Tatjana Burchardt

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.

Erectile dysfunction is a marker for cardiovascular complications and psychological functioning in men with hypertension.

The aim of this study was to investigate the incidence of cardiovascular complications in hypertensive patients with erectile dysfunction (ED). An anonymous questionnaire was mailed to 467 and received from 104 hypertensive male patients. Despite the low response rate of 22%, the following interesting findings could be observed: 70.6% of the patients who responded suffered from ED. The hypertensive patients with ED had significantly higher prevalence of cardiovascular complications (P<0.05). The correlation between depression and low quality of life as well as between ED and low sexual satisfaction was also statistically significant (P=0.05). ED in hypertensive patients can be considered as a marker for cardiovascular complications in this patient group.

Microvessel density as a predictor of PSA recurrence after radical prostatectomy. A comparison of CD34 and CD31.

Whether prostate cancer recurrence can be predicted by microvessel density (MVD) measurements is controversial. One reason for the lack of agreement may be the differing antibodies used to determine MVD. We evaluated MVD using 2 different antibodies against endothelial cells, CD31 and CD34, on 102 patients who underwent radical prostatectomy without adjuvant hormonal therapy. The tumors from these cases were identified, and areas with the highest Gleason pattern were immunostained. Average MVD determined by CD31 (MVD/CD31) staining was significantly lower than that obtained by MVD/CD34 staining (60.1 vs 80.3). By using Kaplan-Meier analysis, prostate-specific antigen (PSA) recurrence was correlated with MVD/CD31 and MVD/CD34. MVD/CD34 and MVD/CD31 were associated strongly with PSA recurrence on a univariate level. However, only MVD/CD34 was an independent predictor of PSA failure. Therefore, some of the confusion about MVD value as a prognostic indicator may be due to the antibodies used.

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.

CAN PERINEURAL INVASION ON PROSTATE NEEDLE BIOPSY PREDICT PROSTATE SPECIFIC ANTIGEN RECURRENCE AFTER RADICAL PROSTATECTOMY

PURPOSE We evaluated the role of perineural invasion identified on prostate needle biopsy as a predictor of prostate specific antigen (PSA) recurrence after radical prostatectomy. MATERIALS AND METHODS Between 1993 and 1998 radical prostatectomy was performed in 319 consecutive patients. Prostate needle biopsies were reviewed in all cases. We compared perineural invasion with other preoperative parameters, including digital rectal examination, PSA and biopsy Gleason score, for the ability to predict PSA recurrence with recurrence defined as any serum PSA level greater than 0.2 ng./ml. RESULTS Perineural invasion was identified on 77 of 319 preoperative prostate biopsies (24%). There was PSA recurrence in 46 patients (14.4%) at a mean followup of 25.4 months (range 0.2 to 62.1). Perineural invasion statistically correlated with PSA recurrence. Kaplan-Meier analysis revealed disease-free survival rates of 24 versus 64% when perineural invasion was and was not present in the prostate biopsy (p = 0.0003, log rank 12.92). Multivariate analysis demonstrated that perineural invasion (p = 0.012) and PSA (p = 0.005) were independent preoperative predictive factors of PSA recurrence. When perineural invasion was compared with postoperative parameters, including disease stage, surgical margins and seminal vesicle invasion, it was not an independent predictor because it closely correlated with tumor stage. CONCLUSIONS Perineural invasion on preoperative prostate needle biopsy is a strong independent predictor of PSA recurrence in patients in whom prostate cancer was treated with radical prostatectomy.

Inhibition of p53 function diminishes androgen receptor-mediated signaling in prostate cancer cell lines

Current therapy for advanced prostate cancer is mainly based on androgen deprivation, although most patients relapse to androgen-insensitive disease. Several mechanisms contributing to androgen-independent growth including alterations in the structure or expression of the androgen receptor (AR) and its cofactors have been identified. Recent evidence suggests that p53 is involved in androgen signaling. The analysis of the effect of p53 on androgen signaling was performed in 22Rv1 and LNCaP prostate cancer cells that express both p53 and AR. The overexpression of p53 diminished the androgenic response in both cell lines in a reporter gene assay. Conversely, the inhibition of p53 by three different p53 inhibitors, Pifithrin-1α (PFT-1α), an inhibitor of p53-dependent transactivation; MDM2, a regulator of p53 expression; and a dominant-negative N-terminally truncated p53 gene also reduced transactivation of androgen-dependent reporter genes. The inactivation of p53 by PFT-1α decreased AR-protein expression in both 22Rv1 and LNCaP cells. Our findings confirm that the overexpression of wild-type p53 decreases androgen function, whereas p53 expression at physiological levels stabilizes AR signaling. Thus, our findings suggest that there is a balance of AR and p53 expression during the androgen-dependent growth of prostate cancer, which is obliterated during further progression of the disease.

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.

Expression of VEGF Splice Variants 144/145 and 205/206 in Adult Male Tissues

Currently, at least five different mRNA species encoding vascular endothelial growth factor‐A (VEGF‐A) have been characterized. These variants result from alternative splicing of the VEGF‐A transcript and encode human isoforms of VEGF protein of 121, 145, 165, 189, and 206 amino acids. In the rat, a similar profile of VEGF‐A splice variants has been described, each encoding one fewer amino acid than the human species. Studies of mammalian tissues have shown that these mRNA isoforms vary in abundance. Whereas VEGF 188/189, 164/165, and 120/121 (rat/human, respectively) are the predominant forms expressed in most tissues and cells examined, VEGF 144/145 and 205/206 are rare variants. Previously, VEGF 144/145 had been detected only in placental and uterine tissues and endometrial carcinoma cell lines, whereas VEGF 205/206 was detected only in fetal liver and placenta. Using an RTPCR technique, cDNA cloning, and sequencing, we have detected and confirmed the presence of mRNA encoding VEGF 144/145 and 205/206 in both adult rat lung and penis. Therefore, these lowabundance VEGF splice variants have a more diverse expression pattern than originally predicted.

Sexual dysfunction is common and overlooked in female patients with hypertension.

Our objective was to investigate sexual activity, behavior, dysfunction, and satisfaction in hypertensive women. Sixty-seven patients with a mean age of 60.4 years completed a detailed questionnaire. Of these women, 81.3% had a sex partner; 42.6% had untreated sexual dysfunction with a duration of more than 5 years in 70.9% and a duration of more than 10 years in 41.7%; 5.3% initiated sexual activity; 36.6% reported less sexual activity than desired; and 54.8% reported sexual activity as important. Our study revealed highly prevalent untreated sexual dysfunction of long duration. It also showed low frequency of sexual activity in spite of the high availability of partners. There was low frequency of initiation of sexual activity. In spite of the high prevalence of sexual dysfunction, more than a third of patients reported sexual activity to be less than desired, and more than half of patients reported sexual activity as important.