Blockade of inhibitors of apoptosis (IAPs) in combination with tumor-targeted delivery of tumor necrosis factor-? leads to synergistic antitumor activity.
ABSTRACT: In the current study, we examined whether the combination of tumor vasculature-targeted gene therapy with adeno-associated virus bacteriophage-tumor necrosis factor-? (AAVP-TNF-?) and/or the orally administered LCL161, an antagonist of inhibitors of apoptosis proteins (IAPs), enhanced antitumor efficacy without systemic toxicity. M21 human melanoma xenografts were grown subcutaneously in nude mice. Mice were treated according to one of four treatment regimens: AAVP-TNF-? alone (AAVP-TNF-? plus sodium acetate-acetic acid (NaAc) buffer) via tail vein injection; LCL161 alone (phosphate-buffered saline (PBS) plus LCL161) via oral gavage; AAVP-TNF-? plus LCL161; and PBS plus NaAc Buffer as a control group. Tumor volume, survival and toxicity were analyzed. AAVP trafficking and TNF-? production in vivo were detected on days 7 and 21 by real-time PCR, enzyme-linked immunosorbent assay and immunofluorescence. The levels of apoptosis and activation of caspases were assessed on days 7 and 21 by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) and immunofluorescence assays. Our results showed that the combination of AAVP-TNF-? and LCL161 significantly inhibited tumor growth and prolonged survival in mice with melanoma xenografts. The combination of AAVP-TNF-? and LCL161 was also significantly more effective than either agent alone, showing a synergistic effect without systemic toxicity.
Project description:We demonstrated sensitization for chemotherapy by Smac mimetic (SM) LCL161, a potent antagonist of inhibitor of apoptosis proteins (IAP), in neuroblastoma (NB). Vinca alkaloids, particularly vincristine (VCR), displayed the strongest impact on inhibition of proliferation and apoptosis induction in combination with LCL161. The underlying signaling pathways remain elusive, though. LCL161 induces a quick degradation of cellular IAP 1 (cIAP-1). Combination of LCL161 with VCR had only marginal effects on X-linked IAP (XIAP) protein expression. Cell death is accompanied by activation of intrinsic (caspase-9 and MMP) and extrinsic (caspase-8) pathways of apoptosis, repression of migratory potential and cell cycle arrest in G2 phase. LCL161-induced cIAP degradation leads to activation of non-canonical and blockade of canonical NF-?B pathways but not induction of apoptosis. Surprisingly NF-?B and TNF-? signaling is negligible for VCR- and VCR/LCL161-induced apoptosis since chemical inhibition of NF-?B using BAY-7085 and PBS-1086, as well as application of TNF-? blocking antibody Humira (adalimumab) has no relevant effect on cell death. Recently formation of a TNF-?-independent complex (ripoptosome) consisting of RIP1, FADD and caspase-8 following IAP inhibition by SM has been described. However, targeting of RIP1 by Necrostatin was not sufficient to influence apoptosis induced by VCR/LCL161.
Project description:Patients with inoperable or unresectable pancreatic neuroendocrine tumors (NETs) have limited treatment options. These rare human tumors often express somatostatin receptors (SSTRs) and thus are clinically responsive to certain relatively stable somatostatin analogs, such as octreotide. Unfortunately, however, this tumor response is generally short-lived. Here we designed a hybrid adeno-associated virus and phage (AAVP) vector displaying biologically active octreotide on the viral surface for ligand-directed delivery, cell internalization, and transduction of an apoptosis-promoting tumor necrosis factor (TNF) transgene specifically to NETs. These functional attributes of AAVP-TNF particles displaying the octreotide peptide motif (termed Oct-AAVP-TNF) were confirmed in vitro, in SSTR type 2-expressing NET cells, and in vivo using cohorts of pancreatic NET-bearing Men1 tumor-suppressor gene KO mice, a transgenic model of functioning (i.e., insulin-secreting) tumors that genetically and clinically recapitulates the human disease. Finally, preclinical imaging and therapeutic experiments with pancreatic NET-bearing mice demonstrated that Oct-AAVP-TNF lowered tumor metabolism and insulin secretion, reduced tumor size, and improved mouse survival. Taken together, these proof-of-concept results establish Oct-AAVP-TNF as a strong therapeutic candidate for patients with NETs of the pancreas. More broadly, the demonstration that a known, short, biologically active motif can direct tumor targeting and receptor-mediated internalization of AAVP particles may streamline the potential utility of myriad other short peptide motifs and provide a blueprint for therapeutic applications in a variety of cancers and perhaps many nonmalignant diseases as well.
Project description:Glioblastoma persists as a uniformly deadly diagnosis for patients and effective therapeutic options are gravely needed. Recently, targeted gene therapy approaches are reemerging as attractive experimental clinical agents. Our ligand-directed hybrid virus of adeno-associated virus and phage (AAVP) is a targeted gene delivery vector that has been used in several formulations displaying targeting ligand peptides to deliver clinically applicable transgenes. Here we compared different constructs side-by-side in a tumor model, an orthotopic model of xenograft human glioblastoma cells stereotactically implanted in immunodeficient mice. We have used divergent therapeutic strategies for two AAVP constructs, both displaying a double-cyclic RGD4C motif ligand specific for alpha V integrins expressed in tumor vascular endothelium, but carrying different genes of interest for the treatment of intracranial xenografted tumors. One construct delivered tumor necrosis factor (TNF), a purely cytotoxic gene for antitumor activity (RGD4C-AAVP-TNF); in the other construct, we delivered Herpes simplex virus thymidine kinase (HSVtk) for in tandem molecular-genetic imaging and targeted therapy (RGD4C-AAVP-HSVtk) utilizing ganciclovir (GCV) for a suicide gene therapy. Both AAVP constructs demonstrated antitumor activity, with damage to the tumor-associated neovasculature and induction of cell death evident after treatment. In addition, the ability to monitor transgene expression with a radiolabeled HSVtk substrate pre and post GCV treatment demonstrated the theranostic potential of RGD4C-AAVP-HSVtk. We conclude that targeted AAVP constructs delivering either cytotoxic TNF or theranostic HSVtk followed by suicide gene therapy with GCV have comparable preclinical efficacy, at least in this standard experimental model. The results presented here provide a blueprint for future studies of targeted gene delivery against human glioblastomas and other brain tumors.
Project description:Smac mimetic compounds (SMCs) are anti-cancer drugs that antagonize Inhibitor of Apoptosis proteins, which consequently sensitize cancer cells to death in the presence of proinflammatory ligands such as tumor necrosis factor alpha (TNF-?). SMCs synergize with the attenuated oncolytic vesicular stomatitis virus (VSV?51) by eliciting an innate immune response, which is dependent on the endogenous production of TNF-? and type I interferon. To improve on this SMC-mediated synergistic response, we generated TNF-?-armed VSV?51 to produce elevated levels of this death ligand. Due to ectopic expression of TNF-? from infected cells, a lower viral dose of TNF-?-armed VSV?51 combined with treatment of the SMC LCL161 was sufficient to improve the survival rate compared to LCL161 and unarmed VSV?51 co-therapy. This improved response is attributed to a bystander effect whereby the spread of TNF-? from infected cells leads to the death of uninfected cells in the presence of LCL161. In addition, the treatments induced vascular collapse in solid tumors with a concomitant increase of tumor cell death, revealing another mechanism by which cytokine-armed VSV?51 in combination with LCL161 can kill tumor cells. Our studies demonstrate the potential for cytokine-engineered oncolytic virus and SMCs as a new combination immunotherapy for cancer treatment.
Project description:Glutamine plus glutamate (Glx), as well as N-acetylaspartate compounds (NAAc, N-acetylaspartate plus N-acetyl-aspartyl-glutamate), a marker of neuronal viability, can be quantified with proton magnetic resonance spectroscopy (1H-MRS). We used 1H-MRS imaging to assess Glx and NAAc, as well as total-choline (glycerophospho-choline plus phospho-choline), myo-inositol and total-creatine (creatine plus phosphocreatine) from an axial supraventricular slab of gray matter (GM, medial-frontal and medial-parietal) and white matter (WM, bilateral-frontal and bilateral-parietal) voxels. Schizophrenia subjects (N = 104) and healthy controls (N = 97) with a broad age range (16 to 65) were studied. In schizophrenia, Glx was increased in GM (P < .001) and WM (P = .01), regardless of age. However, with greater age, NAAc increased in GM (P < .001) but decreased in WM (P < .001) in schizophrenia. In patients, total creatine decreased with age in WM (P < .001). Finally, overall cognitive score correlated positively with WM neurometabolites in controls but negatively in the schizophrenia group (NAAc, P < .001; and creatine [only younger], P < .001). We speculate the results support an ongoing process of increased glutamate metabolism in schizophrenia. Later in the illness, disease progression is suggested by increased cortical compaction without neuronal loss (elevated NAAc) and reduced axonal integrity (lower NAAc). Furthermore, this process is associated with fundamentally altered relationships between neurometabolite concentrations and cognitive function in schizophrenia.
Project description:The cellular inhibitors of apoptosis (cIAP) 1 and 2 are amplified in about 3% of cancers and have been identified in multiple malignancies as being potential therapeutic targets as a result of their role in the evasion of apoptosis. Consequently, small-molecule IAP antagonists, such as LCL161, have entered clinical trials for their ability to induce tumor necrosis factor (TNF)-mediated apoptosis of cancer cells. However, cIAP1 and cIAP2 are recurrently homozygously deleted in multiple myeloma (MM), resulting in constitutive activation of the noncanonical nuclear factor (NF)-?B pathway. To our surprise, we observed robust in vivo anti-myeloma activity of LCL161 in a transgenic myeloma mouse model and in patients with relapsed-refractory MM, where the addition of cyclophosphamide resulted in a median progression-free-survival of 10 months. This effect was not a result of direct induction of tumor cell death, but rather of upregulation of tumor-cell-autonomous type I interferon (IFN) signaling and a strong inflammatory response that resulted in the activation of macrophages and dendritic cells, leading to phagocytosis of tumor cells. Treatment of a MM mouse model with LCL161 established long-term anti-tumor protection and induced regression in a fraction of the mice. Notably, combination of LCL161 with the immune-checkpoint inhibitor anti-PD1 was curative in all of the treated mice.
Project description:Bacteriophage (phage), which are viruses that infect bacteria only, have shown promise as vehicles for targeted cancer gene therapy, albeit with poor efficiency. Recently, we generated an improved version of phage vectors by incorporating cis genetic elements of adeno-associated virus (AAV). This novel AAV/phage hybrid (AAVP) efficiently delivered systemically administered therapeutic genes to various tumor targets by displaying an integrin tumor-targeting ligand on the phage capsid. However, inherent limitations in bacteriophage mean that these AAVP vectors still need to be improved. One of the limitations of AAVP in mammalian cells may be its susceptibility to proteasomal degradation. The proteasome is upregulated in cancer and it is known that it constitutes a barrier to gene delivery by certain eukaryotic viruses. We report here that inhibition of proteasome improved targeted reporter gene delivery by AAVP in cancer cells in vitro and in tumors in vivo after intravenous vector administration to tumor-bearing mice. We also show enhanced targeted tumor cell killing by AAVP upon proteasome inhibition. The AAVP particles persisted significantly in cancer cells in vitro and in tumors in vivo after systemic administration, and accumulated polyubiquitinated coat proteins. Our results suggest that the proteasome is indeed a barrier to tumor targeting by AAVP and indicate that a combination of proteasome-inhibiting drugs and AAVP should be considered for clinical anticancer therapy.
Project description:Continuous treatment with organic nitrates causes nitrate tolerance and endothelial dysfunction, which is involved with protein kinase C (PKC) signal pathway and NADPH oxidase activation. We determined whether chronic administration with nitroglycerine compromises the protective effects of propofol against tumor necrosis factor (TNF-) induced toxicity in endothelial cells by PKC- ?2 dependent NADPH oxidase activation. Primary cultured human umbilical vein endothelial cells were either treated or untreated with TNF- ? (40 ng/mL) alone or in the presence of the specific PKC- ?2 inhibitor CGP53353 (1 ?M)), nitroglycerine (10 ?M), propofol (100 ?M), propofol plus nitroglycerin, or CGP53353 plus nitroglycerine, respectively, for 24 hours. TNF-? increased the levels of superoxide, Nox (nitrate and nitrite), malondialdehyde, and nitrotyrosine production, accompanied by increased protein expression of p-PKC-?2, gP91phox, and endothelial cell apoptosis, whereas all these changes were further enhanced by nitroglycerine. CGP53353 and propofol, respectively, reduced TNF-? induced oxidative stress and cell toxicity. CGP53353 completely prevented TNF- ? induced oxidative stress and cell toxicity in the presence or absence of nitroglycerine, while the protective effects of propofol were neutralized by nitroglycerine. It is concluded that nitroglycerine comprises the protective effects of propofol against TNF-? stimulation in endothelial cells, primarily through PKC-?2 dependent NADPH oxidase activation.
Project description:The previously developed adeno-associated virus/phage (AAVP) vector, a hybrid between M13 bacteriophage (phage) viruses that infect bacteria only and human Adeno-Associated Virus (AAV), is a promising tool in targeted gene therapy against cancer. AAVP can be administered systemically and made tissue specific through the use of ligand-directed targeting. Cancer cells and tumor-associated blood vessels overexpress the αν integrin receptors, which are involved in tumor angiogenesis and tumor invasion. AAVP is targeted to these integrins via a double cyclic RGD4C ligand displayed on the phage capsid. Nevertheless, there remain significant host-defense hurdles to the use of AAVP in targeted gene delivery and subsequently in gene therapy. We previously reported that histone deacetylation in cancer constitutes a barrier to AAVP. Herein, to improve AAVP-mediated gene delivery to cancer cells, we combined the vector with selective adjuvant chemicals that inhibit specific histone deacetylases (HDAC). We examined the effects of the HDAC inhibitor C1A that mainly targets HDAC6 and compared this to sodium butyrate, a pan-HDAC inhibitor with broad spectrum HDAC inhibition. We tested the effects on melanoma, known for HDAC6 up-regulation, and compared this side by side with a normal human kidney HEK293 cell line. Varying concentrations were tested to determine cytotoxic levels as well as effects on AAVP gene delivery. We report that the HDAC inhibitor C1A increased AAVP-mediated transgene expression by up to ~9-fold. These findings indicate that selective HDAC inhibition is a promising adjuvant treatment for increasing the therapeutic value of AAVP.
Project description:AIM:To investigate whether high-mobility group box 1 (HMGB1) Boxb exacerbates BALB/c mice corneal immune responses and inflammatory through the Toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)-dependent signaling pathway in Aspergillus fumigatus (A. fumigatus) keratitis. METHODS:The mice corneas were pretreated with phosphate buffer saline (PBS), Boxb before A. fumigatus infection. The abdominal cavity extracted macrophages were pretreated with PBS, Boxb, TLR4 inhibitor (CLI-095), Dimethyl sulfoxide (DMSO) separately before A. fumigatus hyphae stimulation. HMGB1 was detected in normal and infected mice corneas and macrophages by real-time reverse transcriptase polymerase chain reaction (RT-PCR), the TLR4, MyD88, interleukin-1? (IL-1?), tumor necrosis factor-? (TNF-?) were detected by Western blot and PCR. RESULTS:In BALB/c mice corneas, the expressions of TLR4, HMGB1, IL-1?, TNF-? were increased after A. fumigatus infection. While pretreatment with Boxb significantly increased the expressions of TLR4, HMGB1, MyD88, IL-1?, TNF-? compared with PBS control after infection. In BALB/c mice abdominal cavity extracted macrophages, pretreatment with Boxb increased the expressions of TLR4, HMGB1, MyD88, IL-1?, TNF-?, while pretreatment with CLI-095 and Boxb significantly decreased the expressions of TLR4, HMGB1, MyD88, IL-1?, TNF-?. CONCLUSION:In A. fumigatus keratitis, Boxb play a pro-inflammatory role in corneal anti-fungi immune response through the HMGB1-TLR4-MyD88 signal pathway.