cytochrome-c-t and Ovarian-Neoplasms

Previously we have shown inhibition of endometrial cancer cell growth with progesterone and calcitriol. However, the mechanisms by which the two agents attenuate proliferation have not been well characterized yet. Herein, we investigated how progesterone and calcitriol induce apoptosis in cancer cells. DNA fragmentation was upregulated by progesterone and calcitriol in ovarian and endometrial cancer cells. Time-dependent treatment of ovarian cancer cells, ES-2, and TOV-21G with progesterone enhanced caspase -8 activity after 12 h, whereas OV-90, TOV-112D, HEC-1A, and HEC-59 cells showed increased activity after 24 h. Caspase 9 activity was increased in all cell lines after 24 h treatment with calcitriol. Pretreatment of cancer cells with a caspase-8 inhibitor (z-IETD-fmk) or caspase-9 inhibitor (Z-LEHD-fmk) significantly attenuated progesterone and calcitriol induced caspase-8 and caspase-9 expression, respectively. The expression of FasL, Fas, FAD, and pro-caspase-8, which constitute the death-inducing signaling complex (DISC), was upregulated in progesterone treated cancer cells. Knockdown of FAS or FADD with specific siRNAs significantly blocked progesterone-induced caspase-8. Cleavage of the BID was not affected by caspase-8 activation suggesting the absence of cross-talk between caspase-8 and caspase-9 pathways. Calcitriol treatment decreased mitochondrial membrane potential and increased the release of cancer cytochrome C. These findings indicate that progesterone induces apoptosis through activation of caspase-8 and calcitriol through caspase-9 activation in cancer cells. A combination of progesterone-calcitriol activates both extrinsic and intrinsic apoptotic pathways in cancer cells.

Topics: Apoptosis; Calcitriol; Caspase 8; Caspase 9; Caspases; Cell Line, Tumor; Cytochromes c; Death Domain Receptor Signaling Adaptor Proteins; Death Domain Superfamily; Endometrial Neoplasms; Fas Ligand Protein; fas Receptor; Female; Humans; In Vitro Techniques; Membrane Potential, Mitochondrial; Ovarian Neoplasms; Progesterone; Signal Transduction

Green tea is a natural compound with anti-neoplastic properties. Paclitaxel (PTX) is a natural anti-tumor medication used to manage patients with advanced ovarian cancer. This manuscript evaluated the cytotoxic effects of green tea extract combined with PTX drug in two human ovarian cancer cell lines (p53-negative cell line, SKOV-3; and mutant type p53 cell line, OVCAR-3) and underlying mechanisms.. The human ovarian cancer cell lines were treated with green tea extract, PTX, and green tea plus PTX for 24 h, and cell viability was assessed using the MTT method. Flow cytometric analyses were carried out to detect apoptosis. For the apoptotic process, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting analysis were applied to study pAkt, Bax, Bcl-2, Cytochrome C (Cyt-C), cleaved-caspase-3, and cleaved-caspase-9 levels after drug treatments.. Our results pointed out that various green tea (25 and 50 µg/ml) concentrations combined with PTX (20 and 40 µg/ml) synergistically inhibited cell viability of cancer cells more than green tea or PTX alone after 24 h of treatment. Also, green tea and PTX combination induced apoptosis in ovarian cancer cells by blocking the phosphorylation of Akt and the expression of Bcl-2 while inducing Bax, Cyt-C, cleaved-caspase 3, and cleaved-caspase 9.. Our results showed that the combination of green tea and PTX could be more potent than the individual drug to induce cytotoxicity and apoptosis in ovarian cancer cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Screening Assays, Antitumor; Drug Synergism; Female; Humans; Mitochondria; Ovarian Neoplasms; Paclitaxel; Plant Extracts; Polyphenols; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Tea

Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational modifications or loss of organelle membrane integrity lead to the movement of proteins from one compartment to another. This movement of proteins or protein translocation, exerts a significant effect on protein function. This is clearly demonstrated in the context of apoptosis wherein the cytoplasmic translocation of the mitochondrial resident protein, cytochrome C, initiates the activation of the intrinsic arm of the apoptotic pathway. Experimentally, protein translocation can be demonstrated by subcellular fractionation and subsequent western blot analysis of the isolated fractions. This chapter describes the step-by-step procedure in obtaining mitochondrial and cytoplasmic fractions from cell pellets and determining their purity and integrity.

Topics: Apoptosis; Blotting, Western; Caspases; Cytochromes c; Cytoplasm; Female; Humans; Mitochondria; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured

Delivery of apoptosis-associated proteins is an attractive approach to treat cancer, but their large molecular sizes and membrane-impermeability require the use of a suitable delivery carrier. As a versatile drug carrier, mesoporous silica nanoparticles (MSNs) have been utilized to transport a variety of therapeutic molecules. However, the use of MSNs for protein delivery has been limited because their conventionally obtainable pore size (ca. 2-3 nm in diameter) is too small to load large-sized biomolecular cargos. In this article, we present surface erosion of MSNs by hydrolytic degradation as a new strategy to obtain a mesoporous colloidal carrier for effective delivery of a bulky apoptosis-inducible protein, cytochrome c (CYT). A series of physicochemical properties of particles were analyzed before and after the hydrolytic surface erosion of pristine small-pored MSNs and the subsequent CYT loading. The results showed that hydrolytic degradation of MSNs imparts beneficial structural features for CYT loading and release, i.e., enlarged pores (up to ~10 nm in diameter) and roughened surface texture, leading to significantly enhanced intracellular delivery of CYT over conventional small-pored MSNs. The present results may offer a useful insight into silica degradability for tuning the internal/external surface characteristics of MSN-based colloidal particles to open a wide range of biomedical applications.

Topics: Biocompatible Materials; Cell Proliferation; Cytochromes c; Drug Carriers; Drug Delivery Systems; Female; Humans; Hydrolysis; Nanoparticles; Ovarian Neoplasms; Silicon Dioxide; Surface Properties; Tumor Cells, Cultured

To investigate whether TRIAP1inhibition affects the ovarian cancer cell resistance to cisplatin (DDP) via the Cyt c/Apaf-1/caspase-9 pathway by in vitro and in vivo experiments.. CCK8 assay was performed to find out how treatment with both TRIAP1 siRNA and DDP affects the cell viability of SKOV3 cells and DDP-resistant human ovarian carcinoma cell line SKOV3/DDP. SKOV3/DDP cells were transfected with control siRNA or TRIAP1 siRNA before 24 h of treatment with DDP (5 μg/mL). Flow cytometry was employed to detect cell apoptosis and Western blot to examine the expressions of Cyt c/Apaf-1/caspase-9 pathway-related proteins. SKOV3/DDP cells transfected with control siRNA or TRIAP1 siRNA were subcutaneously injected into BALB/c-nu/nu nude mice followed by the intraperitoneal injection of DDP (4 mg/kg). Cyt c/Apaf-1/caspase-9 pathway in transplanted tumors was detected by immunohistochemistry.. TRIAP1 expression declined in SKOV3 cells when compared with SKOV3/DDP cells. The proliferation rate was lower in SKOV3/DDP cells transfected with TRIAP1 siRNA combined with treatment of DDP (1, 2, 4, 6, 8, 16, 32 μg/mL) than in those transfected with control siRNA. Moreover, the TRIAP1 siRNA group had an increased SKOV3/DDP cell apoptosis rate with the activation of the Cyt c/Apaf-1/caspase-9 pathway. During DDP treatment, nude mice in TRIAP1 siRNA group had slower growth and smaller size of transplanted tumor than those in control siRNA group, with increased expression of Cyt c, Apaf-1, and caspase-9.. TRIAP1 inhibition may enhance the sensitivity of SKOV3/DDP cells to cisplatin via activation of the Cyt c/Apaf-1/caspase-9 pathway.

Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Caspase 9; Cell Line, Tumor; Cisplatin; Cytochromes c; Down-Regulation; Drug Resistance, Neoplasm; Female; Humans; Intracellular Signaling Peptides and Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Ovarian Neoplasms; RNA Interference; RNA, Small Interfering; Signal Transduction; Transplantation, Heterologous

Ovarian cancer is the leading cause of gynaecology related cancer death worldwide. It is often diagnosed with an advanced stage. Apoptosis is a process of programmed cell death controlled by cell cycle machinery and several signaling pathways. Plant-derived compounds have received an increased interest in the treatment of cancer. Quercetin is a flavonoid present in fruits and vegetables which possess anticancer properties. Several studies have been demonstrated that quercetin induces apoptosis in various cancers. However, the apoptotic role of quercetin in metastatic ovarian cancer has not been extensively studied. In the present study, we investigated the apoptotic effect of quercetin on human metastatic ovarian cancer PA-1 cell line. Quercetin treatment (0-200 μM) for 24h decreases PA-1 cells viability in a dose-dependent manner. The effective dose was identified as 50 and 75 μM based on MTT assay. Quercetin induces apoptosis in metastatic ovarian cancer cells which were confirmed by AO/EtBr dual staining, DAPI staining and DNA fragmentation assay. Molecules involved in the intrinsic apoptotic pathway were altered by quercetin. Interestingly, antiapoptotic molecules such as Bcl-2, Bcl-xL were decreased while proapoptotic molecules such as caspase-3, caspase-9, Bid, Bad, Bax and cytochrome c were increased in the quercetin-treated PA-1 cells. Our results indicate that quercetin induces mitochondrial-mediated apoptotic pathway and thus it inhibits the growth of metastatic ovarian cancer cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; DNA Fragmentation; Female; Humans; Neoplasm Metastasis; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; Quercetin

Since their discovery, liposomes have been widely employed in biomedical research. These nano-size spherical vesicles consisting one or few phospholipid bilayers surrounding an aqueous core are capable of carrying a wide variety of bioactive compounds, including drugs, peptides, nucleic acids, proteins and others. Despite considerable success achieved in synthesis of liposome constructs containing bioactive compounds, preparation of ligand-targeted liposomes comprising large quantities of encapsulated proteins that are capable of affecting pathological cells still remains a big challenge. Here we described a novel method for preparation of small (80-90 nm in diameter) unilamellar liposomes containing very large quantities (thousands of protein molecules per liposome) of heme-containing cytochrome c, highly fluorescent mCherry and highly toxic PE40 (Pseudomonas aeruginosa Exotoxin A domain). Efficient encapsulation of the proteins was achieved through electrostatic interaction between positively charged proteins (at pH lower than pI) and negatively charged liposome membrane. The proteoliposomes containing large quantities of mCherry or PE40 and functionalized with designed ankyrin repeat protein (DARPin)_9-29, which targets human epidermal growth factor receptor 2 (HER2) were shown to specifically stain and kill in sub-nanomolar concentrations HER2-positive cells, overexpressing HER2, respectively. Specific staining and eradication of the receptor-positive cells demonstrated here makes the DARPin-functionalized liposomes carrying large quantities of fluorescent and/or toxic proteins a promising candidate for tumor detection and therapy.

Topics: Adenocarcinoma; ADP Ribose Transferases; Animals; Ankyrin Repeat; Bacterial Toxins; Breast Neoplasms; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Cytochromes c; Exotoxins; Female; Heme; Humans; Liposomes; Luminescent Proteins; Ovarian Neoplasms; Particle Size; Pseudomonas aeruginosa Exotoxin A; Receptor, ErbB-2; Red Fluorescent Protein; Virulence Factors

Ovarian carcinoma (OC) patients encounter the severe challenge of clinical management owing to lack of screening measures, chemoresistance and finally dearth of non-toxic therapeutics. Cancer cells deploy various defense strategies to sustain the tumor microenvironment, among which deregulated apoptosis remains a versatile promoter of cancer progression. Although recent research has focused on identifying agents capable of inducing apoptosis in cancer cells, yet molecules efficiently breaching their survival advantage are yet to be classified. Here we identify lectin, Sambucus nigra agglutinin (SNA) to exhibit selectivity towards identifying OC by virtue of its specific recognition of α-2, 6-linked sialic acids. Superficial binding of SNA to the OC cells confirm the hyper-sialylated status of the disease. Further, SNA activates the signaling pathways of AKT and ERK1/2, which eventually promotes de-phosphorylation of dynamin-related protein-1 (Drp-1). Upon its translocation to the mitochondrial fission loci Drp-1 mediates the central role of switch in the mitochondrial phenotype to attain fragmented morphology. We confirmed mitochondrial outer membrane permeabilization resulting in ROS generation and cytochrome-c release into the cytosol. SNA response resulted in an allied shift of the bioenergetics profile from Warburg phenotype to elevated mitochondrial oxidative phosphorylation, altogether highlighting the involvement of mitochondrial dysfunction in restraining cancer progression. Inability to replenish the SNA-induced energy crunch of the proliferating cancer cells on the event of perturbed respiratory outcome resulted in cell cycle arrest before G2/M phase. Our findings position SNA at a crucial juncture where it proves to be a promising candidate for impeding progression of OC. Altogether we unveil the novel aspect of identifying natural molecules harboring the inherent capability of targeting mitochondrial structural dynamics, to hold the future for developing non-toxic therapeutics for treating OC.

Topics: Apoptosis; Cell Survival; Cytochromes c; Cytosol; Dynamins; Female; GTP Phosphohydrolases; Humans; Membrane Potential, Mitochondrial; Microscopy, Confocal; Microtubule-Associated Proteins; Mitochondria; Mitochondrial Dynamics; Mitochondrial Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Ovarian Neoplasms; Plant Lectins; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Ribosome Inactivating Proteins; S Phase Cell Cycle Checkpoints; Signal Transduction

Cleistopholine is a natural alkaloid present in plants with numerous biological activities. However, cleistopholine has yet to be isolated using modern techniques and the mechanism by which this alkaloid induces apoptosis in cancer cells remains to be elucidated.. This study aims to isolate cleistopholine from the roots of Enicosanthellum pulchrum by using preparative-HPLC technique and explore the mechanism by which this alkaloid induces apoptosis in human ovarian cancer (CAOV-3) cells in vitro from 24 to 72 h. This compound may be developed as an anticancer agent that induces apoptosis in ovarian cancer cells.. Cytotoxicity was assessed via the cell viability assay and changes in cell morphology were observed via the acridine orange/propidium iodide (AO/PI) assay. The involvement of apoptotic pathways was evaluated through caspase analysis and multiple cytotoxicity assays. Meanwhile, early and late apoptotic events via the Annexin V-FITC and DNA laddering assays, respectively. The mechanism of apoptosis was explored at the molecular level by evaluating the expression of specific genes and proteins. In addition, the proliferation of CAOV-3-cells treated with cleistopholine was analysed using the cell cycle arrest assay.. The IC50 of cleistopholine (61.4 µM) was comparable with that of the positive control cisplatin (62.8 µM) at 24 h of treatment. Apoptos is was evidenced by cell membrane blebbing, chromatin compression and formation of apoptotic bodies. The initial phase of apoptosis was detected at 24 h by the increase in Annexin V-FITC binding to cell membranes. A DNA ladder was formed at 48 h, indicating DNA fragmentation in the final phase of apoptosis. The mitochondria participated in the process by stimulating the intrinsic pathway via caspase 9 with a reduction in mitochondrial membrane potential (MMP) and an increase in cytochrome c release. Cell death was further validated through the mRNA and protein overexpression of Bax, caspase 3 and caspase 9 in the treated cells compared with the untreated cells. In contrast, Bcl-2, Hsp70 and survivin decreased in expression upon cleistopholine treatment. Cell cycle was arrested at the G0/G1 phase and cell population percentage significantly increased to 43.5%, 45.4% and 54.3% in time-dependent manner in the cleistopholine-treated CAOV-3 cells compared with the untreated cells at 24, 48 and 72 h respectively.. The current study indicated that cleistopholine can be a potential candidate as a new drug to treat ovarian cancer disease.

Topics: Angiogenesis Inhibitors; Annexin A5; Annonaceae; Anthraquinones; Antineoplastic Agents, Phytogenic; Apoptosis; Aza Compounds; Caspase 9; Caspases; Cell Cycle; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cytochromes c; DNA Fragmentation; Female; Humans; Membrane Potential, Mitochondrial; Mitochondria; Ovarian Neoplasms; Phytotherapy; Plant Extracts; Plant Roots

Artonin E is a prenylated flavonoid isolated from the stem bark of Artocarpus elasticus Reinw.(Moraceae). This study aimed to investigate the apoptotic mechanisms induced by artonin E in a metastatic human ovarian cancer cell line SKOV-3 in vitro. MTT assay, clonogenic assay, acridine orange and propidium iodide double staining, cell cycle and annexin V analyses were performed to explore the mode of artonin E-induced cell death at different time points. DNA laddering, activation of caspases-3, -8, and -9, multi-parametric cytotoxicity-3 analysis by high-content screening, measurement of reactive oxygen species generation, and Western blot were employed to study the pathways involved in the apoptosis. MTT results showed that artonin E inhibited the growth of SKOV-3 cells, with IC50 values of 6.5±0.5 μg/mL after 72 h treatment, and showed less toxicity toward a normal human ovarian cell line T1074, with IC50 value of 32.5±0.5 μg/mL. Results showed that artonin E induced apoptosis and cell cycle arrest at the S phase. This compound also promoted the activation of caspases-3, -8, and -9. Further investigation into the depletion of mitochondrial membrane potential and release of cytochrome c revealed that artonin E treatment induced apoptosis via regulation of the expression of pro-survival and pro-apoptotic Bcl-2 family members. The expression levels of survivin and HSP70 proteins were also down regulated in SKOV-3 cells treated with artonin E. We propose that artonin E induced an antiproliferative effect that led to S phase cell cycle arrest and apoptosis through dysregulation of mitochondrial pathways, particularly the pro- and anti-apoptosis signaling pathways.

Topics: Apoptosis; Artocarpus; Blotting, Western; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; DNA Fragmentation; Enzyme Activation; Female; Flavonoids; Flow Cytometry; Humans; Inhibitory Concentration 50; Mitochondria; Molecular Structure; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; S Phase Cell Cycle Checkpoints

In the course of screening for novel cell cycle inhibitors and apoptotic inducers, CR389, elucidated as 5-(1H-benzoimidazol-2-yl)-1H-pyridin-2-one, was generated as a new hit compound. Flow cytometric analysis and western blots of PA-1 cells treated with 40 micrometer CR389 revealed an appreciable cell cycle arrest at the G2/M phase through direct inhibition of the CDK1 complex. In addition, activation of p53 via phosphorylation at Ser15 and subsequent up-regulation of p21(CIP1) showed that CR389 also induces p53-dependent-p21(CIP1)-mediated cell cycle arrest. Furthermore, apoptotic induction in 40 micrometer CR389-treated PA-1 cells is associated with the release of cytochrome c from mitochondria through up-regulation of the proapoptotic Bax protein, which results in the activation of procaspase-9 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP). Accordingly, CR389 seems to have multiple mechanisms of antiproliferative activity through p53-mediated pathways against human ovarian cancer cells. Therefore, we conclude that CR389 is a candidate therapeutic agent for the treatment of human ovarian cancer via the activation of p53.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Benzimidazoles; Caspases; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Line, Tumor; Cytochromes c; Female; G2 Phase; Humans; Mitochondria; Ovarian Neoplasms; Poly(ADP-ribose) Polymerases; Pyridones; Tumor Suppressor Protein p53

9-Oxo-(10E,12E)-octadecadienoic acid (9-EE-KODE), which is isolated from the calyx of eggplants, exhibits cytotoxic activity against human ovarian cancer (HRA) cells. The aim of the present study is to clarify the action mechanism of 9-EE-KODE leading to cell death. After the treatment of 9-EE-KODE in HRA cells, we found intracellular DNA fragmentation, surface-exposure of phosphatidylserine in the outer cell membrane, and increased caspase-3/7 activities in the HRA cells. The dissipation of mitochondrial membrane potential, release of cytochrome c from mitochondria to cytosol, down-regulation of Bcl-2, and up-regulation of Bax levels were also found in 9-EE-KODE-treated cells in a dose-dependent manner. These results demonstrated that 9-EE-KODE induced apoptosis in HRA cells via the mitochondrial regulation pathway.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Caspase 3; Cell Line, Tumor; Cytochromes c; DNA Fragmentation; Drug Screening Assays, Antitumor; Female; Flowers; Humans; Linolenic Acids; Membrane Potential, Mitochondrial; Mitochondria; Ovarian Neoplasms; Solanum melongena

Butein is a polyphenol, one of the compounds of chalcones, which are flavonoids that are widely biosynthesized in plants, and exhibits different pharmacological activities. Plants containing butein have been used in Chinese traditional medicine. Recently, it has been reported that butein suppresses proliferation and triggers apoptosis in various human cancer cells in vitro and in vivo. The aim of this study was to investigate its pro-apoptotic effect and mechanisms in two cultured human ovarian cancer cells (ES-2 and TOV-21G). The effects of butein on cell viability were assessed by a MTT assay at 3, 10, 30, and 100 μ/M. The apoptotic pathway related factors, including the mitochondrial transmembrane potential (MTP), cytochrome c, caspase cascade, and Bcl-2 family proteins, were examined. MTT assay revealed that butein was cytotoxic to both ovarian cancer cells in a dose- and time-dependent manner. JC-1 flow cytometry, cytochrome c, and caspase activity assays revealed that butein damaged the MTP, increased the level of cytosol cytochrome c and the activities of caspase-3, -8, and -9 in the two ovarian cancer cells. Western blot analysis revealed that butein down-regulated the anti-apoptotic proteins Bcl-2 and Bcl-xL and increased the pro-apoptotic proteins Bax and Bad. These findings suggest that butein-induced apoptosis in ovarian cancer cells via the activation of both extrinsic and intrinsic pathways. In addition, butein also down-regulated the expressions of the inhibitor of apoptosis (IAP) proteins, XIAP, survivin, CIAP-1, and CIAP-2. This indicates that the inhibition of IAP proteins was also involved in butein-induced apoptosis. The results of our study suggest that butein may be a promising anticancer agent in treating ovarian cancer.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Baculoviral IAP Repeat-Containing 3 Protein; bcl-X Protein; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chalcones; Cytochromes c; Dose-Response Relationship, Drug; Female; Gene Expression; Humans; Inhibitor of Apoptosis Proteins; Membrane Potential, Mitochondrial; Molecular Targeted Therapy; Ovarian Neoplasms; Phytotherapy; Proto-Oncogene Proteins c-bcl-2; Survivin; Ubiquitin-Protein Ligases; X-Linked Inhibitor of Apoptosis Protein

To explore if a natural isothiocyanate, sulforaphene (SFE), sensitizes ovarian cancer cells to the chemotherapy drug cisplatin (CDDP).. We studied reactive oxygen species (ROS), mitochondrial membrane depolarization and cell-cycle distribution in two ovarian cancer cell lines SKOV3 and SNU 8 treated with SFE and cisplatin. We further analyzed the expression of caspases 3, 8, and 9, Phosphoinositide 3-kinase (PI3K) and Phosphatase and tensin homolog (PTEN) by western blotting.. SFE sensitized cells to cisplatin by enhancing ROS and mitochondrial membrane depolarization that released cytochrome c and activated caspase 9 and caspase 3 in the mitochondrial pathway. It also inhibited extrinsic pathway protein caspase 8, growth-related protein PI3K and further activated PTEN in combination with cisplatin.. SFE synergistically inhibited proliferation and induced apoptosis of SKOV3 and SNU8 cells in combination with cisplatin by activating multiple apoptotic pathways. Therefore, we suggest sulforaphene as a chemo-enhancing adjuvant to improve the efficacy of cisplatin in ovarian cancer treatment.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspases; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cytochromes c; Drug Synergism; Enzyme Activation; Female; Humans; Isothiocyanates; Membrane Potential, Mitochondrial; Mitochondria; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; PTEN Phosphohydrolase; Reactive Oxygen Species

The purpose of this study was to observe the apoptosis of A2780 cells transfected with the recombinant plasmid of pcDNA-Bax and to observe the release of cytochrome C from the mitochondria.. The recombinant plasmid of pcDNA-Bax was constructed and transfected into A2784 cells. The Hoechst 33258 stain method was applied to evaluate the apoptosis of the transfected cells and MTT mothod was used to test the cell viability. Western blot analysis was performed to determine the overexpression of Bax and the release of cytochrome C from the mitochondria.. The recombinant plasmid of pcDNA-Bax was successfully constructed by using endonuclease digestion and the sequence analysis. The apoptosis of A2780 cells was induced after transfected with pcDNA3. 1-Bax as demonstrated with Hoechst staining. The cell viability were decreased in the pcDNA3. 1-Bax transfected group by MTT assay. The release of cytochrome C from the mitochondria was observed when using Western blotting analysis. And the caspase-9 and the caspase-3 were activated.. Our data suggestted that Bax exhibited potent pro-apoptotic activity against the ovarian cancer cells. This study is a foundation for the further research in the pro-apoptotic activity of Bax.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Female; Humans; Mitochondria; Ovarian Neoplasms; Transfection

The purpose of this study was to provide a detailed explanation of the mechanism of bisanthracycline,?WP 631 in comparison to doxorubicin (DOX), a first generation anthracycline, currently the most widely used pharmaceutical in clinical oncology. Experiments were performed in SKOV-3 ovarian cancer cells which are otherwise resistant to standard drugs such as cis-platinum and adriamycin. As attention was focused on the ability of WP 631 to induce apoptosis, this was examined using a double staining method with Annexin V and propidium iodide probes, with measurement of the level of intracellular calcium ions and cytosolic cytochrome c. The western blotting technique was performed to confirm PARP cleavage. We also investigated the involvement of caspase activation and DNA degradation (comet assay and immunocytochemical detection of phosphorylated H2AX histones) in the development of apoptotic events. WP 631 demonstrated significantly higher effectiveness as a pro-apoptotic drug than DOX. This was evident in the higher levels of markers of apoptosis, such as the externalization of phosphatidylserine and the elevated level of cytochrome c. An extension of incubation time led to an increase in intracellular calcium levels after treatment with DOX. Lower changes in the calcium content were associated with the influence of WP 631. DOX led to the activation of all tested caspases, 8, 9 and 3, whereas WP 631 only induced an increase in caspase 8 activity after 24h of treatment and consequently led to the cleavage of PARP. The lack of active caspase 3 had no outcome on the single and double-stranded DNA breaks. The obtained results show that WP 631 was considerably more genotoxic towards the investigated cell line than DOX. This effect was especially visible after longer times of incubation. The above detailed studies indicate that WP 631 generates early apoptosis and cell death independent of caspase-3, detected at relatively late time points. The observed differences in the mechanisms of the action of WP631 and DOX suggest that this bisanthracycline can be an effective alternative in ovarian cancer treatment.

Topics: Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Calcium; Caspases; Comet Assay; Cytochromes c; Daunorubicin; DNA Damage; Female; Flow Cytometry; Histones; Humans; Ovarian Neoplasms; Phosphorylation; Tumor Cells, Cultured

Pseudolaric acid B (PAB) is a diterpene acid isolated from the root and trunk bark of Pseudolarix kaempferi Gordon (Pinaceae). Recent studies have reported that PAB exhibits cytotoxic effects in several cancer cell lines. In the present study, we assessed its antitumor activity and molecular mechanisms in HO-8910 and A2780 ovarian cancer cells in vitro. We found that PAB reduced cell viability and induced apoptosis in a dose- and time-dependent manner in HO-8910 and A2780 human ovarian cancer cells. The induction of apoptosis was also accompanied by the regulation of Bcl-2 and XIAP family proteins, cytochrome c and Apaf-1. Moreover, we observed that PAB treatment resulted in the activation of caspase-3 and -9, which may partly explain the anticancer activity of PAB. Collectively, the present study for the first time suggests that PAB enhances apoptosis of HO-8910 and A2780 cells through regulation of Bcl-2 and IAP family proteins. Moreover, the triggering of caspase-3 and -9 activation mediated apoptotic induction. Our results suggest that PAB may be a new therapeutic option for the treatment of ovarian cancers.

Topics: Apoptosis; Apoptotic Protease-Activating Factor 1; Benzoquinones; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Line, Tumor; Cell Survival; Cytochromes c; Diterpenes; Enzyme Activation; Female; Humans; Microscopy, Electron, Transmission; Mitochondria; Oligopeptides; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; X-Linked Inhibitor of Apoptosis Protein

In this work we investigated the mode of cell death induced by WP 631, a novel anthracycline antibiotic, in the ovarian cancer cell line (OV-90) derived from the malignant ascites of a patient diagnosed with advanced disease. The effects were compared with those of doxorubicin (DOX), a first generation anthracycline. The ability of WP 631 to induce apoptosis and necrosis was examined by double staining with Annexin V and propidium iodide, measurements of the level of intracellular calcium ions and cytochrome c, PARP cleavage. We also investigated the possible involvement of the caspases activation, DNA degradation (comet assay) and intracellular reactive oxygen species (ROS) production in the development of the apoptotic events and their significance for drug efficiency. The results obtained clearly demonstrate that antiproliferative capacity of WP 631 in tested cell line was a few times greater than that of DOX. Furthermore, ovarian cancer cells treated with WP 631 showed a higher mean level of basal DNA damage in comparison to DOX. In conclusion, WP 631 is able to induce caspase - dependent apoptosis in human ovarian cancer cells. Obtained results suggested that WP 631 may be a candidate for further evaluation as chemotherapeutic agents for human cancers.

Topics: Anthracyclines; Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Calcium; Caspases; Cell Line, Tumor; Cell Survival; Comet Assay; Cytochromes c; Daunorubicin; DNA Damage; Doxorubicin; Female; Humans; Membrane Potential, Mitochondrial; Ovarian Neoplasms; Phosphatidylserines; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species

The molecular mechanisms underlying epothilone B (EpoB) induced apoptosis were investigated in SKOV-3 human ovarian cancer cells. The aim of this research was to compare EpoB's, which belongs to the new class of anticancer drugs, with paclitaxel's (PTX) ability to induce apoptosis. The mode of cell death was assessed colorimetrically, fluorimetrically and by immunoblot analyses through measuring DNA fragmentation, the level of intracellular calcium, the level of cytochrome c, TRAIL, the cleavage of poly(ADP-ribose) polymerase (PARP) and the activation of caspase-9, -8 and -3. EpoB leads to an increase of the cytosolic level of cytochrome c after 4 h of cell treatment. After 24 and 48 h of cell treatment the level of intracellular calcium also increased by about 21% and 24% respectively. Moreover, EpoB, similarly to PTX, promoted the expression of TRAIL in lymphocytes, although high TRAIL expression on tumor cells was detected only after adding EpoB to SKOV-3 cells. EpoB mediates caspases-8 and -3 activation, which is independent of the reduction in the amount of caspase-9. Epitope-specific monoclonal and polyclonal antibodies revealed characteristic apoptotic changes that included cleavage of the 116 kDa PARP polypeptide to 25 kDa fragments. The results of our study show that EpoB induces mainly the extrinsic pathway.

Topics: Antineoplastic Agents; Apoptosis; Calcium; Caspases; Cell Line, Tumor; Cytochromes c; DNA Damage; Edetic Acid; Epothilones; Female; Gene Expression Regulation; Humans; Ovarian Neoplasms; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Trypsin

The purpose of this study was to determine the influence of autophagy on cisplatin-induced ovarian cancer SKOV3/DDP cell line death through regulation of the expression of the autophagy gene, Beclin 1, and to explore the potential mechanism underlying the relationship between autophagy and apoptosis. When compared with a blank control group, the proportion of apoptotic cells undergoing Beclin 1 interfering increased significantly after cisplatin treatment, accompanied by reduction in mitochondrial membrane potential, increase in activities of caspase-9/3 and cytoplasmic cytochrome C, elevation of Bax expression, and reduction in Bcl-2 expression. However, the proportion of apoptotic cells with Beclin 1 overexpression reduced. These findings suggest that Beclin 1 plays an important role in the regulation of potent antitumor activity through a mitochondrial-dependent pathway in SKOV3/DDP cell line, and inhibition of Beclin 1 expression may become a new target for the sensitization therapy of ovarian cancer with cisplatin.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Autophagy; bcl-2-Associated X Protein; Beclin-1; Caspases; Cell Line, Tumor; Cisplatin; Cytochromes c; Female; Humans; Membrane Potential, Mitochondrial; Membrane Proteins; Microtubule-Associated Proteins; Mitochondria; Molecular Targeted Therapy; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Transfection

Multidrug resistance (MDR) remains one of the major obstacles in chemotherapy, potentially rendering a multitude of drugs ineffective. Previously, we have demonstrated that mitochondrial targeting of DNA damaging agents is a promising tool for evading a number of common resistance factors that are present in the nucleus or cytosol. In particular, mitochondria-targeted chlorambucil (mt-Cbl) has increased potency and activity against resistant cancer cells compared to the parent compound chlorambucil (Cbl). However, it was found that, due to its high reactivity, mt-Cbl induces a necrotic type of cell death via rapid nonspecific alkylation of mitochondrial proteins. Here, we demonstrate that by tuning the alkylating activity of mt-Cbl via chemical modification, the rate of generation of protein adducts can be reduced, resulting in a shift of the cell death mechanism from necrosis to a more controlled apoptotic pathway. Moreover, we demonstrate that all of the modified mt-Cbl compounds effectively evade MDR resulting from cytosolic GST-μ upregulation by rapidly accumulating in mitochondria, inducing cell death directly from within. In this study, we systematically elucidated the advantages and limitations of targeting alkylating agents with varying reactivity to mitochondria.

Topics: Alkylating Agents; Antineoplastic Agents; Apoptosis; Cell Death; Cell Line, Tumor; Cell Nucleus; Cell Survival; Chlorambucil; Colorimetry; Cytochromes c; Cytosol; DNA Damage; Drug Delivery Systems; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Glutathione Transferase; HeLa Cells; Humans; Inhibitory Concentration 50; Mitochondria; Necrosis; Ovarian Neoplasms

The polyphenolic flavonoid silymarin that is the milk thistle extract has been found to possess an anti-cancer effect against various human epithelial cancers. In this study, to explore the regulative effect of silymarin on human ovarian cancer line A2780s and PA-1 cells, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assay and flow cytometry were respectively used to determine the inhibitory effect of silymarin on the both cell lines, and to measure their cell cycle progression. Apoptosis induction and mitochondrial membrane potential damage were separately detected by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling assay and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide staining. Additionally, western blotting was applied to determine cytochrome C release and expression levels of p53, p21, p27, p16, CDK2, Bax, Bcl-2, procaspase-9, procaspase-3, cleaved caspase-9 and caspase-3 proteins. The activity of caspase-9 and caspase-3 was measured using Caspase-Glo-9 and Caspase-Glo-3 assay. The results indicated that silymarin effectively suppressed cell growth in a dose- and time-dependent manner, and arrested cell cycle progression at G1/S phase in A2780s and PA-1 cells via up-regulation of p53, p21, and p27 protein expression, and down-regulation of CDK2 protein expression. Additionally, silymarin treatment for 24h at 50 and 100µg/ml resulted in a reduction of mitochondrial membrane potential and cytochrome C release, and significantly induced apoptosis in A2780s and PA-1 cells by increasing Bax and decreasing Bcl-2 protein expression, and activation of caspase-9 and caspase-3. Therefore, silymarin is a possible potential candidate for the prevention and treatment of ovarian cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Cytochromes c; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; Humans; Membrane Potential, Mitochondrial; Ovarian Neoplasms; Proto-Oncogene Proteins c-bcl-2; Silymarin; Tumor Suppressor Protein p53; Up-Regulation

In the present study, we downregulated FANCF expression by small interfering RNA (siRNA) in OVCAR ovarian cancer cells to address the effects of decreased FANCF expression on the function of the Fanconi anemia (FA)/breast cancer susceptibility gene (BRCA) pathway. Furthermore, we investigated whether this method increases the sensitivity of OVCAR3 cells to adriamycin (ADM) and the possible mechanism(s). We found that silencing of FANCF inactivated the FA/BRCA pathway by decreasing the monoubiquitination and focus formation of FANCD2 and reduced the function of the FA/BRCA pathway, resulting in the inhibition of cell proliferation, increased cell apoptosis and DNA damage in OVCAR3 cells. Moreover, we observed that silencing of FANCF enhanced the antiproliferative effect of ADM in OVCAR3 cells and increased ADM intracellular accumulation consequently sensitizing OVCAR3 cells to ADM. Furthermore, silencing of FANCF increased cell apoptosis of OVCAR3 cells which was caused by decreased mitochondrial membrane potential (MMP)-induced DNA damage, activated Jun N-terminal kinase (JNK), increased release of cytochrome c, increased expression of cleaved caspase-3 and poly(ADP-ribose) polymerase (PARP) dependent on JNK activation following treatment of ADM. Collectively, we confirm that silencing of FANCF sensitizes OVCAR3 ovarian cancer cells to ADM, suggesting that FANCF may serve as a potential target for therapeutic strategies in the treatment of ovarian cancer.

Topics: Apoptosis; BRCA1 Protein; Caspase 3; Cell Line, Tumor; Cell Proliferation; Combined Modality Therapy; Cytochromes c; DNA Damage; Down-Regulation; Doxorubicin; Fanconi Anemia; Fanconi Anemia Complementation Group F Protein; Female; Humans; JNK Mitogen-Activated Protein Kinases; Membrane Potential, Mitochondrial; Ovarian Neoplasms; Poly(ADP-ribose) Polymerases; RNA Interference; RNA, Small Interfering; Signal Transduction

Brefeldin A induces apoptosis in various cancer cells; however, the apoptotic process in cancer cells exposed to brefeldin A remains unclear. In addition, it is unclear whether brefeldin A-induced apoptosis is mediated by the formation of reactive oxygen species. Furthermore, the effect of brefeldin A on the invasion and migration of human epithelial ovarian cancer cells has not been studied. Therefore, we investigated the effect of brefeldin A on apoptosis, cell adhesion and migration using the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. The results suggest that brefeldin A may induce apoptotic cell death in ovarian carcinoma cell lines by activating the mitochondrial pathway and the caspase-8- and Bid-dependent pathways. The apoptotic effect of brefeldin A seems to be mediated by formation of reactive oxygen species and depletion of GSH, which results in the activation of apoptotic caspases. Brefeldin A inhibited foetal bovine serum-induced adhesion and migration of OVCAR-3 cells. Brefeldin A may prevent the foetal bovine serum-induced cell adhesion and migration by limiting the focal adhesion kinase-dependent activation of cytoskeletal-associated components.

Topics: Apoptosis; BH3 Interacting Domain Death Agonist Protein; Brefeldin A; Carcinoma, Ovarian Epithelial; Caspase 8; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Survival; Cytochromes c; DNA Damage; DNA Fragmentation; Focal Adhesion Protein-Tyrosine Kinases; Humans; Membrane Potential, Mitochondrial; Mitochondria; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Reactive Oxygen Species

The epothilones, a new class of microtubule-targeting agents, seem to be a very promising alternative to the current strategy of cancer treatment. We have analyzed the aspects of epothilone B (Epo B) on cellular metabolism of tumor (OV-90) and normal (MM 14) ovarian cells. The observed effects were compared with those of paclitaxel (PTX), which is now a standard for the treatment of ovarian cancer. The results provide direct evidence that Epo B is considerably more cytotoxic to human OV-90 ovarian cancer cells than PTX. We have found, that antitumor efficacy of this new drug is related to its apoptosis-inducing ability, which was confirmed during measurements typical markers of the process. Epo B induced changes in morphology of cells, mitochondrial membrane potential and cytochrome c release. Also a slight increase of the intracellular calcium level was observed. Moreover, we have found that ROS production, stimulated by Epo B, is directly involved in the induction of apoptosis via mitochondrial pathway.

Topics: Animals; Antineoplastic Agents; Apoptosis; Calcium; Cell Line, Tumor; Cell Shape; Cytochromes c; Dose-Response Relationship, Drug; Epothilones; Female; Humans; Membrane Potential, Mitochondrial; Mice; Mitochondria; Ovarian Neoplasms; Ovary; Paclitaxel; Reactive Oxygen Species; Signal Transduction; Time Factors; Tubulin Modulators

PNAS-4, a novel pro-apoptotic gene, was activated during the early response to DNA damage. Previous studies have shown that hPNAS-4 can inhibit tumor growth when over-expressed in ovarian cancer cells. However, the underlying action mechanism remains elusive. In this work, we found that hPNAS-4 expression was significantly increased in SKOV3 cells when exposed to cisplatin, methyl methanesulfonate or mitomycin C, and that its overexpression could induce proliferation inhibition, S phase arrest and apoptosis in A2780s and SKOV3 ovarian cancer cells. The S phase arrest caused by hPNAS-4 was associated with up-regulation of p21. p21 is p53-dispensable and correlates with activation of ERK, and activation of the Cdc25A-Cdk2-Cyclin E/Cyclin A pathway, while the pro-apoptotic effects of hPNAS-4 were mediated by activation of caspase-9 and -3 other than caspase-8, and accompanied by release of AIF, Smac and cytochrome c into the cytosol. Taken together, these data suggest a new mechanism by which hPNAS-4 inhibits proliferation of ovarian cancer cells by inducing S phase arrest and apoptosis via activation of Cdc25A-Cdk2-Cyclin E/Cyclin A axis and mitochondrial dysfunction-mediated caspase-dependent and -independent apoptotic pathways. To our knowledge, we provide the first molecular evidence for the potential application of hPNAS-4 as a novel target in ovarian cancer gene therapy.

Topics: Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; Carbon-Nitrogen Lyases; Caspase 3; Caspase 8; Caspase 9; cdc25 Phosphatases; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cyclin A; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; DNA Damage; Enzyme Activation; Female; Humans; Intracellular Signaling Peptides and Proteins; Methyl Methanesulfonate; Mitochondrial Proteins; Mitomycin; Ovarian Neoplasms; S Phase Cell Cycle Checkpoints; Up-Regulation

Hsp90 inhibitor geldanamycin and parthenolide have been shown to induce apoptosis in cancer cells. However, the combined effect of geldanamycin and parthenolide on epithelial ovarian cancer cells has not been studied. In respect of cell death process, we investigated the promoting effect of parthenolide on geldanamycin-induced apoptosis in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. Geldanamycin induced a decrease in Bid, Bcl-2, Bcl-xL and survivin protein levels; an increase in Bax and tumour suppressor p53 levels; loss of the mitochondrial transmembrane potential; cytochrome c release; activation of caspases (-8, -9 and -3); cleavage of PARP-1; and increase in the reactive oxygen species formation. Parthenolide enhanced geldanamycin-induced changes in the apoptosis-related protein levels, reactive oxygen species formation, nuclear damage and cell death. The combined effect was inhibited by the addition of oxidant scavengers. The results suggest that parthenolide may potentiate the apoptotic effect of geldanamycin on ovarian carcinoma cell lines by the activation of the caspase-8- and Bid-dependent pathway and the mitochondria-mediated apoptotic pathway. The apoptosis-promoting effect seems to be mediated by the stimulatory effect on the formation of reactive oxygen species.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2-Associated X Protein; Benzoquinones; Blotting, Western; Carcinoma, Ovarian Epithelial; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Female; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Mitochondria; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Reactive Oxygen Species; Sesquiterpenes; Tumor Suppressor Protein p53

Previously we have shown that interferon (IFN)-α induced apoptosis is predominantly mediated by the upregulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) via the caspase-8 pathway. It was also shown that recruitment of mitochondria in IFN-α induced apoptosis involves the cleavage of BH3 interacting domain death agonist (Bid) to truncated Bid (tBid). In the present study, we demonstrate that tBid induced by IFN-α2a activates mitochondrial Bak to trigger the loss of mitochondrial membrane integrity, consequently causing release of apoptosis-inducing factor (AIF) in ovarian cancer cells, OVCAR3. AIF translocates from the mitochondria to the nucleus and induces nuclear fragmentation and cell death. Both a small molecule Bid inhibitor (BI-6C9) or Bid-RNA interference (RNAi) preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated IFN-α2a-induced cell death. Cell death induced by tBid was inhibited by AIF-RNAi, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that BI-6C9 did not prevent the release of cytochrome c from mitochondria to cytosol, while the release of AIF was prevented. In conclusion, IFN-α2a-induced apoptosis is mediated via the mitochondria-associated pathway involving the cleavage of Bid followed by AIF release that involves Bak activation and translocation of AIF from the mitochondria to the nucleus in OVCAR3 cells.

Topics: Apoptosis; Apoptosis Inducing Factor; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Caspase 9; Cell Line, Tumor; Cell Survival; Cytochromes c; Female; Gene Knockdown Techniques; Humans; Interferon-alpha; Membrane Potential, Mitochondrial; Ovarian Neoplasms; Protein Multimerization; Protein Transport; RNA Interference

Rhizoma Paridis (dried root and rhizome) has been an essential ingredient in traditional Chinese herbal medicine. In the past decade, active components of Rhizoma Paridis - the Paris saponins have emerged as promising anti-cancer agents. Among these saponins, polyphyllin D (Paris saponin (PS) I), has been extensively studied and proposed to be a potent antitumor agent. In this study, we continue to establish the efficacy and mechanisms underlying the cytotoxic effects of the steroidal PS members, namely formosanin C (PSII) in ovarian cancer treatment. We isolated PSII and evaluated its effects on a panel of ten human cell lines. Isolated PSII has potent inhibitory effects on the growth of tumor cells without deleterious effects to different normal cell types or benign neoplastic derived cells. While PSII, PSI, and etoposide are effective promoting agents for cell cycle arrest and apoptosis, PSII appeared to be marginally more potent than the later two in inhibiting SKOV3 cell growth. In PSII-treated SKOV3 cells, there was an elevation in proapoptotic elements including Bax, cytosolic cytochrome c, activated-caspase-3, and activated-caspase-9. The treatment also reduced extracellular signal-regulated kinase (ERK1/2) phosphorylation and anti-apoptotic Bcl-2 expression. We also assessed the antitumor efficacy of intraperitoneal administration of PSII in human SKOV3 ovarian cancer xenografts in athymic mice. PSII treatment significantly inhibited the growth of xenograft tumors relative to controls by 70% (p < 0.05). These findings demonstrated that, in addition to the unique selectivity against cancer cells, PSII is a potent antitumor molecule that may be developed as a cancer therapeutic agent.

Topics: Animals; Apoptosis; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Diosgenin; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Female; Flow Cytometry; Humans; Mice; Ovarian Neoplasms; Phosphorylation; Rhizome; Saponins; Xenograft Model Antitumor Assays

Some mutations in FOXL2 result in premature ovarian failure accompanied by blepharophimosis, ptosis, epicanthus inversus syndrome type I disease, and FOXL2-null mice exhibit developmental defects in granulosa cells. Recently, FOXL2 c.402C>G, a new somatic mutation that leads to a p.C134W change, was found in the majority of adult-type ovarian granulosa cell tumors (GCTs). In this study, we investigated the possible mechanisms by which the C134W mutation contributes to the development of GCTs. Wild-type (WT) and mutant FOXL2 displayed differential apoptotic activities. Specifically, WT FOXL2 induced significant granulosa cell death, but the mutant exhibited minimal cell death. The FOXL2-induced apoptotic response was greatly dependent on caspase 8, BID and BAK because the depletion of any of these three proteins inhibited FOXL2 from eliciting the full apoptotic response. Activation of caspase 8 and subsequent increased production of truncated BID, and oligomerization of BAK, and release of cytochrome c were all associated with the apoptosis induced by WT FOXL2 expression. In contrast, the mutant FOXL2 was unable to elicit the full array of apoptotic signaling responses. In addition, we found differential TNF-R1 (tumor necrosis factor-receptor 1) and Fas (CD95/APO-1) upregulation between the WT and the mutant, and the silencing of TNF-R1 or Fas and the blockage of the death signaling mediated by TNF-R1 or Fas using TNF-Fc or Fas-Fc, respectively, resulted in significant attenuations of FOXL2-induced apoptosis. Moreover, granulosa cells that expressed either WT FOXL2 or mutant exhibited distinct cell death sensitivities on activation of death receptors and deprivation of serum. Thus, the differential activities of FOXL2 and its mutant may partially account for the pathophysiology of GCT development.

Topics: Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; BH3 Interacting Domain Death Agonist Protein; Caspase 8; Cell Line, Tumor; Cytochromes c; fas Receptor; Female; Forkhead Box Protein L2; Forkhead Transcription Factors; Granulosa Cell Tumor; Humans; Mice; Mutation; Ovarian Neoplasms; Receptors, Tumor Necrosis Factor, Type I; Up-Regulation

Twelve Pt(II) complexes with cis-PtP(2)S(2) pharmacophores (where P(2) refers to two monodentate or one bidentate phosphane ligand and S(2) is a dithiolato ligand) were prepared, characterized and evaluated as potential antiproliferative agents. The various compounds were first studied from the structural point of view; afterward, their solubility properties as well as their solution behaviour were analyzed in detail. Antiproliferative properties were specifically evaluated against A2780 human ovarian carcinoma cells, either resistant or sensitive to cisplatin. For comparison purposes similar studies were carried out on four parent cis-dichloro bisphosphane Pt(II)complexes. On the whole, the cis-PtP(2)S(2) compounds displayed significant antiproliferative properties while the cis-PtP(2)Cl(2) (cis-dichloro bisphosphane Pt(II)) compounds revealed quite poor biological performances. To gain further insight into the molecular mechanisms of these bisphosphane Pt(II) compounds, the reactions of selected complexes against the model protein cytochrome c were investigated by ESI-MS and their adduct formation explored. A relevant reactivity with cyt c was obtained only for cis-PtP(2)Cl(2) compounds, whereas cis-PtP(2)S(2) compounds turned out to be nearly unreactive. The obtained results are interpreted and discussed in the frame of the current knowledge of anticancer platinum compounds and their structure-activity-relationships. The observation of appreciable antiproliferative effects for the relatively inert cis-PtP(2)S(2) compounds strongly suggests that these compounds will undergo specific activation within the cellular environment.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cytochromes c; Female; Humans; Ligands; Magnetic Resonance Spectroscopy; Molecular Structure; Organoplatinum Compounds; Ovarian Neoplasms; Phosphines; Phosphorus; Platinum Compounds; Protein Binding; Spectrophotometry, Ultraviolet; Structure-Activity Relationship; Sulfur

Histone deacetylase inhibitors and proteasome inhibitor are all emerging as new classes of anticancer agents. We chose TSA and PS-341 to identify whether they have a synergistic efficacy on human ovarian cancer cells. After incubated with 500 nM TSA or/and 40 nM PS-341, we found that combined groups resulted in a striking increase of apoptosis and G2/M blocking rates, no matter in A2780, cisplatin-sensitive ovarian cancer cell line OV2008 or its resistant variant C13*. This demonstrated that TSA interacted synergistically with PS-341, which raised the possibility that combined the two drugs may represent a novel strategy in ovarian cancer.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Boronic Acids; Bortezomib; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Drug Resistance, Neoplasm; Drug Synergism; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Ovarian Neoplasms; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; RNA Interference; Time Factors

Histone deacetylase inhibitor-induced apoptosis in cancer cells may be mediated by the Ras/Raf/MEK/ERK and protein kinase B/Akt signaling pathways. However, inhibition of ERK and Akt activity has different effects on proliferation and apoptosis in cancer cells. We assessed and compared the inhibitory effects of Akt and ERK pathways on the apoptotic effect of trichostatin A using the human epithelial carcinoma cell lines OVCAR-3 and SK-OV-3. Trichostatin A induced nuclear damage, decrease in Bid and Bcl-2 protein levels, increase in Bax levels, cytochrome c release, activation of caspases (8, 9, and 3) and increase in tumor suppressor p53 levels. Akt inhibitor potentiated trichostatin A-induced apoptosis-related protein activation and cell death, whereas ERK inhibitor exhibited an additive toxic effect. These results suggest that the Akt and ERK inhibitors may have a differential effect on trichostatin A-induced apoptosis in human epithelial ovarian carcinoma cell lines. Akt inhibitor may potentiate the apoptotic effect of trichostatin A on ovarian carcinoma cell lines by increasing the activation of the caspase-8-dependent pathway and the mitochondria-mediated cell death pathway, leading to caspase activation. In contrast, ERK inhibitor may exhibit an additive toxic effect on trichostatin A toxicity by increasing apoptosis-related protein activation.

Topics: Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Carcinoma, Ovarian Epithelial; Caspase 8; Cell Line, Tumor; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Inositol Phosphates; Microscopy, Fluorescence; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53

Ovarian cancer is the number one cause of death from gynecologic malignancy. A defective p53 pathway is a hallmark of ovarian carcinoma. The p53 mutation correlates significantly with resistance to platinum-based chemotherapy, early relapse and shortened overall survival in ovarian cancer patients. PUMA (p53 upregulated modulator of apoptosis), a BH3-only Bcl-2 family protein, was recently identified as a transcriptional target of p53 and a potent apoptosis inducer in various cancer cells. In this study, we showed that the induction of PUMA by cisplatin was abolished in p53-deficient SKOV3 cells. Elevated expression of PUMA-induced apoptosis and sensitized A2780s and SKOV3 ovarian cancer cells to cisplatin, and the combination of PUMA and low-dose cisplatin, significantly suppressed xenograft tumor growth in vivo through enhanced induction of apoptosis compared with treatment with PUMA or cisplatin alone. The effects of PUMA were mediated by enhanced caspase activation and release of cytochrome c and Smac (second mitochondria-derived activator of caspase) into the cytosol. Furthermore, PUMA chemosensitized intrinsically resistant SKOV3 cells to cisplatin through downregulation of B-cell lymphoma-extra large (Bcl-x(L)) and myeloid cell leukemia sequence 1 (Mcl-1). PUMA-mediated Bcl-x(L) downregulation mainly happened at the transcription level, whereas PUMA-induced Mcl-1 down-regulation was associated with caspase-dependent cleavage and proteasome-mediated degradation. To our knowledge, these data suggest a new mechanism by which overexpression of PUMA enhances sensitivity of SKOV3 cells to cisplatin by lowering the threshold set simultaneously by Bcl-x(L) and Mcl-1. Taken together, our findings indicate that PUMA is an important modulator of therapeutic responses of ovarian cancer cells and is potentially useful as a chemosensitizer in ovarian cancer therapy.

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Caspases; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Down-Regulation; Drug Resistance, Neoplasm; Enzyme Activation; Female; Gene Deletion; Humans; In Situ Nick-End Labeling; Intracellular Signaling Peptides and Proteins; Mitochondrial Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Ovarian Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Treatment Outcome; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Study of the mechanisms of apoptosis in tumor cells is an important field of tumor therapy and cancer molecular biology. Apoptosis triggered by activation of the mitochondrial-dependent caspase pathway represents the main programmed cell death mechanism. The mitochondrial-dependent apoptosis pathway is activated by various intracellular stresses that induce permeabilization of the mitochondrial membrane, leading to cytochrome C release. This study was to investigate the anti-tumor effects of Dioscin from traditional Chinese anti-snake venom medicine Paris chinensis (PCD) and correlated mechanisms regarding apoptosis in human ovarian cancer SKOV3 cells.. Cell viability was analyzed by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl-tetrazolium bromide (MTT) assay. Cell apoptosis was evaluated by flow cytometry and Laser Scanning Confocal Microscope (LSCM) using Annexin-V/PI staining. Intracellular calcium ions were detected using fluorescence microscopy. The expression of apoptosis-related proteins cytochrome C and caspase-3 was measured by immunohistochemical staining.. PCD had an anti-proliferation effect on human ovarian cancer SKOV3 cells in a dose- and time-dependent manner. After treatment with PCD, the apoptotic rate significantly increased, and accompanied with the increased levels of caspase-3 and cytochrome C protein in SKOV3 cells. Morphological changes typical of apoptosis were also observed with LSCM by Annexin V/PI staining. Moreover, intracellular calcium accumulation occurred in PCD-treated cells.. The molecular determinants of inhibition of cell proliferation as well as apoptosis of PCD may be associated with the activation of Ca2+-related m itochondrion pathway in SKOV3 cells.

Topics: Apoptosis; Calcium; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Diosgenin; Drugs, Chinese Herbal; Female; Humans; Mitochondria; Mitochondrial Membranes; Ovarian Neoplasms; Permeability; Trillium

Antrodia camphorata is a Chinese herb. Recently, several reports demonstrated that it had growth-inhibiting effects on some cancer cells. In this study, we investigated whether the crude extract of A. camphorata could inhibit the growth of ovarian cancer cells and examined the possible mechanisms involved. We also examined whether the cytotoxic effect of paclitaxel on ovarian cancer cells would be affected by A. camphorata.. Two human ovarian cancer cell lines, SKOV-3 and TOV-21G, were treated with A. camphorata (3-300 μg/mL). An MTT assay was used to test its cytotoxic effect. The apoptosis-related factors including the activity of caspase-3, -8, and -9 and the cytochrome c level released from mitochondria were analyzed. The expression of Bcl-2 family proteins (Bcl-2, Bcl-xL, Bax, Bim, Bad, and Bak) was examined by Western blot analysis. Cell lines were further treated with paclitaxel or paclitaxel plus A. camphorata to examine the cytotoxic efficiency.. The MTT assay revealed that A. camphorata was cytotoxic to both the ovarian cancer cells in a dose- and time-dependent manner. Activities of caspase-3, -8, and -9 and release of mitochondrial cytochrome c increased in both ovarian cancer cell lines with increased dose of A. camphorata. Western blot analysis of Bcl-2 family proteins revealed an increased expression of Bad in SKOV-3 cells, whereas increased expression of Bim and Bak and decreased expression of Bcl-xL were noted in TOV-21G cells. In addition, the cytotoxic effect of paclitaxel on SKOV-3 and TOV-21G cells was increased significantly with the addition of A. camphorata (P < 0.01) by MTT assay.. These in vitro results suggest that A. camphorata causes a cytotoxic effect on ovarian cancer cells through the induction of apoptosis. It may also enhance the antitumor effect of paclitaxel. Further studies with the ultimate goal of conducting clinical trials are warranted.

Topics: Antineoplastic Agents, Phytogenic; Antrodia; Apoptosis; Caspases; Cell Line, Tumor; Cytochromes c; Drug Therapy, Combination; Drugs, Chinese Herbal; Female; Humans; Ovarian Neoplasms; Paclitaxel; Proto-Oncogene Proteins c-bcl-2

The apoptotic effect of fluoxetine (FLX), an antidepressant, against human epithelial ovarian cancer cell lines OVCAR-3 and SK-OV-3 was investigated in relation to the mitochondria-mediated cell death process and nuclear factor (NF)-kappaB activation. FLX-induced mitochondrial membrane permeability change and formation of reactive oxygen species, leading to cell death. FLX-induced increase in mitochondrial Bax levels, decrease in cytosolic Bid and Bcl-2 levels, loss of the mitochondrial transmembrane potential, cytochrome c release, caspase-3 activation and up-regulation of p53. Oxidant scavengers and Bay 11-7085 [an inhibitor of nuclear factor kappaB (NF-kappaB) activation] prevented the FLX-induced cell death, increase in phosphorylated inhibitory kappaB-alpha and NF-kappaB p65 levels, and binding of NF-kappaB p65 to DNA. Results from this study suggest that FLX may exhibit apoptotic effect against ovarian cancer cell lines by inducing the mitochondrial membrane permeability change, which leads to cytochrome c release and subsequent caspase-3 activation, through reactive oxygen species-dependent activation of NF-kappaB.

Topics: Apoptosis; Caspase 3; Cell Death; Cell Line, Tumor; Cytochromes c; Female; Fluoxetine; Humans; Membrane Potential, Mitochondrial; NF-kappa B; Ovarian Neoplasms; Reactive Oxygen Species; Selective Serotonin Reuptake Inhibitors

Cancer-upregulated gene 2 (CUG2) was originally identified as a potential oncogene commonly up-regulated in various human cancers. Recently, CUG2 was also identified as a new member of a centromere protein complex, important in the formation of a functional kinetochore complex. Presently, we report the pro-apoptotic effect of CUG2 when this gene was overexpressed in the SKOV-3 human ovarian cancer cell line. Apoptotic cell death mediated by CUG2 overexpression was independently demonstrated using cell viability determination, flow cytometry analysis, chromosome fragmentation assay, and the cleavage of the death substrate poly(ADP-ribose) polymerase. Moreover, activation of caspase-3 and -8 and the cytoplasmic translocation of mitochondrial cytochrome c were evident upon CUG2 expression. Apoptotic cell death was also observed during early development of zebrafish when CUG2 was overexpressed in zebrafish embryo. We propose that high expression of CUG2 induces apoptotic cell death.

Topics: Animals; Apoptosis; Caspases; Cell Line, Tumor; Cell Survival; Chromosomal Proteins, Non-Histone; Cytochromes c; Female; Gene Expression Regulation, Neoplastic; Humans; Nuclear Proteins; Ovarian Neoplasms; Zebrafish

This study investigates the antitumor effects and functional mechanism of resveratrol-bovine serum albumin nanoparticles (RES-BSANP) on human primary ovarian carcinoma cells in nude mice. An implanted tumor model was established by injecting a suspension of the human primary ovarian cancer cell SKOV(3) into the subcutaneous tissue of nude mice. The tumor-bearing mice (n = 32) were randomly divided into 8 groups, which received intraperitoneal injections of normal saline (0.9%, 0.5 mL), BSA (1.5 mg/kg, 0.5 mL), or RES-BSANP or RES (200, 100, and 50 mg/kg, 0.5 mL), respectively, once a week for 4 weeks. The in vivo antitumor efficacy was evaluated by measurement of tumor volume, whereas morphological alterations were observed by transmission electron microscope (atomic force microscopy); TUNEL assays and immunoblotting for apoptotic and cell proliferation proteins were carried out to elucidate the possible mechanism. RES-BSANP was found to exhibit certain highly desirable characteristics such as innocuity, better dispersity, and water solubility; it affected the in vivo tissue/organ distribution of RES in a remarkable manner. The administration of RES-BSANP significantly retarded the growth of carcinomas in nude mice from the third week onwards, and the inhibition rate was markedly higher than in mice treated with RES (52.43% vs. 46.34%, p < 0.05), without causing weight loss (p > 0.05). Simultaneously, apoptotic and necrotic morphological characteristics were observed with electron microscopy in the tumor tissues of treated mice. TUNEL staining revealed that the tumors from RES-BSANP-treated mice exhibited a similar apoptotic index as RES control tumors. Western blot analysis of the protein expression profiles revealed that part of the mechanism may be mediated by triggering the release of cytochrome c from the intermembrane space and upregulating the expression of caspase-9 and caspase-3, suggesting that the mitochondrial apoptotic pathway was being activated.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Caspase 3; Caspase 9; Cattle; Chromatography, High Pressure Liquid; Cytochromes c; Female; Humans; In Situ Nick-End Labeling; Injections, Subcutaneous; Mice; Mice, Nude; Mitochondria; Nanoparticles; Ovarian Neoplasms; Resveratrol; Serum Albumin, Bovine; Signal Transduction; Stilbenes; Tissue Distribution; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

To study the effect of resveratrol bovine serum albumin nanoparticles on SKOV3 cell line and its mechanisms.. The morphological changes of the cells exposed to the nanoparticles were observed by apoptotic body/cell nucleus DNA staining under inverted microscope and fluorescence microscope, and the pathway of cell death was determined by phosphatidylserine translocation. Western blotting was performed to detect the activation of cyto.c, caspase-3 and caspase-9.. DNA ladder was detected with gel electrophoresis and the cell death was partially inhibited by the pan-caspase inhibitor Z-VAD-FMK. Gel electrophoresis displayed both DNA ladder and smear in RES-BSANP exposed groups, while DNA ladder disappeared in Z-VAD-FMK group and only the smear was left. Cyto.c in the cytoplasm was released at 2 h, while the expression of caspase-9 protein reached the peak level at 4 h and caspase-3 expression was obvious enhanced at 8 h. At 4 h, caspase-9 expression in the cells exposed to 100 µmol/L RES-BSANP was decreased significantly as compared to the cells treated with 50 µmol/L RES-BSANP (P>0.05).. RES-BSANP can induce the necrosis and apoptosis of SKOV3 cells via either caspase-dependent or caspase-independent pathways.

Topics: Apoptosis; Caspase 3; Caspase 9; Cell Death; Cell Line, Tumor; Cytochromes c; Female; Humans; Nanoparticles; Ovarian Neoplasms; Resveratrol; Stilbenes

MDM4 is a key regulator of p53, whose biological activities depend on both transcriptional activity and transcription-independent mitochondrial functions. MDM4 binds to p53 and blocks its transcriptional activity; however, the main cytoplasmic localization of MDM4 might also imply a regulation of p53-mitochondrial function. Here, we show that MDM4 stably localizes at the mitochondria, in which it (i) binds BCL2, (ii) facilitates mitochondrial localization of p53 phosphorylated at Ser46 (p53Ser46(P)) and (iii) promotes binding between p53Ser46(P) and BCL2, release of cytochrome C and apoptosis. In agreement with these observations, MDM4 reduction by RNA interference increases resistance to DNA-damage-induced apoptosis in a p53-dependent manner and independently of transcription. Consistent with these findings, a significant downregulation of MDM4 expression associates with cisplatin resistance in human ovarian cancers, and MDM4 modulation affects cisplatin sensitivity of ovarian cancer cells. These data define a new localization and function of MDM4 that, by acting as a docking site for p53Ser46(P) to BCL2, facilitates the p53-mediated intrinsic-apoptotic pathway. Overall, our results point to MDM4 as a double-faced regulator of p53.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma; Cell Line, Tumor; Cells, Cultured; Cisplatin; Cytochromes c; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mitochondria; Ovarian Neoplasms; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases

The present study was designed to determine the effects of artemisinin (ARS) and its derivatives on human ovarian cancer cells, to evaluate their potential as novel chemotherapeutic agents used alone or in combination with a conventional cancer chemotherapeutic agent, and to investigate their underlying mechanisms of action. Human ovarian cancer cells (A2780 and OVCAR-3), and immortalized non-tumourigenic human ovarian surface epithelial cells (IOSE144), were exposed to four ARS compounds for cytotoxicity testing. The in vitro and in vivo antitumour effects and possible underlying mechanisms of action of dihydroartemisinin (DHA), the most effective compound, were further determined in ovarian cancer cells. ARS compounds exerted potent cytotoxicity to human ovarian carcinoma cells, with minimal effects on non-tumourigenic ovarian surface epithelial (OSE) cells. DHA inhibited ovarian cancer cell growth when administered alone or in combination with carboplatin, presumably through the death receptor- and, mitochondrion-mediated caspase-dependent apoptotic pathway. These effects were also observed in in vivo ovarian A2780 and OVCAR-3 xenograft tumour models. In conclusion, ARS derivatives, particularly DHA, exhibit significant anticancer activity against ovarian cancer cells in vitro and in vivo, with minimal toxicity to non-tumourigenic human OSE cells, indicating that they may be promising therapeutic agents for ovarian cancer, either used alone or in combination with conventional chemotherapy.

Topics: Apoptosis; Artemisinins; Carboplatin; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cytochromes c; Drug Screening Assays, Antitumor; Female; Humans; Membrane Potential, Mitochondrial; Mitochondrial Membranes; Models, Biological; Ovarian Neoplasms

Several studies in the last years evidenced that deregulation of proapoptotic and antiapoptotic pathways are key players in the onset and maintenance of chemoresistance in advanced ovarian cancers. To characterize the signaling events and molecules involved in the acquisition of cisplatin resistance, we used the human ovarian cancer cell line A2780 and its derivative cisplatin-resistant subline A2780 CIS. We found that the mitochondrial intrinsic apoptotic pathway, induced by cis-dichlorodiammineplatinum (CDDP) in A2780 wild-type cells, was compromised in the resistant subline CIS. The analysis of expression of proteins involved in mitochondria-dependent apoptosis revealed a role of Bax and p73 but not p53. Indeed, we found that CDDP treatment induced the up-regulation of p53 in both sensitive and resistant A2780 cell lines. By contrast, p73 and Bax expressions were compromised in resistant cells. Pretreatment of resistant A2780 CIS cells with the histone deacetylase inhibitor trichostatin A overcomes apoptosis resistance to CDDP by restoring both p73 and Bax but not p53 expression. Altogether, these data indicate that p73, but not p53, is involved in the regulation of apoptosis susceptibility to cisplatin in A2780 ovarian cancer cells and evidence a key contribution of histone deacetylase activation in the acquisition of chemotherapy resistance in human ovarian cancer cells.

Topics: Apoptosis; Base Sequence; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cisplatin; Cytochromes c; DNA-Binding Proteins; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Enzyme Activation; Female; Humans; Hydroxamic Acids; Membrane Potential, Mitochondrial; Mitochondria; Molecular Sequence Data; Nuclear Proteins; Ovarian Neoplasms; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Up-Regulation

We examined the antiproliferation effect of Jaceosidin (4', 5, 7-trihydroxy-3', 6-dimethoxyflavone) isolated from the herb of Artemisia vestita Wall on several human cancer cell lines. Jaceosidin significantly reduced the proliferation of CAOV-3, SKOV-3, HeLa, and PC3 cells in a concentration-dependent manner. A time-dependent inhibition was also observed in CAOV-3 cells by Jaceosidin. By flow cytometric analysis, we found that Jaceosidin treatment resulted in an increased apoptosis in CAOV-3 cells. The cells treated with Jaceosidin exhibited a decreased mitochondrial membrane potential. Jaceosidin also increased the level of cleaved caspase-9 and induced the cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP), while caspase-3 inhibitor Z-DEVD-FMK significantly reversed the proapoptotic effect of Jaceosidin in CAOV-3 cells. Moreover, Jaceosidin elevated the level of cytochrome c in cytosol. These findings suggest that the anticancer effect of Jaceosidin may be contributed by an induction of apoptosis involving cytochrome c release from mitochondria to cytosol.

Topics: Apoptosis; Caspase 3; Caspase 9; Caspase Inhibitors; Cell Proliferation; Cytochromes c; Female; Flavonoids; HeLa Cells; Humans; Male; Membrane Potential, Mitochondrial; Mitochondria; Oligopeptides; Ovarian Neoplasms; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms

Duchesnea indica (Andr.) Focke has been commonly used to treat cancer in Asian countries of centuries, and more recently, has been shown to possess anticancer properties in vivo and in vitro. However, little is known about the underlying mechanism of its anticancer action. In the present study, we investigated the effect of Duchesnea phenolic fraction (DPF) on SKOV-3 ovarian cancer cells to provide insights into the mechanisms of growth suppression involved in DPF-mediated apoptosis and cell cycle arrest.. Cytotoxic activity of DPF on SKOV-3 cells was determined using MTT assay, apoptosis (AO/EB staining, DNA fragmentation, FACS), caspase-3 activation and cell cycle analysis studies. The role of the molecules in apoptosis and cell cycle regulation was analyzed by Western blot and RT-PCR.. DPF significantly inhibited SKOV-3 cell proliferation in a dose-dependent manner and markedly induced apoptosis evidenced by characteristic apoptotic morphological changes, nuclear DNA fragmentation and sub-G1 peak. DPF suppressed Bcl-2 levels, enhanced Bax levels and Bax/Bcl-2 ratio, and simultaneously translocated Bax to mitochondria followed by mitochondrial release of cytochrome c into the cytosol and activation of effector caspase-3. Furthermore, DPF provoked S phase arrest in SKOV-3 cells with down-regulation of cyclin A, E, D1 and CDK2.. DPF exhibits cytotoxicity towards human ovarian cancer SKOV-3 cells through induction of apoptosis via mitochondrial pathway and arresting cell cycle progression in S phase. All together, these data sustain our contention that DPF has anticancer properties and merits further investigation as a potential therapeutic agent.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Cycle; Cell Cycle Proteins; Cell Growth Processes; Cell Line, Tumor; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Female; HeLa Cells; Humans; Mitochondria; Ovarian Neoplasms; Phenol; Plant Extracts; Proto-Oncogene Proteins c-bcl-2; Rosaceae

Epithelial ovarian carcinoma (EOC) remains a highly lethal malignancy. Despite the progress in surgical and therapeutic strategies, resistance to chemotherapy is still a major concern. Cytotoxic therapies mediate killing of cancer cells by activating the intrinsic mitochondrial apoptotic pathway, and p53 status is a key factor in determining the efficacy of apoptotic signaling. The extrinsic (CD95) death receptor-dependent signaling pathway also contributes to the efficacy of cancer therapy. We previously showed that EOC are generally resistant to CD95-dependent apoptosis. In p53 wild-type EOC tumors, CD95-mediated apoptosis is impaired at the receptor level by the long form of cellular FLICE-inhibitory protein, whereas this mechanism does not account for resistance in tumors with mutated p53 (p53mu). In the present study, we examined both intrinsic and death receptor-dependent apoptotic signaling in p53mu OVCAR3 EOC cell line, showing that these cells are less susceptible to cisplatin treatment as compared with p53 wild-type EOC cells and also resist CD95-mediated apoptosis due to inefficient formation of the death-inducing signaling complex and weak mitochondrial signal amplification. However, pretreatment of OVCAR3 cells with clinically relevant cisplatin concentrations significantly improved receptor-dependent apoptotic signaling by up-modulating CD95 receptor expression and increasing death-inducing signaling complex formation efficiency. The synergy of cisplatin pretreatment and CD95 triggering in inducing cell death was also shown in p53mu tumor cells derived from ascitic fluid of advanced-stage EOC patients. These findings support the effectiveness of a combined therapeutic treatment able to sensitize cancer cells to apoptosis even when p53 is functionally inactivated.

Topics: Antineoplastic Agents; Apoptosis; Ascitic Fluid; Blotting, Western; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspases; Cell Line, Tumor; Cisplatin; Cystadenocarcinoma, Serous; Cytochromes c; Death Domain Receptor Signaling Adaptor Proteins; Drug Synergism; fas Receptor; Female; Humans; Immunoprecipitation; Membrane Microdomains; Membrane Potential, Mitochondrial; Mutation; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Poly(ADP-ribose) Polymerases; Receptors, Death Domain; Signal Transduction; Tumor Suppressor Protein p53

Cisplatin is an effective antitumor agent for the treatment of several carcinomas. However, the development of resistance to cisplatin represents a serious clinical problem. The effects of auranofin, a gold(I) compound clinically used as an antirheumatic agent, on cisplatin-sensitive (2008) and-resistant (C13*) cancer cells were studied. Auranofin is more effective than cisplatin in decreasing cell viability and its action is particularly marked in C13* cells, indicating that no cross-resistance occurs. Furthermore, auranofin is able to permeate C13* cells more efficiently than 2008 cells. Treatment with auranofin determines a consistent release of cytochrome c in both cell lines, while cisplatin is effective only in sensitive cells. Both auranofin and cisplatin induce apoptosis in 2008 cells, while in C13* cells only auranofin is effective. Apoptosis is accompanied by an increased production of hydrogen peroxide that, however, is inhibited by N-acetyl-L-cysteine. In resistant cells, H(2)O(2) production is counteracted by a large overexpression of thioredoxin reductase that constitutes the preferred target of the inhibitory action of auranofin. This specific effect of auranofin might rationalize its ability in overcoming cisplatin resistance in human ovarian cancer cells.

Topics: Acetylcysteine; Antineoplastic Agents; Antirheumatic Agents; Apoptosis; Auranofin; Cell Line, Tumor; Cisplatin; Cytochromes c; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Female; Humans; Mitochondria; Ovarian Neoplasms; Thioredoxin-Disulfide Reductase

Src tyrosine kinase has been found to be overexpressed and activated in a high proportion of ovarian cancers and ovarian cancer cell lines. Furthermore, Src activation is associated with activation of growth and survival signaling pathways. The present study was conducted in order to determine the effects of Src inhibition on ovarian cancer cell survival in response to chemotherapeutic agents. Inhibition of Src, either pharmacologically or through expression of a Src dominant-negative fusion construct, enhanced the cytotoxicity of two different classes of chemotherapeutics: paclitaxel and cisplatinum, in both mouse and human ovarian cancer cells. Interestingly, Src inhibition also restored sensitivity to drug-resistant ovarian cancer cells. The increased cytotoxicity in response to Src inhibition was associated with a large increase in processing and activation of caspase-3. The activation of caspase-3 seems to be independent of cytochrome c release and caspase-9 activation. The present study indicates that Src tyrosine kinase may provide an important target for small molecule inhibition in ovarian cancer.

Topics: Animals; Antineoplastic Agents, Phytogenic; Caspase 3; Caspase 9; Caspases; Cisplatin; Cytochromes c; Drug Resistance, Neoplasm; Enzyme Activation; Female; Humans; Mice; Ovarian Neoplasms; Paclitaxel; Protein Kinase Inhibitors; Pyrimidines; src-Family Kinases; Tumor Cells, Cultured

Topics: Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cytochromes c; Cytosol; Down-Regulation; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Ligands; Membrane Glycoproteins; Mitochondria; Ovarian Neoplasms; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

IFN-gamma has direct anti-proliferative effects on ovarian cancer cell lines and tumour cells isolated from ovarian cancer ascites. The aim of this study was to further elucidate the mechanisms involved. An IFN-gamma-mediated cell cycle blockade was detectable in synchronised cell populations. Apoptosis, which was caspase dependent, was also induced. When caspase activity was blocked, the anti-proliferative effect of IFN-gamma was only partially reduced indicating independent roles for both growth inhibition and apoptosis in its actions. We have demonstrated involvement of the intrinsic apoptotic pathway; IFN-gamma treatment resulted in mitochondrial membrane depolarisation, cytochrome c release into the cytosol and activation of caspase 9. Cytochrome c release was blocked by the presence of a general caspase inhibitor, suggesting a role for caspases upstream of the mitochondria. One candidate is caspase 8, which was also activated in cells treated with IFN-gamma. Levels of Bid, a pro-apoptotic molecule that can mediate mitochondrial membrane permeabilisation when cleaved by caspase 8, were also decreased and indicated a potential link between these two pathways in IFN-gamma-induced apoptosis. Furthermore, together with cisplatin, IFN-gamma exerted a more powerful anti-proliferative effect.

Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Caspase Inhibitors; Cell Line, Tumor; Cell Proliferation; Cisplatin; Cytochromes c; Female; Humans; Interferon-gamma; Membrane Potentials; Mitochondria; Ovarian Neoplasms; Poly(ADP-ribose) Polymerases; Recombinant Proteins

We are currently conducting clinical trials of E1A gene therapy for patients with ovarian cancer. The adenovirus type 5 E1A gene suppresses growth of ovarian cancer cells that overexpress HER-2/neu (HER2) and growth of some--but not all--that express low HER2. In HER2-overexpressing cells, suppression by E1A is predominantly by down-regulation of HER2, but the mechanism in low HER2-expressing cells is not fully understood. The adenoviral E1B protein has sequential and functional homology to Bcl-2 and prolongs the viability of adenovirus host cells by inhibiting E1A-induced apoptosis. Bcl-2 is overexpressed in ovarian cancer and participates in chemoresistance; we hypothesized that Bcl-2 inhibits E1A-induced apoptosis leading to resistance to E1A gene therapy. E1A suppressed colony formation of ovarian cancer cells that express low levels of Bcl-2 and HER2 (OVCAR-3 and OVCA 433), but enhanced colony formation in low HER2-, high Bcl-2-expressing ovarian cancer cells (2774 and HEY). Treating 2774 or HEY cells with antisense oligonucleotide Bcl-2 (Bcl-2-ASO) did not reduce cell viability. E1A combined with Bcl-2-ASO led to significant decreases in cell viability resulting from increased apoptosis relative to cells treated with E1A alone (P < 0.05). The increase in apoptosis was partly due to cytochrome c release and subsequently caspase-9 activation by Bcl-2-ASO. Finally, in an ovarian cancer xenograft model, treatment with Bcl-2-ASO did not prolong survival, but E1A plus Bcl-2-ASO did (P < 0.001). In conclusion, ovarian tumors overexpressing Bcl-2 may not respond well to E1A gene therapy, but treatment with a combination of E1A and Bcl-2-ASO may overcome this resistance.

Topics: Adenovirus E1B Proteins; Animals; Apoptosis; Caspase 9; Caspases; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Female; Genetic Therapy; Humans; Mice; Mice, Nude; Oligonucleotides, Antisense; Ovarian Neoplasms; Receptor, ErbB-2; Thionucleotides; Transfection; Xenograft Model Antitumor Assays

Ovarian carcinoma is a highly lethal malignancy that often becomes resistant to chemotherapy. Alterations in apoptotic signals and p53 status contribute to drug resistance, and CD95-mediated apoptosis is also deficient in resistant cells. We analyzed the mechanism of resistance to CD95-mediated apoptosis in ovarian carcinoma cell lines differing in p53 status.. CD95-mediated apoptosis was induced by agonistic anti-CD95 antibody, and the apoptotic cascade was monitored with biochemical and functional assays.. CD95-mediated apoptosis was blocked in human ovarian cancer cells. In cell lines with wild-type p53, treatment with the protein synthesis inhibitor cycloheximide (CHX) together with anti-CD95 overcame the resistance, suggesting the presence of a labile inhibiting protein. Indeed, the labile protein cellular FLICE-inhibitory protein long form (c-FLIP(L)) was found to block caspase-8 recruitment to the death-inducing signaling complex (DISC), and sensitization of cells by CHX was due to c-FLIP(L) down-modulation at the DISC level. Down-regulation of c-FLIP(L) with antisense oligonucleotides increased CD95-mediated apoptosis as in cells sensitized by CHX, demonstrating the direct involvement of c-FLIP(L) in apoptosis resistance. Removal of c-FLIP(L) block at DISC level allowed full activation of the mitochondrial pathway and, eventually, apoptosis in wild-type p53 cells, whereas in cells with mutated p53, c-FLIP(L) involvement in CD95-mediated apoptosis resistance appeared to be irrelevant. Immunohistochemical analysis of an ovarian tumor tissue array revealed c-FLIP(L) expression in samples with no p53 accumulation (P = 0.034), and a significant (P = 0.037) inverse relationship between c-FLIP(L) and p53 expression levels was also observed in 27 epithelial ovarian cancer specimens with known p53 status.. The inhibitory protein c-FLIP(L) is involved in resistance to CD95-mediated apoptosis in ovarian carcinoma cells with wild-type p53.

Topics: Apoptosis; Blotting, Western; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 8; Caspases; Cell Death; Cell Line, Tumor; Cycloheximide; Cytochromes c; DNA Fragmentation; Down-Regulation; Drug Resistance; Enzyme Activation; fas Receptor; Female; Flow Cytometry; Humans; Immunohistochemistry; Immunoprecipitation; Intracellular Signaling Peptides and Proteins; Lymphocytes; Mitochondria; Oligonucleotides, Antisense; Ovarian Neoplasms; Protein Synthesis Inhibitors; Subcellular Fractions; Time Factors; Transfection; Tumor Suppressor Protein p53