Integrative multi-omics analysis depicts the methylome and hydroxymethylome in recurrent bladder cancers and identifies biomarkers for predicting PD-L1 expression.

BACKGROUND: Urinary bladder cancer (UBC) is a common malignancy of the urinary tract; however, the mechanism underlying its high recurrence and responses to immunotherapy remains unclear, making clinical outcome predictions difficult. Epigenetic alterations, especially DNA methylation, play important roles in bladder cancer development and are increasingly being investigated as biomarkers for diagnostic or prognostic predictions. However, little is known about hydroxymethylation since previous studies based on bisulfite-sequencing approaches could not differentiate between 5mC and 5hmC signals, resulting in entangled methylation results. METHODS: Tissue samples of bladder cancer patients who underwent laparoscopic radical cystectomy (LRC), partial cystectomy (PC), or transurethral resection of bladder tumor (TURBT) were collected. We utilized a multi-omics approach to analyze both primary and recurrent bladder cancer samples. By integrating various techniques including RNA sequencing, oxidative reduced-representation bisulfite sequencing (oxRRBS), reduced-representation bisulfite sequencing (RRBS), and whole exome sequencing, a comprehensive analysis of the genome, transcriptome, methylome, and hydroxymethylome landscape of these cancers was possible. RESULTS: By whole exome sequencing, we identified driver mutations involved in the development of UBC, including those in FGFR3, KDMTA, and KDMT2C. However, few of these driver mutations were associated with the down-regulation of programmed death-ligand 1 (PD-L1) or recurrence in UBC. By integrating RRBS and oxRRBS data, we identified fatty acid oxidation-related genes significantly enriched in 5hmC-associated transcription alterations in recurrent bladder cancers. We also observed a series of 5mC hypo differentially methylated regions (DMRs) in the gene body of NFATC1, which is highly involved in T-cell immune responses in bladder cancer samples with high expression of PD-L1. Since 5mC and 5hmC alternations are globally anti-correlated, RRBS-seq-based markers that combine the 5mC and 5hmC signals, attenuate cancer-related signals, and therefore, are not optimal as clinical biomarkers. CONCLUSIONS: By multi-omics profiling of UBC samples, we showed that epigenetic alternations are more involved compared to genetic mutations in the PD-L1 regulation and recurrence of UBC. As proof of principle, we demonstrated that the combined measurement of 5mC and 5hmC levels by the bisulfite-based method compromises the prediction accuracy of epigenetic biomarkers.

Activation hypoxia inducible factor-1α gene affected the tumor microenvironment and induced recurrence and invasion of bladder cancer in vitro.

OBJECTIVE: This work aimed to investigate the molecular mechanism of the activation of hypoxia-inducible factors under different low oxygen partial pressures. METHODS: Strictly follow in vitro aseptic culture of bladder cancer cell line UMUC3 and when the cells grow in the logarithmic phase, culture the cells under different low oxygen partial pressures. Among these groups, two groups of cells were transfected with small interfering-hypoxia inducible factor 1α (si-HIF-1α) liposome plasmids to silencing the HIF-1α expression. RESULTS: Cell cloning experiment showed that HIF-1α will increase cell adhesion and proliferation under hypoxia. Matrigel angiogenesis experiment showed that hypoxia has a negative impact on the angiogenesis of tumor cells. Cell scratch test indicated that hypoxia has a greater impact on the migration ability of cancer cells, and HIF-1α has a significant impact on the migration process. Cell invasion test proved that hypoxia has a greater impact on the invasion ability of cancer cells, and HIF-1α has a great impact on the invasion process. CONCLUSION: HIF-1α can target the regulatory gene vascular endothelial growth factor to promote tumor cell proliferation, migration, invasion, neovascularization and lymph node metastasis.

OBJETIVO: Por lo tanto, este trabajo investiga el mecanismo molecular de la activación de factores inducibles por hipoxia bajo diferentes presiones parciales de oxígeno bajas. MÉTODOS: Siga estrictamente el cultivo aséptico in vitro de la línea celular de cáncer de vejiga UMUC3 y cuando las células crezcan en la fase logarítmica, cultive las células bajo diferentes presiones parciales de oxígeno bajas. Entre estos grupos, se transfectaron dos grupos de células con plásmidos de liposomas si-HIF-1α para silenciar la expresión de HIF-1α. RESULTADOS: El experimento de clonación celular mostró que HIF-1α aumentará la adhesión y proliferación celular bajo hipoxia. El experimento de angiogénesis de Matrigel mostró que la hipoxia tiene un impacto negativo en la angiogénesis de las células tumorales. La prueba de raspado celular indicó que la hipoxia tiene un mayor impacto en la capacidad de migración de las células cancerosas, y HIF-1α tiene un impacto significativo en el proceso de migración. La prueba de invasión celular demostró que la hipoxia tiene un mayor impacto en la capacidad de invasión de las células cancerosas y HIF-1α tiene un gran impacto en el proceso de invasión. CONCLUSIÓN: HIF-1α puede dirigirse al gen regulador vascular endothelial growth factor para promover la proliferación, migración, invasión, neovascularización y metástasis en los ganglios linfáticos de las células tumorales.

CDH1 overexpression predicts bladder cancer from early stage and inversely correlates with immune infiltration.

BACKGROUND: Bladder cancer (BC) seriously endangers public health, but effective biomarkers for BC diagnosis, particularly in the early stage, are still lacking. Identification of reliable biomarkers associated with early-stage BC is of great importance to early treatment and an improved outcome. METHODS: Differentially expressed genes (DEGs) were identified using four publicly available early-stage BC gene-expression profiles. Protein-protein interaction (PPI) and survival analysis for hub genes was evaluated. The correlation between methylation of genes and prognosis was evaluated using the MethSurv database. Co-expressed genes were explored using Cancer Cell Line Encyclopedia database and the corresponding expression were assessed in vitro. The competing endogenous RNA network and the immune cell infiltration in BC were generated using data of The Cancer Genome Atlas. RESULTS: Ten hub genes of the 213 integrated DEGs were identified, including CDH1, IGFBP3, PPARG, SDC1, EPCAM, ACTA2, COL3A1, TPM1, ACTC1, and ACTN1. CDH1 appeared to increase from tumor initiation stage and negatively correlated with methylation. Six methylated sites in CDH1 indicated a good prognosis and one site indicated an aberrant prognosis. High CDH1 expression was negatively correlated with infiltrations by most immune cells, such as plasmacytoid dendritic cells (pDCs), regulatory T cells, macrophages, neutrophils, DCs, and natural killer cells. CDH1 was highly positively correlated with EPCAM and appeared to be directly regulated by miR-383. CONCLUSIONS: The identified oncogenic alterations provide theoretical support for the development of novel biomarkers to advance early-stage BC diagnosis and personalized therapy.

Decreased Sirt3 contributes to cyclic production of reactive oxygen species and islet ß-cell apoptosis in high glucose conditions.

BACKGROUND: Reactive oxygen species (ROS) plays a vital role in the apoptosis of islet ß-cells in type 2 diabetes mellitus (T2DM). Sirt3 (Sirtuin 3, a deacetylase) and FoxO1 (a transcription factor) might be involved in ROS production. This study was to investigate mechanism of ROS production and ß-cell apoptosis in T2DM. METHODS: Oxidative stress and apoptosis in islets of db/db mice and high glucose cultured ß-cells were observed by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay and western blotting. Then, H2O2 was used to ascertain the effect of ROS on the expression of Sirt3. Meanwhile, FoxO1, antioxidant enzymes - catalase (CAT) and manganese superoxide dismutase (MnSOD) and ß-cell apoptosis were also determined by western blotting. Finally, Sirt3 was knocked down to evaluate the effect on oxidative stress and apoptosis of ß-cells. RESULTS: Under high glucose environment, enhanced ROS made a decrease of Sirt3 expression, which increased acetylation of FoxO1, thus reduced the expression of its target proteins -MnSOD and CAT, and further significantly increased ROS levels. Increased ROS finally led to the apoptosis of ß-cells. CONCLUSION: Down-regulation of Sirt3 plays an important role in the cyclic production of ROS and ß-cell apoptosis. Targeting Sirt3 may be favorable for the treatment of T2DM.

Quercetin ameliorates oxidative stress­induced cell apoptosis of seminal vesicles via activating Nrf2 in type 1 diabetic rats.

It was known that diabetes may affect the male reproductive function by inhibiting the secretion of male accessory glands including seminal vesicles. Increased cell apoptosis induced by oxidative stress is thought to be an important pathological change in the seminal vesicles in diabetic patients. Quercetin is a potent anti-oxidative bioflavonoid. In this study, we explore the effect of quercetin on cell apoptosis of seminal vesicles and its underlying mechanism. The STZ-induced type 1 diabetic rat model was established. Three doses (low, medium and high) of quercetin were administrated to the STZ-induced type 1 diabetic rats for 4 months. Fasting blood glucose, the fructose in seminal plasma, total antioxidant capacity (T-AOC) and malondialdehyde (MDA) in seminal vesicles were determined by colorimetric method. Nuclear transcription factor- Nrf2 was observed by immunofluorescent staining. Biomarkers related to cell apoptosis, such as Bcl-2, Bax and cleaved -Caspase3 were measured by Western blotting and immumohistochemical staining. The body weight and seminal vesicle weight indexes were also determined. The results showed that T-AOC and Nrf2 were decreased, the levels of MDA were increased, the cleaved Caspase-3 was increased and the ratio of Bax to BCL-2 was decreased in seminal vesicles of diabetic rats, along with the severe hyperglycemia. When diabetic rats were treated by quercetin for 4 months, all the indexes were reversed at different degree except the fasting blood glucose. Our results suggested that quercetin could ameliorate oxidative stress­induced cell apoptosis of seminal vesicles via inhibiting Nrf2 in type 1 diabetic rats, which indicated that quercetin could be used for preventing lesions of seminal vesicles in type 1 diabetes.

Quercetin protects islet ß-cells from oxidation-induced apoptosis via Sirt3 in T2DM.

OBJECTIVES: Sirt3 may regulate ROS production and might be involved in ß-cell apoptosis, which plays an important role in the progression of type 2 diabetes mellitus (T2DM). Quercetin is a potent anti-oxidative bioflavonoid, but its effects on T2DM remain to be explored. This study aimed to investigate the effects of quercetin on ß-cell apoptosis and explore its mechanisms. MATERIALS AND METHODS: The effects of quercetin were conducted on db/db mice and INS1 cells. Fasting blood glucose was determined by the colorimetric method, serum insulin was measured by enzyme-linked immunosorbent assay (ELISA). Meanwhile, Sirt3 in INS1 cells was knocked down by plasmid transfection. The antioxidant proteins (SOD2 and CAT), apoptosis proteins (cleaved Caspase-3, Bax, and BCL-2), and Sirt3 protein in pancreases and INS1 cells were determined by western blotting. RESULTS: When INS1 cells and diabetic mice were treated with quercetin, the levels of SOD2, CAT, and Sirt3 proteins were increased, the levels of cleaved Caspase-3 and the ratio of Bax to BCL-2 were decreased at different degrees, along with reduced blood glucose levels and elevated insulin levels in diabetic mice. When Sirt3 was knocked down in INS1 cells, increase of two antioxidants and decrease of cell apoptosis generated by quercetin could not occur. CONCLUSION: Quercetin protected islet ß-cells from oxidation-induced apoptosis via Sirt3 in T2DM, which would be beneficial to develop new strategies for preventing ß-cell failure in T2DM.

Preoperative Fibrinogen-Albumin Ratio, Potential Prognostic Factors for Bladder Cancer Patients Undergoing Radical Cystectomy: A Two-Center Study.

BACKGROUND: We conducted a two-center study to investigate the prognostic value of preoperative fibrinogen-albumin ratio (FAR) in patients undergoing radical cystectomy (RC). METHODS: The clinical and survival data of 267 patients with bladder cancer (BCa) treated with RC were collected, of which 140 patients from Xuzhou Central Hospital were divided into training set and 127 patients from The Second Affiliated Hospital of Nantong University were divided into validation set. X-tile software was used to obtain the optimal cut-off values for preoperative platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR) and FAR. Receiver operating characteristic (ROC) curves were used to compare the predictive ability of PLR, NLR, FAR and modified Glasgow prognostic score (mGPS). Kaplan-Meier curves were used to assess overall survival (OS) and progression-free survival (PFS) of patients in different FAR groups. Univariate and multivariate Cox regression were used to assess patients' independent risk factors, and R software was used to construct prognostic nomograms. RESULTS: In the training set, the optimal cut-off values for PLR, NLR and FAR were 76.76, 3.97 and 0.08, respectively. Both in the training and validation sets, FAR had better ability to predict OS and PFS than PLR and NLR, and patients in the higher FAR group had worse OS and PFS. In the multivariate Cox regression analysis, FAR was an independent risk factor for OS [hazard ratio (HR) 3.569, 95% confidence interval (CI): 1.015-12.546, P=0.047] and PFS [HR 5.071, 95% CI: 1.394-18.451, P=0.014]. In addition, FAR-based prognostic nomograms had high predictive ability than TNM staging. CONCLUSION: Preoperative FAR is an independent prognostic factor for OS and PFS in BCa patients treated with RC, and a high FAR predicted a poor prognosis. In addition, a prognostic nomogram based on FAR can better predict individual survival.

Antitumor mechanism of MAP30 in bladder cancer T24 cells, and its potential toxic effects in mice.

To investigate the antitumor mechanism of MAP30 in human bladder cell line (T24) and its potential toxic effects in mice.  In this study, the biological behavior of MAP30's influence on bladder cell was investigated to reveal the antitumor mechanism and role of MAP30 in bladder cancer. MAP30 gene sequence optimized by gene synthesis codon was inserted into the prokaryotic expression vector pET-28a to produce a large amount of target protein in Escherichia coli. The protein product was obtained after purification. Membrane hydration method was used to prepare MAP30 liposome in order to enhance its membrane permeability. The effects of MAP30 on the viability, apoptosis and migration of T24 cell were assessed using 3­(4,5­dimethyl­thiazol­2­yl)­2,5­diphenyl­2H­tetrazolium bromide (MTT), flow cytometric and TUNEL assays, respectively. Mice were transfected with bladder cancer cells for 48 h. The expressions of apoptotic and non-apoptotic proteins were determined using Western blotting. Changes in tumor volume and occurrence of metastasis were assessed using luciferase assay. After 7 days, liver and kidney were excised for histological examination. The levels of reactive oxygen species (ROS), malondialdehyde (MDA), and reduced glutathione (GSH), and activities of catalase and glutathione peroxidase (GPx) were determined in serum or homogenate using enzyme-linked immunosorbent assay (ELISA). The yield of MAP30 after purification was significantly increased. The results of MTT assay showed that MAP30 significantly and concentration-dependently inhibited the proliferation and migration of T24 cells (p < 0.05). The prepared liposomes had uniform hydrated particle size of 132.6 nm, with encapsulation efficiency of 78 %. The inhibitory effect of MAP30 liposome on T24 cells was significantly higher than that of MAP30, and MAP30 significantly increased the number of apoptotic cells (p < 0.05). Western blotting showed that MAP30 significantly promoted the expression of caspase 3 (p < 0.05), but did not significantly affect the expressions of bcl-2 and bax (p > 0.05). It also significantly down-regulated the expressions of NF-kB, JNK and MMP2 (p < 0.05). Tumor formation was significantly inhibited, and tumor volume reduced in bladder cancer-bearing mice after treatment with MAP30 (p < 0.05). Histological examination showed that MAP30 induced mild histological changes in the liver and kidney of mice, and significantly increased the level of MDA at day 1 (p < 0.05). It also significantly and time-dependently increased ROS, but reduced GSH levels and activities of catalase and GPx (p < 0.05). However, MAP30 had no significant effect on DNA (p > 0.05). The apoptotic effect of MAP30 in T24 cells is mediated via activation of caspase-3 signaling pathway. The protein produces mild histological changes in the liver and kidney of mice, but has no significant effect on DNA.

Comprehensive gene expression analysis after ERH gene knockdown in human bladder cancer T24 cell lines.

INTRODUCTION: In this article, we utilized Ingenuity® Pathway Analysis (IPA®) bioinformatics analysis software and Metascape® bioinformatics analysis website tools to analyse the possible mechanism of ERH affecting tumourigenesis (proliferation and apoptosis) in bladder cancer (BC) T24 cells. METHODS: The ERH gene was knocked down, and BC T24 cells were divided into ERH normal and knockdown groups. Affymetrix® gene expression microarrays were performed to obtain a differentially expressed gene list (DEGL) between the 2 groups. IPA® data analyses contain five modules: disease and function analysis, upstream analysis, regulator effects analysis, canonical pathway analysis and molecular network analysis. The results of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were analysed by Metascape®. RESULTS: The results of the gene expression profiling chip and the DEGL showed that 344 genes were upregulated and 254 genes were downregulated. The IPA® and Metascape® pathway analyses showed that the ERH gene may affect proliferation and apoptosis by affecting the apoptosis, cell cycle, Toll-like receptor (TLR), NF-κB or TGF-beta signalling pathways. Upstream analysis determined that the ERH gene may regulate TNF and NK-κB in the BC T24 cell lines. The ERH gene may be involved in the "cell death and survival" molecular network in BC T24 cells. ERH may be a regulator of KITLG through TNF. CONCLUSIONS: The ERH gene may affect apoptosis through the TLR, NF-κB, TNF or TGF-beta signalling pathways in BC T24 cells, and may be a regulator of KITLG to ultimately activate the growth of malignant tumours.

Accessory spleen after splenectomy mimicking adrenal tumor: a case report.

Accessory spleen is a congenital anomaly, those asymptomatic generally do not need surgical intervention. Retroperitoneal heterotopic accessory spleens are often misdiagnosed, especially in cases that have undergone splenectomy for various reasons. In these cases, most patients are received unnecessary resection for misdiagnosed as adrenal gland tumors, ganglioblastomas or paragangliomas preoperatively. We report on a case of accessory spleen mimicking a left adrenal tumor. A 47-year-old man who had undergone splenectomy 25 years ago was referred to our department because of hypertension, CT scan revealed a mass about 4 cm in the left adrenal gland. The mass was confirmed nonfunctional through hormonal evaluation of the adrenal gland. We found the mass was not originated from the adrenal gland intraoperation even they were close together. Histopathologic examination of the surgical specimen revealed an accessory spleen. The differences between the case and other accessory spleen patients include a history of hypertension, the location of the mass and a history of splenectomy, and these also are the main reasons for our misdiagnosis. This case remind us that an accessory spleen should be considered for the biochemically inactive mass in left adrenal area, even the splenectomy has been performed before.

Effects of Free Anthraquinones Extract from the Rhubarb on Cell Proliferation and Accumulation of Extracellular Matrix in High Glucose Cultured-Mesangial Cells.

Diabetic nephropathy (DN) is the leading cause of end-stage failure of the kidney, but the efficacy of currently available strategies for the prevention of DN remains unsatisfactory. In this study, we investigated the effects of free anthraquinones (FARs) extract, which was extracted from the rhubarb and purified by macroporous resin DM130 with gradient mixtures of ethanol/water as the lelution solvents, in high glucose-cultured glomerular mesangial cells (MCs). The cell proliferation was determined by CCK-8 assay, the levels of TGF-ß1, CTGF, ColIV and FN proteins in the supernatant of MCs were measured by ELISA assays, and the mRNA levels of these four genes were detected by RT-PCR. The results showed that the increased proliferation of MCs, the mRNA levels and protein expression of TGF-ß1, CTGF, ColIV and FN induced by high glucose were inhibited after the treatment with the FARs extract. This indicated that FARs extract could inhibit cell proliferation and the expression of main extracellular matrix induced by high glucose in MCs. The FARs extract exhibited potential values for prophylaxis and therapy of DN.

Re-transurethral resection treatment for non-invasive bladder tumor.

The objective of this study was to compare surgical treatments for non-invasive bladder tumor. Hundred and forty patients with non-invasive bladder tumor were studied. Seventy-three patients were treated by transurethral resection of bladder tumor (TURBT) and Repeated-Transurethral Resection of Bladder Tumor (R-TURBT), while 67 by partial cystectomy. Operation time, blood loss, postoperative complications, and postoperative recurrence rate were better in the TURBT+R-TURBT group compared with the partial cystectomy group. Further, TURBT+R-TURBT offers advantages, such as simple surgical manipulation, less trauma, faster recovery, repeatedly performable procedure, and safety. In conclusion, this is an optimal therapy for treatment of non-invasive bladder tumor.

[Elective microscopic resection of dorsal penile nerves for primary premature ejaculation: a clinical observation].

OBJECTIVE: To evaluate the effect of elective microscopic resection of dorsal penile nerves in the treatment of primary premature ejaculation (PPE). METHODS: Seventy-eight PPE patients received elective microscopic resection of dorsal penile nerves, 5 branches in 9 cases, 6 in 17, 7 in 15, 8 in 14, 9 in 8, 10 in 6, 11 in 6, and 12 in 3. The patients were followed up for 12 months, and their intravaginal ejaculation latency time (IELT) and sexual intercourse satisfaction scores were recorded before and after treatment. RESULTS: Compared with the baseline, the IELT was significantly prolonged after surgery ([0.86 +/- 0.32] vs [6.65 +/- 3.9] min, P < 0.01), and the sexual intercourse satisfaction scores of the patients were dramatically increased (7.32 +/- 2.52 vs 12.32 +/- 3.76, P < 0.01), so were those of their sexual partners (4.46 +/- 1.36 vs 12.73 +/- 1.45, P < 0.01). CONCLUSION: Elective microscopic resection of dorsal penile nerves is safe and effective for the treatment of PPE.

Target expression of Staphylococcus enterotoxin A from an oncolytic adenovirus suppresses mouse bladder tumor growth and recruits CD3+ T cell.

We recently engineered an oncolytic adenovirus (PPE3-SEA) that expresses the superantigen, Staphylococcus enterotoxin A (SEA), and that has enhanced tumor specificity because the telomerase reverse transcriptase and hypoxia-inducible factor promoters regulate expression of E1A and E1B genes, respectively. Here, we evaluated the PPE3-SEA adenovirus anti-tumor activity against MB49 mouse bladder cancer cell proliferation in vitro and in vivo. PPE3-SEA infection of murine MB49 cells in vitro induced cytopathic effects, and significant expression of SEA mRNA and protein, as measured by RT-PCR and western blot, respectively. Subcutaneous MB29 bladder tumors were established in syngeneic C57BL/6 mice. After 10 days, tumors were injected with either oncolytic virus or PBS. Tumor dimensions were measured on days 1, 3, 5, 7, 9, and 11 post-treatment and tumor volumes were calculated. One of eight PPE3-SEA-treated mice had no tumor by day 9. PPE3-SEA treated group had significantly lower mean tumor volume beginning on day 5 post-treatment (p < 0.01), more fibrous tissue in the tumor, and increased presence of infiltrating CD3+ T cells than those of the control group. Gross appearance and histologic sections from the livers and kidneys of the PPE3-SEA-treated group were similar to those of the control group. In conclusion, oncolytic adenoviruses can provide a novel delivery vehicle for SEA to tumor sites, and PPE3-SEA warrants further study as a potential anti-tumor agent for bladder cancer.

The transurethral seminal vesiculoscopy in the diagnosis and treatment of the seminal vesicle disease.

To develop a transurethral endoscopy technique of the transurethral seminal vesiculoscopy to examine and treat seminal vesicle disease. A total of 61 patients with seminal vesicle disease were diagnosed and treated with the transurethral seminal vesiculoscopy through the distal seminal tracts and vesicles. 58 cases were successfully treated using transurethral seminal vesiculoscopy via the seminal vesicles. The operation took 25 ~ 85 min, with an average of (35.6) mins. In this group, seven cases were diagnosed as ejaculatory orifice cyst, 14 cases had blood clots in the seminal vesicles, and nine patients had stones in the seminal vesicles. All patients were treated properly. Follow-up occurred at 3 months, with two cases showing post-operative discomfort in perineal region. One patient had recurrence with seminal vesiculitis, which improved with treatment. Four infertile patients had a significant increase in sperm count and ejaculation volume and two of these patients were able to naturally inseminate within seven to 18 months post-surgery. This approach enables a new endoscopic technique with the transurethral seminal vesiculoscopy to diagnose and treat seminal vesicle disease through the normal anatomic pathway which can be easily performed with few post-operative complications.

Mechanism of monoclonal antibody-coupled Staphylococcus superantigen-A induced apoptosis in human bladder cancer cells.

To investigate the mechanism of apoptosis induced by BDI-1 monoclonal antibody (MAb) coupled Staphylococcal superantigen-A (SEA) in human bladder cancer cell line BIU-87. Human PBMC (effector cells) mediated cytotoxic killing of BIU-87 cells (target cells) was studied by culturing the BIU-87 cells in the presence of effector cells plus medium after their activation by treatment with SEA-targeted by MAb, SEA alone or vehicle (control). Proliferation and apoptosis of BIU-87 cells was measured after the treatments. Expression of Bax and Bcl-2 and cytokine concentration in co-culture supernatants were detected by Western blot and ELISA, respectively. Proliferation of MAb-targeted SEA BIU-87 cells decreased significantly (P < 0.05) as compared to control and SEA groups. Flow cytometry revealed apoptosis in SEA alone and more prominently in targeted-SEA treated in BIU-87 cells, which is significantly more than in controI cells (P < 0.05). In addition, Western blot analysis indicated that the ratio of Bax/Bcl-2 significantly increased by targeted SEA treatment, even at low concentration, as compared to cells treated with SEA alone or control cells (P < 0.05). However, there were no significant differences in IL-2, TNF-α and IFN-γ levels in the culture medium between SEA and targeted SEA groups, even though they are several folds higher than in control cells. SEA targeting by MAb significantly increases apoptosis in BIU-87 cells, possibly through the up-regulation of proapoptotic protein Bax and down regulation of antiapoptotic protein, Bcl-2.

[Correlation of testis/sperm specific thioredoxin-1, 2 and 3 with male reproduction].

Sptrx-1, 2 and 3 are a series of thioredoxins specifically expressed in the testis/sperm. They play a significant role structurally and functionally in the process of spermiogenesis. The genesis and mutation of sptrx-1, 2 and 3 are correlated to male reproduction. Taking sptrx-1, 2 and 3 as the target of study and treatment will open up a new field in the clinical study of male reproduction.