Dar-Shih Hsieh

THE EXPRESSION OF NEUROPEPTIDES IN HYPERPLASTIC AND MALIGNANT PROSTATE TISSUE AND ITS POSSIBLE CLINICAL IMPLICATIONS

PURPOSE We characterized the incidence and pattern of distribution of neuroendocrine differentiated tumor cells in prostatic hyperplastic and carcinomatous tissue, correlated neuroendocrine differentiation with prostate specific antigen (PSA) and assessed whether neuroendocrine cells have value as an independent indicator of poor prognosis in patients with prostate carcinoma. MATERIALS AND METHODS We immunohistochemically evaluated hyperplastic and carcinomatous prostate specimens for chromogranin A, neuron specific enolase and serotonin expressing tumor cells. The expression of various markers in cells was analyzed and correlated with tumor DNA ploidy, disease grade and stage, PSA and clinical course in patients with prostate cancer. RESULTS Enrolled in our study were 31 patients with hyperplastic prostate tissue and 30 with prostatic carcinoma. Followup in cancer cases was 1 to 9 years (mean 3.7). During followup 9 patients (30%) died of cancer. We noted DNA content aneuploidy in 5 cases (16.7%) of prostate carcinoma. Chromogranin A, neuron specific enolase and serotonin were expressed in 80%, 43% and 77% of cases of prostate carcinoma and in 29%, 10% and 36% of hyperplastic tissue, respectively. Larger prostates had no higher content of various neuroendocrine cells than smaller prostates. There was higher expression of neuropeptides in carcinomatous than in hyperplastic tissue. Of the 3 peptides chromogranin A was significantly related to all parameters, including Gleason score, tumor stage, PSA and patient survival. In addition to PSA, neuron specific enolase was also closely associated with other clinicopathological parameters. Serotonin was significantly related to patient survival only but we noted no correlation with Gleason score, tumor stage or PSA. In regard to factors predictive of patient prognosis expression of the 3 neuropeptides in tumor cells, Gleason score, tumor stage and PSA were closely related to patient survival in this study CONCLUSIONS The growth of hyperplastic prostate tissue is related to neuroendocrine cell activity. The chromogranin A marker has the highest expression in prostate cancer. Neuroendocrine cells may represent an independent indicator of poor prognosis in patients with prostate carcinoma.

Correlation of CD44v5 Expression with Invasiveness and Prognosis in Renal Cell Carcinoma

BACKGROUND AND PURPOSE The expression of specific CD44 isoforms is associated with metastasis and poor prognosis in human malignant tumors. We retrospectively investigated the expression of the CD44 variant isoform v5 (CD44v5) in human renal cell carcinoma (RCC) specimens by immunohistochemistry. METHODS We studied the expression of CD44v5 immunohistochemistry in archival tissue specimens from 72 RCC patients (47 men and 25 women) with a mean age of 60.1 years (range, 30 to 83 years) who underwent resection in our hospital from 1986 to 1996. The patients were divided into non-invasive (pT1, pT2, and pT3a; n = 49) and invasive groups (pT3b, pT3c, and any T with N+; n = 23). Among the 72 patients, 67 were followed for at least 60 months (up to 190 months) after radical nephrectomy. Twenty nine renal specimens, including 15 of normal renal parenchyma and 14 of inflammatory or immune renal diseases, were used as controls. RESULTS All control group tissues expressed CD44v5. CD44v5 expression was detected in 66 of 72 RCC specimens (91.7%). In general, the expression of CD44v5 was higher in the non-invasive group [47/49 (95.9%) vs 19/23 (82.6%), p < 0.05]. Five cases were lost to follow-up. Thirty three of 67 patients (49.3%) died of RCC-related causes during the follow-up period. The CD44v5 non-expression group had a higher mortality rate than the expression group [4/5 (80%) vs 29/62 (46.8%), p < 0.001]. The 5-year and 10-year survival rates were significantly higher in patients with CD44v5 expression than in those without (p < 0.001). CONCLUSIONS This study found that CD44v5 expression was greater in early stage than in advanced stage RCC and was associated with tumor progression and long-term survival. Although the survival analysis showed both tumor stage and CD44v5 expression were significant prognostic factors in RCC, only tumor stage remained an independent factor.

INCREASING EXPRESSION OF GST-pi, MIF, AND ID1 GENES IN CHEMORESISTANT PROSTATE CANCER CELLS

The differential expression of genes and related proteins of multidrug resistance in chemoresistant prostate cancer cell lines were elucidated in this study. RNA extracted from doxorubicin-resistant rat prostate cancer (PCa) cells (AT3/ADR1000) and native PCa cells was hybridized to expression arrays containing cDNAs from 588 known genes. Differential expression of selected genes was confirmed by quantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. Protein contents were measured by fluorescent flow cytometry and immunoblotting. Localization of selected proteins in cells was observed by immunocytochemical staining. Up-regulation of eleven genes and down-regulation of one single gene were displayed in the chemoresistant prostate cancer cells. Overexpression of mRNAs in macrophage migration inhibitory factor (MIF), DNA binding protein inhibitor 1 (ID1), and glutathione S-transferase-pi (GST-pi) were confirmed by gene-specific RT-PCR. Protein over-expression of GST-pi, MIF, and ID1 in resistant cells were 3.3-, 1.5-, and 1.5-fold to native cells, respectively. Immunocytochemistry revealed that GST-pi, MIF, and ID1 were present primarily in the cytoplasm of tumor cells, but ID1 also could be found in the nucleus. AT3/ADR1000 drug-resistant PCa cells displayed significantly increased expression of GST-pi, MIF, and ID1 proteins when compared with native PCa cells. It indicates these genes may play a role in drug resistance of prostate cancer.

MODULATION OF MDR-1 GENE BY MIF AND GSTpi WITH DRUG RESISTANCE GENERATION IN HORMONE INDEPENDENT PROSTATE CANCER

The expression of MIF and GSTpi were upregulated in prostate cancer cells with mulitdrug resistant phenotype. The aim of this study is to determine the relationship between these genes and multidrug resistance (mdr-1) gene in acquired multidrug resistance of prostate cancer. The expression of MIF, GSTpi and gp-170 in multidrug resistant (MDR) subline or native cells were determined using flow cytometry and western blotting. The mRNA level of various genes was analyzed with RT-PCR method. The chemosensitivity of tumor cells and stable transfectants to paclitaxel was measured using MTT (tetrazolium bromide) assay. The protein levels of MIF, GSTpi and gp-170 increased in MDR sublines of prostate cancer when compared with their parental cells. The MIF and GSTpi stable transfectants expressed higher MIF and GSTpi protein levels than their parental cells in western blotting analysis, respectively. The expression of mdr-1 gene and the production of pg-170 were also increased in either MIF or GSTpi stable transfectants when compared with vector control by using RT-PCR and flow cytometric analysis. The MTT results demonstrated that the increased chemoresistance was correlated with the increased production of gp-170 protein in either MIF or GSTpi transfectants. The upregulation of MIF and GSTpi during the development of acquired drug resistance of hormone independent prostate cancer may simultaneously and partially modulate the activation of gp-170.

Synergism of Fas-mediated apoptosis and tumor necrosis factor on adriamycin cytotoxicity to transitional cell carcinoma.

Objective: To explore the accurate Fas antigenic expression in transitional cell carcinoma (TCC) cells and and compare its synergistic modulation on adriamycin cytotoxicity with α-tumor necrosis factor (TNF-α). Methods: The expression of Fas antigen in normal urothelium and TCC cell lines was measured by flow cytometry. The Fas/Fas ligand reaction-induced cytotoxic changes in tumor cells were evaluated by microculture MTT technique. The synergism efficacy of Fas-mediated apoptosis and TNF-α on adriamycin cytotoxicity of TCC cells was analyzed. The Fas/Fas ligand-induced apoptosis in tumor cells was studied by annexin-V apoptotic cell expression and DNA ladder fragmentation analysis. Results: 75% of TCC cell lines showed Fas antigen expression. The Fas expression was not influenced by in vitro growth density of tumor cells. Apoptosis of TCC cells was observed during 48 h of treatment after activation of the Fas/Fas ligand pathway. TNF-α had a higher cytotoxicity activity on TCC cells than Fas ligand (17 vs. 0.7%) while combination of both reagents had 30% increased synergistic cytotoxicity. The increasing rate of apoptotic body after 3 days of treatment was 74% in adriamycin, 32% in Fas ligand, 60% in TNF-α, 69% in Fas and adriamycin combination and Fas and TNF-α combination, 103% in combination of TNF-α and adriamycin, and 136% in combination of these three reagents individually. In the DNA ladder analysis, Fas ligand had a 1.5-fold increase of DNA fragmentation when compared with adriamycin while TNF-α had a 4.4-fold increase and triple combination had a 5.5-fold increase after 3 days of treatment. Conclusions: Although the Fas antigen expression is common in TCC cells and apoptosis can be activated by the Fas/Fas ligand pathway activation, the synergistic cytotoxicity of adriamycin by the Fas/Fas ligand pathway is lower than that of TNF-α.

Redistribution of androgen receptors in acquired hormone-refractory prostate cancer cells.

The dynamic translocation of androgen receptors (ARs) in prostate cancer cells after hormone conversion was studied. The prostate cancer cell line LNCaP was converted into androgen-independent sublines after long-term treatment with 5α-reductase inhibitor and steroid-depleted medium. Immunohistochemical, immunofluorescent staining and laser scanning microscopy were used to observe the redistribution and serial translocation of ARs in these tumor cells. The androgen-independent tumor cells (LNCaP/Fin and LNCaP/HR) grew slower than native cells with fibroblastic-like characteristics. On immunohistochemical and immunofluorescent double staining, translocation and exocytosis of ARs were noted in androgen- independent tumor cells much more markedly than in native cells. Furthermore, laser-scanning microscopy revealed serial image changes of AR vesicle shifting and exocytosis in androgen-converted tumor cells. Translocation and exocytosis processes were observed in androgen-independent prostate cancer cells. ARs lose partly normal cellular biologic role during hormone manipulation.