Fernando Ferrer

Vascular Endothelial Growth Factor (VEGF) Expression in Human Prostate Cancer: In Situ and in Vitro Expression of VEGF by Human Prostate Cancer Cells

PURPOSE A growing body of literature supports the role of angiogenesis in the development and spread of a variety of human cancers including prostate cancer (Pca). Angiogenesis is controlled by chemical signals known as angiogenic factors (AF) however, little is known about angiogenesis factors in prostate cancer. We evaluated the in situ and in vitro expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor, in archival prostate cancer specimens and prostate cancer cell cultures during unstimulated and cytokine stimulated conditions. METHODS Ex-vivo studies involved immunohistochemical analysis for VEGF expression and distribution in 25 archival specimens including, prostate cancer, benign prostatic hyperplasia (BPH) and normal prostate tissue. In-vitro studies utilized prostate cancer cells (DU-145) grown in culture and stimulated with cytokines thought to induce VEGF (i.e. IL-1 alpha, IL-1 beta, TNF-alpha and TNF-beta). Cell culture supernatants were analyzed by ELISA for VEGF levels. RESULTS Immunohistochemical studies demonstrated that in 20 of 25 specimens prostate cancers cells stained positively for VEGF. BPH and normal prostate cells displayed little staining for VEGF. DU-145 prostate cancer cells produced low levels of VEGF in unstimulated conditions. Induction of DU-145 cells with cytokines resulted in differential stimulation whereby TNF was a potent inducer of VEGF, and IL-1 produced lesser but statistically significant increases in VEGF expression. CONCLUSIONS Our immunohistochemical results indicate that significant levels of VEGF are present in prostate cancer, but not in BPH or normal prostate cells in-vivo. In-vitro studies suggest that differential regulation of angiogenesis factor expression by IL-1 and TNF occurs in prostate cancer. Identifying the angiogenesis factors involved in prostate cancer growth and understanding their regulation will lead to the development of anti-angiogenic strategies useful for diagnostic studies and therapeutic interventions.

Angiogenesis and prostate cancer : In vivo and in vitro expression of angiogenesis factors by prostate cancer cells

OBJECTIVES Recently, it was confirmed that angiogenesis is important in the development and spread of a variety of human cancers, including prostate cancer (PCa). Tumor neovascularization is thought to be controlled by chemical signals, known as angiogenic factors (AF). To date, little is known regarding the existence and role of AF in PCa. We previously reported on vascular endothelial growth factor (VEGF) in PCa. Currently, we compare VEGF expression with that of interleukin-8 (IL-8), another putative regulator of angiogenesis. We evaluated the expression of these two important AF in PCa and explored the role of inflammatory cytokines IL-1 and tumor necrosis factor (TNF) in their regulation. METHODS Ex vivo studies involved previously reported immunohistochemical analysis for VEGF and recent evaluation of IL-8 expression and distribution in archival tissue samples of PCa, benign prostatic hyperplasia (BPH), and normal prostate tissue. In vitro studies used PCa cells (DU-145) grown in culture and stimulated with cytokines thought to induce VEGF and IL-8 (ie, IL-1 alpha, IL-1 beta, TNF-alpha, and TNF-beta). After 24 hours, with or without cytokines, cell culture supernatants were analyzed by enzyme-linked immunosorbent assay or radioimmunoassay for VEGF or IL-8 levels. RESULTS Immunohistochemical studies of prostate tissue showed that PCa cells stained positively for VEGF and IL-8. Benign prostatic hyperplasia and normal prostate cells displayed little staining for either AF. Low levels of VEGF and IL-8 were produced by unstimulated DU-145 cells. Induction of DU-145 cells with cytokines resulted in differential stimulation whereby TNF was the predominant inducer of VEGF, whereas IL-1 was the predominant inducer of IL-8. CONCLUSIONS Our results indicate that significant levels of VEGF and IL-8 are present in PCa, but not BPH or normal prostate cells in vivo. In vitro studies suggest that differential regulation of AF expression occurs in PCa. Because IL-1 and TNF are present in the PCa tumor microenvironment, it is likely that differential regulation of AF also occurs in human PCa and contributes to differential tumor growth and metastasis.

Expression of vascular endothelial growth factor receptors in human prostate cancer

OBJECTIVES We recently reported the expression and cytokine regulation of vascular endothelial growth factor (VEGF) in human prostate cancer (PCa). VEGF exerts its angiogenic and pro-tumorigenic properties by way of two high affinity receptors, fms-like tyrosine kinase 1 (FLT-1) and fetal liver kinase 1 (FLK-1). We hypothesized that these receptors are expressed and control VEGF functions in the PCa microenvironment. Herein, we evaluate the expression of these receptors in ex vivo PCa tissue, benign prostatic hypertrophy (BPH) tissue, and cultured PCa cell lines. METHODS Ex vivo PCa specimens were obtained from patients undergoing radical retropubic prostatectomy. Specimens were selected to contain both PCa and BPH tissue (n = 15). Immunohistochemical analysis using antihuman FLT-1 and FLK-1 was performed and specimens were analyzed to characterize the expression and distribution of both receptors. Immunocytochemical analysis for FLT-1 and FLK-1 was also performed on cultured PCa cell lines (DU-145 and LNCaP). RESULTS PCa cells expressed the VEGF receptor FLT-1 in 100% of specimens evaluated. Expression of FLK-1 was variable and related to tumor grade; high-grade tumors displayed little or no FLK-1 expression. Vascular endothelial cells (VECs) within areas of PCa consistently expressed both FLT-1 and FLK-1 receptors. FLT-1 and FLK-1 were both expressed in BPH tissue. FLT-1 was expressed in the glandular epithelial cells in BPH, but in most cases FLK-1 was localized specifically to the basal cell layer of hypertrophic glands. FLT-1, but not FLK-1, was expressed by the DU-145 and LNCaP cell lines. CONCLUSIONS Although they are differentially expressed, both FLT-1 and FLK-1 are present in PCa and BPH. Expression of receptors on VECs of tumor vessels supports the well-established role of VEGF in paracrine stimulation of VECs in the tumor microenvironment. The expression of FLT-1 and FLK-1 on tumor cells themselves suggests a potential autocrine function for VEGF (such as regulating tumor cell proliferation). These findings imply that a novel dual role may exist for VEGF, such that it is involved in tumor cell activation (autocrine), in addition to paracrine actions whereby it regulates endothelial cell functions and subsequent neovascular development.

The G Protein–Coupled Receptor S1P2 Regulates Rho/Rho Kinase Pathway to Inhibit Tumor Cell Migration

Sphingosine 1-phosphate (S1P) is a lysophospholipid that exerts a variety of responses in cells such as proliferation, migration, and survival. These effects are mediated by G protein-coupled receptors on the cell surface (S1P1-5), which activate downstream signaling intermediates such as Rac and Rho GTPases. Mechanisms of S1P action in human glioblastoma cells are not well defined. S1P receptors (1-5) and S1P-metabolizing enzymes were expressed in three human glioblastoma cell lines. S1P had a profound and differential effect on glioblastoma cell migration. U87 cells treated with S1P showed a significant increase in migration, whereas U118 and U138 cell lines were strongly inhibited. S1P-mediated inhibition correlated with S1P2 receptor expression. FTY720-P, an S1P analogue that binds all S1P receptors except S1P2, did not inhibit glioblastoma cell migration. Overexpression of S1P2 further suppressed migration, and blockage of S1P2 mRNA expression by small interfering RNA reversed the inhibitory effect. Contrary to previous reports showing bimodal regulation of Rac activity and migration by S1P2 receptor stimulation, both Rac1 and RhoA GTPases were activated by S1P treatment in native cells and cells overexpressing S1P2. Treatment of U118 cells with the Rho-associated protein kinase (ROCK) inhibitor Y-27632 restored migration suggesting that ROCK-dependent mechanisms are important. Actin staining of S1P stimulated U118 cells overexpressing beta-galactosidase resulted in pronounced stress fiber formation that was exacerbated by S1P2 overexpression, partially blocked by S1P1, or totally abolished by pretreatment with Y-27632. These data provide evidence of a novel mechanism of S1P inhibition of tumor cell migration via Rho kinase-dependent pathway.

PELVIC FLOOR MUSCLE RETRAINING FOR PEDIATRIC VOIDING DYSFUNCTION USING INTERACTIVE COMPUTER GAMES

PURPOSE We evaluated a new noninvasive outpatient method of pelvic muscle retraining in children using computer game assisted biofeedback. MATERIALS AND METHODS All patients in whom voiding dysfunction was confirmed by history, uroflowmetry-electromyography and voiding cystourethrography were enrolled in a pelvic floor muscle retraining program. Patients received a pretreatment, mid treatment and posttreatment survey instrument documenting subjective improvement, including the frequency of diurnal enuresis, nocturnal enuresis, constipation and encopresis. Pretreatment and posttreatment simultaneous uroflowmetry surface electrode electromyography was performed and post-void residual urine volume was determined in all patients. RESULTS A total of 8 boys and 33 girls 5 to 11 years old (mean age 7.2) completed therapy and were available for evaluation. These patients completed 2 to 11 (average 6) hourly treatment sessions. Followup was 3 to 15 months (average 7). At the midterm evaluation improvement in nocturnal enuresis was reported by 57% of the patients, diurnal enuresis by 84%, constipation by 83% and encopresis by 91%. End treatment evaluation revealed improvement in nocturnal enuresis by 90% of patients, diurnal enuresis by 89%, constipation by 100% and encopresis by 100%. Uroflowmetry-electromyography patterns improved in 42% of the patients and post-void residual urine decreased in 57%. Comparison of initial to end recorded millivoltage pelvic floor muscle values demonstrated that 56% of the patients had lower resting tone at the beginning of the session after completing therapy and 78% had improved contracting tone after performing Kegel exercises, as proved by increased microvoltage values. Initial uroflowmetry-electromyography revealed certain categories of cases, including a flattened voiding curve with a hyperactive pelvic floor and low post-void residual urine in 40%, a flattened voiding curve with a hyperactive pelvic floor and high post-void residual urine in 40%, a staccato voiding curve with a hyperactive pelvic floor and low post-void residual urine in 3%, and a staccato voiding curve with a hyperactive pelvic floor and high post-void residual urine in 17%. Of the girls 91% presented with the classic spinning top deformity on voiding cystourethrography. A total of 22 patients presented with a significant history of recurrent urinary tract infections, and infection developed in 3 during treatment and followup. Vesicoureteral reflux in 14 patients resolved during treatment in 3, reimplantation was performed in 1 and 10 are still being observed. CONCLUSIONS A program of conservative medical management with computer game assisted pelvic floor muscle retraining resulted in significant subjective improvement in continence, constipation and encopresis as well as objective improvement in uroflowmetry-electromyography, post-void residual urine volume and the microvoltage value of pelvic floor muscles in the majority of patients with dysfunctional voiding.

A comparison of open vs laparoscopic adrenalectomy

AbstractBackground: To compare the outcome of patients who underwent laparoscopic transabdominal adrenalectomy (LA) with those who had open adrenalectomy (OA). Methods: A retrospective review of consecutive adrenalectomies performed by a single surgical team at a university hospital. Outcome measurements were operative time, operative blood loss, procedure-related complications, postoperative stay, and return to regular activity. Results: Twenty-nine adrenalectomies were done in 23 patients during a 54-month period. There were 12 OAs performed in nine patients and 17 LAs were done in 14 patients. Both groups were similar in their demographics and their indications for operation. All attempted LAs were successfully completed. The mean operative time was longer for LA than for OA (289 vs 201 min; p= 0.042). Resumption of oral intake (1.0 vs 3.0 days; p= 0.002), postoperative hospital stay (3.0 vs 7.9 days; p= 0.002), and return to regular activity (8.9 vs 14.6 days; p= 0.002) were significantly shorter after LA than after OA. There were no postoperative deaths and there was no difference in operative blood loss between the two groups. Procedure-related complications occurred in three patients having LA and in five patients having OA. Conclusions: Patients having LA had longer operative procedures but shorter hospital stays and faster return to normal activity than patients having OA. Procedure-related complications for LA were due to bleeding into the retroperitoneum or abdominal wall. Significant postoperative cardiac and respiratory complications occurred only in the OA group.

Comparison of outcomes based on treatment algorithms for rhabdomyosarcoma of the bladder/prostate: combined results from the Children's Oncology Group, German Cooperative Soft Tissue Sarcoma Study, Italian Cooperative Group, and International Society of Pediatric Oncology Malignant Mesenchymal Tumors Committee

The purpose of this study was to determine patient characteristics and outcomes for bladder/prostate (BP) rhabdomyosarcoma (RMS) using an international cohort of prospectively treated patients comparing different treatment algorithms. Data were collected from 379 patients (1979–1998) treated on protocol; Intergroup Rhabdomyosarcoma Study, IRS‐IV (n = 239 patients), International Society of Pediatric Oncology Malignant Mesenchymal Tumors (MMT) Committee MMT‐84 and ‐89 (n = 74), Italian Cooperative Group, RMS‐79 and RMS‐88 Studies (n = 37) or German Cooperative Soft Tissue Sarcoma Study CWS‐91 protocols (n = 29). A total of 322 (85%) patients had localized embryonal RMS (ERMS) and 27 had metastatic disease. Thirty patients (21 local disease; 9 metastatic) had nonembryonal BP RMS. Patients with localized ERMS had large tumors (64% >5 cm) that were invasive (54%) with uninvolved regional lymph nodes (N0, 93%). The 5‐year failure‐free survival (FFS) was 75% and the overall survival (OS) was 84%, with 89% of deaths attributed to disease. Treatment failures were usually local disease recurrence (60%). Predictors of FFS included T‐stage (invasiveness), size, and histology. FFS was decreased for patients not receiving initial radiotherapy but this did not translate into a decreased OS. The 21 patients with localized nonembryonal BP RMS had a FFS and OS of 47%. The 36 patients with metastatic disease were more likely to be older and had large tumors that were invasive with alveolar histology and regional lymph node involvement. The 5‐year FFS and OS were 41 and 44%, respectively. In conclusion, the majority of BP RMS patients had localized ERMS with a resultant good prognosis using current treatment algorithms. There were differences in FFS between treatment protocols but this did not result in an altered OS.

S1P/S1P1 signaling stimulates cell migration and invasion in Wilms tumor.

Sphingosine-1-phosphate (S1P) is an important regulator of cellular functions via interaction with its receptors S1P(1-5). To date, nothing is known about the S1P receptor expression and the effects of S1P signaling in Wilms tumor. In this study, we found ubiquitous expression of S1P receptors in Wilms tumor specimens and cell lines. We demonstrated that S1P(1) acted as a promigratory modulator by employing S1P(1) antagonist VPC44116, S1P(1) siRNA and adenoviral transduction in Wilms tumor cells. Further, we clarified that S1P(1)-mediated migration occurred via Gi coupling and activation of PI3K and Rac1. In addition, S1P stimulated WiT49 cell invasion through S1P(1)/Gi signaling pathway. We consider that targeting S1P(1) may be a point of therapeutic intervention in Wilms tumor.

Clinical and molecular analysis of XX sex reversed patients.

PURPOSE The XX male syndrome presents with a spectrum of clinical appearances from phenotypic male individuals to true hermaphrodites. Previous reports established the sex determining region of the Y chromosome (SRY) gene as the testis determining factor. However, a subset of XX sex reversed male individuals exists without a translocation of SRY deoxyribonucleic acid (DNA) material to the X chromosome. In addition to clinical or endocrinological criteria, Y DNA probe studies, and radiological and surgical evaluation as indicated are necessary for an accurate diagnosis. MATERIALS AND METHODS We evaluated 5 XX sex reversed patients (2 true hermaphrodites and 3 male individuals) by physical examination, pedigree analysis, endocrinological testing, molecular analysis of Y DNA, radiological studies and surgery (exploration and/or biopsy). RESULTS All patients were SRY gene negative. Two patients were siblings. Complete endocrinological testing was negative in all cases. Two patients had a normal male phenotype. Radiological findings confirmed by cystoscopy or laparoscopy revealed a utricle, vesicoureteral reflux, and cervix and uterus in various patients. Gonadal biopsy showed ovotestes or ovary and testis in the 2 true hermaphrodites. The 3 XX male individuals had normal immature testes on biopsy. CONCLUSIONS Categories of XX sex reversal include classic XX male individuals with normal phenotypes, nonclassic XX male individuals with sexual ambiguity and XX true hermaphrodites. Simple translocation of the SRY gene to the X chromosome does not always account for testicular differentiation and a male phenotype. The masculinization of our patients in the absence of SRY suggests an alteration of 1 or more downstream Y, X or autosomal testis determining genes. We present another theory for male sex determination, including a downstream gene on the X chromosome in which expression is influenced by X inactivation. Y DNA genomic analysis, radiological studies and laparoscopic evaluation with gonadal biopsy as appropriate are recommended for complete assessment and treatment of these intersex patients.

Bilateral laparoscopic adrenalectomy for adrenocorticotropic dependent Cushing's syndrome.

PURPOSE We report our experience with bilateral laparoscopic adrenalectomy for total adrenal ablation in patients with Cushings syndrome. MATERIALS AND METHODS Four women (mean age 63 years) with Cushings syndrome secondary to nonlocalized ectopic adrenocorticotropic hormone production in 3 and pituitary microadenoma after failed transsphenoidal ablation in 1 underwent bilateral transabdominal laparoscopic adrenalectomy. Preoperatively risk was III or IV according to the American Society of Anesthesiologists classification. RESULTS In all cases bilateral laparoscopic adrenalectomy was successfully performed. Operative time ranged from 375 to 475 minutes (mean 404) and mean blood loss was 162 cc. All patients resumed oral intake on postoperative day 1, mean number of postoperative parentral narcotic doses was 2.25 and mean postoperative hospital stay was 5.75 days (range 3 to 8). Complications included an abdominal wall hematoma. All patients resumed baseline activity by postoperative day 14. CONCLUSIONS Our experience in 4 cases of Cushings syndrome suggests that bilateral laparoscopic adrenalectomy is a safe and effective alternative to open adrenalectomy. Further experience with this technique will likely decrease operative time, and confirm the benefit of a decreased hospital stay and convalescence.