title: Proteasome inhibitor MG132 sensitizes HPV-positive human cervical cancer cells to rhTRAIL-induced apoptosis.
abstract: 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.
authors.affiliation: Department of Gynecological Oncology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
authors.forename: Brigitte M T
authors.initials: BM
authors.lastname: Hougardy
authors.forename: John H
authors.initials: JH
authors.lastname: Maduro
authors.forename: Ate G J
authors.initials: AG
authors.lastname: van der Zee
authors.forename: Derk Jan A
authors.initials: DJ
authors.lastname: de Groot
authors.forename: Fiona A J
authors.initials: FA
authors.lastname: van den Heuvel
authors.forename: Elisabeth G E
authors.initials: EG
authors.lastname: de Vries
authors.forename: Steven
authors.initials: S
authors.lastname: de Jong
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000502:physiology*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000502:physiology*; D027383:Papillomaviridae / Q000472:pathogenicity; D030361:Papillomavirus Infections / Q000150:complications*; D061988:Proteasome Inhibitors; D034622:RNA Interference; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor / Q000502:physiology; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*; D016159:Tumor Suppressor Protein p53; D002583:Uterine Cervical Neoplasms / Q000473:pathology* / Q000821:virology
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D007976:Leupeptins; D008562:Membrane Glycoproteins; D061988:Proteasome Inhibitors; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C105190:TNFRSF10A protein, human; C505639:TNFRSF10B protein, human; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde