Improving anticancer efficacy of (-)-epigallocatechin-3-gallate gold nanoparticles in murine B16F10 melanoma cells.

(-)-Epigallocatechin-3-gallate (EGCG), the major bioactive constituent in green tea, has been reported to effectively inhibit the formation and development of tumors. To maximize the effectiveness of EGCG, we attached it to nanogold particles (EGCG-pNG) in various ratios to examine in vitro cytotoxicity and in vivo anti-cancer activity. EGCG-pNG showed improved anti-cancer efficacy in B16F10 murine melanoma cells; the cytotoxic effect in the melanoma cells treated with EGCG-pNG was 4.91 times higher than those treated with EGCG. The enhancement is achieved through mitochondrial pathway-mediated apoptosis as determined by annexin V assay, JC-10 staining, and caspase-3, -8, -9 activity assay. Moreover, EGCG-pNG was 1.66 times more potent than EGCG for inhibition of tumor growth in a murine melanoma model. In the hemolysis assay, the pNG surface conjugated with EGCG is most likely the key factor that contributes to the decreased release of hemoglobin from human red blood cells.

The preparation and characterization of gold-conjugated polyphenol nanoparticles as a novel delivery system.

Nanogold particles are commonly used in nanomedicine. We generated physical nanogold (pNG) conjugated with different ratios of epigallocatechin-3-gallate (EGCG) and evaluated its physicochemical properties, antioxidant activity, and cytotoxicity in vitro as well as anticancer activity in vivo. Results showed that the EGCG-pNG conjugates were successfully prepared at ratios between 23:1 and 23:5, with the percentage of EGCG content increasing with the EGCG:pNG ratio from 23:1 (2.0% ± 0.02%) to 23:5 (28% ± 0.3%). EGCG-pNG particles at ratios of 23:1 and 23:5 demonstrated significantly decreased size from 500 to 20 nm and decreasing zeta potentials of 21 mV to -22 mV, respectively. At a ratio of 23:2.5, the EGCG-pNG particles (27% EGCG, 50 nm in size, zeta potential of -8 mV) showed longer EGCG activity half-life (110 days vs 5 hours), controlled release (2 hours vs 30 minutes), and higher antioxidant activity (four times), as well as inhibition of tumor cell growth, than controls. The present study indicated that EGCG-pNG possesses promising therapeutic potential, based on its strong free-radical scavenging and anticancer activities.

The treatment of bladder cancer in a mouse model by epigallocatechin-3-gallate-gold nanoparticles.

(--)-Epigallocatechin-3-gallate (EGCG), an active ingredient in green tea, was known to effectively inhibit formation and development of tumors. However, excessive uptake of EGCG was also known to cause cytotoxicity to normal cells. In this study, EGCGs that were physically attached onto the surface of nanogold particles (pNG) was confirmed by scanning electron microscopy. The anticancer activity of the EGCG-adsorbed pNG was investigated in C3H/HeN mice subcutaneously implanted with MBT-2 murine bladder tumor cells. EGCG-pNG was confirmed to inhibit tumor cell growing by means of cell apoptosis. The mechanism that EGCG-pNG mediates tumor apoptosis was uncovered to activate the caspase cascade through the Bcl-family proteins in the mitochondrial pathway. Additionally, the mechanism that tumors were suppressed by injecting EGCG-pNG directly into the tumor site was determined to be through downregulation of VEGF, whereas that by oral administration of EGCG was through reversing immune suppression upon cancer progression. In this assessment, the prepared EGCG-pNG was confirmed to be more effective than free EGCG in inhibiting bladder tumor in model mice.

Possibility of spontaneous seeding of transitional cell carcinoma of the ureter in renal tubules: another mechanism of transitional cell carcinoma dissemination.

Cancer cell seeding inside the urinary tract always has been considered one possible mechanism of the multicentric origin of transitional cell carcinoma (TCC). However, there is still no direct clinical evidence to prove that the natural seeding of TCC is a real event. To our knowledge, we report the first case of spontaneous seeding of TCC of the ureter in the renal tubules of a hydronephrotic kidney. The TCC nature of the intratubular tumor cells has been confirmed by the morphological appearance of them after hematoxylin and eosin staining and positive p53 immunohistochemical staining.

Increasing expression of extracellular matrix metalloprotease inducer in renal cell carcinoma: tissue microarray analysis of immunostaining score with clinicopathological parameters.

AIM: Renal tumor cell invasion is responsible for both local tissue destruction and distant metastasis. Invasion is largely mediated by matrix metalloproteases that are thought to be induced by tumor cell-derived extracellular matrix metalloprotease inducer (EMMPRIN) in surrounding fibroblasts. We hypothesized that EMMPRIN and matrix metalloproteinase-9 (MMP-9) are over-expressed in renal cell carcinoma. METHODS: Immunohistochemical analysis of EMMPRIN and MMP-9 was performed in tissue microarrays of 79 renal cell carcinomas including 12 cases of chromophobe renal cell carcinoma (ChRCC), 53 cases of clear cell renal cell carcinoma (CRCC), 8 cases of papillary renal cell carcinoma (PRCC), and 6 cases of carcinoma of the collecting ducts of Bellini (CoRCC). RESULTS: All renal cell carcinomas showed significant immunohistochemical expression of EMMPRIN. The EMMPRIN score in ChRCC (321+/-21) was significantly higher than in other histological subtypes of RCC (166+/-19 for CRCC; 276+/-24 for PRCC; 98+/-17 for CoRCC). MMP-9 was mainly expressed in tumor stromal cells and not in non-cancerous fibrovascular regions. The percent positive staining of MMP-9 at the invasive front of tumor cells was significantly higher in CRCC than in ChRCC, PRCC, or CoRCC. Higher EMMPRIN scores in CRCC were associated with shorter survival time, and correlated with higher T staging and nuclear grading. CONCLUSIONS: Our findings demonstrate for the first time that EMMPRIN is over-expressed in renal cell carcinomas. Increased expression of EMMPRIN in tumor cells is associated with poor prognosis of patients with CRCC.

Increasing expression of extracellular matrix metalloprotease inducer in ovary tumors: tissue microarray analysis of immunostaining score with clinicopathological parameters.

Ovary cancer invasion is responsible for both local tissue destruction and distant metastasis. Invasion is largely mediated by matrix metalloproteases that are thought to be induced by tumor cell-derived extracellular matrix metalloprotease inducer (EMMPRIN) in surrounding fibroblasts. We hypothesized that EMMPRIN isoverexpressed in ovary tumors. Immunohistochemical analysis of EMMPRIN was performed in tissue microarrays of ovary neoplasms including 84 cases of serous adenocarcinoma, 23 cases of mucinous adenocarcinoma, 10 cases of endometrioid adenocarcinoma, 12 cases of yolk sac tumor, 12 cases of clear cell carcinoma, 8 cases of dysgerminoma, 8 cases of granulosa cell tumor, 6 cases of transitional cell carcinoma, and 6 cases of Brenner tumor. All malignant ovary tumors showed significant immunohistochemical expression of EMMPRIN. The EMMPRIN scores in malignant ovary tumors were significantly higher than their nontumor counterparts (313+/-28 for serous adenocarcinoma; 308+/-25 for mucinous adenocarcinoma; 187+/-19 for endometrioid adenocarcinoma; 265+/-23 for yolk sac tumors; 87+/-13 for clear cellcarcinoma; 126+/-15 for dysgerminoma; 243+/-26 for granulosa cell tumor; 87+/-16 for transitional cell carcinoma). The EMMPRIN score was significantly higher in serous adenocarcinomas than in serous adenomas and serous borderline tumors and was correlated with nodal stage. Our findings show for the first time that EMMPRIN is overexpressed in all malignant ovary tumors.

Primary retroperitoneal abscess complicated with septic arthritis of the hip.

Primary retroperitoneal abscess complicated with septic arthritis of the hip is an unusual disease. The insidious and occult nature of abscess coexistent with arthritis causes diagnostic delays, prolonged sepsis, and considerably higher morbidity and mortality. We herein present a case of gouty arthritis and avascular necrosis of the femoral head in a 41-year-old woman who complained of fever, right flank pain, body weight loss, swelling over her right lower limb, and 2 weeks of pain in the right hip. The computed tomographic scan showed a huge abscess (about 32 x 10 x 8 cm) over the right posterior pararenal space, with swelling of the right psoas, iliac, and obturator muscles. During surgery, the abscess was drained and sequestrectomy of the right hip was performed. Cultures of pus from the retroperitoneum and right hip showed Escherichia coil and Staphylococcus aureus. We review the literature and discuss possible causes.

Expression of serine protease matriptase in renal cell carcinoma: correlation of tissue microarray immunohistochemical expression analysis results with clinicopathological parameters.

Serine protease matriptase (matriptase) cleaves and activates proteins implicated in the progression of cancer and represents a potential therapeutic target. Immunohistochemical analysis of matriptase was performed in tissue microarrays of 168 renal cell carcinomas (RCCs). All subtypes of RCC showed significant immunohistochemical expression of matriptase. In contrast, no expression occurred in areas of RCC with sarcomatous differentiation (SRCC) and in normal collecting tubules. The matriptase scores were significantly higher in papillary RCC (341+/-28) and clear cell RCC with granular cell differentiation (GRCC; 324+/-27) than in other histologic subtypes of RCC. In GRCC, matriptase scores were correlated with TNM staging and nuclear grading. Matriptase was overexpressed in all subtypes of RCC, and matriptase scores could distinguish between conventional clear cell RCC, GRCC, and SRCC.

Increasing expression of serine protease matriptase in ovarian tumors: tissue microarray analysis of immunostaining score with clinicopathological parameters.

Matriptase is a type II transmembrane serine protease expressed by cells of surface epithelial origin, including epithelial ovarian tumor cells. Matriptase cleaves and activates proteins implicated in the progression of cancer and represents a potential prognostic and therapeutic target. The aim of this study was to examine the expression of matriptase in ovarian tumors and to assign clinicopathological correlations. Immunohistochemical analysis of matriptase was performed in tissue microarrays of 164 ovarian neoplasms including 84 serous adenocarcinomas, 23 mucinous adenocarcinomas, 10 endometrioid adenocarcinomas, six yolk sac tumors, 12 clear cell carcinomas, six dysgerminomas, eight granulosa cell tumors, four transitional cell carcinomas, five fibromas, and six Brenner tumors. All ovarian tumors except the fibromas and Brenner tumors showed significant expression of matriptase. The matriptase scores were significantly higher in the tumors than in their nontumor counterparts (304+/-26 for serous adenocarcinoma; 361+/-28 for mucinous adenocarcinoma; 254+/-17 for endometrioid adenocarcinoma; 205+/-19 for yolk sac tumor; 162+/-16 for clear cell carcinoma; 109+/-11 for dysgerminoma; 105+/-9 for granulosa cell tumor; and 226+/-18 for transitional cell carcinoma). Matriptase scores in serous adenocarcinoma were correlated with TNM stage and FIGO stage. Our findings demonstrate for the first time that matriptase is overexpressed in many malignant ovarian tumors. It may be a novel biomarker for diagnosis and treatment of malignant ovarian tumors.

Utility of FDG-PET for investigating unexplained serum AFP elevation in patients with suspected hepatocellular carcinoma recurrence.

UNLABELLED: The aim of this study was to evaluate the potential role of positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) in patients with unexplained rising serum alpha-fetoprotein (AFP) levels after the treatment of hepatocellular carcinoma (HCC). PATIENTS AND METHODS: Thirty-one FDG-PET studies were performed in 26 patients (age range, 45-83; 21 men and 5 women), who had undergone either surgical resection or interventional therapy for HCC, but were subsequently noted to have high AFP serum levels on routine follow-up examinations, although imaging studies and physical examinations were normal. The FDG-PET results were correlated with histological findings, as well as long-term radiological and clinical follow-up (shortest follow-up period after FDG-PET was 6 months). RESULTS: FDG-PET was abnormal in 22 of the 31 studies (71.0%) among the 26 patients. Intrahepatic lesions were detected in 20 of a total 30 lesions (66.7%) in 18 studies of FDG-PET among 26 patients. Ten FDG-PET studies among 9 patients identified one intrahepatic lesion, while 3 studies among 3 patients identified more than one intrahepatic lesion. Extrahepatic metastases were found in 9/31 studies of FDG-PET (29.0%) among 8 patients. These metastatic foci, composed of increased FDG accumulation, were identified in several locations; lung (4 studies among 4 patients), bone (2 studies among 2 patients) and the peritoneum (4 studies among 3 patients). Overall, FDG-PET for detecting HCC recurrence demonstrated 22 true-positives, 8 false-negatives, 1 true-negative and 0 false-positive results., The sensitivity, specificity and accuracy of FDG-PET for detecting HCC recurrence was 73.3%, 100% and 74.2%, respectively. CONCLUSION: When conventional examinations are normal, FDG-PET is a valuable imaging tool in patients who have rising AFP levels after HCC treatment. FDG-PET whole-body scan also provides an important and valuable imaging study for detecting extrahepatic metastasis.

Modulation of mitogen-activated protein kinase cascades by differentiation-1 protein: acquired drug resistance of hormone independent prostate cancer cells.

PURPOSE: The inhibitor of differentiation-1 protein (Id-1) is over expressed in multidrug resistance prostate cancer cells. We determined the effect of Id-1 expression and its underlying pathways on the development of multidrug resistance in prostate cancer. MATERIALS AND METHODS: AT3 cells were transfected with the Id-1 gene or a blank vector. Id-1 mRNA expression was determined by reverse transcriptase-polymerase chain reaction and Id-1 protein content was detected by immunoblot and flow cytometry. Cellular cytotoxicity was determined by MTT (microculture 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Sigma Chemical Co., St. Louis, Missouri). The activation and expression of mitogen-activated protein kinase (MAPK) were measured by transactivation assay and Western blotting, respectively. RESULTS: Id-1 overproduction drove AT3 cells to become resistant to chemotherapeutic agents but did not induce mdr-1 gene expression. The p38MAPK and c-jun N-terminal kinase (JNK) pathways were suppressed, which correlated with increased Id-1 expression. No significant change in extracellular signal-regulated kinase (ERK) activation was observed in Id-1 transfectants compared with that of AT3 or vector control. Treatment of Id-1 expressing cells with p38MAPK and JNK inhibitors resulted in decreased doxorubicin induced apoptosis. In contrast, Id-1 expressing cells treated with ERK inhibitor made cells more sensitive to drug induced apoptosis. CONCLUSIONS: Up-regulation of Id-1 was found in prostate cancer multidrug resistant cells. Sustained ERK activation, and JNK and p38MAPK inhibition by Id-1 in cells may confer drug resistance. These changes in MAPKs could be a mechanism for the acquisition of multidrug resistance in prostate cancer.

The association of Id-1, MIF and GSTpi with acquired drug resistance in hormone independent prostate cancer cells.

Most patients with prostate cancer respond to androgen ablation therapy, but the tumor eventually progresses to an androgen-independent stage with multiple anti-cancer drug resistance. Novel therapeutic strategies for hormone independent prostate cancer need to be developed. The genes for Id-1, MIF and GSTpi were up-regulated in drug resistant cells of hormone independent prostate cancer cells using cDNA microarray gene expression analysis. In this study we aimed to investigate whether constitutive overexpression of these candidate genes in cells can affect cellular resistance to chemotherapeutic agents. The mRNA expression was determined by reverse transcriptase-polymerase chain reaction, and the Id-1, MIF and GSTpi protein contents in these cell lines were measured by Western blotting. Susceptibility of the cells to chemotherapeutic agents including doxorubicin, paclitaxel and cyclophosphamide was determined by microculture tetrazolium bromide assay. The analysis of apoptosis was assessed by annexin V assay. The cytotoxity assay results demonstrated that cells overexpressing the Id-1 gene increased in their resistance to doxorubicin, paclitaxel and cyclophosphamide. MIF expression can drive cells to increase in their resistance to paclitaxel, and GSTpi expression confers drug resistance to doxorubicin and cyclophosphamide. The induction of mdr1 gene expression was noted in up-regulation of MIF and GSTpi. Our findings suggest that overproduction of the Id-1, MIF and GSTpi proteins and coinduction of mdr-1 play an important role in the development of acquired drug resistance to various drugs of prostate cancer cells.

Antitumor effects of recombinant BCG and interleukin-12 DNA vaccines on xenografted murine bladder cancer.

OBJECTIVES: To evaluate the antitumor effects of recombinant bacille Calmette-Guérin (BCG) DNA (multi-rBCG) and murine interleukin-12 DNA (mIL-12) vaccines on xenografted MBT-2 murine bladder tumors. METHODS: Treatment with combined multi-rBCG and mIL-12 was examined in syngeneic C3H/HeN mice and athymic nude mice. The delivery efficiency of multi-rBCG expression was detected by flow cytometry. Inhibition of tumor growth was monitored, and antitumor effects were evaluated after one dose of electroporation immunogenetherapy, with measurement of cytokines and phenotyping of infiltrating lymphocytes in tumors. RESULTS: In vivo expression of multi-rBCG was efficient and reached a maximum on day 7 after electroporation. Treatment with multi-rBCG plus mIL-12 significantly inhibited tumor growth in C3H/HeN mice, with increased production of Th1-type cytokines, including interferon-gamma and IL-12. Treatment with multi-rBCG and/or mIL-12 in C3H/HeN mice induced infiltration of CD4+/CD8+ T cells and expansion of natural killer cells within tumors. By contrast, however, athymic nude mice treated in the same way showed no significant immune cells within tumors and died of the fast growing tumors. CONCLUSIONS: Electroporation using multi-rBCG plus mIL-12 could be effective immunotherapy for existing bladder cancer. The antitumor effects correlated with the elicitation of Th1 lymphocytes and natural killer cell-mediated cytotoxic immune responses.

Treatment of bladder carcinomas using recombinant BCG DNA vaccines and electroporative gene immunotherapy.

Intravesical immunotherapy with live Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the treatment of choice for superficial bladder cancers. Nevertheless, a significant proportion of patients do not respond to this therapy, and adverse effects are common. Here, we report the cloning of recombinant mycobacterial DNA vaccines and demonstrate the ability of multicomponent and multisubunit DNA vaccines to enhance Th1-polarized cytokine-mediated responses as well as effector cell responses. Splenocytes from immunized groups of mice were restimulated in vitro and examined for cytotoxicity against murine bladder tumur (MBT-2) cells. We used four combined recombinant BCG DNA vaccines (poly-rBCG) for electroporative gene immunotherapy (EPGIT) in vivo, and found that tumor growth was significantly inhibited and mouse survival was prolonged. Increased immune cell infiltration and induction of apoptosis were noted after treatment with poly-rBCG alone, with the murine interleukin-12 (mIL-12) vaccine alone, and-most significantly-with the poly-rBCG+mIL-12 vaccine combination. Electroporation of poly-rBCG+mIL-12 resulted in complete tumor eradication in seven of eight mice (P<.01) within 28 days. Thus, EPGIT using multicomponent multisubunit BCG is highly effective in the treatment of bladder cancer. This approach presents new possibilities for the treatment of bladder cancer using recombinant BCG DNA vaccines.

Immunotherapy for bladder cancer using recombinant bacillus Calmette-Guerin DNA vaccines and interleukin-12 DNA vaccine.

PURPOSE: We investigated the efficacy of recombinant bacillus Calmette-Guerin (BCG) DNA (poly-rBCG) and murine interleukin (IL)-12 (mIL-12) vaccines in inducing T helper 1 polarized cytokines and suppressing bladder tumor growth in mice. MATERIALS AND METHODS: Four mycobacteria candidate genes (Ag85A, Ag85B, Mpt64 and PstS3) were cloned, fused with ESAT6 and ligated into eukaryotic expression vectors. Combined poly-rBCG and mIL-12 vaccines were transferred into a murine bladder tumor model. The efficiency of gene expression was detected using Western blotting, flow cytometry and semiquantitative reverse transcriptase-polymerase chain reaction. Systemic cytokine responses, tumor growth and cumulative survival rates were monitored. RESULTS: Transfected bladder cancer cells showed high in vitro and in vivo expression of the recombinant subcomponents. Mice with tumors injected with poly-rBCG plus mIL-12 produced serum interferon-gamma significantly within 21 days but no significant elevations in tumor necrosis factor-alpha, IL-2, IL-4 or IL-5 were found. On day 28 after electroporation the growth of MBT-2 implants treated with poly-rBCG, mIL-12 or poly-rBCG plus mIL-12 was significantly inhibited. The cumulative survival of mice treated with poly-rBCG plus mIL-12 was significantly higher than that of the other 3 groups. CONCLUSIONS: Highly immunopotent recombinant vaccines of bacillus Calmette-Guerin DNA were produced that elicited T helper 1 immune responses with a high serum interferon-gamma level, inhibited tumor growth and prolonged the survival of tumor bearing mice. Thus, electroporation immunogene therapy using poly-rBCG plus mIL-12 may be an attractive regimen for the treatment of bladder cancer.

Modulation of prostate carcinoma cell growth and apoptosis by chromogranin A.

PURPOSE: We elucidated the effect and possible pathways of chromogranin A in regulating prostatic cancer cell growth. MATERIALS AND METHODS: Chromogranin A expression in prostatic cancer cell lines were detected with immunofluorescence flow cytometry (FCM) and the inhibition of cell growth due to chromogranin A antibody was measured using microculture tetrazolium. Cell cycle and RNA changes were evaluated with acridine orange FCM. Intracellular chromogranin A variations were detected with FCM, as were apoptotic changes, p53, Fas and tumor necrosis factor-alpha. Apoptosis and caspase-3 expression of tumor cells was assessed with dual immunohistochemical staining. RESULTS: Increased chromogranin A expression was observed in PC-3, DU145 and LNCap cells independent of hormone dependence. Dose and time dependent growth inhibition occurred at 12 hours. Chromogranin A antibody arrested PC3 cells in the S-phase immediately after treatment. The number of G0/G1 and G2/M cells subsequently decreased. PC3 tumor cells had transiently increased RNA at 12 hours with a marked decrease at 48 hours. Decreasing chromogranin A expression started at 12 hours and was most prominent at 48 hours. Apoptotic cells markedly increased at 12 hours with an increase in p53, Fas and tumor necrosis factor-alpha (Fas more than the others). Increased apoptotic cell and caspase-3 expression was observed on immunohistochemical stains. CONCLUSIONS: Chromogranin A is an important neuropeptide regulating the growth of prostate cancer cells. Specific antibodies can suppress its function through apoptotic pathways (Fas and caspase-3), leading to programmed cell death. Chromogranin A antibody mediated apoptosis may be a specific alternative treatment for prostate cancer.

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.

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 alpha-tumor necrosis factor (TNF-alpha). 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-alpha 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-alpha 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-alpha, 69% in Fas and adriamycin combination and Fas and TNF-alpha combination, 103% in combination of TNF-alpha 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-alpha 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-alpha.