Arsenic Trioxide Combining Leflunomide Activates Nrf2-ARE-HO-1 Signaling Pathway and Protects Heart Xenografts.

OBJECTIVE: To investigate the molecular mechanisms underlying the effects of arsenic trioxide (As2O3) in combination with leflunomide on the hamster-to-rat heart xenotransplant. METHODS: Transplantation of LVG hamster hearts to Lewis rats was performed by anastomosis of vessels in the neck using end-to-end anastomosis with a non-suture cuff technique. Four groups of recipient rats (n=6 in each) were treated with normal saline (control), As2O3 [5 mg/(kg·day) intraperitoneally], leflunomide [5 mg/(kg·d) orally], or leflunomide [5 mg/(kg·d)+As2O3 [5 mg/(kg·d)] in combination. Donor hearts and/or rat spleens were harvested and analyzed 4 days after transplantation. Quantitative reverse-transcription polymerase chain reaction and Western blot analysis were performed to detect the expression of the nuclear factor erythroid-derived factor 2-related factor (Nrf2) and its target gene heme oxygenase-1 (HO-1), Treg cell marker fork-head Box P3 (FOXP3), apoptosis-associated proteins Bcl-2, Bax, and cleaved caspase-3. Immunohistochemical staining was used to detect the levels of inflammatory natural killer cell and macrophage infiltration, intercellular cell adhesion molecule-1 (ICAM-1) and complement C3. RESULTS: Expression of Nrf2-ARE-HO-1 signaling pathway was upregulated in heart xenografts in rats treated with As2O3 plus leflunomide compared with control rats or rats treated with either drug alone (P<0.01), and this was accompanied by an increased Treg cells in the recipient rat spleen (P<0.01). In contrast, the expressions of Bax, cleaved caspase-3, ICAM-1, and complement C3, and infiltration of inflammatory cells in the xenografts were inhibited by As2O3 plus leflunomide treatment (P<0.01). CONCLUSION: Combination treatment with As2O3 and leflunomide protected hamster heart-xenografts in recipient rats.

Nucleobindin-2 enhances the epithelial-mesenchymal transition in renal cell carcinoma.

Nucleobindin 2 (NUCB-2) is a multifunctional protein that contains several functional domains and is associated with a wide variety of biological processes, such as food intake and energy homeostasis. NUCB-2 has been demonstrated to be associated with worse malignant outcomes and cell migration in breast and prostate cancer. However, to the best of our knowledge, its clinical and biological significance in renal cell carcinoma remains unknown. In the present study, tissue specimens from 68 patients with renal cell carcinoma and 10 normal controls were collected for NUCB-2 mRNA and protein assays. The NUCB-2 level in the patients with renal cell cancer was significantly increased compared with the normal control patients. NUCB-2-knockout in the renal cancer cell line SK-RC-52 inhibited migration and invasion. In addition, the expression levels of molecules associated with epithelial-mesenchymal transition (EMT), including E-cadherin, ß-catenin, Slug and Twist, were affected by NUCB-2 suppression and the zinc finger E-box binding to homeobox 1 (ZEB1)-dependent pathway. The AMP-dependent protein kinase (AMPK)/target of rapamycin complex (mTORC) 1 signaling pathway participates in the regulation of NUCB-2-mediated metastasis and EMT. Suppression of NUCB-2 also inhibited tumor nodule formation in a murine renal cell carcinoma tumor model. In summary, NUCB-2 increased migration, invasion and EMT in renal cell carcinoma cells through the AMPK/TORC1/ZEB1 pathway in vitro and in vivo.

As2 O3 combined with leflunomide prolongs heart xenograft survival via suppressing the response of Th1, Th2, and B cells in a rat model.

Xenotransplantation remits the severe shortage of human organs and tissues for transplantation, which is a problem that severely limits the application of transplantation to the treatment of human disease. However, severe immune rejection significantly limits the efficacy of xenotransplantation. In this study, we systematically investigated the immunosuppressive effect and mechanism of action of As2 O3 and leflunomide using a hamster-to-rat heart xenotransplantation model. We initially examined heart xenograft survival following As2 O3 and leflunomide treatment alone or combined treatment. We found that treatment with As2 O3 combined with leflunomide can significantly prolong the survival of heart xenograft by inhibiting Th1 and Th2 differentiation and reducing the production of IgG and IgM. Interestingly, As2 O3 and leflunomide showed low toxicity to the organs of the recipient. Taken together, these observations indicate that treatment with As2 O3 combined with leflunomide may be a promising immunosuppressive schedule for xenotransplantation.

Antitumor effects of mutant endostatin are enhanced by Bcl-2 antisense oligonucleotides in UM-UC-3 bladder cancer cell line.

BACKGROUND: Endostatin is a potent inhibitor of tumor angiogenesis. In the preliminary studies, we developed a mutant endostatin containing Arg-Gly-Asp-Arg-Gly-Asp (RGDRGD) sequences. In this study, we compared the antitumor effects of mutant endostatin and Bcl-2 antisense oligonucleotides both in combination and individually. METHODS: The artificially synthesized Bcl-2 ASODN (antisense oligonucleotides) included a translation-initiation site and was transfected into the bladder cancer cells by Lipofectamine. Cell growth was investigated by the tumor cell growth chart, MTT assay, caspase-3 activity detection assay, AO/EB fluorescein stain, and the annexin V-FITC apoptosis detection assay. In the in vivo study, UM-UC-3 bladder cancer cells were subcutaneously implanted into nude mice and the growth of tumor was examined. The ultrastructure of the tumor tissues in the treated and control groups were observed. RESULTS: The cell growth chart showed that the cell population of the treated combination group decreased by 52.04% compared to the control group. The inhibition rate of the treated combination group was (79.66 ± 6.79)%, whereas those of the individual ASODN and ES groups were (53.39 ± 3.22)% and (50.22 ± 5.46)% respectively. In the caspase-3 activity detection using AO/EB fluorescein stain and annexin V-FITC apoptosis detection assay, the co-inhibitory effect was higher than the individual inhibitory effects (P < 0.05). There were significant differences in the inhibition of the solid tumor growth in the in vivo study. CONCLUSIONS: Our findings indicated that Bcl-2 antisense oligonucleotides enhance the antitumor effects of mutant endostatin both in vitro and in vivo. We noted the synergistic effects of Bcl-2 antisense oligonucleotides combined with mutant endostatin.

Ultrasound-guided minimally invasive percutaneous nephrolithotomy in flank position for management of complex renal calculi.

OBJECTIVES: To evaluate the safety and efficacy of performing ultrasound-guided minimally invasive percutaneous nephrolithotomy (MPCNL) in the flank position for the management of complex renal calculi. Percutaneous nephrolithotomy is usually performed with the patient in the prone position under fluoroscopic guidance; however, this position, and guidance method have some limitations. METHODS: From January 2007 to December 2009, 93 patients (101 kidneys) with complex renal calculi underwent ultrasound-guided MPCNL in the flank position. RESULTS: The mean age of the patients was 45.3 years (range 29-71). The calculi-free rate in the patients who underwent a single procedure was 78.2% (79 of 101 kidneys). The average operative duration was 82.6 minutes (range 45-190). Although the perioperative blood loss was not significantly different between single-tract and double-tract MPCNL (P = .087, F = 2.981), the calculi-free rate was significantly greater in the patients who underwent double-tract MPCNL than in those who underwent single-tract MPCNL (P = .027, chi-square = 4.873). Perioperative blood transfusions were not required in any patient. Similarly, ureteral calculi due to percutaneous nephrolithotomy were not observed. Secondary renal hemorrhage occurred in 3 patients who had undergone single-tract MPCNL and 1 underwent nephrectomy. CONCLUSIONS: The results of our study have shown that ultrasound-guided MPCNL with the patient in the flank position is safe and effective for treating complex renal calculi, without the side effects of radiation to the patient and surgeon. Double-tract MPCNL is suitable for complex renal calculi and, in some cases, is required to increase the calculi-free rate. The insertion of twin ureteral catheters before lithotripsy might be helpful in avoiding residual ureteral calculi after percutaneous nephrolithotomy.

[Effects of irrigating fluid absorption in percutaneous nephrolithotripsy].

OBJECTIVE: To determine the hemodynamic status, fluid-electrolyte changes and complications associated with irrigation time in percutaneous nephrolithotripsy. METHODS: A total of 68 renal calculi patients (31 males and 37 females) were recruited. The lateral recumbent percutaneous nephrolithotripsy was operated with Ho laser under ultrasonic guidance. 0.9% NaCI was used as perfusion fluid. The following items were recorded: mean arterial blood pressure (MAP), heart rate, central venous pressure (CVP), hemoglobin, sodium, potassium and chloride; perfusion time during operation; peri-operative and post-operative complications. RESULTS: (1) Peri-operative and post-operative conditions: the average operative time was 83.1 +/- 22.21 minutes. Two cases stopped because of bleeding after puncture and the tube of stoma was placed for stone clearance of the second time. There was more bleeding in 11 patients, but the operations were continued with blood transfusion and close monitoring. Two operations ceased because of a premunition of congestive heart failure. Nine patients needed post-operative blood transfusion and 18 had a post-operative fever. One patient bled in and around the tube and had a peri-renal infection a week later. (2) Changes of observation parameters: there was no significant difference in CVP, heart rate, hemoglobin, sodium, potassium and chloride (P > 0.05). The post-perfusion value of MAP increased (P < 0.05) especially in the cases of more bleeding and long time of irrigation. Peri-operative and post-operative injection of furosemide could reduce the CVP value. The average irrigation time in the fever group was longer than the non-fever group (P < 0.05) and the CVP value of the fever group was higher than the non-fever group (P < 0.05). CONCLUSION: Low pressure and short time of perfusion are safe in clinical practice. Congestive heart failure after the perfusion and the occurrence of post-operative infections are difficult to avoid when there are a long time of irrigation and more bleeding during operation.

[Treatment of ureteral obstruction by holmium: YAG laser endoureterotomy: a report of 18 cases].

OBJECTIVE: To investigate the clinical value and safety of holmium: YAG laser endoureterotomy in the treatment of ureteral obstruction. METHODS: Holmium: YAG laser endoureterotomy, with the laser optic fiber 550 microm in diameter and the output power of 3.5 Watt, via ureteroscopy, was performed on 18 patients ureteral obstruction, 8 males and 10 females, aged 52.1 (34-67), 11 with the stricture in the upper segment (complete obstruction in 4 cases), 5 in the middle segment, and 2 in lower segment; and 6 cases complicated with ureteral calculus. Postoperatively, an orthopedic ureteral stent (a 6-Fr double-J ureteral stent with a movable 5 cm length 9-Fr orthopedic cannula) was remained indwelling for 3-6 months. Follow-up was conducted for 10.7 (2-14) months. RESULTS: The operative duration was 32 (25-70) minutes. One patient underwent failed endoureterotomy and was turned to percutaneous nephroscopy. Success was achieved in 16 patients. The glomerular filtration rate (GFR) of these affected kidneys increased from 16.4+/-6.9 ml/min ante-operatively to 24.9+/-8.2 ml/min (P<0.01) postoperatively. One kidney was resected because of non-function, with GFR of 2 ml/min and intractable pyelitis. No recurrence of ureteral stricture was observed. CONCLUSION: Holmium: YAG laser endoureterotomy with insertion of orthopedic ureteral stent is an efficient and safe treatment for ureteral strictures with minimal invasion, less complications and easy recovery. This operation should be performed with a thorough preparation and severely restricted indication.

Mutational analysis of proto-oncogene Dbl on Xq27 in testicular germ cell tumors reveals a rare SNP in a patient with bilateral undescended testis.

OBJECTIVES: An abundance of X chromosomes in testicular germ cell tumors (TGCTs), and a candidate TGCTs susceptibility gene (TGCT1) on Xq27 highlight the potential involvement of X chromosomes in TGCT pathogenesis. However, the TGCT1 on Xq27 has so far not been identified. We hypothesized that a somatic mutation of dbl oncogene on Xq27 may play a role for the development of TGCTs. METHODS: We have screened 41 TGCT tissues for dbl mutations using single-strand conformation polymorphism (SSCP) analysis. These tissues are composed of 25 seminomatous TGCTs tissues and 16 non-seminomatous TGCTs tissues, including two cases with a rhabdomyosarcoma component. RESULTS: Somatic mutations were not detected in the 25 exons of dbl in these TGCTs. However, we found a rare single nucleotide polymorphism (SNP) (T to C nucleotide change) within intron 22 in one out of the 41 TGCTs cases (2%). Furthermore, the sample with the rare SNP was identified as the sole TGCTs case associated with bilateral undescended testis in our series. CONCLUSIONS: Our results indicate that proto-oncogene dbl is not a major target for sporadic TGCTs. However, the rare SNP in dbl may affect the susceptibility to undescended testis. Determining the frequency of this SNP in patients with various types of undescended testis in different ethnic groups is a warranted study.

[Optimization of extracting method and inhibitory action of recombinant human endostatin in bladder cancer].

OBJECTIVE: To explore the optimal method for protein expression in rhES (recombinant human endostatin) and study the anti-tumor activities of rhES in solid tumor and established cell line. METHODS: Different IPTG concentrations, the timing of adding IPTG into the culture medium and the different time of expression were employed to explore the optimized conditions of protein expression. Activity examination: (1) animal experiment: nude mice bearing subcutaneous cancer in treated group and controlled group were observed. (2) cellular experiment: the inhibitory effect of rhES in T-24 established cell line were observed by MTT assay and cancer cell growth curve. RESULTS: The expression of rhES protein was 58.65%. Of all the E. coli body proteins, the protein purity after purification was 96.22%. Activity examination indicated that rhES could inhibit the growth of solid tumor and the established cell line. In animal experiment, the tumor inhibition rate was 66.8%. Cell experiment: the 50% inhibitory concentration (IC(50)) was 22 microg/ml. The cell population decreased 58.75% than the control group at Day 7 in the tumor cell growth curve. CONCLUSION: A high expression and activity of rhES protein can be obtained by the optimized expression conditions. rhES can inhibit the cellular growth in both solid tumor and established cell line of bladder cancer.

[Effects of 4-hydroxytamoxifen on prostate smooth muscle cells: an in vitro experiment].

OBJECTIVE: To investigate the effects of 4- hydroxytamoxifen (OHT) on the proliferation and apoptosis of prostate smooth muscle cells and the expression of estrogen receptor (ER) and androgen receptor (AR). METHODS: Prostate smooth muscle cells were isolated from the resected specimens of prostate glands of 10 patients with benign prostatic hypertrophy (BPH), cultured, and exposed to estradiol (E(2)), diethylstilbestrol (DES), and OHT of different concentrations (1 x 10(-8) - 1 x 10(-5) mol/L) or mixture of E(2) (1 x 10(-8) - 1 x 10(-6) mol/L) with OHT (1 x 10(-7) mol/L). Flow cytometry was used to test the proliferation and apoptosis of the cells, and immunocytochemistry was used to test the expression of estrogen and androgen receptors. RESULTS: E(2) and DES promoted the proliferation of the prostate smooth muscle cells in a certain concentration range, but not dose-dependently, and OHT at the concentration of 1 x 10(-8) mol/L slightly increased the G(2)-M peak rate of the prostate smooth muscle cells, but suppressed the G(2)-M peak rate dose-dependently when its concentration was >or= 1 x 10(-7) mol/L (P < 0.05) and this suppression effect was dose-dependently (r = -0.312, P = 0.011). E(2) at the concentration >or= 1 x 10(-5) mol/L and DES at the concentration >or= 1 x 10(-6) mol/L slightly promoted the apoptosis of the prostate smooth muscle cells, but not dose-dependently, and OHT at the concentrations from 1 x 10(-8) mol/L to 1 x 10(-5) mol/L promoted the apoptosis of the prostate smooth muscle cells dose-dependently (r = 0.363, P = 0.021) and this effect could not be reversed by administration of E(2) at the concentration 1 x 10(-8) - 1 x 10(-6) mol/L (P > 0.05). E(2), DES, and OHT of different concentrations all increased the ERalpha and AR positive staining rates of the prostate smooth muscle cells (all P < 0.05). CONCLUSIONS: OHT suppresses the proliferation and promotes the apoptosis of prostate smooth muscle cells, and these functions do not depend on the estrogen receptor pathway. Low blood OHT concentration after oral administration of TAM and up-regulation of estrogen receptors by OHT may be the caused of the inefficiency of TAM for treatment of BPH.