ABSTRACT
The simultaneous delivery of multiple cancer drugs in combination therapies to achieve optimal therapeutic effects in patients can be challenging. This study investigated whether co-encapsulation of the BH3-mimetic ABT-737 and the topoisomerase I inhibitor camptothecin (CPT) in PEGylated polymeric nanoparticles (NPs) was a viable strategy for overcoming their clinical limitations and to deliver both compounds at optimal ratios. We found that thrombocytopenia induced by exposure to ABT-737 was diminished through its encapsulation in NPs. Similarly, CPT-associated leukopenia and gastrointestinal toxicity were reduced compared with the administration of free CPT. In addition to the reduction of dose-limiting side effects, the co-encapsulation of both anticancer compounds in a single NP produced synergistic induction of apoptosis in both in vitro and in vivo colorectal cancer models. This strategy may widen the therapeutic window of these and other drugs and may enhance the clinical efficacy of synergistic drug combinations.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Biphenyl Compounds/administration & dosage , Camptothecin/administration & dosage , Colorectal Neoplasms/drug therapy , Drug Compounding/methods , Nitrophenols/administration & dosage , Sulfonamides/administration & dosage , Animals , Antineoplastic Combined Chemotherapy Protocols/chemistry , Antineoplastic Combined Chemotherapy Protocols/toxicity , Apoptosis/drug effects , Biphenyl Compounds/chemistry , Biphenyl Compounds/toxicity , Camptothecin/chemistry , Camptothecin/toxicity , Cell Line, Tumor , Colorectal Neoplasms/physiopathology , Drug Synergism , Humans , Male , Mice , Mice, Inbred C57BL , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Nanoparticles/toxicity , Nitrophenols/chemistry , Nitrophenols/toxicity , Piperazines/administration & dosage , Piperazines/chemistry , Piperazines/toxicity , Sulfonamides/chemistry , Sulfonamides/toxicity , Thrombocytopenia/etiologyABSTRACT
Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages the DEDs of procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices, respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD's α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.
Subject(s)
CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Caspase 8/metabolism , Fas-Associated Death Domain Protein/metabolism , Blotting, Western , Chromatography, Gel , HCT116 Cells , Humans , Immunoprecipitation , Protein BindingABSTRACT
Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFα) signaling for their activity. As circulating TNFα expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFα. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFα promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA.
Subject(s)
Antineoplastic Agents/pharmacology , CASP8 and FADD-Like Apoptosis Regulating Protein/physiology , Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Caspase 3/metabolism , Caspase 8/metabolism , Cell Line, Tumor , Cisplatin/pharmacology , Down-Regulation/drug effects , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Drug Synergism , Enzyme Activation , Humans , Inhibitor of Apoptosis Proteins/metabolism , Mesothelioma , Molecular Mimicry , Pleural Neoplasms , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , VorinostatABSTRACT
FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-specific inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as efficient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as 'FLIP inhibitors'.