benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Urinary-Bladder-Neoplasms

The aim of this study was to explore the correlation between hepatocyte cell adhesion molecule (hepaCAM) and SMAD family member 2/3 (SMAD2/3) in bladder carcinoma, and the involvement of the SMAD2/3 pathway in hepaCAM-induced tumor apoptosis. Immunohistochemistry was used to measure hepaCAM and p-SMAD2/3 protein levels in bladder cancer tissues. Flow cytometry and Hoechst staining were used to study the effect of hepaCAM on cellular apoptosis. Western blot was employed to determine the expression of hepaCAM and SMAD2/3/caspase pathway molecules using a hepaCAM overexpression adenovirus, a caspase inhibitor (Z-VAD-FMK), and a SMAD2/3 activator (transforming growth factor (TGF)-β1), respectively. Translocation of p-SMAD2/3 was measured by immunofluorescence and western blot. HepaCAM proteins were significantly decreased (P < 0.05), while p-SMAD2/3 proteins were remarkably increased (P < 0.05) in bladder carcinoma compared to adjacent tissues. However, the low hepaCAM and high p-SMAD2/3 were not statistically associated with clinicopathological characteristics of the patients. A negative linear correlation between hepaCAM and p-SMAD2/3 was observed according to Pearson analysis (r = -0.712/-0.724, P = 0.008/0.011). Overexpression of hepaCAM activated caspase 3/8/9 and downregulated poly-ADP ribose polymerase (PARP) and p-SMAD2/3. Treatment of bladder cancer cells with Z-VAD-FMK + hepaCAM significantly downregulated procaspase 3/8/9 and PARP and induced cellular apoptosis, compared with that using Z-VAD-FMK alone. Similarly, combined treatment of TGF-β1 + hepaCAM significantly downregulated p-SMAD2/3, procaspase 3/8/9, and PARP and induced apoptosis of bladder cancer cells, compared with TGF-β1 alone. Overexpression of hepaCAM prevented the p-SMAD2/3 translocation from the cytoplasm to the nucleus in bladder cancer cells BIU-87 and T24. Our findings uncover that the p-SMAD2/3 pathway is critical for hepaCAM-induced cancer cell apoptosis and provide valuable insights for current and future Ad-hepaCAM and p-SMAD2/3 clinical trials.

Topics: Aged; Amino Acid Chloromethyl Ketones; Apoptosis; Carcinoma, Transitional Cell; Caspases; Cell Cycle Proteins; Cell Line, Tumor; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Neoplasm Proteins; Neoplasm Staging; Poly(ADP-ribose) Polymerases; Protein Transport; Proteins; Recombinant Fusion Proteins; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta1; Urinary Bladder Neoplasms

Diallyl disulfide (DADS), one of the major components of garlic (Allium sativum), is well known to have chemopreventative activity against human cancer such as colon, lung and skin. But the exact mechanism of the action is still unclear. In this study, we investigated how DADS--induced cell cycle arrest and apoptosis in T24 human bladder cancer cells in vitro. Apoptosis induction, cell viability, cell cycle arrest, caspases-3, -9 activity and gene expression were measured to determine their variation by flow cytometric assay, western blot, and determination of caspase-3 activity, PCR and cDNA microarray. There are significant differences in cell death (decreased viable cells then increased the amounts of apoptosis) of T24 cells that were detected between DADS (5-75 microM) treated and untreated groups. A significant increase was found in apoptosis induction when cells were treated with DADS (50 microM) compared to without DADS treated groups. DADS also promoted caspase-3 activity after exposure for 1, 3, 6, 12, and 24 h, which led to induce apoptosis. DADS also increased the product of intracellular hydrogen peroxide. Furthermore, the DADS-induced apoptosis on T24 cells was blocked by the broad-spectrum caspase inhibitor, z-VAD-fmk and antioxidant (catalase). DADS also increased cyclin E and decreased CDK2 gene expression which may lead to the G2/M arrest of T24 cells.

Topics: Allyl Compounds; Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Caspase 3; Caspases; CDC2-CDC28 Kinases; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinase 2; Disulfides; DNA Fragmentation; DNA, Neoplasm; Enzyme Activation; Flow Cytometry; Humans; Hydrogen Peroxide; Indicators and Reagents; Oligonucleotide Array Sequence Analysis; Poly Adenosine Diphosphate Ribose; Reverse Transcriptase Polymerase Chain Reaction; Trypan Blue; 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

Selective estrogen receptor modulator is a proven agent for chemoprevention and chemotherapy of cancer. Raloxifene, a mixed estrogen agonist/antagonist, was developed to prevent osteoporosis and potentially reduce the risk of breast cancer. In this study, we examined the effect of raloxifene on the TSU-PR1 cell line. This cell line was originally reported to be a prostate cancer cell line, but recently it has been shown to be a human bladder transitional cell carcinoma cell line. The TSU-PR1 cell line contains high levels of estrogen receptor beta. Following treatment with raloxifene, evidence of apoptosis, including change in nuclear morphology, DNA fragmentation, and cytochrome c release, was observed in a dose-dependent manner in the TSU-PR1 cells (10(-9) to 10(-6) m range). We observed no detectable change in the steady-state levels of Bax, Bcl-2, and Bcl-X(L) following raloxifene treatment. However, raloxifene induced caspase-dependent cleavage of BAD to generate a 15-kDa truncated protein. Overexpression of a double mutant BAD resistant to caspase 3 cleavage blocked raloxifene-induced apoptosis. These results demonstrate that raloxifene induces apoptosis through the cleavage of BAD in TSU-PR1 cells. This molecular mechanism of apoptosis suggests that raloxifene may be a therapeutic agent for human bladder cancer.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; bcl-Associated Death Protein; bcl-X Protein; Carrier Proteins; Caspase 3; Caspases; Cell Division; Cell Membrane; Cell Nucleus; Cycloheximide; Cytochrome c Group; DNA Fragmentation; Dose-Response Relationship, Drug; Estrogen Receptor Modulators; Humans; In Situ Nick-End Labeling; Membrane Potentials; Mitochondria; Phosphorylation; Protein Binding; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-bcl-2; Raloxifene Hydrochloride; Retroviridae; Time Factors; Tumor Cells, Cultured; Urinary Bladder Neoplasms