CD44 Protein Expression Research Articles

The Novel HDAC Inhibitor Chidamide Synergizes with Rituximab to Inhibit DLBCL Tumor Growth in Vitro and In Vivo By up-Regulating CD20 Expression

Background: Histone deacetylases (HDACs) are crucial proteins for supporting tumorigenesis. HDACs reverse chromatin acetylation and alter transcription of oncogenes and tumor suppressor genes by removing acetyl groups from histones. HDAC inhibitors are considered as promising anti-cancer drugs, particularly in combination with other standard treatment regimens. Chidamide is the world first oral HDAC inhibitor which selectively inhibits class I HDAC1, HDAC2, and HDAC3 as well as class IIb HDAC10. Chidamide has been approved by China FDA in 2015 for the treatment of relapsed or refractory peripheral T-cell lymphoma.Diffuse large B-cell lymphoma (DLBCL) is the most aggressive form of B-cell lymphoma. Treatment with R-CHOP i.e. Rituximab (the anti-CD20 monoclonal antibody) plus CHOP (Cyclophosphamide, doxorubicin, vincristine, and prednisone) has significantly improved clinical outcome for DLBCL patients. However, treatment-induced deacetylation of CD20 gene and consequently down-regulation of CD20 protein expression causes an acquired resistance to further treatment with R-CHOP.We hypothesize that inhibition of HDACs by Chidamide could overcome Rituximab-mediated down-regulation of CD20 and facilitate Rituximab-induced DLBCL tumor growth inhibition. The aim of this study is to determine the synergistic effect of Chidamide and Rituximab in the treatment of DLBCL in vitro and in vivo.Methods: The levels of CD20 (MS4A1) mRNA expression and clinical outcomes in patients with DLBCL treated either with R-CHOP or CHOP were obtained from the Gene Expression Omnibus (GEO) repository (NCBI GSE 10846). The association of CD20 expression with overall survival (OS) was analyzed by Cox regression analysis and the cut-off point was calculated by the X-tile software. CD20 protein surface expression and Rituximab-induced cell death were analyzed by flow cytometry. The IC50s of Chidamide and the synergisms with Rituximab (10 µg/ml) on five DLBCB cell lines (OCI-LY3, OCI-LY7, Su-DHL6, Su-DHL8, and Su-DLH10) were determined by MTT test after cells were treated with a range of concentrations of Chidamide with or without Rituximab for 24 hours. The synergism was calculated using ComboSyn software to obtain the combination index (CI). For in vivo experiments, the human DLBCL cell line OCI-LY7 were injected to 6 weeks BALB/C nude mice to develop xenograft DLBCL mice models. After tumors were palpable, mice were divided into four groups and injected with NaCl (control), Rituximab, Chidamide and Rituximab plus Chidamide daily for three weeks. The tumor volumes were monitored frequently during the treatment.Results: In R-CHOP treated cohort (n=233), higher expression of CD20 expression (n=137) is significantly associated with superior clinical outcomes compared with lower CD20 expression (n=96) with P=0.0038, HR=0.4753, 95% CI=0.274-0.779. However, the levels of CD20 have no effect on clinical outcome in DLBCL patients treated with CHOP (n=183). The levels of CD20 protein surface expression on five DLBCL cell lines were significantly and positively correlated with the sensitivities of cells to Rituximab-induced cell death (P=0.0018, R=0.88). HDAC1, HDCA2 and HDCA3 proteins were detected in these DLBCL cell lines. Treatment with Rituximab significantly reduced CD20 surface expression but treatment with Chidamide significantly increased CD20 surface expression in DLBCL cells. The CI numbers for combined treatment with Chidamide and Rituximab were either <0.01 (very strong synergism) or <0.3 (strong synergism), indicating that Chidamide significantly synergized Rituximab-induced cell death. For in vivo assay, treatment with either Rituximab or Chidamide alone slightly but not significantly reduced tumor volume. Combination with Chidamide and Rituximab significantly inhibited tumor growth in DLBCL xenograft mice (P<0.0001). Mice with combined treatment showed significantly prolonged survival compared with other groups.Conclusions: our data demonstrate for the first time that inhibition of HDACs by Chidamide significantly synergized Rituximab-induced tumor growth inhibition in vitro and in vivo. We propose that CD20 surface expression should be used clinically to evaluate treatment response in patients with DLBCL. Chidamide is a promising sensitizer for the treatment of DLBCL with R-CHOP. DisclosuresNo relevant conflicts of interest to declare.

The low protein diet affects the nonspecific inflammatory response of middle-aged and old mice through mTOR.

To explore the effect of low protein diet on nonspecific inflammatory changes in mice during aging and related mechanisms. Thirty-two 14-month-old female KM mice were randomly divided into 4 groups: control group, low protein group, high protein group, high protein + rapamycin group. Hematoxylin-eosin (HE) staining was performed to observe the pathological changes of the liver. Immunohistochemistry of liver sections was performed to detect the expression of CD68 protein. HE staining of colon sections was performed to observe intestinal lymphocyte infiltration. The percentage of spleen CD4+ T and CD8+ T cells was detected by flow cytometry. The mTOR expression in the liver was detected by Western blot and immunohistochemistry. Compared with the control group, HE staining of liver tissue sections in high-protein group showed the cytoplasm of hepatocytes was loose and disordered, and the hepatic sinus was significantly expanded. Immunohistochemistry of the liver showed a significant increase in CD68 protein expression. Colorectal HE staining showed extensive lymphocyte infiltration. The number of CD4+ T and CD8+ T cells in spleen flow cytometry was significantly decreased (*p < 0.05). Western blot and immunohistochemistry detected a significant increase in mTOR expression in the liver (*p < 0.05, *p < 0.05). In the High Protein+Rapamycin group and Low-protein group, the time-dependent changes were reduced, the numbers of CD4+ T cell and CD8+ T cell in the spleen were significantly increased (*p < 0.05) and the expression of mTOR was significantly reduced (*p < 0.05). Low-protein diet is beneficial for delaying the non-specific inflammatory changes of liver and intestines in middle-aged and aged mice, and this effect may be achieved through down-regulation of mTOR.

Albumin induces CD44 expression in glomerular parietal epithelial cells by activating extracellular signal-regulated kinase 1/2 pathway.

De novo expression of CD44 in glomerular parietal epithelial cells (PECs) leads to a prosclerotic and migratory PEC phenotype in glomerulosclerosis. However, the regulatory mechanisms underlying CD44 expression by activated PECs remain largely unknown. This study was performed to examine the mediators responsible for CD44 induction in glomerular PECs in association with diabetes. CD44 expression and localization were evaluated in the glomeruli of Zucker diabetic rat kidneys and primary cultured PECs upon albumin stimulation. Real-time polymerase chain reaction confirmed an albuminuria-associated upregulation of the CD44 gene in the glomeruli of diabetic rats. Immunostaining analysis of diabetic kidneys further revealed an increase in CD44 in hypertrophic PECs, which often contain albumin-positive vesicles. Losartan treatment significantly attenuated albuminuria and lowered CD44 protein levels in the diabetic kidneys. In primary cultured rat PECs, rat serum albumin (0.25-1 mg/ml) caused a dose-dependent upregulation of CD44, claudin-1, and megalin protein expression, which was accompanied by an activation of extracellular signal-regulated kinase1/2 (ERK1/2) signaling. Albumin-induced CD44 and claudin-1 expression were greatly suppressed in the presence of the ERK1/2 inhibitor, U0126. In addition, knockdown of megalin by small interfering RNA interference in PECs resulted in a significant reduction of albumin-induced CD44 and claudin-1 proteins. Taken together, our results demonstrate that albumin induces CD44 expression by PECs via the activation of the ERK signaling pathway, which is partially mediated by endocytic receptor megalin.

Morphology and morphometry of preantral follicles, and immunolocalization of angiogenic factors in ovarian tissue from the neotropical primate Sapajus apella.

SummaryOvarian biopsies from five health adult monkeys were collected by exploratory laparotomy. Preantral follicles (primordial, primary, and secondary) were classified as normal or degenerated and submitted to morphometric analysis in which granulosa cell counts and the areas of follicles, oocytes, and oocyte nuclei were measured. Ovarian fragments were also immunolabelled for the quantitative analysis of VEGFA and CD31 protein expression in the ovarian tissue and in the preantral follicles. In total, 213 preantral follicles was examined for morphometry and morphological classification. From this total, 20 (9.4%) were follicles enclosing two or more oocytes, i.e. multi-oocyte follicles (MOFs). From the 193 follicles enclosing only one oocyte, 46.3% were classified as primordial, 24,1% as transition, 23.3% as primary, and 6.3% as secondary follicles. The mean number of granulosa cells surrounding primordial, transition, primary, and secondary follicles was 9.2, 12.1, 18.7, and 45.3, respectively. Increase in oocyte diameter was observed from primary to secondary follicles, while the oocyte nucleus increased only when follicles reached the secondary stage. The expression of CD31 was strong in vessels, corpus luteum, and in normal oocytes and granulosa cells from preantral follicles at all developmental stages. Likewise, VEGFA expression was observed in vessels and preantral follicles (granulosa cells, the oocyte and the oocyte nucleus). We characterized the morphology, and morphometry and expression of angiogenic factors in normal and atretic preantral follicles from Sapajus apella. This description can support the analysis of follicular quality and survival after procedures such as transplantation and cryopreservation.

The relationship between HER2 overexpression and angiogenesis in gastric cancer.

In gastric cancer, HER2 protein overexpression is considered to be conducive to the higher proliferation activity of the tumor cells. Tumor formation is associated with angiogenesis in order to secure an abundant supply of oxygen and glucose to cancer cells. The aim of the study was to assess if HER2 overexpression is related to higher microvessel density (MVD) in the tumor stroma.The archival samples of primary tumor from 144 consecutive patients that underwent gastric resection for cancer between August 1, 2006 and December 31, 2013 in the Department of Oncological Surgery of Medical University of Gdańsk were analyzed. CD34 was used as a marker of MVD in the tumor stroma. Both CD34 and HER2 protein expressions were tested by immunohistochemistry.The assays were unsuccessful to estimate HER2 in 10 cases and CD34 in 14 cases due to technical reasons. The results were obtained for 128 patients. HER2 0 and HER2 1+ were considered negative, while HER2+ and HER2 3+ were recognized as positive. Mean MVD (mean number of vessels in the visual field) was 32.4 (median 29.5). Microvessel density was insignificantly higher in HER2 positive tumors. The slight difference was also seen between IHC 2+ and 3+ groups. The differences did not reach the level of statistical significance.Statistical analysis performed in our study did not reveal the significant relationship between HER2 overexpression on the tumor cells and MVD in the tumor stroma.

1866P - Association of the rs1883832 variant of CD40 with NSCLC risk and overall survival

Background: During the last few years a growing number of studies has attempted to shed light on the role of CD40 (Tumor Necrosis Factor Receptor Superfamily Member 5) in non-small-cell lung cancer (NSCLC), which remains the leading cause of cancer-related deaths worldwide. The aim of the current study was to investigate the clinical relevance of CD40 functional single nucleotide polymorphism (SNP) rs1883832 (-1C/T) with susceptibility to NSCLC, the clinicopathological parameters, relapse and survival rates of NSCLC patients, as well as with the protein expression of CD40. Methods: CD40 SNP rs1883832 was genotyped in 268 randomly selected NSCLC patients and 279 age- and gender-matched healthy donors. Patients were under observation during a five-year period. Immunohistochemical analysis for CD40 was performed on 106 NSCLC tumor tissue samples. All the participants were Greeks with Caucasian origin. Results: Genotype frequencies of rs1883832 (CC, CT, and TT) were significantly different between healthy controls and patients. CC homozygotes had higher risk for NSCLC compared to T allele carriers in univariate (P < 0.001), as well as in multivariate analysis (P = 0.006). In addition, rs1883832 was related to overall survival. More specifically, CT heterozygotes had worse clinical outcome after two-, three- and five-year observation compared to TT and CC homozygotes (P = 0.015, P = 0.005 and P = 0.017, respectively). Stratifying according to histological subtype, this association was observed only in patients with adenocarcinomas (P = 0.028) and not in patients with squamous- and large-cell carcinomas. Furthermore, taking into consideration disease stage, worse survival for CT heterozygotes was observed in stage II patients and not in patients of other stages (P = 0.016). Moreover, the variant had also strong association with brain metastases, with T allele carriers developing more often metastatic disease in CNS (P = 0.018). Interestingly, rs1883832 was related to CD40 protein expression in malignant cells (P < 0.001) as well as in stromal cells (P = 0.004). Conclusions: The present findings suggest that investigated SNP rs1883832 may be a useful and independent biomarker in NSCLC. However, more studies are needed in order to further demonstrate their role in NSCLC. Legal entity responsible for the study: University of Patras. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

CD36 Promotes Podocyte Apoptosis by Activating the Pyrin Domain-Containing-3 (NLRP3) Inflammasome in Primary Nephrotic Syndrome.

BackgroundCD36 plays a critical role in many sterile inflammatory diseases, including type 2 diabetes mellitus, atherosclerosis, and primary nephrotic syndrome. This study investigated whether CD36 activates the nucleotide-binding domain leucine-rich repeat-containing family, pyrin domain-containing-3 (NLRP3) inflammasome and promotes podocytes apoptosis in primary nephrotic syndrome.Material/MethodsThe mouse podocyte cell line MPC5 was used as a model. mRNA and protein expression of CD36 and NLRP3 was quantified by real-time PCR and Western blotting, respectively. Levels of caspase-1 activity and total cholesterol were determined using commercial kits. Intracellular lipid droplets were detected by Oil Red O staining. CD36 expression was also examined in nephrotic mouse kidney tissue by immunohistochemistry and immunofluorescence. Intracellular lipid droplet was examined by Oil Red O staining.ResultsCD36 expression was increased in nephrotic mouse kidney tissue. Treatment with interleukin-1β increased expression of CD36 and total cholesterol in MPC5 cells. Moreover, this treatment increased expression of NLRP3 and the percentage of apoptotic cells, both of which were inhibited by co-treatment with an anti-CD36 antibody.ConclusionsCD36 might play an important role in podocyte apoptosis by activating the NLRP3 inflammasome in primary nephrotic syndrome.

Expression of stem cell markers CD44 and Lgr5 in colorectal cancer and its relationship with lymph node and liver metastasis

Objective: To investigate the relationship between the expression of stem cell markers CD44 and Lgr5 and the clinicopathological features, lymph node and liver metastasis, and to analyse the value of both in evaluating the prognosis of colorectal cancer. Methods: A total of 90 cases of colorectal cancer, 20 cases of adjacent tissues, 30 cases of lymph node metastases, 28 cases of cancer nodules and 30 cases of liver metastasis of colorectal cancer were collected from Department of Pathology of the Second Hospital of Tianjin Medical University from January 2011 to June 2016. The expression of CD44 and Lgr5 protein was detected by immunohistochemistry, and the relationship between them and clinical pathological data, lymph node metastasis and liver metastasis were analyzed. Results: The positive expression rates of CD44 and Lgr5 in colorectal cancer tissues were 68.9% and 62.2%, which were significantly higher than 30.0% and 15.0% of adjacent tissues(P=0.001 and P<0.001). The expression of CD44 and Lgr5 in colorectal cancer was associated with histological differentiation, depth of tumor invasion, lymph node metastasis, soft tissue nodules, hepatic metastasis, and TNM staging(P=0.021, 0.032, 0.030, 0.011, 0.015, 0.007 and P=0.011, 0.027, 0.017, 0.008, 0.011, 0.021). The positive rates of CD44 and Lgr5 expression in lymph node metastases, cancer nodules and liver metastases (93.3%, 89.3%, 90.0%, and 83.3%, 89.3%, 86.7%) were higher than those in the primary lesions(68.9% and 62.2%)(P=0.007, 0.032, 0.022 and P=0.033, 0.007, 0.013). There is a positive correlation between the expression of CD44 and Lgr5 in colorectal cancer(r=0.615, P<0.001), and both positive expression of CD44 and Lgr5 is associated with histological differentiation, lymph node metastasis and cancer nodules, liver metastasis and TNM staging(P=0.005, 0.003, 0.003, 0.026, 0.014). The Kaplan-Meier survival curve showed that the survival rate of positive expression of CD44 and both positive expression of CD44 and Lgr5 in patients with colorectal cancer was low(P=0.030 and 0.001). Log-rank univariate survival analysis showed that histological differentiation, depth of invasion, lymph node metastasis, liver metastasis, and clinical stage were the influencing factors of postoperative survival time(P=0.035, 0.035, 0.018, 0.016, 0.004). Cox proportional hazards regression model multivariate analysis showed that both positive expression of CD44 and Lgr5, lymph node metastasis, liver metastasis, and clinical stage were independent factors affecting the prognosis of patients with colorectal cancer(P=0.004, 0.044, 0.031, 0.008). Conclusions: The expression of CD44 and Lgr5 is related to the malignant biological behavior of colorectal cancer, and they are important factors in promoting local and distant metastases. Both positive expression of them have a hint of bad prognosis.

CD38 is methylated in prostate cancer and regulates extracellular NAD+

BackgroundCancer cell metabolism requires sustained pools of intracellular nicotinamide adenine dinucleotide (NAD+) which is maintained by a balance of NAD+ hydrolase activity and NAD+ salvage activity. We recently reported that human prostate cancer can be initiated following oncogene expression in progenitor-like luminal cells marked by low expression of the NAD+-consuming enzyme CD38. CD38 expression is reduced in prostate cancer compared to benign prostate, suggesting that tumor cells may reduce CD38 expression in order to enhance pools of NAD+. However, little is known about how CD38 expression is repressed in advanced prostate cancer and whether CD38 plays a role in regulating NAD+ levels in prostate epithelial cells.MethodsCD38 expression, its association with recurrence after prostatectomy for clinically localized prostate cancer, and DNA methylation of the CD38 promoter were evaluated in human prostate tissues representing various stages of disease progression. CD38 was inducibly over-expressed in benign and malignant human prostate cell lines in order to determine the effects on cell proliferation and levels of NAD+ and NADH. NAD+ and NADH were also measured in urogenital tissues from wild-type and CD38 knockout mice.ResultsCD38 mRNA expression was reduced in metastatic castration-resistant prostate cancer compared to localized prostate cancer. In a large cohort of men undergoing radical prostatectomy, CD38 protein expression was inversely correlated with recurrence. We identified methylation of the CD38 promoter in primary and metastatic prostate cancer. Over-expression of wild-type CD38, but not an NAD+ hydrolase-deficient mutant, depleted extracellular NAD+ levels in benign and malignant prostate cell lines. However, expression of CD38 did not significantly alter intracellular NAD+ levels in human prostate cell lines grown in vitro and in urogenital tissues isolated from wild-type and CD38 knockout mice.ConclusionsCD38 protein expression in prostate cancer is associated with risk of recurrence. Methylation results suggest that CD38 is epigenetically regulated in localized and metastatic prostate cancer tissues. Our study provides support for CD38 as a regulator of extracellular, but not intracellular, NAD+ in epithelial cells. These findings suggest that repression of CD38 by methylation may serve to increase the availability of extracellular NAD+ in prostate cancer tissues.

Alterations of Hyaluronan Metabolism in Acute Coronary Syndrome: Implications for Plaque Erosion

BackgroundSuperficial erosion currently causes at least one-third of acute coronary syndromes (ACS), and its incidence is increasing. Yet, the underlying mechanisms in humans are still largely unknown. ObjectivesThe authors sought to assess the role of hyaluronan (HA) metabolism in ACS. MethodsPeripheral blood mononuclear cells were collected from ACS (n = 66), stable angina (SA) (n = 55), and control (CTRL) patients (n = 45). The authors evaluated: 1) gene expression of hyaluronidase 2 (HYAL2) (enzyme degrading high-molecular-weight HA to its proinflammatory 20-kDa isoform) and of CD44v1, CD44v4, and CD44v6 splicing variants of HA receptor; and 2) HYAL2 and CD44 protein expression. Moreover, they compared HYAL2 and CD44 gene expression in ACS patients with plaque erosion (intact fibrous cap and thrombus) and in ACS patients with plaque rupture, identified by optical coherence tomography analysis. ResultsGene expression of HYAL2, CD44v1, and CD44v6 were significantly higher in ACS as compared with SA (p = 0.003, p < 0.001, and p = 0.033, respectively) and CTRL subjects (p < 0.001, p < 0.001, and p = 0.009, respectively). HYAL2 protein expression was significantly higher in ACS than in SA (p = 0.017) and CTRL (p = 0.032), whereas no differences were found in CD44 protein expression. HYAL2 and CD44v6 gene expression was significantly higher in patients with plaque erosion than in those with plaque rupture (p = 0.015 and p = 0.029, respectively). ConclusionsHYAL2 and CD44v6 splicing variants seem to play an important role in ACS, in particular when associated with plaque erosion. After further validation, HYAL2 might represent a potentially useful biomarker for the noninvasive identification of this mechanism of coronary instability.

Hyaluronic acid, CD44 and RHAMM regulate myoblast behavior during embryogenesis

Hyaluronic acid (HA) is an extracellular matrix (ECM) component that has been shown to play a significant role in regulating muscle cell behavior during repair and regeneration. For instance, ECM remodeling after muscle injury involves an upregulation in HA expression that is coupled with skeletal muscle precursor cell recruitment. However, little is known about the role of HA during skeletal muscle development. To gain insight into the way in which HA mediates embryonic myogenesis, we first determined the spatial distribution and gene expression of CD44, RHAMM and other HA related proteins in embryonic day (E)10.5 to E12.5 murine forelimbs. While HA and CD44 expression remained high, RHAMM decreased at both the protein (via immunohistochemistry) and RNA (via qPCR) levels. Next, we determined that 4-methylumbelliferone-mediated knockdown of HA synthesis inhibited the migration and proliferation of E11.5/E12.5 forelimb-derived cells. Then, the influence of CD44 and RHAMM on myoblast and connective tissue cell behavior was investigated using antibodies against these receptors. Anti-RHAMM, but not anti-CD44, significantly decreased the total distance myogenic progenitors migrated over 24 h, whereas both inhibited connective tissue cell migration. In contrast, anti-CD44 inhibited the proliferation of connective tissue cells and muscle progenitors, but anti-RHAMM had no effect. However, when myoblasts and connective tissue cells were depleted of CD44 and RHAMM by shRNA, motility and proliferation were significantly inhibited in both cells indicating that blocking cell surface-localized CD44 and RHAMM does not have as pronounced effect as global shRNA-mediated depletion of these receptors. These results show, for the first time, the distribution and activity of RHAMM in the context of skeletal muscle. Furthermore, our data indicate that HA, through interactions with CD44 and RHAMM, promotes myogenic progenitor migration and proliferation. Confirmation of the role of HA and its receptors in directing myogenesis will be useful for the design of regenerative therapies that aim to promote the restoration of damaged or diseased muscle.

Inhibition of the CD36 receptor reduces visceral fat accumulation and improves insulin resistance in obese mice carrying the BDNF-Val66Met variant

Obesity-induced metabolic dysfunctions increase the risk for vascular diseases, including type II diabetes and stroke. Managing obesity is of interest to address the worldwide health problem; however, the role of genetic variability in human obesity development and specific targets for obesity-related metabolic disease have not been thoroughly studied. A SNP in the brain-derived neurotropic factor (BDNF) gene that results in the substitution of a valine with a methionine at codon 66 (Val66Met) occurs with a high frequency in humans. This study addressed the effect of genetic variability in developing obesity and the efficacy of the inhibition of cluster of differentiation 36 (CD36), a multifunctional receptor implicated in obesity and insulin resistance, in WT mice and mice with the BDNF Val66Met variant. CD36 inhibition by salvionolic acid B (SAB) in diet-induced obese WT mice reduced visceral fat accumulation and improved insulin resistance. The benefit of SAB was abrogated in CD36 knockout mice, showing the specificity of SAB. In addition, mice with the Val66Met variant in both alleles (BDNFM/M) fed a high-fat diet exhibited extreme obesity with increased CD36 gene and protein levels in macrophages. Chronic SAB treatment in BDNFM/M mice significantly decreased visceral fat accumulation and improved insulin resistance. Notably, the effect of SAB was greater in the extremely obese BDNFM/M mice compared with the WT mice. The study demonstrated a link between BDNF Val66Met and elevated CD36 expression and suggested that CD36 inhibition may be a potential strategy to improve metabolic dysfunctions and to normalize risk factors for vascular diseases in the obese population.

High-Content Screening of a Taiwanese Indigenous Plant Extract Library Identifies Syzygium simile leaf Extract as an Inhibitor of Fatty Acid Uptake.

Non-alcoholic fatty liver disease (NAFLD) has become the most common liver disease in the recent decades in both developed and developing countries, and is predicted to be the major etiology for liver transplantation in the next decade. Thus, pharmacological strategies to treat NAFLD are urgently needed. Natural products are considered an excellent source for drug discovery. By utilizing an image-based high-throughput screening with a library containing 3000 Taiwanese indigenous plant extracts, we discovered that the extract of Syzygium simile leaves (SSLE) has an anti-lipid droplet (LD) accumulation effect in hepatic cell lines. Analyses of the expression profile of genes involved in lipid metabolism revealed that SSLE suppressed the mRNA expression of CD36, fatty acid translocase. In agreement with this observation, we showed that SSLE inhibited CD36 protein expression and fatty acid uptake and has only limited effects on pre-formed LDs. Moreover, SSLE reduced LD accumulation and CD36 expression in enterocyte and macrophage cell lines. In conclusion, our findings suggest that SSLE could serve as a potential source for the discovery of novel therapeutic modalities for NAFLD and that the suppression of CD36 expression and fatty acid uptake could contribute to the lipid-lowering effect of SSLE.

MiR-98 modulates macrophage polarization and suppresses the effects of tumor-associated macrophages on promoting invasion and epithelial\u2013mesenchymal transition of hepatocellular carcinoma

BackgroundTumor-associated macrophages (TAMs) are generally recognized as a promoter of tumor progression. miR-98 has been shown to suppress the proliferation, migration, invasion and epithelial–mesenchymal transition (EMT) of hepatocellular carcinoma (HCC) cells. Here, we aim to investigate the role of miR-98-mediated macrophage polarization in HCC progression.MethodsHuman blood monocytes were isolated from healthy male donors and incubated with culture medium collected from HepG2 cells for 7 days. The phenotype of the macrophages was detected. The protein expression was detected by Western blot. Levels of cytokines secreted in culture medium were measured using the specific enzyme-linked immunosorbent assay kits. To explore the role of miR-98 in HCC-conditioned TAMs, HCC cells HepG2 and SMMC7721 were cultured with conditioned medium from HCC-conditioned TAMs that had been transfected with miR-98 mimic/inhibitor. Cell proliferation, migration and invasion assays were performed.ResultsHCC-conditioned TAMs possessed M2-like phenotype, including increased protein expression of CD163 and TNF-αlow, IL-1βlow, TGF-βhigh and IL-10high phenotype. HCC-conditioned TAMs also promoted proliferation, migration, invasion and EMT of HepG2 and SMMC7721 cells. Furthermore, miR-98 modulated macrophage polarization from M2 to M1 in HCC-conditioned TAMs, as evidenced by the alteration of M1- or M2-related cytokines. Moreover, miR-98 mimic significantly suppressed the HCC-conditioned TAMs-mediated promotion of cell migration, invasion and EMT in HepG2 and SMMC7721 cells compared with negative control, whereas miR-98 inhibitor exerted reversed effects.ConclusionsmiR-98 may play a vital role in regulating macrophage polarization, thereby suppressing the TAMs-mediated promotion of invasion and EMT in HCC.

Abstract CT087: Early tumor immune microenvironment (TME) modulation by the BRAF inhibitor (BRAFi) dabrafenib (D) and/or the MEK inhibitor (MEKi) trametinib (T) in patients (pts) with BRAF V600E/K-mutant melanoma in the COMBAT trial

Abstract Several trials are evaluating combinations of BRAFi/MEKi and immunotherapy (IO) treatment (Tx) in melanoma. This descriptive biomarker study explored tumor cell and TME modification during BRAFi, MEKi or combination Tx in pts with BRAF V600E/K–mutant unresectable or metastatic melanoma and their potential impact on biomarkers implicated in response to IO. 48 pts enrolled and were randomized 1:1:1 to receive D+T, D, or T (n = 16 each). In the D and T arms, pts received D+T after 8 wk of D or T alone. Tumor biopsies were collected at baseline (BL), wk 2, 8, and 10, and at disease progression. Phosphorylated ERK (pERK), PD-L1, CD8, and CD68 protein expression was analyzed by IHC. RNA expression was assessed by NanoString immune profiling of 770 genes covering adaptive, innate, and humoral immune responses. The primary objectives were to evaluate pERK reduction from BL and characterize safety and efficacy in each Tx arm with changes in pERK H-score. At the final data cutoff, 21 pts (44%) completed the study, including 3 deaths and disease progression. Across Tx arms serious adverse events were reported in 19 pts (40%); no new safety signals were observed. In the biomarker population (pts with BL and ≥ 1 on-Tx biopsy; n = 42), the number of evaluable samples decreased with time due to insufficient tumor material (often due to good tumor response). Due to this limitation, the correlation between biomarker changes and clinical response could not be analyzed, but early biomarker changes are reported. pERK H-score, a MAPK pathway activity marker, decreased from BL to wk 2 in 13 of 17 pts (D, 4/5 pts; T, 6/7 pts; D+T, 3/5 pts) and from BL to wk 8 in 5 of 8 pts (D, 2/3 pts; T, 2/3 pts; D+T, 1/2 pts). IHC showed an early increase in CD8+ and CD68+ cells in the center of tumors compared with the periphery in all 3 arms. Innate immune response, chemotaxis, MHC class II antigen presentation, and complement pathway genes were upregulated. RNA expression of PD-L1, IFNγ pathway genes, and immunosuppressive cytokines IL-6 and CCL2 were slightly increased. Intriguingly, both M1 (IRF5) and M2 (CD163) macrophage genes were upregulated, with an increase of protumoral cytokines (eg, VEGF, TNF) and the MDSC-related cytokine CSF-1. These results indicate that D and T, alone or combined, induced early modification of the melanoma TME, with a tendency to recruit cytotoxic CD8+ cells and increase antigen presentation, which may, however, be offset by induction of immunosuppressive events. Early induction of complement pathways is a novel finding that needs further investigation as it can be both cytotoxic and cytoprotective for tumor cells. These findings provide additional rationale for evaluation of IO and/or agents targeting MDSC following or combined with BRAFi/MEKi in BRAF-mutant melanoma. Additional efficacy and safety analyses will be presented. Citation Format: Caroline Robert, Shensi Shen, Delphine Allard, Ana Arance Fernández, Caroline Dutriaux, Egbert de Jong, Matthew Squires, Jean-Jacques Grob. Early tumor immune microenvironment (TME) modulation by the BRAF inhibitor (BRAFi) dabrafenib (D) and/or the MEK inhibitor (MEKi) trametinib (T) in patients (pts) with BRAF V600E/K-mutant melanoma in the COMBAT trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT087.

Effects of Tumor Necrosis Factor α (TNF-α) and Interleukina 10 (IL-10) on Intercellular Cell Adhesion Molecule-1 (ICAM-1) and Cluster of Differentiation 31 (CD31) in Human Coronary Artery Endothelial Cells

BackgroundThe aim of this study was to investigate the effects of TNF-α and IL-10 on the expression of ICAM-1 and CD31 in human coronary artery endothelial cells (HCAEC).Material/MethodsHCAEC was treated with 0, 2.5 μg/l, 5 μg/l, and 10 μg/l of TNF-α for 2 h, 6 h, and 10 h, and with 0 μg/l, 10 μg/l, 100 μg/l, and 200 μg/l of IL-10 for 5 h, 10 h and 15 h, respectively. RNA inference of TNF-αR was performed with siRNA. Real-time PCR, Western blot analysis, and ELSA were used to detect the mRNA level and protein level of ICAM-1 and CD31.ResultTNF-α significantly increased the mRNA and protein expression of ICAM-1 (P<0.05), and 2.5 μg/l TNF-α had the most obvious effect. RNAi of TNF-αR reduced the induction of TNF-α on the mRNA and protein expression of ICAM-1 (P<0.05). TNF-α significantly decreased the CD31 in the supernatant (P<0.05), and 2.5 μg/l TNF-α had the most obvious effect. IL-10 significantly decreased the ICAM-1 protein level. IL-10 decreased the mRNA expression and the protein expression of CD31. The effect on mRNA was not significant (P>0.05), while the effect on the protein expression was significant (P<0.05).ConclusionsTNF-α and IL-10 treatment can affect the expression of ICAM-1 and CD31 in HCAEC.

Lipocalin-Type Prostaglandin D2 Synthase (L-PGDS) Knockout Mice Exhibits Hepatosteatosis Mediated by Enhanced Cd36 Hepatic Expression as a Result of Hyperinsulinemia

Hepatosteatosis is strongly associated with hyperinsulinaemia and obesity. Previously, we demonstrated accelerated atherosclerosis and impaired glucose tolerance in L-PGDS knockout (KO) mice. Interestingly, L-PGDS KO mice also exhibit hyperinsulinemia on high fat diet in a time-dependent manner. In this study, we investigated the role of L-PGDS on liver pathophysiology using L-PGDS KO mice compared to control C57BL/6 mice) (n=6/group). Mice were kept on high fat diet (60% fat) for 14 weeks and all parameters were measured initially and at 4-, 8- and 14-weeks post high fat diet. Data were analyzed by unpaired t-test or one-way ANOVA where appropriate with p&amp;lt;0.05 deemed significant. Fasting insulin, blood glucose and calculated HOMA-IR were determined. KO mice showed significantly higher HOMA-RI 19.3± 4.62 and 19.7±4.74 at 8 and 14 weeks, respectively, compared to the C57BL/6 mice, which were 7.73±1.14 and 10.1±1.87 at 8 and 14 weeks, respectively. Hepatic Cd36 protein expression was significantly increased at 14 weeks in KO mice compared to the control. Liver steatosis directly correlated with elevated serum triglyceride levels, which significantly increased to 81.7±3.24 and 82.8±1.63 mg/dl at 8 and 14 week,s respectively, compared to the initial level of 64.1±5.66 mg/dl Control serum triglyceride levels were 61.1±3.49 and 73.6±3.2 mg/dl at 8 and 14 weeks, respectively. Furthermore, immunohistochemistry staining of lipid showed severely more liver steatosis in KO mice at all-time points when compared to control mice. Interestingly, KO mice always weighed less compared to the control mice at all-time points, which suggests that L-PGDS partially or fully contributes towards the development of hepatosteatosis independent of body weight. We conclude that L-PGDS is an essential component of hepatic metabolic substrate utilization and may play a significant role in diabetes-related steatosis. Disclosure S. Kumar: None. T. Palaia: None. C. Hall: None. J. Lee: None. M. Stevenson: None. L. Ragolia: None.

Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages

BackgroundAs crucial regulators of the immune response against pathogens, macrophages have been extensively shown also to be important players in several diseases, including cancer. Specifically, breast cancer macrophages tightly control the angiogenic switch and progression to malignancy. ID4, a member of the ID (inhibitors of differentiation) family of proteins, is associated with a stem-like phenotype and poor prognosis in basal-like breast cancer. Moreover, ID4 favours angiogenesis by enhancing the expression of pro-angiogenic cytokines interleukin-8, CXCL1 and vascular endothelial growth factor. In the present study, we investigated whether ID4 protein exerts its pro-angiogenic function while also modulating the activity of tumour-associated macrophages in breast cancer.MethodsWe performed IHC analysis of ID4 protein and macrophage marker CD68 in a triple-negative breast cancer series. Next, we used cell migration assays to evaluate the effect of ID4 expression modulation in breast cancer cells on the motility of co-cultured macrophages. The analysis of breast cancer gene expression data repositories allowed us to evaluate the ability of ID4 to predict survival in subsets of tumours showing high or low macrophage infiltration. By culturing macrophages in conditioned media obtained from breast cancer cells in which ID4 expression was modulated by overexpression or depletion, we identified changes in the expression of ID4-dependent angiogenesis-related transcripts and microRNAs (miRNAs, miRs) in macrophages by RT-qPCR.ResultsWe determined that ID4 and macrophage marker CD68 protein expression were significantly associated in a series of triple-negative breast tumours. Interestingly, ID4 messenger RNA (mRNA) levels robustly predicted survival, specifically in the subset of tumours showing high macrophage infiltration. In vitro and in vivo migration assays demonstrated that expression of ID4 in breast cancer cells stimulates macrophage motility. At the molecular level, ID4 protein expression in breast cancer cells controls, through paracrine signalling, the activation of an angiogenic programme in macrophages. This programme includes both the increase of angiogenesis-related mRNAs and the decrease of members of the anti-angiogenic miR-15b/107 group. Intriguingly, these miRNAs control the expression of the cytokine granulin, whose enhanced expression in macrophages confers increased angiogenic potential.ConclusionsThese results uncover a key role for ID4 in dictating the behaviour of tumour-associated macrophages in breast cancer.

A Snake Venom-Secreted Phospholipase A2 Induces Foam Cell Formation Depending on the Activation of Factors Involved in Lipid Homeostasis.

MT-III, a snake venom GIIA sPLA2, which shares structural and functional features with mammalian GIIA sPLA2s, activates macrophage defense functions including lipid droplet (LDs) formation, organelle involved in both lipid metabolism and inflammatory processes. Macrophages (MΦs) loaded with LDs, termed foam cells, characterize early blood vessel fatty-streak lesions during atherosclerosis. However, the factors involved in foam cell formation induced by a GIIA sPLA2 are still unknown. Here, we investigated the participation of lipid homeostasis-related factors in LD formation induced by MT-III in macrophages. We found that MT-III activated PPAR-γ and PPAR-β/δ and increased the protein levels of both transcription factors and CD36 in macrophages. Pharmacological interventions evidenced that PPAR-γ, PPAR-β/δ, and CD36 as well as the endoplasmic reticulum enzymes ACAT and DGAT are essential for LD formation. Moreover, PPAR-β/δ, but not PPAR-γ, is involved in MT-III-induced PLIN2 protein expression, and both PPAR-β/δ and PPAR-γ upregulated CD36 protein expression, which contributes to MT-III-induced COX-2 expression. Furthermore, production of 15-d-PGJ2, an activator of PPARs, induced by MT-III, was dependent on COX-1 being LDs an important platform for generation of this mediator.

Expression of CD98 in colorectal cancer and its relationship with CD147

Objective To investigate the protein exp peopletracellular metalloproteinase inducer (CD147) in human colorectal cancer tissues. Methods EnVision immunohistochemical method was used to detect the expression of CD98 protein in 70 cases of colorectal cancer and their adjacent tissues. Western blotting was used to detect the expression of CD98 and CD147 protein in HCT116 cells after transfection with CD98 small interfering RNA (siRNA) and negative control. Results The positive rate of CD98 in colorectal cancer and the adjacent tissues was 57.1% (40/70) and 40.0% (28/70), which was statistically significant (P=0.042). The expression of CD98 in colorectal cancer was closely correlated with tumor stage (P=0.017) and lymph node metastasis (P=0.017), but not with gender (P=0.125), age (P=0.935), distant metastasis (P=0.464), tumor differentiation (P=0.995). There was a significant positive correlation between the expression of CD98 and CD147 in colorectal cancer (r=0.471, P=0.001). The expression of CD98 and CD147 protein was obviously decreased in HCT116 cells after transfection with CD98 siRNA, compared with negative control. Conclusion The expression of CD98 is closely related to the invasion and metastasis of colorectal cancer. CD98 may regulate the invasion and metastasis of colorectal cancer by up-regulating the expression of CD147. Key words: Colorectal cancer; CD147; CD98; Immunohistochemistry; Cell transfection