leupeptins and Ovarian-Neoplasms

Ovarian cancer is a disease with the highest mortality in gynecologic malignancies. Activation of STAT3 pathway is well known to be associated with tumor progression and metastasis in a number of cancers, including ovarian cancer. Therefore, STAT3 may be an ideal target for ovarian cancer treatment.. The present study aims to determine the antitumor activity of STAT3 inhibitor Napabucasin as a single agent or in combination with proteasome inhibitor MG-132 in ovarian cancer cells.. MTT was performed to determine the anti-proliferative effect of Napabucasin on ovarian cancer SKOV-3 cells. The involved anti-tumor mechanism was explored by flow cytometry, qRTPCR and western blot. MDC staining and tandem mRFP-GFP-LC3 fluorescence microscopy were used to analyze the autophagy-inducing capability of Napabucasin with or without MG-132. The combinational anticancer effect of Napabucasin and MG-132 was evaluated according to Chou and Talalay's method (1984).. Napabucasin showed obvious tumor-inhibitory effects against SKOV-3 cells. Treatment by Napabucasin arrested cell cycle progression in G2/M phase. Mechanistically, elevated expression of p21 may contribute to the blockade of the cell cycle. Moreover, we demonstrated that Napabucasin induced autophagy in SKOV-3 cells by using various assays, including MDC staining, autophagic flux examination, and detection of the autophagy markers. In addition, a combination of Napabucaisin with MG-132 exhibited a significant synergistic anti-proliferative effect, probably by inducing apoptosis through a mitochondria-dependent pathway. The two compounds induced pro-survival autophagies, and co-treatment with autophagy inhibiter might further enhance their antitumor effects.. Napabucasin alone or in combination with MG-132 might be promising treatment strategy for ovarian cancer patients.

Topics: Apoptosis; Benzofurans; Carcinoma, Ovarian Epithelial; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cysteine Proteinase Inhibitors; Drug Synergism; Female; G2 Phase Cell Cycle Checkpoints; Humans; Leupeptins; Naphthoquinones; Ovarian Neoplasms; Proteasome Endopeptidase Complex; Proteasome Inhibitors; STAT3 Transcription Factor

Tumour metastasis, the spread of cancer cells from the original tumour site followed by growth of secondary tumours at distant organs, is the primary cause of cancer-related deaths and remains poorly understood. Here we demonstrate that inhibition of CDK4/6 blocks breast tumour metastasis in the triple-negative breast cancer model, without affecting tumour growth. Mechanistically, we identify a deubiquitinase, DUB3, as a target of CDK4/6; CDK4/6-mediated activation of DUB3 is essential to deubiquitinate and stabilize SNAIL1, a key factor promoting epithelial-mesenchymal transition and breast cancer metastasis. Overall, our study establishes the CDK4/6-DUB3 axis as an important regulatory mechanism of breast cancer metastasis and provides a rationale for potential therapeutic interventions in the treatment of breast cancer metastasis.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Movement; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Disease Models, Animal; Endopeptidases; Female; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; Liver Neoplasms; Lung Neoplasms; MCF-7 Cells; Mice; Ovarian Neoplasms; Piperazines; Pyridines; RNA, Small Interfering; Signal Transduction; Snail Family Transcription Factors; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

Platinum-based combination chemotherapy after surgery is considered a standard treatment; therefore, any recent drug development should be new, effective, and low toxic, and should have a synergistic effect with platinum. This study aimed to observe the growth of SKOV3 cells after treatment with cisplatin by combining with carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132) and to investigate the effect of the relationship between MG132 and cisplatin combination.. Cell growth was detected by methyl thiazolyl tetrazolium assay after treatment with MG132 at 0.5, 1.5, 2.5, 3.5, and 5.0 μg/mL concentrations for 24, 48, and 72 hours; with cisplatin at 1.0, 2.0, 3.0, 4.0, and 5.0 μg/mL concentrations; and with combination with MG132 at 1.5 μg/mL for 24 hours. The apoptotic rates of cells were detected by a flow cytometer after cisplatin treatment at 1.0, 2.0, 3.0, and 4.0 μg/mL concentrations and that combined with MG132 at 1.5 μg/mL concentration for 12, 24, and 36 hours. A total of 20 BALB/c (nu/nu) female nude mice (age, 4-6 weeks; body weight, 17-19 g) were divided into 4 groups: control, MG132, cisplatin, and combination groups. The expression of Caspase3 and Beclin1 was detected by Western blot analysis and reverse transcription-polymerase chain reaction after treatment with 3.0 μg/mL of the cisplatin group and combined treatment with 1.5 μg/mL of MG132 group for 24 hours, respectively.. Methyl thiazolyl tetrazolium assay demonstrated the inhibitory rates, and the flow cytometery showed that the apoptotic rates in the combination group were higher than those in the cisplatin group (P < 0.01). Western blot analysis and reverse transcription-polymerase chain reaction detected that Caspase3 and Beclin1 at a relative quantity in the combination group were higher than those in the cisplatin group (P < 0.05).. MG132 has a synergistic antitumor effect by combining with cisplatin, and it is expected to be an effective antitumor drug for platinum-resistant refractory ovarian cancer.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Ovarian Epithelial; Cell Growth Processes; Cell Line, Tumor; Cisplatin; Cysteine Proteinase Inhibitors; Drug Synergism; Female; Humans; Leupeptins; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Glandular and Epithelial; Ovarian Neoplasms; Random Allocation; Xenograft Model Antitumor Assays

The antitumor activity of an inhibitor of 26S proteasome bortezomib (Velcade) has been observed in various malignancies, including colon cancer, prostate cancer, breast cancer, and ovarian cancer. Bortezomib has been proposed to stimulate autophagy, but scientific observations did not always support this. Interactions between ERK activity and autophagy are complex and not completely clear. Autophagy proteins have recently been shown to regulate the functions of ERK, and ERK activation has been found to induce autophagy. On the other hand, sustained activation of ERK has also been shown to inhibit the maturation step of the autophagy process. In this study, we sought to identify the mechanism of autophagy regulation in cancer cells treated with bortezomib. Our results indicate that bortezomib blocked the autophagic flux without inhibiting the fusion of the autophagosome and lysosome. In ovarian cancer, as well as endometrial cancer and hepatocellular carcinoma cells, bortezomib inhibited protein degradation in lysosomes by suppressing cathepsins, which requires the participation of ERK phosphorylation, but not JNK or p38. Our findings that ERK phosphorylation reduced cathepsins further explain how ERK phosphorylation inhibits the autophagic flux. In conclusion, bortezomib may induce ERK phosphorylation to suppress cathepsin B and inhibit the catalytic process of autophagy in ovarian cancer and other solid tumors. The inhibition of cisplatin-induced autophagy by bortezomib can enhance chemotherapy efficacy in ovarian cancer. As we also found that bortezomib blocks the autophagic flux in other cancers, the synergistic cytotoxic effect of bortezomib by abolishing chemotherapy-related autophagy may help us develop strategies of combination therapies for multiple cancers.

Topics: Antineoplastic Agents; Autophagy; Boronic Acids; Bortezomib; Cell Line, Tumor; Cell Survival; Cisplatin; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression Regulation, Neoplastic; Humans; Leupeptins; MAP Kinase Kinase 4; Ovarian Neoplasms; p38 Mitogen-Activated Protein Kinases; Phagosomes; Phosphorylation; Proteasome Inhibitors; Pyrazines; Signal Transduction

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is a central negative regulator of the PI3K/AKT signaling cascade and suppresses cell survival as well as cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. In the present study, we have investigated the regulation of PTEN during cisplatin induced apoptosis in A2780, A270-CP (cisplatin resistant), OVCAR-3 and SKOV3 ovarian cancer cell lines.. Cells were treated with 10μM of cisplatin for 24h. Transcript and protein levels were analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting, respectively. Immunofluorescence microscopy was used to assess the intracellular localization of PTEN. Proteasome inhibitor and various caspases inhibitors were used to find the mechanism of PTEN degradation.. PTEN protein levels were found to be decreased significantly in A2780 cells; however, there was no change in PTEN protein levels in A2780-CP, OVCAR-3 and SKOV3 cells with cisplatin treatment. The decrease in PTEN protein was accompanied with an increase in the levels of AKT phosphorylation (pAKT) in A2780 cells and a decrease of BCL-2. Cisplatin treatment induced the activation/cleavage of caspase-3, -6, -7, -8, -9 in all cell lines tested in this study except the resistant variant A2780-CP cells. In A2780 cells, restoration of PTEN levels was achieved upon pre-treatment with Z-DEVD-FMK (broad range caspases inhibitor) and not with MG132 (proteasome inhibitor) and by overexpression of BCL-2, suggesting that caspases and BCL-2 are involved in the decrease of PTEN protein levels in A2780 cells.. The decrease in pro-apoptotic PTEN protein levels and increase in survival factor pAKT in A2780 ovarian cancer cells suggest that cisplatin treatment could further exacerbate drug resistance in A2780 ovarian cancer cells.

Topics: Antineoplastic Agents; Apoptosis; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Enzyme Activation; Female; Humans; Leupeptins; Oligopeptides; Ovarian Neoplasms; Phosphorylation; Proteasome Inhibitors; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; PTEN Phosphohydrolase

In the present study we have explored the sensitivity of ovarian cancer cells to TRAIL and proteasome inhibitors. Particularly, we have explored the capacity of proteasome inhibitors to bypass TRAIL resistance of ovarian cancer cells. For these studies we have used the A2780 ovarian cancer cell line and its chemoresistant derivatives A2780/DDP and A2780/ADR, providing evidence that: (i) the three cell lines are either scarcely sensitive (A2780 and A2780/ADR) or moderately sensitive (A2780/DDP) to the cytotoxic effects of TRAIL; (ii) the elevated c-FLIP expression observed in ovarian cancer cells is a major determinant of TRAIL resistance of these cells; (iii) proteasome inhibitors (PS-341 or MG132) are able to exert a significant pro-apoptotic effect and to greatly enhance the sensitivity of both chemosensitive and chemoresistant A2780 cells to TRAIL; (iv) proteasome inhibitors damage mitochondria through stabilization of BH3-only proteins, Bax and caspase activation and significantly enhance TRAIL-R2 expression; (v) TRAIL-R2, but not TRAIL-R1, mediates the apoptotic effects of TRAIL on ovarian cancer cells. Importantly, studies on primary ovarian cancer cells have shown that these cells are completely resistant to TRAIL and proteasome inhibitors markedly enhance the sensitivity of these cells to TRAIL. Given the high susceptibility of ovarian cancer cells to proteasome inhibitors, our results further support the experimental use of these compounds in the treatment of ovarian cancer.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Boronic Acids; Bortezomib; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspases; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Female; Humans; Leupeptins; Membrane Proteins; Mitochondria; Ovarian Neoplasms; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrazines; Receptors, TNF-Related Apoptosis-Inducing Ligand; RNA, Small Interfering; TNF-Related Apoptosis-Inducing Ligand

Proteasome inhibitors sensitize tumor cells to DNA-damaging agents, including ionizing radiation (IR), and DNA cross-linking agents (melphalan and cisplatin) through unknown mechanisms. The Fanconi anemia pathway is a DNA damage-activated signaling pathway, which regulates cellular resistance to DNA cross-linking agents. Monoubiquitination and nuclear foci formation of FANCD2 are critical steps of the Fanconi anemia pathway. Here, we show that proteasome function is required for the activation of the Fanconi anemia pathway and for DNA damage signaling. Proteasome inhibitors (bortezomib and MG132) and depletion of 19S and 20S proteasome subunits (PSMD4, PSMD14, and PSMB3) inhibited monoubiquitination and/or nuclear foci formation of FANCD2, whereas depletion of DSS1/SHFM1, a subunit of the 19S proteasome that also directly binds to BRCA2, did not inhibit FANCD2 monoubiquitination or foci formation. On the other hand, DNA damage-signaling processes, such as IR-induced foci formation of phosphorylated ATM (phospho-ATM), 53BP1, NBS1, BRCA1, FANCD2, and RAD51, were delayed in the presence of proteasome inhibitors, whereas ATM autophosphorylation and nuclear foci formation of gammaH2AX, MDC1, and RPA were not inhibited. Furthermore, persistence of DNA damage and abrogation of the IR-induced G(1)-S checkpoint resulted from proteasome inhibition. In summary, we showed that the proteasome function is required for monoubiquitination of FANCD2, foci formation of 53BP1, phospho-ATM, NBS1, BRCA1, FANCD2, and RAD51. The dependence of specific DNA damage-signaling steps on the proteasome may explain the sensitization of tumor cells to DNA-damaging chemotherapeutic agents by proteasome inhibitors.

Topics: Ataxia Telangiectasia Mutated Proteins; Blotting, Western; Boronic Acids; Bortezomib; BRCA1 Protein; Cell Cycle Proteins; Cells, Cultured; DNA Damage; DNA Repair; DNA-Binding Proteins; Fanconi Anemia; Fanconi Anemia Complementation Group D2 Protein; Female; Flow Cytometry; Gamma Rays; HeLa Cells; Humans; Leupeptins; Microscopy, Fluorescence; Nuclear Proteins; Ovarian Neoplasms; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Serine-Threonine Kinases; Pyrazines; Rad51 Recombinase; RNA-Binding Proteins; RNA, Small Interfering; Signal Transduction; Tumor Suppressor Proteins; Ubiquitin

The ubiquitin-proteasome system (UPS) mediates targeted protein degradation. Notably, the UPS determines levels of key checkpoint proteins controlling apoptosis and proliferation by controlling protein half-life. Herein, we show that ovarian carcinoma manifests an overstressed UPS by comparison with normal tissues by accumulation of ubiquitinated proteins despite elevated proteasome levels. Elevated levels of total ubiquitinated proteins and 19S and 20S proteasome subunits are evident in both low-grade and high-grade ovarian carcinoma tissues relative to benign ovarian tumors and in ovarian carcinoma cell lines relative to immortalized surface epithelium. We find that ovarian carcinoma cell lines exhibit greater sensitivity to apoptosis in response to proteasome inhibitors than immortalized ovarian surface epithelial cells. This sensitivity correlates with increased cellular proliferation rate and UPS stress rather than absolute proteasome levels. Proteasomal inhibition in vitro induces cell cycle arrest and the accumulation of p21 and p27 and triggers apoptosis via activation of caspase-3. Furthermore, treatment with the licensed proteasome inhibitor PS-341 slows the growth of ES-2 ovarian carcinoma xenograft in immunodeficient mice. In sum, elevated proliferation and metabolic rate resulting from malignant transformation of the epithelium stresses the UPS and renders ovarian carcinoma more sensitive to apoptosis in response to proteasomal inhibition.

Topics: Animals; Apoptosis; Boronic Acids; Bortezomib; Caspases; Cell Division; Cell Line, Tumor; Female; G2 Phase; Humans; Leupeptins; Mice; Mice, Nude; Oligopeptides; Ovarian Neoplasms; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; Ubiquitin; Xenograft Model Antitumor Assays

The purpose of this study was to examine ovarian cancer cells for the expression of decorin, a proteoglycan component of the cell matrix that can inhibit cancer cell growth.. Cultured ovarian cancer cells and surgically excised tumors were examined by immunohistochemistry and Western blot analysis for decorin expression. Reverse transcription-polymerase chain reaction analysis was used to analyze cultured cells for decorin transcripts.. We detected decorin transcripts in two ovarian cancer cell lines by reverse transcription-polymerase chain reaction analysis. However, no decorin was found in conditioned culture medium from those cell lines. Cells treated with the proteasome inhibitor MG132 showed strong perinuclear staining with a decorin-specific monoclonal antibody by immunohistochemistry. Also, Western blot analysis showed the presence of a ladder of decorin-specific bands that were intensified by treatment with MG132, suggesting that de novo synthesized decorin was degraded by the ubiquitination pathway. The decorin component of tumor stroma was previously shown to contain high levels of chondroitin sulfate as opposed to dermatan sulfate side chains, and those molecules contained unusually high levels of O- and 6-sulfate linkages. We provided immunohistochemical evidence that these chondroitin sulfate side chains may have been produced by myofibroblasts.. Decorin protein expression was not detected in ovarian cancer cells. Decorin transcripts were produced and probably translated, but the protein was probably degraded by the ubiquitination pathway. We present evidence that stromal decorin of ovarian tumors was made by myofibroblasts. We also propose that decorin may be a tumor suppressor gene that is inactivated during epithelial cell development.

Topics: Actins; Antineoplastic Agents; Blotting, Western; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Decorin; DNA Primers; Endometriosis; Extracellular Matrix Proteins; Female; Humans; Immunoenzyme Techniques; Leupeptins; Multienzyme Complexes; Muscle, Smooth; Ovarian Neoplasms; Proteasome Endopeptidase Complex; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

The ubiquitin-proteasome pathway plays a critical role in the degradation of several proteins involved in the cell cycle. Dysregulation of this pathway leads to inhibition of cellular proliferation and the induction of apoptosis. Ubiquitination and its downstream consequences have been investigated intensively as targets for the development of drugs for tumour therapy. Here we have investigated the mechanism of apoptosis induced by the proteasome inhibitors MG-132, lactacystin and calpain inhibitor I (ALLN), in the HEK 293 cell line and the ovarian cancer cell lines SKOV3 and OVCAR3. We have found strong caspase-3-like and caspase-6-like activation upon treatment of HEK 293 cells with MG-132. Using a tricistronic expression vector based on a tetracycline-responsive system we generated stable SKOV3 nd OVCAR3 cell lines with inducible expression of pro-caspase-3. Induction of pro-caspase-3 expression in normally growing cells does not induce apoptosis. However, in the presence of the proteasome inhibitors MG-132, lactacystin or ALLN we found that cells overexpressing pro-caspase-3 are rapidly targeted for apoptosis. Our results demonstrate that pro-caspase-3 can sensitise ovarian cancer cells to proteasome inhibitor-induced apoptosis, and a combination of these approaches might be exploited for therapy of ovarian and other cancers.

Topics: Acetylcysteine; Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Cell Division; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Enzyme Induction; Female; Flow Cytometry; G2 Phase; Glycoproteins; Humans; Immunoblotting; Isoenzymes; Leupeptins; Ovarian Neoplasms; Transfection

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

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

We have investigated the adhesive properties and invasiveness of cells of the human ovarian carcinoma line, NIH:OVCAR-3, in vitro. OVCAR-3 cells exhibited a similar rate of adhesion to all substrates tested including laminin, fibronectin, and collagens I and IV. The synthetic peptide YIGSR-NH2, which corresponds to an attachment site in laminin, inhibited the adhesion of the cells to laminin, but not to fibronectin. In contrast, a GRGDS-NH2 peptide blocked adhesion to fibronectin but not to laminin. OVCAR-3 cells invaded and formed branched colonies on Matrigel. Colony formation was retarded by both YIGSR-NH2 and GRGDS-NH2 peptides. Serine protease inhibitors and human recombinant TIMP, the tissue inhibitor of metalloproteases, inhibited ovarian tumor cell invasion while a synthetic collagenase IV inhibitor (SC-44463) had no effect. These studies suggest that metalloproteases other than collagenase IV may be important for the invasive activity of ovarian cancer cells. It is possible that synthetic peptides with antiadhesive cellular activity and certain antiproteases could be used to control the progressive colonization and invasion of peritoneal surfaces by malignant ovarian cancer cells.

Topics: Amides; Amino Acid Sequence; Aminocaproates; Antineoplastic Agents; Benzamidines; Biocompatible Materials; Cell Adhesion; Cell Line; Chemotaxis; Collagen; Dose-Response Relationship, Drug; Drug Combinations; Female; Glycoproteins; Humans; In Vitro Techniques; Laminin; Leupeptins; Metalloendopeptidases; Molecular Sequence Data; Neoplasm Invasiveness; Oligopeptides; Ovarian Neoplasms; Pepstatins; Polyamines; Protease Inhibitors; Proteoglycans; Tissue Inhibitor of Metalloproteinases; Tumor Cells, Cultured; Tyrosine

Topics: Cell Line; Cholesterol; Chromatography, Gel; Female; Humans; Iodine Radioisotopes; Leupeptins; Lipoproteins, HDL; Ovarian Neoplasms; Trypsin Inhibitors; Uterine Neoplasms; Vaginal Neoplasms