Haitao Niu

Effect of TLR4 and B7-H1 on Immune Escape of Urothelial Bladder Cancer and its Clinical Significance

BACKGROUND/AIM Toll-like receptor 4 (TLR4) and B7-H1, both normally expressed restricted to immune cells, are found to be aberrantly expressed in a majority of human tumors and may play important roles in regulation of tumor immunity. It has been shown that urothelial bladder cancer (UBC) patients can manifest tumoral immune escape which may be a potential critical factor in tumor pathogenesis and progression. However, so far, the mechanisms of UBC-related immune escape have not been clarified. The aim of this study was to investigate the effect of TLR4 and B7-H1 on immune escape of UBC. METHODS Bladder cancer T24 cells were pre-incubated with LPS and co-cultured with tumor specific CTLs. CTL cytotoxicity and apoptosis rates were measured by MTT assay and flow cytometry, respectively. The effects of an ERK inhibitor on B7-H1 expression and CTL cytotoxicity against T24 cells were also evaluated. In addition, TLR4, B7-H1 and PD-1 protein expression was analyzed by immunohistochemistry in 60 UBC specimens and 10 normal urothelia. RESULTS TLR4 activation protected T24 cells from CTL killing via B7-H1 overexpression. However PD98059, an inhibitor of ERK, enhanced CTL killing of T24 cells by reducing B7-H1 expression. TLR4 expression was generally decreased in UBC specimens, while B7-H1 and PD-1 were greatly overexpressed. Moreover, expression of both B7-H1 and PD-1 was significantly associated with UICC stage and WHO grade classification. CONCLUSIONS TLR4 and B7-H1 may contribute to immune escape of UBC. Targeting B7-H1 or the ERK pathway may offer new immunotherapy strategies for bladder cancer.

Inhibitory effect of valproic acid on bladder cancer in combination with chemotherapeutic agents in vitro and in vivo

Histone deacetylase inhibitors (HDACIs) are a promising class of drugs that act as antiproliferative agents by promoting differentiation and inducing apoptosis. Valproic acid (VPA) is an HDACI that has been widely used as an anti-convulsant and shows promise as a chemotherapeutic drug for a number of tumor cells. The present study aimed to investigate the inhibitory effect of VPA on the viability of bladder cancer cells and its synergistic effect with chemotherapeutic agents in vitro and in vivo. The cell viability of human bladder cancer cell lines following treatment with VPA and/or VPA in combination with mitomycin C, cisplatin (DDP) and adriamycin were determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Hoechst staining was used to observe the morphology of the apoptotic cells. Survivin protein and acetylated histone H3 levels were quantified using western blot analysis. The in vivo tumor growth inhibition of VPA was determined in rats with N-methyl-N-nitrosourea-induced bladder cancer. VPA significantly inhibited the growth of the bladder cancer cells in a concentration- and time-dependent manner. Furthermore, improved results were achieved for tumor inhibition when VPA was combined with chemotherapeutic agents in vitro and in vivo. Survivin expression decreased and acetylated histone H3 expression increased in the bladder cancer cells following the treatment with VPA. Intravesical injections of VPA were able to inhibit tumor progression when combined with DDP. In conclusion, VPA acts as an HDACI that has a direct anticancer effect and markedly enhances the action of several chemotherapy agents. VPA may sensitize bladder cancer to anticancer drugs by downregulating survivin expression.

Genome-wide association analysis identifies three new risk loci for gout arthritis in Han Chinese

Gout is one of the most common types of inflammatory arthritis, caused by the deposition of monosodium urate crystals in and around the joints. Previous genome-wide association studies (GWASs) have identified many genetic loci associated with raised serum urate concentrations. However, hyperuricemia alone is not sufficient for the development of gout arthritis. Here we conduct a multistage GWAS in Han Chinese using 4,275 male gout patients and 6,272 normal male controls (1,255 cases and 1,848 controls were genome-wide genotyped), with an additional 1,644 hyperuricemic controls. We discover three new risk loci, 17q23.2 (rs11653176, P=1.36 × 10−13, BCAS3), 9p24.2 (rs12236871, P=1.48 × 10−10, RFX3) and 11p15.5 (rs179785, P=1.28 × 10−8, KCNQ1), which contain inflammatory candidate genes. Our results suggest that these loci are most likely related to the progression from hyperuricemia to inflammatory gout, which will provide new insights into the pathogenesis of gout arthritis.

Cardioprotective Effect of Licochalcone D against Myocardial Ischemia/Reperfusion Injury in Langendorff-Perfused Rat Hearts

Flavonoids are important components of ‘functional foods’, with beneficial effects on cardiovascular function. The present study was designed to investigate whether licochalcone D (LD) could be a cardioprotective agent in ischemia/reperfusion (I/R) injury and to shed light on its possible mechanism. Compared with the I/R group, LD treatment enhanced myocardial function (increased LVDP, dp/dt max, dp/dt min, HR and CR) and suppressed cardiac injury (decreased LDH, CK and myocardial infarct size). Moreover, LD treatment reversed the I/R-induced cleavage of caspase-3 and PARP, resulting in a significant decrease in proinflammatory factors and an increase in antioxidant capacity in I/R myocardial tissue. The mechanisms underlying the antiapoptosis, antiinflammation and antioxidant effects were related to the activation of the AKT pathway and to the blockage of the NF-κB/p65 and p38 MAPK pathways in the I/R-injured heart. Additionally, LD treatment markedly activated endothelial nitric oxide synthase (eNOS) and reduced nitric oxide (NO) production. The findings indicated that LD had real cardioprotective potential and provided support for the use of LD in myocardial I/R injury.

A bladder cancer microenvironment simulation system based on a microfluidic co-culture model

A tumor microenvironment may promote tumor metastasis and progression through the dynamic interplay between neoplastic cells and stromal cells. In this work, the most representative and significant stromal cells, fibroblasts, endothelial cells, and macrophages were used as vital component elements and combined with bladder cancer cells to construct a bladder cancer microenvironment simulation system. This is the first report to explore bladder cancer microenvironments based on 4 types of cells co-cultured simultaneously. This simulation system comprises perfusion equipment, matrigel channel units, a medium channel and four indirect contact culture chambers, allowing four types of cells to simultaneously interact through soluble biological factors and metabolites. With this system, bladder cancer cells (T24) with a tendency to form a ‘reticular’ structure under 3 dimensional culture conditions were observed in real time. The microenvironment characteristics of paracrine interactions and cell motility were successfully simulated in this system. The phenotype change process in stromal cells was successfully reproduced in this system by testing the macrophage effector molecule Arg-1. Arg-1 was highly expressed in the simulated tumor microenvironment group. To develop “precision medicine” in bladder cancer therapy, bladder cancer cells were treated with different clinical ‘neo-adjuvant’ chemotherapy schemes in this system, and their sensitivity differences were fully reflected. This work provides a preliminary foundation for neo-adjuvant chemotherapy in bladder cancer, a theoretical foundation for tumor microenvironment simulation and promotes individual therapy in bladder cancer patients.

T-cell immunoglobulin mucin-3 expression in bladder urothelial carcinoma: Clinicopathologic correlations and association with survival

T cell immunoglobulin mucin‐3 (Tim‐3) was initially recognized as a pivotal immune checkpoint inhibitor that maintains immune homeostasis and tolerance. Recently, Tim‐3 has been demonstrated to play an important role in tumor‐associated immune suppression and aberrant Tim‐3 expression has been reported in several human malignancies. However, the role of Tim‐3 in bladder urothelial carcinoma (BUC) remains largely unknown. The present study aims to investigate Tim‐3 expression in BUC and analyze correlations with clinicopathologic outcomes and postoperative survival.

High-Frequency Variant p.T60M in NaCl Cotransporter and Blood Pressure Variability in Han Chinese

Objective: Frequent studies have confirmed that homozygous or compound heterozygous loss-of-function mutation p.Thr60Met in NaCl cotransporter (NCC) lead to the salt-wasting Gitelman’s syndrome (GS) of hypotension. The finding that Thr60 is a key SPAK/OSR1 phosphorylation site on NCC also raises the possible importance of Thr60 in regulating the activity of NCC and blood pressure (BP). However, the association of heterozygous NCC mutation p.Thr60Met and BP has not yet been studied. Methods: We collected 38 heterozygous mutation p.Thr60Met carriers, respectively, from 14 GS families confirmed by our previous studies and 1,000 unrelated Han Chinese, and matched them pairwise (sex, age ±2 years and BMI ±1) with 38 unrelated healthy controls. BP and biochemistry data were obtained. Student’s t test and χ2 test were used to compare the differences between these study subjects with the heterozygous variant p.T60M and the controls. p < 0.05 was considered statistically significant. Results p.Thr60Met carriers had markedly lower BP (systolic 110.3 ± 13.5 vs. 119.1 ± 15.0 mm Hg; diastolic 70.2 ± 7.0 vs. 75.4 ± 8.2 mm Hg, p < 0.01) than controls. p.Thr60Met heterozygotes had higher fasting plasma glucose concentration (5.35 ± 0.73 vs. 4.95 ± 0.69 mmol/l, p < 0.01). 14 carriers versus 6 control subjects had impaired fasting glucose (36.8 vs. 15.8%, p < 0.01), and p.Thr60Met carriers had higher 24-hour urinary sodium excretion than controls but not significantly (170.2 ± 35.6 vs. 159.5 ± 39.0 mmol, p = 0.10). Conclusions: The NCC mutation p.Thr60Met carriers in Han populations have markedly lower BP and slightly higher fasting plasma glucose compared with normal controls.

Overexpression of monocarboxylate anion transporter 1 and 4 in T24-induced cancer-associated fibroblasts regulates the progression of bladder cancer cells in a 3D microfluidic device.

Stromal fibroblasts are essential for tumor proliferation and invasion. Here we presented a 3-dimensional (3D) microfluidic co-culture device to reconstruct an in vivo-like tumor microenvironment for investigation of the interactions of cancer-associated fibroblasts (CAFs) and bladder cancer cells. With this device, we verified that the cytokines secreted by bladder cancer cells T24 effectively transform the fibroblasts into CAFs. Compared to fibroblasts, the CAFs, which undergo the aerobic glycolysis, showed higher ability to produce lactate and provide energy for bladder cancer cell proliferation and invasion. We also demonstrated that this kind of tumor-promoting effect was associated with the upregulation of monocarboxylate anion transporter 1 (MCT1) and MCT4 expression in CAFs. We concluded that MCT1 and MCT4 are involved in bladder cancer cell proliferation and invasiveness. Moreover, this 3D microfluidic co-culture device allows for the assay to characterize various cellular events in a single device sequentially, facilitating a better understanding of the interactions among heterotypic cells in a sophisticated microenvironment.

Stromal proteome expression profile and muscle-invasive bladder cancer research

BackgroundTo globally characterize the cancer stroma expression profile of muscle-invasive transitional cell carcinoma and to discuss the cancer biology as well as biomarker discovery from stroma. Laser capture micro dissection was used to harvest purified muscle-invasive bladder cancer stromal cells and normal urothelial stromal cells from 4 paired samples. Two-dimensional liquid chromatography tandem mass spectrometry was used to identify the proteome expression profile. The differential proteins were further analyzed using bioinformatics tools and compared with the published literature.ResultsWe identified 868/872 commonly expressed proteins and 978 differential proteins from 4 paired cancer and normal stromal samples using laser capture micro dissection coupled with two-dimensional liquid chromatography tandem mass spectrometry. 487/491 proteins uniquely expressed in cancer/normal stroma. Differential proteins were compared with the entire list of the international protein index (IPI), and there were 42/42 gene ontology (GO) terms exhibited as enriched and 8/5 exhibited as depleted in cellular Component, respectively. Significantly altered pathways between cancer/normal stroma mainly include metabolic pathways, ribosome, focal adhesion, etc. Finally, descriptive statistics show that the stromal proteins with extremes of PI and MW have the same probability to be a biomarker.ConclusionsBased on our results, stromal cells are essential component of the cancer, biomarker discovery and network based multi target therapy should consider neoplastic cells itself and corresponding stroma as whole one.

The status, limitation and improvement of adoptive cellular immunotherapy in advanced urologic malignancies

In recent years, immunotherapy has been gradually established as the fourth frequently adopted antitumor therapy, following surgery, chemotherapy and radiotherapy, for advanced urologic malignancies with an improved understanding of theoretical basis, such as molecular biology and immunology. Thereinto, adoptive cellular immunotherapy (ACI) has become one of the hotspots, which comprises a variety of treatment approaches, such as TIL, CIK cell, γδ T cell, CAR-engineered T cell and Allogeneic stem cell transplantation (alloSCT). Although preclinical efficacy has been demonstrated remarkably, clinical trials could not consistently show the benefit due to multi-factors in complex immunosuppressive microenvironment in vivo compared to that of in vitro. Here we review some timely aspects of ACI for advanced urologic malignancies, and describe the current status and limitation of immunotherapy from the cellular level. Its our expectation to provide prompting consideration of novel combinatorial ACI strategies and a resurgence of interest in ACI for advanced urologic malignancies.