Omar Hayek

TRANSDIFFERENTIATION OF PROSTATE CANCER CELLS TO A NEUROENDOCRINE CELL PHENOTYPE IN VITRO AND IN VIVO

PURPOSE To better understand the source of neuroendocrine cells associated with human prostate cancer progression, we studied the ability of a cultured prostate cancer cell line, LNCaP, to transdifferentiate into neuroendocrine-like cells in vitro and in vivo. MATERIALS AND METHODS Cyclic AMP concentrations were measured in extracts of LNCaP cells cultured in the presence of normal or hormone-deficient medium (containing charcoal-stripped serum) with the use of an immunoassay. Quantitative RT-PCR procedures were used to determine whether hormone depletion affects TGF-beta2 mRNA expression. Western blotting procedures (for neuron specific enolase [NSE]) were used to determine whether TGF-beta2 supplementation or antibody neutralization might affect the ability of cultured LNCaP cells to transdifferentiate to neuroendocrine-like cells. Finally, tumors formed from LNCaP cells xenografted into male nude mice were evaluated for the presence of neuroendocrine cells (prior and subsequent to castration of the host mouse) using an immunohistochemical stain for chromogranin A. RESULTS LNCaP cells cultured in a hormone-deficient medium have a mean 9-fold increase in cyclic AMP (p = 0.02) and a significant decline in the expression of TGF-beta2 mRNA when compared with cells grown in normal medium. Supplementation or depletion of TGF-beta2 did not affect the neuroendocrine conversion of LNCaP cells as assessed by NSE expression patterns. LNCaP tumors growing in castrated male nude mice were found to have significantly increased numbers of chromogranin A positive neuroendocrine cells (46/high powered field) when compared with tumors growing in intact male mice (3/high powered field) (p = 0.0038). CONCLUSIONS Exposure of LNCaP cells to a hormone deficient medium drastically increased cyclic AMP production and this may identify the biochemical pathway through which hormone depletion induces a neuroendocrine conversion of prostate cancer cells. Hormone depletion also reduced TGF-beta2 mRNA expression and this finding was consistent with our inability to demonstrate any effect of TGF-beta2 on neuroendocrine conversion in vitro. Finally, our demonstration of increased neuroendocrine cells found in LNCaP tumors growing in castrated immunodeficient mice suggests that the neuroendocrine cells associated with advanced human prostate tumors in vivo, arise from prostate cancer cells through the transdifferentiation process.

Salvage cryotherapy for recurrent prostate cancer after radiation therapy: the Columbia experience.

OBJECTIVES Cryotherapy of the prostate represents a potential treatment for localized recurrent prostate cancer after radiation therapy. We report our experience and evaluate the predictive factors for prostate-specific antigen (PSA) recurrence. METHODS Between October 1994 and April 1999, 43 patients underwent salvage cryoablation. All patients had biopsy-proven recurrent prostate cancer without seminal vesicle invasion, negative bone scans, and negative lymph node dissection. Patients had received 3 months of combined hormonal therapy before cryosurgery. Biochemical recurrence-free survival (bRFS) was defined as a PSA value less than 0.1 ng/mL. RESULTS Complications included incontinence (9%), obstruction (5%), urethral stricture (5%), rectal pain (26%), urinary infection (9%), scrotal edema (12%), and hematuria (5%). The mean follow-up was 21.9 months (range 1.2 to 54). Twenty-six patients (60%) reached a serum PSA nadir less than 0.1 ng/mL, 16 (37%) had a PSA less than 4 ng/mL, and 1 (3%) had a PSA less than 10 ng/mL. The bRFS rate was 79% at 6 months and 66% at 12 months. The bRFS rate was higher for patients who had an undetectable postcryotherapy PSA than for patients who did not reach a PSA less than 0. 1 ng/mL (73% versus 30%, P = 0.0076). Using multivariate analysis, a PSA nadir greater than 0.1 ng/mL was an independent predictor of PSA recurrence. CONCLUSIONS Current salvage cryotherapy of the prostate can result in undetectable serum PSA levels with low morbidity. Our data support the current safety and efficacy profile. We believe that cryotherapy is a viable option in the treatment of patients who have biopsy-proven local failure after radiation therapy for prostate cancer. Further refinements in technique and equipment may enhance cryosurgical results.

Herbal therapy PC-SPES: in vitro effects and evaluation of its efficacy in 69 patients with prostate cancer.

PURPOSE We investigate the potential use of the phytotherapeutic PC-SPES to treat human prostate cancer, and evaluate its in vivo and in vitro activity, and clinical efficacy. MATERIALS AND METHODS PC-SPES was evaluated for its ability to induce apoptosis on prostate cancer cell lines LNCaP, PC3 and DU145. The effect of oral PC-SPES on growth of PC3 tumors present in male immunodeficient mice was studied. A total of 30 male nude mice were divided in 5 groups. In groups 1 control and 2 full dose therapy was started the same day of the tumor injection. In groups 3 control, 4 half dose and 5 full dose PC-SPES therapy was initiated 1 week after tumor injection. A total of 69 patients with prostate cancer were treated with 3 capsules of 320 mg. PC-SPES daily. Serum prostate specific antigen (PSA) responses and side effects were evaluated. RESULTS All of the cultured prostate cancer cell lines had a significant dose dependent induction of apoptosis following exposure to an alcoholic PC-SPES extract. Immunodeficient mice xenografted with the PC3 cell line had reduced tumor volume compared with sham treated controls when they were treated with a PC-SPES extract from the time of tumor cell implantation (931 +/- 89 versus 1,424 +/- 685 mm.3, p not significant) but not when the treatment was begun 1 week after tumor cell implantation. The testis, prostate, bladder and seminal vesicles of the treated mice were significantly reduced in weight compared with the sham treated animals. Of the patients with prostate cancer 82% had decreased serum PSA 2 months, 78% 6 months and 88% 12 months after treatment with PC-SPES. Side effects in the treated patient population included nipple tenderness in 42% and phlebitis requiring heparinization in 2%. CONCLUSIONS An extract of the phytotherapeutic agent PC-SPES proved to be active in inducing apoptosis of hormone sensitive and insensitive prostate cancer cells in vitro, and in suppressing the growth rate of a hormone insensitive prostate cancer cell line in vivo. The overwhelming majority of patients with prostate cancer treated with the agent experienced a decrease in serum PSA but also demonstrated a side effect profile comparable to estrogen treatment.

CASTRATION INDUCES ACUTE VASOCONSTRICTION OF BLOOD VESSELS IN THE RAT PROSTATE CONCOMITANT WITH A REDUCTION OF PROSTATIC NITRIC OXIDE SYNTHASE ACTIVITY

PURPOSE Previous studies demonstrating a rapid and drastic reduction of blood flow to the rat prostate gland resulting from castration caused us to consider the influence of castration on the state of vascular constriction and on the activity of the vascular tone-regulating factors (nitric oxide synthase and cyclic GMP) in the rat prostate. MATERIALS AND METHODS Sections of ventral prostate glands obtained from intact and castrated rats were analyzed for the mean areas within smooth muscle-coated blood vessels using a computerized microscopic image analysis system. Nitric oxide synthase (NOS) levels were measured in prostatic extracts from unoperated or castrated rats using an enzyme assay system that measures conversion of 3H-L-arginine to citruline. Cyclic GMP levels were measured in prostatic extracts from unoperated or castrated rats using a competitive radioimmunoassay system. RESULTS The mean area within ventral prostate smooth muscle-coated blood vessels was reduced 39% at 24 hours after castration (p = 0.039) and 47.7% at 48 hours after castration (p = 0.039). NOS activity measured in prostatic extracts was reduced 38% at 24 hours (p = 0.0012) and 51.6% at 36 hours after castration (p = 0.0001) compared with the control group of noncastrated rats. Finally, prostatic cGMP levels were reduced 55.8% (p = 0.0018) at 36 hours after castration when compared with controls rats. CONCLUSION Within 24 hours after castration, the lumenal areas of smooth muscle-coated blood vessels in the rat prostate gland were found to be significantly reduced. This vasoconstriction was associated with a significant reduction of prostatic NOS activity as well as a reduction in the prostatic levels of the NOS co-factor, cGMP. Thus, acute vasoconstriction is a prominent early event associated with rat prostate regression in response to castration and likely contributes to the regression of the tissue.

Vascular endothelial growth factor-A expression in the rat ventral prostate gland and the early effects of castration.

Blood flow to the rat ventral prostate gland is drastically reduced during the very early period after castration, and this reduction coincides with the appearance of striking degenerative changes within the prostatic vascular system. These early effects on the prostate vascular system are likely to be important for the subsequent regression of the ventral prostate that occurs in response to castration. Since the endothelial cells of the ventral prostate do not express androgen receptor protein (AR), we proposed that these early effects might be indirectly mediated by changes in the local expression of vascular regulatory factors. In order to evaluate whether vascular endothelial growth factor‐A (VEGF‐A) might be among the primary mediators of these effects, we measured expression of VEGF‐A mRNA and protein in the rat ventral prostate gland prior to and within the first 3 days after castration.

Acute increase in blood flow to the rat bladder subsequent to partial bladder outlet obstruction.

Partial obstruction of the rat bladder outlet initiates a multi‐step process during which the bladder progressively loses its functional ability. The first step in this progression is bladder hypertrophy; the organ dramatically increases in size and weight to compensate for the effects of obstruction. Unoperated female rats, age‐matched, sham‐obstructed rats, and rats that received a partial bladder outlet obstruction were studied. During the first 24 hours after partial bladder outlet obstruction, relative bladder blood flow was measured using a fluorescent microsphere infusion technique and laser Doppler imaging. Nitric oxide synthase (NOS) activities of control and obstructed rat bladder tissues were determined using an enzymatic assay that measures the conversion of 3H‐L‐arginine to 3H‐L‐citrulline. Using the microsphere infusion technique, a significant increase in blood flow to the obstructed rat bladder was observed during the first 24 hours after partial bladder outlet obstruction. Relative bladder blood flow increased approximately sixfold at 4 and 6 hours post‐obstruction and remained elevated through 24 hours of obstruction. Sham operations (evaluated after 6 hours after surgery) resulted in a minor increase in blood flow that did not reach statistical significance. Relative blood flow to the spleen, measured in the same rats, was not significantly changed. Laser Doppler measurements also identified a significant increase in rat bladder blood flow after outlet obstruction and showed that increased blood flow could be detected as early as 1 hour post‐obstruction. Interestingly, despite the significant differences in bladder blood flow between control and early post‐obstructed rat bladders, NOS activities of control and obstructed rat bladders were comparable. The increase in bladder blood flow precedes the urothelial, fibroblast and smooth muscle cell hyperplasia, and the smooth muscle hypertrophy that occurs after obstruction. We propose that, in response to surgical induction of partial outlet obstruction, acute up‐regulation of bladder blood flow may be an initiating factor for subsequent bladder cell proliferation and smooth muscle hypertrophy. Neurourol. Urodynam. 19:195–208, 2000.

Blood-based RT-PCR assays of MN/CA9 or PSMA: clinical application in renal cancer patients

OBJECTIVES To report a survey of blood-based RNAs obtained from common groups of control and renal cancer patients for expression of both MN/CA9 and prostate-specific membrane antigen (PSMA) messenger RNAs. METHODS Reverse transcription polymerase chain reaction (RT-PCR) assays for MN/CA9 and PSMA were performed on RNAs extracted from 81 blood samples (59 patients with renal cancer, 7 with benign tumors, and 15 control volunteers). The results of these assays were statistically analyzed to determine whether a positive result (individually or combined) correlates with any tumor characteristics. RESULTS Neither MN/CA9 nor PSMA amplification products were detected in the RNAs from peripheral blood samples of the 15 control volunteers and from the 7 patients with benign renal tumor (sensitivity 100%). MN/CA9 alone was detected in 11 (19%) of 59 samples and PSMA alone in 12 (20%) of 59 samples from patients with renal cancer. PSMA positivity was significantly correlated with vascular invasion of the primary tumor. Expression of one or both of these molecular tumor markers was detected in 21 (36%) of 59 renal cancer patients. When combined, the results of the MN/CA9 and PSMA RT-PCR tests were found to be highly associated with vascular invasion in nephrectomy specimens (sensitivity 67%, specificity 77%, odds ratio = 6.89, P = 0.002). CONCLUSIONS Combination of RT-PCR assays for MN/CA9 and PSMA provides a sensitive blood test for molecular detection of clear cell carcinoma of the kidney and its potential for vascular invasion. Further testing of this assay will be required to evaluate its efficacy in the diagnosis, screening, and follow-up of patients with kidney cancer.

Evaluation of the Incidence of Bladder Perforation After Transurethral Bladder Tumor Resection in a Residency Setting

PURPOSE To evaluate prospectively the actual bladder perforation incidence during transurethral resection of bladder tumor (TURB) performed by residents and to identify possible predisposing factors to such condition. PATIENTS AND METHODS Thirty-four patients with bladder tumor were submitted to TURB in our academic institution in April 2006, and were prospectively studied. Procedures were all done by senior residents under an attending direct supervision. All patients had a cystograms performed after the procedure by the injection of 400 mL of saline-diluted contrast solution with low-pressure infusion through the Foley catheter. The cystograms were evaluated blindly by a single radiologist. All patients were examined by cystoscopy and/or CT every 3 months for the first 2 years postoperatively. RESULTS The cystogram showed contrast leaking compatible with bladder perforation in 17 (50%) cases. None of the perforations were recognized intraoperatively by the surgeon. All perforations were extraperitoneal and managed conservatively. There was no significant correlation between the incidence of bladder perforation and the patient age (p = 0.508), the tumor stage (p = 0.998), the tumor grade (p = 0.833), the number of lesions (p = 0.394), and the tumor size (p = 0.651). The only factor that had impact on the development of bladder perforation was tumor localization at the bottom of the bladder (p = 0.035; OR, 6750; 95% CI, 1.14, 39.8). CONCLUSION Asymptomatic perforations of the bladder wall occur very frequently after a TURB procedure performed by residents in training and, most of the time, are not noticed by the surgeon. Localization of the tumor at bladder dome was the only factor that negatively influenced perforation rates.

The necessity of a second prostate biopsy cannot be predicted by PSA or PSA derivatives (density or free:total ratio) in men with prior negative prostatic biopsies.

Serum prostate specific antigen, prostate specific antigen density and free:total prostate specific antigen are known to be useful for determining the risk of prostate cancer in patients undergoing prostate cancer screening. The patient with a positive biopsy presents no future prostate specific antigen dilemma. Those with negative biopsies often go on to numerous repeat biopsies. Our goal was to establish criteria that could be used to identify patients who will require repeat prostate biopsies (possibly false negative initial biopsy), while not exposing the low risk population (probable true negative initial biopsy) to additional invasive procedures. Between March 1991 and March 1998, 148 patients who had a biopsy for an elevated prostate specific antigen value (4.1-10.0) or an altered digital rectal examination, had no cancer found in the specimen. From these, 51 (34.4%) had repeated biopsies, while the others persisted on close follow-up. We examined their serum prostate specific antigen, prostate specific antigen density and free:total prostate specific antigen value, as well as their age and histology results of the initial and repeat biopsy, to determine if any predictor of the need for a repeat biopsy could be identified. Eight (15.7%) from 51 men who had repeat biopsy had prostate cancer detected. Forty three (84.3%) patients persisted with a negative biopsy, despite filling the criteria for re-biopsy. Multivariate analysis failed to identify any significant predictors of prostate cancer in the repeat biopsy group. Despite initial success, the prostate specific antigen derivatives and free:total prostate specific antigen have not safely limited the number of biopsies performed for an abnormal prostate specific antigen (4.1-10.0). Neither prostate specific antigen density nor free:total prostate specific antigen predicted the need for repeat biopsy in this specific group. The results of this ongoing study demonstrate that to date, prostate specific antigen and prostate specific antigen derivatives can not be utilized to determine which patients will be at high risk for requiring repeat prostate biopsy. All patients must be closely monitored for evidence of a change in status from benign to malignant disease, and new markers for this purpose are urgently needed.

Vascular anatomy of the rat ventral prostate

The rat ventral prostate gland is a model tissue to study the effects of androgenic steroids on prostate cells. Recent reports suggest that the prostatic vascular system is a primary target of androgen action in this tissue. In order to better understand how the vascular system of the ventral prostate supports the tissue in an androgenically normal adult male rat we utilized a variety of microscopic imaging techniques to more fully characterize its structural anatomy and its interaction with other prostatic cell types.