leupeptins and Uterine-Cervical-Neoplasms

HDAC3 is involved in deacetylation of histone and non-histone proteins, having a key role in the regulation of gene transcription and also in the process of tumorgenesis. However, how HDAC3 is regulated in cancer remains largely unclear. Here, we showed that PIWIL2 can interact with HDAC3, leading to stabilization of HDAC3 from ubiquitin-mediated degradation by competitive association with E3 ubiquitin ligase Siah2. Furthermore, we found that expression of PIWIL2 enhanced HDAC3 activity via CK2α. PIWIL2 facilitated the interaction between HDAC3 and CK2α, thus exhibiting a promotion on the HDAC3 phosphorylation by CK2α. Further work showed that PIWIL2 could promote cell proliferation and suppress cell apoptosis via regulating HDAC3. Our present study firstly revealed that PIWIL2 can play a role in HDAC3-mediated epigenetic regulation on cancer cell proliferation and apoptosis. These findings provide a novel insight into the roles of PIWIL2 in tumorigenesis.

Topics: Antibodies; Apoptosis; Argonaute Proteins; Casein Kinase II; Cell Line, Tumor; Female; Histone Deacetylases; Humans; Leupeptins; Nuclear Proteins; Phosphorylation; Protein Binding; Proteolysis; RNA Interference; RNA, Small Interfering; Ubiquitin-Protein Ligases; Ubiquitination; Uterine Cervical Neoplasms

The E6 oncoprotein can interfere with the ability of infected cells to undergo programmed cell death through the proteolytic degradation of proapoptotic proteins such as p53, employing the proteasome pathway. Therefore, inactivation of the proteasome through MG132 should restore the activity of several proapoptotic proteins. We investigated whether in HPV16 E6-expressing keratinocytes (KE6 cells), the restoration of p53 levels mediated by MG132 and/or activation of the CD95 pathway through apoptosis antigen-1 (APO-1) antibody are responsible for the induction of apoptosis. We found that KE6 cells underwent apoptosis mainly after incubation for 24 h with MG132 alone or APO-1 plus MG132. Both treatments activated the extrinsic and intrinsic apoptosis pathways. Autophagy was also activated, principally by APO-1 plus MG132. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in the elevation of p53 protein levels and its phosphorylation in Ser46 and Ser20; the p53 protein was localized mainly at nucleus after treatment with MG132 or APO-1 plus MG132. In addition, induction of its transcriptional target genes such as p21, Bax and TP53INP was observed 3 and 6 h after treatment. Also, LC3 mRNA was induced after 3 and 6 h, which correlates with lipidation of LC3B protein and induction of autophagy. Finally, using pifithrin alpha we observed a decrease in apoptosis induced by MG132, and by APO-1 plus MG132, suggesting that restoration of APO-1 sensitivity occurs in part through an increase in both the levels and the activity of p53. The use of small molecules to inhibit the proteasome pathway might permit the activation of cell death, providing new opportunities for CC treatment.

Topics: Antibodies; Apoptosis; Autophagy; bcl-2-Associated X Protein; Benzothiazoles; Carrier Proteins; Cyclin-Dependent Kinase Inhibitor p21; fas Receptor; Female; Heat-Shock Proteins; Humans; Keratinocytes; Leupeptins; Oncogene Proteins, Viral; Proteasome Endopeptidase Complex; Proteolysis; Repressor Proteins; Toluene; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

Treatment of recurrent or advanced cervical cancer is still limited, and new therapeutic choices are needed for improving prognosis and quality of life of patients. Because human papilloma virus (HPV) infection is critical in cervical carcinogenesis, with the E6 and E7 oncogenes of HPV degrading tumor suppressor proteins through the ubiquitin proteasome system, the inhibition of the ubiquitin proteasome system appears to be an ideal target to suppress the growth of cervical tumors. Herein, we focused on the ubiquitin proteasome inhibitor MG132 (carbobenzoxy-Leu-Leu-leucinal) as an anticancer agent against cervical cancer cells, and physically incorporated it into micellar nanomedicines for achieving selective delivery to solid tumors and improving its in vivo efficacy. These MG132-loaded polymeric micelles (MG132/m) showed strong tumor inhibitory in vivo effect against HPV-positive tumors from HeLa and CaSki cells, and even in HPV-negative tumors from C33A cells. Repeated injection of MG132/m showed no significant toxicity to mice under analysis by weight change or histopathology. Moreover, the tumors treated with MG132/m showed higher levels of tumor suppressing proteins, hScrib and p53, as well as apoptotic degree, than tumors treated with free MG132. This enhanced efficacy of MG132/m was attributed to their prolonged circulation in the bloodstream, which allowed their gradual extravasation and penetration within the tumor tissue, as determined by intravital microscopy. These results support the use of MG132 incorporated into polymeric micelles as a safe and effective therapeutic strategy against cervical tumors.

Topics: Animals; Apoptosis; Cell Line, Tumor; Female; Leupeptins; Membrane Proteins; Mice; Micelles; Proteasome Inhibitors; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Uterine Cervical Neoplasms; Xenograft Model Antitumor Assays

Inaccessibility of drugs to poorly vascularized strata of tumor is one of the limiting factors in cancer therapy. With the advent of bystander effect (BE), it is possible to perpetuate the cellular damage from drug-exposed cells to the unexposed ones. However, the role of infiltrating tumor-associated macrophages (TAMs), an integral part of the tumor microenvironment, in further intensifying BE remains obscure. In the present study, we evaluated the effect of mitomycin C (MMC), a chemotherapeutic drug, to induce BE in cervical carcinoma. By using cervical cancer cells and differentiated macrophages, we demonstrate that MMC induces the expression of FasL via upregulation of PPARγ in both cell types (effector cells) in vitro, but it failed to induce bystander killing in cervical cancer cells. This effect was primarily owing to the proteasomal degradation of death receptors in the cervical cancer cells. Pre-treatment of cervical cancer cells with MG132, a proteasomal inhibitor, facilitates MMC-mediated bystander killing in co-culture and condition medium transfer experiments. In NOD/SCID mice bearing xenografted HeLa tumors administered with the combination of MMC and MG132, tumor progression was significantly reduced in comparison with those treated with either agent alone. FasL expression was increased in TAMs, and the enhanced level of Fas was observed in these tumor sections, thereby causing increased apoptosis. These findings suggest that restoration of death receptor-mediated apoptotic pathway in tumor cells with concomitant activation of TAMs could effectively restrict tumor growth.

Topics: Animals; Bystander Effect; Cell Proliferation; Fas Ligand Protein; Female; Gene Expression Regulation, Neoplastic; HeLa Cells; Humans; In Situ Nick-End Labeling; Leupeptins; Macrophages; Mice, Inbred NOD; Mice, SCID; Mitomycin; PPAR gamma; Tumor Microenvironment; Uterine Cervical Neoplasms

To investigate whether altered energy metabolism induces the Warburg effect and results in tumor malignancy, the respiratory enzyme citrate synthase (CS) was examined, silenced, and the effects analyzed. In human cervical carcinoma cells, RNAi-mediated CS knockdown induced morphological changes characteristic of the epithelial-mesenchymal transition (EMT). This switch accelerated cancer cell metastasis and proliferation in in vitro assays and in vivo tumor xenograft models. Notably, CS knockdown cells exhibited severe defects in respiratory activity and marked decreases in ATP production, but great increases in glycolytic metabolism. This malignant progression was due to activation of EMT-related regulators; altered energy metabolism resulted from deregulation of the p53/TIGAR and SCO2 pathways. This phenotypic change was completely reversed by p53 reactivation via treatment with proteasome inhibitor MG132 or co-knockdown of E3 ligase HDM2 and partially suppressed by ATP treatment. This study directly links the Warburg effect to tumor malignancy via induction of the EMT phenotype.

Topics: Adenosine Triphosphate; Animals; Apoptosis Regulatory Proteins; Carrier Proteins; Cell Line, Tumor; Citrate (si)-Synthase; Disease Progression; Energy Metabolism; Epithelial-Mesenchymal Transition; Female; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins; Mice; Mice, Inbred NOD; Mitochondrial Proteins; Molecular Chaperones; Phenotype; Phosphoric Monoester Hydrolases; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-mdm2; RNA Interference; RNA, Small Interfering; Signal Transduction; Transplantation, Heterologous; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

Successful biochemical studies of the natural products belactosin A and C as well as their more stable acylated derivatives have proved them to be powerful proteasome inhibitors and thereby potential candidates as pharmacologically relevant active compounds. In order to understand their structure-biological activity relations in detail and to find ways of improving their biological activity, four new modified belactosin congeners have been synthesized and tested. One of them (compound 6) turned out to be a more potent inhibitor against HeLa cells than the known proteasome inhibitor MG132.

Topics: Acylation; Animals; Antineoplastic Agents; Blotting, Western; Crystallography, X-Ray; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Female; Fibroblasts; HeLa Cells; Humans; Leupeptins; Mice; Mice, Transgenic; Models, Molecular; Molecular Structure; Peptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Stereoisomerism; Structure-Activity Relationship; Uterine Cervical Neoplasms

Human papillomavirus (HPV) infection is considered a necessary step for the development of cervical cancer, and >95% of all cervical cancers have detectable HPV sequences. The authors of this report recently demonstrated the efficacy of radioimmunotherapy (RIT) targeting viral oncoprotein E6 in the treatment of experimental cervical cancer. They hypothesized that the pretreatment of tumor cells with various agents that cause cell death and/or elevation of E6 levels would increase the accumulation of radiolabeled antibodies to E6 in cervical tumors.. HPV type 16 (HPV-16)-positive CasKi cells were treated in vitro with up to 6 grays of external radiation, or with the proteasome inhibitor MG-132, or with unlabeled anti-E6 antibody C1P5; and cell death was assessed. The biodistribution of (188)Re-labeled C1P5 antibody was determined in both control and radiation MG-132-treated CasKi tumor-bearing nude mice.. (188)Re-C1P5 antibody demonstrated tumor specificity, very low uptake, and fast clearance from the major organs. The amount of tumor uptake was enhanced by MG-132 but was unaffected by pretreatment with radiation. In addition, in vitro studies demonstrated an unanticipated effect of unlabeled antibody on the amount of cell death, a finding that was suggested by the authors' previous in vivo studies in a CasKi tumor model.. The current results indicated that pretreatment of cervical tumors with the proteasome inhibitor MG-132 and with unlabeled antibody to E6 can serve as a means to generate nonviable cancer cells and to elevate the levels of target oncoproteins in the cells for increasing the accumulation of targeted radiolabeled antibodies in tumors. These results favor the further development of RIT for cervical cancers targeting viral antigens.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Female; Humans; Leupeptins; Mice; Mice, Nude; Oncogene Proteins, Viral; Radioimmunotherapy; Radioisotopes; Repressor Proteins; Rhenium; Uterine Cervical Neoplasms

To investigate the effect of the ubiquitin-proteasome pathway inhibitor MG132 on the natural resistance to cisplatin in the human cervical cancer line (HCE1) multicellular spheroid (HCE1/MCS) model and to probe it if MG132 could reverse the HCE1/MCS resistance to cisplatin, as well as the possible mechanism of drug resistance.. (1) HCE1/MCS was obtained using liquid overlay and rotating technique. (2) Four groups were established (MG132 group, cisplatin group, MG132+cisplatin group, the control group). Cell viability were measured by trypan blue exclusion assay. Cell cycle and apoptosis were detected by flow cytometry. (3) The expression of nuclear factor (NF) kappaB of both HCE1 monolayer cells (HCE1/MC) and HCE1/MCS was detected by western blot, and the expression of B cell lymphoma/leukemia-2 (bcl-2) was detected by immunohistochemistry.. (1) HCE1/MCS was established successfully. (2) Cell inhibited rate of HCE1/MC and HCE1/MCS was: in MG132 group, (11.67+/-2.34)% vs (10.78+/-1.17)% (P>0.05); in MG132+cisplatin group, (92.67+/-2.52)% vs (91.33+/-2.18)% (P>0.05); in cisplatin group, (45.01+/-7.44)% vs (9.45+/-5.98)% (P<0.05). (3) The rate of apoptosis of HCE1/MC and HCE1/MCS were: in MG132 group, 8.14% and 5.97%; in MG132+cisplatin group, 99.01% and 95.22%; in cisplatin group, 33.61% and 0.88%. (4) The expression level of NF-kappaB and the high expression rate of bcl-2 were: in HCE1/MCS of control group, 0.67 and 60%; in HCE1/MCS of cisplatin group, 0.85 and 83%; in HCE1/MCS of MG132 group, 0.39 and 20%; in HCE1/MCS of MG132+cisplatin group, 0.47 and 33%.. (1) HCE1/MCS present natural resistance to cisplatin and may become a good model for the study of cervical cancer drug resistance in vitro. (2) MG132 could induce the inhibition and apoptosis of HCE1/MCS cells and partially reverse the natural resistance of HCE1/MCS to cisplatin, of which partially reverse the natural resistance may be in relation to the down-regulation of NF-kappaB and bcl-2 expression.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Survival; Cisplatin; Down-Regulation; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Leupeptins; NF-kappa B; Proto-Oncogene Proteins c-bcl-2; Spheroids, Cellular; Tumor Cells, Cultured; Uterine Cervical Neoplasms

HPV16 is associated with ~50% of all cervical cancers worldwide. The E6 and E7 genes of oncogenic HPV types, such as HPV16, are necessary for the HPV transforming function and tumorogenesis making them ideal targets for novel treatments. Radioimmunotherapy employs systemically administered radiolabeled monoclonal antibodies (mAbs) that bind to tumor-associated antigens. Previously we demonstrated in mice that radioimmunotherapy targeting viral antigens with mAb to HPV16 E6 suppressed CasKi cervical tumors expressing high levels of E6 (~600 copies of HPV per cell). However, that study opened the question whether radioimmunotherapy can suppress the growth of cervical tumors with low E6 and E7 expression, such as may be seen in patients.. We evaluated the expression of E6 in patients' tumors and in the SiHa cell line expressing low levels of E6 and E7 (1-2 copies of HPV per cell) and found them comparable. We initiated SiHa tumors in nude mice, radiolabeled C1P5 mAb to E6 with a beta-emitter 188-Rhenium (¹⁸⁸Re) and treated tumor-bearing mice with: (1) 200 μCi ¹⁸⁸Re-C1P5 alone; (2) proteasome inhibitor MG132 alone; (3) MG132 followed by 200 μCi ¹⁸⁸Re-C1P5; (4) unlabeled C1P5; (5) 200 μCi ¹⁸⁸Re-18B7 (isotype-matching control mAb); (6) no treatment. ¹⁸⁸Re-C1P5 alone and in combination with MG-132 significantly retarded tumor growth compared to all control groups.. Our data demonstrate the possibility to suppress tumor growth by targeting viral antigens even in cervical tumors with low E6 expression and provide additional evidence for the potential usefulness of radioimmunotherapy targeting HPV-related antigens in the clinic.

Topics: Animals; Antibodies, Monoclonal; Blotting, Western; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Female; Humans; Leupeptins; Mice; Mice, Nude; Neoplasms, Experimental; Oncogene Proteins, Viral; Radioimmunotherapy; Repressor Proteins; Uterine Cervical Neoplasms

MG132 (carbobenzoxy-Leu-Leu-leucinal) is a peptide aldehyde, which effectively blocks the proteolytic activity of the 26S proteasome complex. We evaluated the effects of MG132 on the growth of human cervix cancer HeLa cells in relation to the cell growth, reactive oxygen species (ROS) and glutathione (GSH) levels. Dose-dependent inhibition of cell growth was observed in HeLa cells with an IC50 of approximately 5 microM MG132 for 24 h. DNA flow cytometric analysis indicated that treatment with MG132 induced S, G2-M or non-specific phase arrests of the cell cycle dose-dependently. Treatment with MG132 induced apoptosis in a dose-dependent manner, as evidenced by sub-G1 cells and annexin V staining cells. Treatment with MG132 also induced the loss of mitochondrial membrane potential in HeLa cells. The intracellular ROS levels including O2*- were significantly increased in MG132-treated cells. Furthermore, the depletion of intracellular GSH content was observed in cells treated with MG132. In conclusion, MG132 inhibited the growth of HeLa cells via inducing the cell cycle arrest as well as triggering apoptosis. The changes of ROS and GSH by MG132 were closely related to apoptosis in HeLa cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Proliferation; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Female; Glutathione; HeLa Cells; Humans; Inhibitory Concentration 50; Leupeptins; Membrane Potential, Mitochondrial; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Reactive Oxygen Species; Uterine Cervical Neoplasms

Development of medical therapies for high-grade cervical intraepithelial neoplasia (CIN II/III) is hampered by the lack of CIN II/III cell lines. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis upon binding to its receptors DR4 or DR5. Proteasome inhibition by MG132 sensitized cervical cancer cell lines to recombinant human (rh)TRAIL. In our study, we aimed to develop an ex vivo model for CIN II/III and to investigate the apoptosis-inducing effect of rhTRAIL and/or MG132 in cervical explants from CIN II/III patients. A short-term ex vivo culture system was optimized for cervical biopsies, in which explants from normal cervix and CIN II/III lesions were exposed to either rhTRAIL (1 microg/ml), MG132 (5 microM) or the combination and compared to untreated explants for apoptosis induction. Normal cervix (n = 90) and CIN II/III (n = 24) explants could be reproducibly put in culture and kept viable for up to 7 days using a transwell membrane system. CIN II/III explants (n = 5) were highly sensitive to rhTRAIL plus MG132 (mean % apoptosis: 91 +/- 5) compared to normal cervix (n = 10) treated with rhTRAIL plus MG132 (mean % apoptosis: 24 +/- 10, p < 0.0001), while monotherapy with either rhTRAIL, MG132 or medium resulted in a mean % apoptosis <10 in both CIN II/III and normal cervix. Our ex vivo model system allows preclinical evaluation of (topical) medical therapies for CIN II/III. A strong synergistic apoptosis-inducing effect of the combination of rhTRAIL and MG132, especially in CIN II/III lesions indicates that rhTRAIL combined with proteasome inhibitors deserves exploration as medical treatment for CIN II/III.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Culture Techniques; Cells, Cultured; Female; Humans; Immunohistochemistry; Leupeptins; Precancerous Conditions; Recombinant Proteins; TNF-Related Apoptosis-Inducing Ligand; Uterine Cervical Dysplasia; Uterine Cervical Neoplasms

Sulindac, a nonsteroidal anti-inflammatory drug (NSAID), induces growth arrest in HeLa cells and causes strong inhibition of the G1 to S transition of the cell cycle in a concentration-dependent manner. The G1 arrest is preceded by suppression of cyclin E and A, inactivation of cdk2, and the complete loss of the viral oncoprotein E7, despite ongoing HPV transcription. As shown by inhibitors specific for cyclooxygenase (COX) 1 and 2 loss of E7 is COX-independent. Moreover, inhibition of the proteasome activity with MG132 partially blocked the ability of sulindac to suppress E7 suggesting that sulindac induces degradation of E7 by the proteasomal pathway. In addition to inhibiting growth, sulindac strongly induces apoptosis, which can be abrogated by using the general caspase inhibitor zVAD-fmk. Unchanged expression of the pro-apoptotic protein Bax and suppression of the anti-apoptotic molecules Bcl-2 and Bcl-x(L) argues for the engagement of the mitochondrial apoptotic pathway. These results support the notion that sulindac is a potent growth inhibitor and inducer of apoptosis on cervical cancer cells in vitro and may offer new perspectives as a chemopreventive or supplementary anti-cervical cancer drug.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Blotting, Northern; Blotting, Western; Cell Cycle; Cyclins; Cyclooxygenase 2; Cyclooxygenase Inhibitors; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Dose-Response Relationship, Drug; Female; G1 Phase; Gene Expression Regulation, Viral; HeLa Cells; Humans; Leupeptins; Meloxicam; Oncogene Proteins, Viral; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proto-Oncogene Proteins c-bcl-2; Pyrazoles; S Phase; Sulfonamides; Sulindac; Thiazines; Thiazoles; Uterine Cervical Neoplasms

Experimental data suggest therapeutic advantage from selective disruption of the hypoxia response. We recently found that the proteasome inhibitor bortezomib decreases tumor carbonic anhydrase IX (CAIX) expression in colon cancer patients and herein report a companion laboratory study to test if this effect was the result of hypoxia-inducible factor (HIF) inhibition. Human cervical (SiHa and Me180) and colon (RKO) carcinoma cell lines were treated with bortezomib or the structurally unrelated proteasome inhibitor MG132 in normoxic and hypoxic conditions in vitro. Two different in vivo experiments investigated bortezomib effects after single dose (2 mg/kg, 24 h) or longer exposure in severe combined immunodeficient mice bearing SiHa xenografts. Treatment with either drug produced accumulation of HIF-1alpha in vitro but strongly inhibited the production of CAIX and vascular endothelial growth factor (VEGF) under hypoxia. This correlated with more than 10-fold reduction in HIF-1 transcriptional activity under hypoxic conditions. A similar effect of bortezomib was seen in vivo, using the nitroimidazole probe EF5 to define regions of tumor hypoxia and a triple immunofluorescence technique to measure the spatial distributions of HIF-1alpha and CAIX. Plasma VEGF levels decreased by approximately 90% during treatment with bortezomib, indicating that this agent can potently inhibit the hypoxia response in tumors.

Topics: Antigens, Neoplasm; Antineoplastic Agents; Boronic Acids; Bortezomib; Carbonic Anhydrase IX; Carbonic Anhydrases; Carcinoma, Squamous Cell; Caspase 3; Cell Hypoxia; Cell Line, Tumor; Cell Nucleus; Colonic Neoplasms; E1A-Associated p300 Protein; Enzyme Activation; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Male; Prostatic Neoplasms; Protease Inhibitors; Protein Binding; Pyrazines; Uterine Cervical Neoplasms; Vascular Endothelial Growth Factor A; Xenograft Model Antitumor Assays

In cervical carcinogenesis, the p53 tumor suppressor pathway is disrupted by HPV (human papilloma virus) E6 oncogene expression. E6 targets p53 for rapid proteasome-mediated degradation. We therefore investigated whether proteasome inhibition by MG132 could restore wild-type p53 levels and sensitize HPV-positive cervical cancer cell lines to apoptotic stimuli such as rhTRAIL (recombinant human TNF-related apoptosis inducing ligand). In a panel of cervical cancer cell lines, CaSki was highly, HeLa intermediate and SiHa not sensitive to rhTRAIL-induced apoptosis. MG132 strongly sensitized HeLa and SiHa to rhTRAIL-induced apoptosis in a caspase-dependent and time-dependent manner. MG132 massively induced TRAIL receptor DR4 and DR5 membrane expression in HeLa, whereas in SiHa only DR5 membrane expression was upregulated from almost undetectable to high levels. Antagonistic DR4 antibody partially inhibited apoptosis induction by rhTRAIL and MG132 in HeLa but had no effect on apoptosis in SiHa. Inhibition of E6-mediated p53 proteasomal degradation by MG132 resulted in elevated levels of active p53 as demonstrated by p53 small interfering RNA (siRNA) sensitive p21 upregulation. Although p53 siRNA partially inhibited MG132-induced DR5 upregulation in HeLa and SiHa, no effect on rhTRAIL-induced apoptosis was observed. MG132 plus rhTRAIL enhanced caspase 8 and caspase 3 activation and concomitant cleavage of X-linked inhibitor of apoptosis (XIAP), particularly in HeLa. In addition, caspase 9 activation was only observed in HeLa. Downregulation of XIAP using siRNA in combination with rhTRAIL induced high levels of apoptosis in HeLa, whereas MG132 had to be added to the combination of XIAP siRNA plus rhTRAIL to induce apoptosis in SiHa. In conclusion, proteasome inhibition sensitized HPV-positive cervical cancer cell lines to rhTRAIL independent of p53. Our results indicate that not only DR4 and DR5 upregulation but also XIAP inactivation contribute to rhTRAIL sensitization by MG132 in cervical cancer cell lines. Combining proteasome inhibitors with rhTRAIL may be therapeutically useful in cervical cancer treatment.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Female; HeLa Cells; Humans; Leupeptins; Membrane Glycoproteins; Papillomaviridae; Papillomavirus Infections; Proteasome Inhibitors; Receptors, TNF-Related Apoptosis-Inducing Ligand; Receptors, Tumor Necrosis Factor; Recombinant Proteins; RNA Interference; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms

Transient hypoxia and subsequent reoxygenation are common phenomena in solid tumors that greatly influence the outcome of radiation therapy. This study was designed to determine how varying cycles of hypoxia/reoxygenation affect the response of cervical carcinoma cells irradiated under oxic and hypoxic conditions and whether this could be modulated by proteasome inhibition.. Plateau-phase SiHa cervical carcinoma cells in culture were exposed to varying numbers of 30-minute cycles of hypoxia/reoxygenation directly before irradiation under oxic or hypoxic conditions. 26S Proteasome activity was blocked by addition of MG-132. Clonogenic survival was measured by a colony-forming assay.. Under oxic conditions, repeated cycles of hypoxia/reoxygenation decreased the clonogenic survival of SiHa cells. This effect was even more pronounced after the inhibition of 26S proteasome complex. In contrast, under hypoxic conditions, SiHa cells were radioresistant, as expected, but this was increased by proteasome inhibition.. Proteasome inhibition radiosensitizes oxygenated tumor cells but may also protect tumor cells from ionizing radiation under certain hypoxic conditions.

Topics: Carcinoma; Cell Hypoxia; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Cytoprotection; Dose-Response Relationship, Radiation; Female; Humans; Leupeptins; Oxygen; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Radiation, Ionizing; Uterine Cervical Neoplasms

Tumor necrosis factor alpha (TNFalpha) induces apoptosis of a variety of tumor cell types. The anti-tumor effect of TNFalpha is often augmented by interferon (IFN) gamma. We hypothesized that IFNalpha, which shares many biological activities with IFNgamma, might also synergize with TNFalpha for the induction of tumor cell death. We tested our hypothesis using ME-180 human cervical cancer cells exposed to either IFNalpha or TNFalpha alone or both. We analyzed the death of ME-180 cells by biochemical and cytological means, and investigated the molecular mechanism underlying cytotoxic synergism between the two cytokines. We found that (i) IFNalpha/TNFalpha synergistically induced apoptosis of ME-180 cells, which was accompanied by activation of caspases-3 and -8; (ii) IFNalpha induced signal transducer and activator of transcription (STAT) 1 phosphorylation, and transfection of phosphorylation-defective STAT1 dominant-negative mutant inhibited IFNalpha/TNFalpha-induced apoptosis; (iii) inhibition of nuclear factor kappaB (NF-kappaB) by proteasome inhibitor MG-132 sensitized ME-180 cells to TNFalpha alone; (iv) IFNalpha treatment attenuated TNFalpha-induced NF-kappaB reporter activity, while it did not inhibit DNA binding of NF-kappaB. Taken collectively, our results indicate that IFNalpha sensitizes ME-180 cells to TNFalpha-induced apoptosis by inhibiting TNFalpha-mediated cytoprotective NF-kappaB activation, and this sensitizing effect of IFNalpha is mediated through a STAT1-dependent pathway.

Topics: Apoptosis; Caspase 3; Caspase 8; Caspase 9; Caspases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytoprotection; DNA-Binding Proteins; Drug Synergism; Female; Humans; Interferon-alpha; Leupeptins; Multienzyme Complexes; NF-kappa B; Proteasome Endopeptidase Complex; Signal Transduction; STAT1 Transcription Factor; Trans-Activators; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Uterine Cervical Neoplasms

High-risk human papillomaviruses (HPVs) are etiologically linked to human cervical and oral cancers. The E6 and E7 oncoproteins encoded by HPV target host cell tumor suppressor proteins. E6 induces proteolysis of p53 through the ubiquitin-proteasome pathway. Recent studies showed that overexpression of E7 caused proteolytic degradation of the tumor suppressor Rb. However, unlike p53, Rb is not regulated by proteolysis in normal cells. In addition, it was unclear whether in its natural context E7 regulates Rb through the ubiquitin-proteasome pathway. Therefore, we sought to determine whether Rb is regulated by the ubiquitin-proteasome pathway in HPV-containing tumor cells. We carried out a detailed analysis in Caski cells, that are derived from HPV-containing cervical cancer tissues. Studies with various protease inhibitors revealed that Rb is regulated specifically by the ubiquitin-proteasome pathway in HPV-containing cervical tumor cells. Several inhibitors of the 26S proteasome significantly increased the level of Rb in the Caski cells. Rb controls cell growth by forming complexes with the E2F-family transcription factors. Surprisingly, in spite of a significant accumulation of the hypophosphorylated form of Rb, no Rb/E2F complex was detectable in the proteasome inhibitor treated cells. Further analysis revealed that there was an increased accumulation of the E7 oncoprotein. We showed that the proteasome inhibitors simultaneously blocked the proteolysis of E7 and Rb, suggesting that E7 is also regulated by the ubiquitin-dependent proteolysis in cervical cancer cells. Taken together, this study suggests that targeted inhibition of Rb proteolysis will be required for restoring Rb function in HPV-containing cervical cancer cells.

Topics: Carcinoma; Cell Cycle Proteins; Cell Line, Transformed; Cell Transformation, Viral; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; E2F Transcription Factors; Female; Humans; Leupeptins; Multienzyme Complexes; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus E7 Proteins; Phosphorylation; Proteasome Endopeptidase Complex; Retinoblastoma Protein; Transcription Factors; Ubiquitins; Uterine Cervical Neoplasms

The E6 proteins derived from tumour associated papillomavirus types target the cellular tumour suppressor protein p53 for ubiquitin mediated degradation. In cell lines derived from cervical tumours the p53 protein is present in very low amounts, but it can be activated by appropriate DNA damaging agents, indicating that functional p53 is present within these lines. Recent studies have also shown that different polymorphic forms of the p53 protein are differentially susceptible to E6 mediated degradation. Therefore we have been interested in analysing the effects of different HPV E6 proteins upon p53 levels in a variety of cervical tumour derived cell lines. We show that inhibition of E6 mediated degradation of p53 frequently results in increased levels of p53 expression. However, there are notable exceptions to this where increased p53 levels are only obtained following DNA damage and proteasome inhibition. We also show in E6 expressing cells, that as well as p53 being targeted for degradation, the localization of p53 to the nucleus is also inhibited, consistent with previous observations which indicate that degradation of p53 is not essential for E6 mediated inhibition of p53 function. These results have important implications for any potential therapies which might aim to block E6 mediated degradation of p53.

Topics: Acetylcysteine; Adenocarcinoma; Animals; Antibiotics, Antineoplastic; Breast Neoplasms; Carcinoma; Cell Nucleus; Cysteine Proteinase Inhibitors; DNA Damage; DNA-Binding Proteins; Female; Fibrosarcoma; Humans; Leupeptins; Mitomycin; Oncogene Proteins, Viral; Papillomaviridae; Polymorphism, Genetic; Repressor Proteins; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Uterine Cervical Neoplasms