RESUMO
B-cell maturation antigen (BCMA) plays a pathobiologic role in myeloma and is a validated target with five BCMA-specific therapeutics having been approved for relapsed/refractory disease. However, these drugs are not curative, and responses are inferior in patients with molecularly-defined high-risk disease, including those with deletion 17p (del17p) involving the tumor suppressor TP53, supporting the need for further drug development. Del17p has been associated with reduced copy number and gene expression of RNA polymerase II subunit alpha (POLR2A) in other tumor types. We therefore studied the possibility that HDP-101, an anti-BCMA antibody drug conjugate (ADC) with the POLR2A poison α-amanitin could be an attractive agent in myeloma, especially with del17p. HDP-101 reduced viability in myeloma cell lines representing different molecular disease subtypes, and overcame adhesion-mediated and both conventional and novel drug resistance. After confirming that del17p is associated with reduced POLR2A levels in publicly available myeloma patient databases, we engineered TP53 wild-type cells with a TP53 knockout (KO), POLR2A knockdown (KD), or both, the latter to mimic del17p. HDP-101 showed potent anti-myeloma activity against all tested cell lines, and exerted enhanced efficacy against POLR2A KD and dual TP53 KO/POLR2A KD cells. Mechanistic studies showed HDP-101 up-regulated the unfolded protein response, activated apoptosis, and induced immunogenic cell death. Notably, HDP-101 impacted CD138-positive but not-negative primary cells, showed potent efficacy against aldehyde dehydrogenase-positive clonogenic cells, and eradicated myeloma in an in vivo cell line-derived xenograft (CDX). Interestingly, in the CDX model, prior treatment with HDP-101 precluded subsequent engraftment on tumor cell line rechallenge in a manner that appeared to be dependent in part on natural killer cells and macrophages. Finally, HDP-101 was superior to the BCMA-targeted ADC belantamab mafodotin against cell lines and primary myeloma cells in vitro, and in an in vivo CDX. Together, the data support the rationale for translation of HDP-101 to the clinic, where it is now undergoing Phase I trials, and suggest that it could emerge as a more potent ADC for myeloma with especially interesting activity against the high-risk del17p myeloma subtype.
RESUMO
Mantle cell lymphoma (MCL) is a haematologic malignancy. The proteasome inhibitor (PI) bortezomib has been approved to treat MCL, but resistance has emerged through mechanisms that remain unclear. This study aimed to explore the mechanism of PI resistance in MCL and identify new targets for this patient subgroup. Carfilzomib-resistant (CR) MCL cell lines and primary samples were used for both in vitro and in vivo experiments to identify gene expression and explore their related signalling pathways. We first identified mucin 20 (MUC20) suppression in carfilzomib-resistant MCL models. MUC20 overexpression sensitized cells to carfilzomib in vitro and in vivo. MUC20 expression was inversely related to activation of c-Met and the downstream p44/42 MAPK pathway. c-Met activation with hepatocyte growth factor (HGF) induced PI resistance, while c-Met inhibition restored PI sensitivity. Carfilzomib resistance and depressed MUC20 expression were associated with enhanced proteasome activity and higher expression of proteassemblin (POMP), a chaperone for catalytically active proteasome assembly. c-Met and POMP were associated through binding and induction of MAPK-regulated ELK1 to the POMP promoter. Our data reveal that c-Met signalling activation enhanced proteasome capacity as a mechanism of PI resistance, and MUC20 expression may be a useful biomarker for PI therapy.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Linfoma de Célula do Manto/metabolismo , Mucinas/metabolismo , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Transdução de Sinais , Animais , Catálise , Linhagem Celular Tumoral , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mucinas/genética , Proteínas Elk-1 do Domínio etsRESUMO
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma; 40% of patients relapse after a complete response or are refractory to therapy. To survive, the activated B-cell (ABC) subtype of DLBCL relies upon B-cell receptor signaling, which can be modulated by the activity of Bruton tyrosine kinase (BTK). Targeting BTK with ibrutinib, an inhibitor, provides a therapeutic approach for this subtype of DLBCL. However, non-Hodgkin lymphoma is often resistant to ibrutinib or acquires resistance soon after exposure. We explored how this resistance develops. We generated 3 isogenic ibrutinib-resistant DLBCL cell lines and investigated the deregulated pathways known to be associated with tumorigenic properties. Reduced levels of BTK and enhanced phosphatidylinositol 3-kinase (PI3K)/AKT signaling were hallmarks of these ibrutinib-resistant cells. Upregulation of PI3K-ß expression was demonstrated to drive resistance in ibrutinib-resistant cells, and resistance was reversed by the blocking activity of PI3K-ß/δ. Treatment with the selective PI3K-ß/δ dual inhibitor KA2237 reduced both tumorigenic properties and survival-based PI3K/AKT/mTOR signaling of these ibrutinib-resistant cells. In addition, combining KA2237 with currently available chemotherapeutic agents synergistically inhibited metabolic growth. This study elucidates the compensatory upregulated PI3K/AKT axis that emerges in ibrutinib-resistant cells.
Assuntos
Linfoma Difuso de Grandes Células B , Fosfatidilinositol 3-Quinase , Tirosina Quinase da Agamaglobulinemia , Linhagem Celular Tumoral , Humanos , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Recidiva Local de Neoplasia , Fosfatidilinositol 3-Quinases/genéticaRESUMO
Intravenous immunoglobulin G (IVIgG) is approved for primary immunodeficiency syndromes but may induce anti-cancer effects, and while this has been attributed to its anti-inflammatory properties, IgG against specific tumor targets may play a role. We evaluated IVIgG alone, and with a Heat shock protein (HSP)-90 or proteasome inhibitor, using multiple myeloma and mantle cell lymphoma (MCL) cells in vitro, and with the proteasome inhibitor bortezomib in vivo. IVIgG inhibited the growth of all cell lines tested, induced G1 cell cycle arrest, and suppressed pro-tumor cytokines including Interleukin (IL)-6, IL-8, and IL-10. Genomic and proteomic studies showed that IVIgG reduced tumor cell HSP70-1 levels by suppressing the ability of extracellular HSP70-1 to stimulate endogenous HSP70-1 promoter activity, and reduced extracellular vesicle uptake. Preparations of IVIgG were found to contain high titers of anti-HSP70-1 IgG, and recombinant HSP70-1 reduced the efficacy of IVIgG to suppress HSP70-1 levels. Combining IVIgG with the HSP90 inhibitor AUY922 produced superior cell growth inhibition and correlated with HSP70-1 suppression. Also, IVIgG with bortezomib or carfilzomib was superior to each single agent, and enhanced bortezomib's activity in bortezomib-resistant myeloma cells. Moreover, IVIgG reduced transfer of extracellular vesicles (EVs) to cells, and blocked transfer of bortezomib resistance through EVs. Finally, IVIgG with bortezomib were superior to the single agents in an in vivo myeloma model. These studies support the possibility that anti-HSP70-1 IgG contained in IVIgG can inhibit myeloma and MCL growth by interfering with a novel mechanism involving uptake of exogenous HSP70-1 which then induces its own promoter.
Assuntos
Proteínas de Choque Térmico HSP70/efeitos dos fármacos , Imunoglobulinas Intravenosas/farmacologia , Linfoma de Célula do Manto/imunologia , Mieloma Múltiplo/imunologia , Animais , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Linhagem Celular Tumoral , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP90/imunologia , Humanos , Camundongos , Camundongos SCID , Inibidores de Proteassoma/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
KRAS, NRAS, and BRAF mutations which activate p44/42 mitogen-activated protein kinase (MAPK) signaling are found in half of myeloma patients and contribute to proteasome inhibitor (PI) resistance, but the underlying mechanisms are not fully understood. We established myeloma cell lines expressing wild-type (WT), constitutively active (CA) (G12V/G13D/Q61H), or dominant-negative (DN) (S17N)-KRAS and -NRAS, or BRAF-V600E. Cells expressing CA mutants showed increased proteasome maturation protein (POMP) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) expression. This correlated with an increase in catalytically active proteasome subunit ß (PSMB)-8, PSMB9, and PSMB10, which occurred in an ETS transcription factor-dependent manner. Proteasome chymotrypsin-like, trypsin-like, and caspase-like activities were increased, and this enhanced capacity reduced PI sensitivity, while DN-KRAS and DN-NRAS did the opposite. Pharmacologic RAF or MAPK kinase (MEK) inhibitors decreased proteasome activity, and sensitized myeloma cells to PIs. CA-KRAS, CA-NRAS, and CA-BRAF down-regulated expression of endoplasmic reticulum (ER) stress proteins, and reduced unfolded protein response activation, while DN mutations increased both. Finally, a bortezomib (BTZ)/MEK inhibitor combination showed enhanced activity in vivo specifically in CA-NRAS models. Taken together, the data support the hypothesis that activating MAPK pathway mutations enhance PI resistance by increasing proteasome capacity, and provide a rationale for targeting such patients with PI/RAF or PI/MEK inhibitor combinations. Moreover, they argue these mutations promote myeloma survival by reducing cellular stress, thereby distancing plasma cells from the apoptotic threshold, potentially explaining their high frequency in myeloma.
Assuntos
Estresse do Retículo Endoplasmático , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Mieloma Múltiplo/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Apoptose/efeitos dos fármacos , Bortezomib/farmacologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , GTP Fosfo-Hidrolases/genética , Humanos , Proteínas de Membrana/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/fisiopatologia , Mutação , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Small molecules targeting the cereblon-containing E3 ubiquitin ligase including thalidomide, lenalidomide, and pomalidomide modulate turnover of downstream client proteins and demonstrate pre-clinical and clinical anti-myeloma activity. Different drugs that engage with cereblon hold the potential of unique phenotypic effects, and we therefore studied the novel protein homeostatic modulator (PHM™) BTX306 with a unique thiophene-fused scaffold bearing a substituted phenylurea and glutarimide. This agent much more potently reduced human-derived myeloma cell line viability, with median inhibitory concentrations in the single nanomolar range versus micromolar values for lenalidomide or pomalidomide, and more potently activated caspases 3/8/9. While lenalidomide and pomalidomide induced greater degradation of Ikaros and Aiolos in myeloma cells, BTX306 more potently reduced levels of GSPT1, eRF1, CK1α, MCL-1, and c-MYC. Suppression of cereblon or overexpression of Aiolos or Ikaros induced relative resistance to BTX306, and this agent did not impact viability of murine hematopoietic cells in an in vivo model, demonstrating its specificity for human cereblon. Interestingly, BTX306 did show some reduced activity in lenalidomide-resistant cell line models but nonetheless retained its nanomolar potency in vitro, overcame bortezomib resistance, and was equipotent against otherwise isogenic cell line models with either wild-type or knockout TP53. Finally, BTX306 demonstrated strong activity against primary CD138-positive plasma cells, showed enhanced anti-proliferative activity in combination with bortezomib and dexamethasone, and was effective in an in vivo systemic model of multiple myeloma. Taken together, the data support further translational studies of BTX306 and its derivatives to the clinic for patients with relapsed and/or refractory myeloma. KEY MESSAGES: BTX306 has a unique thiophene-fused scaffold bearing phenylurea and glutarimide. BTX306 is more potent against myeloma cells than lenalidomide or pomalidomide. BTX306 overcomes myeloma cell resistance to lenalidomide or bortezomib in vitro. BTX306 is active against primary myeloma cells, and shows efficacy in vivo.
Assuntos
Antineoplásicos/farmacologia , Bortezomib/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Lenalidomida/farmacologia , Proteostase/efeitos dos fármacos , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Camundongos , Mieloma Múltiplo , Ubiquitina-Proteína Ligases/antagonistas & inibidoresRESUMO
Three proteasome inhibitors have garnered regulatory approvals in various multiple myeloma settings; but drug resistance is an emerging challenge, prompting interest in blocking upstream components of the ubiquitin-proteasome pathway. One such attractive target is the E1 ubiquitin-activating enzyme (UAE); we therefore evaluated the activity of TAK-243, a novel and specific UAE inhibitor. TAK-243 potently suppressed myeloma cell line growth, induced apoptosis, and activated caspases while decreasing the abundance of ubiquitin-protein conjugates. This was accompanied by stabilization of many short-lived proteins, including p53, myeloid cell leukemia 1 (MCL-1), and c-MYC, and activation of the activating transcription factor 6 (ATF-6), inositol-requiring enzyme 1 (IRE-1), and protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) arms of the ER stress response pathway, as well as oxidative stress. UAE inhibition showed comparable activity against otherwise isogenic cell lines with wild-type (WT) or deleted p53 despite induction of TP53 signaling in WT cells. Notably, TAK-243 overcame resistance to conventional drugs and novel agents in cell-line models, including bortezomib and carfilzomib resistance, and showed activity against primary cells from relapsed/refractory myeloma patients. In addition, TAK-243 showed strong synergy with a number of antimyeloma agents, including doxorubicin, melphalan, and panobinostat as measured by low combination indices. Finally, TAK-243 was active against a number of in vivo myeloma models in association with activation of ER stress. Taken together, the data support the conclusion that UAE inhibition could be an attractive strategy to move forward to the clinic for patients with relapsed and/or refractory multiple myeloma.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteassoma/farmacologia , Enzimas Ativadoras de Ubiquitina/antagonistas & inibidores , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Sinergismo Farmacológico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Estresse Oxidativo/efeitos dos fármacos , Terapia de Salvação/métodos , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismoRESUMO
PURPOSE: Waldenström's macroglobulinemia is an incurable lymphoproliferative disorder driven by an L265P mutation in the myeloid differentiation primary response gene 88 (MYD88), which activates downstream NF-κB signaling through the Myddosome. As this pathway depends in part on activity of interleukin-1 receptor-associated kinases (IRAKs)-1 and -4, we sought to evaluate the potential of the IRAK1/4 inhibitor R191 in preclinical models. EXPERIMENTAL DESIGN: Patient-derived cell lines and primary samples were used in both in vitro and in vivo experiments to model Waldenström's macroglobulinemia and its response to IRAK1/4 inhibitors. RESULTS: R191 induced a dose- and time-dependent reduction in viability of BCWM.1 and MWCL-1 Waldenström's cell lines, and suppressed activation of IRAK1/4. This was associated with cell-cycle arrest at G0-G1, reduced levels of cyclin-dependent kinases 4 and 6, and induction of apoptosis in cell lines and primary patient samples. Further downstream, R191 exposure led to reduced activation of NF-κB, and of protein kinase B/Akt/mammalian target of rapamycin signaling, whereas expression of a constitutively active Akt mutant induced R191 resistance. Gene expression profiling and gene set enrichment analysis revealed a signature consistent with inhibition of c-Myc and activation of the endoplasmic reticulum stress response. In both subcutaneous and systemic murine models of Waldenström's, R191 showed antitumor activity. Finally, the activity of R191 was enhanced when it was combined with novel chemotherapeutics such as bortezomib, afuresertib, and ibrutinib. CONCLUSIONS: Taken together, these data support the translation of R191 as an approach to target IRAK1/4 to the clinic for patients with Waldenström's macroglobulinemia.
Assuntos
Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Macroglobulinemia de Waldenstrom/metabolismo , Animais , Apoptose/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Sobrevivência Celular , Modelos Animais de Doenças , Sinergismo Farmacológico , Retículo Endoplasmático/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Macroglobulinemia de Waldenstrom/tratamento farmacológico , Macroglobulinemia de Waldenstrom/etiologiaRESUMO
Bromodomain and extraterminal (BET) domain containing protein (BRD)-4 modulates the expression of oncogenes such as c-myc, and is a promising therapeutic target in diverse cancer types. We performed pre-clinical studies in myeloma models with bi-functional protein-targeting chimeric molecules (PROTACs) which target BRD4 and other BET family members for ubiquitination and proteasomal degradation. PROTACs potently reduced the viability of myeloma cell lines in a time-dependent and concentration-dependent manner associated with G0/G1 arrest, reduced levels of CDKs 4 and 6, increased p21 levels, and induction of apoptosis. These agents specifically decreased cellular levels of downstream BRD4 targets, including c-MYC and N-MYC, and a Cereblon-targeting PROTAC showed downstream effects similar to those of an immunomodulatory agent. Notably, PROTACs overcame bortezomib, dexamethasone, lenalidomide, and pomalidomide resistance, and their activity was maintained in otherwise isogenic myeloma cells with wild-type or deleted TP53. Combination studies showed synergistic interactions with dexamethasone, BH3 mimetics, and Akt pathway inhibitors. BET-specific PROTACs induced a rapid loss of viability of primary cells from myeloma patients, and delayed growth of MM1.S-based xenografts. Our data demonstrate that BET degraders have promising activity against pre-clinical models of multiple myeloma, and support their translation to the clinic for patients with relapsed and/or refractory disease.
Assuntos
Antineoplásicos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Proteínas/metabolismo , Motivos de Aminoácidos/efeitos dos fármacos , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Proteínas Nucleares/metabolismo , Domínios Proteicos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Mucormycosis is a destructive invasive mold infection afflicting patients with diabetes and hematologic malignancies. Patients with diabetes are often treated with statins, which have been shown to have antifungal properties. We sought to examine the effects of statins on Rhizopus oryzae, a common cause of mucormycosis. METHODS: Clinical strains of R. oryzae were exposed to lovastatin, atorvastatin, and simvastatin and the minimum inhibitory concentrations (MICs) were determined. R. oryzae germination, DNA fragmentation, susceptibility to oxidative stress, and ability to damage endothelial cells were assessed. We further investigated the impact of exposure to lovastatin on the virulence of R. oryzae RESULTS: All statins had MICs of >64 µg/mL against R. oryzae Exposure of R. oryzae to statins decreased germling formation, induced DNA fragmentation, and attenuated damage to endothelial cells independently of the expression of GRP78 and CotH. Additionally, R. oryzae exposed to lovastatin showed macroscopic loss of melanin, yielded increased susceptibility to the oxidative agent peroxide, and had attenuated virulence in both fly and mouse models of mucormycosis. CONCLUSIONS: Exposure of R. oryzae to statins at concentrations below their MICs decreased virulence both in vitro and in vivo. Further investigation is warranted into the use of statins as adjunctive therapy in mucormycosis.
Assuntos
Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Mucormicose/tratamento farmacológico , Rhizopus/efeitos dos fármacos , Virulência/efeitos dos fármacos , Animais , Atorvastatina/farmacologia , Atorvastatina/uso terapêutico , Dípteros/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Feminino , Humanos , Lovastatina/farmacologia , Lovastatina/uso terapêutico , Camundongos , Testes de Sensibilidade Microbiana , Sinvastatina/farmacologia , Sinvastatina/uso terapêutico , Esporos Fúngicos/efeitos dos fármacos , TexasRESUMO
Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) colonize cystic fibrosis (CF) patient airways. Pa culture filtrates inhibit Af biofilms, and Pa non-CF, mucoid (Muc-CF) and nonmucoid CF (NMuc-CF) isolates form an ascending inhibitory hierarchy. We hypothesized this activity is mediated through apoptosis induction. One Af and three Pa (non-CF, Muc-CF, NMuc-CF) reference isolates were studied. Af biofilm was formed in 96 well plates for 16 h ± Pa biofilm filtrates. After 24 h, apoptosis was characterized by viability dye DiBAc, reactive oxygen species (ROS) generation, mitochondrial membrane depolarization, DNA fragmentation and metacaspase activity. Muc-CF and NMuc-CF filtrates inhibited and damaged Af biofilm (p<0.0001). Intracellular ROS levels were elevated (p<0.001) in NMuc-CF-treated Af biofilms (3.7- fold) compared to treatment with filtrates from Muc-CF- (2.5- fold) or non-CF Pa (1.7- fold). Depolarization of mitochondrial potential was greater upon exposure to NMuc-CF (2.4-fold) compared to Muc-CF (1.8-fold) or non-CF (1.25-fold) (p<0.0001) filtrates. Exposure to filtrates resulted in more DNA fragmentation in Af biofilm, compared to control, mediated by metacaspase activation. In conclusion, filtrates from CF-Pa isolates were more inhibitory against Af biofilms than from non-CF. The apoptotic effect involves mitochondrial membrane damage associated with metacaspase activation.
Assuntos
Apoptose/fisiologia , Aspergillus fumigatus/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Fibrose Cística/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Aspergillus fumigatus/metabolismo , Fragmentação do DNA , Humanos , Membranas Mitocondriais/metabolismo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Mortality associated with mucormycosis remains high despite current antifungals. Iron-starvation strategies have been shown to have promising activity against Mucorales. We hypothesized that iron starvation enhances apoptosis in Rhizopus oryzae. Apoptosis was characterized in R. oryzae transformed with RNAi plasmid targeting FTR1 expression (iron permease mutant) or empty plasmid grown in iron rich (0.125% FeCl3) and iron depleted media (YNB+1mM ferrozine and 1 mM ascorbic acid). Increased apoptosis was observed with dihydrorhodamine-123 and rhodamine-123 staining in the iron starved mutant FTR1 when compared to empty plasmid, followed by increased extracellular ATP levels. In addition, DNA fragmentation and metacaspase activity were prominent in FTR1. In contrast, Rhizopus strains grown in iron-rich medium displayed minimal apoptosis. Our results demonstrate a metacaspase dependent apoptotic process in iron deprived condition and further support the role of iron starvation strategies as an adjunct treatment for mucormycosis, a mechanism by which iron starvation affects R. oryzae.
Assuntos
Apoptose , Ferro/metabolismo , Rhizopus/genética , Rhizopus/fisiologia , Caspases/metabolismo , Meios de Cultura , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Potencial da Membrana Mitocondrial , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Mutação , Espécies Reativas de Oxigênio/metabolismo , Rhizopus/ultraestruturaRESUMO
We hypothesized that the cell wall inhibitor micafungin (MICA) induces apoptosis in both MICA-susceptible (MICA-S) and MICA-non-susceptible (MICA-NS) Candida parapsilosis. Antifungal activity and apoptosis were analyzed in MICA-S and MICA-NS C. parapsilosis strains following exposure to micafungin for 3 h at 37°C in RPMI 1640 medium. Apoptosis was characterized by detecting phosphatidylserine externalization (PS), plasma membrane integrity, reactive oxygen species (ROS) generation, mitochondrial membrane potential changes, adenosine triphosphate (ATP) release, and caspase-like activity. Apoptosis was detected in MICA exposed (0.25 to 1 mg/L) susceptible C. parapsilosis strains and was associated with apoptosis of 20-52% of analyzed cells versus only 5-30% of apoptosis in MICA-NS cells exposed to micafungin (0.5 to 2 mg/L; P = 0.001). The MICA antifungal activity was correlated with apoptotic cells showing increased dihydrorhodamine-123 staining (indicating ROS production), Rh-123 staining (decreased mitochondrial membrane potential), elevated ATP, and increased metacaspase activity. In conclusion, MICA is pro-apoptotic in MICA-S cells, but still exerts apoptotic effects in MICA -NS C. parapsilosis.
RESUMO
The incidence of mucormycosis has dramatically increased in immunocompromised patients. Moreover, the array of cellular targets whose inhibition results in fungal cell death is rather limited. Mitochondria have been mechanistically identified as central regulators of detoxification and virulence in fungi. Our group has previously designed and developed a proteolytically-resistant peptidomimetic motif D(KLAKLAK)2 with pleiotropic action ranging from targeted (i.e., ligand-directed) activity against cancer and obesity to non-targeted activity against antibiotic resistant gram-negative rods. Here we evaluated whether this non-targeted peptidomimetic motif is active against Mucorales. We show that D(KLAKLAK)2 has marked fungicidal action, inhibits germination, and reduces hyphal viability. We have also observed cellular changes characteristic of apoptosis in D(KLAKLAK)2-treated Mucorales cells. Moreover, the fungicidal activity was directly correlated with vacuolar injury, mitochondrial swelling and mitochondrial membrane depolarization, intracellular reactive oxygen species accumulation (ROS), and increased caspase-like enzymatic activity. Finally, these apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine indicating mechanistic pathway specificity. Together, these findings indicate that D(KLAKLAK)2 makes Mucorales exquisitely susceptible via mitochondrial injury-induced apoptosis. This prototype may serve as a candidate drug for the development of translational applications against mucormycosis and perhaps other fungal infections.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mucorales/efeitos dos fármacos , Peptidomiméticos/farmacologia , Esporos Fúngicos/efeitos dos fármacos , Acetilcisteína/farmacologia , Motivos de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/síntese química , Apoptose/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Mitocôndrias/metabolismo , Mucorales/isolamento & purificação , Mucorales/metabolismo , Mucormicose/microbiologia , Peptidomiméticos/síntese química , Espécies Reativas de Oxigênio/agonistas , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Esporos Fúngicos/metabolismoRESUMO
The high mortality rate of mucormycosis with currently available monotherapy has created interest in studying novel strategies for antifungal agents. With the exception of amphotericin B (AMB), the triazoles (posaconazole [PCZ] and itraconazole [ICZ]) are fungistatic in vitro against Rhizopus oryzae . We hypothesized that growth at a high temperature (42°C) results in fungicidal activity of PCZ and ICZ that is mediated through apoptosis. R. oryzae had high MIC values for PCZ and ICZ (16 to 64 µg/ml) at 25°C; in contrast, the MICs for PCZ and ICZ were significantly lower at 37°C (8 to 16 µg/ml) and 42°C (0.25 to 1 µg/ml). Furthermore, PCZ and ICZ dose-dependent inhibition of germination was more pronounced at 42°C than at 37°C. In addition, intracellular reactive oxygen species (ROS) increased significantly when fungi were exposed to antifungals at 42°C. Characteristic cellular changes of apoptosis in R. oryzae were induced by the accumulation of intracellular reactive oxygen species. Cells treated with PCZ or ICZ in combination with hyperthermia (42°C) exhibited characteristic markers of early apoptosis: phosphatidylserine externalization visualized by annexin V staining, membrane depolarization visualized by bis-[1,3-dibutylbarbituric acid] trimethine oxonol (DiBAC) staining, and increased metacaspase activity. Moreover, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and DAPI (4',6-diamidino-2-phenylindole) staining demonstrated DNA fragmentation and condensation, respectively. The addition of N-acetylcysteine increased fungal survival, prevented apoptosis, reduced ROS accumulation, and decreased metacaspase activation. We concluded that hyperthermia, either alone or in the presence of PCZ or ICZ, induces apoptosis in R. oryzae. Local thermal delivery could be a therapeutically useful adjunct strategy for these refractory infections.
Assuntos
Antifúngicos/farmacologia , Apoptose/efeitos dos fármacos , Itraconazol/farmacologia , Espécies Reativas de Oxigênio/agonistas , Rhizopus/efeitos dos fármacos , Triazóis/farmacologia , Acetilcisteína/farmacologia , Anfotericina B/farmacologia , Caspases/metabolismo , Sequestradores de Radicais Livres/farmacologia , Temperatura Alta , Humanos , Potenciais da Membrana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Fosfatidilserinas/metabolismo , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Rhizopus/crescimento & desenvolvimento , Rhizopus/isolamento & purificação , Rhizopus/metabolismoRESUMO
The incidence of mucormycosis has increased drastically in immunocompromised patients. Also the array of targets whose inhibition results in Mucorales death is limited. Recently, researchers identified mitochondria as important regulators of detoxification and virulence mechanisms in fungi. In this context, targeting the mitochondrial respiratory chain may provide a new platform for antifungal development. We hypothesized that targeting respiratory pathways potentiates triazoles activity via apoptosis. We found that simultaneous administration of antimycin A (AA) and benzohydroxamate (BHAM), inhibitors of classical and alternative mitochondrial pathways respectively, resulted in potent activity of posaconazole (PCZ) and itraconazole (ICZ) against Rhizopus oryzae. We observed cellular changes characteristic of apoptosis in R. oryzae cells treated with PCZ or ICZ in combination with AA and BHAM. The fungicidal activity of this combination against R. oryzae was correlated with intracellular reactive oxygen species accumulation (ROS), phosphatidylserine externalization, mitochondrial membrane depolarization, and increased caspase like activity. DNA fragmentation and condensation assays also revealed apoptosis of R. oryzae cells. These apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine. Taken together, these findings suggest that the use of PCZ or ICZ in combination with AA and BHAM makes R. oryzae exquisitely sensitive to treatment with triazoles via apoptosis. This strategy may serve as a new model for the development of improved or novel antifungal agents.
Assuntos
Antifúngicos/farmacologia , Antimicina A/farmacologia , Ácidos Hidroxâmicos/farmacologia , Itraconazol/farmacologia , Mitocôndrias/metabolismo , Rhizopus/efeitos dos fármacos , Triazóis/farmacologia , Apoptose , Caspases/metabolismo , Citocromos c/metabolismo , Fragmentação do DNA , DNA Fúngico/genética , Sinergismo Farmacológico , Transporte de Elétrons , Proteínas Fúngicas/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Rhizopus/metabolismoRESUMO
We report herein the synthesis and biological evaluation of bile acid dimers linked through 1,2,3-triazole and bis-beta-lactam. The dimers were synthesized using 1,3-dipolar cycloaddition reaction of diazido bis-beta-lactams , and terminal alkynes derived from cholic acid/deoxycholic acid in the presence of Cu(i) catalyst (click chemistry). These novel molecules were evaluated in vitro for their antifungal and antibacterial activity. Most of the compounds exhibited significant antifungal as well as antibacterial activity against all the tested fungal and bacterial strains. Moreover, their in vitro cytotoxicities towards HEK-293 and MCF-7 cells were also established.
Assuntos
Ácido Cólico/síntese química , Ácido Cólico/farmacologia , Ácido Desoxicólico/síntese química , Ácido Desoxicólico/farmacologia , Triazóis/química , beta-Lactamas/química , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/toxicidade , Antifúngicos/síntese química , Antifúngicos/química , Antifúngicos/farmacologia , Antifúngicos/toxicidade , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ácido Cólico/química , Ácido Cólico/toxicidade , Ácido Desoxicólico/química , Ácido Desoxicólico/toxicidade , Dimerização , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Tetrapeptides derived from glycine and beta-alanine were hooked at the C-3beta position of the modified cholic acid to realize novel linear tetrapeptide-linked cholic acid derivatives. All the synthesized compounds were tested against a wide variety of microorganisms (gram-negative bacteria, gram-positive bacteria and fungi) and their cytotoxicity was evaluated against human embryonic kidney (HEK293) and human mammary adenocarcinoma (MCF-7) cell lines. While relatively inactive by themselves, these compounds interact synergistically with antibiotics such as fluconazole and erythromycin to inhibit growth of fungi and bacteria, respectively, at 1-24 microg/mL. The synergistic effect shown by our novel compounds is due to their inherent amphiphilicity. The fractional inhibitory concentrations reported are comparable to those reported for Polymyxin B derivatives.
Assuntos
Antibacterianos/síntese química , Ácido Cólico/química , Peptídeos/química , Antibacterianos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Química Farmacêutica/métodos , Desenho de Fármacos , Eritromicina/farmacologia , Fluconazol/farmacologia , Glicina/química , Humanos , Modelos Químicos , Conformação Molecular , Polimixina B/análogos & derivados , Polimixina B/farmacologia , beta-Alanina/químicaRESUMO
A series of 2-amino-5-oxo-4-phenyl-5,6,7,8-tetrahydroquinoline-3-carbonitrile and various analogues have been synthesized in excellent isolated yields starting from various arylidenemalononitrile and 3-amino-2-cyclohexen-1-one in 1-propanol as solvent at reflux temperature in the absence of any added catalyst. All the synthesized compounds were evaluated for their antifungal activity. The relationship between functional group variation and biological activity of the evaluated compounds is discussed in the article.