RESUMO
Ovarian cancer continues to be the most lethal among gynecological malignancies and the major cause for cancer-associated mortality among women. Limitations of current ovarian cancer therapeutics is highlighted by the high frequency of drug-resistant recurrent tumors and the extremely poor 5-year survival rates. Zinc oxide nanoparticles (ZnO-NPs) have shown promise in various biomedical applications including utility as anti-cancer agents. Here, we describe the synthesis and characterization of physical properties of ZnO-NPs of increasing particle size (15 nm - 55 nm) and evaluate their benefits as an ovarian cancer therapeutic using established human ovarian cancer cell lines. Our results demonstrate that the ZnO-NPs induce acute oxidative and proteotoxic stress in ovarian cancer cells leading to their death via apoptosis. The cytotoxic effect of the ZnO-NPs was found to increase slightly with a decrease in nanoparticle size. While ZnO-NPs caused depletion of both wild-type and gain-of-function (GOF) mutant p53 protein in ovarian cancer cells, their ability to induce apoptosis was found to be independent of the p53-mutation status in these cells. Taken together, these results highlight the potential of ZnO-NPs to serve as an anti-cancer therapeutic agent for treating ovarian cancers independent of the p53 mutants of the cancer cells.
RESUMO
BACKGROUND: The androgen-regulated homeodomain transcription factor NKX3.1 plays roles in early prostate development and functions as a prostate-specific tumor suppressor. Decreased expression of NKX3.1 protein is common in primary prostate cancer. Discordance between NKX3.1 mRNA and protein levels during prostate carcinogenesis suggested a key role for post-transcriptional modifications in regulating NKX3.1 protein levels in prostate epithelial cells. Subsequent studies revealed NKX3.1 to be modified post-translationally at multiple sites. METHODS: We reviewed published literature to identify and summarize post-translational modifications and structural elements critical in regulating NKX3.1 stability and levels in prostate epithelial cells. RESULTS: NKX3.1 is modified post-translationally at multiple sites by different protein kinases. These modifications together with several structural determinants were identified to play an important role in NKX3.1 stability and biology. CONCLUSIONS: In this review, we provide a comprehensive overview of the known post-translational modifications and structural features that impact NKX3.1. Defining factors that regulate NKX3.1 in prostate epithelial cells will extend our understanding of molecular changes that may contribute to prostate cancer initiation and progression.
Assuntos
Proteínas de Homeodomínio/metabolismo , Neoplasias da Próstata/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Fatores de Transcrição/metabolismo , Genes Supressores de Tumor/fisiologia , Humanos , Masculino , Fosforilação , Neoplasias da Próstata/patologia , Estabilidade Proteica , UbiquitinaçãoRESUMO
MYC levels are tightly regulated in cells, and deregulation is associated with many cancers. In this report, we describe the existence of a MYC-protein kinase A (PKA)-polo-like kinase 1 (PLK1) signaling loop in cells. We report that sequential MYC phosphorylation by PKA and PLK1 protects MYC from proteasome-mediated degradation. Interestingly, short term pan-PKA inhibition diminishes MYC level, whereas prolonged PKA catalytic subunit α (PKACα) knockdown, but not PKA catalytic subunit ß (PKACß) knockdown, increases MYC. We show that the short term effect of pan-PKA inhibition on MYC is post-translational and the PKACα-specific long term effect on MYC is transcriptional. These data also reveal distinct functional roles among PKA catalytic isoforms in MYC regulation. We attribute this effect to differential phosphorylation selectivity among PKA catalytic subunits, which we demonstrate for multiple substrates. Further, we also show that MYC up-regulates PKACß, transcriptionally forming a proximate positive feedback loop. These results establish PKA as a regulator of MYC and highlight the distinct biological roles of the different PKA catalytic subunits.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Domínio Catalítico , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Chlorocebus aethiops , Humanos , Dados de Sequência Molecular , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Quinase 1 Polo-LikeRESUMO
Loss of NKX3.1 is an early and consistent event in prostate cancer and is associated with increased proliferation of prostate epithelial cells and poor prognosis. NKX3.1 stability is regulated post-translationally through phosphorylation at multiple sites by several protein kinases. Here, we report the paradoxical stabilization of the prostate-specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim-1 in prostate cancer cells. Pharmacologic Pim-1 inhibition using the small molecule inhibitor CX-6258 decreased steady state levels and half-life of NKX3.1 protein but mRNA was not affected. This effect was reversed by inhibition of the 26S-proteasome, demonstrating that Pim-1 protects NKX3.1 from proteasome-mediated degradation. Mass spectrometric analyses revealed Thr89, Ser185, Ser186, Ser195, and Ser196 as Pim-1 phospho-acceptor sites on NKX3.1. Through mutational analysis, we determined that NKX3.1 phosphorylation at Ser185, Ser186, and within the N-terminal PEST domain is essential for Pim-1-mediated stabilization. Further, we also identified Lys182 as a critical residue for NKX3.1 stabilization by Pim-1. Pim-1-mediated NKX3.1 stabilization may be important in maintaining normal cellular homeostasis in normal prostate epithelial cells, and may maintain basal NKX3.1 protein levels in prostate cancer cells.
Assuntos
Proteínas de Homeodomínio/metabolismo , Próstata/enzimologia , Próstata/patologia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Azepinas/farmacologia , Linhagem Celular Tumoral , Análise Mutacional de DNA , Meia-Vida , Humanos , Indóis/farmacologia , Lisina/metabolismo , Masculino , Modelos Biológicos , Proteínas Mutantes/metabolismo , Especificidade de Órgãos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Estabilidade Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Especificidade por Substrato/efeitos dos fármacosRESUMO
Ovarian theca androgen production is regulated by the pituitary LH and intrafollicular factors. Enhanced androgen biosynthesis by theca cells contributes to polycystic ovary syndrome (PCOS) in women, but the ovarian consequences of elevated androgens are not completely understood. Our study documents the molecular events that are altered in the theca and stromal cells of mice exposed to high androgen levels, using the nonaromatizable androgen DHT. Changes in ovarian morphology and function were observed not only in follicles, but also in the stromal compartment. Genome-wide microarray analyses revealed marked changes in the ovarian transcriptome of DHT-treated females within 1 week. Particularly striking was the increased expression of vascular cell adhesion molecule 1 (Vcam1) specifically in the NR2F2/COUPTF-II lineage theca cells, not granulosa cells, of growing follicles and throughout the stroma of the androgen-treated mice. This response was mediated by androgen receptors (ARs) present in theca and stromal cells. Human theca-derived cultures expressed both ARs and NR2F2 that were nuclear. VCAM1 mRNA and protein were higher in PCOS-derived theca cells compared with control theca and reduced markedly by the AR antagonist flutamide. In the DHT-treated mice, VCAM1 was transiently induced by equine chorionic gonadotropin, when androgen and estrogen biosynthesis peak in preovulatory follicles, and was potently suppressed by a superovulatory dose of human chorionic gonadotropin. High levels of VCAM1 in the theca and interstitial cells of DHT-treated mice and in adult Leydig cells indicate that there may be novel functions for VCAM1 in reproductive tissues, including the gonads.
Assuntos
Di-Hidrotestosterona , Hiperandrogenismo/metabolismo , Folículo Ovariano/metabolismo , Ovário/metabolismo , Células Estromais/metabolismo , Células Tecais/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Fator II de Transcrição COUP/metabolismo , Feminino , Hiperandrogenismo/induzido quimicamente , Camundongos , Receptores Androgênicos/metabolismoRESUMO
Gain-of-function p53 mutants such as p53-R175H form stable aggregates that accumulate in cells and play important roles in cancer progression. Selective degradation of gain-of-function p53 mutants has emerged as a highly attractive therapeutic strategy to target cancer cells harboring specific p53 mutations. We identified a small molecule called MCB-613 to cause rapid ubiquitination, nuclear export, and degradation of p53-R175H through a lysosome-mediated pathway, leading to catastrophic cancer cell death. In contrast to its effect on the p53-R175H mutant, MCB-613 causes slight stabilization of p53-WT and has weaker effects on other p53 gain-of-function mutants. Using state-of-the-art genetic and chemical approaches, we identified the deubiquitinase USP15 as the mediator of MCB-613's effect on p53-R175H, and established USP15 as a selective upstream regulator of p53-R175H in ovarian cancer cells. These results confirm that distinct pathways regulate the turnover of p53-WT and the different p53 mutants and open new opportunities to selectively target them.
Assuntos
Regulação Neoplásica da Expressão Gênica , Lisossomos/metabolismo , Neoplasias Ovarianas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Linhagem Celular Tumoral , Cicloeximida/química , Feminino , Células HEK293 , Humanos , Células MCF-7 , Mutação , Plasmídeos/metabolismo , Receptores de Esteroides/metabolismoRESUMO
Targeted intracellular protein degradation in eukaryotes is largely mediated by the proteasome. Here, we report the formation of an alternative proteasome isoform in human cells, previously found only in budding yeast, that bears an altered subunit arrangement in the outer ring of the proteasome core particle. These proteasomes result from incorporation of an additional α4 (PSMA7) subunit in the position normally occupied by α3 (PSMA4). Assembly of "α4-α4" proteasomes depends on the relative cellular levels of α4 and α3 and on the proteasome assembly chaperone PAC3. The oncogenic tyrosine kinases ABL and ARG and the tumor suppressor BRCA1 regulate cellular α4 levels and formation of α4-α4 proteasomes. Cells primed to assemble α4-α4 proteasomes exhibit enhanced resistance to toxic metal ions. Taken together, our results establish the existence of an alternative mammalian proteasome isoform and suggest a potential role in enabling cells to adapt to environmental stresses.
Assuntos
Evolução Biológica , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína BRCA1/metabolismo , Cloreto de Cádmio/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Elucidating kinase-substrate relationships is critical for understanding how phosphorylation affects signal transduction and regulatory cascades. Using the alpha catalytic subunit of protein kinase CK2 (CK2alpha) as a paradigm, we developed an in-gel method to facilitate identification of physiologic kinase substrates. In this approach, the roles of kinase and substrate in a classic in-gel kinase assay are reversed. In the reverse in-gel kinase assay (RIKA), a kinase is copolymerized in a denaturing polyacrylamide gel used to resolve a tissue or cell protein extract. Restoration of kinase activity and substrate structure followed by an in situ kinase reaction and mass spectrometric analyses results in identification of potential kinase substrates. We demonstrate that this method can be used to profile both known and novel human and mouse substrates of CK2alpha and cAMP-dependent protein kinase (PKA). Using widely available straightforward technology, the RIKA has the potential to facilitate discovery of physiologic kinase substrates in any biological system.