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

D-116883 (Aeterna Zentaris GmbH, Frankfurt, Germany) is an orally effective drug that acts via inhibition of phosphatidylinositol 3-kinase (PI3K). The PI3K/AKT signal transduction pathway is involved in ovarian cancer tumorigenesis. Phosphatase and Tensin homolog (PTEN) loss and other activating mutations frequently contribute to the activation of this pathway. We tested whether D-116883 exerts cytostatic effects in in vitro models of ovarian cancer and analyzed the induced programmed cell death.. We evaluated the potency of D-116883 in four ovarian carcinoma cell lines with different cellular assays. The effects of D-116883 on cell proliferation was analysed by crystal-violet staining and tetrazolium salt [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT] assay. The capacity for anchorage-independent growth was analyzed in two ovarian carcinoma cell lines without and with D-116883 addition by using the soft agar assay. Fluorescence activated cell sorting (FACS) cell cycle analyses were performed. Cells were incubated with multicaspase inhibitor benzyloxycarbonyl-val-ala-asp(OMe)-fluoromethylketone (zVAD) and inhibitor of necroptosis necrostatin.. Growth inhibition occurred in all ovarian carcinoma cell lines studied (A2780, A2780cis, OAW42 and SKOV3) in a micromolar range (IC(50)<1 μM). By using soft agar assay, a reduced capacity for anchorage-independent growth, a hallmark of tumor cells, caused by D-116883 was demonstrated. Cell cycle analyses showed that D-116883 dose-dependently increased apoptotic cells. Multicaspase inhibitor zVAD and inhibitor of necroptosis necrostatin did not abrogate the growth-inhibiting effect of the compound.. PI3K inhibitor D-116883 showed substantial cytotoxic effects in various in vitro models of ovarian cancer. Our results make D-116883 a good candidate for further ovarian cancer research including in vivo experiments.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Cell Cycle; Cell Line, Tumor; Dose-Response Relationship, Drug; Female; Humans; Imidazoles; Indoles; Ovarian Neoplasms; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt

We analyzed the anti-tumor effect and the mechanism of action of perifosine, an orally active alkylphospholipid AKT inhibitor using in vitro models of human ovarian cancer.. Ovarian cancer cells OAW42, PA-1, SKOV3, and A2780 as well as platinum resistant A2780cis cells were incubated with increasing concentrations of perifosine, with and without multi-caspase inhibitor zVAD-FMK. The effect of a combined treatment with cisplatin and perifosine was investigated in OAW42, SKOV3, A2780 and A2780cis cells. Cytotoxic effects of perifosine were analyzed using crystal violet staining, FACS analysis of DNA content as well as Annexin V/propidium iodide-double staining. The effect of perifosine on AKT phosphorylation was determined by Western blotting.. Perifosine displayed anti-tumor activity in all five cell lines, which increased time-dependently. While IC(50) values at 24 h were >40 μM, IC(50) values after 72 h decreased to 10 μM in OAW42 and 25 μM in PA-1 and 30 μm in SKOV3 cells. In platinum resistant A2780cis cells perifosine showed good antiproliferative activity (IC(50) = 3 μm). At adequate doses, perifosine increased cytotoxic effects of cisplatin in OAW42, A2780 and A2780cis cell. Anti-tumor activity of perifosine was not confined to a specific phase of the cell cycle and could not be decreased by the pan-caspase inhibitor zVAD-FMK. AnnexinV/propidium iodide-double staining after treatment with perifosine was not indicative of classical apoptosis. AKT phosphorylation was dose-dependently inhibited by perifosine.. Perifosine showed substantial cytotoxic effects in various in vitro models of ovarian cancer. Since anti-tumor effects were not confined to platinum-sensitive cells perifosine seems to be a good candidate for clinical studies in patients especially with platinum resistant ovarian cancer.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Carcinoma; Cell Line, Tumor; Cisplatin; Cysteine Proteinase Inhibitors; Drug Resistance, Neoplasm; Female; Humans; Ovarian Neoplasms; Phosphorylcholine; Proto-Oncogene Proteins c-akt

HIV patients taking antiretroviral protease inhibitors have a lower incidence of infection-associated malignancies, leading to the hypothesis that these drugs have antineoplastic activity. Given the need for novel treatment approaches in ovarian cancer, we sought to determine whether the protease inhibitor saquinavir has antineoplastic activity in ovarian cancer cell lines, and to elucidate the mechanism through which this occurs.. A panel of ovarian cancer cell lines was treated with saquinavir. The effect of saquinavir on cell growth, viability, apoptotic and non-apoptotic cell death was determined. Stimulation of endoplasmic reticulum stress (ERS) response was assessed by immunoblotting for ERS regulators GRP78 and ATF6. Induction of autophagy was assessed using transmission electron microscopy (TEM), and confocal microscopy was performed to demonstrate changes in green fluorescent protein-labeled LC3 expression patterns.. Saquinavir induced cell death in chemosensitive and chemoresistant ovarian cancer cells in a time- and dose-dependent manner. Saquinavir treatment resulted in caspase-dependent apoptosis and caspase-independent cell death characterized by induction of ERS and autophagy. Cellular morphology assessed by TEM revealed apoptotic, autophagic, and necrotic cell death.. Saquinavir is an FDA-approved agent for the treatment of HIV, and our data suggest that it may also have clinical application in the treatment of ovarian cancer. Saquinavir induces endoplasmic reticulum stress, autophagy, and apoptosis in ovarian cancer cells. Given the challenges of chemoresistance in ovarian cancer, saquinavir may have particular benefit in the treatment of chemoresistant tumors that may respond to the induction of caspase-independent cell death by mechanisms such as autophagy.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Apoptosis; Autophagy; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Female; HIV Protease Inhibitors; Humans; Ovarian Neoplasms; Saquinavir

TRAIL induced apoptosis of tumor cells is currently entering phase II clinical settings, despite the fact that not all tumor types are sensitive to TRAIL. TRAIL resistance in ovarian carcinomas can be caused by a blockade upstream of the caspase 3 signaling cascade. We explored the ability of restriction endonucleases to directly digest DNA in vivo, thereby circumventing the caspase cascade. For this purpose, we delivered enzymatically active endonucleases via the cationic amphiphilic lipid SAINT-18((R)):DOPE to both TRAIL-sensitive and insensitive ovarian carcinoma cells (OVCAR and SKOV-3, respectively). Functional nuclear localization after delivery of various endonucleases (BfiI, PvuII and NucA) was indicated by confocal microscopy and genomic cleavage analysis. For PvuII, analysis of mitochondrial damage demonstrated extensive apoptosis both in SKOV-3 and OVCAR. This study clearly demonstrates that cellular delivery of restriction endonucleases holds promise to serve as a novel therapeutic tool for the treatment of resistant ovarian carcinomas.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 3; Cell Line, Tumor; DNA Fragmentation; Endonucleases; Enzyme Inhibitors; Female; Fibroblasts; Histones; Humans; Ovarian Neoplasms; Phosphatidylethanolamines; Pyridinium Compounds; Signal Transduction; TNF-Related Apoptosis-Inducing Ligand

AEZS 112 is an orally active small molecule anticancer drug which inhibits the polymerization of tubulin at low micromolar concentrations. The current study investigates the anti-tumor effect and the mechanism of action of AEZS 112 in in vitro models of human ovarian and endometrial cancers. Four human ovarian and 2 endometrial cancer cell lines were incubated with increasing concentrations of AEZS 112 with and without multi-caspase inhibitor zVAD-FMK for 72 hours. Cytotoxic effects of AEZS 112 were analyzed using crystal violet staining, FACS analysis of DNA content as well as Annexin V/propidium iodide-double staining. AEZS 112 displayed anti-tumor activity in all six cell lines. The EC50 determined after 72-h incubation for Ishikawa and HEC 1A was 0.0312 and 0.125 microm, respectively. The EC50 was 5 microm for SKOV 3 cells, 1 microm for 0.5 microm for OAW 42 cells, 0.125 microm for OvW 1 cells and 0.0312 microm for PA 1 cells. Cytotoxic effects of AEZS 112 could not be abrogated by caspase inhibition with pan-caspase inhibitor zVAD-fmk. Annexin V/propidium iodide-double staining after treatment with AEZS 112 was indicative of necrosis-like cell death. AEZS 112 dose-dependently increased non-vital hypodiploid cells and the cytotoxic effect was least pronounced in G2 phase of the cell cycle, indicating cell death during mitosis, as determined by FACS analysis. The orally active small molecule tubulin inhibitor AEZS 112 showed anti-tumor activity in human ovarian and endometrial cancer cell lines at low micromolar concentrations, which could not be abrogated by caspase inhibition and is therefore a good candidate for in vivo studies in these tumors.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Caspase Inhibitors; Cell Cycle; Cell Line, Tumor; Endometrial Neoplasms; Female; Humans; Ovarian Neoplasms; Poly(ADP-ribose) Polymerases; Tubulin Modulators

The isoflavones biochanin A ( 1a), genistein ( 1b), and daidzein ( 4) at concentrations >20 microM inhibit cell growth of various cancer cell lines. To enhance the antiproliferative activities of these compounds, we synthesized three analogs, 2-[3-carboxy-(6-tert-butoxycarbonylamino)-hexylamino-propyl]-7,5-dihydroxy-4'-methoxyisoflavone ( 3a), 2-[3-[N-[6-(tert-butoxycarbonyl)-aminohexyl]]-caboxamidopropyl]-5,7,4'-trihydroxyisoflavone ( 3b), and 5-{2-[3-(4-hydroxy-phenyl)-4-oxo-4 H-chromen-7-yloxy]-acetylamino}-pentyl)-carbamic acid tert-butyl ester ( 6). When cancer cells expressing predominantly estrogen receptor mRNA of the beta- relative to alpha-subtype were treated with 3a, 3b, or 6, DNA synthesis was inhibited in a dose-dependent manner, ranging from 15 to 3000 nmol/L, with little inhibitory effect in normal vascular smooth muscle cells. Compound 6 was the most potent one, and its antiproliferative effect in cancer cells was modulated by estrogen and by the apoptosis inhibitor Z-VADFK. When tested in vivo, compound 6 decreased tumor volume of ovarian xenografts by 50%, with no apparent toxicity. Compound 6 may be a promising agent for therapy of cancer either alone or in combination with chemotherapeutic agents.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; Carbamates; Cell Line, Tumor; Chromones; Colonic Neoplasms; DNA; Drug Interactions; Estrogen Antagonists; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Humans; Isoflavones; Male; Mice; Mice, Nude; Muscle, Smooth, Vascular; Neoplasm Transplantation; Ovarian Neoplasms; Ovary; Prostate; RNA, Messenger; Transplantation, Heterologous

We investigated the antitumoral efficacy, endocrine consequences, and molecular mechanisms underlying cell death induced by the Hecate-chorionic gonadotropin (CG)beta conjugate, a fusion protein of a 23-amino acid lytic peptide Hecate with a 15-amino acid (81-95) fragment of the human CGbeta chain. Transgenic (TG) mice expressing the inhibin alpha-subunit promoter (inhalpha)/Simian Virus 40 T-antigen (Tag) transgene, developing luteinizing hormone (LH) receptor (R) expressing Leydig and granulosa cell tumors, and wild-type control littermates were treated either with vehicle, Hecate, or Hecate-CGbeta conjugate for 3 weeks. Hecate-CGbeta conjugate treatment reduced the testicular and ovarian tumor burden (P < .05), whereas a concomitant increase (testis; P < .05) or no change (ovary) in tumor volumes occured with Hectate treatment. A drop in serum progesterone, produced by the tumors, and an increase in LH levels occured in Hecate-CGbeta treated mice, in comparison with vehicle and Hecate groups, providing further support for the positive treatment response. Hecate-CGbeta conjugate induced a rapid and cell-specific membrane permeabilization of LHR-expressing cells in vitro, suggesting a necrotic mode of cell death without activation of apoptosis. These results prove the principle that the Hecate-CGbeta conjugate provides a novel specific lead into gonadal somatic cell cancer therapy by targeted destruction of LHR-expressing tumor cells.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Blotting, Northern; Caspase 3; Caspases; Cell Death; Cell Line, Tumor; Cell Separation; Chorionic Gonadotropin, beta Subunit, Human; Disease Models, Animal; Enzyme Activation; Female; Flow Cytometry; Granulosa Cell Tumor; Humans; Leydig Cell Tumor; Male; Melitten; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Fluorescence; Necrosis; Ovarian Neoplasms; Progesterone; Promoter Regions, Genetic; Protein Structure, Tertiary; Receptors, LH; Recombinant Fusion Proteins; Testicular Neoplasms; Time Factors

Progesterone (P4) has been implicated as a protective factor for epithelial ovarian cancers, yet little is known about its mechanism of action. We previously reported that pregnancy-equivalent doses of P4 inhibited the growth of normal and malignant human ovarian surface epithelial (HOSE) cells. Here, we investigated how P4-induced cell death in two immortalized normal (HOSE 642, HOSE 12-12) and two malignant (OVCA 429, OVCA 432) HOSE cell lines. The exposure of HOSE or OVCA cell cultures to 10(-6) M P4 induced time-dependent increases in early and late apoptotic cells and activation of caspase-8 and -3, but not that of caspase-9. A general caspase inhibitor Z-VAD effectively blocked the P4-induced cell death in a dose-dependent manner. Comparable levels of Fas mRNA and protein were expressed in HOSE and OVCA cell lines, and these levels were unaffected by P4. In contrast, levels of FasL mRNA and protein were higher in OVCA cells than in HOSE cells. Interestingly, the hormone enhanced levels of FasL mRNA and protein in HOSE cells, but lowered their levels in OVCA cells. The exposure of HOSE or OVCA cells to an activating anti-Fas antibody induced cell loss, whereas treatment of cells with a blocking anti-FasL antibody reduced the P4-induced cell loss. Cotreatment of cells with the activating anti-Fas antibody and P4 produced additive effects on cell loss. These results reveal for the first time that P4 induces apoptosis in HOSE and OVCA cells via activation of a caspase-8-initiated Fas/FasL signaling pathway. They also demonstrate differential P4-regulation of FasL expression between HOSE and OVCA cells.

Topics: Adenocarcinoma; Adult; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 8; Caspase 9; Caspases; Cell Division; Dose-Response Relationship, Drug; Drug Therapy, Combination; Enzyme Activation; Enzyme Inhibitors; Epithelial Cells; Fas Ligand Protein; Female; Gene Expression Regulation, Neoplastic; Humans; Membrane Glycoproteins; Middle Aged; Ovarian Neoplasms; Ovary; Progesterone; Signal Transduction; Tumor Cells, Cultured

There is a need to enhance the efficacy of genetic prodrug activation therapy using herpes simplex virus thymidine kinase (tk) and ganciclovir (GCV) following disappointing results in early clinical trials. tk/GCV has been shown to lead to the activation of caspase-3, a potent executor of apoptosis. We demonstrate that co-expression of pro-caspase-3 with tk/GCV leads to enhanced cell death in ovarian carcinoma cells in vitro. Following transfection with recombinant adenoviral vectors encoding tk, GCV treatment leads to greater cell death in pro-caspase-3-expressing clones of SKOV3 and IGROV1 than control cells, as well as more rapid activation of caspase-3 and more rapid cleavage of PARP. Flow cytometry suggests that there is a greater degree of S-phase block in the pro-caspase-3-expressing clones than in control cells following treatment with tk/GCV. None of these effects is seen following transfection with a control adenovirus that does not encode tk. The increased cell death, early caspase-3 activation and PARP cleavage, and flow cytometric changes seen in pro-caspase-3-expressing cells can be partially inhibited by treatment with benzyloxycarbonyl-val-ala-asp fluoromethylketone, a synthetic caspase inhibitor. Our data suggest that co-expression of pro-caspase-3 may lead to a significant enhancement of the efficacy of tk/GCV therapy.

Topics: Adenoviridae; Amino Acid Chloromethyl Ketones; Antiviral Agents; Apoptosis; Blotting, Western; Caspase 3; Caspase Inhibitors; Caspases; Cell Survival; Cysteine Proteinase Inhibitors; DNA Primers; Drug Resistance, Neoplasm; Female; Flow Cytometry; Ganciclovir; Genetic Therapy; Genetic Vectors; Green Fluorescent Proteins; Humans; L-Lactate Dehydrogenase; Luminescent Proteins; Ovarian Neoplasms; Poly(ADP-ribose) Polymerases; Reverse Transcriptase Polymerase Chain Reaction; Simplexvirus; Thymidine Kinase; Transfection; Tumor Cells, Cultured

Although activated Ras proteins are usually associated with driving growth and transformation, they may also induce senescence, apoptosis, and terminal differentiation. The subversion of these anti-neoplastic effects during Ras-dependent tumor development may be as important as the acquisition of the pro-neoplastic effects. None of the currently identified potential Ras effector proteins can satisfactorily explain the apoptotic action of Ras. Consequently, we have sought to identify novel Ras effectors that may be responsible for apoptosis induction. By examining the EST data base, we identified a potential Ras association domain in the tumor suppressor RASSF1. We now show that RASSF1 binds Ras in a GTP-dependent manner, both in vivo and directly in vitro. Moreover, activated Ras enhances and dominant negative Ras inhibits the cell death induced by transient transfection of RASSF1 into 293-T cells. This cell death appears to be apoptotic in nature, as RASSF1-transfected 293-T cells exhibit membrane blebbing and can be rescued by the addition of a caspase inhibitor. Thus, the RASSF1 tumor suppressor may serve as a novel Ras effector that mediates the apoptotic effects of oncogenic Ras.

Topics: 3T3 Cells; Alternative Splicing; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Down-Regulation; Female; Genes, Tumor Suppressor; Guanosine Triphosphate; Humans; Mice; Neoplasm Proteins; Ovarian Neoplasms; Protein Binding; Protein Structure, Tertiary; ras Proteins; Recombinant Fusion Proteins; RNA, Messenger; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins

To date, eight neurodegenerative disorders, including Huntington's disease and dentatorubral-pallidoluysian atrophy, have been identified to be caused by expansion of a CAG repeat coding for a polyglutamine (polyQ) stretch. It is, however, unclear how polyQ expansion mediates neuronal cell death observed in these disorders. Here, we have established a tetracycline-regulated expression system producing 19 and 56 repeats of glutamine fused with green fluorescent protein. Induced expression of the 56 polyQ, but not of the 19 polyQ stretch caused marked nuclear aggregation and apoptotic morphological changes of the nucleus. In vitro enzyme assays and Western blotting showed that polyQ56 expression sequentially activated initiator and effector caspases, such as caspase-8 or -9, and caspase-3, respectively. Furthermore, using cell-permeable fluorogenic substrate, the activation of caspase-3-like proteases was demonstrated in intact cells with aggregated polyQ. This is the first direct evidence that the expression of extended polyQ activates caspases and together with the previous findings that some of the products of genes responsible for CAG repeat diseases are substrates of caspase-3 indicates an important role of caspases in the pathogenesis of these diseases.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Nucleus; Enzyme Activation; Female; Green Fluorescent Proteins; Humans; Luminescent Proteins; Neurodegenerative Diseases; Oligopeptides; Ovarian Neoplasms; Peptides; Poly(ADP-ribose) Polymerases; Protein Binding; Recombinant Fusion Proteins; Transfection; Tumor Cells, Cultured

Treatment of NIH-OVCAR-3 cells with paclitaxel, a microtubule-stabilizing agent, induces mitotic arrest and apoptosis, but also Bcl-2 phosphorylation. We report here that Bcl-2 phosphorylation precedes Bcl-2 down-regulation and that both events are closely associated with mitotic arrest, but are not sufficient for paclitaxel to trigger apoptosis. Indeed, when paclitaxel-treated cells were induced to exit mitosis in the presence of 2-aminopurine, Bcl-2 phosphorylation and Bcl-2 down-regulation were both inhibited. In contrast, when apoptosis was inhibited by a caspase inhibitor or Bcl-2 over-expression, Bcl-2 phosphorylation and down-regulation still occurred. Furthermore, we show that Bcl-2 is degraded in mitosis by the proteasome-dependent pathway since Bcl-2 down-regulation is inhibited by proteasome inhibitors such as MG132, Lactacystin and LLnL. Taken together these results indicate that mitotic spindle damage results in post-translational modifications of Bcl-2 by phosphorylation and degradation.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Apoptosis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Female; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Humans; Leupeptins; Microtubules; Mitosis; Multienzyme Complexes; Ovarian Neoplasms; Paclitaxel; Phosphorylation; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Virulence Factors, Bordetella

We have previously demonstrated that a new farnesyltransferase inhibitor, FPT inhibitor III, triggers apoptosis in human ovarian cancer cells. Here, we report that induction of apoptotic cell death in PA-1 ovarian cancer cells by FPT inhibitor III was accompanied by the activation of interleukin-1 #-converting enzyme (ICE)-like proteases, which have recently been renamed as caspases. The caspase inhibitor, ZVAD-FMK, which inhibits a number of caspase family proteases, blocked FPT inhibitor III-induced apoptotic cell death in a dose-dependent manner. Additionally, cleavage of poly(ADP-ribose) polymerase, an identified in vivo substrate for caspase family proteases, in FPT inhibitor III-treated cells was confirmed by immunoblotting. Our results suggest that the caspase family proteases are involved in the induction of apoptosis triggered by FPT inhibitor III.

Topics: Alkyl and Aryl Transferases; Amino Acid Chloromethyl Ketones; Apoptosis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Farnesyltranstransferase; Female; Humans; Hydrolysis; Ovarian Neoplasms; Poly(ADP-ribose) Polymerases; Tumor Cells, Cultured