Muhammad Choudhury

Overexpression of the Heme Oxygenase Gene in Renal Cell Carcinoma

Heme oxygenase (HO) activity has been implicated in the regulation of renal function and cell growth in normal and disease states. Expression of HO genes has been shown to regulate important hemoprotein(s) such as cytochrome P450. In the present study, HO activity was measured in samples of human adenocarcinoma, jux-tatumor, and normal renal tissues. The samples were histologically examined to verify the malignant and normal nature. HO activity was 4-fold higher in the adenocarcinoma than in either normal or juxtatumor tissues. We designed a reverse transcriptase-polymerase chain reaction (RT-PCR) method to assess the presence of HO-1 and HO-2 mRNA in biopsy samples of various human renal tissues. Total RNA from renal samples was reverse transcribed and amplified simultaneously by PCR using specific primers for HO-1 and HO-2. Results show that both HO-1 and HO-2 mRNAs were expressed in all renal tissues examined and that HO-1 appeared to be amplified more than HO-2. Northern blot analysis revealed that HO-1 mRNA was elevated by several-fold in adenocarcinoma compared with juxtatumor or normal tissues. In contrast, no differences in HO-2 mRNA levels were observed using either RT-PCR or Northern blot. Cytochrome P450 arachidonic acid epoxygenase and ω-hydroxylase activities were markedly reduced in the tumor tissues, whereas, in the juxtatumor tissue, cytochrome P450 ω-hydroxylase activity was significantly increased. Northern blot analysis using cytochrome P450 cDNA probe 4A2 cDNA for the ω-hydroxylase gene family revealed that mRNA levels for ω-hydroxylase transcripts were significantly decreased in the adenocarcinoma compared with juxtatumor. The decrease in cytochrome P450 4All mRNA levels correlated with a decrease in the arachidonic acid ω-hydroxylation metabolite, 20-HETE. The production of 20-HETE was significantly higher in juxtatumor in agreement with ω-hydroxylase mRNA. Higher levels of HO-1 may be a contributing factor for the undetectable levels of cytochrome P450 arachidonic acid metabolites, 20-HETE, in the adenocarcinoma. Our results suggest that increased generation of mitogenic activities by ω-hydroxylase and 20-HETE in the juxtatumor may be a contributing factor in the development and growth of neoplastic tissues, and the induction of HO in the tumor tissue may be an attempt to limit oxidative injury caused by the cytochrome P450 metabolites and other oxidative stress.

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.

Establishment of new human prostatic cancer cell line (JCA-I)

The establishment of a new human prostatic cancer cell line is described. This cell line was derived from a poorly to moderately differentiated prostatic adenocarcinoma. It has been maintained in tissue culture for fourteen months and has been passed fifty-two times. This cell line has an ability to form colonies in soft agar suspension cultures, and also is transplantable to nude mice. Tumors grown in nude mice revealed a poorly differentiated adenocarcinoma with positive PSA staining. Acid phosphatase activity was detected in freeze-thawed cells by enzymatic assay. A karyotype analysis demonstrated aneuploidy with a model chromosomal number of 69 and six marker chromosomes.

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.

Malignant renal tumor with extension to the inferior vena cava

BACKGROUND Management of malignant renal tumors involving the inferior vena cava (IVC) depends on tumor extension within the cava. METHODS Of 295 patients treated for renal cancer, propagation of tumor mass through the renal vein to IVC was seen in 22 (7%) patients. Cephalad extension of the tumor was suprarenal: infrahepatic in 12, retrohepatic in 6, and within the right atrium in 4 patients. All patients had radical nephrectomy, cavotomy, and complete resection of tumors except 1 with diffuse peritoneal metastasis. RESULTS Twenty-one patients had curative resections. No operative deaths and no instances of pulmonary embolism or exsanguination occurred. Seventeen patients were alive at 2 years and 12 at 5 years, resulting in 77% and 55% survival rates, respectively. CONCLUSIONS An aggressive approach for vena cava involvement from malignant renal neoplasms resulted in prevention of tumor embolus, minimization of blood loss, and maintenance of venous return to the heart.

Urologic complications of high-dose chemotherapy and bone marrow transplantation

OBJECTIVE This study reviews the incidence and management of cyclophosphamide-induced hemorrhagic cystitis in a group of patients who received high-dose chemotherapy and bone marrow transplantation. METHODS The records of 217 consecutive patients undergoing bone marrow transplantation were reviewed. The incidence, degree, and management of hematuria in this group of pancytopenic and immunocompromised patients were recorded. RESULTS Despite prophylaxis, cystitis developed in 58 of these 217 patients (27%). In 12 patients (6%) the cystitis was severe. These patients had gross hematuria, clot retention, and drop in hematocrit necessitating blood transfusion. These patients were managed with continuous bladder irrigation, alum irrigation, and when less aggressive approach was unsuccessful, with intravesical formalin instillation. Alum irrigation was used in 5 patients, and was successful in only 1 patient. Six patients required intravesical formalin instillation to control the hematuria. Formalin solution 2-5% was instilled initially. When lower-concentration formalin failed, 5-10% formalin was used progressively. CONCLUSIONS Patients with bone marrow transplantation in whom severe hemorrhagic cystitis develops should be managed aggressively early. Intravesical formalin appears to be the most effective regimen in controlling profuse, persistent hematuria.

Cavitron ultrasonic surgical aspirator. Applications in urologic surgery.

The Cavitron Ultrasonic Surgical Aspirator is a unique modality for precise tissue removal which results in increased visibility, reduced bleeding, and shorter operating time when dealing with vascular lesions or organs. Herein, we present our experience in a dog laboratory as well as in a clinical setting with this new instrument. The mechanism of action, proper surgical technique required, and further application in urologic surgery are discussed.

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.

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.