noise immediate and noise 4-week groups exhibited increased auditory thresholds at 4, 8, 16, and 32 kHz frequencies. The genes encoding the pro-inflammatory cytokines TNF-α, IL6, IL1β, and NF- κB were increased 3.74, 1.63, 6.42, and 6.23-fold in the noise immediate group, respectively (P = 0.047, 0.043, 0.044, and 0.041). RAGE mRNA expression was elevated 1.42-fold in the noise 4-week group (P = 0.032). Cytosolic RAGE expression was increased 1.76 and 6.99-fold in the noise immediate and noise 4-week groups, respectively (P = 0.04 and 0.03). Nuclear RAGE expression was comparable between the noise and control groups. matrix metalloproteinase 9 (MMP9) inhibition reduced cytosolic RAGE expression in the noise immediate group (P = 0.004).ConclusionsNoise exposure increased the expression of cytosolic RAGE in the auditory cortex and upregulated pro-inflammatory genes, but this response could be alleviated by MMP9 inhibition.

Upregulation of antioxidant and autophagy pathways via NRF2 activation protects spinal cord neurons from ozone damage.

Ozone therapy can relieve multiple types of pain but exhibits potential neurotoxicity, the mechanism of which is unclear. The present study aimed to identify the role of nuclear factor (erythroid-derived-2)-related 2 (NRF2) in preventing spinal cord injury caused by ozone overdose. Primary neuronal cells were extracted from newborn Wistar rats and authenticated by immunofluorescence using anti-microtubule-associated protein 2 as a cell type-specific marker. Cell viability assay with different ozone concentrations (0, 10, 20, 30 and 40 µg/ml) was used to determine the concentration that caused primary neuron injury; 30 min of 40 µg/ml ozone therapy notably decreased cell viability to 71%. In order to test the effects of ozone, the cells were divided into five treatment groups [0-, 30- and 40 µg/ml ozone, tert-butylhydroquinone (tBHQ) + 40 µg/ml ozone (T40) and tBHQ (T0)]. Cells in the T40 and T0 groups received 40 µmol/l tBHQ on the fifth day of SCN cultivation. Reverse transcription-quantitative PCR and western blotting showed that protein expression levels of heme oxygenase-1 (HO-1) and mRNA expression levels of HO-1 and NRF2 were decreased. NRF2, ubiquitin-binding protein p62 and microtubule-associated proteins 1A/1B light chain 3B expression levels were decreased following treatment with 40 µg/ml ozone. Immunofluorescence showed that NRF2 nuclear expression levels also decreased following 40 µg/ml ozone treatment. However, cells in the T40 group did not display decreased NRF2 nuclear expression levels. Normal/Apoptotic/Necrotic Cell Detection kit revealed that necrosis rate increased following treatment with 40 µg/ml ozone; however, the T40 group did not exhibit this increased necrosis. At 40 µg/ml, ozone increased spinal cord neuron (SCN) death in vitro. Moreover, treatment with 40 µg/ml ozone damaged SCNs. The p62/NRF2/antioxidant response element pathway prevented such injury. tBHQ activated this pathway, upregulated autophagy and increased local nuclear NRF2 concentration, thus enhancing the antioxidant system to protect SCNs from injury caused by high concentrations of ozone.

Inducible high level expression of a variant ΔD19A,D58A-ferredoxin-hydrogenase fusion increases photohydrogen production efficiency in the green alga Chlamydomonas reinhardtii

Oxygenic photosynthetic organisms use solar energy to split water into protons, electrons, and oxygen. Unicellular green algae like Chlamydomonas reinhardtii have evolved the additional ability to reduce protons to produce hydrogen (H2) via chloroplast hydrogenases (HYDA1 and HYDA2) under light-driven anaerobic conditions. In wild type cells, H2 production is limited by proton and electron supply to HYDA; most electrons are diverted to ferredoxin-NADP+-reductase. To enhance electron supply to HYDA and improve H2 production, variant ferredoxins with reduced ferredoxin-NADP+-reductase affinity have been demonstrated in vitro. Ferredoxin-hydrogenase fusions have also been tested, but achieving robust expression in vivo has been challenging. To improve efficiency, we here combine (1) a ferredoxin-HYDA fusion containing (2) a mutant FDX1 ferredoxin (D19A, D58A) with lowered affinity for ferredoxin-NADP+-reductase, utilising (3) a strongly inducible lhcbm9 promoter for controllable expression, and (4) a transgene construct containing introns for higher nuclear expression levels. Fusion constructs with 15 (pDK6-15) or 25 (pDK6-25) amino acid linkers were expressed in the nucleus of a HYDA1/HYDA2 knockout strain. Under illuminated, sulfur-deprived conditions, the variant ferredoxin-HYDA fusion strains yielded 3.0 and 4.6-fold more H2 than the wild type background strain over a 6-day period (typical purity range attained; 92–97%), confirming that the variant ferredoxin fused to HYDA is functional in vivo, strongly inducible, expresses at a similar level to the native HYDA gene, and significantly increases light-driven H2 production over wild type levels, likely by reducing losses to other electron acceptors such as ferredoxin-NADP+-reductase.