Simvastatin-Mediated Nrf2 Activation Induces Fetal Hemoglobin and Antioxidant Enzyme Expression to Ameliorate the Phenotype of Sickle Cell Disease.

Sickle cell disease (SCD) is a pathophysiological condition of chronic hemolysis, oxidative stress, and elevated inflammation. The transcription factor Nrf2 is a master regulator of oxidative stress. Here, we report that the FDA-approved oral agent simvastatin, an inhibitor of hydroxymethyl-glutaryl coenzyme A reductase, significantly activates the expression of Nrf2 and antioxidant enzymes. Simvastatin also induces fetal hemoglobin expression in SCD patient primary erythroid progenitors and a transgenic mouse model. Simvastatin alleviates SCD symptoms by decreasing hemoglobin S sickling, oxidative stress, and inflammatory stress in erythroblasts. Particularly, simvastatin increases cellular levels of cystine, the precursor for the biosynthesis of the antioxidant reduced glutathione, and decreases the iron content in SCD mouse spleen and liver tissues. Mechanistic studies suggest that simvastatin suppresses the expression of the critical histone methyltransferase enhancer of zeste homolog 2 to reduce both global and gene-specific histone H3 lysine 27 trimethylation. These chromatin structural changes promote the assembly of transcription complexes to fetal γ-globin and antioxidant gene regulatory regions in an antioxidant response element-dependent manner. In summary, our findings suggest that simvastatin activates fetal hemoglobin and antioxidant protein expression, modulates iron and cystine/reduced glutathione levels to improve the phenotype of SCD, and represents a therapeutic strategy for further development.

Targeting TREM1 augments antitumor T cell immunity by inhibiting myeloid-derived suppressor cells and restraining anti-PD-1 resistance.

The triggering receptor expressed on myeloid cell 1 (TREM1) plays a critical role in development of chronic inflammatory disorders and the inflamed tumor microenvironment (TME) associated with most solid tumors. We examined whether loss of TREM1 signaling can abrogate the immunosuppressive TME and enhance cancer immunity. To investigate the therapeutic potential of TREM1 in cancer, we used mice deficient in Trem1 and developed a novel small molecule TREM1 inhibitor, VJDT. We demonstrated that genetic or pharmacological TREM1 silencing significantly delayed tumor growth in murine melanoma (B16F10) and fibrosarcoma (MCA205) models. Single-cell RNA-Seq combined with functional assays during TREM1 deficiency revealed decreased immunosuppressive capacity of myeloid-derived suppressor cells (MDSCs) accompanied by expansion in cytotoxic CD8+ T cells and increased PD-1 expression. Furthermore, TREM1 inhibition enhanced the antitumorigenic effect of anti-PD-1 treatment, in part, by limiting MDSC frequency and abrogating T cell exhaustion. In patient-derived melanoma xenograft tumors, treatment with VJDT downregulated key oncogenic signaling pathways involved in cell proliferation, migration, and survival. Our work highlights the role of TREM1 in cancer progression, both intrinsically expressed in cancer cells and extrinsically in the TME. Thus, targeting TREM1 to modify an immunosuppressive TME and improve efficacy of immune checkpoint therapy represents what we believe to be a promising therapeutic approach to cancer.

The Oxysterol Receptor EBI2 Links Innate and Adaptive Immunity to Limit IFN Response and Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with abnormal activation of the immune system. Recent attention is increasing about how aberrant lipid and cholesterol metabolism is linked together with type I interferon (IFN-I) signaling in the regulation of the pathogenesis of SLE. Here, a metabonomic analysis is performed and increased plasma concentrations of oxysterols, especially 7α, 25-dihydroxycholesterol (7α, 25-OHC), are identified in SLE patients. The authors find that 7α, 25-OHC binding to its receptor Epstein-Barr virus-induced gene 2 (EBI2) in macrophages can suppress STAT activation and the production of IFN-ß, chemokines, and cytokines. Importantly, monocytes/macrophages from SLE patients and mice show significantly reduced EBI2 expression, which can be triggered by IFN-γ produced in activated T cells. Previous findings suggest that EBI2 enhances immune cell migration. Opposite to this effect, the authors demonstrate that EBI2-deficient macrophages produce higher levels of chemokines and cytokines, which recruits and activates myeloid cells,T and B lymphocytes to exacerbate tetramethylpentadecane-induced SLE. Together, via sensing the oxysterol 7α, 25-OHC, EBI2 in macrophages can modulate innate and adaptive immune responses, which may be used as a potential diagnostic marker and therapeutic target for SLE.

Nrf2 Drives Hepatocellular Carcinoma Progression through Acetyl-CoA-Mediated Metabolic and Epigenetic Regulatory Networks.

Correlations between the oxidative stress response and metabolic reprogramming have been observed during malignant tumor formation; however, the detailed mechanism remains elusive. The transcription factor Nrf2, a master regulator of the oxidative stress response, mediates metabolic reprogramming in multiple cancers. In a mouse model of hepatocellular carcinoma (HCC), through metabolic profiling, genome-wide gene expression, and chromatin structure analyses, we present new evidence showing that in addition to altering antioxidative stress response signaling, Nrf2 ablation impairs multiple metabolic pathways to reduce the generation of acetyl-CoA and suppress histone acetylation in tumors, but not in tumor-adjacent normal tissue. Nrf2 ablation and dysregulated histone acetylation impair transcription complex assembly on downstream target antioxidant and metabolic regulatory genes for expression regulation. Mechanistic studies indicate that the regulatory function of Nrf2 is low glucose dependent, the effect of which is demolished under energy refeeding. Together, our results implicate an unexpected effect of Nrf2 on acetyl-CoA generation, in addition to its classic antioxidative stress response regulatory activity, integrates metabolic and epigenetic programs to drive HCC progression. IMPLICATIONS: This study highlights that Nrf2 integrates metabolic and epigenetic regulatory networks to dictate tumor progression and that Nrf2 targeting is therapeutically exploitable in HCC treatment.

VEGFR-3 signaling restrains the neuron-macrophage crosstalk during neurotropic viral infection.

Upon recognizing danger signals produced by virally infected neurons, macrophages in the central nervous system (CNS) secrete multiple inflammatory cytokines to accelerate neuron apoptosis. The understanding is limited about which key effectors regulate macrophage-neuron crosstalk upon infection. We have used neurotropic-virus-infected murine models to identify that vascular endothelial growth factor receptor 3 (VEGFR-3) is upregulated in the CNS macrophages and that virally infected neurons secrete the ligand VEGF-C. When cultured with VEGF-C-containing supernatants from virally infected neurons, VEGFR-3+ macrophages suppress tumor necrosis factor α (TNF-α) secretion to reduce neuron apoptosis. Vegfr-3ΔLBD/ΔLBD (deletion of ligand-binding domain in myeloid cells) mice or mice treated with the VEGFR-3 kinase inhibitor exacerbate the severity of encephalitis, TNF-α production, and neuron apoptosis post Japanese encephalitis virus (JEV) infection. Activating VEGFR-3 or blocking TNF-α can reduce encephalitis and neuronal damage upon JEV infection. Altogether, we show that the inducible VEGF-C/VEGFR-3 module generates protective crosstalk between neurons and macrophages to alleviate CNS viral infection.

The Transcription of ZIP9 Is Associated With the Macrophage Polarization and the Pathogenesis of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common digestive system cancers (DSCs) with a poor prognosis. Zinc-regulated transporter (ZRT)/iron-regulated transporter (IRT) like protein transporters (ZIPs) encode membrane transport proteins, which are responsible for the absorption of zinc and play important roles in the pathogenesis of various human cancers. Tumor-associated macrophages (TAMs) are important participants in the regulation of tumor microenvironment and the development of HCC. Individual role of each ZIP involved in hepatocarcinogenesis remains elusive. In this study, the transcription patterns of ZIPs in the DSCs were screened firstly through GEPIA2 database. Interestingly, the analysis of the DSCs data showed the distinct mRNA levels of ZIPs between DSCs tissues and healthy controls. Notably, the transcription levels of ZIP2, ZIP5, ZIP8, ZIP9 and ZIP14 were decreased significantly in the tissues of human liver cancer compared to paracarcinoma liver tissues. To further confirm the mRNA transcriptional changes of Zips in HCC, N-Nitrosodiethylamine (DEN) combined with carbon tetrachloride (CCl4) inducing mouse model of HCC were established. Consistently, the mRNA levels of Zip2, Zip9, and Zip14 in liver tissues of HCC induced mice were also decreased compared with the healthy controls. In addition, mouse peritoneal elucidated macrophages (PEMs)-derived M1/M2 macrophages in vitro, as well as human patients of HCC-derived TAMs, were used to examine the transcription levels of ZIPs. Our results showed that both Zip2 and Zip9 were up-regulated in M2-polarized macrophages. Zip2 transcript was also up-regulated M1-polarized macrophages, but Zip9 was slightly down-regulated. TAMs generated from human liver cancer tissues also displayed a decrease in ZIP9 transcription compared to paracarcinoma tissues. To further explore the role of Zip9 in M1/M2 polarization, the siRNA knockdown results revealed that Zip9, but not Zip2, could promote M2 macrophage polarization and impair M1 macrophage polarization. Mechanistically, Zip9 enhances phosphorylated STAT6 to promote M2 macrophage polarization but suppresses the phosphorylation of IκBα/ß to inhibit M1 macrophage polarization. Together, our results indicate that ZIP9 may involve in macrophages polarity in HCC development and may be a potent new biomarker for the diagnosis of HCC.

Hypermethylated LTR retrotransposon exhibits enhancer activity.

LTR retrotransposons are repetitive DNA elements comprising ∼10% of the human genome. They are silenced by hypermethylation of cytosines in CpG dinucleotides and are considered parasitic DNA serving no useful function for the host genome. However, hypermethylated LTRs contain enhancer and promoter sequences and can promote tissue-specific transcription of cis-linked genes. To resolve the apparent paradox of hypermethylated LTRs possessing transcriptional activities, we studied the ERV-9 LTR retrotransposon located at the 5' border of the transcriptionally active ß-globin gene locus in human erythroid progenitor and erythroleukemia K562 cells. We found that the ERV-9 LTR, containing 65 CpGs in 1.7 kb DNA, was hypermethylated (with > 90% methylated CpGs). Hypermethylated LTR possessed transcriptional enhancer activity, since in vivo deletion of the LTR by CRISPR-cas9 suppressed transcription of the globin genes by > 50%. ChIP-qPCR and ChIP-seq studies showed that the hypermethylated LTR enhancer spanning recurrent CCAATCG and GATA motifs associated respectively with key transcription factors (TFs) NF-Y and GATA-1 and -2 at reduced levels, compared with the unmethylated LTR in transfected LTR-reporter gene plasmids. Electrophoretic mobility shift assays with methylated LTR enhancer probe showed that the methylated probe bound both NF-Y and GATA-1 and -2 with lower affinities than the unmethylated enhancer probe. Thus, hypermethylation drastically reduced, but did not totally abolish, the binding affinities of the enhancer motifs to the key TFs to assemble the LTR-pol II transcription complex that activated transcription of cis-linked genes at reduced efficiency.

[Pylorus-preserving gastrectomy in treating middle-third early gastric cancer].

Compared with distal gastrectomy, pylorus-preserving gastrectomy is less invasive which can decrease incidence of dumping syndrome, diarrhea and body weight lost, cholecystitis and gallstone, reflux gastritis and esophagitis and remnant gastric cancer. Based on new Japanese Gastric Cancer Treatment Guideline and new progression in the world, we give a review mainly basic characteristics, indications, operation details and short- and long-time outcomes after pylorus-preserving gastrectomy.

NF-Y recruits both transcription activator and repressor to modulate tissue- and developmental stage-specific expression of human γ-globin gene.

The human embryonic, fetal and adult ß-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.

Expression of the co-signaling molecules CD40-CD40L and their growth inhibitory effect on pancreatic cancer in vitro.

We investigated the expression of the co-signalling molecule CD40 in pancreatic cancer and the growth inhibitory effect of the recombinant soluble human CD40 ligand (rshCD40L) in pancreatic cancer cell lines. Twenty-six cases of pancreatic cancer tissues and corresponding paratumoral normal tissues were immunohistochemically analyzed for CD40 expression. The association of CD40 expression with clinicopathological parameters, including clinical stage, pathological grade, invasion and metastasis, were statistically analyzed. The serum sCD40 levels in pancreatic cancer patients were examined by ELISA. The expression of CD40 in the pancreatic cancer cell lines Panc-1, Aspc-1 and Miapaca-2 was examined by RT-PCR and flow cytometry. The growth inhibitory activity of rshCD40L on pancreatic cancer cell lines was determined by MTT assay. Tumor cell apoptosis was detected by TUNEL and Annexin V/PI double staining method. CD40 was positive both on the membrane and in the cytoplasm of tumor cells, 69.2% (18/26) of the cases were positive for CD40. CD40 expression was significantly higher in pancreatic cancer tissues compared to adjacent normal tissues (P<0.05). High CD40 expression was associated with TNM stage and lymph node metastasis (both P<0.05). Patients with pancreatic cancer have higher serum sCD40L levels (3.53 ± 0.70 ng/ml) compared to healthy subjects (1.81 ± 0.48 ng/ml, P<0.05). rshCD40L significantly inhibited the proliferation of the pancreatic cancer cell lines and induced apoptosis in these cell lines. The co-signaling molecule CD40 is highly expressed in pancreatic cancer tissues and cell lines and rshCD40L is a potential tool for antitumor therapies.

Effects of cyclin-dependent kinase 8 specific siRNA on the proliferation and apoptosis of colon cancer cells.

BACKGROUND: To investigate the expression of cyclin-dependent kinase 8 (CDK8) and ß-catenin in colon cancer and evaluate the role of CDK8 in the proliferation, apoptosis and cell cycle progression of colon cancer cells, especially in HCT116 cell line. METHODS: Colon cancer cell line HCT116 was transfected with small interfering RNA (siRNA) targeting on CDK8. After CDK8-siRNA transfection, mRNA and protein expression levels of CDK8 and ß-catenin were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot assay in HCT116 cells. Cell proliferation was measured by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide Methylthiazolyl tetrazolium (MTT) assay, and cell cycle distribution and apoptosis were analyzed by flow cytometry analysis (FACS). CDK8 and ß-catenin protein levels were also examined by real-time PCR and immunohistochemistry (IHC) in colon cancer tissues and adjacent normal tissues. RESULTS: After CDK8 specific siRNA transfection, mRNA and protein expression levels of CDK8 and ß-catenin in HCT116 cells were noticeably decreased (P < 0.05). CDK8 specific siRNA transfection inhibited HCT116 cells' proliferation and facilitated their apoptosis significantly (P < 0.05). In addition, the proportion of HCT116 cells in the G0/G1 phase was remarkably increased after CDK8-siRNA transfection (P < 0.05). The expression levels of CDK8 and ß-catenin in adjacent normal tissues were lower than in tumor tissues (P < 0.05). Moreover, the expression of CDK8 was correlated with the expression of ß-catenin in both tumor and adjacent normal tissues (P < 0.05). CONCLUSIONS: CDK8 and ß-catenin were expressed in colon cancer at a high frequency. CDK8 specific siRNA transfection down-regulated the expression of CDK8 in colon cancer cells, which was also associated with a decrease in the expression of ß-catenin Moreover, CDK8 specific siRNA inhibited the proliferation of colon cancer cells, promoted their apoptosis and arrested these cells in the G0/G1 phase. Interference of CDK8 might be an effective strategy through ß-catenin regulation of colon cancer.

Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth.

BACKGROUND: Pancreatic cancer is a highly malignant tumor affecting an ever increasing number of patients with a mean 5-year survival rate below 4%. Therefore, gene therapy for cancer has become a potential novel therapeutic modality. In this study we sought to determine the inhibitory effects of adenovirus-mediated human interleukin-24 (AdhIL-24) on pancreatic cancer. METHODS: Human interleukin-24 gene was cloned into replication-defective adenovirus specific for patu8988 tumor cells by virus recombination technology. Reverse transcription-polymerase chain reaction and Western blotting analysis were used to determine the expression of human interleukin-24 mRNA in patu8988 cells in vitro. Induction of apoptosis by overexpression of human interleukin-24 in patu8988 cells was determined by flow cytometry. In vivo efficacy of adenoviral delivery of human interleukin-24 was assessed in nude mice (n = 10 for each group) bearing patu8988 pancreatic cancer cell lines by determining inhibition of tumor growth, endothelial growth factor and CD34 expression, and intratumoral microvessel density (MVD). RESULTS: The recombinant adenovirus vector AdVGFP/IL-24 was constructed with a packaged recombinant retrovirus titer of 1.0 x 10(10) pfu/ml and successfully expressed of both mRNA and protein in patu8988 cells. The AdVGFP/IL-24 induced apoptosis of patu8988 tumor cells in vitro and significantly inhibited tumor growth in vivo (P < 0.05). The intratumoral MVD decreased significantly in the treated tumors (P < 0.05). CONCLUSION: The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.

Inhibition of pancreatic carcinoma cell growth in vitro by DPC4 gene transfection.

AIM: To detect the expression of DPC4 in malignant and non-malignant specimens of human pancreas, and observe the inhibition of retroviral pLXSN containing DPC4 on pancreatic carcinoma cells in vitro. METHODS: The expression of DPC4 was determined in 40 pancreatic adenocarcinoma and 36 non-malignant pancreatic specimens by reverse-transcriptase polymerase chain reaction (RT-PCR) and immunohisto-chemistry. Furthermore, we constructed retroviral vectors containing DPC4, which then infected the pancreatic carcinoma cell line BxPC-3. Cell growth in vitro after being infected was observed, and the vascular endothelial growth factor (VEGF) mRNA level in the daughter cells was determined by semi-quantitative PCR assay. RESULTS: The RT-PCR assay showed a positive rate of DPC4 mRNA in 100% (36/36) of normal specimens, compared to 40% (16/40) in adenocarcinoma specimens. The regional and intense positive cases of DPC4 expression in adenocarcinoma detected by immunohistochemistry were 10 and four, whereas it was all positive expression in normal tissues. There was a significant difference of DPC4 expression between them. The stable expression of DPC4 in the pancreatic carcinoma cells BxPC-3 could be resumed by retroviral vector pLXSN transfection, and could inhibit cell growth in vitro. Rather, DPC4 could decrease VEGF mRNA transcription levels. CONCLUSION: The deletion of DPC4 expression in pancreatic carcinoma suggests that loss of DPC4 may be involved in the development of pancreatic carcinoma. The retroviral vector pLXSN containing DPC4 can inhibit the proliferation of pancreatic carcinoma cells, and down-regulate the level of VEGF.

Role of CD80 in stimulating T lymphocyte activation.

AIM: To observe biological characteristics of hepatocarcinoma cells before and after CD80 transfection and to compare the effect of CD80-transfected hepatocarcinoma cells on T lymphocyte activation. METHODS: Retro virus vector carrying CD80 gene was transfected into HepG2 cells to establish CD80-transfected hepatocarcinoma cells (HepG2/hCD80). Flow cytometry (FCM) was performed to detect CD80 expression in the transfected cells. RT-PCR was used to evaluate CD80 expression at mRNA level. In the presence of anti-CD3 mAb, the proliferation of T lymphocyte was observed by MTT. Meanwhile, the expression of activated molecule marker CD25 was analyzed through FCM. RESULTS: A stable cell line HepG2/hCD80 expressing the human CD80 was established. Growth curve showed that the molecule CD80 could obviously decrease the growth of tumor cells. HepG2/hCD80 was evidenced to have a potency to enhance T cell proliferation and upregulate CD25 expression. CONCLUSION: CD80 transfection can lower malignant phenotype of hepatocarcinoma cells. CD80 transfection has a down-regulatory effect to activated T cells in vitro.

The long terminal repeat (LTR) of ERV-9 human endogenous retrovirus binds to NF-Y in the assembly of an active LTR enhancer complex NF-Y/MZF1/GATA-2.

The solitary ERV-9 long terminal repeat (LTR) located upstream of the HS5 site in the human beta-globin locus control region exhibits prominent enhancer activity in embryonic and erythroid cells. The LTR enhancer contains 14 tandemly repeated subunits with recurrent CCAAT, GTGGGGA, and GATA motifs. Here we showed that in erythroid K562 cells these DNA motifs bound the following three transcription factors: ubiquitous NF-Y and hematopoietic MZF1 and GATA-2. These factors and their target DNA motifs exhibited a hierarchy of DNA/protein and protein/protein binding affinities: NF-Y/CCAAT > NF-Y/GATA-2 > NF-Y/MZF1 > MZF1/GTGGGGA; GATA-2/GATA. Through protein/protein interactions, NF-Y bound at the CCAAT motif recruited MZF1 and GATA-2, but not Sp1 and GATA-1, and stabilized their binding to the neighboring GTGGGGA and GATA sites to assemble a novel LTR enhancer complex, NF-Y/MZF1/GATA-2. In the LTR-HS5-epsilonp-GFP plasmid integrated into K562 cells, mutation of the CCAAT motif in the LTR enhancer to abolish NF-Y binding inactivated the enhancer, closed down the chromatin structure of the epsilon-globin promoter, and silenced transcription of the green fluorescent protein gene. The results indicated that NF-Y bound at the CCAAT motifs assembled a robust LTR enhancer complex, which could act over the intervening DNA to remodel the chromatin structure and to stimulate the transcription of the downstream gene locus.