RESUMO
Ruthenium(II/III) metal complexes have been widely recognized as the alternative chemotherapeutic agents to overcome the drug resistance and tumor recurrence associated with platinum derivatives. In this work, a novel ruthenium(II) triazine complex namely, 1 ([Ru(bdpta)(tpy)]2+) was synthesized and spectroscopically characterized. Drug resistant cancer stem cells (CSCs) were used to evaluate the cytotoxicity of Ru(II) complex 1. The complex 1 showed a greater cytotoxic potential with IC50 values lower than that of cisplatin. The intracellular localization assay confirmed that the complex 1 was effectively distributed into mitochondria as well as endoplasmic reticulum (ER), and executed a ROS-mediated calcium and Bax/Bak dependent intrinsic apoptosis. Interestingly, direct interaction between complex 1 and glucose regulated protein 78 (GRP78), a protein associated with drug resistance caused the ROS-mediated ubiquitination of GRP78. Notably, western blot and confocal microscopy analysis confirmed that complex 1 significantly reduced the protein levels of GRP78. Dose-dependent in vivo antitumor efficacy against CD133+HCT-116 CSCs derived tumor xenograft further validated that complex 1 could be an effective chemotherapeutic agent.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Complexos de Coordenação/uso terapêutico , Proteínas de Choque Térmico/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Rutênio/uso terapêutico , Triazinas/uso terapêutico , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Complexos de Coordenação/análogos & derivados , Complexos de Coordenação/farmacologia , Chaperona BiP do Retículo Endoplasmático , Feminino , Células HEK293 , Proteínas de Choque Térmico/química , Humanos , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Simulação de Acoplamento Molecular , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Espécies Reativas de Oxigênio/metabolismo , Rutênio/química , Rutênio/farmacologia , Triazinas/química , Triazinas/farmacologiaRESUMO
Degradation of cyclic adenosine mono phosphate (cAMP) by phosphodiesterase-4B (PDE-4B) in the inflammatory cells leads to elevated expression of inflammatory cytokines in inflammatory cells. Suppression of cytokines has proved to be beneficial in the treatment of atopic dermatitis (AD). Henceforth, application of PDE4B specific inhibitor to minimize the degradation of cAMP can yield better results in the treatment of AD. PDE4B specific inhibitor with a limited side effect is highly warranted. Herein, we synthesized a novel PDE4 inhibitor, compound 2 comprising catecholopyrimidine core functionalized with trifluoromethyl (-CF3) group. PDE4B inhibitory potential and specificity of novel compounds were evaluated by PDE inhibitor assay. In vivo efficacy of the compounds was analyzed using DNCB-induced NC/Nga mice. IgE, CD4+ T-helper cell infiltration, and cytokine profiles were analyzed by ELISA and immunohistochemistry techniques. Toluidine blue staining was performed for mast cell count. PDE4 inhibitor assay confirmed that compound 2 specifically inhibits PDE4B. In vivo analysis with DNCB-induced NC/Nga mice confirmed that compound 2 suppressed the levels of pro-inflammatory cytokines such as TNF-α, IL-4, IL-5, and IL-17. Furthermore, compound 2 significantly reduced the infiltrative CD4+ T-helper cells, mast cells and IgE levels in atopic tissue. The in vitro and in vivo data suggested that compound 2 specifically inhibit the PDE4B and the symptoms of the AD in atopic mice. Compound 2 might constitute a good candidate molecule for the treatment of AD.
RESUMO
Selective inhibition of phosphodiesterase (PDE) 4B favorably suppresses the synthesis of inflammatory cytokines and subsequently arrest the development of atopic dermatitis via modulating the intracellular cAMP levels. Considering the side effects of corticosteroids, selective PDE4 inhibition could constitute an effective alternative therapy for the treatment of atopic dermatitis (AD). In this study, a series of novel catechol based compounds bearing pyrimidine as the core have been synthesized and screened for the PDE4 inhibitory properties. The PDE4 selectivity of the active compounds over other PDEs has been investigated. Compound 23 bearing pyrimidine core functionalized with catechol, pyridine and trifluoromethyl groups can effectively inhibit the PDE4B with IC50 value in nanomolar range (IC50â¯=â¯15⯱â¯0.4â¯nM). Compound 23 exhibited seven fold higher selectivity towards PDE4B over PDE4D. Molecular Docking study confirmed its stronger affinity towards catalytic domain of PDE4B. In-vivo analysis confirmed that compound 23 effectively alleviated the symptoms of atopic dermatitis in DNCB-treated Balb/c mice by suppressing the synthesis of inflammatory mediators such as TNF-α, and Ig-E. Taken together, this study suggested that compound 23 could be an effective PDE4 inhibitor for the potential treatment of AD.
Assuntos
Catecóis/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Dermatite Atópica/tratamento farmacológico , Desenho de Fármacos , Inibidores da Fosfodiesterase 4/farmacologia , Pirimidinas/farmacologia , Animais , Catecóis/síntese química , Catecóis/química , Cristalografia por Raios X , Dermatite Atópica/metabolismo , Dermatite Atópica/patologia , Relação Dose-Resposta a Droga , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Estrutura Molecular , Inibidores da Fosfodiesterase 4/síntese química , Inibidores da Fosfodiesterase 4/química , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
Primary cell cultures mimic the physiology and genetic makeup of in-vivo tissue of origin, nonetheless, a complication in the derivation and propagation of primary cell culture limits its use in biological research. However, in-vitro models using primary cells might be a complement model to mimic in vivo response. But, conventional techniques such as western blot and PCR employed to study the expression and activation of proteins requires a large number of cells, hence repeated establishment and maintenance of primary culture are unavoidable. Quantum dot (Q-dot) and acousto-optic tunable filters (AOTF) based multiplex imaging system is a viable alternative choice to evaluate multiple signaling molecules by using a small number of cells. Q-dots have broad excitation and narrow emission spectra, which allows to simultaneously excite multiple Q-dots by using single excitation wavelength. The use of AOTF in the fluorescence detection system enables to scan the fluorescence emission intensity of a Q-dot at their central wavelength, this phenomenon effectively avoids spectral overlap among the neighboring Q-dots. When Q-dots are conjugated with antibodies it acts as effective sensing probes. To validate this, the expression pattern of p-JNK-1, p-GSK3ß, p-IRS1ser, p-IRS1tyr, p-FOXO1, and PPAR-γ, involved in the insulin resistance were concurrently monitored in adipocyte and HepG2 co-cell culture model. The observed results clearly indicate that PPAR-γ is the critical component in the development of insulin resistance. Moreover, the results proved that developed Q-dot based AOTF imaging methodology is a sensible choice to concurrently monitor multiple signaling molecules with limited cell population.
Assuntos
Nanoestruturas , Óptica e Fotônica , Pontos Quânticos , Adipócitos/metabolismo , Fenômenos Fisiológicos Celulares , Cor , Células Hep G2 , Humanos , Resistência à Insulina , PPAR gama/metabolismoRESUMO
The existence of a well-established drug resistance mechanism in cancer stem cells (CSC) complicates the cancer treatment. Clusterin (CLU) plays a key role in maintaining the integrity of endoplasmic reticulum (ER) during drug-induced stress. Hence, silencing the CLU could significantly reduce the inherent drug resistance mechanism of CSC. The combination of drug-induced cytotoxicity, as well as the suppression of drug resistance in CSC, could circumvent the recurrence capability of the tumor. In the present study, camptothecin (CPT)-induced apoptosis and necrosis in CSC with and without siCLU treatment were simultaneously measured using Qdot-based total internal reflection fluorescence microscope (TIRF). In addition, to elucidate the mechanism of CPT-induced cytotoxicity in CLU-suppressed CSC, expression of Bcl-2, Bax, Bak, and PARP and mitochondrial permeability transition pore (MPTP) were studied. EC50 values of CPT-induced apoptosis and necrosis were significantly reduced (p < 0.01) in CLU-suppressed MCF-7 and CSC. Significantly increased MPTP (p < 0.001) and cytosolic Ca2+ (p < 0.001) were observed in CPT-treated CLU-suppressed CSC as compared to the normal CSC. Elevated expression of Bax, Bak, and cleaved PARP and reduced expression of Bcl-2 and cytosolic ATP were observed in CPT-treated CLU-suppressed CSC. Observed results indicate that silencing the expression of CLU could improve the anticancer efficacy of CPT at 128.4-nM concentration by equally inducing necrotic signals along with apoptosis. Furthermore, the developed high content TIRF assay based on the CLU-suppressed CSC could be an ideal and beneficial tool for rapidly analyzing the cytotoxicity of anti-cancer agents.
Assuntos
Antineoplásicos/farmacologia , Camptotecina/farmacologia , Clusterina/genética , Dinâmica Mitocondrial/genética , Necrose/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Células MCF-7 , Dinâmica Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/genética , Poro de Transição de Permeabilidade MitocondrialRESUMO
Cadmium, a heavy metal pollutant, causes cancer. The existence of cancer stem cells (CSCs) in tumors is widely considered to be the reason for the recurrence and treatment failure of cancer. Increasing evidence has confirmed that under certain conditions non-CSCs could be converted into CSCs. The impact of cadmium on the development of CSC lineage in the bulk tumor cell population is not yet studied. The aim of this study was to evaluate the effect of cadmium on the conversion of non-CSCs to CSCs and the identification of CSCs based on the concurrent monitoring of multiple CSC markers. High-content monitoring of molecular markers was performed using quantum dot (QD) nanoprobes and an acousto-optical tunable filter (AOTF)-based imaging device. Cadmium treatment significantly increased the CSC population in MCF-7 and HepG2 cell lines. The cadmium-induced CSCs were identified by a concurrent analysis of stem-cell markers, namely, CD44, CD24, CD133, and ALDH1. Moreover, increased m-RNA expression of CD44, ALDH1, and CD133 and protein expression of p-Ras, p-Raf-1, p-MEK-1, and p-ERK-1 were observed in the cadmium-treated MCF-7 and HepG2 cells. This study demonstrates that cadmium induces the gene expression of CSC markers in the breast and liver cancer cell lineage and promotes the conversion of non-CSCs to CSCs.
RESUMO
Elevated expression of drug efflux pumps such as multidrug resistant protein-1 (MDR1/ABCB1) and multidrug resistance associated protein-1 (MRP1/ABCC1) in cancer stem cells (CSCs) among a bulky tumor cell population was attributed to drug resistance. For the first time, we have quantitatively evaluated the cytotoxic profile of camptothecin (CPT) against the CSC. In the present study, a Qdot based total internal reflection fluorescence (TIRF) detection system effectively interpreted that drug resistance to CPT was reduced in the CSC under ABCB1 inhibited conditions. This study revealed that quantitative finding of the EC50 value for apoptosis and necrosis in correlation with the ABC inhibitor and CSC population using TIRF could provide more details of the anti-cancer efficacy of chemotherapeutic agents.
Assuntos
Camptotecina/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Imagem Molecular/métodos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos Fitogênicos/administração & dosagem , Humanos , Células MCF-7 , Microscopia de Fluorescência/métodos , Microscopia de Interferência/métodos , Células-Tronco Neoplásicas/patologia , Pontos QuânticosRESUMO
Elevated expression of epidermal growth factor receptor (EGFR) is reported to be associated with poor prognosis in breast cancer. EGFR subtype identification plays a crucial role in deciding the drug combination to treat the cancer patients. Conventional application of immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) produces more discordance results in EGFR subtype identification of cancer specimens. The present study is designed to develop an analytical method for simultaneous identification of cell surface biomarkers and quantitative estimation of drug efficacy in cancer specimens. For this study, we have utilized a total internal reflection fluorescence microscope (TIRFM), Qdot molecular probes and chemotherapeutic agent camptothecin (CPT)-treated breast cancer cell lines namely MCF-7, SK-BR-3 and JIMT-1. Highly sensitive detection signals with low background noise generated from the evanescent field excitation of TIRFM make it a highly suitable tool to detect the cell surface biomarkers in living cells. Moreover, single wavelength excitation of Qdot probes offers multicolour imaging with strong emission brightness. In the present study, TIRF high-content imaging system simultaneously showed the expression pattern of EGFRs and EC50 value for CPT-induced apoptosis and necrosis in MCF-7, SK-BR-3 and JIMT-1 cancer cell lines.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Receptores ErbB/metabolismo , Genes erbB-2 , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , HumanosRESUMO
Cancer stem cells (CSCs), known as tumor initiating cells, have become a critically important issue for cancer therapy. Although much research has demonstrated the induction of hepato cellular carcinoma by aflatoxin B1, the formation of hepatocellular CSCs and their quantitative determination is hardly reported. In this work, it was found that hepatocellular CSCs were produced from HepG2 cells by aflatoxin B1-induced mutation, and their amount was quantitatively determined using crosstalk-eliminated multicolor cellular imaging based on quantum dot (Qdot) nanoprobes and an acousto-optical tunable filter (AOTF). Hepatocellular CSCs were acquired via magnetic bead-based sorting and observed using concurrent detection of three different markers: CD133, CD44, and aldehyde dehydrogenase1 (ALDH1). The DNA mutation of HepG2 cells caused by aflatoxin B1 was quantitatively observed via absorbance spectra of aflatoxin B1-8, 9-epoxide-DNA adducts. The percentages of hepatocellular CSCs formed in the entire HepG2 cells were determined to be 9.77±0.65%, 10.9±1.39%, 11.4±1.32%, and 12.8±0.7%, respectively, at 0 µM, 5 µM, 10 µM, and 20 µM of aflatoxin B1. The results matched well with those obtained utilizing flow cytometry. This study demonstrates that aflatoxin mediated mutation induced the conversion of hepatic cancer cell to hepatic CSCs by using a Qdot based constructed multicolor cellular imaging system.