William J.S. Huang

INHIBITORY EFFECTS OF DIGITALIS ON THE PROLIFERATION OF ANDROGEN DEPENDENT AND INDEPENDENT PROSTATE CANCER CELLS

PURPOSE Digitalis or cardiac glycosides have been noted to induce tumor static or oncolytic effects in various types of cancer. We evaluated the effects and underlying mechanisms of cardiac glycosides, including digoxin, digitoxin and ouabain, on the proliferation of hormone dependent and independent prostate cancer cell lines. MATERIALS AND METHODS Cell proliferation of the 3 human prostate cancer cell lines LNCaP, DU145 and PC3 was measured by 3-(4,5-dimethylthiazol-2-yle)2,5-diphenyltetralozium bromide (Sigma Chemical Co., St. Louis, Missouri) colorimetric assay. The cytotoxic effects of digitalis on prostate cancer cells were determined by lactate dehydrogenase measurements of the culture medium. Intracellular Ca2+ was measured by a dual wavelength spectrometer system. The percent of apoptotic cells after digitalis treatment was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling and flow cytometry. RESULTS Digoxin, digitoxin and ouabain significantly inhibited the proliferation of LNCaP, DU145 and PC3 cells at a dose of 1 or 10 microM. after 1 to 4 days of culture. Cytotoxicity of digitalis on the DU145 and LNCaP cells was dose dependent but cytotoxicity was not obvious in PC3. Digitalis (1 microM.) significantly increased intracellular Ca2+ in LNCaP and DU145 after 12 hours of culture but PC3 cells needed a 24-hour treatment to show any effect. In the apoptosis measurement digitalis at a dose of 1 and 10 microM. also significantly increased the percent of apoptotic cells in the LNCaP, DU145 and PC3 cell lines. Normal control human glomerular epithelial cells showed no response to digitalis treatment at all tested doses. CONCLUSIONS Digitalis may inhibit the proliferation of prostate cancer cell lines, although the 3 cell lines showed varied sensitivity to digitalis. These effects are possibly the result of a mechanism involving sustained elevation of the concentration of intracellular Ca2+ and of apoptosis.

Inhibitory effects of evodiamine on the growth of human prostate cancer cell line LNCaP

Evodiamine, isolated from a Chinese herbal drug named Wu‐Chu‐Yu, possesses many biological functions. Recently, it has been reported that Wu‐Chu‐Yu exerts an antiproliferative effect on several cancers. Prostate carcinoma initially occurs as an androgen‐dependent tumor and is the second leading cause of cancer death in American males. In the present study, the effect of evodiamine on the growth of androgen‐dependent prostate cancer cell line LNCaP in vitro was examined. Based on [3‐(4,5‐dimethylthiazol‐2‐yle)2,5‐diphenyltetrazolium bromide] (MTT) assay, evodiamine significantly inhibited the growth of LNCaP cells in a concentration‐dependent manner. A significant and concentration‐dependent inhibitory effect of evodiamine on LNCaP cell growth was observed at 24 hr and persisted for 96 hr. The examination of lactate dehydrogenase (LDH) assay showed that the cytotoxic effects of evodiamine on LNCaP cells were concentration dependent. Furthermore, we examined the influences of evodiamine on cell death and cell cycle. The flow cytometric analysis of evodiamine‐treated cells indicated a block of G2/M phase and an elevated level of DNA fragmentation. The G2/M arrest reached a maximum at 24 hr after evodiamine treatment. The G2/M arrest was accompanied by an elevated p34cdc2 kinase activity and an increase in the protein expression of cyclin B1 and phosphorylated form of p34cdc2 (Thr 161). Examination of TUNEL showed that evodiamine‐induced apoptosis was observed at 24 hr and extended for 72 hr. Evodiamine elevated caspase‐3, and caspase‐9 activities and the processing of caspase‐3 and caspase‐9. These results suggested that evodiamine inhibits the growth of prostate cancer cell line, LNCaP, through an accumulation of cell cycle at G2/M phase and an induction of apoptosis.

Haplotypes, Loss of Heterozygosity, and Expression Levels of Glycine N-Methyltransferase in Prostate Cancer

Purpose: Glycine N-methyltransferase (GNMT) affects genetic stability by regulating DNA methylation and interacting with environmental carcinogens. In a previous study, we showed that GNMT acts as a susceptibility gene for hepatocellular carcinoma. Here, we report on our efforts to characterize the haplotypes, loss of heterozygosity (LOH), and expression levels of the GNMT in prostate cancer. Experimental Design: Peripheral blood mononuclear cell DNA collected from 326 prostate cancer patients and 327 age-matched controls was used to determine GNMT haplotypes. Luciferase reporter constructs were used to compare the promoter activity of different GNMT haplotypes. GNMT LOH rates in tumorous specimens were investigated via a comparison with peripheral blood mononuclear cell genotypes. Immunohistochemical staining was used to analyze GNMT expression in tissue specimens collected from 5 normal individuals, 33 benign prostatic hyperplasia patients, and 45 prostate cancer patients. Results: Three major GNMT haplotypes were identified in 92% of the participants: A, 16GAs/DEL/C (58%); B, 10GAs/INS/C (19.9%); and C, 10GAs/INS/T (14.5%). Haplotype C carriers had significantly lower risk for prostate cancer compared with individuals with haplotype A (odds ratio, 0.68; 95% confidence interval, 0.48-0.95). Results from a phenotypic analysis showed that haplotype C exhibited the highest promoter activity (P < 0.05, ANOVA test). In addition, 36.4% (8 of 22) of the prostatic tumor tissues had LOH of the GNMT gene. Immunohistochemical staining results showed abundant GNMT expression in normal prostatic and benign prostatic hyperplasia tissues, whereas it was diminished in 82.2% (37 of 45) of the prostate cancer tissues. Conclusions: Our findings suggest that GNMT is a tumor susceptibility gene for prostate cancer.

Measurement of Testicular Volume in Smaller Testes: How Accurate Is the Conventional Orchidometer?

The aim of this study was to evaluate the accuracy of different methods, including the Seager orchidometer (SO) and ultrasonography (US), for assessing testicular volume of smaller testes (testes volume less than 18 mL). Moreover, the equations used for the calculations--the Hansen formula (length [L] x width [W](2) x 0.52, equation A), the prolate ellipsoid formula (L x W x height [H] x 0.52, equation B), and the Lambert equation (L x W x H x 0.71, equation C)--were also examined and compared with the gold standard testicular volume obtained by water displacement (Archimedes principle). In this study, 30 testes from 15 men, mean age 75.3 (+/-8.3) years, were included. They all had advanced prostate cancer and were admitted for orchiectomy. Before the procedure, all the testes were assessed using SO and US. The dimensions were then input into each equation to obtain the volume estimates. The testicular volume by water displacement was 8.1 +/- 3.5 mL. Correlation coefficients (R(2)) of the 2 different methods (SO, US) to the gold standard were 0.70 and 0.85, respectively. The calculated testicular volumes were 9.2 +/- 3.9 mL (measured by SO, equation A), 11.9 +/- 5.2 mL (measured by SO, equation C), 7.3 +/- 4.2 mL (measured by US, equation A), 6.5 +/- 3.3 mL (measured by US, equation B) and 8.9 +/- 4.5 mL (measured by US, equation C). Only the mean size measured by US and volume calculated with the Hansen equation (equation A) and the mean size measured by US and volume calculated with the Lambert equation (equation C) showed no significant differences when compared with the volumes estimated by water displacement (mean difference 0.81 mL, P = .053, and 0.81 mL, P = .056, respectively). Based on our measurements, we categorized testicular volume by different cutoff values (7.0 mL, 7.5 mL, 8.0 mL, and 8.5 mL) to calculate a new constant for use in the Hansen equation. The new constant was 0.59. We then reexamined the equations using the new 0.59 constant, and found that the equation Volume (V) = L x W(2) x 0.59 was the best for describing testicular volume among our subjects (difference between the new equation and the gold standard of water displacement = 0.19 mL, P = .726). We also found that US was more precise in measuring testicular dimensions. We propose a new formula, V = L x W(2) x 0.59, to assess the volumes of smaller testes.

Effects of hyperprolactinemia on testosterone production in rat Leydig cells

The pathogenesis of hyperprolactinemia (hyperPRL) induced hypogonadism has been suggested to be related with a dysfunction of hypothalamus–pituitary–testis axis. While the direct inhibitory effects of prolactin (PRL) on testosterone (T) release have been demonstrated, the mechanism is still unclear. Our previous study demonstrated a diminished T release in the testicular interstitial cells (TICs) from the anterior pituitary (AP)‐grafted rats as compared with the control, and the pattern was in agreement with the in vivo model. However, TICs incubation cannot totally represent the response of the Leydig cells. Therefore, a Percoll gradient purified Leydig cell model was adopted to explore the response of T release under similar challenges in this study to investigate the effects of hyperPRL on the Leydig cells per se. HyperPRL in male rats was induced by grafting rat AP under the renal capsule. The control animals were grafted with rat brain cortex tissue (CX). Six weeks after grafting, the rats were sacrificed. Either TICs or Leydig cells were isolated, respectively, for in vitro incubation and challenge. Challenge drugs included human chorionic gonadotropin (hCG, 0.05 IU/ml), steroidogenic precursors (25‐OH‐cholesterol, 10−6 M; pregnenolone, 10−6 M), forskolin (an anenylyl cyclase activator, 10−4 M) and 8‐bromo‐3′:5′ cyclic adenosine monophosphate (cAMP) (8‐Br‐cAMP 10−4 M). T released by TICs or Leydig cells was determined by radioimmunoassay. The TICs from the AP‐grafted rats showed lower levels of T release than the control group while the purified Leydig cells demonstrated a reverse pattern in response to challenges of hCG, steroidogenic precursors, forskolin and 8‐Br‐cAMP. In hyperPRL rats, a paradoxical pattern of T release between TICs and purified Leydig cells is observed. The purified Leydig cells from AP‐grafted rats demonstrated a higher level amount of T release than the control after stimulation. The phenomenon can be attributed to the change of Leydig cell sensitivity to the stimulation after the effects of chronic hyperPRL. Moreover, another possibility is the role played by other interstitial cells to modulate steroidogenesis in Leydig cells. J. Cell. Biochem. 80:313–320, 2001.

Correlation between serum prostate specific antigen and prostate volume in Taiwanese men with biopsy proven benign prostatic hyperplasia.

PURPOSE We studied the correlation between serum prostate specific antigen and the volume of different zones of the prostate in Taiwanese men with biopsy proven benign prostatic hyperplasia. MATERIALS AND METHODS A total of 233 patients with a mean age of 71.4 years (range 42 to 89), serum prostate specific antigen less than 10 ng/ml and pathologically confirmed benign prostatic hyperplasia were enrolled in this study. Total prostate and transitional zone volumes were measured with transrectal ultrasonography. Peripheral zone volume was determined by subtracting transitional zone volume from total prostate volume. Correlations between patient age, total serum prostate specific antigen and the volume of each prostate zone were analyzed with the Pearson correlation coefficient. A linear regression model was used to determine the relationship between prostate specific antigen and prostate volume. The prostate specific antigen-prostate volume relationship in our patients was compared with published data on white and Japanese men. RESULTS Age did not significantly correlate with serum prostate specific antigen and prostate volume. Serum prostate specific antigen significantly correlated with the volume of each prostate zone. After log transformation the Pearson correlation coefficient between total prostate specific antigen and the volume of the whole prostate gland, the transitional zone and the peripheral zone were 0.369, 0.377 and 0.272, respectively (p <0.001). Taiwanese men had lower prostate volume per unit prostate specific antigen comparing with white men, while the prostate specific antigen-total prostate volume relationship between Taiwanese and Japanese men was similar. CONCLUSIONS In Taiwanese men with biopsy proven benign prostatic hyperplasia the volume of each prostate zone has significantly correlates with serum prostate specific antigen. The prostate specific antigen-total prostate volume relationship in Taiwanese men is different from that in white men. However, the prostate specific antigen-total prostate volume relationship between Taiwanese and Japanese men is similar.

Retroperitoneoscopy-assisted nephroureterectomy for the management of upper urinary urothelial cancer

SummaryTraditionally, transitional cell carcinoma of the upper urinary tract needs a flank incision to remove the kidney and a lower abdominal incision to remove the ureter and bladder cuff. We report the surgical techniques and the initial clinical experience of retroperitoneoscopy-assisted nephroureterectomy for the treatment of this disease. Seven patients (6 males and 1 female; mean age 64.3 years, range 47-75 years) with the pre-operative diagnosis of upper urinary tract tumour underwent retroperitoneoscopy-assisted nephroureterectomy. The operation was performed first by retroperito-neoscopic nephrectomy, dissection of the lower third ureter and bladder cuff excision were performed with the traditional open method. The whole specimen with intact urothelium was removed through the lower abdominal incisional wound. We have successfully applied this technique for six patients with urothelial tumours. In one case, this technique had to be converted to open nephroureterectomy due to severe perirenal adhe...

Common variants at 8q24 are associated with prostate cancer risk in Taiwanese men.

Recently, independent genome‐wide scans have found multiple genetic variants at 8q24 to be associated with prostate cancer risk. This study was performed to determine whether two of the variants more strongly associated with prostate cancer risk in European and American populations, specifically rs16901979 and rs6983561, were also associated with prostate cancer risk in Taiwanese men.

Regulation of testosterone secretion by prolactin in male rats.

The goal of this study was to characterize the mechanism by which hyperprolactinemia alters testosterone production in rat testicular interstitial cells (TICs). Hyperprolactinemia was induced by grafting 2 anterior pituitary (AP) glands under the subcapsular space of the kidney in experimental rats. Control rats were grafted with brain cortex (CX). Six weeks post‐grafting, rats were challenged with human chorionic gonadotropin (hCG) then, the changes in either plasma testosterone or luteinizing hormone was measured. Additionally, TICs were isolated and challenged in vitro with hCG or prolactin, and the testosterone release measured by radioimmunoassay. Further investigation in signal transduction as intracellular 3′:5′ cyclic adenosine monophosphate (cAMP) production was observed under a regulation of forskolin or SQ22536. After the challenge of hCG or GnRH, the AP‐grafted rats showed a suppressed response in testosterone release as compared to those in the CX‐grafted group. The in vitro data from the AP‐grafted rats compared to the CX‐grafted animals showed a diminished response in testosterone release upon hCG stimulation. Administration of forskolin or SQ22536 disclosed dysfunction of adenylate cyclase in TICs from the AP‐grafted rats. When 8‐Br‐cAMP was incubated with TICs, the testosterone production was lower in the AP‐grafted compared to the CX‐grafted group. These results suggest that in addition to adenylate cyclase dysfunction, inefficiency of post‐cAMP pathways are also involved in the hypogonadism elicited by hyperprolactinemia in rats. J. Cell. Biochem. 74:111–118, 1999.

Direct needle insufflation for pneumoretroperitoneum: anatomic confirmation and clinical experience

OBJECTIVES The feasibility and safety of direct needle insufflation to create pneumoretroperitoneum was assessed by an imaging study and clinical experience. METHODS A total of 10 patients without previous retroperitoneal surgery or diseases received computed tomography scans of the retroperitoneum 2 cm above the iliac crest. Distances between quadratus lumborum and colon (Q-C distance) were measured in the supine and lateral positions. Changes of Q-C distance were calculated when the patient was changed from the supine to the lateral position. Operative charts on 38 retroperitoneoscopic procedures were collected prospectively to assess complications related to direct needle insufflation, which was performed by inserting a 14 G Veress needle blindly along the posterior axillary line 2 cm above the iliac crest. RESULTS Q-C distance increased from 8.7 to 27.3 mm (left side) and 4.6 to 18.1 mm (right side) when the patient was changed from the supine to the lateral position, both P values < 0.05. An average distance of 23 mm between colon and quadratus lumborum was found when patients were lying laterally. The misplacement of a Veress needle was encountered in 1 patient, in which a prefascia insufflation resulted in conversion of the endoscopic procedure. Needle puncture caused no visceral or great vessel injury. CONCLUSIONS Significant anterior movement of the colon was found when patients were changed from the supine to the lateral position. It provided a window for inserting the Veress needle blindly into the retroperitoneum. The high success rate (97%) and low complication rate of direct needle insufflation were found in actual clinical applications. We considered needle insufflation a safe and effective method of establishing a pneumoretroperitoneum for any retroperitoneoscopic procedure.