cytochrome-c-t and Urinary-Bladder-Neoplasms

Cancer is one of the major causes of human deaths at present. It is the leading cause of deaths in developed countries. Moreover, Circular RNAs (circRNAs) have been discovered to play important roles in tumor genesis and development and are abnormally expressed in bladder cancer .. The present study aims to investigate the anti-cancer effects of circ 001418 on bladder carcinoma and its possible mechanism.. Quantitative PCR (qPCR) and gene chip were used to measure the circ 001418 expression. Cell proliferation and transfer, apoptosis and caspase-8 and caspase-3 activity levels were measured using MTT, Transwell assay, Flow cytometry. Caspase-3 and 9 activity levels, EphA2, cytochrome c and FADD protein expression, were detected using Western blotting.. The expression of circ 001418 was increased in patients with bladder carcinoma. Over-expression of circ 001418 promoted cell proliferation and transfer, and reduced apoptosis in vitro model of bladder carcinoma. Down-regulation of Circ 001418 inhibited cell proliferation and transfer, and induced apoptosis in vitro model of bladder carcinoma. Meanwhile, over-expression of circ 001418 induced EphA2 and cytochrome c protein expression, and suppressed FADD protein expression in vitro model of bladder carcinoma by the suppression of miR-1297. MiR-1297 reduced the pro-cancer effect of circ 001418 on apoptosis of bladder carcinoma.. Results showed that circRNA 001418 promoted cell growth and metastasis of bladder carcinoma via EphA2 by miR-1297.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Fas-Associated Death Domain Protein; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Receptor, EphA2; RNA, Circular; Signal Transduction; Urinary Bladder; Urinary Bladder Neoplasms

Polydopamine-coated branched Au-Ag nanoparticles (Au-Ag@PDA NPs) exhibit good structural stability, biocompatibility, and photothermal performance, along with potential anticancer efficacy. Here, we investigated the cytotoxicity of Au-Ag@PDA NPs against human bladder cancer cells (T24 cells) in vitro and in vivo, as well as the underlying molecular mechanisms of photothermal therapy-induced T24 cell death.. T24 cells were treated with different doses of Au-Ag@PDA NPs followed by 808 nm laser irradiation, and the effects on cell proliferation, cell cycle, apoptosis, and autophagy were analyzed. To confirm the mechanisms of inhibition, real-time PCR and Western blot analysis were used to evaluate markers of cell cycle, apoptosis, autophagy, and the AKT/ERK signaling pathway. Moreover, we evaluated the effects of the treatment on mitochondrial membrane potential and ROS generation to confirm the underlying mechanisms of inhibition. Finally, we tested the T24 tumor inhibitory effects of Au-Ag@PDA NPs plus laser irradiation in vivo using a xenograft mouse model.. Au-Ag@PDA NPs, with appropriate laser irradiation, dramatically inhibited the proliferation of T24 cells, altered the cell cycle distribution by increasing the proportion of cells in the S phase, induced cell apoptosis by activating the mitochondria-mediated intrinsic pathway, and triggered a robust autophagy response in T24 cells. Moreover, Au-Ag@PDA NPs decreased the expression of phosphorylated AKT and ERK and promoted the production of ROS that function upstream of apoptosis and autophagy. In addition, Au-Ag@PDA NP-mediated photothermolysis also significantly suppressed tumor growth in vivo.. This preclinical study can provide a mechanistic basis for Au-Ag@PDA NP-mediated photothermal therapy toward promotion of this method in the clinical treatment of bladder cancer.

Topics: Animals; Apoptosis; Autophagy; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Gold; Humans; Hyperthermia, Induced; Indoles; Membrane Potential, Mitochondrial; Metal Nanoparticles; Mice, Nude; Phototherapy; Polymers; Reactive Oxygen Species; Signal Transduction; Silver; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Bladder cancer (urothelial carcinoma) is a common malignancy characterized by high recurrence rates and intense clinical follow-up, indicating the necessity for more effective therapies. Current treatment regimens include intra-vesical administration of mitomycin C (MMC) for non-muscle invasive disease and systemic cisplatin for muscle-invasive or metastatic disease. Hyperthermia, heating a tumor to 40-44°C, enhances the efficacy of these chemotherapeutics by various modes of action, one of which is inhibition of DNA repair via homologous recombination. Here, we explore whether ex vivo assays on freshly obtained bladder tumors can be applied to predict the response towards hyperthermia.. The cytochrome C release assay (apoptosis) and the RAD51 focus formation assay (DNA repair) were first established in the bladder cancer cell lines RT112 and T24 as measurements for hyperthermia efficiency, and subsequently tested in freshly obtained bladder tumors (n = 59).. Hyperthermia significantly increased the fraction of apoptotic cells after cisplatin or MMC treatment in both RT112 and T24 cells and in most of the bladder tumors (8/10). The RAD51 focus formation assay detected both morphological and numerical changes of RAD51 foci upon hyperthermia in the RT112 and T24 cell lines. In 64% of 37 analyzed primary bladder tumor samples, hyperthermia induced similar morphological changes in RAD51 foci.. The cytochrome C assay and the RAD51 focus formation assay are both feasible on freshly obtained bladder tumors, and could serve to predict the efficacy of hyperthermia together with cytotoxic agents, such as MMC or cisplatin.

Topics: Cell Line, Tumor; Cisplatin; Cytochromes c; Humans; Hyperthermia, Induced; Mitomycin; Urinary Bladder Neoplasms

Heat shock protein-70kDa (Hsp70) is a member of molecular chaperone family, involved in the proper folding of various proteins. Hsp70 is important for tumor cell survival and is also reported to be involved in enhancing the drug resistance of various cancer types. Hsp70 controls apoptosis both upstream and downstream of the mitochondria by regulating the mitochondrial membrane permeabilization (MMP) and apoptosome formation respectively. In the present study, we have elucidated the role of Hsp70 in Gambogic acid (GA) induced apoptosis in bladder cancer cells. We observed that functional inhibition of Hsp70 by Pifithrin-μ switches GA induced caspase dependent (apoptotic) cell death to caspase independent cell death. However, this cell death was not essentially necrotic in nature, as shown by the observations like intact plasma membranes, cytochrome-c release and no significant effect on nuclear condensation/fragmentation. Inhibition of Hsp70 by Pifithrin-μ shows differential effect on MMP. GA induced MMP and cytochrome-c release was inhibited by Pifithrin-μ at 12h but enhanced at 24h. Pifithrin-μ also reverted back GA inhibited autophagy which resulted in the degradation of accumulated ubiquitinated proteins. Our results demonstrate that Hsp70 plays an important role in GA induced apoptosis by regulating caspase activation. Therefore, inhibition of Hsp70 may hamper with the caspase dependent apoptotic pathways induced by most anti-cancer drugs and reduce their efficacy. However, the combination therapy with Pifithrin-μ may be particularly useful in targeting apoptotic resistant cancer cells as Pifithrin-μ may initiate alternative cell death program in these resistant cells.

Topics: Aged; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Autophagy; Carcinoma; Caspases; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme Activation; Female; HSP70 Heat-Shock Proteins; Humans; Male; Membrane Potential, Mitochondrial; Middle Aged; Necrosis; Proteolysis; Reactive Oxygen Species; Signal Transduction; Sulfonamides; Tumor Cells, Cultured; Ubiquitination; Urinary Bladder Neoplasms; Xanthones

Pachymic acid (PA) is a lanostane-type triterpenoid derived from Poria cocos mushroom that possess various biological effects such as anti-cancer, antiinflammatory and anti-metastasis effects. In this study, we investigated the anti-cancer effects of PA in EJ bladder cancer cells. The results showed that PA significantly inhibited proliferation of EJ cells in a dose-dependent manner. PA induced accumulation of sub-G1 DNA content (apoptotic cell population), apoptotic bodies and chromatin condensation and DNA fragmentation in EJ cells in a dose-dependent manner. PA also induces activation of caspase-3, -8 and -9, and subsequent cleavage of poly (ADP-ribose) polymerase, and significantly suppressed the inhibitor of apoptosis protein family proteins in a dose-dependent manner. Furthermore, PA activates Bid and induced the loss of mitochondrial membrane potential (ΔΨm ) with up-regulated pro-apoptotic proteins (Bax and Bad), down-regulated anti-apoptotic proteins (Bcl-2 and Bcl-xL) and cytochrome c release. In turn, PA increased the generation of reactive oxygen species (ROS); also, the ROS production was blocked by N-acetyl-L-cysteine. The expressions of TNF-related apoptosis inducing ligand and death receptor 5 were up-regulated by PA in a dose-dependent manner, suggesting extrinsic pathway also involved in PA-induced apoptosis. This study provides evidence that PA might be useful in the treatment of human bladder cancer.

Topics: Acetylcysteine; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Caspase 3; Caspases; Cytochromes c; DNA Fragmentation; Down-Regulation; Humans; Membrane Potential, Mitochondrial; Phospholipases A; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; TNF-Related Apoptosis-Inducing Ligand; Triterpenes; Up-Regulation; Urinary Bladder Neoplasms

To investigate the effects of ellagic acid on the growth inhibition of TSGH8301 human bladder cancer cells in vitro, cells were incubated with various doses of ellagic acid for different time periods. The phase-contrast microscope was used for examining and photographing the morphological changes in TSGH8301 cells. Flow cytometric assay was used to measure the percentage of viable cells, cell cycle distribution, apoptotic cells, ROS, mitochondrial membrane potential (ΔΨm), Ca(2+) , caspase-9 and -3 activities in TSGH8301 cells after exposure to ellagic acid. Western blotting was used to examine the changes of cell cycle and apoptosis associated proteins levels. Results indicated that ellagic acid induced morphological changes, decreased the percentage of viable cells through the induction of G0/G1 phase arrest and apoptosis, and also showed that ellagic acid promoted ROS and Ca(2+) productions and decreased the level of ΔΨm and promoted activities of caspase-9 and -3. The induction of apoptosis also confirmed by annexin V staining, comet assay, DAPI staining and DNA gel electrophoresis showed that ellagic acid induced apoptosis and DNA damage in TSGH8301 cells. Western blotting assay showed that ellagic acid promoted p21, p53 and decreased CDC2 and WEE1 for leading to G0/G1 phase arrest and promoting BAD expression, AIF and Endo G, cytochrome c, caspase-9 and -3 for leading to apoptosis in TSGH8301 cells. On the basis of these observations, we suggest that ellagic acid induced cytotoxic effects for causing a decrease in the percentage of viable cells via G0/G1 phase arrest and induction of apoptosis in TSGH8301 cells.

Topics: Annexin A5; Antineoplastic Agents; Apoptosis; Caspase 3; Caspase 9; Cell Cycle; Cell Line, Tumor; Cytochromes c; DNA Damage; Ellagic Acid; Endoplasmic Reticulum Stress; G1 Phase Cell Cycle Checkpoints; Humans; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species; Resting Phase, Cell Cycle; Signal Transduction; Urinary Bladder Neoplasms

We examined the expression of anti-apoptotic genes (XIAP and Bcl-2) and apoptotic genes (cytochrome c, caspase-9, Apaf-1) in tissue samples of patients with superficial bladder cancer. Thirty-two bladder cancer tissue samples (8 papillary urothelial neoplasm of low malignant potential, 10 low-grade, and 14 high-grade) and 8 normal bladder tissue samples from necropsy were used for the study of gene expression by real-time PCR analysis. Analysis of the expression of apoptotic gene constituents of an apoptosome demonstrated an increase in Apaf-1 expression in the three tumor grades when compared with the control (P < 0.01, P < 0.05, and P < 0.01), low expression of caspase-9 in all groups (P < 0.05), and an increase in cytochrome c expression in all tumor grades in relation to the control, although without statistically significant difference. The expression of anti-apoptotic genes revealed an increase in XIAP expression in all tumor grades in relation to the control, although without statistically significant difference, and low expression of Bcl-2 in all tumor grades and the control (P < 0.05). The results proved that there is low evidence of apoptotic activity by the intrinsic pathway, demonstrated by the low expression of caspase-9 and considerable increase in XIAP expression, which may render these genes potential therapeutic targets in bladder cancer treatment.

Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 9; Cytochromes c; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Proto-Oncogene Proteins c-bcl-2; Real-Time Polymerase Chain Reaction; Signal Transduction; Urinary Bladder Neoplasms

2-(3-methoxyphenyl)-6,7-methylenedioxoquinolin-4-one (MMEQ) is a novel synthesized compound, and this study investigated the effects of MMEQ on molecular signal pathways of the induction of apoptosis in TSGH8301 human bladder cancer cells. The studies included examining the effects of morphological changes by contrast-phase microscope, the percentage of viable cells, cell cycle distribution mitochondria membrane potential (ΔΨm), ROS and caspase activities were examined by flow cytometry, apoptotic cells were examined by DAPI staining and the changes of associated apoptosis proteins levels were examined by Western blotting. Release of apoptotic factors from mitochondria was examined by confocal laser microscope. Our results showed that MMEQ caused morphological changes and inhibited the cell growth of TSGH8301 cells in a time- and dose-dependent manner. MMEQ induced G2/M arrest through the promotion of chk1, chk2 and cdc25c in TSGH8301 cells. MMEQ caused a marked increase in the percentage of DNA damage and apoptosis as characterized by DAPI and DNA fragmentation. The specific inhibitors of caspase-8, -9, and -3 blocked MMEQ-induced growth inhibition action. A remarkable loss of ΔΨm and increase in ROS production were observed after a 24-h treatment. MMEQ promoted the levels of caspase-3, caspase-8, caspase-9, Bax, Bcl-xs, decreased the levels of Bcl-2 and Bid and then led to dysfunction of ΔΨm, following the releases of cytochrome c, AIF and Endo G from mitochondria to cytosol and nuclei, and finally caused cell apoptosis. In conclusions, these molecular mechanisms provide insight into MMEQ-caused growth inhibition, G2/M arrest and apoptotic cell death in TSGH8301 cells.

Topics: Apoptosis; Apoptosis Inducing Factor; bcl-2-Associated X Protein; Benzodioxoles; Caspases; cdc25 Phosphatases; Cell Cycle Checkpoints; Cell Division; Cell Line, Tumor; Checkpoint Kinase 1; Checkpoint Kinase 2; Cytochromes c; DNA Damage; DNA Fragmentation; G2 Phase; Humans; Membrane Potential, Mitochondrial; Mitochondria; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Quinolones; Reactive Oxygen Species; Signal Transduction; Urinary Bladder Neoplasms

Resveratrol shows chemopreventive activity in a variety of human cancers by targeting mitochondria and triggering apoptosis. The purpose of this study was to investigate the antitumor action of resveratrol in bladder cancer and its underlying mechanism.. Using two different bladder cell lines, BTT739 and T24, the cytotoxicity of resveratrol were determined by MTT assay. The apoptosis induced by resveratrol was assayed by transferase dUTP nick end labeling staining. To show whether the mitochondrial dysfunction involved in the effects of resveratrol, mitochondrial function was detected by mitochondrial membrane potential, reactive oxygen species production and adenosine 5'-triphosphate content. In addition, the markers of apoptosis in the intrinsic mitochondrial-dependent pathway were analyzed by the release of cytochrome c and the activities of caspase-9 and caspase-3.. Resveratrol effectively decreased cell viability and induced apoptosis in a concentration- and time-dependent manner. In addition, resveratrol significantly disrupted the mitochondrial membrane potential in both intact cells and isolated mitochondria. Resveratrol also increased reactive oxygen species production and reduced adenosine 5'-triphosphate concentrations. Western blot analysis showed that resveratrol provoked the release of cytochrome c from mitochondria to the cytosol. Furthermore, resveratrol significantly promoted the activation of caspase-9 and caspase-3.. These findings suggest that resveratrol efficiently triggers apoptosis in bladder cancer cells through the intrinsic mitochondrial-dependent pathway, which is associated with mitochondrial dysfunction. Resveratrol might have great pharmacological promise in the treatment of bladder cancer.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Drug Screening Assays, Antitumor; Enzyme Activation; Humans; Membrane Potential, Mitochondrial; Mice; Reactive Oxygen Species; Resveratrol; Stilbenes; Urinary Bladder Neoplasms

Sulforaphane (SFN), an isothiocyanate that exists exclusively in cruciferous vegetables, may be the most promising preventive agent for bladder cancer (BC) to date. We previously observed that SFN dramatically inhibits human BC T24 cells in vitro. Our hypothesis is that SFN may attenuate BC growth. To test our hypothesis, we investigated the effect of SFN on human BC UM-UC-3 cell xenografts implanted into athymic mice. Sulforaphane extract was routinely prepared in our laboratory, and its content was measured with high-performance liquid chromatography. Athymic mice were injected subcutaneously with a UM-UC-3 cell suspension (2.0×10(6) cells/200 μL per mouse) and randomly divided into 2 groups. The positive control group was orally gavaged with water, and the treatment group was orally administered SFN from broccoli sprout (12 mg/kg body weight) for 5 weeks. At the end of the experiment, tumor tissues were harvested and processed for hematoxylin and eosin staining and immunohistochemistry. The average tumor volume decreased from 4.1±1.67 cm(3) in the positive control mice to 1.5±0.72 cm(3) in the SFN-treated mice, evidencing an inhibitory rate of 63%. The SFN extract also reduced the appearance of tumors, including karyopyknosis and angiogenesis. Sulforaphane extract induced caspase 3 and cytochrome c expression but reduced the expression of survivin. Sulforaphane extract retards the growth of UM-UC-3 xenografts in vivo, confirming its future potential in BC therapy.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Brassica; Caspase 3; Cell Line, Tumor; Cytochromes c; Humans; Inhibitor of Apoptosis Proteins; Isothiocyanates; Male; Mice; Mice, Nude; Phytotherapy; Plant Extracts; Repressor Proteins; Sulfoxides; Survivin; Thiocyanates; Thymus Gland; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Sinulariolide, an isolated compound from the soft coral Sinularia flexibilis, possesses the anti-proliferative, anti-migratory and apoptosis-inducing activities against the TSGH bladder carcinoma cell. The anti-tumor effects of sinulariolide were determined by 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, cell migration assay and flow cytometry, respectively. Sinulariolide inhibited the growth and migration of bladder carcinoma cells in a dose-dependent manner, as well as induced both early and late apoptosis as determined by the flow cytometer. Also, the sinulariolide-induced apoptosis is related to the mitochondrial-mediated apoptosis via caspase-dependent pathways, elucidated by the loss of mitochondrial membrane potential, release of cytochrome C, activation of caspase-3/-9, Bax and Bad, as well as suppression of Bcl-2/Bcl-xL/Mcl-1. Detection of the PARP-1 cleaved product suggested the partial involvement of caspase-independent pathways. Moreover, inhibition of p38MAPK activity leads to the rescue of the cell cytotoxicity of sinulariolide-treated TSGH cells, indicating that the p38MAPK pathway is also involved in the sinulariolide-induced cell apoptosis. Altogether, these results suggest that sinulariolide induces apoptosis against bladder cancer cells through mitochondrial-related and p38MAPK pathways.

Topics: Animals; Anthozoa; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cytochromes c; Diterpenes; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Membrane Potential, Mitochondrial; Mitochondria; p38 Mitogen-Activated Protein Kinases; Time Factors; Urinary Bladder Neoplasms

Intravesical chemotherapy is often used to prevent the recurrence of superficial bladder cancer after transurethral resection. A search for more effective and less toxic intravesical agents is urgently needed. We previously found the in vitro apoptotic effects of silibinin, a natural flavonoid, on high-risk bladder carcinoma cells. Here, we further explored the underlying mechanisms and examined the intravesical efficacy in the prevention and treatment of bladder cancer. Human bladder carcinoma cell line 5637, which has the same molecular features of high-risk superficial bladder cancer, was used as the model system in vitro and in vivo. Autochthonous rat model of bladder cancer induced by intravesical N-methyl-N-nitrosourea (MNU) was used to investigate its intravesical efficacy. Exposure of 5637 cells to silibinin resulted in growth inhibition and induction of caspase-dependent and -independent apoptosis, which was associated with disruption of mitochondrial membrane potential and selective release of cytochrome c, Omi/HtrA2, and apoptosis-inducing factor (AIF) from mitochondria. Silibinin also downregulated survivin and caused nuclear translocation of AIF. Oral silibinin suppressed the growth of 5637 xenografts, which was accompanied with the activation of caspase-3, downregulation of survivin, and increased translocation of AIF. Furthermore, intravesical silibinin effectively inhibited the carcinogenesis and progression of bladder cancer in rats initiated by MNU by reducing the incidence of superficial and invasive bladder lesions without any side effects, which was accompanied with proapoptotic effects. These findings identify the in vitro and in vivo antitumor efficacy of silibinin, and suggest silibinin as an effective and novel intravesical agent for bladder cancer.

Topics: Administration, Intravesical; Animals; Antioxidants; Apoptosis; Apoptosis Inducing Factor; Carcinoma, Transitional Cell; Cell Line, Tumor; Cell Survival; Cytochromes c; Down-Regulation; Female; High-Temperature Requirement A Serine Peptidase 2; Humans; Inhibitor of Apoptosis Proteins; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Mitochondrial Proteins; Rats; Rats, Sprague-Dawley; Serine Endopeptidases; Silybin; Silymarin; Survivin; Urinary Bladder Neoplasms; Xenograft Model Antitumor Assays

Allyl isothiocyanate (AITC) occurs in many commonly consumed cruciferous vegetables and exhibits significant anti-cancer activities. Available data suggest that it is particularly promising for bladder cancer prevention and/or treatment. Here, we show that AITC arrests human bladder cancer cells in mitosis and also induces apoptosis. Mitotic arrest by AITC was associated with increased ubiquitination and degradation of α- and β-tubulin. AITC directly binds to multiple cysteine residues of the tubulins. AITC induced mitochondrion-mediated apoptosis, as shown by cytochrome c release from mitochondria to cytoplasm, activation of caspase-9 and caspase-3, and formation of TUNEL-positive cells. Inhibition of caspase-9 blocked AITC-induced apoptosis. Moreover, we found that apoptosis induction by AITC depended entirely on mitotic arrest and was mediated via Bcl-2 phosphorylation at Ser-70. Pre-arresting cells in G(1) phase by hydroxyurea abrogated both AITC-induced mitotic arrest and Bcl-2 phosphorylation. Overexpression of a Bcl-2 mutant prevented AITC from inducing apoptosis. We further showed that AITC-induced Bcl-2 phosphorylation was caused by c-Jun N-terminal kinase (JNK), and AITC activates JNK. Taken together, this study has revealed a novel anticancer mechanism of a phytochemical that is commonly present in human diet.

Topics: Allyl Compounds; Apoptosis; Caspase 3; Caspase 9; Cell Line, Tumor; Cytochromes c; Enzyme Activation; G1 Phase; Humans; Isocyanates; JNK Mitogen-Activated Protein Kinases; Mitochondria; Mitosis; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Tubulin; Ubiquitination; Urinary Bladder Neoplasms

Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root, has demonstrated anti-cancer properties. In the present study, we found that decursin inhibited cell viability in cultured human urinary bladder cancer 235J cells and colon cancer HCT116 cells. The inhibited proliferation was due to apoptotic induction, because both cells treated with decursin dose-dependently showed a sub-G1 phase accumulation and an increased cytoplasmic DNA-histone complex. Cell death caused by decursin was also associated with the down-regulation of anti-apoptotic factor Bcl-2 and the up-regulation of pro-apoptotic molecules cytochrome c, caspase 3 and Bax. Treatment of both types of cancer cells with decursin resulted in G1-phase cell cycle arrest, as revealed by FACS analyses. In addition, decursin increased protein levels of p21WAF1 with a decrease in cyclins and cyclin dependent kinases (CDKs). Furthermore, decursin induced the activation of extracellular signal-regulated kinases (ERK) in both cancer cell lines, with the notable exceptions of c-Jun N-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase. Finally, pretreatment with ERK-specific inhibitor PD98059 reversed decursin-induced p21WAF1 expression and decursin-inhibited cell growth. Thus, these findings suggest that decursin has potential therapeutic efficacy for the treatment of bladder and colon cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Benzopyrans; Butyrates; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Down-Regulation; Drug Screening Assays, Antitumor; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; G1 Phase; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Protein Kinase Inhibitors; Up-Regulation; Urinary Bladder Neoplasms

Human A(3) adenosine receptor (A(3)AR) plays an essential role in several physiopathological processes. Thus far, A(3)AR-selective ligands have been evaluated as anti-inflammation and anticancer therapeutic agents. Among these ligands, truncated thio-Cl-IB-MECA is a newly reported antagonist, and its function has not been studied.. Cell viability was measured by MTS assay. Cell cycle progression was analysed by propidium iodide (PI) flow cytometric assay. The apoptotic effects were investigated by Hoechst staining and annexin V-FITC/PI staining. The signal-transduction mechanism was explored by Western blot.. Truncated thio-Cl-IB-MECA induced the growth arrest of T24 cells at sub-G(1) phase and provoked apoptosis but not necrosis. Apoptotic death was mediated by the activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK).. Since truncated thio-Cl-IB-MECA induces anti-proliferation and apoptotic effects via ERK and JNK activation, it may function as an anticancer agent in human bladder cancer cells.

Topics: Adenosine; Adenosine A3 Receptor Antagonists; Apoptosis; bcl-2-Associated X Protein; Caspases; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; G1 Phase; Humans; MAP Kinase Kinase 4; Proto-Oncogene Proteins c-bcl-2; Urinary Bladder Neoplasms

This study investigated the anticancer activity of Magnolia officinalis on urinary bladder cancer in vitro and in vivo, and elucidated the mechanism of its activity. An aqueous extract of M. officinalis inhibited cell viability and DNA synthesis in cultured human urinary bladder cancer 5637 cells. Inhibition of proliferation was the result of apoptotic induction, because FACS analyses of 5637 cells treated with M. officinalis showed a sub-G1 phase accumulation. M. officinalis extract also increased cytoplasmic DNA-histone complex dose-dependently. These inhibitory effects were associated with the upregulation of proapoptotic molecules Bax, cytochrome c and caspase 3. Treatment of 5637 cells with M. officinalis extract suppressed the expression of matrix metalloproteinase 2 (MMP-2) and MMP-9, as revealed by zymographic and immunoblot analyses. When M. officinalis extract was given to mice simultaneously with the carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine, which induces urinary bladder tumors, the size of the induced tumors was smaller. Finally, histological data indicated that the histological grade of carcinoma and the depth of invasion were dramatically decreased by treatment with M. officinalis extract in mice with N-butyl-N-(4-hydroxybutyl) nitrosamine-induced urinary bladder tumors. In conclusion, the findings showed that M. officinalis extract exhibited potential chemopreventive activity against urinary bladder tumor in vitro and in vivo.

Topics: Animals; Anticarcinogenic Agents; Apoptosis; bcl-2-Associated X Protein; Butylhydroxybutylnitrosamine; Carcinoma, Transitional Cell; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA; Dose-Response Relationship, Drug; Humans; Magnolia; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Nucleic Acid Synthesis Inhibitors; Plant Extracts; Urinary Bladder Neoplasms

Taxol is the most commonly used agent for salvage chemotherapy in transitional cell carcinoma of the urothelium. We examined mechanisms responsible for taxol resistance by using T24 human bladder carcinoma cells.. We used an inhibitor and an activator of the phosphatidylinositol-3 kinase-Akt pathway in cell survival and caspase-3 assays, an HPLC method for determining released cytochrome c and immunoblotting for detecting protein phosphorylation.. Activation of Akt increased paclitaxel resistance by increasing Bad phosphorylation, leading to decreased release of mitochondrial cytochrome c and caspase-3-mediated apoptosis. On the other hand, inhibition of Akt prevented paclitaxel resistance by enhancing the effects of paclitaxel on Bad phosphorylation, mitochondrial cytochrome c release and caspase-3-mediated apoptosis, besides diminishing or abolishing the opposing effects of Akt activation.. Akt-mediated Bad phosphorylation plays an important role in preservation of mitochondrial membrane systems leading to paclitaxel resistance in T24 cells.

Topics: Antineoplastic Agents, Phytogenic; bcl-Associated Death Protein; Carcinoma, Transitional Cell; Caspase 3; Chromones; Cytochromes c; Drug Resistance, Neoplasm; Enzyme Activation; Humans; Mitochondrial Membranes; Morpholines; Oncogene Protein v-akt; Paclitaxel; Phosphatidylinositol 3-Kinases; Phosphorylation; Urinary Bladder Neoplasms

The efficacy of cisplatin in cancer chemotherapy is limited by the development of resistance. To elucidate the molecular basis of resistance to cisplatin, we compared cisplatin-induced apoptotic responses of the parental human bladder cancer cell line, T24 and its resistant subclone, T24R2. In T24 cells, cisplatin induce apoptosis and the activation of caspase-8, -9 and -3 and poly(ADP-ribose) polymerase cleavage. The expression levels of Fas, FasL, and FADD were not changed by the treatment with cisplatin. Furthermore, neither Fas-neutralizing antibody nor dominant negative mutant of FADD affected cisplatin-induced apoptosis. Western blot analysis of subcellular fractions showed that cisplatin induced redistribution of Bax and cytochrome c. Thus, cisplatin causes apoptosis in a death receptor-independent and mitochondria-dependent fashion in T24 cells. In contrast, overexpressed Bcl-2 protein inhibited cisplatin-induced Bax translocation and its downstream events in T24R2. Downregulation of Bcl-2 by RNAi potentiated the redistribution of Bax and cytochrome c and reversed cisplatin-resistance. Our results indicate that upregulation of Bcl-2 contributes to the development of cisplatin-resistance and usage of siRNA which targets the Bcl-2 gene may offer a potential tool to reverse the resistance to cisplatin in bladder cancer.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Line, Tumor; Cisplatin; Cytochromes c; Drug Resistance, Neoplasm; Enzyme Activation; Humans; Mitochondria; Protein Transport; Proto-Oncogene Proteins c-bcl-2; RNA Interference; Up-Regulation; Urinary Bladder Neoplasms

Silibinin, a natural flavonolignan, induces apoptosis in human bladder transitional-cell papilloma RT4 cells both in vitro and in vivo; however, mechanisms of such efficacy are not completely identified. Here, we studied the mechanisms involved in silibinin-induced apoptosis of RT4 cells having intact p53. Silibinin increased p53 protein level together with its increased phosphorylation at serine 15, activated caspase cascade and caused Bid cleavage for apoptosis. Silibinin-caused p53 activation was mediated via ATM-Chk2 pathway, which in turn induced caspase 2-mediated apoptosis. Pifithrin-alpha, a p53 inhibitor, reversed silibinin-induced caspase activation including caspase 2; however, caspase 2 inhibitor also reversed p53 phosphorylation suggesting a bidirectional regulation between them. Further, silibinin caused a rapid translocation of p53 and Bid into mitochondria leading to increased permeabilization of mitochondrial membrane and cytochrome c release into the cytosol. JNK1/2 activation was observed as a connecting link for p53-mediated caspase 2 activation. Interestingly, silibinin-induced apoptosis was mediated, in part, via Cip1/p21 cleavage by caspase, which was reversed by Cip1/p21 siRNA. Together, these results suggested the novel mechanisms for apoptosis induction by silibinin involving p53-caspase 2 activation and caspase-mediated cleavage of Cip1/p21.

Topics: Apoptosis; Benzothiazoles; Caspase 2; Cell Line, Tumor; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Models, Biological; Silybin; Silymarin; Toluene; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms

Consumption of the traditional kava preparation was reported to correlate with low and uncustomary gender ratios (more cancer in women than men) of cancer incidences in three kava-drinking countries: Fiji, Vanuatu, and Western Samoa. We have identified flavokawain A, B, and C but not the major kavalactone, kawain, in kava extracts as causing strong antiproliferative and apoptotic effect in human bladder cancer cells. Flavokawain A results in a significant loss of mitochondrial membrane potential and release of cytochrome c into the cytosol in an invasive bladder cancer cell line T24. These effects of flavokawain A are accompanied by a time-dependent decrease in Bcl-x(L), a decrease in the association of Bcl-x(L) to Bax, and an increase in the active form of Bax protein. Using the primary mouse embryo fibroblasts Bax knockout and wild-type cells as well as a Bax inhibitor peptide derived from the Bax-binding domain of Ku70, we showed that Bax protein was, at least in part, required for the apoptotic effect of flavokawain A. In addition, flavokawain A down-regulates the expression of X-linked inhibitor of apoptosis and survivin. Because both X-linked inhibitor of apoptosis and survivin are main factors for apoptosis resistance and are overexpressed in bladder tumors, our data suggest that flavokawain A may have a dual efficacy in induction of apoptosis preferentially in bladder tumors. Finally, the anticarcinogenic effect of flavokawain A was evident in its inhibitory growth of bladder tumor cells in a nude mice model (57% of inhibition) and in soft agar.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Transitional Cell; Caspase 3; Caspase 9; Caspases; Cell Growth Processes; Chalcone; Cytochromes c; Flavonoids; Humans; Inhibitor of Apoptosis Proteins; Kava; Membrane Potentials; Mice; Mice, Nude; Microtubule-Associated Proteins; Mitochondria; Neoplasm Proteins; Plant Extracts; Poly(ADP-ribose) Polymerases; Proteins; Proto-Oncogene Proteins c-bcl-2; Survivin; Urinary Bladder Neoplasms; X-Linked Inhibitor of Apoptosis Protein; Xenograft Model Antitumor Assays

Many isothiocysanates (ITC) are promising cancer-preventive agents, and induction of apoptosis is one of their underlying mechanisms of action. We recently found that caspase-9 was preferentially activated over other initiator caspases in human bladder cancer UM-UC-3 cells. We report here that caspase-9 activation is the major step leading to ITC-induced apoptosis in this cell line. More importantly, our results show that caspase-9 activation by the ITCs may result primarily from mitochondrial damage. Four common naturally occurring ITCs were studied, including allyl ITC, benzyl ITC (BITC), phenethyl ITC (PEITC), and sulforaphane. BITC and PEITC showed more potent mitochondria-damaging ability than the other two ITCs, correlating well with their stronger apoptosis-inducing potentials. Furthermore, BITC and PEITC damaged both the outer and inner mitochondrial membranes. Use of isolated mitochondria allowed us to establish that ITCs, and more importantly their major intracellular derivatives (glutathione conjugates) at concentrations that are readily achievable in cells, damage mitochondria, leading to the collapse of mitochondrial trans-membrane potential and release of cytochrome c. The mitochondria-damaging potencies of the ITCs correlate well with their lipophilicities. Bcl-2 family members are known to influence the stability of mitochondrial membrane. Our results show that the ITCs caused phosphorylation of Bcl-2, induced mitochondrial translocation of Bak, and disrupted the association of Bcl-xl with both Bak and Bax in mitochondrial membrane, indicating that ITC-induced mitochondrial damage results at least in part from modulation of select Bcl-2 family members.

Topics: Anticarcinogenic Agents; Apoptosis; Caspase 9; Caspases; Cytochromes c; Enzyme Activation; Humans; Intracellular Membranes; Isothiocyanates; Membrane Potentials; Mitochondria; Phosphorylation; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Urinary Bladder Neoplasms

Bladder cancer is a common cancer with high risk of recurrence and mortality. Intravesicle chemotherapy after trans-urethral resection is required to prevent tumor recurrence and progression. It has been known that antioxidants enhance the antitumor effect of bacillus Calmette-Guerin (BCG), the most effective intravesical bladder cancer treatment. Capsaicin, the major pungent ingredient in genus Capsicum, has recently been tried as an intravesical drug for overactive bladder and it has also been shown to induce apoptotic cell death in many cancer cells. In this study, we investigated the apoptosis-inducing effect and alterations in the cellular redox state of capsaicin in MBT-2 murine bladder tumor cells. Capsaicin induced apoptotic MBT-2 cell death in a time- and dose-dependent manner. The capsaicin-induced apoptosis was blocked by the pretreatment with Z-VAD-fmk, a broad-range caspase inhibitor, or Ac-DEVD-CHO, a caspase-3 inhibitor. In addition to the caspase-3 activation, capsaicin also induced cytochrome c release and decrease in Bcl-2 protein expression with no changes in the level of Bax. Furthermore, capsaicin at the concentration of inducing apoptosis also markedly reduced the level of reactive oxygen species and lipid peroxidation, implying that capsaicin may enhance the antitumor effect of BCG in bladder cancer treatment. These results further suggest that capsaicin may be a valuable intravesical chemotherapeutic agent for bladder cancers.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Capsaicin; Caspase 3; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Cytochromes c; Cytosol; DNA Fragmentation; Electrophoresis, Agar Gel; Enzyme Activation; Flow Cytometry; Mice; Mitochondria; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Time Factors; Urinary Bladder Neoplasms