Targeting hydrogen sulphide signaling in breast cancer.

INTRODUCTION: Hydrogen sulphide (H2S) has been established as a key member of the gasotransmitters family that recently showed a pivotal role in various pathological conditions including cancer. OBJECTIVES: This study investigated the role of H2S in breast cancer (BC) pathogenesis, on BC immune recognition capacity and the consequence of targeting H2S using non-coding RNAs. METHODS: Eighty BC patients have been recruited for the study. BC cell lines were cultured and transfected using validated oligonucleotide delivery system. Gene and protein expression analysis was performed using qRT-PCR, western blot and flow-cytometry. In-vitro analysis for BC hallmarks was performed using MTT, BrdU, Modified Boyden chamber, migration and colony forming assays. H2S and nitric oxide (NO) levels were measured spectrophotometrically. Primary natural killer cells (NK cells) and T cell isolation and chimeric antigen receptor transduction (CAR T cells) were performed using appropriate kits. NK and T cells cytotoxicity was measured. Finally, computational target prediction analysis and binding confirmation analyses were performed using different software and dual luciferase assay kit, respectively. RESULTS: The H2S synthesizing enzymes, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), exhibited elevated levels in the clinical samples that correlated with tumor proliferation index. Knock-down of CBS and CSE in the HER2+ BC and triple negative BC (TNBC) cells resulted in significant attenuation of BC malignancy. In addition to increased susceptibility of HER2+ BC and TNBC to the cytotoxic activity of HER2 targeting CAR T cells and NK cells, respectively. Transcriptomic and phosphoprotein analysis revealed that H2S signaling is mediated through Akt in MCF7, STAT3 in MDA-MB-231 and miR-155/ NOS2/NO signaling in both cell lines. Lastly, miR-4317 was found to function as an upstream regulator of CBS and CSE synergistically abrogates the malignancy of BC cells. CONCLUSION: These findings demonstrate the potential role of H2S signaling in BC pathogenesis and the potential of its targeting for disease mitigation.

Staphylococcus aureus induces cell-surface expression of immune stimulatory NKG2D ligands on human monocytes.

Staphylococcus aureus is among the leading causes of bacterial infections worldwide. The pathogenicity and establishment of S. aureus infections are tightly linked to its ability to modulate host immunity. Persistent infections are often associated with mutant staphylococcal strains that have decreased susceptibility to antibiotics; however, little is known about how these mutations influence bacterial interaction with the host immune system. Here, we discovered that clinical S. aureus isolates activate human monocytes, leading to cell-surface expression of immune stimulatory natural killer group 2D (NKG2D) ligands on the monocytes. We found that expression of the NKG2D ligand ULBP2 (UL16-binding protein 2) is associated with bacterial degradability and phagolysosomal activity. Moreover, S. aureus-induced ULBP2 expression was linked to altered host cell metabolism, including increased cytoplasmic (iso)citrate levels, reduced glycolytic flux, and functional mitochondrial activity. Interestingly, we found that the ability of S. aureus to induce ULBP2 and proinflammatory cytokines in human monocytes depends on a functional ClpP protease in S. aureus These findings indicate that S. aureus activates ULBP2 in human monocytes through immunometabolic mechanisms and reveal that clpP inactivation may function as a potential immune evasion mechanism. Our results provide critical insight into the interplay between the host immune system and S. aureus that has evolved under the dual selective pressure of host immune responses and antibiotic treatment. Our discovery of an immune stimulatory pathway consisting of human monocyte-based defense against S. aureus suggests that targeting the NKG2D pathway holds potential for managing persistent staphylococcal infections.

Values of early diagnosis and prognosis evaluation of peripheral blood UL16 binding protein 2 expression in patient with colorectal cancer / 中国医师进修杂志

Objective@#To detect the level of peripheral blood UL16 binding protein 2 (ULBP2) in patients with colorectal cancer, and study its value on early diagnosis and prognosis evaluation.@*Methods@#Eighty patients with colorectal cancer (colorectal cancer group) and 60 healthy subjects (healthy control group) from May 2016 to May 2019 in Affiliated Dongfeng Hospital, Hubei University of Medicine were selected. Serum expression level of ULBP2 was detected by enzyme-linked immunosorbent assay. The diagnostic efficacy of serum ULBP2 in colorectal cancer was evaluated by receiver operating characteristic (ROC) curve. The influencing factors of survival in patients with colorectal cancer were analyzed by Cox regression model. Kaplan-Meier method was used for drawing the survival curve, and log-rank test method was used for comparison.@*Results@#The serum ULBP2 level in colorectal cancer group was significantly higher than that in healthy control group: (85.52 ± 12.18) ng/L vs. (66.20 ± 8.28) ng/L, and the serum ULBP2 level of stage Ⅰ to Ⅱ in colorectal cancer group was also significantly higher than that in healthy control group: (76.44 ± 7.56) ng/L vs. (66.20 ± 8.28) ng/L, and there were statistical differences (P<0.05). ROC curve analysis result showed that the optimal cut off value of serum ULBP2 for colorectal cancer diagnosis was 79.53 ng/L, and area under curve (AUC) was 0.869, with a sensitivity of 73.75% and a specificity of 91.67%; the optimal cut off value of serum ULBP2 for stage Ⅰ to Ⅱ colorectal cancer diagnosis was 71.86 ng/L, and AUC was 0.827, with a sensitivity of 78.57%, and a specificity of 78.33%. According to the median serum ULBP2 level, the patients were divided into ULBP2 high expression (ULBP2>85.52 ng/L, 38 cases) and ULBP2 low expression (42 cases). The serum expression level of ULBP2 was related to lymph node metastasis and tissue differentiation (P < 0.05). Univariate Cox regression analysis result showed that lymph node metastasis, distant metastasis, tissue differentiation and serum ULBP2 were risk factors of poor prognosis in patients with colorectal cancer (P<0.01 or <0.05); multivariate Cox regression analysis result showed that serum ULBP2 was the independent risk factor of poor prognosis in patients with colorectal cancer (HR = 0.194, 95% CI 0.077 to 0.490, P = 0.001). The median survival time in patients with serum ULBP2 high expression was significantly shorter than that in patients with serum ULBP2 low expression (28 months vs. 50 months), and there was statistical difference (P<0.05).@*Conclusions@#Serum ULBP2 can be used as an indicator for early diagnosis and prognostic evaluation in patients with colorectal cancer.

Values of early diagnosis and prognosis evaluation of peripheral blood UL16 binding protein 2 expression in patient with colorectal cancer / 中国医师进修杂志

Objective To detect the level of peripheral blood UL16 binding protein 2 (ULBP2) in patients with colorectal cancer,and study its value on early diagnosis and prognosis evaluation.Methods Eighty patients with colorectal cancer (colorectal cancer group) and 60 healthy subjects (healthy control group) from May 2016 to May 2019 in Affiliated Dongfeng Hospital,Hubei University of Medicine were selected.Serum expression level of ULBP2 was detected by enzyme-linked immunosorbent assay.The diagnostic efficacy of serum ULBP2 in colorectal cancer was evaluated by receiver operating characteristic (ROC) curve.The influencing factors of survival in patients with colorectal cancer were analyzed by Cox regression model.Kaplan-Meier method was used for drawing the survival curve,and log-rank test method was used for comparison.Results The serum ULBP2 level in colorectal cancer group was significantly higher than that in healthy control group:(85.52 ± 12.18) ng/L vs.(66.20 ± 8.28) ng/L,and the serum ULBP2 level of stage Ⅰ to Ⅱ in colorectal cancer group was also significantly higher than that in healthy control group:(76.44 ± 7.56) ng/L vs.(66.20 ± 8.28) ng/L,and there were statistical differences (P ＜ 0.05).ROC curve analysis result showed that the optimal cut off value of serum ULBP2 for colorectal cancer diagnosis was 79.53 ng/L,and area under curve (AUC) was 0.869,with a sensitivity of 73.75％ and a specificity of 91.67％;the optimal cut off value of serum ULBP2 for stage Ⅰ to Ⅱ colorectal cancer diagnosis was 71.86 ng/L,and AUC was 0.827,with a sensitivity of 78.57％,and a specificity of 78.33％.According to the median serum ULBP2 level,the patients were divided into ULBP2 high expression (ULBP2 ＞ 85.52 ng/L,38 cases) and ULBP2 low expression (42 cases).The serum expression level of ULBP2 was related to lymph node metastasis and tissue differentiation (P ＜ 0.05).Univariate Cox regression analysis result showed that lymph node metastasis,distant metastasis,tissue differentiation and serum ULBP2 were risk factors of poor prognosis in patients with colorectal cancer (P ＜ 0.01 or ＜ 0.05);multivariate Cox regression analysis result showed that serum ULBP2 was the independent risk factor of poor prognosis in patients with colorectal cancer (HR =0.194,95％ CI 0.077 to 0.490,P =0.001).The median survival time in patients with serum ULBP2 high expression was significantly shorter than that in patients with serum ULBP2 low expression (28 months vs.50 months),and there was statistical difference (P ＜ 0.05).Conclusions Serum ULBP2 can be used as an indicator for early diagnosis and prognostic evaluation in patients with colorectal cancer.

Effect of Clarithromycin on the Expression of UL16-Binding Protein 2 in Human Cells.

BACKGROUND: Clarithromycin is a macrolide antibiotic that possesses anti-inflammatory and immunomodulatory properties. Although recent data suggests that macrolide antibiotics enhance Pseudomonas aeruginosa clearance from the lung, involving natural killer (NK) T cells in this process by activating the NKG2D-NKG2D ligand system, the precise underlying mechanism is still unclear. In this study, we examined the effect of clarithromycin on a potent NKG2D ligand, UL16-binding protein 2 (ULBP2), in the lung and its shedding mechanism. METHODS: The gene expressions of ULBP2 and the shredder proteinases of ULBP2, a disintegrin and metalloproteinase domain 10 (ADAM10) and ADAM17, were measured using real-time PCR. The cell surface ULBP2 expression was measured by flow cytometry. The amount of solubilized ULBP2 (sULBP2) was measured using an ELISA. The activity of ADAM17 was examined by measurement of fluorescence intensity from the fluorescence resonance energy transfer peptide substrate cleaved by ADAM17. RESULTS: Clarithromycin significantly induced transcription of ULBP2 and ADAM17 in both A549 and LCSC #2 cells, which endogenously express minimal and abundant levels of ULBP2, respectively. However, there was no significant change on transcription of ADAM10. The same tendency was observed when LCSC #2 cells were treated with tumor necrosis factoralpha processing inhibitor-2 to inhibit ADAM17 activity. The amount of sULBP2 was significantly decreased in both A549 and LCSC #2 cells by treatment with clarithromycin. Finally, clarithromycin significantly inhibited the activity of ADAM17 in LCSC #2 cells. CONCLUSION: These findings suggest that clarithromycin induces ULBP2 expression and reduces the amount of sULBP2, by possibly inhibiting the activity of the potent ULBP2-shedding enzyme ADAM17. Because these changes in ULBP2 and sULBP2 levels could activate NKT cells, this finding might indicate a novel mechanism by which clarithromycin improves the clearance of P. aeruginosa in chronic respiratory diseases.

Combined detection of serum UL16-binding protein 2 and macrophage inhibitory cytokine-1 improves early diagnosis and prognostic prediction of pancreatic cancer.

Pancreatic cancer (PC) is the fourth leading cause of cancer-related mortality in the United States. There is no effective serum biomarker for the early diagnosis of PC at present. Although serum UL16-binding protein 2 (ULBP2) and macrophage inhibitory cytokine-1 (MIC-1) levels are reported to be elevated in PC patients, the diagnostic and prognostic value of ULBP2 and MIC-1 alone or in combination remains unknown. The aim of the present case-control study was to compare the diagnostic value of ULBP2, MIC-1 and carbohydrate antigen 19-9 (CA19-9) in 359 serum samples, consisting of 152 cases of PC, 20 cases of pre-pancreatic cancer, 91 cases of chronic pancreatitis (CP) and 96 normal controls (NC). All patients were followed up for a median of 2 years. It was found that the serum levels of ULBP2, MIC-1 and CA19-9 were significantly higher in the PC patients compared with those in the NC group. In distinguishing PC from the CP, the highest sensitivity and specificity were ULBP2 (0.878) and CA19-9 (0.816), respectively. The area under the receiver operating characteristic curve of ULBP2 was 0.923, which was the highest of the three biomarkers. MIC-1 was the optimal choice for the diagnosis of early-stage PC (area under the curve, 0.831). Overall, MIC-1 in combination with ULBP2 improved the diagnostic accuracy in differentiating PC from CP and NC. In addition, a higher level of MIC-1 was correlated with a poorer prognosis, as calculated by the Kaplan-Meier test (P=0.039). Patients with serum MIC-1 levels of ≥1,932 ng/ml had a median survival time of 15.62±2.44 months (mean ± standard deviation) vs. 18.66±2.43 months in patients with a lower level of MIC-1. Overall, combined detection of serum MIC-1 and ULBP2 improved the diagnostic accuracy in differentiating PC from CP and NC, and serum MIC-1 level alone was a predictor of survival in the patients with PC.