CD4+CCR8+ Tregs in ovarian cancer: a potential effector Tregs for immune regulation.

BACKGROUND: Tregs are key drivers of immunosuppression in solid tumors. As an important chemokine receptor on Tregs, the regulatory effect of CCR8 on tumor immunity has received more and more attention. However, the current research on CCR8 in the immune microenvironment of ovarian cancer has not been clear. METHODS: Bioinformatics analysis was used to compare the transcriptome differences between CD4+ T cells in the peripheral circulation and infiltrated in ovarian tumor tissues. RT-PCR was used to detect the expression levels of chemokine receptor-related differential genes on CD4+ T cells in peripheral blood and ovarian tumor tissues. Multiparameter flow cytometry was used to detect the proportion and phenotypic characteristics of CD4+CCR8+ Tregs and CD4+CCR8- Tregs in different sample types. The expression level of CCR8 ligands was detected at multiple levels. To explore the important role of CCR8-CCL1 and CCR8-CCL18 axis in the migration and invasion of CD4+CCR8+ Tregs into ovarian tumor tissues by establishing a chemotaxis system in vitro. RESULTS: In this study, significantly different gene expression profiles were found between peripheral circulating CD4+ T cells and infiltrating CD4+ T cells in ovarian tumor tissues, in which chemokine-chemokine receptor signaling pathway was significantly enriched in all three groups of differential genes. The expression level of CCR8 in infiltrating CD4+ T cells of ovarian cancer tissue was significantly higher than that in peripheral blood of healthy controls and ovarian cancer patients, and high expression of CCR8 was significantly correlated with advanced tumor stage and poor differentiation. CD4+CCR8+ Tregs are the main type of infiltrating CD4+ Tregs in ovarian tumor tissues, which have stronger immunosuppressive phenotypes, secrete more inhibitory cytokines and have stronger proliferation ability. The ligands CCL1 and CCL18 corresponding to CCR8 were significantly overexpressed in ovarian tumor tissues, and the CCR8-CCL1 and CCR8-CCL18 axis played a key role in the migration and infiltration of CD4+CCR8+ Tregs into ovarian tumor tissues. CONCLUSIONS: The results of this study may help to understand the phenotypic characteristics and recruitment process of Tregs in the tumor, and provide new ideas for improving the immunosuppressive status of the ovarian cancer microenvironment.

Clinical Application of Prostatic Exosomal Protein and Prostate-Specific Antigen Levels in the Detection of Prostate-Related Diseases.

OBJECTIVE: Our aim was to study the use of prostatic exosomal protein (PSEP) and prostate-specific antigen (PSA) in diagnosis of prostate-related diseases. METHODS: A total of 54 cases of acute prostatitis (AP), 72 cases of chronic prostatitis (CP), and 36 cases of prostate cancer (PCa) were enrolled. Levels of PSEP and PSA were analyzed. RESULTS: The positive rate and level of PSEP in CP was highest (both P < .05). The total PSA (tPSA) level in PCa was the highest (P < .05), followed by AP and CP. The free PSA (fPSA) level was lowest in CP (P < .05); fPSA/tPSA in AP was the highest (P < .05). The PSEP level in type II CP was higher than in type IIIa and type IIIb (both P < .05), and it was higher in type IIIa than in type IIIb (P < .05). The tPSA level in type IIIb was the lowest in the 3 types (both P < .05). The fPSA/tPSA in type IIIb was the highest in the 3 types (P < .05). CONCLUSION: The PSEP combined with PSA better distinguishes prostate-related diseases.

Individualized Correction of the Interference of Hemolysis on Glycated Albumin Determined by the Ketamine Oxidase Method.

OBJECTIVE: To investigate the effect of hemolysis on glycated albumin (GA) levels, as determined by the ketamine oxidase method. METHODS: GA levels and the hemolysis index were determined in nonhemolyzed serum and hemolyzed serum from corresponding patients. We developed an equation to correct the interference of hemolysis on GA, using multiple regression analysis. RESULTS: The degree of hemolysis was negatively correlated with GA levels (R2 = 0.9500). A correction equation for GA (corrected GA = 2.703 × OD of hemolysis + 1.044 × measured GA -0.906) can revert GA concentrations of hemolyzed specimens to values that were not significantly different from the GA concentration of corresponding nonhemolyzed specimens. The bias of GA concentrations before and after correction was statistically significantly different (P <.01). CONCLUSIONS: Our results indicate that the level of GA measured through the ketamine oxidase method is negatively affected by hemolysis. The individualized correction of GA results provides increased accuracy in hemolyzed specimens.

Autoimmune pancreatitis masquerading as pancreatic cancer: case report of a Chinese man.

Autoimmune pancreatitis (AIP) is a type of chronic pancreatitis with an autoimmune basis, characterized by infiltrating lymphocytes and plasma cells and fibrosis. Imaging examination revealed pancreatic enlargement and irregular stenosis of the pancreatic duct, and laboratory inspection showed elevated serum IgG4 level. Effectiveness of glucocorticoids (hormone) management is a remarkable feature of this disease. It is reported that both AIP and pancreatic cancer show a marked predilection in older men, and AIP is easily misdiagnosed as pancreatic cancer which leads to unnecessary surgery. Today we present a case of type I AIP in a 64-year-old Chinese old man.

Changes in regulatory T cells in patients with ovarian cancer undergoing surgery: Preliminary results.

Regulatory T cells (Treg) suppress immune responses in patients with cancer. Surgery is the most effective therapeutic strategy for ovarian cancer (OC). However, the interplay between the Treg population and surgical resection remains unclear. 61 patients with OC who received no prior treatment were enrolled in the study. Treg percentages were characterized from peripheral blood mononuclear cells. We investigated CD4+CD25+, CD4+CD25+Foxp3+, CD8+CD28-, and CD8+Foxp3+ Tregs in OC patients and their postoperative changes using flow cytometry. Treg percentages were significantly higher in OC patients than those in benign ovarian tumors (BOT) and healthy controls. Higher percentages of Tregs were found in patients with stage III/IV than stage I/II OC. Treg percentages were significantly decreased postoperatively. The postoperative Treg percentages in patients with stage I/II OC were similar to those in BOT patients, while postoperative Treg percentages in patients with stage III/IV OC remained higher. Tregs were markedly lower on postoperative day (POD) 3 than preoperatively. They increased slightly after 7days, but remained lower than preoperative levels. These data suggested that Tregs continued to decline from POD 7 to POD 30. Treg percentages are correlated with the tumor burden and could be a key factor in monitoring the immunological status of patients with OC.

Gene expression profiling of CD8+ T cells induced by ovarian cancer cells suggests a possible mechanism for CD8+ Treg cell production.

OBJECTIVES: The aim of this study was to investigate a possible mechanism of CD8+ regulatory T-cell (Treg) production in an ovarian cancer (OC) microenvironment. MATERIALS AND METHODS: Agilent microarray was used to detect changes in gene expression between CD8+ T cells cultured with and without the SKOV3 ovarian adenocarcinoma cell line. QRT-PCR was performed to determine glycolysis gene expression in CD8+ T cells from a transwell culturing system and OC patients. We also detected protein levels of glycolysis-related genes using Western blot analysis. RESULTS: Comparing gene expression profiles revealed significant differences in expression levels of 1420 genes, of which 246 were up-regulated and 1174 were down-regulated. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis indicated that biological processes altered in CD8+ Treg are particularly associated with energy metabolism. CD8+ Treg cells induced by co-culture with SKOV3 had lower glycolysis gene expression compared to CD8+ T cells cultured alone. Glycolysis gene expression was also decreased in the CD8+ T cells of OC patients. CONCLUSIONS: These findings provide a comprehensive bioinformatics analysis of DEGs in CD8+ T cells cultured with and without SKOV3 and suggests that metabolic processes may be a possible mechanism for CD8+ Treg induction.

Tumor-associated macrophages promote invasion via Toll-like receptors signaling in patients with ovarian cancer.

Tumor-associated macrophages (TAMs) derived from peripheral blood monocytes recruit into tumor microenvironment and display functions associated with tumor progression. The mechanisms by which TAMs display roles that associated with the invasion ability of ovarian cancer have not been well investigated. In our research, we found abundant TAMs infiltrate in ovarian cancer compared with benign ovarian tumor tissues. Levels of matrix metalloproteinase (MMP)-2, MMP-9 and MMP-10, and Toll-like receptors (TLRs) signaling proteins were evaluated in ovarian cancer. The high level of TAMs was associated with metastasis and advance of patients with ovarian cancer. TAMs and ovarian cancer cell line SKOV3 were cocultured in vitro, MMPs level and the invasion ability of SKOV3 cells were significantly up-regulated. The coculture process was correlated with the activation of TLRs signaling and downstream nuclear factor (NF)-κB p65 and microtubule-associated proteins (MAPs) kinases pathway in SKOV3. In addition, pre-incubation with TLRs signaling inhibitors remarkably suppressed invasion ability of SKOV3. Levels of TLRs signaling pathways proteins were also down-regulated in this blocking process. These findings demonstrated that TAMs promoted up-regulation of MMP-2, MMP-9 and MMP-10 expressions and enhanced ovarian cancer cells invasion via TLRs signaling pathway. We conclude that TAMs could enhance ovarian cancer cells invasion and ultimately promote ovarian cancer progression.

TGF-ß1 contributes to CD8+ Treg induction through p38 MAPK signaling in ovarian cancer microenvironment.

CD8+ regulatory T cells (Tregs) contribute to cancer progression and immune evasion. We previously reported that CD8+ Tregs could be induced in vitro by co-culture of CD8+ T cells with the OC cell lines SKOV3/A2780. Here, we described the role of TGF-ß1 in CD8+ Treg induction by the OC microenvironment. OC patients expressed high levels of TGF-ß1, as did the co-culture supernatant from CD8+ T cells and SKOV3. Additionally, TGF-ß1 levels were positively correlated with CD8+ Treg percentages in OC. Neutralization experiments, cytokine studies and proliferation assays revealed that the in vitro-induced CD8+Tregs depended at least partially on up-regulated expression of TGF-ß1 to exert their suppressive function. CD8+ T cells cultured with SKOV3 exhibited marked activation of p38 MAPK than CD8+ T cells cultured alone, which could be inhibited by TGF-ß1-neutralizing antibody. Moreover, the p38 specific inhibitor SB203580 dose-dependently blocked the TGF-ß1 activated conversion of CD8+ T cells into CD8+ Tregs. These data suggested that in vitro-induction of CD8+ Tregs depended in part on TGF-ß1 activation of p38 MAPK signaling. Therefore, p38 MAPK could be a therapeutic target in OC anti-tumor immunotherapy.

Analysis of CD8+ Treg cells in patients with ovarian cancer: a possible mechanism for immune impairment.

Regulatory T (Treg) cells may participate in mediating a suppressive microenvironment that blunts successful anti-tumor immunotherapy. Recent studies show that CD8(+) Treg cells might impede effective immune responses to established tumors. However, there is limited research regarding CD8(+) Treg cells in ovarian cancer (OC) patients. Here, we investigated CD8(+) Treg cells in OC patients and their in vitro induction. The immunohistochemistry of tumor-infiltrating lymphocytes revealed a significant correlation between the intratumoral CD8(+) T cells and the forkhead box p3 (Foxp3)(+) cells in the intraepithelial and stromal areas of advanced OC tissues. We examined the expression of Treg markers in CD8(+) T cells from the peripheral blood and fresh tumor tissues of OC patients using flow cytometry. Our results indicated an increase in the CD8(+) Treg cell subsets of OC patients compared with those in patients with benign ovarian tumors and healthy controls, including an increased expression of CD25, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and Foxp3 and decreased CD28 expression. To demonstrate whether the tumor microenvironment could convert CD8(+) effector T cells into suppressor cells, we used an in vitro transwell culturing system. Compared with the CD8(+) T cells cultured alone, the CD8(+) Treg cells induced in vitro by coculture with SK-OV-3/A2780 showed increased CTLA-4 and Foxp3 expression and decreased CD28 expression. In addition, the in vitro-induced CD8(+) Treg cells inhibited nai¨ve CD4(+) T-cell proliferation, which was partially mediated through TGF-ß1 and IFN-γ. Our study suggests that CD8(+) Treg cells were increased in OC patients and could be induced in vitro, which may be the way that tumors limit antitumor immunity and evade immune surveillance.

miR-638 is a new biomarker for outcome prediction of non-small cell lung cancer patients receiving chemotherapy.

MicroRNAs (miRNAs), a class of small non-coding RNAs, mediate gene expression by either cleaving target mRNAs or inhibiting their translation. They have key roles in the tumorigenesis of several cancers, including non-small cell lung cancer (NSCLC). The aim of this study was to investigate the clinical significance of miR-638 in the evaluation of NSCLC patient prognosis in response to chemotherapy. First, we detected miR-638 expression levels in vitro in the culture supernatants of the NSCLC cell line SPC-A1 treated with cisplatin, as well as the apoptosis rates of SPC-A1. Second, serum miR-638 expression levels were detected in vivo by using nude mice xenograft models bearing SPC-A1 with and without cisplatin treatment. In the clinic, the serum miR-638 levels of 200 cases of NSCLC patients before and after chemotherapy were determined by quantitative real-time PCR, and the associations of clinicopathological features with miR-638 expression patterns after chemotherapy were analyzed. Our data helped in demonstrating that cisplatin induced apoptosis of the SPC-A1 cells in a dose- and time-dependent manner accompanied by increased miR-638 expression levels in the culture supernatants. In vivo data further revealed that cisplatin induced miR-638 upregulation in the serum derived from mice xenograft models, and in NSCLC patient sera, miR-638 expression patterns after chemotherapy significantly correlated with lymph node metastasis. Moreover, survival analyses revealed that patients who had increased miR-638 levels after chemotherapy showed significantly longer survival time than those who had decreased miR-638 levels. Our findings suggest that serum miR-638 levels are associated with the survival of NSCLC patients and may be considered a potential independent predictor for NSCLC prognosis.

Activation of Toll-like receptors signaling in non-small cell lung cancer cell line induced by tumor-associated macrophages.

BACKGROUND: Inflammation is often linked with the progress and poor outcome of lung cancer. The understanding of the relationship between tumor-associated macrophages (TAMs) and lung cancer cells involves in the underlying mechanism of inflammatory cytokine production. Toll-like receptors (TLRs) are engaged in promoting the production of pro-inflammatory cytokines and play an important role in tumor immunology. METHODS: To investigate the mechanisms by which TAMs influence the production of pro-inflammatory cytokines in lung cancer cells, we established an in vitro coculture system using TAMs and human non-small cell lung cancer (NSCLC) cell line SPC-A1. Levels of interleukin (IL)-1ß, IL-6 and IL-8 in SPC-A1 were evaluated by RT-PCR and cytometric bead array assay after being cocultured with TAMs. Expression changes of TLRs and TLRs signaling pathway proteins in SPC-A1 were further confirmed by RT-PCR and western blot. The level changes of IL-1ß, IL-6 and IL-8 in SPC-A1 were also detected after the stimulation of TLRs agonists. RESULTS: We found that the phenotype markers of TAMs were highly expressed after stimulating human monocyte cell line THP-1 by phorbol-12-myristate-13-acetate (PMA). Higher mRNA and supernate secretion levels of IL-1ß, IL-6 and IL-8 were detected in SPC-A1 after being cocultured with TAMs. We also found that TLR1, TLR6 and TLR7 were up-regulated in SPC-A1 in the coculture system with TAMs. Meanwhile, TLRs signaling pathway proteins were also significantly activated. Moreover, pre-treatment with agonist ligands for TLR1, TLR6 and TLR7 could dramatically promote inductions of IL-1ß, IL-6 and IL-8. CONCLUSIONS: These findings demonstrated that TAMs may enhance IL-1ß, IL-6 and IL-8 expressions via TLRs signaling pathway. We conclude that TAMs contribute to maintain the inflammation microenvironment and ultimately promote the development and progression of lung cancer.

Expression and function of Toll-like receptors in peripheral blood mononuclear cells from patients with ovarian cancer.

Inflammation has been implicated in the initiation and progression of ovarian cancer (OC), the underlying mechanisms of which are still unclear. We hypothesized that the abnormal expression of Toll-like receptors (TLRs), which were potential activators of nuclear factor-kappa B p65 (NF-κB p65), could promote inflammation and tumorigenesis in OC. In this study, we characterized the expression of TLRs in peripheral blood mononuclear cells (PBMCs) and found TLR2 and TLR6 mRNAs levels to be higher in PBMCs from OC patients than in those from benign disease (BC) or healthy normal controls (NC). Flow cytometry analysis showed that TLR1, TLR2 and TLR6 were highly expressed in monocytes from OC patients, but not in those from control subjects. Consistently, inflammatory cytokines interleukin (IL)-1ß and IL-6 were up-regulated in PBMCs from OC patients upon stimulation with Pam3CSK4 (TLR1 ligand) and HKLM (TLR2 ligand), compared with unstimulated PBMCs. Stimulation of PBMCs with TLR ligands led to activation of downstream signaling molecules in TLRs (MyD88, TRAF6, TANK, NF-κB p65 and p-NF-κB p65). We also discovered that SK-OV-3-secreted factors were potent PBMCs activators, leading to the production of IL-1ß, IL-6 and IL-8 through activation of TLRs and downstream signaling molecules in PBMCs. Before coculturing with SK-OV-3, pretreatment of THP-1 cells or PBMCs with monoclonal antibodies against TLR1, TLR2 or TLR6 inhibited the production of IL-1ß and IL-6 and activation of MyD88, TRAF6, TANK, NF-κB p65 and p-NF-κB p65. Our results provided new evidence that TLR1, TLR2 and TLR6 signaling was linked with inflammation in OC microenvironment.

Singly protonated dehydronorcantharidin silver coordination polymer induces apoptosis of lung cancer cells via reactive oxygen species-mediated mitochondrial pathway.

Silver complexes have been shown to possess antimicrobial and anticancer properties. Ag-SP-DNC, a novel silver and singly protonated dehydronorcantharidin complex, was synthesized in our previous study. In this study, we offer evidence that Ag-SP-DNC elicits a reactive oxygen species (ROS)-mediated mitochondrial apoptosis in lung cancer cells. Ag-SP-DNC inhibited the growth of A549 cells by inducing G2/M phase cell cycle arrest and apoptosis. Ag-SP-DNC induced apoptosis was associated with the levels of intracellular ROS. The further study revealed that Ag-SP-DNC disrupted the mitochondrial membrane potential, induced the caspase-3 activation and led to the translocation of apoptosis inducing factor and endonucleaseG to the nucleus. These findings have important implications for the development of silver complexes for anticancer applications.