E2F1 Levels Research Articles

Modified Liangfu granule exhibits anti-cancer effects in gastric cancer by regulating apoptosis-related proteins and genes

Abstract Objective To investigate the anti-cancer effects and mechanism of modified Liangfu granule (MLFG) in BGC-823 gastric cancer cells. Methods The drug serum was extracted from abdominal aorta of Wistar rats treated by cyclophosphamide, dioscin, MLFG (high-, medium-, and low-dose), respectively. MTS assay was performed to detect the effect of different concentrations of MLFG and dioscin on cell growth. The effect of MLFG on cellular apoptosis was detected by flow cytometry. Western blot was used to examine Bcl-2 and Akt in BGC-823 cells treated with MLFG. Quantitative real-time polymerase chain reaction assay was performed to determine the expression level of caspase-3, E2F1 and E2F3 genes in cells treated by MLFG- and dioscin-containing serum. Results MLFG- and dioscin-containing serum inhibited the cell proliferation of BGC-823 cells. MLFG induced gastric carcinoma cell apoptosis and inhibited the expression of Bcl-2 and Akt in a dose-dependent manner. MLFG also significantly induced the gene expression of caspase-3 and downregulated E2F1 and E2F3 gene expression. Conclusion The effect of MLFG and dioscin on inhibiting cell proliferation and induction of apoptosis of gastric cancer cells might be related to the regulation of Bcl-2 and Akt proteins and the expression of caspase-3, E2F1 and E2F3 genes.

E2F1 activates MFN2 expression by binding to the promoter and decreases mitochondrial fission and mitophagy in HeLa cells.

Mitofusin-2 (MFN2) is primarily involved in mitochondrial fusion and participates in diverse biological processes. Several reports show that MFN2 is a target of different miRNAs; however, the transcriptional regulation of MFN2 has not been extensively studied. To gain insight into the transcriptional regulation of MFN2, we expressed E2F transcription factor 1 (E2F1) exogenously and observed that it increased the endogenous expression of MFN2 by binding to its putative promoter region. Although the levels of E2F1 were shown to vary during the cell cycle, the expression of MFN2 and its regulator SP1 did not change throughout the different phases, suggesting that E2F1 regulates MFN2 in a cell-cycle-independent manner. In the cell-cycle phases, where the expression of E2F1 was reduced, SP1 might act in its place to regulate the expression of MFN2. We showed that E2F1 and SP1 are present as a complex on the promoter of MFN2 during the S-phase as well as in E2F1 overexpressing cells, suggesting that they may regulate the expression of MFN2 synergistically. Furthermore, we found that E2F1 modulated mitochondrial fusion and mitophagy, likely through regulation of MFN2. Bioinformatic analysis revealed that several potential targets of E2F1 are localized in mitochondria and associated with autophagy. Collectively, these data identify the E2F1-MFN2 axis as a regulator of mitochondrial morphology and mitophagy, suggesting a potential therapeutic target for the treatment of mitochondrial disorders.

Rewiring E2F1 with classical NHEJ via APLF suppression promotes bladder cancer invasiveness

BackgroundBladder cancer progression has been associated with dysfunctional repair of double-strand breaks (DSB), a deleterious type of DNA lesions that fuel genomic instability. Accurate DSB repair relies on two distinct pathways, homologous recombination (HR) and classical non-homologous end-joining (c-NHEJ). The transcription factor E2F1 supports HR-mediated DSB repair and protects genomic stability. However, invasive bladder cancers (BC) display, in contrast to non-invasive stages, genomic instability despite their high E2F1 levels. Hence, E2F1 is either inefficient in controlling DSB repair in this setting, or rewires the repair apparatus towards alternative, error-prone DSB processing pathways.MethodsRT-PCR and immunoblotting, in combination with bioinformatics tools were applied to monitor c-NHEJ factors status in high-E2F1-expressing, invasive BC versus low-E2F1-expressing, non-invasive BC. In vivo binding of E2F1 on target gene promoters was demonstrated by ChIP assays and E2F1 CRISPR-Cas9 knockdown. MIR888-dependent inhibition of APLF by E2F1 was demonstrated using overexpression and knockdown experiments, in combination with luciferase assays. Methylation status of MIR888 promoter was monitored by methylation-specific PCR. The changes in invasion potential and the DSB repair efficiency were estimated by Boyden chamber assays and pulse field electrophoresis, correspondingly.ResultsHerein, we show that E2F1 directly transactivates the c-NHEJ core factors Artemis, DNA-PKcs, ligase IV, NHEJ1, Ku70/Ku80 and XRCC4, but indirectly inhibits APLF, a chromatin modifier regulating c-NHEJ. Inhibition is achieved by miR-888-5p, a testis-specific, X-linked miRNA which, in normal tissues, is often silenced via promoter methylation. Upon hypomethylation in invasive BC cells, MIR888 is transactivated by E2F1 and represses APLF. Consequently, E2F1/miR-888/APLF rewiring is established, generating conditions of APLF scarcity that compromise proper c-NHEJ function. Perturbation of the E2F1/miR-888/APLF axis restores c-NHEJ and ameliorates cell invasiveness. Depletion of miR-888 can establish a ‘high E2F1/APLF/DCLRE1C’ signature, which was found to be particularly favorable for BC patient survival.ConclusionSuppression of the ‘out-of-context’ activity of miR-888 improves DSB repair and impedes invasiveness by restoring APLF.

Abstract 5071: Palbociclib inhibits the stemness of mammary epithelial cells in premalignant tissues of MMTV-erbB2 transgenic mice

Abstract Palbociclib is the first CDK4/6-Cyclin D inhibitor approved by the FDA for ER+/Her2(erbB2)-breast cancer treatment. With the success of the drug in hormone-positive breast cancer, it is encouraging to explore more options of Palbociclib as an anticancer agent. erbB2 is a receptor tyrosine kinase (RTK) frequently overexpressed in breast cancer. The molecular hallmarks of erbB2+ breast cancer include the upregulation of cyclin D. The aim of this study was to explore the potential of Palbociclib as a preventive agent using MMTV-erbB2 transgenic mice, with a focus on the stemness of mammary epithelial cells (MECs) in the premalignant tissues. We first tested the in vivo effect of Palbociclib on tumor cells on MMTV-erbB2 mammary tumors using syngeneic tumor graft models. The animals bearing grafted tumors were treated with saline (control), low (75 mg/kg/day) and high (150 mg/kg/day) Palbociclib via oral gavage every 3 days for 29 days. We found that Palbociclib significantly inhibited tumor growth in a concentration dependent manner. To test the effect of Palbociclib on MEC stemness in premalignant mammary tissues, the animals at 9 wks of age were treated with saline, low and high (0, 75, 150 mg/kg/day) doses of Palbociclib every 3 days for 4 wks. We found the efficiency of both primary and secondary mammosphere formation of MECs from drug treated mice was significantly inhibited, suggesting the inhibition of mammary stemness in the premalignant tissues. Consistently, results from 3D cultures showed that colony numbers of the cells from drug treated mice were also lower than the control. Using CD24 and CD49f as markers, flow cytometry was performed to analyze the effect of Palbociclib on MEC subpopulations, including luminal, basal, and stromal populations. We showed that the percentage of luminal cells and putative mammary reconstitution units (MRUs) in drug treated mice was significantly inhibited. We also found that the percentage of CD61+/CD49+ cells, which are enriched with luminal progenitor cells and the origin of tumor initiation cells of this model, in Palbociclib treated tissues were also inhibited. The data suggest that Palbociclib induces MEC reprogramming in the premalignant tissues. Moreover, we found that the protein levels of pRb, E2F1, cyclin D1, c-myc, and cdc2 in cell cycle regulation, and pAKT, pERK, pmTOR, and p4EBP1 in the RTK pathway, pER, and DVL2 and LRP6 in the Wnt pathway were significantly downregulated in Palbociclib treated tissues, which was more evident in the high dose group. Taken together, our data indicate that Palbociclib inhibits MEC proliferation and stemness in the premalignant mammary tissues. Downregulation of cell cycle regulators and the signaling in ER, RTK, and Wnt pathway plays a critical role in this process. Our data support further investigation of Palbociclib in the prevention of ER+/ErbB2+ breast cancer. Citation Format: Amanda B. Parris, Yongxuan Liu, Zhikun Ma, Erin W. Howard, Xiaoshan Feng, Xiaohe Yang. Palbociclib inhibits the stemness of mammary epithelial cells in premalignant tissues of MMTV-erbB2 transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5071.

LncRNA NEAT1 facilitates melanoma cell proliferation, migration, and invasion via regulating miR-495-3p and E2F3.

Melanoma contributes a lot to skin cancer-related deaths. lncRNAs are implicated in various diseases, including melanoma. lncRNA NEAT1 is frequently dysregulated and can play important roles in multiple cancers. Nevertheless, little has been studied about the function of NEAT1 in melanoma progression. In our present research, we displayed NEAT1 was overexpressed in melanoma cells. A series of functional assays showed that overexpression of NEAT1 promoted the proliferation, migration, and invasion of melanoma cells. By contrast, NEAT1 knockdown obviously restrained melanoma cell progression. Mechanistically, it was revealed that NEAT1 could directly bind with miR-495-3p, which led to a negative effect on miR-495-3p levels. In addition, miR-495-3p was significantly decreased in melanoma cells. Furthermore, E2F3 was postulated as the target of miR-495-3p and overexpression of this miR could suppress the levels of E2F3. Meanwhile, it was exhibited that melanoma cell proliferation, migration, and invasion induced by E2F3 silence was abrogated by miR-495-3p. Moreover, an in vivo xenograft nude mice model was established using A375 cells and it was indicated that NEAT1 promoted melanoma progression in vivo via regulating the miR-495-3p/E2F3 axis. In conclusion, we suggest that NEAT1 exerts an oncogenic effect on melanoma development via inhibition of miR-495-3p and induction of E2F3. NEAT1 might serve as a crucial prognostic biomarker of melanoma.

Whole-Genome and Transcriptional Analysis of Treatment-Emergent Small-Cell Neuroendocrine Prostate Cancer Demonstrates Intraclass Heterogeneity.

Therapeutic resistance in metastatic castration-resistant prostate cancer (mCRPC) can be accompanied by treatment-emergent small-cell neuroendocrine carcinoma (t-SCNC), a morphologically distinct subtype. We performed integrative whole-genome and -transcriptome analysis of mCRPC tumor biopsies including paired biopsies after progression, and multiple samples from the same individual. t-SCNC was significantly less likely to have amplification of AR or an intergenic AR-enhancer locus, and demonstrated lower expression of AR and its downstream transcriptional targets. Genomic and transcriptional hallmarks of t-SCNC included biallelic loss of RB1, elevated expression levels of CDKN2A and E2F1, and loss of expression of the AR and AR-responsive genes including TMPRSS2 and NKX3-1. We identified three tumors that converted from adenocarcinoma to t-SCNC and demonstrate spatial and temporal intrapatient heterogeneity of metastatic tumors harboring adenocarcinoma, t-SCNC, or mixed expression phenotypes, with implications for treatment strategies in which dual targeting of adenocarcinoma and t-SCNC phenotypes may be necessary. IMPLICATIONS: The t-SCNC phenotype is characterized by lack of AR enhancer gain and loss of RB1 function, and demonstrates both interindividual and intraindividual heterogeneity.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/17/6/1235/F1.large.jpg.

Contextual role of E2F1 in suppression of melanoma cell motility and invasiveness.

The general transcription factor E2F1 reportedly functions in a protumorigenic manner in several cancer models. We show that the genetic context of cancer cells influence E2F1's role to impede the protumorigenic role. Thirty to fifty percent of melanoma patients carry mutant BRAF with about 90% of mutant BRAF melanomas being V600E mutation. Tissue microarrays from melanoma patients were used to establish an association between E2F1 and BRAFV600E . We show for the first time that low E2F1 levels in BRAFV600E melanomas are associated with lymph node metastasis. Genetic manipulation of E2F1 in BRAFV600E and BRAFwt cells were used to determine its role in malignant melanoma progression by examining effects on migration and invasion. E2F1-mediated negative regulation of myosin light chain kinase (MYLK) increased migration and invasion in BRAFV600E cells by phosphorylating myosin light chain and increased stress fiber formation. We show that E2F1 inhibits extracellular signal-regulated kinase (ERK) activation in BRAFV600E cells and provide evidence for a negative feedback loop between E2F1 and ERK in these cells. This study shows for the first time that E2F1 has a cancer protective role in oncogenic BRAF-activated melanoma cells and that loss of E2F1 can allow disease progression through a novel mechanism of E2F1-mediated MYLK regulation. This study has implications for oncogenic BRAF-activated tumors and resistance to targeted oncogenic BRAF therapy.

Mortalin (GRP75/HSPA9) Promotes Survival and Proliferation of Thyroid Carcinoma Cells.

We previously reported that upregulation of mortalin (HSPA9/GRP75), the mitochondrial HSP70 chaperone, facilitates tumor cell proliferation and survival in human medullary thyroid carcinoma (MTC), proposing mortalin as a novel therapeutic target for MTC. In this report, we show that mortalin is also upregulated in other thyroid tumor types, including papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), and anaplastic thyroid carcinoma (ATC), and that mortalin depletion can effectively induce growth arrest and cell death in human PTC (TPC-1), FTC (FTC133), and ATC (8505C and C643) cells in culture. Intriguingly, mortalin depletion induced varied effects on cell cycle arrest (G0/G1 phase arrest in TPC-1 and C643, G2/M phase arrest in 8505C, and mild G2/M phase arrest with increased sub-G0/G1 population in FTC133) and on the levels of TP53, E2F-1, p21CIP1, p27KIP1, and poly (ADP-ribose) polymerase cleavage in these cells, suggesting that thyroid tumor cells respond to mortalin depletion in a cell type-specific manner. In these cells, we also determined the efficacy of triphenyl-phosphonium-carboxy-proxyl (Mito-CP) because this mitochondria-targeted metabolism interfering agent exhibited similar tumor suppressive effects as mortalin depletion in MTC cells. Indeed, Mito-CP also induced robust caspase-dependent apoptosis in PTC and ATC cell lines in vitro, exhibiting IC50 lower than PLX4032 in 8505C cells and IC50 lower than vandetanib and cabozantinib in TPC-1 cells. Intriguingly, Mito-CP-induced cell death was partially rescued by mortalin overexpression, suggesting that Mito-CP may inactivate a mechanism that requires mortalin function. These findings support the significance of mortalin and mitochondrial activity in a broad spectrum of thyroid cancer.

Modulation of the cell cycle regulating transcription factor E2F1 pathway by the proteasome following amino acid starvation

The proteasome is one of the main catalytic machineries of eukaryotic cells responsible for protein degradation, and is known to be regulated during several cellular stress conditions. Recent studies suggest that the activity of the proteasome is modulated following mTOR inhibition. However, it is not clear how this process affects the proteome. In the present study, we investigated the role of the proteasome in the modulation of the proteome of HeLa cells following amino acid starvation, a stress known to inhibit mTOR activity. We used label-free quantitative proteomics to identify proteins regulated by the proteasome in starved cells. We found that nearly 50% of the proteins the level of which decreased significantly during starvation stress, could be rescued by addition of the proteasome inhibitor MG132. This suggests a key role for the proteasome in reshaping the proteome under starvation. Importantly, the expression of several of these proteins is known to be dependent on the transcription factor E2F1. Further investigation of E2F1 level showed that this transcription factor along with several other proteins involved in its pathway are regulated by the proteasome upon amino acids starvation.

RETRACTED: Comprehensive Analysis of the Expression and Prognosis for E2Fs in Human Breast Cancer

E2F transcription factors (E2Fs), a group of genes that encode a family of transcription factors, have been identified as being involved in the tumor progression of various cancer types. Increasing experimental evidence indicates that E2Fs are implicated in breast cancer tumorigenesis. However, the diverse expression patterns and prognostic values of eight E2Fs have yet to be analyzed. Herein we investigated the transcriptional and survival data of E2Fs in patients with breast cancer from the Oncomine, Gene Expression Profiling Interactive Analysis (GEPIA), Kaplan-Meier Plotter, and cBioPortal databases. We found that the expression levels of E2F1-3 and 5-8 were higher in breast cancer tissues than in normal breast tissues, whereas the expression level of E2F4 was lower in the former than in the latter. The expression levels of E2F2, 5, 7, and 8 were correlated with advanced tumor stage. Survival analysis using the Kaplan-Meier Plotter database revealed that the high transcription levels of E2F1-3, 5, 7, and 8 were associated with lowrelapse-free survival in all of the patients with breast cancer. Conversely, high E2F4 and E2F6 levels predicted high relapse-free survival in these patients. This study implied that E2F1-3, 5, 7, and 8 are potential targets of precision therapy for patients with breast cancer and that E2F4 and 6 are new biomarkers for the prognosis of breast cancer.

MiR-221 promotes lens epithelial cells apoptosis through interacting with SIRT1 and E2F3

MicroRNAs (miRNAs) have been regarded as potential modulators in varying ocular diseases, including age-related cataract (ARC). However, the roles of miR-221 in ARC progression and its underlying mechanism remain poorly understood. In this study, human lens epithelial cell line (SRA01/04) was used to investigate the potential function of miR-221 in vitro. The expressions of miR-221, sirtuin-1 (SIRT1) and E2F transcription factor 3 (E2F3) were measured in ARC tissues by quantitative real-time polymerase chain reaction and western blotting, respectively. To investigate the effect of miR-221, SIRT1 and E2F3 on cell apoptosis, SRA01/04 cells were transfected with miR-221 inhibitor, negative control inhibitor, pcDNA3.1-SIRT1 overexpression vector, pcDNA3.1-E2F3 overexpression vector or pcDNA3.1 empty vector. After the transfection, cell viability was detected in SRA01/04 cells at 0, 24, 48 or 72 h by cell counting kit-8 assay. Cell apoptosis was evaluated in transfected SRA01/04 cells by flow cytometry and western blotting at 72 h. The interaction between miR-221 and SIRT1 or E2F3 was probed by luciferase activity and RNA immunoprecipitation assays. Results showed that high expression of miR-221 was exhibited in ARC tissues compared with that in normal samples and associated with Lens Opacities Classification System III grades. Knockdown of miR-221 promoted cell viability and inhibited apoptosis in SRA01/04 cells. Moreover, both of SIRT1 and E2F3 levels were directly targeted by miR-221 and down-regulated in ARC tissues. Besides, overexpression of SIRT1 or E2F3 increased cell viability and suppressed apoptosis in SRA01/04 cells, which was reversed by addition of miR-221. We concluded that miR-221 promoted lens epithelial cells apoptosis through regulating SIRT1 and E2F3, providing a novel biomarker for treatment of ARC.

MicroRNA-26a inhibits multiple myeloma cell growth by suppressing cyclin-dependent kinase 6 expression.

MicroRNA-26a (miR-26a) has been reported to be involved in the tumorigenesis of several tumors, but its biological function and molecular mechanism in multiple myeloma (MM) are still unknown. In this study, we found that overexpression of miR-26a obviously inhibited MM cell growth, and delayed tumor growth in xenografts. Further studies showed that overexpression of miR-26a induced cell cycle arrest at G0/G1 phase in MM cells. MiR-26a mimic down-regulated the expression levels of CDK6 and E2F1, but up-regulated p53 and p21 expression. In contrast, overexpression of CDK6 decreased the effect of miR-26a mimic on MM cell survival. Moreover, miR-26a targeted CDK6 mRNA and thus suppressed CDK6 protein expression. Overexpression of miR-26a also enhanced the cytotoxic action of doxorubicin against MM. These results demonstrated that miR-26a was involved in the development of MM through regulating CDK6 signaling pathway, and indicated that miR-26a could be as a novel target for anti-tumor therapy in clinic as a single strategy or in combination with other anti-tumor drugs in MM.

E2F1 mediates the downregulation of POLD1 in replicative senescence

POLD1, the catalytic subunit of DNA Pol δ, plays an important role in DNA synthesis and DNA damage repair, and POLD1 is downregulated in replicative senescence and mediates cell aging. However, the mechanisms of age-related downregulation of POLD1 expression have not been elucidated. In this study, four potential CpG islands in the POLD1 promoter were found, and the methylation levels of the POLD1 promoter were increased in aging 2BS cells, WI-38 cells and peripheral blood lymphocytes, especially at a single site, CpG 36, in CpG island 3. Then, the transcription factor E2F1 was observed to bind to these sites. The binding affinity of E2F1 for the POLD1 promoter was found to show age-related attenuation and was confirmed to be positively regulated by the E2F1 level and negatively regulated by POLD1 promoter methylation. Moreover, cell senescence characteristics were observed in the cells transfected with shRNA-E2F1 and could contribute to the downregulation of POLD1 induced by the E2F1 decline. Collectively, these results indicated that the attenuation of the binding affinity of E2F1 for the POLD1 promoter, mediated by an age-related decline in E2F1 and increased methylation of CpG island 3, downregulates POLD1 expression in aging.

The Effect of Different Glucose Concentrations on E2F1 Level and Cell Proliferation

The Effect of Different Glucose Concentrations on E2F1 Level and Cell Proliferation

E2F1 copy number variations contribute to spermatogenic impairment and cryptorchidism by increasing susceptibility to heat stress.

Copy number variations (CNVs) play an important role in the onset of several diseases, and recently research focused on the relationship between these structural variants and diseases of the reproductive tract, including male infertility and cryptorchidism. To evaluate the contribution of copy number variations of E2F1 gene to idiopathic male infertility and the factors influencing expression of this gene. We performed a retrospective study on 540 subjects recruited from September 2014 to February 2015. TaqMan CNV assay was used to analyze E2F1 CNV. Real-time PCR was used to assess E2F1 and HSP70 expression level in heat stressed and transfected cells with three E2F1 copies. We found a significant difference in the frequency of altered E2F1 copies in patients (12/343, 3.5%) compared with controls (0/197) (p=0.005). Six patients with E2F1 CNV had history of cryptorchidism, but the prevalence between men with idiopathic infertility (6/243, 2.5%) and infertile men with history of cryptorchidism (6/100, 6.0%) was not statistically different (p=0.1). E2F1 expression increased under heat stress conditions, especially in cells carrying more copies of gene and this was associated with increased expression of HSP70. Our data suggest that an abnormal E2F1 expression caused by multiple copies of E2F1 gene predisposes to the onset of infertility and that the risk further increases if subjects with altered E2F1 copies have stressful conditions, such as heat stress or history of cryptorchidism. This study shows a link between E2F1 CNV and male infertility, suggesting that the increased risk of spermatogenic impairment associated with higher E2F1 copies might be due to higher susceptibility to stressful conditions.

Chromatin remodeling factor lymphoid-specific helicase links with Epstein-Barr virus associated the follicular germinal center B cell lymphomas.

We assessed the frequency of epigenetic lesions, including lymphoid-specific helicase (LSH), 5-hydroxymethylcytosine (5-hmC) and E2F1, and the possible correlations among molecular findings, phenotype, clinical features, and outcome. We investigated 181 paraffin-embedded B-cell lymphoma samples using immunohistochemistry and in situ hybridization. The levels of Ki67, LSH, 5-hmC, and E2F1 were all increased in germinal center B-cell lymphomas when compared with those in normal lymph nodes, and LSH was highly expressed in diffuse large B-cell lymphomas (DLBCLs) and Burkitt lymphomas (BLs) that were positive for Epstein-Barr virus (EBV) infection, indicating that LSH is linked to EBV infection in DLBCL and BL. Interestingly, LSH was mainly localized in the germinal centers of lymph nodes whereas 5-hmC staining localized to areas surrounding the germinal centers. These findings indicate a critical role for LSH as a biomarker and therapeutic target in follicular germinal center B-cell lymphoma.

Inhibitor of polyamine catabolism MDL72.527 restores the sensitivity to doxorubicin of monocytic leukemia Thp-1 cells infected with human cytomegalovirus

Leukemic cells from different patients exhibit different sensitivity to anticancer drugs including doxorubicin (DOX). Resistance to chemotherapy decreases efficacy of the treatment and promotes cancer recurrence and metastases. One of the approaches to overcome drug resistance includes E2F1-mediated regulation of the р73 protein that belongs to the р53 family. Its ΔNp73 isoform exhibits pro-oncogenic effects, and TAp73 – anti-oncogenic effects. Human cytomegalovirus (HCMV), often found in tumors, suppresses pro-apoptotic pathways and E2F1/p73 in particular. The activity of E2F1 and p73 transcription factors is linked to metabolism of biogenic polyamines. Therefore, it could be suggested that compounds that target polyamine-metabolizing enzymes can sensitize HCMV-infected hematological malignancies to doxorubicin.Here we report that HCMV infection of ТНР-1 monocytic leukemic cells considerably elevates E2F1 levels and shifts the balance between the р73 isoforms towards ΔNp73 leading to survival of DOX-treated leukemic cells. In contrast, MDL72.527, an inhibitor of polyamine catabolism, decreases ΔNp73/ТАр73 ratio and thus restores sensitivity of the cells to DOX. Our findings indicate the combination of doxorubicin and MDL72.527 may present a novel strategy for therapy of leukemia in patients with and without HCMV infection.

Ethanolic extract of Cordyceps cicadae exerts antitumor effect on human gastric cancer SGC-7901 cells by inducing apoptosis, cell cycle arrest and endoplasmic reticulum stress

Ethnopharmacological relevanceCordyceps cicadae (Miq.) Massee is a traditional Chinese medicine that has been used for approximately 1600 years in China. C. cicadae, a member of the Cordyceps genus, exerts a therapeutic effect on many diseases, such as cancer. ObjectiveThis study aimed to evaluate the antineoplasmic activity of C. cicadae and to identify its molecular mechanism of cell death. Materials and methodsThe toxicity of the ethanolic extract of C. cicadae (EEC) against different cancer cell lines was determined through MTT assay. Human gastric cancer SGC-7901 cells were treated with EEC for 48 h. Cell morphology was examined by using an Olympus phase-contrast microscope. The cell apoptosis was quantified through Annexin V-FITC/PI staining. Cells were stained with PI and then subjected to flow cytometry for the investigation of cell cycle status. Cells were subjected to mitochondrial membrane potential (MMP) assay after incubation with JC-1 probes and to intracellular Ca2+ measurement through flow cytometry after incubation with Fluo-3 AM fluorescent probes. Western blot analysis was conducted to quantify the expression of proteins related to apoptosis, cell cycle and endoplasmic reticulum stress. High-performance liquid chromatography (HPLC) analysis was performed to analyse the biological activity components of EEC. ResultsEEC suppressed the proliferation of SGC-7901 cells and induced the development of abnormal morphological features in a dose-dependent manner. Flow cytometry results indicated that EEC treatment caused cell apoptosis and arrested the cell cycle in the S phase. In addition, EEC treatment triggered MMP depolarization and Ca2+ overloading in the cytosol of SGC-7901 cells. Western blot analysis demonstrated that EEC increased Bax, AIF, caspase-8, caspase-6 and caspase-3 activities and decreased Bcl-2 activity. The release of cytochrome c from mitochondria was associated with mitochondrial dysfunction, which was caused by the activation of the cell surface receptor Fas and the cleavage of PARP. EEC-induced S phase arrest was associated with the up-regulation of E2F1, cyclin A2, cyclin E and p53 expression levels and the down-regulation of CDK2 expression. In addition, EEC increased the expression of endoplasmic reticulum stress-related proteins, such as calpain-1, caspase-12 and caspase-9. HPLC assay results suggested that EEC contained adenine, uridine, adenosine and N6-(2-Hydroxyethyl)-adenosine. ConclusionEEC inhibited the proliferation of SGC-7901 cells by inducing caspase-dependent apoptosis, arresting the cell cycle in the S phase and increasing endoplasmic reticulum stress. This study revealed that C. cicadae is a potential natural source of anticancer drugs.

Peripheral blood E2F1 mRNA in depression and following electroconvulsive therapy

The E2F transcription factors are a group of proteins that bind to the promotor region of the adenovirus E2 gene. E2F1, the first family member to be cloned, is linked to functions including cell proliferation and apoptosis, DNA repair, cell senescence and metabolism. We recently performed a deep sequencing study of micro-RNA changes in whole blood following ECT. Two micro-RNAs (miR-126-3p and miR-106a-5p) were identified and gene targeting analysis identified E2F1 as a shared target of these miRNAs. To our knowledge, no studies have examined E2F1 mRNA levels in patients with depression. Peripheral blood E2F1 mRNA levels were therefore examined in patients with depression, compared to healthy controls, and the effects of a course of ECT on peripheral blood E2F1 mRNA was investigated. Depressed patient and healthy control groups were balanced on the basis of age and sex. E2F1 mRNA levels were significantly lower in depressed patients in comparison to controls (p = .009) but did not change with ECT. There was no relationship between baseline E2F1 levels and depression severity, response to treatment, presence of psychosis or polarity of depression. There were no significant correlations between E2F1 levels and mood scores based on the HAM-D24. These results indicate that reduced peripheral blood E2F1 mRNA could be a trait feature of depression.

MicroRNA Dysregulation in Pulmonary Arteries from Chronic Obstructive Pulmonary Disease. Relationships with Vascular Remodeling.

Pulmonary vascular remodeling is an angiogenic-related process involving changes in smooth muscle cell (SMC) homeostasis, which is frequently observed in chronic obstructive pulmonary disease (COPD). MicroRNAs (miRNAs) are small, noncoding RNAs that regulate mRNA expression levels of many genes, leading to the manifestation of cell identity and specific cellular phenotypes. Here, we evaluate the miRNA expression profiles of pulmonary arteries (PAs) of patients with COPD and its relationship with the regulation of SMC phenotypic change. miRNA expression profiles from PAs of 12 patients with COPD, 9 smokers with normal lung function (SK), and 7 nonsmokers (NS) were analyzed using TaqMan Low-Density Arrays. In patients with COPD, expression levels of miR-98, miR-139-5p, miR-146b-5p, and miR-451 were upregulated, as compared with NS. In contrast, miR-197, miR-204, miR-485-3p, and miR-627 were downregulated. miRNA-197 expression correlated with both airflow obstruction and PA intimal enlargement. In an in vitro model of SMC differentiation, miR-197 expression was associated with an SMC contractile phenotype. miR-197 inhibition blocked the acquisition of contractile markers in SMCs and promoted a proliferative/migratory phenotype measured by both cell cycle analysis and wound-healing assay. Using luciferase assays, Western blot, and quantitative PCR, we confirmed that miR-197 targets the transcription factor E2F1. In PAs from patients with COPD, levels of E2F1 were increased as compared with NS. In PAs of patients with COPD, remodeling of the vessel wall is associated with downregulation of miR-197, which regulates SMC phenotype. The effect of miR-197 on PAs might be mediated, at least in part, by the key proproliferative factor, E2F1.