RESUMO
Introduction: Adoption of allogeneic T cells directly supplements the number of T cells and rapidly induces T-cell immunity, which has good efficacy for treating some tumors and immunodeficiency diseases. However, poor adoptive T-cell engraftment and graft-versus-host disease (GVHD) limit the application of these methods. Alloreactive T-cell clones were eliminated from the donor T-cell repertoire, and the remaining T-cell clones were prepared as Tscm for T-cell adoptive treatment to reconstruct recipient T-cell immunity without GVHD. Methods: The subjects in this study included three different strains of mice. Lymphocytes from mice (C57BL/6) were used as the donor T-cell repertoire, from which the Tscm allo-reactive T cell clone was depleted (ATD-Tscm). This was confirmed by showing that the Tscm was not responsive to the alloantigen of the recipient (BALB/c). To prepare ATD-Tscm cells, we used recipient lymphocytes as a simulator, and coculture of mouse and recipient lymphocytes was carried out for 7 days. Sorting of non-proliferative cells ensured that the prepared Tscm cells were nonresponsive. The sorted lymphocytes underwent further expansion by treatment with TWS119 and cytokines for an additional 10 days, after which the number of ATD-Tscm cells increased. The prepared Tscm cells were transferred into recipient mice to observe immune reconstitution and GVHD incidence. Results: Our protocol began with the use of 1×107 donor lymphocytes and resulted in 1 ×107 ATD-Tscm cells after 17 days of preparation. The prepared ATD-Tscm cells exhibited a nonresponse upon restimulation of the recipient lymphocytes. Importantly, the prepared ATD-Tscm cells were able to bind long and reconstitute other T-cell subsets in vivo, effectively recognizing and answering the "foreign" antigen without causing GVHD after they were transferred into the recipients. Discussion: Our strategy was succeeded to prepare ATD-Tscm cells from the donor T-cell repertoire. The prepared ATD-Tscm cells were able to reconstitute the immune system and prevent GVHD after transferred to the recipients. This study provides a good reference for generating ATD-Tscm for T-cell adoptive immunotherapy.
Assuntos
Doença Enxerto-Hospedeiro , Camundongos Endogâmicos C57BL , Animais , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Camundongos , Linfócitos T/imunologia , Camundongos Endogâmicos BALB C , Imunoterapia Adotiva/métodos , Transplante Homólogo , Transferência Adotiva/métodos , Reconstituição Imune , Modelos Animais de DoençasRESUMO
The poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) Olaparib is a widely used targeted therapy for a variety of solid tumors with homologous recombination deficiency (HRD) caused by mutation of BRCA1/2 or other DNA repair genes. The anti-tumor activity of Olaparib has been largely attributed to its ability to inhibit PARP enzymes and block DNA single-strand break (SSB) repair, which eventually leads to the most detrimental DNA damage, double-strand breaks (DSB), in HRD cells. Although PARPi was found to induce p53-dependent cell death, the underlying molecular mechanism remains incompletely understood. Here, we report that Olaparib treatment leads to p53 stabilization and activation of its downstream target genes in a dose- and time-dependent manner. Mechanistically, Olaparib triggers nucleolar stress by inhibiting biosynthesis of the precursor of ribosomal RNAs (pre-rRNA), resulting in enhanced interaction between ribosomal proteins (RPs), RPL5 and RPL11, and MDM2. Consistently, knockdown of RPL5 and RPL11 prevents Olaparib-induced p53 activation. More importantly, Olaparib efficiently suppresses breast and colorectal cancer cell survival and proliferation through activation of p53. Altogether, our study demonstrates that Olaparib activates the nucleolar stress-RPs-p53 pathway, suggesting rRNA biogenesis as a novel target for PARPi.
RESUMO
Cysteine (Cys) as a vital antioxidant molecule and an effective biomarker for illness, plays an essential role in physiological functions and pathological processes. Extensive work has been done to explore the physiological functions of Cys and develop probes for detection of biothiols. However, the challenge to differentiate Cys from glutathione and homocystine remains. In this work, we constructed a novel near-infrared (NIR) probe, termed TMN-Cys, using TMN-NH2 and thionoesters. The probe could selectively detect Cys over homocysteine and glutathione in solution. It displayed a large Stokes shift (210 nm) upon treatment with Cys, and its detection limit was as low as 79 nM. Moreover, this probe showed low toxicity and was successfully employed in monitoring endogenous Cys in living cells and mice.
Assuntos
Cisteína , Corantes Fluorescentes , Animais , Glutationa , Homocisteína , Limite de Detecção , CamundongosRESUMO
The application of laccase-mediator-based catalysis is limited owing to the high cost of laccases and mediators and the potential toxicity of free mediators. Here, a novel biocatalyst (Im-LMS) was fabricated by immobilizing both laccase and a mediator (2,2'-azino-bis-[3-ethylbenzothiazoline]-6-sulfonic acid) on layered double hydroxide/alginate biohybrid beads. The catalytic activity of Im-LMS was evaluated for dye decolorization using malachite green. The decolorization yields of malachite green by Im-LMS and the free laccase-mediator system were 92% within 120 min and 90% within 90 min. Malachite green solution was detoxified completely after biodegradation by Im-LMS. Following eight reuse cycles of Im-LMS for dye treatment, a decolorization yield of 79% was obtained. The activity of Im-LMS was almost completely stable after being stored for 10 days. The recyclability and stability of Im-LMS will be helpful for reducing the running cost and potential toxicity associated with mediators to facilitate practical applications.
Assuntos
Alginatos/química , Aspergillus oryzae/enzimologia , Enzimas Imobilizadas/química , Proteínas Fúngicas/química , Lacase/química , Corantes de Rosanilina/química , Ácidos Sulfônicos/químicaRESUMO
OBJECTIVE: To promote targeting specificity of anti-CD47 agents, we have constructed a novel bispecific antibody fusion protein against EGFR and CD47, which may minimize the "off-target" effects caused by CD47 expression on red blood cells. RESULTS: The novel bispecific antibody fusion protein, denoted as Bi-SP could simultaneously bind to EGFR and CD47 and exhibited potent phagocytosis-stimulation effects in vitro. Bi-SP treatment with a low dose more effectively inhibited tumor growth than either EGFR-targeting antibody, Pan or the SIRPα variant-Fc (SIRPαV-Fc) in the A431 xenograft tumor model. In addition, the treatment with Bi-SP produced less red blood cell (RBC) losses than the SIRPαV-Fc treatment, suggesting its potential use for minimizing RBC toxicity in therapy. CONCLUSIONS: Bi-SP with improved therapeutic index has the potential to treat CD47+ and EGFR+ cancers in clinics.
Assuntos
Anticorpos Biespecíficos/administração & dosagem , Antineoplásicos Imunológicos/administração & dosagem , Antígeno CD47/antagonistas & inibidores , Carcinoma/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Animais , Anticorpos Biespecíficos/farmacologia , Antineoplásicos Imunológicos/farmacologia , Carcinoma/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Humanos , Camundongos , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Índice Terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Dithiocarbamate has been tested for its effective anti-tumor activity, but the underlying mechanism remains unclear. We previously prepared a novel diththiocarbamate derivative, DpdtC with an ability of catalase inhibition. Here, we for the first time investigated the growth inhibition effects of DpdtC on HER2-amplified cancer cells and elucidated its mechanism of action. Results showed that DpdtC exerted the potent anti-tumor effects against HER2-overexpressed SK-OV-3 and SK-BR-3 cells, especially on SK-OV-3 cells with a higher NDRG1 level, which was also confirmed in the SK-OV-3 xenograft model. Interestingly, we observed that NDRG1 was up-regulated, while membrane expression of HER2 was regressed in SK-OV-3 cells upon DpdtC treatment. In agreement, silencing endogenous NDRG1 also increased the expression of HER2 in SK-OV-3 cells, while overexpressing NDRG1 decreased HER2 expression in SK-BR-3 cells. Furthermore, our results showed the formation of the EGFR/HER2 heterodimer was attenuated and phosphorylation of ERK1/2 was inhibited in SK-OV-3 cells when treated with DpdtC. Collectively, these observations demonstrated that NDRG1 plays an important role in mediating the inhibition effects of DpdtC in HER2-overexpressed cancer cells via selective targeting of the HER2-ERK1/2 pathway. Hence, our investigation suggests that up-regulation of NDRG1 by DpdtC is a promising therapeutic approach in HER2-overexpressed cancers.
Assuntos
Proteínas de Ciclo Celular/genética , Ditiocarb/análogos & derivados , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Receptor ErbB-2/genética , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Animais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ditiocarb/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Fosforilação/efeitos dos fármacosRESUMO
Cancer cells have higher demand of iron and copper ions for growth, disturbing the metal's homeostasis can inhibit proliferation of cancer cell. Dithiocarbamates possessing excellent metal chelating ability and antitumor activity are considered as candidates in chelation therapy, however, their antitumor molecular mechanisms remain to be elucidated. In the present study, a dithiocarbamate derivative, di-2-pyridylhydrazone dithiocarbamate s-acetic acid (DpdtaA) was prepared to address the issue whether the molecular mechanism behind biological behavior showed by dithiocarbamate was p53 mediated. The proliferation inhibition assay showed that DpdtaA exhibited excellent antiproliferative effect for hepatocellular carcinoma (IC50= 3.0±0.4 µM for HepG2, 6.1±0.6 µM for Bel-7402 cell). However, in the presence of copper ion, the antiproliferative activity of DpdtaA significantly attenuated (~3-fold for HepG2) due to formation of copper chelate. The ROS assay revealed that the antiproliferative activity of DpdtaA correlated with ROS generation. Western blotting demonstrated that DpdtaA could upregulate p53 via down-regulating the Mdm2, accordingly leading to changes of bcl family proteins, indicating that a p53-dependent intrinsic apoptosis was partly involved. Simulation from molecular docking hinted that DpdtaA could disrupt interaction between p53 and Mdm2, indicating the disruption might also contribute to the upregulation of p53. The alternations in lysosome membrane permeability and acidic vacuoles as well as LC3-II upregulation indicated that autophagy was involved. The copper addition led to significantly attenuate biological activity of DpdtaA, with few dithiocarbamates, but the mechanism in apoptosis induction was not altered except for weaker ability.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Hidrazonas/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Tiocarbamatos/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Quelantes/farmacologia , Cobre/metabolismo , Células Hep G2 , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
The bioactivity of drugs is attributed to their interaction with biological molecules, embodied in either their direct or indirect influence on enzyme activity and conformation. Di-2-pyridylketone hydrazine dithiocarbamate (DpdtC) exhibits significant antitumor activity in our preliminary study. We speculated that its activity may partly stem from enzyme inhibition due to strong metal chelating ability. To this end, we assessed its effect on catalase from erythrocytes and found evidence of inhibition, which was further confirmed by ROS determination in vivo. Thus, detailing the interaction between the agent and catalase via spectroscopic methods and molecular docking was required to obtain information on both the dynamics and thermodynamic parameters. The Lineweaver-Burk plot implied an uncompetitive pattern between DpdtC and catalase from beef liver, and IC50 = â¼7 µM. The thermodynamic parameters from fluorescence quenching measurements indicated that DpdtC could bind to catalase with moderate affinity (Ka = approximately 104 M-1). CD spectra revealed that DpdtC could significantly disrupt the secondary structure of catalase. Docking studies indicated that DpdtC bound to a flexible region of catalase, involving hydrogen bonds and salt bond; this was consistent with thermodynamic results from spectral investigations. Our data clearly showed that catalase inhibition of DpdtC was not due to direct chelation of iron from heme (killing), but through an allosteric effect. Thus, it can be concluded that the antiproliferative activity of DpdtC is partially attributed to its catalase inhibition.
Assuntos
Catalase/antagonistas & inibidores , Catalase/química , Pirazóis/química , Tiocarbamatos/química , Tiocarbamatos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Ativação Enzimática/efeitos dos fármacos , Humanos , Cinética , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
The successful encapsulation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), a well-known laccase mediator, within a mesoporous metal-organic framework sample (i.e., MIL-100(Fe)) was achieved using a one-pot hydrothermal synthetic method. The as-prepared ABTS@MIL-100(Fe) was characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, nitrogen sorption, and cyclic voltammetry (CV). Our ABTS@MIL-100(Fe)-based electrode exhibited an excellent electrochemical response, indicating that MIL-100(Fe) provides an appropriate microenvironment for the immobilization and electroactivity of ABTS molecules. ABTS@MIL-100(Fe) was then evaluated as an immobilized laccase mediator for dye removal using indigo carmine (IC) as a model dye. Through the application of laccase in combination with a free (ABTS) or immobilized (ABTS@MIL-100(Fe)) mediator, decolorization yields of 95% and 94%, respectively, were obtained for IC after 50 min. In addition, following seven reuse cycles of ABTS@MIL-100(Fe) for dye treatment, a decolorization yield of 74% was obtained. Dye decolorization occurred through the breakdown of the chromophoric group by the Laccase/ABTS@MIL-100(Fe) system, and a catalytic mechanism was proposed. We therefore expect that the stability, reusability, and validity of ABTS@MIL-100(Fe) as a laccase mediator potentially render it a promising tool for dye removal, in addition to reducing the high running costs and potential toxicity associated with synthetic mediators.
Assuntos
Benzotiazóis/química , Ferro/química , Lacase/química , Ácidos Sulfônicos/química , Catálise , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
Hierarchical copper shells anchored on magnetic nanoparticles were designed and fabricated to selectively deplete hemoglobin from human blood by immobilized metal affinity chromatography. Briefly, CoFe2O4 nanoparticles coated with polyacrylic acid were first synthesized by a one-pot solvothermal method. Hierarchical copper shells were then deposited by immobilizing Cu2+ on nanoparticles and subsequently by reducing between the solid CoFe2O4@COOH and copper solution with NaBH4. The resulting nanoparticles were characterized by scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectrometry, X-ray photoelectron spectroscopy, and vibrating sample magnetometry. The particles were also tested against purified bovine hemoglobin over a range of pH, contact time, and initial protein concentration. Hemoglobin adsorption followed pseudo-second-order kinetics and reached equilibrium in 90 min. Isothermal data also fit the Langmuir model well, with calculated maximum adsorption capacity 666 mg g-1. Due to the high density of Cu2+ on the shell, the nanoparticles efficiently and selectively deplete hemoglobin from human blood. Taken together, the results demonstrate that the particles with hierarchical copper shells effectively remove abundant, histidine-rich proteins, such as hemoglobin from human blood, and thereby minimize interference in diagnostic and other assays.
Assuntos
Cobre/química , Hemoglobinas/análise , Hemoglobinas/isolamento & purificação , Nanopartículas de Magnetita/química , Resinas Acrílicas/química , Adsorção , Animais , Bovinos , Histidina/química , Humanos , Concentração de Íons de Hidrogênio , Cinética , Magnetismo , Magnetometria , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nanocompostos/química , Espectroscopia Fotoeletrônica , Proteínas/química , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Thiosemicarbazones display significant antitumor activity and their copper complexes also exhibit enhanced biological activities in most situations, but the underlying mechanism is poorly understood. Therefore, investigation of the mechanism involved in the change upon chelation is required to extend our understanding of the effects of thiosemicarbazones. In the present study, the inhibitory effect of 2-pyridinecarboxaldehyde thiosemicarbazone (PCT) and its copper complex (PCT-Cu) on cell proliferation was investigated. The copper chelate exhibited a 3- to 10-fold increase in antitumor activity (with an IC50 <5 µM). The results showed that both PCT and PCT-Cu induced reactive oxygen species (ROS) generation in vitro and in vivo, caused cellular DNA fragmentation, depolarization of the mitochondrial membrane and cell cycle arrest. Western blotting showed that both PCT and PCT-Cu induced apoptosis. Upregulation of GRP78 in HepG2 cells following treatment with the agents indicated that endoplasmic reticulum (ER) stress occurred. Furthermore calcium release was revealed in this study, suggesting that PCT and PCT-Cu disturbed calcium homeostasis. It was noted that PCT-Cu sensitized thapsigarginstimulated calcium release from the ER, which was correlated with the ROS level they induced, implying that the antitumor activity of PCT and PCT-Cu partly stemmed from calcium mobilization, a situation that was reported in few studies. Our findings may significantly contribute to the understanding of the antiproliferative effect of the derivatives of thiosemicarbazones along with their antitumor mechanism.
Assuntos
Antineoplásicos/farmacologia , Cálcio/metabolismo , Carcinoma Hepatocelular/patologia , Neoplasias Colorretais/patologia , Cobre/química , Piridinas/química , Tiossemicarbazonas/farmacologia , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Chaperona BiP do Retículo Endoplasmático , Citometria de Fluxo , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiossemicarbazonas/química , Células Tumorais CultivadasRESUMO
This response refers to.
Assuntos
Dicroísmo Circular , Simulação de Acoplamento Molecular , Animais , Bovinos , DNA/química , Humanos , Albumina Sérica/química , Espectrometria de Fluorescência , Termodinâmica , TiossemicarbazonasRESUMO
The use of chelators for cancer treatment has been an alternative option. Dithiocarbamates have recently attracted considerable attention owning to their diverse biological activities; thus, the preparation of new dithiocarbamate derivatives with improved antitumor activity and selectivity as well as probing the underlying molecular mechanism are required. In this study, di-2-pyridylhydrazone dithiocarbamate S-propionic acid (DpdtpA) and its copper complex were prepared and characterized, and its antiproliferative activity was evaluated. The proliferation inhibition assay showed that DpdtpA exhibited excellent antiproliferative effect in hepatocellular carcinoma (IC50 = 1.3 ± 0.3 µM for HepG2, and 2.5 ± 0.6 µM for Bel-7402). However, in the presence of copper ion, the antiproliferative activity of DpdtpA was dramatically attenuated (20-30 fold) owing to the formation of copper chelate. A preliminarily mechanistic study revealed that reactive oxygen species (ROS) generation mediated the antiproliferative activity of DpdtpA, and accordingly induced apoptosis, DNA cleavage, and autophagy. Surprisingly, the cytotoxicity of DpdtpA copper complex (DpdtpA-Cu) was also involved in ROS generation; however, a paradoxical relation between cellular ROS level and cytotoxicity was observed. Further investigation indicated that DpdtpA could induce cell cycle arrest at the S phase; however, DpdtpA-Cu lacked this effect, which explained the difference in their antiproliferative activity.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Cobre/química , Tiocarbamatos/síntese química , Tiocarbamatos/farmacologia , Antineoplásicos/química , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Células Hep G2 , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Hidrazonas/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Tiocarbamatos/químicaRESUMO
Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) exhibits significant antitumor activity. However, the mechanism of its pharmacological interaction with human serum albumin (HSA) and DNA remains poorly understood. Here, we aimed to elucidate the interactions of Dp44mT with HSA and DNA using MTT assays, spectroscopic methods, and molecular docking analysis. Our results indicated that addition of HSA at a ratio of 1:1 did not alter the cytotoxicity of Dp44mT, but did affect the cytotoxicity of the Dp44mT-Cu complex. Data from fluorescence quenching and UV-VIS absorbance measurements demonstrated that Dp44mT could bind to HSA with a moderate affinity (Ka = approximately 104 M(-1)). CD spectra revealed that Dp44mT could slightly disrupt the secondary structure of HSA. Dp44mT could also interact with Ct-DNA, but had a moderate binding constant (KEB = approximately 104 M(-1)). Docking studies indicated that the IB site of HSA, but not the IIA and IIIA sites, could be favorable for Dp44mT and that binding of Dp44mT to HSA involved hydrogen bonds and hydrophobic force, consistent with thermodynamic results from spectral investigations. Thus, the moderate binding affinity of Dp44mT with HSA and DNA partially contributed to its antitumor activity and may be preferable in drug design approaches.
Assuntos
DNA/metabolismo , Albumina Sérica/metabolismo , Tiossemicarbazonas/metabolismo , Animais , Sítios de Ligação , Bovinos , Diferenciação Celular/efeitos dos fármacos , Dicroísmo Circular , Complexos de Coordenação/química , Complexos de Coordenação/toxicidade , Cobre/química , DNA/química , Células Hep G2 , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , Ligação Proteica , Estrutura Terciária de Proteína , Albumina Sérica/química , Espectrometria de Fluorescência , Termodinâmica , Tiossemicarbazonas/químicaRESUMO
The drug, di-2-pyridylketone-2-pyridine carboxylic acid hydrazone (DPPCAH) and its copper complex (DPPCAH-Cu) exhibit significant antitumor activity. However, the mechanism of their pharmacological interaction with the biological molecule bovine serum albumin (BSA) remains poorly understood. The present study elucidates the interactions between the drug and BSA through MTT assays, spectroscopic methods and molecular docking analysis. Our results indicate that BSA could attenuate effect on the cytotoxicity of DPPCAH, but not DPPCAH-Cu. Data from fluorescence quenching measurements demonstrated that both DPPCAH and DPPCAH-Cu could bind to BSA, with a reversed effect on the environment of tryptophan residues in polarity. CD spectra revealed that the DPPCAH-Cu exerted a slightly stronger effect on the secondary structure of BSA than DPPCAH. The association constant of DPPCAH with BSA was greater than that of DPPCAH-Cu. Docking studies indicated that the binding of DPPCAH to BSA involved a greater number of hydrogen bonds compared to DPPCAH-Cu. The calculated distances between bound ligands and tryptophans in BSA were in agreement with fluorescence resonance energy transfer results. Thus, the binding affinity of the drug (DPPCAH or DPPCAH-Cu) with BSA partially contributes to its antitumor activity; the greater the drug affinity is to BSA, the less is its antitumor activity.
Assuntos
Cobre/química , Hidrazonas/farmacologia , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Animais , Antineoplásicos , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Dicroísmo Circular , Células Hep G2 , Humanos , Hidrazonas/química , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Espectrometria de FluorescênciaRESUMO
Many anticancer drugs used in the clinical have potent metal chelating ability. The formed metal complex(es) may exhibit improved (or antagonistic) antitumor activity. However, the underlying mechanism has received limited attention. Therefore, investigation of the mechanism involved in the change upon chelation is required to extend our understanding of the effects of various drugs. In the present study, the proliferation inhibition effect of benzaldehyde nitrogen mustard-2-pyridine carboxylic acid hydrazone (BNMPH) and its copper complex on tumor cell lines was investigated. The copper chelate exhibited almost a 10-fold increase in antitumor activity (with IC50 <5 µM). The results showed that both BNMPH and its copper complex induced reactive oxygen species (ROS) generation, and caused upregulation of caspase 8 and Bax as well as the downregulation of Bcl-2, indicating that apoptosis was involved in the cytotoxic effects. DNA fragmentation noted in the comet assay further supported ROS involvement. The present study indicated that BNMPH and its copper complex effectively induced S phase arrest and the cell cycle arrest was associated with the downregulation of cyclin D1. The formation of acidic vesicular organelles (AVOs) and an increase in cleaved LC3-II demonstrated that autophagy occurred in the HepG2 cells treated with the agents. Taken together, BNMPH and its copper complex exhibited proliferation inhibition via apoptosis, cell cycle arrest and autophagy, which was dependent on ROS. The enhanced antitumor activity of the copper complex was due to its redox-cycling ability, but the mechanism was not altered compared to BNMPH. Our findings may significantly contribute to the understanding of the anti-proliferative effect of BNMPH and its copper complex.
Assuntos
Antineoplásicos/administração & dosagem , Autofagia/efeitos dos fármacos , Hidrazonas/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Compostos de Mostarda Nitrogenada/administração & dosagem , Caspase 8/biossíntese , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Complexos de Coordenação/administração & dosagem , Cobre/administração & dosagem , Fragmentação do DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Espécies Reativas de Oxigênio/metabolismo , Proteína X Associada a bcl-2/biossínteseRESUMO
Iron depletion and stimulation of iron-dependent free radical damage is a rapidly developing field for chelation therapy, but the iron mobilization from ferritin by chelators has received less attention. In this study, the di-2-pyridylketone 2-pyridine carboxylic acid hydrazone (DPPCAH) and its copper complex was prepared and characterized by NMR and MS spectra. The proliferation inhibition assay showed that both DPPCAH and its copper complex exhibited selectively proliferation inhibition for HepG2 (IC50, 4.6 ± 0.2 µM for DPPACH and 1.3 ± 0.2 µM for its copper complex), but less inhibition for HCT-116 cell line (IC50, >100 µM for DPPACH and 7.8 ± 0.4 µM for its copper complex). The mechanistic studies revealed that DPPACH could remove iron from ferritin in a oxygen-catalytic manner, and contributed to redox activity of labile iron pool (LIP), that is less reported for the chelators that possess significant biological activity. The reactive oxygen species (ROS) generation and DNA cleavage assay in vitro and in vivo showed that both DPPACH-Fe(II) and DPPACH-Cu were redox-active species, indicating that ROS may mediate their antitumor activity. Further study revealed that both DPPACH and its copper complex displayed certain degree of inhibition of type II topoisomerase (Top) which contributed to their antitumor activity. Thus, the mechanism that iron mobilization by DPPACH from ferritin contributed to LIP was proposed, and both DPPACH and its copper complex were involved in ROS generation and Top II inhibition for their antitumor activities.
Assuntos
Proliferação de Células/efeitos dos fármacos , Hidrazonas/administração & dosagem , Neoplasias Hepáticas/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismo , Catálise , Cobre/química , DNA Topoisomerases/efeitos dos fármacos , Células Hep G2 , Humanos , Hidrazonas/química , Ferro/química , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Oxigênio/química , Inibidores da Topoisomerase/administração & dosagem , Inibidores da Topoisomerase/químicaRESUMO
In the present study, 2-pyridinecarboxaldehyde 2-pyridinecarboxylic acid hydrazone (PPAH) was prepared and its antitumor activity was evaluated. The inhibition of proliferation of PPAH against the HepG2 and HCT-116 cell lines was less effective, yet in the presence of copper ions, the mixture demonstrated excellent antitumor activity (IC50 at 2.75±0.30 µM for the HepG2 cell line, and 1.90±0.20 µM for the HCT-116 cell line, respectively) and the new active species was confirmed to be a PPAH copper complex with a 1:1 ratio by spectral analysis. The excellent antitumor activity of the copper complex prompted us to investigate the underlying mechanism. RT-PCR was performed to detect the changes in the expression of apoptotic genes induced by PPAH and its copper complex. However, no changes were observed when the cells were treated by the agents for 24 or 48 h, indicating that ROS were unlikely involved. Cell cycle analysis showed that both PPAH and its copper complex led to S phase arrest of the cells. The sDNA relaxation assay revealed that the PPAH-copper complex displayed dual topoisomerase inhibition for type I and II. The data suggest that the inhibition of proliferation exhibited by the PPAH copper complex may stem from its dual topoisomerase inhibition, which is rarely observed for a metal complex.
Assuntos
Proliferação de Células/efeitos dos fármacos , Cobre/administração & dosagem , Hidrazonas/administração & dosagem , Neoplasias/tratamento farmacológico , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Cobre/química , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Células Hep G2 , Humanos , Hidrazonas/síntese química , Neoplasias/patologia , Piridinas/administração & dosagem , Piridinas/síntese químicaRESUMO
The Mannich base containing ciprofloxacin and kojic acid structural units was prepared and evaluated in antitumor activity. The enhancement in antitumor activity was observed both from the Mannich base (IC(50): 103.3±5.0 µM for HepG2, 87.9±8.0 µM for HCT-116 cell) and its copper complex (IC(50): 11.5±1.8 µM for HepG2, 44.4±2.5 µM for HCT-116 cell) compared to the ciprofloxacin and kojic acid. The mechanistic studies via RT-PCR, cell cycle analysis, mitochondrial membrane potential measurement, inhibition of topoisomerase and molecular docking indicated that there is a different molecular mechanism between the Mannich base and its copper complex. The cytotoxicity of the Mannich base was involved in apoptosis, cell cycle arrest, depolarization of mitochondrial membrane and weaker topoisomerase II inhibition, but the copper complex exerted its cytotoxicity mainly through dual topoisomerase inhibition, especially stabilizing the intermediate of cleavage DNA-topoisomerase complex.