RESUMO
BACKGROUND: Flowers which imitate insect oviposition sites probably represent the most widespread form of floral mimicry, exhibit the most diverse floral signals and are visited by two of the most speciose and advanced taxa of insect - beetles and flies. Detailed comparative studies on brood-site mimics pollinated exclusively by each of these insect orders are lacking, limiting our understanding of floral trait adaptation to different pollinator groups in these deceptive systems. METHODS: Two closely related and apparent brood-site mimics, Typhonium angustilobum and T. wilbertii (Araceae) observed to trap these distinct beetle and fly pollinator groups were used to investigate potential divergence in floral signals and traits most likely to occur under pollinator-mediated selection. Trapped pollinators were identified and their relative abundances enumerated, and thermogenic, visual and chemical signals and morphological traits were examined using thermocouples and quantitative reverse transcription-PCR, reflectance, gas chromatography-mass spectrometry, floral measurements and microscopy. KEY RESULTS: Typhonium angustilobum and T. wilbertii were functionally specialized to trap saprophagous Coleoptera and Diptera, respectively. Both species shared similar colour and thermogenic traits, and contained two highly homologous AOX genes (AOX1a and AOX1b) most expressed in the thermogenic tissue and stage (unlike pUCP). Scent during the pistillate stage differed markedly - T. angustilobum emitted a complex blend of sesquiterpenes, and T. wilbertii, a dung mimic, emitted high relative amounts of skatole, p-cresol and irregular terpenes. The species differed significantly in floral morphology related to trapping mechanisms. CONCLUSIONS: Functional specialization and pollinator divergence were not associated with differences in anthesis rhythm and floral thermogenic or visual signals between species, but with significant differences in floral scent and morphological features, suggesting that these floral traits are critical for the attraction and filtering of beetle or fly pollinators in these two brood-site mimics.
Assuntos
Araceae , Odorantes , Animais , Flores , Insetos , PolinizaçãoRESUMO
Physachenolide C (1) is a 17ß-hydroxywithanolide natural product with a unique anticancer potential, as it exhibits potent and selective in vitro antiproliferative activity against prostate cancer (PC) cells and promotes TRAIL-induced apoptosis of renal carcinoma (RC) and poly I:C-induced apoptosis of melanoma cells. To explore the effect of ring A/B modifications of physachenolide C (1) on these biological activities, 23 of its natural and semisynthetic analogues were evaluated. Analogues 4-23 were prepared by chemical transformations of a readily accessible compound, physachenolide D (2). Compound 1 and its analogues 2-23 were evaluated for their antiproliferative activity against PC (LNCaP and 22Rv1), RC (ACHN), and melanoma (M14 and SK-MEL-28) cell lines and normal human foreskin fibroblast (HFF) cells. Most of the active analogues had selective and potent activity in reducing cell number for PC cell lines, some showing selectivity for androgen-independent and enzalutamide-resistant 22Rv1 cells compared to androgen-dependent LNCaP cells. Analogues with IC50s below 5.0 µM against ACHN cells, when tested in the presence of TRAIL, showed a significantly increased ability to reduce cell number, and those analogues active against the M14 and SK-MEL-28 cell lines exhibited enhanced activity when combined with poly I:C. These data provide additional structure-activity relationship information for 17ß-hydroxywithanolides and suggest that selective activities of some analogues may be exploited to develop natural products-based tumor-specific agents for cancer chemotherapy.
Assuntos
Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Imunoterapia , Neoplasias Renais/terapia , Melanoma/terapia , Neoplasias da Próstata/tratamento farmacológico , Vitanolídeos/uso terapêutico , Humanos , Masculino , Neoplasias da Próstata/patologia , Vitanolídeos/químicaRESUMO
The selective killing of cancer cells without toxicity to normal nontransformed cells is an idealized goal of cancer therapy. Thus, there has been much interest in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a protein that appears to selectively kill cancer cells. TRAIL has been reported to trigger apoptosis and under some circumstances, an alternate death signaling pathway termed necroptosis. The relative importance of necroptosis for cell death induction in vivo is under intensive investigation. Nonetheless, many cancer cells (particularly those freshly isolated from cancer patients) are highly resistant to TRAIL-mediated cell death. Therefore, there is an underlying interest in identifying agents that can be combined with TRAIL to improve its efficacy. There are numerous reports in which combination of TRAIL with standard antineoplastic drugs has resulted in enhanced cancer cell death in vitro. However, many of these chemotherapeutic drugs are nonspecific and associated with adverse effects, which raise serious concerns for cancer therapy in patients. By contrast, natural products have been shown to be safer and efficacious alternatives. Recently, a number of studies have suggested that certain natural products when combined with TRAIL can enhance cancer cell death. In this review, we highlight molecular pathways that might be targeted by various natural products to promote cell death, and focus on our recent work with withanolides as TRAIL sensitizers. Finally, we will suggest synergistic approaches for combining active withanolides with various forms of immunotherapy to promote cancer cell death and an effective antitumor immune response.
Assuntos
Produtos Biológicos/farmacologia , Caspase 8/metabolismo , Morte Celular/genética , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Humanos , Neoplasias/patologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologiaRESUMO
Increasing evidence supports the hypothesis that cancer stem cells (CSCs) are resistant to antiproliferative therapies, able to repopulate tumor bulk, and seed metastasis. NK cells are able to target stem cells as shown by their ability to reject allogeneic hematopoietic stem cells but not solid tissue grafts. Using multiple preclinical models, including NK coculture (autologous and allogeneic) with multiple human cancer cell lines and dissociated primary cancer specimens and NK transfer in NSG mice harboring orthotopic pancreatic cancer xenografts, we assessed CSC viability, CSC frequency, expression of death receptor ligands, and tumor burden. We demonstrate that activated NK cells are capable of preferentially killing CSCs identified by multiple CSC markers (CD24(+)/CD44(+), CD133(+), and aldehyde dehydrogenase(bright)) from a wide variety of human cancer cell lines in vitro and dissociated primary cancer specimens ex vivo. We observed comparable effector function of allogeneic and autologous NK cells. We also observed preferential upregulation of NK activation ligands MICA/B, Fas, and DR5 on CSCs. Blocking studies further implicated an NKG2D-dependent mechanism for NK killing of CSCs. Treatment of orthotopic human pancreatic cancer tumor-bearing NSG mice with activated NK cells led to significant reductions in both intratumoral CSCs and tumor burden. Taken together, these data from multiple preclinical models, including a strong reliance on primary human cancer specimens, provide compelling preclinical evidence that activated NK cells preferentially target cancer cells with a CSC phenotype, highlighting the translational potential of NK immunotherapy as part of a combined modality approach for refractory solid malignancies.
Assuntos
Antígenos CD/imunologia , Imunidade Celular , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Células-Tronco Neoplásicas/imunologia , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Células Matadoras Naturais/patologia , Camundongos , Camundongos Endogâmicos NOD , Neoplasias/patologia , Células-Tronco Neoplásicas/patologiaRESUMO
Investigation of aeroponically grown Physalis peruviana resulted in the isolation of 11 new withanolides, including perulactones I-L (1-4), 17-deoxy-23ß-hydroxywithanolide E (5), 23ß-hydroxywithanolide E (6), 4-deoxyphyperunolide A (7), 7ß-hydroxywithanolide F (8), 7ß-hydroxy-17-epi-withanolide K (9), 24,25-dihydro-23ß,28-dihydroxywithanolide G (10), and 24,25-dihydrowithanolide E (11), together with 14 known withanolides (12-25). The structures of 1-11 were elucidated by the analysis of their spectroscopic data, and 12-25 were identified by comparison of their spectroscopic data with those reported. All withanolides were evaluated for their cytotoxic activity against a panel of tumor cell lines including LNCaP (androgen-sensitive human prostate adenocarcinoma), 22Rv1 (androgen-resistant human prostate adenocarcinoma), ACHN (human renal adenocarcinoma), M14 (human melanoma), SK-MEL-28 (human melanoma), and normal human foreskin fibroblast cells. Of these, the 17ß-hydroxywithanolides (17-BHWs) 6, 8, 9, 11-13, 15, and 19-22 showed selective cytotoxic activity against the two prostate cancer cell lines LNCaP and 22Rv1, whereas 13 and 20 exhibited selective toxicity for the ACHN renal carcinoma cell line. These cytotoxicity data provide additional structure-activity relationship information for the 17-BHWs.
Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/farmacologia , Neoplasias Renais/tratamento farmacológico , Physalis/química , Neoplasias da Próstata/tratamento farmacológico , Vitanolídeos/isolamento & purificação , Vitanolídeos/farmacologia , Antineoplásicos Fitogênicos/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Melanoma/tratamento farmacológico , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Physalis/crescimento & desenvolvimento , Relação Estrutura-Atividade , Vitanolídeos/químicaRESUMO
Fas ligand expression in certain tumors has been proposed to contribute to immunosuppression and poor prognosis. However, immunotherapeutic approaches may elicit the Fas-mediated elimination of immunosuppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) within tumors that represent major obstacles for cancer immunotherapy. Previously, we showed that IL-2 and agonistic CD40 Ab (αCD40) elicited synergistic antitumor responses coincident with the efficient removal of Tregs and MDSCs. We demonstrate in this study in two murine tumor models that Treg and MDSC loss within the tumor microenvironment after IL-2/αCD40 occurs through a Fas-dependent cell death pathway. Among tumor-infiltrating leukocytes, CD8(+) T cells, neutrophils, and immature myeloid cells expressed Fas ligand after treatment. Fas was expressed by tumor-associated Tregs and immature myeloid cells, including MDSCs. Tregs and MDSCs in the tumor microenvironment expressed active caspases after IL-2/αCD40 therapy and, in contrast with effector T cells, Tregs significantly downregulated Bcl-2 expression. In contrast, Tregs and MDSCs proliferated and expanded in the spleen after treatment. Adoptive transfer of Fas-deficient Tregs or MDSCs into wild-type, Treg-, or MDSC-depleted hosts resulted in the persistence of Tregs or MDSCs and the loss of antitumor efficacy in response to IL-2/αCD40. These results demonstrate the importance of Fas-mediated Treg/MDSC removal for successful antitumor immunotherapy. Our results suggest that immunotherapeutic strategies that include exploiting Treg and MDSC susceptibility to Fas-mediated apoptosis hold promise for treatment of cancer.
Assuntos
Antineoplásicos/farmacologia , Antígenos CD40/antagonistas & inibidores , Interleucina-2/farmacologia , Células Mieloides/imunologia , Neoplasias Experimentais/terapia , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia , Receptor fas/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Morte Celular/imunologia , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Células Mieloides/patologia , Neoplasias Experimentais/genética , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Linfócitos T Reguladores/patologia , Microambiente Tumoral/genética , Receptor fas/genéticaRESUMO
An important characteristic of cancer is that the disease can overcome the surveillance of the immune system. A possible explanation for this resistance arises from the ability of tumor cells to block the tumoricidal activity of host immune cells such as natural killer (NK) cells by inducing the localized accumulation of regulatory T (Treg) cells. Evidence exists that components in commonly consumed foods including vitamins A, D, and E, water-soluble constituents of mushrooms, polyphenolics in fruits and vegetables, and n-3 fatty acids in fish oil can modulate NK cell activities, Treg cell properties, and the interactions between those two cell types. Thus, it is extremely important for cancer prevention to understand the involvement of dietary components with the early stage dynamics of interactions among these immune cells. This review addresses the potential significance of diet in supporting the function of NK cells, Treg cells, and the balance between those two cell types, which ultimately results in decreased cancer risk.
Assuntos
Dieta , Células Matadoras Naturais/imunologia , Neoplasias/imunologia , Neoplasias/prevenção & controle , Linfócitos T Reguladores/imunologia , Animais , Citocinas/imunologia , Ácidos Graxos Ômega-3/imunologia , Humanos , Neoplasias/dietoterapia , Polifenóis/imunologia , Vitamina A/imunologia , Vitamina D/imunologiaRESUMO
BACKGROUND: Increasing studies implicate cancer stem cells (CSCs) as the source of resistance and relapse following conventional cytotoxic therapies. Few studies have examined the response of CSCs to targeted therapies, such as tyrosine kinase inhibitors (TKIs). We hypothesized that TKIs would have differential effects on CSC populations depending on their mechanism of action (anti-proliferative vs. anti-angiogenic). METHODS: We exposed human sarcoma cell lines to sorafenib, regorafenib, and pazopanib and assessed cell viability and expression of CSC markers (ALDH, CD24, CD44, and CD133). We evaluated survival and CSC phenotype in mice harboring sarcoma metastases after TKI therapy. We exposed dissociated primary sarcoma tumors to sorafenib, regorafenib, and pazopanib, and we used tissue microarray (TMA) and primary sarcoma samples to evaluate the frequency and intensity of CSC markers after neoadjuvant therapy with sorafenib and pazopanib. Parametric and non-parametric statistical analyses were performed as appropriate. RESULTS: After functionally validating the CSC phenotype of ALDHbright sarcoma cells, we observed that sorafenib and regorafenib were cytotoxic to sarcoma cell lines (P < 0.05), with a corresponding 1.4 - 2.8 fold increase in ALDHbright cells from baseline (P < 0.05). In contrast, we observed negligible effects on viability and CSC sub-populations with pazopanib. At low doses, there was progressive CSC enrichment in vitro after longer term exposure to sorafenib although the anti-proliferative effects were attenuated. In vivo, sorafenib improved median survival by 11 days (P < 0.05), but enriched ALDHbright cells 2.5 - 2.8 fold (P < 0.05). Analysis of primary human sarcoma samples revealed direct cytotoxicity following exposure to sorafenib and regorafenib with a corresponding increase in ALDHbright cells (P < 0.05). Again, negligible effects from pazopanib were observed. TMA analysis of archived specimens from sarcoma patients treated with sorafenib demonstrated significant enrichment for ALDHbright cells in the post-treatment resection specimen (P < 0.05), whereas clinical specimens obtained longitudinally from a patient treated with pazopanib showed no enrichment for ALDHbright cells (P > 0.05). CONCLUSIONS: Anti-proliferative TKIs appear to enrich for sarcoma CSCs while anti-angiogenic TKIs do not. The rational selection of targeted therapies for sarcoma patients may benefit from an awareness of the differential impact of TKIs on CSC populations.
Assuntos
Inibidores da Angiogênese/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Células-Tronco Neoplásicas/metabolismo , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Sarcoma/tratamento farmacológico , Família Aldeído Desidrogenase 1 , Inibidores da Angiogênese/uso terapêutico , Animais , Antígenos CD/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Indazóis , Isoenzimas/metabolismo , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos NOD , Terapia Neoadjuvante , Células-Tronco Neoplásicas/efeitos dos fármacos , Niacinamida/farmacologia , Niacinamida/uso terapêutico , Compostos de Fenilureia/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Retinal Desidrogenase/metabolismo , Sarcoma/secundário , Sorafenibe , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Análise Serial de Tecidos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cucurbitacins B and D were among the compounds identified as sensitizers of cancer cells to TRAIL-mediated apoptosis in a high-throughput screen. Therefore a series of cucurbitacins was further investigated for TRAIL sensitization and possible mechanisms of action. A total of six cucurbitacins promoted TRAIL-induced apoptosis (B, I, E, C, D, and K) and one (P) was inactive. Sensitization of renal adenocarcinoma cells to TRAIL was apparent after as little as 1-4 h pretreatment and did not require continued presence of cucurbitacin. Active cucurbitacins induced caspase-8 activation only after subsequent TRAIL addition and caspase activation was required for apoptosis suggesting amplified proximal signaling from TRAIL death receptors. Cucurbitacin-sensitized TRAIL-induced cytotoxicity was inhibited by N-acetyl cysteine. Structure-activity relationship analysis in comparison to published studies suggests that TRAIL-sensitizing and general cytotoxic activities of cucurbitacins may be decoupled. Cucurbitacins are reported to be inhibitors of STAT3 activation. However, their TRAIL-sensitizing activity is STAT3-independent. Treatment of renal carcinoma cells with active cucurbitacins produced rapid and dramatic changes in cell morphology and cytoskeletal organization (also prevented by NAC). Therefore, cucurbitacins may be useful as tools for investigating the molecular mechanism(s) of action of TRAIL sensitizers, particularly with regard to temporal aspects of sensitization and modulation of TRAIL signaling by cell morphology, and could form the basis for future therapeutic development in combination with TRAIL death receptor agonists.
Assuntos
Apoptose/efeitos dos fármacos , Carcinoma de Células Renais/fisiopatologia , Cucurbitacinas/farmacologia , Neoplasias Renais/fisiopatologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
The natural killer (NK) type of aggressive large granular lymphocytic (LGL) leukemia is a fatal illness that pursues a rapid clinical course. There are no effective therapies for this illness, and pathogenetic mechanisms remain undefined. Here we report that the survivin was highly expressed in both aggressive and chronic leukemic NK cells but not in normal NK cells. In vitro treatment of human and rat NK-LGL leukemia cells with cell-permeable, short-chain C6-ceramide (C6) in nanoliposomal formulation led to caspase-dependent apoptosis and diminished survivin protein expression, in a time- and dose-dependent manner. Importantly, systemic intravenous delivery of nanoliposomal ceramide induced complete remission in the syngeneic Fischer F344 rat model of aggressive NK-LGL leukemia. Therapeutic efficacy was associated with decreased expression of survivin in vivo. These data suggest that in vivo targeting of survivin through delivery of nanoliposomal C6-ceramide may be a promising therapeutic approach for a fatal leukemia.
Assuntos
Ceramidas/farmacologia , Leucemia Linfocítica Granular Grande/terapia , Lipossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Nanopartículas/química , Animais , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ceramidas/uso terapêutico , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Humanos , Leucemia Linfocítica Granular Grande/tratamento farmacológico , Leucemia Linfocítica Granular Grande/enzimologia , Leucemia Linfocítica Granular Grande/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/patologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ratos , Ratos Endogâmicos F344 , Indução de Remissão , Survivina , Resultado do TratamentoRESUMO
Barleria alluaudii and Diospyros maritima were both investigated as part of an ongoing search for synergistic TRAIL (tumor necrosis factor-α-related apoptosis-inducing ligand) sensitizers. As a result of this study, two naphthoquinone epoxides, 2,3-epoxy-2,3-dihydrolapachol (1) and 2,3-epoxy-2,3-dihydro-8-hydroxylapachol (2), both not previously isolated from natural sources, and the known 2-methylanthraquinone (3) were identified from B. alluaudii. Time-dependent density functional theory (TD-DFT) calculations of electronic circular dichroism (ECD) spectra were utilized to establish the absolute configuration of 1 and 2. Additionally, five known naphthoquinone derivatives, maritinone (4), elliptinone (5), plumbagin (6), (+)-cis-isoshinanolone (7), and ethylidene-6,6'-biplumbagin (8), were isolated from D. maritima. Compounds 1, 2, and 4-6 showed varying levels of synergy with TRAIL. Maritinone (4) and elliptinone (5) showed the highest synergistic effect, with more than a 3-fold increase in activity observed with TRAIL than with compound alone.
Assuntos
Acanthaceae/química , Antraquinonas/isolamento & purificação , Diospyros/química , Naftoquinonas/isolamento & purificação , Ligante Indutor de Apoptose Relacionado a TNF/efeitos dos fármacos , Antraquinonas/química , Antraquinonas/farmacologia , Madagáscar , Estrutura Molecular , Naftoquinonas/química , Naftoquinonas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfaRESUMO
The extrinsic apoptosis pathway is triggered by the binding of death ligands of the tumor necrosis factor (TNF) family to their appropriate death receptors (DRs) on the cell surface. One TNF family member, TNF-related apoptosis-inducing ligand (TRAIL or Apo2L), seems to preferentially cause apoptosis of transformed cells and can be systemically administered in the absence of severe toxicity. Therefore, there has been enthusiasm for the use of TRAIL or agonist antibodies to the TRAIL DR4 and DR5 in cancer therapy. Nonetheless, many cancer cells are very resistant to TRAIL apoptosis in vitro. Therefore, there is much interest in identifying compounds that can be combined with TRAIL to amplify its apoptotic effects. In this review, I will provide a brief overview of apoptosis signaling by TRAIL and discuss apoptosis-sensitizing agents, focusing mainly on the proteasome inhibitor bortezomib (VELCADE) and some novel sensitizers that we have recently identified. Alternative ways to administer TRAIL or DR agonist antibodies as therapeutic agents will also be described. Finally, I will discuss some of the gaps in our understanding of TRAIL apoptosis signaling and suggest some research directions that may provide additional information for optimizing the targeting of the extrinsic apoptosis pathway for future cancer therapy.
Assuntos
Apoptose/imunologia , Imunoterapia , Terapia de Alvo Molecular , Neoplasias/terapia , Ligante Indutor de Apoptose Relacionado a TNF/uso terapêutico , Adjuvantes Imunológicos/uso terapêutico , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Ácidos Borônicos/farmacologia , Ácidos Borônicos/uso terapêutico , Bortezomib , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias/imunologia , Neoplasias/patologia , Inibidores de Proteassoma , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Receptores do Fator de Necrose Tumoral/imunologia , Transdução de Sinais/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/agonistasRESUMO
IL-27 exerts antitumor activity in murine orthotopic neuroblastoma, but only partial antitumor effect in disseminated disease. This study demonstrates that combined treatment with IL-2 and IL-27 induces potent antitumor activity in disseminated neuroblastoma metastasis. Complete durable tumor regression was achieved in 90% of mice bearing metastatic TBJ-IL-27 tumors treated with IL-2 compared with only 40% of mice bearing TBJ-IL-27 tumors alone and 0% of mice bearing TBJ-FLAG tumors with or without IL-2 treatment. Comparable antitumor effects were achieved by IL-27 protein produced upon hydrodynamic IL-27 plasmid DNA delivery when combined with IL-2. Although delivery of IL-27 alone, or in combination with IL-2, mediated pronounced regression of neuroblastoma metastases in the liver, combined delivery of IL-27 and IL-2 was far more effective than IL-27 alone against bone marrow metastases. Combined exposure to IL-27 produced by tumor and IL-2 synergistically enhances the generation of tumor-specific CTL reactivity. Potentiation of CTL reactivity by IL-27 occurs via mechanisms that appear to be engaged during both the initial sensitization and effector phase. Potent immunologic memory responses are generated in mice cured of their disseminated disease by combined delivery of IL-27 and IL-2, and depletion of CD8(+) ablates the antitumor efficacy of this combination. Moreover, IL-27 delivery can inhibit the expansion of CD4(+)CD25(+)Foxp3(+) regulatory and IL-17-expressing CD4(+) cells that are otherwise observed among tumor-infiltrating lymphocytes from mice treated with IL-2. These studies demonstrate that IL-27 and IL-2 synergistically induce complete tumor regression and long-term survival in mice bearing widely metastatic neuroblastoma tumors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/imunologia , Interleucina-2/imunologia , Interleucinas/imunologia , Ativação Linfocitária/efeitos dos fármacos , Neuroblastoma/imunologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias da Medula Óssea/tratamento farmacológico , Neoplasias da Medula Óssea/secundário , Sinergismo Farmacológico , Citometria de Fluxo , Interferon gama/imunologia , Interleucina-2/administração & dosagem , Interleucinas/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Masculino , Camundongos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/secundário , Linfócitos T Citotóxicos/imunologiaRESUMO
Screening for sensitizers of cancer cells to TRAIL-mediated apoptosis identified a natural product of the 17ß-hydroxywithanolide (17-BHW) class, physachenolide C (PCC), as a promising hit. In this study, we show that PCC was also able to sensitize melanoma and renal carcinoma cells to apoptosis in response not only to TRAIL, but also to the synthetic polynucleotide poly I:C, a viral mimetic and immune activator, by reducing levels of antiapoptotic proteins cFLIP and Livin. Both death receptor and TLR3 signaling elicited subsequent increased assembly of a proapoptotic ripoptosome signaling complex. Administration of a combination of PCC and poly I:C in human M14 melanoma xenograft and a syngeneic B16 melanoma model provided significant therapeutic benefit as compared with individual agents. In addition, PCC enhanced melanoma cell death in response to activated human T cells in vitro and in vivo in a death ligand-dependent manner. Biochemical mechanism-of-action studies established bromo and extraterminal domain (BET) proteins as major cellular targets of PCC. Thus, by targeting of BET proteins to reduce antiapoptotic proteins and enhance caspase-8-dependent apoptosis of cancer cells, PCC represents a unique agent that can potentially be used in combination with various immunotherapeutic approaches to promote tumor regression and improve outcome. SIGNIFICANCE: These findings demonstrate that PCC selectively sensitizes cancer cells to immune-mediated cell death, potentially improving the efficacy of cancer immunotherapies. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3374/F1.large.jpg.
Assuntos
Produtos Biológicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Imunoterapia/métodos , Melanoma Experimental/tratamento farmacológico , Poli I-C/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Vitanolídeos/farmacologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Proliferação de Células , Quimioterapia Combinada , Feminino , Humanos , Indutores de Interferon/farmacologia , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Single and combination cytokines offer promise in some patients with advanced cancer. Many spontaneous and experimental cancers naturally express ligands for the lectin-like type-2 transmembrane stimulatory NKG2D immunoreceptor; however, the role this tumor recognition pathway plays in immunotherapy has not been explored to date. Here, we show that natural expression of NKG2D ligands on tumors provides an effective target for some cytokine-stimulated NK cells to recognize and suppress tumor metastases. In particular, interleukin (IL)-2 or IL-12 suppressed tumor metastases largely via NKG2D ligand recognition and perforin-mediated cytotoxicity. By contrast, IL-18 required tumor sensitivity to Fas ligand (FasL) and surprisingly did not depend on the NKG2D-NKG2D ligand pathway. A combination of IL-2 and IL-18 stimulated both perforin and FasL effector mechanisms with very potent effects. Cytokines that stimulated perforin-mediated cytotoxicity appeared relatively more effective against tumor metastases expressing NKG2D ligands. These findings indicate that a rational choice of cytokines can be made given the known sensitivity of tumor cells to perforin, FasL, and tumor necrosis factor-related apoptosis-inducing ligand and the NKG2D ligand status of tumor metastases.
Assuntos
Citocinas/uso terapêutico , Imunoterapia/métodos , Células Matadoras Naturais/imunologia , Metástase Neoplásica/terapia , Receptores Imunológicos/metabolismo , Animais , Apoptose/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Proteína Ligante Fas , Citometria de Fluxo , Interleucina-12/imunologia , Interleucina-12/metabolismo , Interleucina-18/imunologia , Interleucina-18/metabolismo , Interleucina-2/imunologia , Interleucina-2/metabolismo , Células Matadoras Naturais/metabolismo , Ligantes , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Proteínas Citotóxicas Formadoras de Poros , Receptores de Células Matadoras Naturais , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Células Tumorais CultivadasRESUMO
Because tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells and plays a critical role in tumor surveillance, its receptor is an attractive target for antibody-mediated tumor therapy. Here we report that a monoclonal antibody (mAb) against the mouse TRAIL receptor, DR5, exhibited potent antitumor effects against TRAIL-sensitive tumor cells in vivo by recruiting Fc receptor-expressing innate immune cells, with no apparent systemic toxicity. Administration of the agonistic anti-DR5 mAb also significantly inhibited experimental and spontaneous tumor metastases. Notably, the anti-DR5 mAb-mediated tumor rejection by innate immune cells efficiently evoked tumor-specific T cell immunity that could also eradicate TRAIL-resistant variants. These results suggested that the antibody-based therapy targeting DR5 is an efficient strategy not only to eliminate TRAIL-sensitive tumor cells, but also to induce tumor-specific T cell memory that affords a long-term protection from tumor recurrence.
Assuntos
Receptores do Fator de Necrose Tumoral/imunologia , Linfócitos T/imunologia , Alanina Transaminase/sangue , Animais , Proteínas Reguladoras de Apoptose , Aspartato Aminotransferases/sangue , Imunidade Celular/imunologia , Imunoterapia/métodos , Neoplasias Mamárias Animais/imunologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Receptores Fc/imunologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Ligante Indutor de Apoptose Relacionado a TNF , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines, which can induce apoptosis in various tumor cells by engaging the receptors, DR4 and DR5. Bortezomib (Velcade) is a proteasome inhibitor that has been approved for patients with multiple myeloma. There is some experimental evidence in preclinical models that bortezomib can enhance the susceptibility of tumors to TRAIL-induced apoptosis. In this study, we investigated the effects of TRAIL-induced death using an agonistic antibody to the TRAIL receptor DR5 (alpha-DR5) in combination with bortezomib administered to mice previously injected with breast cancer cells (TUBO). This combination had some beneficial therapeutic effect, which was significantly enhanced by the co-administration of a Toll-like receptor 9 agonist (CpG). In contrast, single agent treatments had little effect on tumor growth. In addition, we evaluated the effect of combination with alpha-DR5, bortezomib, and CpG in the prevention/treatment of spontaneous mammary tumors in Balb-neuT mice. In this model, which is more difficult to treat, we observed dramatic antitumor effects of alpha-DR5, bortezomib and CpG combination therapy. Since such a mouse model more accurately reflects the immunological tolerance that exists in human cancer, our results strongly suggest that these combination strategies could be directly applied to the therapy for cancer patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Animais , Anticorpos Monoclonais/administração & dosagem , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Ácidos Borônicos/administração & dosagem , Bortezomib , Linhagem Celular Tumoral , Sinergismo Farmacológico , Feminino , Masculino , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Oligodesoxirribonucleotídeos/administração & dosagem , Pirazinas/administração & dosagem , Ratos , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Receptor Toll-Like 9/agonistas , Resultado do TratamentoRESUMO
Dissociating graft-versus-tumor (GVT) effect from acute graft-versus-host disease (GVHD) still remains a great challenge in allogeneic bone marrow transplantation (allo-BMT). Bortezomib, a proteasome inhibitor, has shown impressive efficacy as a single agent in patients with hematologic malignancies but can result in toxicity when administered late after allogeneic transplantation in murine models of GVHD. In the current study, the effects of T-cell subsets and their associated cytokines on the efficacy of bortezomib in murine allogeneic BMT were investigated. Increased levels of serum tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) were observed after allo-BMT and continuous bortezomib administration. Bortezomib-induced GVHD-dependent mortality was preventable by depletion of CD4(+) but not CD8(+) T cells from the donor graft. The improved survival correlated with markedly reduced serum TNFalpha but not IFNgamma levels. Transfer of Tnf(-/-) T cells also protected recipients from bortezomib-induced GVHD-dependent toxicity. Importantly, prolonged administration of bortezomib after transplantation of purified CD8(+) T cells resulted in enhanced GVT response, which was dependent on donor CD8(+) T cell-derived IFNgamma. These results indicate that decreased toxicity and increased efficacy of bortezomib in murine allo-BMT can be achieved by removal of CD4(+) T cells from the graft or by inhibiting TNFalpha.
Assuntos
Transplante de Medula Óssea/métodos , Ácidos Borônicos/administração & dosagem , Citocinas/metabolismo , Pirazinas/administração & dosagem , Subpopulações de Linfócitos T/imunologia , Linfócitos T/transplante , Animais , Transplante de Medula Óssea/efeitos adversos , Bortezomib , Feminino , Efeito Enxerto vs Leucemia , Efeito Enxerto vs Tumor , Interferon gama , Depleção Linfocítica , Camundongos , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transplante Homólogo , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Casearia arguta was investigated as part of the ongoing search for synergistic TRAIL (tumor necrosis factor-α-related apoptosis-inducing ligand) sensitizers. As a result of this study, argutins A-H, eight new highly oxygenated clerodane diterpenes, were isolated from the plant Casearia arguta collected in Guatemala. The modified Mosher ester method was utilized to establish the absolute configuration of argutins A and F. Each of the argutins showed varying levels of synergy with TRAIL. Argutin B showed the highest TRAIL sensitization; the synergistic effect of argutin B and TRAIL together was 3-fold greater than argutin B alone.
Assuntos
Casearia/química , Diterpenos Clerodânicos/isolamento & purificação , Diterpenos Clerodânicos/farmacologia , Plantas Medicinais/química , Ligante Indutor de Apoptose Relacionado a TNF/efeitos dos fármacos , Diterpenos Clerodânicos/química , Guatemala , Estrutura Molecular , Folhas de Planta/químicaRESUMO
We have developed a high-throughput screen (HTS) to search for novel molecules that can synergize with TRAIL, thus promoting apoptosis of ACHN renal tumor cells in a combinatorial fashion. The HTS detects synthetic compounds and pure natural products that can pre-sensitize the cancer cells to TRAIL-mediated apoptosis, yet have limited toxicity on their own. We have taken into account the individual effects of the single agents, versus the combination, and have identified hits that are synergistic, synergistic-toxic, or additive when combined with TRAIL in promoting tumor cell death. Preliminary mechanistic studies indicate that a subset of the synergistic TRAIL sensitizers act very rapidly to promote cleavage and activation of caspase-8 following TRAIL binding. Caspase-8 is an apical enzyme that initiates programmed cell death via the extrinsic apoptotic pathway. Thus, these TRAIL sensitizers may potentially reduce resistance of tumor cells to TRAIL-mediated apoptosis. Two representative sensitizers were found to increase levels of p53 but did not inhibit the proteasome, suggesting that early DNA damage-sensing pathways may be involved in their mechanisms of action.