Treatment Of T24 Cells Research Articles

The Effects of Orientin on Proliferation and Apoptosis of T24 Human Bladder Carcinoma Cells Occurs Through the Inhibition of Nuclear Factor-kappaB and the Hedgehog Signaling Pathway.

BackgroundOrientin is a flavone isolated from medicinal plants used in traditional Chinese medicine (TCM), which suppresses the growth of cancer cells in vitro. The effects of orientin in bladder cancer cells remains unknown. This study aimed to investigate the effect of orientin on proliferation and apoptosis of T24 human transitional cell bladder carcinoma cells in vitro in the presence of an agonist and an inhibitor of nuclear factor-kappaB (NF-κB).Material/MethodsT24 cells were cultured and divided into four study groups: an untreated control group; a group treated with 100 μM orientin; a group treated with 100 μM orientin with NF-κB agonist, phorbol 12-myristate 13-acetate (PMA); and a group treated with 100 μM orientin and the NF-κB inhibitor, IκBα. The MTT assay was performed to assess cell viability, and flow cytometry evaluated the cell cycle. The expression of proteins in the Hedgehog signaling pathway and inflammatory cytokines were determined by Western blot and enzyme-linked immunosorbent assay (ELISA).ResultsOrientin inhibited the proliferation of T24 cells, caused cell cycle arrest, reduced cell viability, and inhibited the expression of inflammatory mediators. Treatment of T24 cells with orientin inhibited the expression of NF-κB and components of the Hedgehog signaling pathway, and the NF-κB agonist, PMA, reversed these effects.ConclusionsTreatment of T24 human bladder carcinoma cells in vitro with orientin inhibited cell proliferation and promoted cell apoptosis by suppressing the Hedgehog signaling pathway and NF-κB.

Induction of mitochondrial-dependent apoptosis in T24 cells by a selenium (Se)-containing polysaccharide from Ginkgo biloba L. leaves

In the present study, a selenium (Se)-containing polysaccharide (Se-GBLP) was isolated and purified from the leaves of Ginkgo biloba L. Se-GBLP was further evaluated for its antitumor activity against human bladder cancer T24 cells together with the possible mechanism of action. Our results showed that treatment of T24 cells with Se-GBLP (50, 100 and 200μg/ml) for 48h significantly inhibited cell viability and induced apoptosis in a dose- dependent manner. This Se-GBLP-induced apoptosis is associated with an increased protein expression of pro-apoptotic Bax, decreased expression of anti-apoptotic Bcl-2, loss of mitochondrial membrane potential, and cleavage of caspase-9, caspase-3 and PARP, suggesting that Se-GBLP-induced apoptosis occurs through the mitochondria-dependent pathway. Se-GBLP therefore merits further investigation as a promising preventive and/or therapeutic agent against human bladder cancer.

MP36-07 ROLE OF ADIPOSE TISSUE IN BLADDER CANCER PROGRESSION

You have accessJournal of UrologyBladder Cancer: Basic Research I1 Apr 2015MP36-07 ROLE OF ADIPOSE TISSUE IN BLADDER CANCER PROGRESSION Nisha Hariharan, Teresa Johnson-Pais, Robert Svatek, Keith Ashcraft, and Robin Leach Nisha HariharanNisha Hariharan More articles by this author , Teresa Johnson-PaisTeresa Johnson-Pais More articles by this author , Robert SvatekRobert Svatek More articles by this author , Keith AshcraftKeith Ashcraft More articles by this author , and Robin LeachRobin Leach More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2015.02.735AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Obesity is associated with an increased risk of disease recurrence in patients with bladder cancer. One potential explanation is that adipose tissue surrounding the bladder may secrete tumor-promoting factors as this has been shown in other solid malignancies. The objective of our study is to elucidate the paracrine interactions between adipose tissue and surrounding bladder tumor as a potential contributor to disease progression. Hence, we characterized potential secreted pro-tumor factors in adipose tissue of obese and non-obese muscle-invasive bladder cancer (MIBC) patients. METHODS Adipose tissue in the abdominal subcutaneous space and surrounding the urinary bladder, urachus, and prostate was collected from normal weight [Body Mass Index (BMI) under 25 kg/m2, n=5], overweight (BMI 25 to 29.9 kg/m2, n=8) and obese (BMI>=30 kg/m2, n=7) patients undergoing radical cystectomy for MIBC. To isolate adipose stromal cells (ASCs), bladder and subcutaneous fat was digested and the stromal vascular fraction was placed into culture-specific media. The adherent ASCs were expanded, plated in 0.5% serum media and the conditioned (CM) was harvested. In addition, whole fat explants were incubated in serum-free media to harvest as whole fat CM. CM was characterized using the Millipore multi-analyte profiling. We evaluated the effect of ASC CM and whole fat CM on T24 cells migration, proliferation, and invasion. RESULTS We found several secreted factors in fat CM including Interleukin-8 and Growth Regulated Oncogene-alpha (GRO-alpha), which were highly elevated across all subjects, irrespective of BMI and fat source. Depot-specific whole fat CM from all the tested subjects increased T24 cell migration, but did not affect T24 cell proliferation or invasion. Treatment of T24 cells with GRO-alpha enhanced their migration and neutralizing antibody against GRO-alpha significantly inhibited migration. Secreted factors in the ASC CM were sufficient to induce T24 cells migration. ASC CM from an obese subject's subcutaneous fat significantly elevated T24 cell migration compared to ASC CM from normal and overweight subject's subcutaneous fat. Notably, we detected high levels of Plasminogen Inhibitor Activator-1 in ASC CM from multiple depots. Ongoing studies are focused on understanding the mechanism by which these factors promote bladder cancer progression in vivo. CONCLUSIONS Secreted factors from adipose cells collected from bladder cancer patient influence bladder tumor cells in vitro. These studies provide the first step in identifying adipose tissue-derived factors that promote bladder tumor progression. © 2015 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 193Issue 4SApril 2015Page: e431 Advertisement Copyright & Permissions© 2015 by American Urological Association Education and Research, Inc.MetricsAuthor Information Nisha Hariharan More articles by this author Teresa Johnson-Pais More articles by this author Robert Svatek More articles by this author Keith Ashcraft More articles by this author Robin Leach More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Diallyl trisulfide-induced apoptosis of bladder cancer cells is caspase-dependent and regulated by PI3K/Akt and JNK pathways

Diallyl trisulfide (DATS) is one of the major organosulfur components of garlic (Allium sativum L.), which inhibits the proliferation of various cancer cells, but the exact mechanisms of this action in human bladder cancer cells still remain largely unresolved. In this study, we investigated how DATS induces apoptosis in T24 human bladder cancer cells in vitro. Treatment of T24 cells with DATS resulted in potent anti-proliferative activity. Additionally, some typical apoptotic characteristics, such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells, were observed. With respect to the mechanism underlying the induction of apoptosis, DATS reduced the expression of anti-apoptotic Bcl-2 and Bcl-xL, and inhibitor of apoptosis protein family proteins, but the expression of pro-apoptotic Bax and death receptor-related proteins was increased compared with the controls. DATS also activated caspase-8 and -9, the respective initiator caspases of the extrinsic and the intrinsic apoptotic pathways. The increase in mitochondrial membrane depolarization was correlated with activation of effector caspase-3 and cleavage of poly-ADP-ribose polymerase, a vital substrate of activated caspase-3. Blockage of caspase activation through treatment with a pan-caspase inhibitor consistently inhibited apoptosis and abrogated growth inhibition in DATS-treated T24 cells. The study further investigated the roles of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) pathways with respect to the apoptotic effect of DATS, and showed that DATS deactivates Akt. Additionally, DATS activates extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK), but not p38 MAPK, in T24 cells. Unlike ERK, JNK inhibitors reversed DATS-induced apoptosis and growth inhibition; however, inhibition of PI3K/Akt notably enhanced the apoptotic action of DATS. The results suggest that the pro-apoptotic activity of DATS is probably regulated by a caspase-dependent cascade through the activation of both intrinsic and extrinsic signaling pathways, which is mediated through the blocking of PI3K/Akt and the activation of the JNK pathway.

Compound K Induces Apoptosis of Bladder Cancer T24 Cells Via Reactive Oxygen Species-Mediated p38 MAPK Pathway

Compound K (CK; 20-O-D-glucopyranosyl-20(S)-protopanaxadiol), a major metabolite of ginsenoside, has been shown to possess several biological activities such as potent antitumor properties. However, the effect of CK on the apoptosis of bladder cancer cells and its underlying mechanisms remain poorly understood. Therefore, we examined the effect of CK on the apoptosis of bladder cancer T 24 cells. Cell counts showed that treatment of T24 cells with CK decreased the cell number in a dose- and time-dependent manner. Flow cytometric analysis revealed that CK could significantly induce apoptosis of T24 cells in vitro. Further, cellular glutathione reduction, accumulation of reactive oxygen species (ROS) were also observed in CK-treated T24 cells. Western blot demonstrated the release of cytochrome c, activation of procaspases-3, procaspases-9, and the change of Bax/Bcl-2 proteins ratio. We also found that the phosphorylation of p38MAPK was increased by CK, while treatment with SB203580 inhibited CK-induced cell apoptosis in T24 cells. The blockage of ROS generation by N-acetylcysteine effectively prevented the apoptosis induction in T24 cells with CK treatment, accompanied by the decrease of activation of p38MAPK. These results suggested that CK induced the apoptosis of bladder cancer T24 cells, which is partially due to ROS generation and p38MAPK activation.

N6-Isopentenyladenosine and its Analogue N6-Benzyladenosine Induce Cell Cycle Arrest and Apoptosis in Bladder Carcinoma T24 Cells

Cytokinins are phytohormones critically involved in the regulation of plant growth and development. They also affect the proliferation and differentiation of animal cells, thus representing new tools to treat diseases that involve dysfunctional cell growth and/or differentiation. Recently, by performing structure-function studies on human cells, we found that only N6-isopentenyladenosine and its benzyl analogue N6-benzyladenosine suppress the clonogenic activity and the growth of different neoplastic cells. We here broaden our studies on bladder carcinoma T24 cells, because, due to the high recurrence rate of bladder cancer, new active molecules are sought to contrast the growth of this tumor. Early events induced by N6-isopentenyladenosine and N6-benzyladenosine are the alteration of T24 cell morphology and the disorganization of the actin cytoskeleton. After 24 h N6-isopentenyladenosine and N6-benzyladenosine inhibit growth by arresting the cells in the G0/G1 phase of the cell cycle. We also show that the two compounds induce apoptosis, an event linked to the activation of caspase 3. Since DNA damage is a prime factor resulting in cell cycle arrest and apoptosis, it is noteworthy that we do not detect any genotoxic effect upon treatment of T24 cells with N6-isopentenyladenosine and N6- benzyladenosine. Because the disruption of actin filaments leads to G1 arrest and is also implicated in apoptosis, we hypothesize that cytoskeletal rearrangement might be responsible for triggering the antiproliferative and proapotpotic effects of N6-isopentenyladenosine and N6- benzyladenosine in T24 cells.

Induction of apoptosis by costunolide in bladder cancer cells is mediated through ROS generation and mitochondrial dysfunction.

Despite the availability of several therapeutic options, a safer and more effective modality is urgently needed for treatment of bladder cancer. Costunolide, a member of sesquiterpene lactone family, possesses potent anticancer properties. In this study, for the first time we investigated the effects of costunolide on the cell viability and apoptosis in human bladder cancer T24 cells. Treatment of T24 cells with costunolide resulted in a dose-dependent inhibition of cell viability and induction of apoptosis which was associated with the generation of ROS and disruption of mitochondrial membrane potential (Δψm). These effects were significantly blocked when the cells were pretreated with N-acetyl- cysteine (NAC), a specific ROS inhibitor. Exposure of T24 cells to costunolide was also associated with increased expression of Bax, down-regulation of Bcl-2, survivin and significant activation of caspase-3, and its downstream target PARP. These findings provide the rationale for further in vivo and clinical investigation of costunolide against human bladder cancer.

Abstract 3599: Aurora kinase inhibitors as a novel targeted drug therapy for bladder cancer

Abstract Introduction: Conventional chemotherapy for invasive bladder cancer has limited efficacy and is generally associated with poor patient prognoses. This study aims to evaluate the potential of targeting the aurora kinases, a family of mitotic regulators, with pharmacologic inhibitors as a novel treatment for bladder cancer. Materials and Methods: RNA microarray and reverse-transcriptase polymerase chain reaction (RT-PCR) were utilized to evaluate expression of genes associated with the mitotic spindle checkpoint, including aurora kinases A and B, in clinical tissue samples of urothelial carcinomas of the bladder and normal urothelium. Human urothelial carcinoma cell lines UM-UC-3 and T24 were treated with the nonspecific aurora kinase inhibitor ZM447439 and the aurora kinase A inhibitor MLN8237, either alone or in combination with gemcitabine or paclitaxel. Effects of drug treatments were evaluated by flow cytometry with propidium iodide staining, immunofluorescence microscopy, MTS proliferation assay, and TUNEL labeling. Results: RNA microarray analysis comparing human tissue specimens of urothelial carcinomas of the bladder (N=10) to normal urothelium (N=8) identified overexpression of 13 gene transcripts related to the mitotic spindle checkpoint, including aurora kinases A and B, in the cancer specimens. Upregulation of these genes was validated by RT-PCR on a separate set of clinical samples (N=4 for each group). Next, the impact of aurora kinase inhibitors on bladder cancer cell lines was assessed. ZM447439 and MLN8237 treatment of UM-UC-3 and T24 cell lines at concentrations of 10 nM to 1 uM induced G2/M cell cycle arrest and aneuploidy in a dose dependent manner. Immunofluorescence microscopy showed abnormal mitotic figures with multipolar spindle apparatuses in treated cells. Both cell lines also exhibited cell death by positive TUNEL staining 48 hours after treatment with either inhibitor. Simultaneous treatment of T24 cells with MLN8237 and paclitaxel and subsequent MTS proliferation assay revealed an antagonistic interaction between paclitaxel and MLN8237, whereas simultaneous treatment with MLN8237 and gemcitabine resulted in an additive effect. Conclusion: Several mitotic spindle checkpoint proteins, including the aurora kinases, are overexpressed in bladder cancer. Our data indicate that aurora kinase inhibitors have significant potential as a novel therapy for bladder cancer, as they induce abnormal mitosis, cell cycle arrest, and eventual death of bladder cancer cells in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3599. doi:10.1158/1538-7445.AM2011-3599

Inhibition of the epidermal growth factor receptor in bladder cancer cells treated with the DNA‐damaging drug etoposide markedly increases apoptosis

To investigate the effect of the epidermal growth factor receptor (EGFR) on the induction of apoptosis by the chemotherapeutic agent etoposide (VP16), and to examine the effect of combining VP16 with gefitinib to see if the cell-survival mechanism can be prevented. The bladder cancer cell lines RT4 and T24, representing low- and high-malignancy grades respectively, were treated with VP16 (10 or 50 microM) and the level of apoptosis determined using a commercial kit. EGFR receptor activity was determined by western blotting using antibodies against phosphorylated EGFR. The EGFR was either activated by heparin-binding (HB)-EGF (1 nM) or inhibited with the specific EGFR inhibitor gefitinib (1 or 5 microM). The pan-caspase inhibitor Z-VAD (30 microM) was used to test the involvement of caspase activity. Treatment of T24 bladder cancer cells with VP16 (50 microM) for 48 h induced phosphorylation of the EGFR and activation of the EGFR prevented the apoptosis induced by VP16. Thus, treatment of T24 cells with 50 microM VP16 for 48 h resulted in 19% apoptosis. However, activation of the EGFR with HB-EGF (1 nM) with VP16 (50 microM) significantly reduced the level of apoptosis by 25% (P < 0.05) showing that activating the EGFR has a cell-survival function. Inhibiting the EGFR with gefitinib (5 microM) blocked the VP16-induced activation of the EGFR. Combined treatment with gefitinib and VP16 resulted in 45% apoptotic cells, i.e. more than double the percentage of apoptotic cells with VP16 alone. This was found in both T24 and RT4 cells. Gefitinib used alone (1 and 5 microM) generated no apoptosis in the cells. Treatment of T24 cells with Z-VAD showed that apoptosis induced by both VP16 alone and VP16 with gefitinib was caspase-mediated. These results suggest that activation of the EGFR induced a cell-survival function when bladder cancer cells were treated with the DNA-damaging drug VP16, and that combined treatment with VP16 and the EGFR inhibitor gefitinib might improve the efficacy of treatment.

Characterization of the oligosaccharide component of α3β1 integrin from human bladder carcinoma cell line T24 and its role in adhesion and migration

Malignant transformation is highly associated with altered expression of cell surface N-linked oligosaccharides. These changes concern integrins, a family of cell surface glycoproteins involved in the attachment and migration of cells on various extracellular matrix proteins. The integrin α 3 β 1 is particularly interesting because of its role in migration and invasion of several types of metastatic tumours. In this study, α 3 β 1 from human bladder T24 carcinoma cells was purified and treated with peptide N-glycosidase F. Then the N-glycans of the α 3 and β 1 subunits were characterized using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). In α 3 β 1 integrin the presence of high-mannose, hybrid and predominantly complex type N-oligosaccharides was shown. Unlike to normal epithelium cells, in both subunits of α 3 β 1 integrin from cancer cells, the sialylated tetraantennary complex type glycan Hex 7HexNAc 6FucSia 4 was present. In a direct ligand binding assay, desialylated α 3 β 1 integrin exhibited significantly higher fibronectin-binding capability than untreated integrin, providing evidence that sialic acids play a direct role in ligand-receptor interaction. Moreover, α 3 β 1 integrin was shown to take part in T24 cell migration on fibronectin: anti- α 3 antibodies induced ca 30% inhibition of wound closure. Treatment of T24 cells with swainsonine reduced the rate of bladder carcinoma cell migration by 16%, indicating the role of β1,6 branched complex type glycans in this process. Our data show that α 3 β 1 integrin function may be altered by glycosylation, that both subunits contribute to these changes, and that glycosylation may be considered a newly found mechanism in the regulation of integrin function.

Induction of apoptosis by Chan Su, a traditional Chinese medicine, in human bladder carcinoma T24 cells

Chan Su is a traditional Chinese medicine prepared from the dried white secretion of the auricular and skin glands of toads, and has been used as an Oriental drug. However, little is known about the effect of Chan Su on the growth of human cancer cells. This study was undertaken to investigate the underlying mechanism of Chan Su-induced apoptosis in a human bladder carcinoma cell line, T24. The effects of this compound were also tested on cyclooxygenase (COX) activity. Treatment of T24 cells with Chan Su resulted in the inhibition of viability and induction of apoptosis in a concentration-dependent manner, which was proved by trypan blue counts, DAPI staining, agarose gel electrophoresis and flow cytometric analysis. Apoptosis of T24 cells by Chan Su was associated with a down-regulation of anti-apoptotic Bcl-2 and Bcl-X(S/L) expression and an up-regulation of pro-apoptotic Bax expression. Chan Su treatment induced the proteolytic activation of caspase-3 and caspase-9, and a concomitant degradation of poly(ADP-ribose)-polymerase and beta-catenin protein. Furthermore, Chan Su decreased the levels of COX-2 mRNA and protein expression without significant changes in the levels of COX-1, which was correlated with an inhibition in prostaglandin E(2) synthesis. Taken together, these findings partially provide novel insights into the possible molecular mechanisms of the anti-cancer activity of Chan Su.

ENHANCED SENSITIVITY OF BLADDER CANCER CELLS TO CISPLATIN MEDIATED CYTOTOXICITY AND APOPTOSIS IN VITRO AND IN VIVO BY THE SELECTIVE CYCLOOXYGENASE-2 INHIBITOR JTE-522

Cyclooxygenase-2 (COX-2) is a key inducible enzyme involved in the production of prostaglandins and its inhibitors have been shown to induce apoptosis in various cancer cells. Several anticancer agents also mediate apoptosis and may share the common intracellular pathways leading to apoptosis with COX-2 inhibitors. We reasoned that combination treatment of bladder cancer cells with COX-2 inhibitors and anticancer agents may result in synergistic apoptosis. We examined whether the selective COX-2 inhibitor JTE-522 (4-(4-cyclohexyl-2-methyloxazol-5-yl)-2-fluorobenzenesulfonamide) synergizes with anticancer agents in cytotoxicity and apoptosis against bladder cancer cells in vitro and in vivo. Cytotoxicity was determined by the microculture tetrazolium dye assay. Combination treatment of T24 bladder cancer cells with JTE-522 and cis-diamminedichloroplatinum (II) (CDDP) resulted in a synergistic cytotoxic effect. Synergy achieved in cytotoxicity with JTE-522 and CDDP was shown to be due to apoptosis. Treatment of T24 cells with JTE-522 decreased expression of the anti-apoptotic molecule Bcl-2. The in vivo significant growth inhibitory effect of JTE-522 and CDDP against the T24 line heterotransplanted in SCID mice was also observed. This study demonstrates that combination treatment of bladder cancer cells with the selective COX-2 inhibitor JTE-522 and CDDP results in synergistic cytotoxicity and apoptosis in vitro and in vivo. These findings support the potential clinical application of a combination of JTE-522 and CDDP for the treatment of bladder cancer as a new form of therapy with more selective cytotoxicity and fewer collateral side effects.

Autocrine/paracrine regulation of apoptosis in epithelial cells by prostaglandin E 2

This study was designed to investigate the effect of IL-1α-induced up-regulation of cyclooxygenase-2 (COX-2) on prostaglandin E 2 (PGE 2) secretion and the subsequent phenotypic effects of PGE 2 on epithelial cells. The effect of IL-1α on COX-2 expression was investigated in the T24 bladder epithelial cell line following treatment with 0, 0.05, 0.5, 1 or 10 ng/ml IL-1α for 1, 2, 4 or 6 h. Quantitative PCR confirmed up-regulation of expression of COX-2 with maximal expression observed following treatment with 0.5 ng/ml IL-1α for 1 h. Co-treatment of the cells with 0.5 ng/ml IL-1α in the presence or absence of 100 ng/ml IL-1 receptor antagonist (RA) abolished the up-regulation in COX-2 expression confirming that the effect of IL-1α is mediated via its membrane-bound receptors. Treatment with 0.5 ng/ml IL-1α resulted in a time-dependent increase in PGE 2 secretion with maximal secretion detected at 24 and 48 h after stimulation with IL-1α. Co-treatment of the cells with IL-1α and IL-1RA or the COX-2 enzyme inhibitor NS398 abolished the IL-1α mediated secretion of PGE 2. Treatment of T24 cells with 100 nM PGE 2 resulted in a significant elevation in cAMP generation confirming the expression of functional PGE 2 receptors. Finally, the effect of exogenous treatment with PGE 2 on apoptosis of T24 cells was assessed using cell death detection ELISA. T24 cells were treated with camptothecin to induce apoptosis in the presence or absence of 50 or 100 nM PGE 2 or 10 μM forskolin. Treatment of T24 cells with increasing doses of camptothecin alone resulted in a significant increase in the induction of apoptosis ( P<0.01). However, co-treatment of the cells with 50 or 100 nM PGE 2 or 10 μM forskolin resulted in the inhibition of induction of the apoptotic pathway by camptothecin. These data demonstrate that PGE 2 inhibits apoptosis of epithelial cells possibly via cAMP-dependent pathway.

ENHANCED SENSITIVITY OF BLADDER CANCER CELLS TO TUMOR NECROSIS FACTOR RELATED APOPTOSIS INDUCING LIGAND MEDIATED APOPTOSIS BY CISPLATIN AND CARBOPLATIN

The development and acquisition of multiple drug resistance in cancer cells are a consequence of cancer chemotherapy and remain a major obstacle in treatment. Therefore, there is an obvious need for alternative approaches, such as immunotherapy and gene therapy. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is one of the tumor necrosis factor ligand families and it selectively induces apoptosis against cancer cells. Several cytotoxic anticancer drugs also mediate apoptosis and may share the common intracellular pathways leading to apoptosis. We reasoned that combination treatment of cancer cells with TRAIL and drugs may overcome this resistance. We evaluated whether bladder cancer cells are sensitive to TRAIL mediated cytotoxicity and whether TRAIL may synergize with anticancer agents in cytotoxicity and apoptosis against bladder cancer cells. Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. Human T24 bladder cancer line was relatively resistant to TRAIL and TRAIL was not cytotoxic against normal bladder cells. Treatment of T24 cells with TRAIL in combination with 5-fluorouracil or mitomycin C did not overcome resistance to these agents. However, treatment of T24 cells with a combination of TRAIL and cisplatin resulted in a synergistic cytotoxic effect. Synergy was also achieved in the cisplatin resistant T24 line (T24/CDDP), 2 other bladder cancer lines and 3 freshly derived bladder cancer cells. The combination of TRAIL and carboplatin resulted in a synergistic cytotoxic effect on T24 cells. However, the combination of TRAIL and trans-diamminedichloroplatinum (II) resulted in an antagonistic cytotoxic effect. The synergy achieved in cytotoxicity with TRAIL and cisplatin was also achieved in apoptosis. Treating T24 cells with cisplatin enhanced the expression of bax but not bcl-2. Incubation of T24 cells with TRAIL increased the intracellular accumulation of cisplatin. This study demonstrates that combination treatment of bladder cancer cells with TRAIL and cisplatin overcomes their resistance. The sensitization obtained with established cisplatin resistant and freshly isolated bladder cancer cells required low subtoxic concentrations of cisplatin, supporting the in vivo potential application of a combination of TRAIL and cisplatin for treating TRAIL resistant and cisplatin resistant bladder cancer.

Caspase involved synergistic cytotoxicity of bcl-2 antisense oligonucleotides and Adriamycin on transitional cell cancer cells

We have previously shown that Bcl-2 expression was negative prognostic factor in transitional cell cancer (TCC), and that TCC cell lines expressing high levels of Bcl-2 are resistant to Adriamycin triggered apoptosis. Here we examined antisense oligonucleotide-mediated downregulation of Bcl-2 expression and its effect on sensitivity to Adriamycin (ADM) treatment in T24 cells. Treatment of T24 cells with 20 μM of bcl-2 antisense phosphorothioate oligodeoxynucleotide (PODN) reduced the Bcl-2 protein level. Combined administration with Adriamycin resulted in synergistic cytotoxicity, accompanied with a 2.4-fold increase in DEVD-specific caspase activity. The finding provides evidence that Bcl-2 expression may be critical for maintaining the drug resistance of TCC. bcl-2 antisense PODN might be useful means for overcoming drug resistance in highly malignant TCC.

Chemoimmunosensitization of the T24 human bladder cancer line to Fas-mediated cytotoxicity and apoptosis by cisplatin and 5-fluorouracil.

Previous studies have demonstrated that cisplatin (CDDP) sensitizes bladder cancer cells to Fas-mediated cytotoxicity and that CDDP also enhances the cytotoxic effect of 5-fluorouracil (5-FU). These agents mediate apoptosis and may share common intracellular pathways leading to cell killing. We reasoned that combination treatment with CDDP, 5-FU and anti-Fas monoclonal antibody (mAb) might overcome the drug-resistance. We investigated whether CDDP, 5-FU and anti-Fas mAb synergize in cytotoxicity and apoptosis against the T24 bladder cancer line. Cytotoxicity was monitored by a one day microculture tetrazolium dye assay. Treatment of T24 cells with anti-Fas mAb in combination with CDDP or 5-FU resulted in a synergistic cytotoxic effect. In addition, combination treatment of T24 cells with CDDP, 5-FU and anti-Fas mAb further enhanced the synergistic cytotoxicity achieved by two agents. The synergy achieved in cytotoxicity with CDDP, 5-FU and anti-Fas mAb was also achieved in apoptosis. The current study demonstrates that combination treatment of bladder cancer cells with CDDP, 5-FU and anti-Fas mAb overcomes their resistance. In addition, the sensitization required low concentrations of CDDP and 5-FU, and thus supporting the potential in vivo application of combination of CDDP, 5-FU and immunotherapy in the treatment of drug-resistant bladder cancer.

Effect of anticancer agents on Fas-mediated cytotoxicity against bladder cancer cells

Fas and Fas ligand play an important role in cytotoxic T lymphocyte-mediated cytotoxicity. Like Fas ligand, anti-Fas monoclonal antibody (mAb) induces apoptosis of cells expressing Fas and mimics tumor necrosis factor-alpha (TNF-alpha) in its cytotoxic activity, but not in regard to other TNF-alpha-mediated activities. Since combination treatment with TNF-alpha and some anticancer chemotherapeutic agents results in synergistic cytotoxicity against various cancer cells, anti-Fas mAb may also synergize with anticancer agents in exerting cytotoxicity. The present study examined this hypothesis using bladder cancer cells. Cytotoxicity was examined by a 1-day microculture tetrazolium dye assay. Treatment of T24 cells with anti-Fas mAb in combination with mitomycin C, methotrexate, or 5-fluorouracil did not overcome their resistance to these agents. However, combination treatment with anti-Fas mAb and adriamycin (ADR) resulted in a synergistic cytotoxic effect on T24 cells, three other bladder cancer lines, and fresh bladder cancer cells derived from four patients. Treatment with ADR enhanced the expression of Fas on T24 cells. The expression of P-glycoprotein was not affected by the antibody-mediated sensitization. This study showed that combination treatment of bladder cancer cells with anti-Fas mAb and ADR can overcome their resistance and that the upregulation of Fas expression by ADR may play a role in the enhanced cytotoxicity.

Doxorubicin sensitizes human bladder carcinoma cells to Fas-mediated cytotoxicity.

The resistance of bladder carcinoma to anticancer chemotherapeutic agents remains a major problem. Hence, several immunotherapeutic approaches have been developed to treat the drug-resistant cancer cells. Fas antigen (Fas) and Fas ligand participate in cytotoxicity mediated by T lymphocytes and natural killer cells. Like Fas ligand, anti-Fas monoclonal antibody (MoAb) induces apoptosis of the cells expressing Fas. This study examined whether bladder carcinoma cells are sensitive to cytotoxicity mediated by anti-Fas MoAb and whether anticancer agents synergize with anti-Fas MoAb in cytotoxicity. Cytotoxicity was determined by a 1-day microculture tetrazolium dye assay. Synergy was assessed by isobolographic analysis. The T24 human bladder carcinoma cell line constitutively expressed the Fas on the cell surface; however, T24 line was resistant to anti-Fas MoAb. Treatment of T24 cells with anti-Fas MoAb in combination with mitomycin C, methotrexate, or 5-fluorouracil did not overcome their resistance to these agents. However, treatment of T24 cells with a combination of anti-Fas MoAb and doxorubicin resulted in a synergistic cytotoxic effect. In addition, the doxorubicin-resistant T24 cells were sensitive to treatment with a combination of anti-Fas MoAb and doxorubicin. Synergy was also achieved in three other bladder carcinoma cell lines and four freshly derived human bladder carcinoma cells. Treatment with anti-Fas MoAb in combination with epirubicin or pirarubicin also resulted in a synergistic cytotoxic effect on T24 cells. The mechanisms of synergy were examined. Anti-Fas MoAb did not affect the intracellular accumulation of doxorubicin, the expression of P-glycoprotein, or the expression of the antioxidant glutathione S-transferase-pi mRNA. However, treatment with doxorubicin enhanced the expression of Fas on T24 cells. This study demonstrated that treatment of bladder carcinoma cells with doxorubicin sensitized the cells to lysis by anti-Fas MoAb. The synergistic effect obtained with established doxorubicin-resistant bladder carcinoma cells and freshly isolated bladder carcinoma cells suggests that drug-resistant bladder carcinoma cells can be sensitized by doxorubicin to Fas- and Fas ligant-mediated cytotoxicity by lymphocytes. Furthermore, the sensitization required low concentrations of doxorubicin, thus supporting the in vivo application of a combination of chemotherapy and immunotherapy in the treatment of drug-resistant and/or immunotherapy-resistant bladder carcinoma.

Enhancement of sensitivity of urinary bladder tumor cells to cisplatin by c-myc antisense oligonucleotide

Tumor cells have different degrees of sensitivity and resistance to anticancer agents. The acquisition of drug resistance is a major concern in cancer treatment. Because the c-myc oncogene has been implicated in the poor prognosis of some human tumors including urinary bladder tumor (UBT), the resistance of such tumors to anticancer drugs may correlate with c-myc expression. The present study tested this hypothesis by examining the effect of c-myc antisense oligonucleotide treatment on the sensitivity of human UBT cells to anticancer chemotherapeutic agents. Cytotoxicity determined by a 1-day microculture tetrazolium dye assay. C-myc mRNA was examined by Northern blot analysis. The T24 human UBT cell line constitutively expresses c-myc mRNA. Treatment of T24 cells with the c-myc antisense oligonucleotide resulted in a significant inhibition of the expression of c-myc mRNA. Treatment of T24 cells with the c-myc antisense oligonucleotide in combination with mitomycin C, Adriamycin, or 5-fluorouracil (5-FU) did not overcome their resistance to these anticancer chemotherapeutic agents. However, combination treatment with the c-myc antisense oligonucleotide and CDDP resulted in a synergistic cytotoxic effect on T24 cells and two freshly derived UBT cells. Further, treatment of CDDP-resistant T24 cells (T24/CDDP) with c-myc antisense oligonucleotide and CDDP reversed the resistance. Pretreatment of T24 cells with either agent and then treatment with the second agent resulted in the same cytotoxic activity as achieved in the presence of the two agents. The combination of c-myc antisense oligonucleotide and carboplatin also resulted in a synergistic cytotoxic effect on T24 cells, and the combination of c-myc antisense oligonucleotide and trans-diamminedichloro-platinum resulted in an additive cytotoxic effect. Incubation of T24 or T24/CDDP cells with the c-myc antisense oligonucleotide increased the intracellular accumulation of CDDP, but not the accumulation of 5-FU. This study demonstrates that combination treatment with c-myc antisense oligonucleotide and CDDP can overcome the CDDP-resistance of UBT cells and that the increased intracellular accumulation of CDDP by c-myc antisense oligonucleotide may play a role in the enhanced cytotoxicity obtained. The synergistic effect obtained with established UBT cells and freshly isolated UBT cells suggests that combination treatment with c-myc antisense oligonucleotide and CDDP may have clinical application in the therapy of CDDP-resistant UBT.

Modulation by Cis-Diamminedichloroplatinum (II) of the Susceptibilities of Human T24 Lined and Freshly Separated Autologous Urinary Bladder Transitional Carcinoma Cells to Peripheral Blood Lymphocytes and Lymphokine Activated Killer Cells

The effects of eis-diamminedichloroplatinum (II) on the susceptibilities of human T24 lined and freshly separated autologous urinary bladder transitional carcinoma cells to lysis by peripheral blood lymphocytes of patients with urinary bladder cancer was analysed in a 12-hour 51Cr release assay. Treatment of T24 cells with cis-diamminedichloroplatinum (II) at 10 μg./ml. for three hours enhanced their susceptibility to peripheral blood lymphocytes and lymphokine activated killer cells. Kinetics studies demonstrated that the enhancement of their susceptibility became noticeable by three hours and continued until 12 hours. The susceptibilities of autologous tumor cells to both large granular lymphocytes and T lymphocytes were also enhanced by treatment of them with cis-diamminedichloroplatinum (II). There was no significant difference in the number of peripheral blood lymphocytes binding to cis-diamminedichloroplatinum (II)-treated T24 cells as compared with untreated T24 cells. Treatment of T24 cells with mitomycin C did not change their natural killer sensitivity. Pretreatment of T24 cells with cis-diamminedichloroplatinum (II) and lysosomotrophic agents (L-leucin-methyl-ester or chloroquine) reduced the enhancement of their susceptibility to natural killer cells by cis-diamminedichloroplatinum (II) alone. On the other hand, pretreatment of peripheral blood lymphocytes with cis-diamminedichloroplatinum (II) had no influence on the cytotoxicity against T24 cells. These results indicate that cis-diamminedichloroplatinum (II) may have an augmenting effect on the susceptibility of tumor cells to the cell-mediated cytotoxicity partly through a modification of cell membrane independently of its antimetabolic activity and this modification may be one of the possible mechanisms responsible for tumor regression after chemotherapy with cis-diamminedichloroplatinum (II).