benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Carcinoma--Renal-Cell

Renal cell carcinoma (RCC) is the most common malignancy in the kidney in the world, and the 5-year overall survival for patients remains poor due to the lack of effective treatment strategies. Although ABT-737, as a Bcl-2/Bcl-xL inhibitor, has recently emerged as a novel cancer therapeutic reagent, apoptosis induced by ABT-737 is often blocked in several types of cancer cells. This study investigated whether the combination of the small-molecule BH3 mimetic ABT-737 and the lysosome inhibitor chloroquine was an effective strategy for treating renal cancer cells. We found that the combination of ABT-737 and chloroquine synergistically decreased cell viability when compared to treatment with either single reagent. Cell apoptosis induced by a combined treatment was markedly inhibited by the caspase inhibitors z-DEVD-FMK and z-VAD-FMK. It was also inhibited by cathepsin inhibitor E-64 and CTSI (cathepsin inhibitor), which suggested that apoptosis was dependent on the cascade of caspase activation and cathepsins released from lysosomes. Furthermore, we found that ABT-737 could increase the cell level of ROS, which triggers cathepsin-mediated cell death and augments the role of chloroquine in cell death. So the combination of ABT-737 and chloroquine was an effective strategy for the treatment of renal cancer cells, and this combined strategy may widen the therapeutic window of ABT-737 and chloroquine as well as enhance the clinical efficacy of synergistic drug combinations.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptosis Regulatory Proteins; Biphenyl Compounds; Carcinoma, Renal Cell; Cell Death; Cell Line, Tumor; Chloroquine; Drug Synergism; Humans; Kidney Neoplasms; Leucine; Nitrophenols; Oligopeptides; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sulfonamides; Thiadiazoles

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received attention as a potential anticancer drug, because it induces apoptosis in a wide variety of cancer cells but not in most normal human cell types. Here, we showed that co-treatment with subtoxic doses of dioscin and TRAIL-induced apoptosis in Caki human renal cancer cells. Treatment of Caki cells with dioscin downregulated c-FLIPL and Bcl-2 proteins in a dose-dependent manner. Dioscin-induced decrease in c-FLIPL protein levels may be caused by the increased protein instability. We also found that dioscin induced downregulation of Bcl-2 at the transcriptional level. Pretreatment with NAC slightly inhibited the expression levels of c-FLIPL downregulated by the treatment of dioscin, suggesting that dioscin is partially dependent on the generation of ROS for downregulation of c-FLIPL. Taken together, the present study demonstrates that dioscin enhances TRAIL-induced apoptosis in human renal cancer cells by downregulation of c-FLIPL and Bcl-2.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Carcinoma, Renal Cell; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 3; Diosgenin; Down-Regulation; Humans; Kidney Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA, Messenger; TNF-Related Apoptosis-Inducing Ligand

Cafestol, one of the major compounds in coffee beans, has been reported for its tumor cell growth inhibitory activity and anti-carcinogenic activity, although the mechanism of action is poorly understood. In the present study, we investigated the effect of cafestol on the apoptotic pathway in human renal Caki cells and other cancer cell lines. Cafestol treatment inhibited Caki cells viability a dose-dependent manner by inducing apoptosis, as evidenced by DNA fragmentation and the accumulation of sub-G1 phase. Cafestol-induced apoptosis is associated with the reduction of mitochondrial membrane potential (MMP), activation of caspase 3, cytochrome c release, and down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1 and cFLIP). Cafestol-induced apoptosis was blocked by pretreatment with broad caspase inhibitor z-VAD-fmk, showing its dependence on caspases. Ectopic expression of Bcl-2 or Mcl-1 in Caki cells attenuates cafestol-induced apoptosis. In addition, we have also shown that cafestol inhibits phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway, and PI3K inhibitor LY29004 significantly increases cafestol-induced apoptosis in Caki cells. Taken together, our results show the activity of cafestol to modulate multiple components in apoptotic response of human renal Caki cells and a potential as a therapeutic agent for preventing cancers such as renal carcinoma.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Carcinoma, Renal Cell; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Chromones; Cytochromes c; Diterpenes; DNA Fragmentation; Down-Regulation; G1 Phase; Humans; Kidney Neoplasms; Membrane Potential, Mitochondrial; Morpholines; Myeloid Cell Leukemia Sequence 1 Protein; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction

Apoptotic DNA fragmentation minimizes the risk of transferring genetic information from apoptotic cancer cells to the neighboring cells. We have reported previously that caspase-deficient human renal cell carcinoma (RCC) lines were almost completely resistant to apoptosis in response to cytotoxic agents. In the present report we examined apoptotic process in caspase competent RCC-91 cells. Apoptosis in RCC-91 cells was accompanied by activation of caspases-3 and -9; cleavage of PARP and DFF45 proteins; typical apoptotic nuclei fragmentation and mitochondrial collapse. Nevertheless, DNA in these cells was not degraded into oligonucleosomal fragments compared to control Jurkat cells. Expression of caspase-activated DNase, DFF40 accountable for characteristic ladder pattern was easily detectable in Jurkat but not renal cancer cells, providing one possible explanation for the lack of oligonucleosomal DNA fragmentation in apoptotic RCC cells. Lack of typical DNA fragmentation indicates a potential threat of transferring genetic information from one tumor cell to another or to the neighboring healthy cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis Regulatory Proteins; Blotting, Western; Carcinoma, Renal Cell; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Membrane Permeability; Cysteine Proteinase Inhibitors; Deoxyribonucleases; DNA Fragmentation; Enzyme Activation; Flow Cytometry; Humans; Intracellular Signaling Peptides and Proteins; Intracellular Space; Jurkat Cells; Kidney Neoplasms; Mitochondria; Nucleosomes; Poly-ADP-Ribose Binding Proteins; Tumor Necrosis Factor-alpha

The caspase family of proteases is speculated to have a crucial role in apoptosis. The effect of treatment with adriamycin (ADR), cisplatin (CDDP), 5-fluorouracil (5-FU), vinblastine (VLB), IFN-alpha, or IFN-gamma on the activation of caspase-3, -6, -8, and -9 in renal cell carcinoma (RCC) cells was investigated, to clarify the mechanisms of chemo- and immunotherapeutic agent-mediated apoptosis. Caspase activity was determined by a quantitative colorimetric assay. Apoptosis was monitored by acridine-orange staining assay. Treatment of ACHN cells with CDDP, VLB, IFN-alpha, or IFN-gamma did not activate caspase-3, but its activity was increased 7.2-fold (p = 0.0001) with ADR and 2.8-fold (p = 0.0385) with 5-FU in comparison with control. Furthermore, when the ADR treatment time was shortened from 24 to 8 or 2 h, the same caspase-3 activation occurred. Activation of caspase-3 was also observed in six freshly isolated human RCC cells after the treatment with ADR. Of the six freshly derived RCC cells treated with 5-FU, caspase-3 activity was increased 3.1-fold (p = 0.0051) and 2.4-fold (p = 0.0346) in two of them, respectively. Epirubicin and pirarubicin, compounds closely related to ADR, also respectively enhanced 4.2-fold (p = 0.0052) and 2.8-fold (p = 0.0147) caspase-3 activity in ACHN cells. The activation of caspase-3 observed with a colorimetric assay was confirmed with immunocytochemical analysis using the anti-active caspase-3 mAb, which specifically recognizes the active form of caspase-3. Furthermore, both active caspase-3 and apoptosis triggered by either ADR or 5-FU were inhibited significantly by the general caspase inhibitor Z-VAD-FMK, or a specific caspase-3 inhibitor DMQD-CHO. These findings provide a mechanistic explanation for anthracyclines and 5-FU induced-apoptosis.

Topics: Amino Acid Chloromethyl Ketones; Antibiotics, Antineoplastic; Apoptosis; Carcinoma, Renal Cell; Caspase 3; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Enzyme Activation; Fluorouracil; Humans; Immunoenzyme Techniques; Interferon-alpha; Interferon-gamma; Kidney Neoplasms; Tumor Cells, Cultured

Activated T cells lyse the murine renal cancer Renca. We have examined the mechanism of tumor cell lysis with the use of T cells derived from C57BL/6, BALB/c, B6.gld, and B6.Pfp-/- mice. C57BL/6 and BALB/c T cells can lyse Renca cells through the use of both granule- and Fas ligand (FasL)-mediated pathways. However, B6.gld T cells predominantly use granule-mediated killing, whereas B6.Pfp-/- T cells use FasL. The lysis of Renca by Pfp-/- T cells is only partially inhibited by the caspase inhibitor ZVAD-FMK, suggesting that caspase-independent signaling is also important for Renca cell lysis. When the reactive oxygen scavenger butylated hydroxyanisole was used alone or in combination with ZVAD-FMK a substantial reduction of Renca lysis was observed. Therefore, the caspase-independent generation of reactive oxygen intermediates in Renca after Fas triggering contributes to the lysis of these cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Apoptosis Regulatory Proteins; Butylated Hydroxyanisole; Carcinoma, Renal Cell; Caspases; Crosses, Genetic; Cytoplasmic Granules; Cytotoxicity, Immunologic; Dipeptides; Enzyme Inhibitors; Fas Ligand Protein; fas Receptor; Free Radical Scavengers; Humans; Interferon-gamma; Ketones; Kidney Neoplasms; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Reactive Oxygen Species; Recombinant Proteins; Signal Transduction; Specific Pathogen-Free Organisms; T-Lymphocytes, Cytotoxic; TNF-Related Apoptosis-Inducing Ligand; Transfection; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha