RESUMO
Heterotrimetic G proteins consist of four subfamilies (Gs, Gi/o, Gq/11, and G12/13) that mediate signaling via G-protein-coupled receptors (GPCRs), principally by receptors binding Gα C termini. G-protein-coupling profiles govern GPCR-induced cellular responses, yet receptor sequence selectivity determinants remain elusive. Here, we systematically quantified ligand-induced interactions between 148 GPCRs and all 11 unique Gα subunit C termini. For each receptor, we probed chimeric Gα subunit activation via a transforming growth factor-α (TGF-α) shedding response in HEK293 cells lacking endogenous Gq/11 and G12/13 proteins, and complemented G-protein-coupling profiles through a NanoBiT-G-protein dissociation assay. Interrogation of the dataset identified sequence-based coupling specificity features, inside and outside the transmembrane domain, which we used to develop a coupling predictor that outperforms previous methods. We used the predictor to engineer designer GPCRs selectively coupled to G12. This dataset of fine-tuned signaling mechanisms for diverse GPCRs is a valuable resource for research in GPCR signaling.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Modelos Biológicos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Feminino , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Masculino , Células PC-3 , Receptores Acoplados a Proteínas G/genéticaRESUMO
Mitochondrial dynamics regulated by mitochondrial fusion and fission maintain mitochondrial functions, whose alterations underline various human diseases. Here, we show that inositol is a critical metabolite directly restricting AMPK-dependent mitochondrial fission independently of its classical mode as a precursor for phosphoinositide generation. Inositol decline by IMPA1/2 deficiency elicits AMPK activation and mitochondrial fission without affecting ATP level, whereas inositol accumulation prevents AMPK-dependent mitochondrial fission. Metabolic stress or mitochondrial damage causes inositol decline in cells and mice to elicit AMPK-dependent mitochondrial fission. Inositol directly binds to AMPKγ and competes with AMP for AMPKγ binding, leading to restriction of AMPK activation and mitochondrial fission. Our study suggests that the AMP/inositol ratio is a critical determinant for AMPK activation and establishes a model in which AMPK activation requires inositol decline to release AMPKγ for AMP binding. Hence, AMPK is an inositol sensor, whose inactivation by inositol serves as a mechanism to restrict mitochondrial fission.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Inositol/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas Quinases Ativadas por AMP/fisiologia , Animais , Linhagem Celular , Humanos , Inositol/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Células PC-3 , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Estresse Fisiológico/fisiologiaRESUMO
Macrophages form a major cell population in the tumor microenvironment. They can be activated and polarized into tumor-associated macrophages (TAM) by the tumor-derived soluble molecules to promote tumor progression and metastasis. Here, we used comparative metabolomics coupled with biochemical and animal studies to show that cancer cells release succinate into their microenvironment and activate succinate receptor (SUCNR1) signaling to polarize macrophages into TAM. Furthermore, the results from in vitro and in vivo studies revealed that succinate promotes not only cancer cell migration and invasion but also cancer metastasis. These effects are mediated by SUCNR1-triggered PI3K-hypoxia-inducible factor 1α (HIF-1α) axis. Compared with healthy subjects and tumor-free lung tissues, serum succinate levels and lung cancer SUCNR1 expression were elevated in lung cancer patients, suggesting an important clinical relevance. Collectively, our findings indicate that the secreted tumor-derived succinate belongs to a novel class of cancer progression factors, controlling TAM polarization and promoting tumorigenic signaling.
Assuntos
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Macrófagos/metabolismo , Metástase Neoplásica/patologia , Receptores Acoplados a Proteínas G/metabolismo , Ácido Succínico/metabolismo , Células A549 , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Células HT29 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células MCF-7 , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Células PC-3 , Transdução de Sinais/fisiologia , Microambiente Tumoral/fisiologiaRESUMO
Cytosolic caspase-8 is a mediator of death receptor signaling. While caspase-8 expression is lost in some tumors, it is increased in others, indicating a conditional pro-survival function of caspase-8 in cancer. Here, we show that tumor cells employ DNA-damage-induced nuclear caspase-8 to override the p53-dependent G2/M cell-cycle checkpoint. Caspase-8 is upregulated and localized to the nucleus in multiple human cancers, correlating with treatment resistance and poor clinical outcome. Depletion of caspase-8 causes G2/M arrest, stabilization of p53, and induction of p53-dependent intrinsic apoptosis in tumor cells. In the nucleus, caspase-8 cleaves and inactivates the ubiquitin-specific peptidase 28 (USP28), preventing USP28 from de-ubiquitinating and stabilizing wild-type p53. This results in de facto p53 protein loss, switching cell fate from apoptosis toward mitosis. In summary, our work identifies a non-canonical role of caspase-8 exploited by cancer cells to override the p53-dependent G2/M cell-cycle checkpoint.
Assuntos
Caspase 8/metabolismo , Núcleo Celular/enzimologia , Proliferação de Células , Pontos de Checagem da Fase G2 do Ciclo Celular , Neoplasias/enzimologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina Tiolesterase/metabolismo , Antineoplásicos/farmacologia , Apoptose , Caspase 8/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/patologia , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Masculino , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Células PC-3 , Estabilidade Proteica , Transdução de Sinais , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Ubiquitina Tiolesterase/genéticaRESUMO
Aberrant expression of programmed death ligand-1 (PD-L1) in tumor cells promotes cancer progression by suppressing cancer immunity. The retinoblastoma protein RB is a tumor suppressor known to regulate the cell cycle, DNA damage response, and differentiation. Here, we demonstrate that RB interacts with nuclear factor κB (NF-κB) protein p65 and that their interaction is primarily dependent on CDK4/6-mediated serine-249/threonine-252 (S249/T252) phosphorylation of RB. RNA-seq analysis shows a subset of NF-κB pathway genes including PD-L1 are selectively upregulated by RB knockdown or CDK4/6 inhibitor. S249/T252-phosphorylated RB inversely correlates with PD-L1 expression in patient samples. Expression of a RB-derived S249/T252 phosphorylation-mimetic peptide suppresses radiotherapy-induced upregulation of PD-L1 and augments therapeutic efficacy of radiation in vivo. Our findings reveal a previously unrecognized tumor suppressor function of hyperphosphorylated RB in suppressing NF-κB activity and PD-L1 expression and suggest that the RB-NF-κB axis can be exploited to overcome cancer immune evasion triggered by conventional or targeted therapies.
Assuntos
Antígeno B7-H1/metabolismo , Neoplasias da Próstata/metabolismo , Proteína do Retinoblastoma/metabolismo , Fator de Transcrição RelA/metabolismo , Evasão Tumoral , Animais , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Quimiorradioterapia/métodos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Células PC-3 , Fosforilação , Neoplasias da Próstata/genética , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/terapia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Tolerância a Radiação , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/imunologia , Transdução de Sinais , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The oxidative pentose phosphate pathway (oxiPPP) contributes to cell metabolism through not only the production of metabolic intermediates and reductive NADPH but also inhibition of LKB1-AMPK signaling by ribulose-5-phosphate (Ru-5-P), the product of the third oxiPPP enzyme 6-phosphogluconate dehydrogenase (6PGD). However, we found that knockdown of glucose-6-phosphate dehydrogenase (G6PD), the first oxiPPP enzyme, did not affect AMPK activation despite decreased Ru-5-P and subsequent LKB1 activation, due to enhanced activity of PP2A, the upstream phosphatase of AMPK. In contrast, knockdown of 6PGD or 6-phosphogluconolactonase (PGLS), the second oxiPPP enzyme, reduced PP2A activity. Mechanistically, knockdown of G6PD or PGLS decreased or increased 6-phosphogluconolactone level, respectively, which enhanced the inhibitory phosphorylation of PP2A by Src. Furthermore, γ-6-phosphogluconolactone, an oxiPPP byproduct with unknown function generated through intramolecular rearrangement of δ-6-phosphogluconolactone, the only substrate of PGLS, bound to Src and enhanced PP2A recruitment. Together, oxiPPP regulates AMPK homeostasis by balancing the opposing LKB1 and PP2A.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Gluconatos/metabolismo , Neoplasias/enzimologia , Proteína Fosfatase 2/metabolismo , Células A549 , Quinases Proteína-Quinases Ativadas por AMP , Animais , Proliferação de Células , Ativação Enzimática , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Células HEK293 , Células HT29 , Humanos , Células K562 , Células MCF-7 , Camundongos Nus , Neoplasias/genética , Neoplasias/patologia , Células PC-3 , Via de Pentose Fosfato , Ligação Proteica , Proteína Fosfatase 2/genética , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ribulosefosfatos/metabolismo , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Carga Tumoral , Quinases da Família src/metabolismoRESUMO
The use of androgen receptor (AR) inhibitors in prostate cancer gives rise to increased cellular lineage plasticity resulting in resistance to AR-targeted therapies. In this study, we examined the chromatin landscape of AR-positive prostate cancer cells post-exposure to the AR inhibitor enzalutamide. We identified a novel regulator of cell plasticity, the homeobox transcription factor SIX2, whose motif is enriched in accessible chromatin regions after treatment. Depletion of SIX2 in androgen-independent PC-3 prostate cancer cells induced a switch from a stem-like to an epithelial state, resulting in reduced cancer-related properties such as proliferation, colony formation, and metastasis both in vitro and in vivo. These effects were mediated through the downregulation of the Wnt/ß-catenin signalling pathway and subsequent reduction of nuclear ß-catenin. Collectively, our findings provide compelling evidence that the depletion of SIX2 may represent a promising strategy for overcoming the cell plasticity mechanisms driving antiandrogen resistance in prostate cancer.
Assuntos
Benzamidas , Plasticidade Celular , Proteínas de Homeodomínio , Nitrilas , Feniltioidantoína , Neoplasias da Próstata , Receptores Androgênicos , Via de Sinalização Wnt , beta Catenina , Animais , Humanos , Masculino , Camundongos , Benzamidas/farmacologia , beta Catenina/metabolismo , beta Catenina/genética , Linhagem Celular Tumoral , Plasticidade Celular/genética , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/genética , Nitrilas/farmacologia , Células PC-3 , Feniltioidantoína/farmacologia , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Elevated circulating levels of growth differentiation factor 15 (GDF15) have been shown to reduce food intake and lower body weight through activation of hindbrain receptor glial-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) in rodents and nonhuman primates, thus endogenous induction of this peptide holds promise for obesity treatment. Here, through in silico drug-screening methods, we found that small molecule Camptothecin (CPT), a previously identified drug with potential antitumor activity, is a GDF15 inducer. Oral CPT administration increases circulating GDF15 levels in diet-induced obese (DIO) mice and genetic ob/ob mice, with elevated Gdf15 expression predominantly in the liver through activation of integrated stress response. In line with GDF15's anorectic effect, CPT suppresses food intake, thereby reducing body weight, blood glucose, and hepatic fat content in obese mice. Conversely, CPT loses these beneficial effects when Gdf15 is inhibited by a neutralizing antibody or AAV8-mediated liver-specific knockdown. Similarly, CPT failed to reduce food intake and body weight in GDF15's specific receptor GFRAL-deficient mice despite high levels of GDF15. Together, these results indicate that CPT is a promising anti-obesity agent through activation of GDF15-GFRAL pathway.
Assuntos
Camptotecina/farmacologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator 15 de Diferenciação de Crescimento/genética , Obesidade/prevenção & controle , Animais , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Camptotecina/farmacocinética , Linhagem Celular , Linhagem Celular Tumoral , Dieta Hiperlipídica/efeitos adversos , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator 15 de Diferenciação de Crescimento/metabolismo , Células HEK293 , Células HL-60 , Humanos , Células MCF-7 , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Obesidade/etiologia , Obesidade/genética , Células PC-3RESUMO
SAMHD1 dNTP hydrolase activity places it at the crossroad of several important biological pathways, such as viral restriction, cell cycle regulation, and innate immunity. Recently, a dNTPase independent function for SAMHD1 in homologous recombination (HR) of DNA double-strand breaks has been identified. SAMHD1 function and activity is regulated by several post-translational modifications, including protein oxidation. Here, we showed that oxidation of SAMHD1 increases ssDNA binding affinity and occurs in a cell cycle-dependent manner during S phase consistent with a role in HR. We determined the structure of oxidized SAMHD1 in complex with ssDNA. The enzyme binds ssDNA at the regulatory sites at the dimer interface. We propose a mechanism that oxidation of SAMHD1 acts as a functional switch to toggle between dNTPase activity and DNA binding.
Assuntos
Modelos Moleculares , Proteína 1 com Domínio SAM e Domínio HD , Oxirredução , Proteína 1 com Domínio SAM e Domínio HD/química , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Ligação Proteica , DNA de Cadeia Simples/metabolismo , Estrutura Terciária de Proteína , Células PC-3 , HumanosRESUMO
Raf Kinase Inhibitory Protein (RKIP) maintains cellular robustness and prevents the progression of diseases such as cancer and heart disease by regulating key kinase cascades including MAP kinase and protein kinase A (PKA). Phosphorylation of RKIP at S153 by Protein Kinase C (PKC) triggers a switch from inhibition of Raf to inhibition of the G protein coupled receptor kinase 2 (GRK2), enhancing signaling by the ß-adrenergic receptor (ß-AR) that activates PKA. Here we report that PKA-phosphorylated RKIP promotes ß-AR-activated PKA signaling. Using biochemical, genetic, and biophysical approaches, we show that PKA phosphorylates RKIP at S51, increasing S153 phosphorylation by PKC and thereby triggering feedback activation of PKA. The S51V mutation blocks the ability of RKIP to activate PKA in prostate cancer cells and to induce contraction in primary cardiac myocytes in response to the ß-AR activator isoproterenol, illustrating the functional importance of this positive feedback circuit. As previously shown for other kinases, phosphorylation of RKIP at S51 by PKA is enhanced upon RKIP destabilization by the P74L mutation. These results suggest that PKA phosphorylation at S51 may lead to allosteric changes associated with a higher-energy RKIP state that potentiates phosphorylation of RKIP at other key sites. This allosteric regulatory mechanism may have therapeutic potential for regulating PKA signaling in disease states.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico , Proteína de Ligação a Fosfatidiletanolamina , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Retroalimentação Fisiológica , Humanos , Masculino , Células PC-3 , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fosforilação , Neoplasias da Próstata/metabolismo , Proteína Quinase C/metabolismo , Transdução de SinaisRESUMO
Confining the activity of a designed protein to a specific microenvironment would have broad-ranging applications, such as enabling cell type-specific therapeutic action by enzymes while avoiding off-target effects. While many natural enzymes are synthesized as inactive zymogens that can be activated by proteolysis, it has been challenging to redesign any chosen enzyme to be similarly stimulus responsive. Here, we develop a massively parallel computational design, screening, and next-generation sequencing-based approach for proenzyme design. For a model system, we employ carboxypeptidase G2 (CPG2), a clinically approved enzyme that has applications in both the treatment of cancer and controlling drug toxicity. Detailed kinetic characterization of the most effectively designed variants shows that they are inhibited by â¼80% compared to the unmodified protein, and their activity is fully restored following incubation with site-specific proteases. Introducing disulfide bonds between the pro- and catalytic domains based on the design models increases the degree of inhibition to 98% but decreases the degree of restoration of activity by proteolysis. A selected disulfide-containing proenzyme exhibits significantly lower activity relative to the fully activated enzyme when evaluated in cell culture. Structural and thermodynamic characterization provides detailed insights into the prodomain binding and inhibition mechanisms. The described methodology is general and could enable the design of a variety of proproteins with precise spatial regulation.
Assuntos
Desenho Assistido por Computador , Desenho de Fármacos , Precursores Enzimáticos , Engenharia de Proteínas , gama-Glutamil Hidrolase , Domínio Catalítico , Desenho de Fármacos/métodos , Precursores Enzimáticos/química , Precursores Enzimáticos/farmacologia , Humanos , Células PC-3 , Engenharia de Proteínas/métodos , gama-Glutamil Hidrolase/química , gama-Glutamil Hidrolase/farmacologiaRESUMO
The transcriptional response to hypoxia is largely regulated by the hypoxia-inducible factors (HIFs), which induce the expression of genes involved in glycolysis, angiogenesis, proliferation, and migration. Virtually all cell culture-based hypoxia experiments have used near-atmospheric (18% O2) oxygen levels as the baseline for comparison with hypoxia. However, this is hyperoxic compared with mammalian tissue microenvironments, where oxygen levels range from 2% to 9% O2 (physioxia). Thus, these experiments actually compare hyperoxia to hypoxia. To determine how the baseline O2 level affects the subsequent response to hypoxia, we cultured PC-3 prostate cancer cells in either 18% or 5% O2 for 2 wk before exposing them to hypoxia (â¼1.1% pericellular O2) for 12-48 h. RNA-seq revealed that the transcriptional response to hypoxia was dependent on the baseline O2 level. Cells grown in 18% O2 before hypoxia exposure showed an enhanced induction of HIF targets, particularly genes involved in glucose metabolism, compared with cells grown in physioxia before hypoxia. Consistent with this, hypoxia significantly increased glucose consumption and metabolic activity only in cells previously cultured in 18% O2, but not in cells preadapted to 5% O2. Transcriptomic analyses also indicated effects on cell proliferation and motility, which were followed up by functional assays. Although unaffected by hypoxia, both proliferation and migration rates were greater in cells cultured in 5% O2 versus 18% O2. We conclude that an inappropriately hyperoxic starting condition affects the transcriptional and metabolic responses of PC-3 cells to hypoxia, which may compromise experiments on cancer metabolism in vitro.NEW & NOTEWORTHY Although human cell culture models have been instrumental to our understanding of the mechanisms involved in the cellular response to hypoxia, in virtually all experiments, cells are routinely cultured in near-atmospheric (â¼18% O2) oxygen levels, which are hyperoxic relative to physiological conditions in vivo. Here, we show for the first time that cells cultured in physiological O2 levels (5% O2) respond differently to subsequent hypoxia than cells grown at 18%.
Assuntos
Regulação Neoplásica da Expressão Gênica , Oxigênio , Neoplasias da Próstata , Humanos , Masculino , Oxigênio/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Neoplasias da Próstata/genética , Hipóxia Celular , Proliferação de Células , Glucose/metabolismo , Células PC-3 , Movimento Celular , Glicólise , Linhagem Celular TumoralRESUMO
BACKGROUND: Prostate cancer (PCa) is the second-leading cause of cancer mortalities in the United States and is the most commonly diagnosed malignancy in men. While androgen deprivation therapy (ADT) is the first-line treatment option to initial responses, most PCa patients invariably develop castration-resistant PCa (CRPC). Therefore, novel and effective treatment strategies are needed. The goal of this study was to evaluate the anticancer effects of the combination of two small molecule inhibitors, SZL-P1-41 (SKP2 inhibitor) and PBIT (KDM5B inhibitor), on PCa suppression and to delineate the underlying molecular mechanisms. METHODS: Human CRPC cell lines, C4-2B and PC3 cells, were treated with small molecular inhibitors alone or in combination, to assess effects on cell proliferation, migration, senescence, and apoptosis. RESULTS: SKP2 and KDM5B showed an inverse regulation at the translational level in PCa cells. Cells deficient in SKP2 showed an increase in KDM5B protein level, compared to that in cells expressing SKP2. By contrast, cells deficient in KDM5B showed an increase in SKP2 protein level, compared to that in cells with KDM5B intact. The stability of SKP2 protein was prolonged in KDM5B depleted cells as measured by cycloheximide chase assay. Cells deficient in KDM5B were more vulnerable to SKP2 inhibition, showing a twofold greater reduction in proliferation compared to cells with KDM5B intact (p < 0.05). More importantly, combined inhibition of KDM5B and SKP2 significantly decreased proliferation and migration of PCa cells as compared to untreated controls (p < 0.005). Mechanistically, combined inhibition of KDM5B and SKP2 in PCa cells abrogated AKT activation, resulting in an induction of both cellular senescence and apoptosis, which was measured via Western blot analysis and senescence-associated ß-galactosidase (SA-ß-Gal) staining. CONCLUSIONS: Combined inhibition of KDM5B and SKP2 was more effective at inhibiting proliferation and migration of CRPC cells, and this regimen would be an ideal therapeutic approach of controlling CRPC malignancy.
Assuntos
Apoptose , Senescência Celular , Histona Desmetilases com o Domínio Jumonji , Neoplasias de Próstata Resistentes à Castração , Proteínas Proto-Oncogênicas c-akt , Proteínas Quinases Associadas a Fase S , Transdução de Sinais , Humanos , Proteínas Quinases Associadas a Fase S/metabolismo , Proteínas Quinases Associadas a Fase S/antagonistas & inibidores , Proteínas Quinases Associadas a Fase S/genética , Masculino , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Histona Desmetilases com o Domínio Jumonji/metabolismo , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Histona Desmetilases com o Domínio Jumonji/genética , Senescência Celular/efeitos dos fármacos , Senescência Celular/fisiologia , Transdução de Sinais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Progressão da Doença , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Células PC-3 , Proteínas Nucleares , Proteínas RepressorasRESUMO
Prostate cancer is a prevalent carcinoma among males, and conventional treatment options are often limited. Cytotoxic chemotherapy, despite its drawbacks, remains a mainstay. We propose a targeted co-delivery approach using nanoscale delivery units for Oncolytic measles virus (OMV) and vincristine (VC) to enhance treatment efficacy. The HA-coated OMV + VC-loaded TCs nanoformulation is designed for targeted oncolytic activity in prostate cancer. The CD44 expression analysis in prostate cancer cell lines indicates a significantly high expression in PC3 cells. The optimization of nanoformulations using Design of Expert (DOE) is performed, and the preparation and characterization of HA-coated OMV + VC-loaded TCs nanoformulations are detailed showing average particle size 397.2 ± 0.01 nm and polydispersity index 0.122 with zeta potential 19.7 + 0.01 mV. Results demonstrate successful encapsulation efficiency with 2.4 × 106 TCID50/Ml and sustained release of OMV and VC from the nanoformulation for up to 72 h. In vitro, assays reveal potent anticancer activity at 10 ± 0.71% cell viability in PC3 cells compared to 73 ± 0.66% in HPrEC and significant morphological changes at 90 µg/ml in dose and time-dependent manner. The co-formulation showed positive cell death 49.5 ± 0.02% at 50 µg PI/ml in PBS and 54.3% cell cycle arrest at the G2/M phase, 8.1% G0/G1 and 5.7% at S phase, with significant mitochondrial membrane potential (MMP) at 50 µg/ml, as assessed by flow cytometry (FACS). The surface-integrating ligand approach enhances the targeted delivery of the oncolytic virus and chemotherapeutic drug, presenting a potential alternative for prostate cancer treatment and suggested that co-administering VC and OMV in a nanoformulation could improve therapeutic outcomes while reducing chemotherapeutic drug doses.
Assuntos
Terapia Viral Oncolítica , Vírus Oncolíticos , Neoplasias da Próstata , Vincristina , Humanos , Masculino , Neoplasias da Próstata/terapia , Neoplasias da Próstata/tratamento farmacológico , Vincristina/farmacologia , Vincristina/administração & dosagem , Terapia Viral Oncolítica/métodos , Linhagem Celular Tumoral , Vírus do Sarampo/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Células PC-3RESUMO
Two new lanthanide-complexes based on the 5-nitropicolinate ligand (5-npic) were obtained and fully characterized. Single-crystal X-ray diffraction revealed that these compounds are isostructural to a Dy-complex, previously published by us, based on dinuclear monomers link together with an extended hydrogen bond network, providing a final chemical formula of [Ln2(5-npic)6(H2O)4]·(H2O)2, where Ln = Dy (1), Gd (2), and Tb (3). Preliminary photoluminescent studies exhibited a ligand-centered emission for all complexes. The potential antitumoral activity of these materials was assayed in a prostatic cancer cell line (PC-3; the 2nd most common male cancerous disease), showing a significant anticancer activity (50-60% at 500 µg·mL-1). In turn, a high biocompatibility by both, the complexes and their precursors in human immunological HL-60 cells, was evidenced. In view of the strongest toxic effect in the tumoral cell line provided by the free 5-npic ligand (~ 40-50%), the overall anticancer complex performance seems to be triggered by the presence of this molecule.
Assuntos
Antineoplásicos , Elementos da Série dos Lantanídeos , Ácidos Picolínicos , Humanos , Elementos da Série dos Lantanídeos/química , Elementos da Série dos Lantanídeos/farmacologia , Ácidos Picolínicos/química , Ácidos Picolínicos/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Complexos de Coordenação/síntese química , Masculino , Ensaios de Seleção de Medicamentos Antitumorais , Modelos Moleculares , Células HL-60 , Cristalografia por Raios X , Estrutura Molecular , Linhagem Celular Tumoral , Células PC-3 , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Sobrevivência Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacosRESUMO
This study demonstrates the potential of sono-photodynamic therapy as an effective approach for enhancing singlet oxygen generation using the synthesized Schiff-base diaxially substituted silicon phthalocyanines. In photochemical studies, the singlet oxygen quantum yields (Φ∆) were determined as 0.43 for Si1a, 0.94 for Q-Si1a, 0.58 for S-Si1a, and 0.49 for B-Sia1. In sono-photochemical studies, the Φ∆ values were reached to 0.67 for Si1a, 1.06 for Q-Si1a, 0.65 for S-Si1a, and 0.67 for B-Sia1. In addition, this study demonstrates the therapeutic efficacy of phthalocyanines synthesized as sensitizers on the PC3 prostate cancer cell line through in vitro experiments. The application of these treatment modalities exhibited notable outcomes, leading to a substantial decrease in cell viability within the PC3 prostate cancer cell line. These findings highlight the potential of utilizing these synthesized phthalocyanines as promising therapeutic agents for prostate cancer treatment.
Assuntos
Sobrevivência Celular , Indóis , Compostos de Organossilício , Neoplasias da Próstata , Bases de Schiff , Oxigênio Singlete , Humanos , Indóis/química , Indóis/farmacologia , Bases de Schiff/química , Bases de Schiff/farmacologia , Masculino , Oxigênio Singlete/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Compostos de Organossilício/química , Compostos de Organossilício/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Ensaios de Seleção de Medicamentos Antitumorais , Células PC-3 , Fotoquimioterapia , Processos Fotoquímicos , Linhagem Celular Tumoral , Estrutura MolecularRESUMO
Prostate cancer is a common type of cancer in men with high incidence and mortality. Our aim was to investigate the effects of oxalipalladium (ox-Pd) on metastatic human prostate cancer PC3 cells and compare them with the effects of oxaliplatin (ox-Pt) (as an approved cancer drug). We synthesized ox-Pd through a new chemical method and used FT-IR, 1H NMR, 13C NMR, and MS analyzes to characterize it. The effects of ox-Pd on PC3 cells viability, apoptosis, cell cycle, migration, and gene expression were examined. Inhibition of topoisomerase IIα activity was investigated by pHOT1 plasmid relaxation and kDNA decatenation assays. Chemical tests showed ox-Pd with the correct composition and structure. For the first time, the exact fragmentation pathway of ox-Pd and its difference with ox-Pt was obtained by MS analysis. Ox-Pd significantly decreased PC3 cell viability with less/no toxicity effect on MHFB-1 normal skin fibroblasts. Wound scratch assay confirmed the strong anti-migratory activity of ox-Pd. According to flow cytometry analysis, this drug increased the number of PC3 cells in late apoptosis and decreased DNA replication and mitosis. Furthermore, pHOT1 plasmid relaxation and kDNA decatenation assays showed that ox-Pd strongly inhibited the catalytic activity of topoisomerase IIα. The expression of topoisomerase IIα, Bcl-2, P21, and survivin was decreased while the expression of Bax and p53 was increased under ox-Pd treatment. We provide the first evidence that ox-Pd exhibits more selective anticancer effects on PC3 cells compared to ox-Pt. Taken together, these data strongly suggest a therapeutic window for ox-Pd in cancer.
Assuntos
Antineoplásicos , Apoptose , Sobrevivência Celular , Neoplasias da Próstata , Humanos , Masculino , Antineoplásicos/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Células PC-3 , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Paládio/farmacologia , Paládio/química , Oxaliplatina/farmacologia , Ciclo Celular/efeitos dos fármacosRESUMO
Prostate cancer is a prevalently detected malignancy with a dismal prognosis. Luteinizing-hormone-releasing-hormone (LHRH) receptors are overexpressed in such cancer cells, to which the LHRH-decapeptide can specifically bind. A lipid-polyethylene glycol-conjugated new LHRH-decapeptide analogue (D-P-HLH) was synthesized and characterized. D-P-HLH-coated and anticancer drug doxorubicin (DX)-loaded solid lipid nanoparticles (F-DX-SLN) were formulated by the cold homogenization technique and characterized by Fourier transform infrared spectroscopy, X-ray diffraction, X-ray photoelectron spectroscopy, differential scanning calorimetry, dynamic light scattering, electron microscopy, entrapment efficiency, and drug-release profile studies. F-DX-SLN allows site-specific DX delivery by reducing the side effects of chemotherapy. Cancer cells could precisely take up F-DX-SLN by targeting specific receptors, boosting the cytotoxicity at the tumor site. The efficacy of F-DX-SLN on PC3/SKBR3 cells by the MTT assay revealed that F-DX-SLN was more cytotoxic than DX and/or DX-SLN. Flow cytometry and confocal microscopic studies further support F-DX-SLNs' increased intracellular absorption capability in targeting LHRH overexpressed cancer cells. F-DX-SLN ensured high apoptotic potential, noticeably larger mitochondrial transmembrane depolarization action, as well as the activation of caspases, a longer half-life, and greater plasma concentration. F-DX-SLN/DX-SLN was radiolabeled with technetium-99m; scintigraphic imaging studies established its tumor selectivity in PC3 tumor-bearing nude mice. The efficacy of the formulations in cancer treatment, in vivo therapeutic efficacy tests, and histopathological studies were also conducted. Results clearly indicate that F-DX-SLN exhibits sustained and superior targeted administration of anticancer drugs, thus opening up the possibility of a drug delivery system with precise control and targeting effects. F-DX-SLN could also provide a nanotheranostic approach with improved efficacy for prostate cancer therapy.
Assuntos
Doxorrubicina , Hormônio Liberador de Gonadotropina , Lipídeos , Nanopartículas , Neoplasias da Próstata , Humanos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/patologia , Masculino , Animais , Hormônio Liberador de Gonadotropina/química , Doxorrubicina/administração & dosagem , Doxorrubicina/farmacologia , Doxorrubicina/química , Nanopartículas/química , Camundongos , Linhagem Celular Tumoral , Lipídeos/química , Camundongos Nus , Portadores de Fármacos/química , Polietilenoglicóis/química , Liberação Controlada de Fármacos , Células PC-3 , Receptores LHRH/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Apoptose/efeitos dos fármacosRESUMO
RATIONALE: Natural variations in the abundance of the stable isotopes of nitrogen (δ15N) and carbon (δ13C) offer valuable insights into metabolic fluxes. In the wake of strong interest in cancer metabolism, recent research has revealed δ15N and δ13C variations in cancerous compared to non-cancerous tissues and cell lines. However, our understanding of natural isotopic variations in cultured mammalian cells, particularly in relation to metabolism, remains limited. This study aims to start addressing this gap using metabolic modulations in cells cultured under controlled conditions. METHODS: Prostate cancer cells (PC3) were cultured in different conditions and their δ15N and δ13C were measured using isotope ratio mass spectrometry. Isotopic variations during successive cell culture passages were assessed and two widely used cell culture media (RPMI and DMEM) were compared. Metabolism was modulated through glutamine deprivation and hypoxia. RESULTS: Successive cell culture passages generally resulted in reproducible δ15N and δ13C values. The impact of culture medium composition on δ15N and δ13C of the cells highlights the importance of maintaining a consistent medium composition across conditions whenever possible. Glutamine deprivation and hypoxia induced a lower δ13C in bulk cell samples, with only the former affecting δ15N. Gaps between theory and experiments were bridged and the lessons learned throughout the process are provided. CONCLUSIONS: Exposing cultured cancer cells to hypoxia allowed us to further investigate the relation between metabolic modulations and natural isotopic variations, while mitigating the confounding impact of changing culture medium composition. This study highlights the potential of natural δ13C variations for studying substrate fluxes and nutrient allocation in reproducible culture conditions. Considering cell yield and culture medium composition is pivotal to the success of this approach.
Assuntos
Isótopos de Carbono , Meios de Cultura , Espectrometria de Massas , Isótopos de Nitrogênio , Humanos , Isótopos de Carbono/análise , Isótopos de Carbono/metabolismo , Isótopos de Nitrogênio/análise , Isótopos de Nitrogênio/metabolismo , Espectrometria de Massas/métodos , Meios de Cultura/química , Meios de Cultura/metabolismo , Glutamina/metabolismo , Glutamina/análise , Neoplasias da Próstata/metabolismo , Masculino , Células PC-3 , Linhagem Celular Tumoral , Técnicas de Cultura de Células/métodosRESUMO
PURPOSE: Radiotherapy (RT) is the primary treatment for prostate cancer (PCa); however, the emergence of castration-resistant prostate cancer (CRPC) often leads to treatment failure and cancer-related deaths. In this study, we aimed to explore the use of microwave hyperthermia (MW-HT) to sensitize PCa to RT and investigate the underlying molecular mechanisms. METHODS: We developed a dedicated MW-HT heating setup, created an in vitro and in vivo MW-HT + RT treatment model for CRPC. We evaluated PC3 cell proliferation using CCK-8, colony experiments, DAPI staining, comet assay and ROS detection method. We also monitored nude mouse models of PCa during treatment, measured tumor weight, and calculated the tumor inhibition rate. Western blotting was used to detect DNA damage repair protein expression in PC3 cells and transplanted tumors. RESULTS: Compared to control, PC3 cell survival and clone formation rates decreased in RT + MW-HT group, demonstrating significant increase in apoptosis, ROS levels, and DNA damage. Lower tumor volumes and weights were observed in treatment groups. Ki-67 expression level was reduced in all treatment groups, with significant decrease in RT + MW-HT groups. The most significant apoptosis induction was confirmed in RT + MW-HT group by TUNEL staining. Protein expression levels of DNA-PKcs, ATM, ATR, and P53/P21 signaling pathways significantly decreased in RT + MW-HT groups. CONCLUSION: MW-HT + RT treatment significantly inhibited DNA damage repair by downregulating DNA-PKcs, ATM, ATR, and P53/P21 signaling pathways, leading to increased ROS levels, aggravate DNA damage, apoptosis, and necrosis in PC3 cells, a well-established model of CRPC.