RESUMO
Activation of compensatory signaling nodes in cancer often requires combination therapies that are frequently plagued by dose-limiting toxicities. Intestinal lymphatic drug absorption is seldom explored, although reduced toxicity and sustained drug levels would be anticipated to improve systemic bioavailability. A potent orally bioavailable multi-functional kinase inhibitor (LP-182) is described with intrinsic lymphatic partitioning for the combined targeting of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways without observable toxicity. We demonstrate selectivity and therapeutic efficacy through reduction of downstream kinase activation, amelioration of disease phenotypes, and improved survival in animal models of myelofibrosis. Our further characterization of synthetic and physiochemical properties for small molecule lymphatic uptake will support continued advancements in lymphatropic therapy for altering disease trajectories of a myriad of human disease indications.
Assuntos
Antineoplásicos , Mielofibrose Primária , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Humanos , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Mielofibrose Primária/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
Established roles for PI3K and MAPK signaling pathways in tumorigenesis has prompted extensive research towards the discovery of small-molecule inhibitors as cancer therapeutics. However, significant compensatory regulation exists between these two signaling cascades, leading to redundancy among survival pathways. Consequently, initial clinical trials aimed at either PI3K or MEK inhibition alone have proven ineffective and highlight the need for development of targeted and innovative therapeutic combination strategies. We designed a series of PI3K inhibitor derivatives wherein a single morpholine group of the PI3K inhibitor ZSTK474 was substituted with a variety of 2-aminoethyl functional groups. Analogs with pendant hydroxyl or methoxy groups maintained low nanomolar inhibition towards PI3Kα, PI3Kγ, and PI3Kδ isoforms in contrast to those with pendant amino groups which were significantly less inhibitory. Synthesis of prototype PI3K/MEK bifunctional inhibitors (6r, 6s) was guided by the structure-activity data, where a MEK-targeting inhibitor was tethered directly via a short PEG linker to the triazine core of the PI3K inhibitor analogs. These compounds (6r, 6s) displayed nanomolar inhibition towards PI3Kα, δ, and MEK (IC50 â¼105-350 nM), and low micromolar inhibition for PI3Kß and PI3Kγ (IC50 â¼1.5-3.9 µM) in enzymatic inhibition assays. Cell viability assays demonstrated superior anti-proliferative activity for 6s over 6r in three tumor-derived cell lines (A375, D54, SET-2), which correlated with inhibition of downstream AKT and ERK1/2 phosphorylation. Compounds 6r and 6s also demonstrated in vivo tolerability with therapeutic efficacy through reduction of kinase activation and amelioration of disease phenotypes in the JAK2V617F mutant myelofibrosis mouse cancer model. Taken together, these results support further structure optimization of 6r and 6s as promising leads for combination therapy in human cancer as a new class of PI3K/MEK bifunctional inhibitors.
Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Morfolinas/química , Fosfatidilinositol 3-Quinases/química , Inibidores de Fosfoinositídeo-3 Quinase/química , Triazinas/química , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Simulação de Acoplamento Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Mielofibrose Primária/tratamento farmacológico , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Relação Estrutura-Atividade , Triazinas/metabolismo , Triazinas/uso terapêuticoRESUMO
BACKGROUND: Mitochondrial dynamics underlies malignant transformation, cancer progression, and response to treatment. Current research presents conflicting evidence for functions of mitochondrial fission and fusion in tumor progression. Here, we investigated how mitochondrial fission and fusion states regulate underlying processes of cancer progression and metastasis in triple-negative breast cancer (TNBC). METHODS: We enforced mitochondrial fission and fusion states through chemical or genetic approaches and measured migration and invasion of TNBC cells in 2D and 3D in vitro models. We also utilized kinase translocation reporters (KTRs) to identify single cell effects of mitochondrial state on signaling cascades, PI3K/Akt/mTOR and Ras/Raf/MEK/ERK, commonly activated in TNBC. Furthermore, we determined effects of fission and fusion states on metastasis, bone destruction, and signaling in mouse models of breast cancer. RESULTS: Enforcing mitochondrial fission through chemical or genetic approaches inhibited migration, invasion, and metastasis in TNBC. Breast cancer cells with predominantly fissioned mitochondria exhibited reduced activation of Akt and ERK both in vitro and in mouse models of breast cancer. Treatment with leflunomide, a potent activator of mitochondrial fusion proteins, overcame inhibitory effects of fission on migration, signaling, and metastasis. Mining existing datasets for breast cancer revealed that increased expression of genes associated with mitochondrial fission correlated with improved survival in human breast cancer. CONCLUSIONS: In TNBC, mitochondrial fission inhibits cellular processes and signaling pathways associated with cancer progression and metastasis. These data suggest that therapies driving mitochondrial fission may benefit patients with breast cancer.
Assuntos
Transformação Celular Neoplásica/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial/fisiologia , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Animais , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/secundário , Carboxiliases/genética , Carboxiliases/metabolismo , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Feminino , Humanos , Imunossupressores/farmacologia , Leflunomida/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Invasividade Neoplásica , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A method was developed to extract and quantify microcystins (MCs) from mouse liver with limits of quantification (LOQs) lower than previously reported. MCs were extracted from 40-mg liver samples using 85:15 (v:v) CH3CN:H2O containing 200 mM ZnSO4 and 1% formic acid. Solid-phase extraction with a C18 cartridge was used for sample cleanup. MCs were detected and quantified using HPLC-orbitrap-MS with simultaneous MS/MS detection of the 135.08 m/z fragment from the conserved Adda amino acid for structural confirmation. The method was used to extract six MCs (MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, and MC-LW) from spiked liver tissue and the MC-LR cysteine adduct (MC-LR-Cys) created by the glutathione detoxification pathway. Matrix-matched internal standard calibration curves were constructed for each MC (R2 ≥ 0.993), with LOQs between 0.25 ng per g of liver tissue (ng/g) and 0.75 ng/g for MC-LR, MC-RR, MC-YR, MC-LA, and MC-LR-Cys, and 2.5 ng/g for MC-LF and MC-LW. The protocol was applied to extract and quantify MC-LR and MC-LR-Cys from the liver of mice that had been gavaged with 50 µg or 100 µg of MC-LR per kg bodyweight and were euthanized 2 h, 4 h, or 48 h after final gavage. C57Bl/6J (wild type, control) and Leprdb/J (experiment) mice were used as a model to study non-alcoholic fatty liver disease. The Leprdb/J mice were relatively inefficient in metabolizing MC-LR into MC-LR-Cys, which is an important defense mechanism against MC-LR exposure. Trends were also observed as a function of MC-LR gavage amount and time between final MC-LR gavage and euthanasia/organ harvest.
Assuntos
Fígado/química , Microcistinas/análise , Animais , Cromatografia Líquida , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microcistinas/farmacocinética , Espectrometria de Massas em TandemRESUMO
Microcystins are potent hepatotoxins that have become a global health concern in recent years. Their actions in at-risk populations with pre-existing liver disease is unknown. We tested the hypothesis that the No Observed Adverse Effect Level (NOAEL) of Microcystin-LR (MC-LR) established in healthy mice would cause exacerbation of hepatic injury in a murine model (Leprdb/J) of Non-alcoholic Fatty Liver Disease (NAFLD). Ten-week-old male Leprdb/J mice were gavaged with 50 µg/kg, 100 µg/kg MC-LR or vehicle every 48 h for 4 weeks (n = 15-17 mice/group). Early mortality was observed in both the 50 µg/kg (1/17, 6%), and 100 µg/kg (3/17, 18%) MC-LR exposed mice. MC-LR exposure resulted in significant increases in circulating alkaline phosphatase levels, and histopathological markers of hepatic injury as well as significant upregulation of genes associated with hepatotoxicity, necrosis, nongenotoxic hepatocarcinogenicity and oxidative stress response. In addition, we observed exposure dependent changes in protein phosphorylation sites in pathways involved in inflammation, immune function, and response to oxidative stress. These results demonstrate that exposure to MC-LR at levels that are below the NOAEL established in healthy animals results in significant exacerbation of hepatic injury that is accompanied by genetic and phosphoproteomic dysregulation in key signaling pathways in the livers of NAFLD mice.
Assuntos
Fígado/efeitos dos fármacos , Microcistinas/toxicidade , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Estresse Oxidativo/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Modelos Animais de Doenças , Progressão da Doença , Relação Dose-Resposta a Droga , Fígado/metabolismo , Fígado/patologia , Masculino , Toxinas Marinhas , Camundongos , Camundongos Endogâmicos , Microcistinas/sangue , Microcistinas/urina , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Tamanho do Órgão/efeitos dos fármacos , Estresse Oxidativo/genética , Proteômica , Análise de Sobrevida , Poluentes Químicos da Água/sangue , Poluentes Químicos da Água/urinaRESUMO
Microcystins (MCs) appear during harmful algal blooms (HABs) in water sources worldwide, and represent a threat for humans and animals ingesting or inhaling MCs from the environment. Herein, treated rice husk (RH) was tested as a recyclable sorbent for removal of six MCs (MC-RR, MC-LR, MC-YR, MC-LA, MC-LF, and MC-LW) from water. RH was refluxed with hydrochloric acid and heated to 250⯰C to produce the sorbent material. Twenty milligrams of treated RH removed >95% of the MCs from a 30â¯mL solution containing 25⯵g/L of each MC. The adsorption of MCs onto RH follows the Freundlich isotherm model (R2â¯≥â¯0.9612) and pseudo-second-order kinetics (R2â¯≥â¯0.9996). More than 90% of MCs were removed within 5â¯min, and >95% were removed at equilibrium (in <40â¯min). Performance of the RH sorbent was evaluated by removing MCs from Lake Erie water collected during an algal bloom in 2017. The total concentration (extracellular plus intracellular) of six tested MCs in lake water ranged from 3.7 to 13,605.9⯵g/L, and removal of MCs by treated RH ranged from 100.0% to 71.8%, respectively. The removal capacity of RH for the six MCs from the lake water sample containing 13,605.9⯵g/L of MCs was 586⯵g per g of treated RH. After being used to extract MCs, the RH was heated to 560⯰C to produce silica nanoparticles. Therefore, treated RH enables rapid and efficient removal of MCs from water and it can be recycled for use as a raw material. Overall, treated RH can contribute to mitigation of environmental and health effects caused by MCs and reduce concerns for toxic waste disposal.
Assuntos
Água Potável/análise , Lagos/análise , Microcistinas/análise , Oryza/química , Poluentes Químicos da Água/análise , Purificação da Água/métodos , Adsorção , Água Potável/microbiologia , Proliferação Nociva de Algas , Lagos/microbiologia , Purificação da Água/instrumentaçãoRESUMO
The protocols for solid-phase extraction (SPE) of six microcystins (MCs; MC-LR, MC-RR, MC-LA, MC-LF, MC-LW, and MC-YR) from mouse urine, mouse plasma, and human serum are reported. The quantification of those MCs in biofluids was achieved using HPLC-orbitrap-MS in selected-ion monitoring (SIM) mode, and MCs in urine samples were also quantified by ultra-HPLC-triple quadrupole-tandem mass spectrometry (UHPLC-QqQ-MS/MS) in multiple reaction monitoring (MRM) mode. Under optimal conditions, the extraction recoveries of MCs from samples spiked at two different concentrations (1 µg/L and 10 µg/L) ranged from 90.4% to 104.3% with relative standard deviations (RSDs) ≤ 4.7% for mouse urine, 90.4-106.9% with RSDs ≤ 6.3% for mouse plasma, and 90.0-104.8% with RSDs ≤ 5.0% for human serum. Matrix-matched internal standard calibration curves were linear with R2 ≥ 0.9950 for MC-LR, MC-RR and MC-YR, and R2 ≥ 0.9883 for MC-LA, MC-LF, and MC-LW. The limits of quantification (LOQs) in spiked urine samples were â¼0.13 µg/L for MC-LR, MC-RR, and MC-YR, and â¼0.50 µg/L for MC-LA, MC-LF, and MC-LW, while the LOQs in spiked plasma and serum were â¼0.25 µg/L for MC-LR, MC-RR, and MC-YR, and â¼1.00 µg/L for MC-LA, MC-LF, and MC-LW. The developed methods were applied in a proof-of-concept study to quantify urinary and blood concentrations of MC-LR after oral administration to mice. The urine of mice administered 50 µg of MC-LR per kg bodyweight contained on average 1.30 µg/L of MC-LR (n = 8), while mice administered 100 µg of MC-LR per kg bodyweight had average MC-LR concentration of 2.82 µg/L (n = 8). MC-LR was also quantified in the plasma of the same mice. The results showed that increased MC-LR dosage led to larger urinary and plasma MC-LR concentrations and the developed methods were effective for the quantification of MCs in mouse biofluids.
Assuntos
Análise Química do Sangue/métodos , Cromatografia Líquida de Alta Pressão , Microcistinas/sangue , Microcistinas/urina , Extração em Fase Sólida , Espectrometria de Massas em Tandem , Urinálise/métodos , Animais , Humanos , CamundongosRESUMO
A novel sorbent material, gold-polypyrrole (Au-PPy) nanocomposite-coated silica, is described for the efficient solid-phase extraction (SPE) of six common microcystins (MCs) well below the recommended United States EPA and World Health Organization (WHO) guidelines. With the optimized SPE protocol, samples spiked with MCs were determined at ng/L concentrations by liquid chromatography-mass spectrometry (LC-MS) in different aqueous sample matrices, including HPLC-grade, tap, and lake water. The average recoveries for all MCs tested in the three water matrices ranged from 94.1-103.2% with relative standard deviations (RSDs) of 1.6-5.4%, which indicated excellent extraction efficiency and reproducibility. Limits of detection (LODs) and limits of quantification (LOQs) for all MCs in both tap and lake water samples were determined to be ≤1.5â¯ng/L and 5.0â¯ng/L, respectively. The Au-PPy nanocomposite-coated sorbent material was reusable for at least three independent MC extractions with a single SPE cartridge in the concentration range of 10-500â¯ng/L. Importantly, off-column selective separation at the sample preparation and preconcentration stage between more hydrophilic and more hydrophobic MCs was achieved by sequential elution through changes in the solvent composition and SPE bed size. Therefore, the Au-PPy nanocomposite-coated silica sorbent is a promising new material for the quantification of MC variants in water samples.