benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Kidney-Neoplasms

Artesunate is a well-known anti-malarial drug originated from artemisinin as a Chinese herb and has been reported to have anti-cancer potential in many cancer cells. In the present study, we examined the efficacy of artesunate against the renal carcinoma Caki cells and explored its mechanism of cytotoxicity. A steep decline in cell viability within 18 h was recorded upon artesunate exposure, but pretreatment of z-VAD-FMK had no effect on the loss of the cell viability by artesunate. On the other hand, necrostatin-1 pretreatment and knockdown of RIP-1 significantly reduced the cytotoxicity of artesunate against Caki cell. Moreover, the generation of mitochondrial ROS prompted by artesunate was found to be the principle mechanism of cell death. Pretreatment with necrostatin-1 or knockdown of RIP-1 inhibited the generation of ROS by artesunate, resulting in the protection of the cells from artesunate toxicity. Moreover, the similar results were observed in the case of other renal carcinoma cell lines (ACHN and A498). The results suggest that artesunate induces the generation of ROS and cell death in RIP1-dependent manner. Therefore, our data suggest that artesunate could induce RIP1-dependent cell death in human renal carcinoma.

Topics: Amino Acid Chloromethyl Ketones; Artemisinins; Artesunate; Cell Death; Cell Line, Tumor; Humans; Imidazoles; Indoles; Kidney Neoplasms; Mitochondria; Neoplasm Proteins; Nuclear Pore Complex Proteins; Reactive Oxygen Species; RNA-Binding Proteins

Fisetin is a natural compound found in fruits and vegetables such as strawberries, apples, cucumbers, and onions. Since fisetin can elicit anti-cancer effects, including anti-proliferation and anti-migration, we investigated whether fisetin induced apoptosis in human renal carcinoma (Caki) cells. Fisetin markedly induced sub-G1 population and cleavage of poly (ADP-ribose) polymerase (PARP), which is a marker of apoptosis, and increased caspase activation. We found that pan-caspase inhibitor (z-VAD-fmk) inhibited fisetin-induced apoptosis. In addition, fisetin induced death receptor 5 (DR5) expression at the transcriptional level, and down-regulation of DR5 by siRNA blocked fisetin-induced apoptosis. Furthermore, fisetin induced p53 protein expression through up-regulation of protein stability, whereas down-regulation of p53 by siRNA markedly inhibited fisetin-induced DR5 expression. In contrast, fisetin induced up-regulation of CHOP expression and reactive oxygen species production, which had no effect on fisetin-induced apoptosis. Taken together, our study demonstrates that fisetin induced apoptosis through p53 mediated up-regulation of DR5 expression at the transcriptional level.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; Carcinoma; Caspase Inhibitors; Cell Line, Tumor; Down-Regulation; Flavonoids; Flavonols; Humans; Kidney Neoplasms; Mice; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Receptors, TNF-Related Apoptosis-Inducing Ligand; Transcription, Genetic; Tumor Suppressor Protein p53; Up-Regulation

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

The prostate-apoptosis-response-gene-4 (Par-4) protein has been shown to function as an effector of cell death in response to various apoptotic stimuli that trigger mitochondria and membrane receptor-mediated cell death pathways. We found that overexpressing Par-4 by stable transfection sensitizes Caki cells to induction of apoptosis by TRAIL and drugs that induce endoplasmic reticulum (ER) stress [thapsigargin (TG), tunicamycin (TU) and etoposide]. Ectopic expression of Par-4 is associated with decreased levels of XIAP protein in TG-treated cells, caused in part by XIAP protein instability and caspase activation. Levels of phospho-Akt are decreased in Caki/Par-4 cells to a significantly greater extent than in Caki/Vector cells by treatment with TG, and this is in turn associated with decreased levels of phospho-PDK1, the kinase upstream of Akt. In conclusion, we provide evidence that ectopic expression of Par-4 sensitizes Caki cells to TG and that XIAP protein instability and inactivation of Akt are important in cellular pathways affected by Par-4.

Topics: Adenocarcinoma, Clear Cell; Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; Caspases; Cell Division; Cell Line, Tumor; Endoplasmic Reticulum; Enzyme Activation; Etoposide; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Phosphorylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyruvate Dehydrogenase Acetyl-Transferring Kinase; Recombinant Fusion Proteins; Thapsigargin; TNF-Related Apoptosis-Inducing Ligand; Transfection; X-Linked Inhibitor of Apoptosis Protein

Renal cell carcinoma is the most common neoplasm occurring in the kidney and is largely resistant to current chemotherapy. Understanding the mechanisms involved in renal carcinoma cell death may lead to novel and more effective therapies. In Cak(i)-1 renal cancer cells, using phosphatidylserine externalization as a marker of apoptosis, the anti-cancer drugs 5-fluorouracil (5-FU), and its pro-drugs, doxifluridine (Dox) and floxuridine (Flox) proceeds via a caspase-dependent mechanism. In contrast, phosphatidylserine externalization produced by staurosporine in the renal cancer cell lines Cak(i)-1 and A-498 proceeds via a caspase-independent mechanism. That is, the pan caspase inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (ZVAD) did not ameliorate annexin V binding, cell shrinkage or changes in nuclear morphology. Subsequent experiments were conducted to determine mediators of phosphatidylserine externalization, using annexin V binding, when caspases were inhibited. Prior treatment of A-498 cells with cathepsin B (CA74 methyl ester), cathespsin D (pepstatin A) or calpain inhibitors (calpeptin, E64d) in the presence or absence of ZVAD did not ameliorate annexin V binding. The endonuclease inhibitor aurintricarboxylic acid (ATA), phospholipase A(2) inhibitor bromoenol lactone (BEL), protein synthesis inhibitor cycloheximide (CH) and chloride channel blockers niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) all had no effect on staurosporine-induced annexin V binding in A-498 cells either in the presence or absence of ZVAD. We also modulated sphingomyelin and the de novo pathways of ceramide synthesis and found no amelioration of staurosporine-induced annexin V binding in A-498 cells either in the presence or absence of ZVAD. These results indicate that 5-FU, Dox and Flox induce externalization of phosphatidylserine during apoptosis in Cak(i)-1 renal cancer cells primarily through a caspase-dependent mechanism and that externalization of phosphatidylserine during apoptosis produced by staurosporine in the renal cancer cell line A-498 is independent of many of the common signaling pathways known to be involved in this process.

Topics: Amino Acid Chloromethyl Ketones; Aniline Compounds; Antimetabolites, Antineoplastic; Apoptosis; Benzoic Acid; Benzylidene Compounds; Calpain; Caspases; Cathepsin B; Cathepsin D; Cell Line, Tumor; Cell Size; Ceramides; Cisplatin; Exocytosis; Fumonisins; Humans; Kidney Neoplasms; Naphthalenes; Niflumic Acid; Nitrobenzoates; Phosphatidylserines; Pyrones; Sphingomyelin Phosphodiesterase; Staurosporine; Triterpenes

Antimycin A, an inhibitor of electron transport in mitochondria, has been used as reactive oxygen species (ROS) generator in the biological system. Here, we investigated the in vitro effect of antimycin A on apoptosis in As4.1 juxtaglomerular cells. Antimycin A efficiently induced apoptosis in As4.1 cells as evidenced by flow cytometric detection of sub-G(1) DNA content, annexin V binding assay and DAPI staining. This apoptotic process was accompanied by loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bcl-2 decrease, caspase-3 activation and PARP cleavage. All of caspase inhibitors tested in this experiment failed to rescue As4.1 cells from antimycin A-induced cell death at the time of 48 h in view of sub-G(1) cells and annexin V positive staining cells. However, with regard to the mitochondrial membrane potential (DeltaPsi(m)), pan caspase inhibitor (Z-VAD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) at the concentration of 25 microM noticeably decreased the loss of mitochondrial membrane potential (DeltaPsi(m)) in antimycin A-treated cells. Taken together, we have demonstrated that antimycin A as an inhibitor of electron transport in mitochondria potently induces apoptosis in As4.1 juxtaglomerular cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antimycin A; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Flow Cytometry; Kidney Neoplasms; Membrane Potential, Mitochondrial; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

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