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1.
Methods Mol Biol ; 2108: 131-146, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31939177

RESUMO

Autophagy is an evolutionarily conserved cellular mechanism in eukaryotes that plays an important role in the maintenance of cellular homeostasis. The autophagy process maintains protein homeostasis by recycling damaged organelles and degrading many long-lived proteins in conjunction with the ubiquitin-proteasome system. Cytokines are low-molecular-weight secreted proteins that regulate a broad range of biological activities. For instance, pro-inflammatory cytokines such as tumor necrosis factor-α (TNFα) induce inflammation, autophagy, and apoptotic cell death. In this chapter, we discuss experimental techniques such as immunoblotting and fluorescence microscopy that can be utilized to measure autophagy in response to TNFα treatment.


Assuntos
Autofagia , Bioensaio/métodos , Fator de Necrose Tumoral alfa/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Citocinas/metabolismo , Humanos , Microscopia de Fluorescência , Complexo de Endopeptidases do Proteassoma/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina
2.
Bioorg Med Chem Lett ; 30(4): 126889, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31902711

RESUMO

Gankyrin is an oncogenic protein involved in various biological processes, such as cellular growth and proliferation. Its overexpression in certain cancers results in an increase of gankyrin-mediated protein-protein interactions (PPIs), leading to cancer proliferation. To date, only one small molecule (cjoc42) has been identified to bind gankyrin, which simultaneously inhibits its interaction with the 26S proteasome. Despite this advance, 2nd generation inhibitors are needed to improve gankyrin binding and cellular efficacy. To this end, an extensive SAR for the aryl sulfonate ester moiety of the cjoc42 scaffold was explored, and showed that substitutions at the 2-, 3-, and 4-positions manifested significant increases in gankyrin binding, resulting in the most potent binders of gankyrin to date. Subsequent cell-based assay evaluation of our derivatives demonstrated antiproliferative activity against pediatric liver cancer cell lines Hep3B and HepG2, which was not previously observed for cjoc42.


Assuntos
Antineoplásicos/química , Benzenossulfonatos/química , Ésteres/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Ácidos Sulfônicos/química , Triazóis/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Benzenossulfonatos/síntese química , Benzenossulfonatos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Triazóis/síntese química , Triazóis/farmacologia
3.
Pharmaceutics ; 11(10)2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31581483

RESUMO

The clinical outcomes of malignant melanoma have improved with the introduction of mitogen-activated protein kinase kinase (MEK) inhibitors. However, off-target toxicities of the MEK inhibitor trametinib (TMB) often result in dose interruption and discontinuation of therapy. The purpose of this study was to anchor a physically stable EphrinA1-mimicking peptide known as YSA (YSAYPDSVPMMS) on TMB-loaded PEGylated nanoliposomes (YTPLs), and evaluate them in BRAFV600E-mutated parent cells (lines A375 and SK-MEL-28) and vemurafenib-resistant cells lines (A375R and SK-MEL-28R) in melanoma. TMB-loaded PEGylated liposomes (TPL) functionalized with nickel-chelated phospholipids were prepared using a modified hydration method. The hydrodynamic diameter and zeta potential values of optimized YTPL were 91.20 ± 12.16 nm and -0.92 ± 3.27 mV, respectively. The drug release study showed TPL did not leak or burst release in 24 h. The hemolysis observed was negligible at therapeutic concentrations of TMB. A differential scanning calorimetry (DSC) study confirmed that TMB was retained in a solubilized state within lipid bilayers. YTPL showed higher intracellular uptake in parental cell lines compared to vemurafenib-resistant cell lines. Western blot analysis and a cytotoxicity study with the EphA2 inhibitor confirmed a reduction in EphA2 expression in resistant cell lines. Thus, EphA2 receptor-targeted nanoliposomes can be useful for metastatic melanoma-specific delivery of TMB.

4.
Exp Cell Res ; 381(2): 288-300, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31128107

RESUMO

Hepatocellular carcinoma (HCC) is one of the leading cancers in the world in incidence and mortality. Current pharmacotherapy of HCC is limited in the number and efficacy of anticancer agents. Metabolic reprogramming is a prominent feature of many cancers and has rekindled interest in targeting metabolic proteins for cancer therapy. Glycogen is a storage form of glucose, and the levels of glycogen have been found to correlate with biological processes in reprogrammed cancer cells. However, the contribution of glycogen metabolism to carcinogenesis, cancer cell growth, metastasis, and chemoresistance is poorly understood. Thus, we studied the processes involved in the inhibition of glycogen metabolism in HCC cells. Pharmacological inhibition of glycogen phosphorylase (GP), a rate-limiting enzyme in glycogen catabolism, by CP-91149 led to a decrease in HCC cell viability. GP inhibition induced cancer cell death through the intrinsic apoptotic pathway. Mitochondrial dysfunction and autophagic adaptations accompanied this apoptosis process whereas endoplasmic reticulum stress, necrosis, and necroptosis were not major components of the cell death. In addition, GP inhibition potentiated the effects of multikinase inhibitors sorafenib and regorafenib, which are key drugs in advanced-stage HCC therapy. Our study provides mechanistic insights into cell death by perturbation of glycogen metabolism and identifies GP inhibition as a potential HCC pharmacotherapy target.


Assuntos
Amidas/farmacologia , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Glicogênio/metabolismo , Indóis/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Amidas/administração & dosagem , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Metabolismo Energético/efeitos dos fármacos , Glicogênio Fosforilase/antagonistas & inibidores , Células Hep G2 , Humanos , Indóis/administração & dosagem , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Piridinas/administração & dosagem , Piridinas/farmacologia , Ratos , Sorafenibe/administração & dosagem , Sorafenibe/farmacologia
5.
PLoS One ; 9(12): e115744, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25548917

RESUMO

OBJECTIVE: Plasma apolipoprotein (apo)D, a ubiquitously expressed protein that binds small hydrophobic ligands, is found mainly on HDL particles. According to studies of human genetics and lipid disorders, plasma apoD levels positively correlate with HDL-cholesterol and apoAI levels. Thus, we tested the hypothesis that apoD was a regulator of HDL metabolism. METHODS & RESULTS: We compared the plasma lipid and lipoprotein profiles of wild-type (WT) C57BL/6 mice with apoD-/- mice on a C57BL/6 background after receiving a high fat-high cholesterol diet for 12 weeks. ApoD-/- mice had higher HDL-cholesterol levels (61±13-apoD-/- vs. 52±10-WT-males; 37±11-apoD-/- vs. 22±2 WT-female) than WT mice with sex-specific changes in total plasma levels of cholesterol and other lipids. Compared to WT, the HDL of apoD-/- mice showed an increase in large, lipid-rich HDL particles and according to size various quantities and sizes of LDL particles. Plasma levels of lecithin:cholesterol acyltransferase in the control and apoD-/- mice were not different, however, plasma phospholipid transfer protein activity was modestly elevated (+10%) only in male apoD-/- mice. An in vivo HDL metabolism experiment with isolated Western-fed apoD-/- HDL particles showed that female apoD-/- mice had a 36% decrease in the fractional catabolic rate of HDL cholesteryl ester. Hepatic SR-BI and LDLR protein levels were significantly decreased; accordingly, LDL-cholesterol and apoB levels were increased in female mice. CONCLUSION: In the context of a high fat-high cholesterol diet, apoD deficiency in female mice is associated with increases in both plasma HDL and LDL-cholesterol levels, reflecting changes in expression of SR-BI and LDL receptors, which may impact diet-induced atherosclerosis.


Assuntos
Apolipoproteínas D/genética , HDL-Colesterol/sangue , LDL-Colesterol/sangue , Dieta Ocidental/efeitos adversos , Animais , Feminino , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Tamanho da Partícula
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