Sensuke Konno

GLYOXALASE I ACTIVITY IN HUMAN PROSTATE CANCER: A POTENTIAL MARKER AND IMPORTANCE IN CHEMOTHERAPY

PURPOSE To provide information on the activity of Gly-I in prostate cancer. MATERIALS AND METHODS We performed qualitative Gly-I assay on prostate tissues. RESULTS Gly-I activity between prostate cancer and noncancerous specimens differed substantially and significantly, although such activity also varied somewhat among cancer specimens. CONCLUSIONS Gly-I activity is indeed higher in cancerous than in noncancerous specimens, suggesting that it may play a role in prostate cancer homeostasis and survival.

Analysis of the secondary structure of hirudin and the mechanism of its interaction with thrombin

Highly purified hirudin with a specific activity of 13,950 antithrombin units/mg was isolated from a commercial preparation by reversed-phase chromatography. The circular dichroism (CD) spectrum of hirudin was investigated and it was found that the spectrum cannot be accounted for solely in terms of the traditional three components of peptide backbone. It was also found that the CD spectrum of the thrombin-hirudin complex was not additive with respect to the individual spectra of thrombin and hirudin. This deviation from additivity was significant between 210 and 225 nm, indicating alterations in the secondary structures of the proteins during complex formation. When thrombin was titrated with hirudin, the spectral deviation from additivity was sigmoidal, suggesting the cooperative nature of the binding process. Gel filtration of the thrombin-hirudin mixture showed no molecular species greater than a 1:1 complex (Mr 45,500), but gel filtration of free hirudin showed a multimeric form (Mr 51,300) under the same experimental conditions. It is concluded that the cooperative nature of the binding process is due to the binding of thrombin molecules to the multimeric form of hirudin. This initial binding occurs with little or no change in the CD spectrum. In the second step, the multiple complex dissociates to form 1:1 complexes, resulting in larger conformational changes and a considerable increase in binding affinity.

First Prize (Tie): Oxidative Renal Cell Injury Induced by Calcium Oxalate Crystal and Renoprotection with Antioxidants: A Possible Role of Oxidative Stress in Nephrolithiasis

PURPOSE Calcium oxalate (CaOx) is one of the key elements for kidney stone formation, but the exact mechanism needs to be defined. CaOx has been shown to cause renal cell injury through oxidative stress, leading to potential crystal deposition in the kidneys. We thus investigated if CaOx crystal would induce such renal cell injury in vitro and also explored how it would be carried out. MATERIALS AND METHODS Renal tubular epithelial LLC-PK(1) cells were employed, and CaOx monohydrate (COM) was used as CaOx crystal in this study. Cytotoxic effects of COM were assessed on cell viability and biochemical parameters, while protective effect of antioxidants against COM was also examined. RESULTS COM demonstrated its cytotoxicity on LLC-PK(1) cells, exhibiting a approximately 35% cell viability reduction with 500 microg/mL COM in 6 hours. This was presumably attributed to oxidative stress, indicated by lipid peroxidation assay, and N-acetylcysteine (NAC), a potent antioxidant, indeed neutralized such COM cytotoxicity. Although COM also induced inactivation of glutathione-dependent enzymes and partial degradation of heat shock protein 90, these adverse effects were completely prevented with NAC. Moreover, such reduced cell viability with COM was rather associated with apoptosis, evidenced by DNA analysis. CONCLUSION COM is cytotoxic to LLC-PK(1) cells through oxidative stress, leading to the cell viability reduction, adverse effects on biochemical parameters, and, consequently, apoptosis. However, NAC effectively averted such severe cytotoxic effects, sustaining the renal cell integrity. Thus, NAC may provide full renoprotection against COM assault, preventing renal cell injury and ultimate stone formation.

The effects of brefeldin A (BFA) on cell cycle progression involving the modulation of the retinoblastoma protein (pRB) in PC-3 prostate cancer cells.

PURPOSE To investigate the effects of brefelding A (BFA) on the growth of the androgen-independent human prostate cancer PC-3 cells, focusing on cell cycle regulation. MATERIALS AND METHODS BFA is a fungal macrocyclic lactone with an antiviral activity. PC-3 cells were cultured with various concentrations of BFA for indicated times and cell growth was monitored at each time point. Cell cycle analysis was performed to explore the mechanism of BFA-induced growth inhibition. To further investigate the cell cycle regulation, cell cycle-controlling factors, such as the retinoblastoma gene product (pRB) and its regulatory components cdk2, cdk4, and cyclin D1, were analyzed by Western immunoblots. RESULTS BFA was a potent growth inhibitor at a concentration of 30 ng./ml., resulting in a > 70% reduction in cell number at 3 days. Cell cycle analysis revealed a cell arrest in the G1 to S phase transition. Western blots further showed that BFA induced dephosphorylation of pRB accompanied by down regulation of cdk2, cdk4, and cyclin D1 expression. The extended pRB dephosphorylation in control cell lysates was also observed by the addition of BFA-treated lysates, but was prevented by the inclusion of phosphatase inhibitors in assay mixtures. CONCLUSION These results suggest that BFA may be a potent cell cycle modulator, which post-translationally regulates pRB phosphorylation possibly by down-regulating cdk2, cdk4, and cyclin D1 and/or by up-regulating a phosphatase(s) capable of dephosphorylating pRB. Thus, BFA-induced growth inhibition in PC-3 cells appears to be at least partially due to the modulation of a pRB-mediated growth pathway.

Holmium laser enucleation for large (greater than 100 mL) prostate glands

Background: To evaluate the holmium laser enucleation of the prostate (HoLEP) using the transurethral soft tissue morcellator (TUSTM), as a primary surgical treatment for symptomatic benign prostatic hyperplasia (BPH) with prostate glands > 100 mL.

ANALYSIS OF CATHEPSIN D FORMS AND THEIR CLINICAL IMPLICATIONS IN HUMAN PROSTATE CANCER

PURPOSE To assess cathepsin D (Cat.D) status in the prostate, we analyzed the different Cat.D forms in human prostate tissues using Western immunoblots. MATERIALS AND METHODS Cell extracts were prepared from prostate tissues (n = 42) obtained from radical prostatectomy, adopting the tissue homogenization method. Expression of the different Cat.D forms was analyzed using Western blots. The catalytic activity of Cat.D was assayed by acid treatment, in which cell extracts were incubated in acidic buffer (pH 3 to 4) at 37C for 1 hour. RESULTS Pathologically confirmed normal (NML), benign prostatic hyperplasia (BPH) and cancer (CAP) specimens all expressed Cat.D, but as two distinct forms. Both NML and BPH predominantly expressed an inactive procathepsin D (Pro.Cat.D), while CAP notably exhibited an active mature Cat.D. The assessment of Cat.D activity, using PSA (prostate specific antigen) as a physiological substrate, showed that such activity was consistently higher in CAP than in NML/BPH specimens. Further studies revealed that the mode of Cat.D activation in CAP specimens appeared to be primarily due to acid-induced autoproteolysis (self-degradation) of mature Cat.D. CONCLUSION This study demonstrates that expression and activity of Cat.D varies among prostate specimens. A greater expression of mature Cat.D with a higher catalytic activity in CAP specimens is the most notable difference from NML/BPH. Therefore, the differential expression/activity of Cat.D forms may be a useful indicator for assessing prostate cancer status.

Methylglyoxal–Induced Apoptosis in Human Prostate Carcinoma: Potential Modality for Prostate Cancer Treatment

Objective: To examine the cellular effects of methylglyoxal (MG), a toxic physiological metabolite, on human prostatic cancer PC–3 cells.Methods: The effects of MG on cell growth and viability were evaluated first, and then its effects on the cell cycle and the glycolytic process were analyzed by Western blots and specific assays. Possible MG–induced apoptosis was also assessed by DNA analysis using agarose gel electrophoresis.Results: MG ≥3 mM caused severe growth inhibition, resulting in nearly 100% cell death by 24h. The time course study revealed that expression of cyclin D1, cdk2, and cdk4 was significantly (>50%) downregulated in 3 h of MG (3 mM) exposure, followed by the dephosphorylation of retinoblastoma protein by 6 h. Both the glyceraldehyde 3phosphate dehydrogenase activity and the cellular lactate level were also reduced by ∼50 and 80%, respectively, following 6–hour MG exposure. Induction of apoptosis by MG was indicated by partial degradation of poly(ADP–ribose) polymerase and further confirmed by discrete DNA fragmentation detected on an agarose gel. Conclusion: MG is capable of inducing apoptosis in prostatic cancer PC–3 cells, due primarily to a blocking of the cell cycle progression (G1 arrest) and glycolytic pathway. Therefore, MG could be a potent apoptosis inducer, which may have a potential for prostate cancer treatment.

Effects of nicotine on cellular proliferation, cell cyclephase distribution, and macromolecular synthesis in human promyelocytic HL-60 leukemia cells

Addition of nicotine causes a dose- and time-dependent inhibition of cell growth in the human promyelocytic HL-60 leukemia cells, with 4 mM nicotine resulting in a 50% inhibition of cellular proliferation after 48-50 h. Accompanying the anticellular effect of nicotine is a significant change in the cell cycle distribution of HL-60 cells. For example, treatment with 4 mM nicotine for 20 h causes an increase in the proportion of G1-phase cells (from 49% to 57%) and a significant decrease in the proportion of S-phase cells (from 41% to 32%). These results suggest that nicotine causes partial cell arrest in the G1-phase which may in part account for its effects on cell growth. To determine whether nicotine changes the cellular uptake/transport to macromolecular precursors, HL-60 cells were treated with 2-6 mM nicotine for 30 h, at the end of which time cells were labeled with [3H]-thymidine, [3H]uridine, [14C]lysine and [35S]methionine, the trichloroacetic acid soluble and insoluble radioactivities from each of the labeling conditions were determined. These studies show that nicotine mainly affects the de novo synthesis of proteins.

Glyoxalase I phenotype as a potential risk factor for prostate carcinoma

OBJECTIVES To elicit a possible link between glyoxalase I (Gly-I), a detoxifying enzyme, and the incidence of prostate cancer (PCa), we investigated Gly-I phenotypic expression in the prostatic tissue and red blood cells (RBCs) from patients with PCa. METHODS Eighty-seven clinical specimens, including 42 PCa tissue samples, 20 RBC samples, and 25 matched pair (prostate and RBC) samples from patients at prostatectomy were examined. The Gly-I phenotypes in these specimens were assessed by nondenaturing starch-polyacrylamide gel electrophoresis. RESULTS Of the 87 patients, 63 (72.4%) were white, 15 (17.2%) were black, and 9 (10.4%) were another ethnicity (eg, Hispanic, Asian, Indian). Three Gly-I phenotypes were detected in these specimens as fast, intermediate, and slow-moving bands on the gel. The fast phenotype was the most common form found in the white (34 [54%] of 63) and black (8 [53.3%] of 15) patients, but the third ethnic group was too small for proper analysis. To validate this finding, the data from the white patients were compared with the Gly-I phenotypic frequencies in U.S. populations. The data analysis confirmed that a higher incidence (54%) of the fast type in our white patients was statistically significant (P <0.0001) compared with its phenotypic frequency of 30.6% in the general U.S. white population. CONCLUSIONS The significantly high frequency (P <0.0001) of the fast Gly-I phenotype was detected among patients with PCa, suggesting it is a potential risk factor for PCa. Whether its increased incidence in whites reflects the lack of sample numbers for other ethnic groups needs additional investigation.

Synergistic potentiation of D-fraction with vitamin C as possible alternative approach for cancer therapy

Maitake D-fraction or PDF is the bioactive extract of maitake mushroom (Grifola frondosa) and its active constituent is the protein-bound polysaccharide (proteoglucan), or more specifically known as β-glucan. PDF has been extensively studied and a number of its medicinal potentials/properties have been unveiled and demonstrated. Those include various physiological benefits ranging from immunomodulatory and antitumor activities to treatment for hypertension, diabetes, hypercholesterolemia, viral infections (hepatitis B and human immunodeficiency virus), and obesity. Particularly, two major biological activities of PDF, immunomodulatory and antitumor activities, have been the main target for scientific and clinical research. To demonstrate and confirm such biological activities, numerous studies have been performed in vitro and in vivo or in clinical settings. These studies showed that PDF was indeed capable of modulating immunologic and hematologic parameters, inhibiting or regressing the cancer cell growth, and even improving quality of life of cancer patients. Synergistic potentiation of PDF with vitamin C demonstrated in vitro is rather interesting and may have clinical implication, because such combination therapy appears to help improve the efficacy of currently ongoing cancer therapies. Recently, intravenous administration of vitamin C has been often used to increase its physiological concentration and this useful procedure may further make this combination therapy feasible. Therefore, PDF may have great potential, either being used solely or combined with other agents, for cancer therapy. Such relevant and detailed studies will be described and discussed herein with a special focus on the combination of PDF and vitamin C as a viable therapeutic option.