RESUMO
The global population of individuals afflicted with diabetes mellitus has been increasing year by year, and this disease poses a serious threat to human health as well as the economies worldwide. Pancreatic or islet transplantations provide one of the most effective and long-term therapies available to treat diabetes, but the scarcity and quality of pancreatic islets limit their use in treatments. Here, we report the development of a one-step, monolayer culture, and chemical-based protocol that efficiently mediates the differentiation of human adipose-derived stem cells (hADSCs) into insulin-producing cells (IPCs). Our data indicate that hADSCs in monolayer culture that are allowed to differentiate into IPCs are superior to those in suspension cultures with respect to insulin secretion capacity (213-fold increase), cell viability (93.5 ± 3.27% vs. 41.67 ± 13.17%), and response to glucose stimulation. Moreover, the expression of genes associated with pancreatic lineage specification, such as PDX1, ISL1, and INS (encoding insulin), were expressed at significantly higher levels during our differentiation protocol (6-fold for PDX1 and ISL1, 11.5-fold for INS). Importantly, in vivo studies demonstrated that transplantation with IPCs significantly mitigated hyperglycemia in streptozotocin-induced diabetic rats. Our results indicate that this one-step, rapid protocol increases the efficiency of IPC generation and that the chemical-based approach for IPC induction may reduce safety concerns associated with the use of IPCs for clinical applications, thereby providing a safe and effective cell-based treatment for diabetes.
Assuntos
Diabetes Mellitus Experimental , Hiperglicemia , Células Secretoras de Insulina , Animais , Diferenciação Celular/fisiologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Humanos , Hiperglicemia/terapia , Insulina/metabolismo , Ratos , Células-Tronco , EstreptozocinaRESUMO
Loss of tumor suppressor activity and upregulation of oncogenic pathways simultaneously contribute to tumorigenesis. Expression of the tumor suppressor, GCIP (Grap2- and cyclin D1-interacting protein), is usually reduced or lost in advanced cancers, as seen in both mouse tumor models and human cancer patients. However, no previous study has examined how cancer cells down-regulate GCIP expression. In this study, we first validate the tumor suppressive function of GCIP using clinical gastric cancer tissues and online database analysis. We then reveal a novel mechanism whereby MEK2 directly interacts with and phosphorylates GCIP at its Ser313 and Ser356 residues to promote the turnover of GCIP by ubiquitin-mediated proteasomal degradation. We also reveal that decreased GCIP stability enhances cell proliferation and promotes cancer cell migration and invasion. Taken together, these findings provide a more comprehensive view of GCIP in tumorigenesis and suggest that the oncogenic MEK/ERK signaling pathway negatively regulates the protein level of GCIP to promote cell proliferation and migration.
Assuntos
Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , MAP Quinase Quinase 2/metabolismo , Sistema de Sinalização das MAP Quinases , Fatores de Transcrição/biossíntese , Proteínas Supressoras de Tumor/biossíntese , Células A549 , Humanos , MAP Quinase Quinase 2/genética , Estabilidade Proteica , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
The epithelial-mesenchymal transition is a major cause of cancer metastasis, and deregulation of the transcription factor, Twist1, is a critical molecular event in the epithelial-mesenchymal transition. The importance of Twist1 protein turnover in this process has not yet been defined. Here, we show that HR23A directly targets the Twist1 protein without changing its gene transcription. Our experiments reveal that: HR23A interacts with Twist1, and this promotes the ubiquitin-mediated proteasomal degradation of Twist1. Depletion of HR23A enhances Twist1 protein levels, epithelial-mesenchymal transition, cancer cell migration and various cancer stemness properties, including the expression of major pluripotency factors, the capacity for tumor-sphere formation in culture and the expression of cancer stem cell surface markers. The increases of these stemness properties are reversed by ectopic expression of HR23A or further knockdown of Twist1 in HR23A-depleted cells. Thus, HR23A-knockdown cells appear to undergo epithelial-mesenchymal transition and take on certain attributes of cancer stemness. Together, our findings indicate that HR23A importantly contributes to regulating Twist1 protein stability, and suggest that altering the stability of Twist1 by modulating HR23A may be a new avenue for therapeutic intervention in cancer.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias Pulmonares/metabolismo , Proteínas Nucleares/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Células A549 , Enzimas Reparadoras do DNA/deficiência , Proteínas de Ligação a DNA/deficiência , Humanos , Neoplasias Pulmonares/patologia , Estabilidade Proteica , Células Tumorais CultivadasRESUMO
BACKGROUND: Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is one of the main pungent components of chili peppers and has been shown to exert various effects on numerous physiological processes. Recent studies have focused on the chemopreventive effects of capsaicin, which can combat growth in various human cancer cell systems. The tribbles-related protein 3 (TRIB3) is evolutionarily conserved from Drosophila to humans. In the latter, TRIB3 is a key determinant in numerous cellular processes, including apoptosis. PURPOSE: The aim of this study was to examine the importance of TRIB3 in the antitumor efficacy of capsaicin in human cancer cells, and further assess potential mechanism(s) underlying the capsaicin-induced upregulation of TRIB3. METHODS: Human cancer cell lines were treated with capsaicin, then evaluated for levels of TRIB3 and molecules related to apoptosis or signaling pathways. The impact of TRIB3 on capsaicin-induced apoptosis was investigated using si-RNA or overexpression of TRIB3. RESULTS: It is the first time to show that TRIB3 is targeted by capsaicin to promote apoptosis. Capsaicin promotes apoptotic cell death by upregulating TRIB3 expression in cancer cells. Overexpression of TRIB3 enhances capsaicin-induced apoptosis, and TRIB3 knockdown experiments demonstrate that the effect of capsaicin in apoptotic cell death is correlated with the induction of TRIB3 in cancer cells. Finally, enhancements in gene expression and protein stability are involved in the capsaicin-induced upregulation of TRIB3. CONCLUSION: Our results show that the capsaicin-induced upregulation of TRIB3 triggers apoptosis and thereby contributes to the suppression of cell growth in cancer cell lines.
RESUMO
Chemotherapeutic agents can upregulate autophagy which contributes to the acquisition of chemoresistance and the recurrence of cancer. The involvement of hHR23A in chemoresistance is unknown. In this study, we provide evidence suggesting that hHR23A may regulate autophagy. Knockdown of hHR23A decreased cell growth and increased the resistance in A549 cells to the DNA-damaging agents, cisplatin and oxaliplatin. Measurement of EGFP-LC3 puncta (a marker of autophagy) revealed that autophagy was increased in hHR23A-depleted cells. This effect was augmented by exposure to cisplatin or oxaliplatin. In contrast, the overexpression of hHR23A reversed the levels of autophagy-related proteins to control levels in hHR23A-knockdown cells. Moreover, we observed direct interactions among hHR23A, Beclin 1, and LC3. Finally, 3-methyladenine (3-MA)-induced inhibition of autophagy was found to reverse the sensitivity of hHR23A-knockdown cells to the tested DNA-damaging agents. These results collectively indicated that hHR23A-depleted cells exhibit enhanced autophagy when treated with DNA-damaging agents, perhaps suggesting a basis for the involvement of hHR23A in the acquired chemoresistance of cancer cells. Our study thus reveals a previously unrecognized autophagic function for hHR23A and suggests that it could be a potential therapeutic target for chemosensitizing resistant cancer cells.
Assuntos
Autofagia/efeitos dos fármacos , Cisplatino/farmacologia , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Compostos Organoplatínicos/farmacologia , Células A549 , Adenina/análogos & derivados , Adenina/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Autofagia/genética , Proteína Beclina-1/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Citometria de Fluxo , Humanos , Immunoblotting , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Oxaliplatina , Ligação Proteica , Interferência de RNARESUMO
Among other functions, the Chk1 protein plays an essential role in coordinating the cellular stress response by determining cell cycle arrest. The levels of Chk1 expression and activity are critical for its functions, especially in cell cycle progression, genomic integrity, cell viability and tissue development. Chk1 protein expression should therefore be tightly controlled both during normal growth and under stress situations. However, it is still unknown how Chk1 protein levels are regulated during normal cell cycle progression. In this study, we show that the effect of hHR23A on Chk1 protein turnover could impact the cell cycle progression observed in hHR23A-knockdown cells. Our results reveal that hHR23A associates with Chk1 through its UBA domains, and that knockdown of hHR23A increases and stabilizes the protein level of Chk1 and its phosphorylation at S347. Knockdown of hHR23A results in proliferation defects and S-phase accumulation. DNA damage reduces the interaction between Chk1 and hHR23A, releasing Chk1 from hHR23A and enhancing S-phase accumulation. Based on these novel findings, we propose that hHR23A acts as a carrier to promote Chk1 degradation through the Ubiquitin Proteasome System. These results strengthen the model in which DNA damage induces Chk1 phosphorylation on chromatin followed by releasing phospho-Chk1 from the chromatin into soluble nucleus and the cytoplasm where Chk1 activates the cell cycle checkpoints; and finally, Chk1 is degraded and checkpoint signaling is terminated.
Assuntos
Pontos de Checagem do Ciclo Celular/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas Quinases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular , Quinase 1 do Ponto de Checagem , Dano ao DNA/genética , Replicação do DNA/genética , Humanos , Fosforilação/genética , Proteínas Quinases/genética , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno/genética , Fase S/genética , Transdução de SinaisRESUMO
The identification of prognostic markers and establishing their value as therapeutic targets improves therapeutic efficacy against human cancers. Ribophorin II (RPN2) has been demonstrated to be a prognostic marker of human cancer, including breast and pancreatic cancers. The present study aimed to evaluate RPN2 expression in gastric cancer and to examine the possible correlation between RPN2 expression and the response of cells to clinical anticancer drugs, which has received little research attention at present. The gastric cancer AGS, TMC-1, SNU-1, TMK-1, SCM-1, MKN-45 and KATO III cell lines were used as a model to elucidate the role of RPN2 in the response of cells to six common chemotherapeutic agents, comprising oxaliplatin, irinotecan, doxorubicin, docetaxel, cisplatin and 5-fluorouricil. The functional role of RPN2 was assessed by silencing RPN2 using small interfering RNA (siRNA), and the cytotoxicity was determined by an MTS assay and analysis of apoptosis. Molecular events were evaluated by western blotting. All the anticancer drugs were found to exert a concentration-dependent decrease on the cell survival rate of each of the cell lines tested, although the RPN2 levels in the various cell lines were not directly correlated with responsiveness to clinical anticancer drugs, based on the calculated IC50 values. siRNA-mediated RPN2 downregulation enhanced cisplatin-induced apoptosis in AGS cells, but did not markedly decrease the cell survival rates of these cells in response to the tested drugs. Furthermore, RPN2 silencing in MKN-45 cells resulted in no additional increase in the cisplatin-induced apoptosis and survival rates. It was also found that RPN2 depletion increased anticancer drug-mediated cytotoxicity in gastric cancer cell lines. However, the predictive value of RPN2 expression in cancer therapy is questionable in gastric cancer models.
RESUMO
Rad23 was identified as a DNA repair protein, although a role in protein degradation has been described. The protein degradation function of Rad23 contributes to cell cycle progression, stress response, endoplasmic reticulum proteolysis, and DNA repair. Rad23 binds the proteasome through a UbL (ubiquitin-like) domain and contains UBA (ubiquitin-associated) motifs that bind multiubiquitin chains. These domains allow Rad23 to function as a substrate shuttle-factor. This property is shared by structurally similar proteins (Dsk2 and Ddi1) and is conserved among the human and mouse counterparts of Rad23. Despite much effort, the regulation of Rad23 interactions with ubiquitinated substrates and the proteasome is unknown. We report here that Rad23 is extensively phosphorylated in vivo and in vitro. Serine residues in UbL are phosphorylated and influence Rad23 interaction with proteasomes. Replacement of these serine residues with acidic residues, to mimic phosphorylation, reduced proteasome binding. We reported that when UbL is overexpressed, it can compete with Rad23 for proteasome interaction and can inhibit substrate turnover. This effect is not observed with UbL containing acidic substitutions, consistent with results that phosphorylation inhibits interaction with the proteasome. Loss of both Rad23 and Rpn10 caused pleiotropic defects that were suppressed by overexpressing either Rad23 or Rpn10. Rad23 bearing a UbL domain with acidic substitutions failed to suppress rad23Δ rpn10Δ, confirming the importance of regulated Rad23/proteasome binding. Strikingly, threonine 75 in human HR23B also regulates interaction with the proteasome, suggesting that phosphorylation is a conserved mechanism for controlling Rad23/proteasome interaction.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Sítios de Ligação/genética , Linhagem Celular Tumoral , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Immunoblotting , Camundongos , Mutação , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Proteólise , Interferência de RNA , Proteínas de Ligação a RNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/genética , Serina/metabolismo , Especificidade por Substrato , Ubiquitina/metabolismoRESUMO
Upregulation of the metastasis-promoting S100A4 protein has been linked to tumor migration and invasion, and clinical studies have demonstrated that significant expression of S100A4 in primary tumors is indicative of poor prognosis. However, the involvement of S100A4 in the drug responsiveness of gastric cancer remains unclear. In the present study, we used gastric cancer cell lines as a model to investigate the involvement of S100A4 in drug responsiveness. We overexpressed S100A4 in AGS and SCM-1 cells, which are characterized by relatively low-level expression of endogenous S100A4, and found that this significantly enhanced cell migration but did not affect cell survival in the presence of six common anticancer drugs. Moreover, in vitro cell proliferation was unchanged. Using RNA interference, we suppressed S100A4 expression in MKN-45 and TMK-1 cells (which are characterized by high-level expression of endogenous S100A4), and found that knockdown of S100A4 markedly attenuated cell motility but did not affect cell survival in the presence of six common anticancer drugs. Further study revealed that a single nucleotide polymorphism (SNP) of S100A4 (rs1803245; c.29A>T), which substitutes an Asp residue with Val (D10V), is localized within the conserved binding surface for Annexin II. Cells overexpressing S100A4D10V showed a significant reduction in cell migration ability, but no change in cell survival, upon anticancer drug treatment. Taken together, our novel results indicate that the expression level of S100A4 does not significantly affect cell survival following anticancer drug treatment. Thus, depending on the cell context, the metastasis-promoting effects of S100A4 may not be positively correlated with anticancer drug resistance in the clinic.
Assuntos
Movimento Celular , Proteínas S100/genética , Neoplasias Gástricas/patologia , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Expressão Gênica , Técnicas de Silenciamento de Genes , Estudos de Associação Genética , Hexosiltransferases , Humanos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteína A4 de Ligação a Cálcio da Família S100 , Proteínas S100/química , Proteínas S100/metabolismo , Neoplasias Gástricas/genéticaRESUMO
BACKGROUND: Many in vitro studies have revealed that the interference of dye molecules in traditional nanoparticle cytotoxicity assays results in controversial conclusions. The aim of this study is to establish an extensive and systematic method for evaluating biological effects of gold nanoparticles in mammalian cell lines. METHODS: We establish the cell-impedance measurement system, a label-free, real-time cell monitoring platform that measures electrical impedance, displaying results as cell index values, in a variety of mammalian cell lines. Cytotoxic effects of gold nanoparticles are also evaluated with traditional in vitro assays. RESULTS: Among the six cell lines, gold nanoparticles induce a dose-dependent suppression of cell growth with different levels of severity and the suppressive effect of gold nanoparticles was indirectly associated with their sizes and cellular uptake. Mechanistic studies revealed that the action of gold nanoparticles is mediated by apoptosis induction or cell cycle delay, depending on cell type and cellular context. Although redox signaling is often linked to the toxicity of nanoparticles, in this study, we found that gold nanoparticle-mediated reactive oxygen species generation was not sustained to notably modulate proteins involved in antioxidative defense system. CONCLUSION: The cell-impedance measurement system, a dye-free, real-time screening platform, provides a reliable analysis for monitoring gold nanoparticle cytotoxicity in a variety of mammalian cell lines. Furthermore, gold nanoparticles induce cellular signaling and several sets of gene expression to modulate cellular physical processes. GENERAL SIGNIFICANCE: The systematic approach, such as cell-impedance measurement, analyzing the toxicology of nanomaterials offers convincing evidence of the cytotoxicity of gold nanomaterials.