RESUMO
Energy band structure of inorganic nano-sonosensitizers is usually optimized by surface decoration with noble metals or metal oxide semiconductors, aiming to enhance interfacial charge transfer, augment spin-flip and promote radical generation. To avoid potential biohazards of metallic elements, herein, metal-free graphitic carbon nitride quantum dots (g-C3 N4 QDs) are anchored onto hollow mesoporous TiO2 nanostructure to formulate TiO2 @g-C3 N4 heterojunction. The direct Z-scheme charge transfer significantly improves the separation/recombination dynamics of electron/hole (e- /h+ ) pairs upon ultrasound (US) stimulation, which promotes the yield of singlet oxygen (1 O2 ) and hydroxyl radicals (·OH). The conjugated g-C3 N4 QDs with peroxidase-mimic activity further react with the elevated endogenous H2 O2 and aggravate oxidative stress. After loading prodrug romidepsin (RMD) in TiO2 @g-C3 N4 , stimulus-responsive drug delivery can be realized by US irradiation. The disulfide bridge of the released RMD tends to be reduced by glutathione (GSH) into a monocyclic dithiol, which arrests cell cycle in G2/M phase and evokes apoptosis through enhanced histone acetylation. Importantly, reactive oxygen species accumulation accompanied by GSH depletion is devoted to deleterious redox dyshomeostasis, leading to augmented systemic oncotherapy by eliciting antitumor immunity. Collectively, this paradigm provides useful insights in optimizing the performance of TiO2 -based nano-sonosensitizers for tackling critical diseases.
Assuntos
Óxidos , Oxirredução , Ultrassonografia , AcetilaçãoRESUMO
Mitochondria are core regulators of tumor cell homeostasis, and their damage has become an arresting therapeutic modality against cancer. Despite the development of many mitochondrial-targeted pharmaceutical agents, the exploration of more powerful and multifunctional medications is still underway. Herein, oxygen vacancy-rich BiO2-x wrapped with CaCO3 (named BiO2-x @CaCO3 /PEG, BCP) is developed for full-fledged attack on mitochondrial function. After endocytosis of BCP by tumor cells, the CaCO3 shell can be decomposed in the acidic lysosomal compartment, leading to immediate Ca2+ release and CO2 production in the cytoplasm. Near-infrared irradiation enhances the adsorption of CO2 onto BiO2-x defects, which enables highly efficient photocatalysis of CO2 -to-CO. Meanwhile, such BiO2-x nanosheets possess catalase-, peroxidase- and oxidase-like catalytic activities under acidic pH conditions, allowing hypoxia relief and the accumulation of diverse reactive oxygen species (ROS) in the tumor microenvironment. Ca2+ overload-induced ion dyshomeostasis, CO-mediated respiratory chain poisoning, ROS-triggered oxidative stress aggravation, and cytosolic hyperoxia can cause severe mitochondrial disorders, which further lead to type I cell death in carcinoma. Not only does BCP cause irreversible apoptosis, but immunogenic cell death is simultaneously triggered to activate antitumor immunity for metastasis inhibition. Collectively, this platform promises high benefits in malignant tumor therapy and may expand the medical applications of bismuth-based nanoagents.
RESUMO
The therapeutic exploration of nano-zirconia semiconductor largely remains untouched in the field of fundamental science to date. Here, a robust nano-sonosensitizer of ZrO2- x @Pt is strategically formulated by in situ growth of Pt nanocrystal onto the surface of oxygen-deficient ZrO2- x . Compared to 3.09 eV of nano-ZrO2- x , the bandgap of ZrO2- x @Pt Schottky junction is narrowed down to 2.74 eV. The band bending and bandgap narrowing enables an enhanced e- /h+ separation in the presence of aPt electron sink, which facilitates a high yield of singlet oxygen (1 O2 ) and hydroxyl radicals (·OH) under ultrasound (US) irradiation. Moreover, nanozyme Pt with catalase-mimic activity can promote 1 O2 generation by relieving the hypoxic tumor microenvironment. Upon further modification of 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH), US-stimulated local thermal shock can disintegrate AIPH to create cytotoxic alkyl radicals (⢠R). US-triggered reactive oxygen species generation and hyperthermia-induced alkyl radical production lead to severe and irreversible tumor cell death. Such combinatorial sonodynamic-thermodynamic therapy benefits the tumor eradication and metastasis inhibition at the animal level, with the aid of immunogenetic cell death and immune checkpoint blockade. Taken together, this proof-of-concept paradigm expands the medical use of nano-zirconia and provides useful insights for its therapeutic perspectives.
Assuntos
Neoplasias , Terapia por Ultrassom , Animais , Catalase/metabolismo , Linhagem Celular Tumoral , Inibidores de Checkpoint Imunológico , Neoplasias/terapia , Estresse Oxidativo , Oxigênio , Platina , Propano , Espécies Reativas de Oxigênio/metabolismo , Oxigênio Singlete , Termodinâmica , Microambiente TumoralRESUMO
OBJECTIVE: To investigate the effects of different concentrations of indium chloride (InCl3) on the proliferation of human lung epithelial (Beas-2B) cells and its potential mechanism. METHODS: Beas-2B cells were exposed to different concentrations of InCl3 (0.3, 1.0, 3.0, 10.0, 30.0, 90.0, 270.0, and 810.0 µmol/L) for 24, 48, and 72 h, respectively. The effects of InCl3 on cell proliferation were determined by the CCK-8 assay. The effects of InCl3 on apoptosis were evaluated using annexin V-PI staining followed by flow cytometry. The level of intracellular reactive oxygen species (ROS) in Beas-2B cells after exposure to InCl3 was determined using 2', 7'-dichlorofluorescein diacetate labeling followed by flow cytometry. RESULTS: Compared with the control group, InCl3 at a relatively low concentration (0.3~3.0 µmol/L) significantly promoted cell proliferation (P < 0.05), while InCl3 at a relatively high concentration (30.0~80.0 µmol/L) significantly inhibited cell proliferation after 72 h (P < 0.05). InCl3 at a concentration of 0.3 µmol/L failed to induce apoptosis within 72 h; however, InCl3 at a concentration of 30.0 or 810.0 µmol/L induced substantial early apoptosis after 72 h. Compared with the control group, cells exposed to 0.3 µmol/L InCl3 showed a slight decrease in the level of intracellular ROS within 72 h, while cells exposed to 30.0 or 810.0 µmol/L InCl3 showed a significant increase in the level of intracellular ROS after 72 h (P < 0.05). CONCLUSION: At a low concentration, InCl3 stimulates cell proliferation by reducing intracellular ROS. However, at a high concentration, InCl3 inhibits cell viability by elevating intracellular ROS and inducing apoptosis.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Índio/toxicidade , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Espécies Reativas de OxigênioRESUMO
BACKGROUND: Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) presents great challenges in the treatment of non-small cell lung cancer (NSCLC) patients, while the mechanisms are still not well understood. The ß-catenin signaling pathway has been found to be associated with chemoresistance and can activate the EGFR and its downstream pathways. This study aimed to investigate the role of ß-catenin in acquired resistance to EGFR-TKIs in NSCLC cell lines. METHODS: The expression and transcriptional activity of ß-catenin were measured in both the NSCLC cell line PC9 and its sub-line PC9/AB(2) which has acquired resistance to gefitinib. Knockdown and overexpression of ß-catenin in the PC9/AB(2) and PC9 cells were performed. The cell survival rate and the activation of the EGFR and its downstream pathways were detected in the two cell lines after transfection. RESULTS: Nuclear translocation of ß-catenin was increased in the PC9/AB(2) cells and the baseline expression of members of the ß-catenin signaling pathway was also higher in the PC9/AB(2) cells. Knocking down the expression of ß-catenin increased the sensitivity of the PC9/AB(2) cells to gefitinib by blocking the activation of the EGFR downstream pathways, while ß-catenin overexpression improved PC9 cells resistance to gefitinib by enhancing the activation of the EGFR and its downstream signaling. CONCLUSION: ß-catenin plays an important role in acquired resistance to EGFR-TKIs in NSCLC cell lines and may be a potential therapeutic target for NSCLC patients who have failed to respond to targeted therapy.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Quinazolinas/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Gefitinibe , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Transdução de Sinais/efeitos dos fármacos , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Traditional Fe-based Fenton reaction for inducing oxidative stress is restricted by random charge transfer without oriental delivery, and the resultant generation of reactive oxygen species (ROS) is always too simplistic to realize a satisfactory therapeutic outcome. Herein, FeNv/CN nanosheets rich in nitrogen vacancies are developed for high-performance redox dyshomeostasis therapy after surface conjugation with polyethylene glycol (PEG) and cyclic Arg-Gly-Asp (cRGD). Surface defects in FeNv/CN serve as electron traps to drive the directional transfer of the excited electrons to Fe atom sites under ultrasound (US) actuation, and the highly elevated electron density promote the catalytic conversion of H2O2 into ·OH. Meanwhile, energy band edges of FeNv/CN favor the production of 1O2 upon interfacial redox chemistry, which is enhanced by the optimal separation/recombination dynamics of electron/hole pairs. Moreover, intrinsic peroxidase-like activity of FeNv/CN contributes to the depletion of reductant glutathione (GSH). Under the anchoring effect of cRGD, PEGylated FeNv/CN can be efficiently enriched in the tumorous region, which is ultrasonically activated for concurrent ROS accumulation and GSH consumption in cytosolic region. The deleterious redox dyshomeostasis not only eradicates primary tumor but also suppresses distant metastasis via antitumor immunity elicitation. Collectively, this study could inspire more facile designs of chalybeates for medical applications.
Assuntos
Peróxido de Hidrogênio , Hiperaldosteronismo , Neoplasias , Nitrilas , Humanos , Espécies Reativas de Oxigênio , Oxirredução , Ultrassonografia , Glutationa , Linhagem Celular TumoralRESUMO
Active polymetallic atomic clusters can initiate heterogeneous catalytic reactions in the tumor microenvironment, and the products tend to cause manifold damage to cell metabolic functions. Herein, bimetallic PtPd atomic clusters (BAC) are constructed by the stripping of Pt and Pd nanoparticles on nitrogen-doped carbon and follow-up surface PEGylation, aiming at efficacious antineoplastic therapy through heterogeneous catalytic processes. After endocytosed by tumor cells, BAC with catalase-mimic activity can facilitate the decomposition of endogenous H2O2 into O2. The local oxygenation not only alleviates hypoxia to reduce the invasion ability of cancer cells but also enhances the yield of â¢O2- from O2 catalyzed by BAC. Meanwhile, BAC also exhibit peroxidase-mimic activity for â¢OH production from H2O2. The enrichment of reactive oxygen species (ROS), including the radicals of â¢OH and â¢O2-, causes significant oxidative cellular damage and triggers severe apoptosis. In another aspect, intrinsic glutathione (GSH) peroxidase-like activity of BAC can indirectly upregulate the level of lipid peroxides and promote ferroptosis. Such deleterious redox dyshomeostasis caused by ROS accumulation and GSH consumption also results in immunogenic cell death to stimulate antitumor immunity for metastasis suppression. Collectively, this paradigm is expected to inspire more facile designs of polymetallic atomic clusters in disease therapy.
Assuntos
Antineoplásicos , Ferroptose , Neoplasias , Humanos , Peróxido de Hidrogênio , Espécies Reativas de Oxigênio , Apoptose , Peroxidases , Antineoplásicos/farmacologia , Catálise , Glutationa , Linhagem Celular Tumoral , Neoplasias/tratamento farmacológico , Microambiente TumoralRESUMO
OBJECTIVE: To detect the inhibitory effect of a p38MAPK inhibitor SB203580 in combination with gefitinib on lung adenocarcinoma cell line PC-9 cells and A549 cells, and its cellular and molecular mechanisms of action. METHODS: MTT test was used to detect the growth inhibition of PC-9 and A549 cells by SB203580 alone and in combination with gefitinib. Cell apoptosis and cell cycles were determined by flow cytometry. The expressions of p38 and phosphorylated -p38 proteins in the two cell lines were analyzed by immunofluorescence microscopy. The associated protein expression was determined by Western-blot. RESULTS: Compared with the SB203580 group and gefitinib group, the growth inhibition and cell apoptosis of PC-9 cells in the SB203580 + gefitinib group were significantly increased (P < 0.05). The inhibition rate of PC-9 cells of 2 µmol/L SB203580 + 0.01 µmol/L gefitinib group was (46.6 ± 2.4)%, significantly higher than that induced by 0.01 µmol/L gefitinib (12.7 ± 1.5%) (P < 0.05). Immunofluorescence microscopy showed a low expression of phosphorylated-p38 protein in A549 cells and high expression in PC-9 cells. Flow cytometry showed that PC-9 cells in the SB203580 + gefitinib group were (77.35 ± 2.83)% at G0/G1 phase, (3.38 ± 0.84)% at S phase, and (19.56 ± 1.99)% at G2/M phase. Western-blotting showed that compared with the control group, the expression of phosphorylated Akt and phospho-p38 proteins in PC-9 cells of the SB203580 + gefitinib group was almost completely suppressed. CONCLUSIONS: The results indicate that the small molecular inhibitor SB203580 can effectively enhance the inhibitory effect of gefitinib on lung adenocarcinoma PC-9 cells. The enhanced inhibitory effect of SB203580 may be correlated with the blockage of p38MAPK signal transduction pathway.
Assuntos
Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Imidazóis/farmacologia , Neoplasias Pulmonares/patologia , Piridinas/farmacologia , Quinazolinas/farmacologia , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Gefitinibe , Humanos , Neoplasias Pulmonares/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
OBJECTIVE: To construct angiogenesis-specific RGD10-NGR9 dual-targeting superparamagnetic iron oxide nanoparticles, and to evaluate its magnetic resonamce imaging (MRI) features in nude mice and potential diagnostic value in tumor MRI. METHODS: Dual-targeting peptides RGD10-NGR9 were designed and synthesized. Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles were synthesized by chemical co-precipitation method and the surface was modified to be hydrophilic by coating with dextran. The dual-targeting peptides RGD10-NGR9 were conjugated to USPIO. Cell binding affinity and up-taking ability of the dual-targeting USPIO nanoparticles to integrin ανß3-APN positive cells were subsequently tested by Prussian blue staining and phenanthroline colorimetry in vitro. The RGD10-NGR9 conjugated with USPIO was injected intravenously into xenograft mice, which were scanned by MRI at predetermined time points. The MRI and contrast-to-noise ratio (CNR) values were calculated to evaluate the ability of dual-targeting USPIO as a potential contrast agent in nude mice. RESULTS: P-CLN-Dextran-USPIO nanoparticles with stable physical properties were successfully constructed. The average diameter of Fe3O4 nanoparticles was 8-10 nm, that of Dextran-USPIO was about 20 nm and P-CLN-Dextran-USPIO had an average diameter about 30 nm. The in vitro studies showed a better specificity of dual-targeting USPIO nanoparticles on proliferating human umbilical vein endothelia cells (HUVEC). In vivo, RGD10-NGR9-USPIO showed a significantly reduced contrast in signal intensity and 2.83-times increased the CNR in the tumor MRI in xenograft mice. CONCLUSION: This novel synthesized RGD10-NGR9 dual-targeting USPIO is with better specific affinity in vitro and in vivo, and might be used as a molecular contrast agent for tumor angiogenesis MRI.
Assuntos
Adenocarcinoma/diagnóstico , Meios de Contraste , Dextranos , Neoplasias Pulmonares/diagnóstico , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Aminopeptidases/análise , Animais , Linhagem Celular Tumoral , Células Cultivadas , Meios de Contraste/química , Dextranos/química , Óxido Ferroso-Férrico/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrina alfaVbeta3/análise , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Nanopartículas de Magnetita/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Oligopeptídeos/química , Tamanho da Partícula , Razão Sinal-RuídoRESUMO
OBJECTIVE: To explore the effect of Wnt signaling suppression on proliferation of non small cell lung cancer to gefitinib, and its related mechanisms. METHODS: PC9 and PC9/AB2 cells of both gefitinib sensitive and resistant were treated with different concentrations of gefitinib, and the proliferation index was measured using CCK8 kit. The members of Wnt signaling pathway were detected by Western blot. Dual luciferase reportor gene assay (TOP Flash) was used to document the transcriptional level of ß-catenin. ß-catenin siRNA was transfected into PC9/AB2 cells to suppress the Wnt signaling transcription, followed by treatment with different concentrations of gefitinib. Western blot was then used to detect the expression of EGFR and its downstream signaling after inhibit the expression of ß-catenin. RESULTS: Treating with different concentrations of gefitinib, the resistance of PC9/AB2 cells to gefitinib was significantly increased (P < 0.05). The members of Wnt signaling expressed at higher level in PC9/AB2 cells than in PC9 cells (t = 24.590, P = 0.000). TOP Flash examination showed that the endogenous transcriptional activity of Wnt signaling was higher in PC9/AB2 cell than that in PC9 cell (t = 4.983, P = 0.008). Compared with the negative control group, apoptotic rate and sensitivity to gefitinib significantly increased in interfered group (P < 0.05). The expression of p-ERK1/2 significantly decreased after Wnt signaling suppression, although other proteins showed no significant alterations. CONCLUSION: Suppressing the activity of Wnt signaling can partly reverse the celluar resistance to gefitinib in non small cell lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares , Quinazolinas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Relação Dose-Resposta a Droga , Gefitinibe , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Quinazolinas/administração & dosagem , beta Catenina/metabolismoRESUMO
To date, the construction of heterogeneous interfaces between sonosensitizers and other semiconductors or noble metals has aroused increasing attention, owing to an enhanced interface charge transfer, augmented spin-flip, and attenuated activation energy of oxygen. Here, a smart therapeutic nanoplatform is constructed by surface immobilization of glucose oxidase (GOx) onto a TiO2@Pt Schottky junction. The sonodynamic therapy (SDT) and starvation therapy (ST) mediated by TiO2@Pt/GOx (TPG) promote systemic tumor suppression upon hypoxia alleviation in tumor microenvironment. The band gap of TiO2@Pt is outstandingly decreased to 2.9 eV, in contrast to that of pristine TiO2. The energy structure optimization enables a more rapid generation of singlet oxygen (1O2) and hydroxyl radicals (â¢OH) by TiO2@Pt under ultrasound irradiation, resulting from an enhanced separation of hole-electron pair for redox utilization. The tumorous reactive oxygen species (ROS) accumulation and GOx-mediated glucose depletion facilitate oxidative damage and energy exhaustion of cancer cells, both of which can be tremendously amplified by Pt-catalyzed oxygen self-supply. Importantly, the combinatorial therapy triggers intense immunogenetic cell death, which favors a follow-up suppression of distant tumor and metastasis by evoking antitumor immunity. Collectively, this proof-of-concept paradigm provides an insightful strategy for highly efficient SDT/ST, which possesses good clinical potential for tackling cancer.
Assuntos
Neoplasias , Terapia por Ultrassom , Humanos , Platina , Microambiente Tumoral , Glucose , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Espécies Reativas de Oxigênio/metabolismo , Glucose Oxidase/farmacologia , Glucose Oxidase/metabolismo , Oxigênio , Linhagem Celular TumoralRESUMO
To construct tumor-targeted nanometer particles as a negative magnetic resonance imaging (MRI) contrast agent. Ultra-small superparamagnetic iron oxide (USPIO) nanometer particles were prepared by one-step chemical precipitation. The covalent bond between cyclic RGD (cRGD) containing an Arg-Gly-Asp sequence targeting integrin-alphavbeta3, and USPIO was conducted by chemical crosslinking. The physico-chemical property of cRGD-USPIO was detected. Prussian blue staining was applied to detect the specific binding capacity of cRGD-USPIO and USPIO to human pulmonary adenocarcinoma A549 cells and human umbilical vein endothelial cells. Subsequently, A549 xenografts in nude mice were established, and intravenous injections of USPIO and cRGD-USPIO into the vena caudalis were performed. The enhancement of cRGD-USPIO against tumor MRI signal was evaluated. The mean hydrodynamic diameter of cRGD-USPIO was 43.97 +/- 10.10 nm and the size of the ferric oxide core was 5-10 nm. The specific saturation magnetization was 59.94 A x m2 x Kg(-1). The cell conjugation assay results indicated that the positive staining of the cRGD-USPIO group was significantly enhanced. The in vivo MRI diagnosis indicated that the cRGD-USPIO tumor signal was significantly reduced compared to that of the USPIO group (P < 0.01). The targeted superparamagnetic iron oxide nanometer particle can be a novel MRI negative contrast agent for more specific tumor early diagnosis.
Assuntos
Meios de Contraste , Compostos Férricos/química , Imageamento por Ressonância Magnética/métodos , Nanopartículas Metálicas , Neoplasias/diagnóstico , Oligopeptídeos/química , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
OBJECTIVE: To study the drug resistance mechanism of non-small cell lung cancer (NSCLC) cell line PC9/AB2 with acquired drug resistance to gefitinib. METHODS: The human lung adenocarcinoma cell line PC9 was cultured in vitro, and was induced by MNNG to obtain the cell line PC9/AB2 with acquired drug resistance to gefitinib. The sensitivity of the cell line PC9 and PC9/AB2 to gefitinib was determined by MTT assay. The effects of gefitinib on cell apoptosis of the 2 cell lines were determined by flow cytometry. The genomic DNA of the 2 cell lines were extracted, and then the exons 19-21 of EGFR gene were amplified by PCR and sequenced. The protein expression of c-MET and integrin beta1 in the 2 cell lines was determined by Western blot method. The adhesion ability and migration ability of the 2 cell lines were determined by adhesion test and scratch assay. RESULTS: (1) The data form MTT and apoptosis detection showed that the IC50 of PC9/AB2 cells was (24.2+/-5.5) micromol/L, 576 times higher than PC9 cells [IC50 (0.04+/-0.01) micromol/L]. Given the same concentration of gefitinib, the apoptosis rate of PC9 cells was 38.48%, while that of PC9/AB2 cells was 2.2%. (2) The results of gene sequencing showed that there was a deletion of 15 bp in both exon 19 of the 2 cell lines, while no T790M mutation occurred. (3) The results from Western blot showed that there was no significant difference in protein expression of c-MET between the 2 cell lines, while the protein expression of integrin beta1 in PC9/AB2 cells was significantly higher than that of the PC9 cells. (4) The result from adhesion test and scratch assay showed that the adhesion ability and migration ability of the PC9/AB2 cells was significantly higher that those of PC9 cells. CONCLUSION: The high expression of integrin beta1 may be associated with acquired drug resistance of NSCLC cell line PC9/AB2 to gefitinib.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Quinazolinas/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Gefitinibe , Humanos , Integrina beta1/metabolismoRESUMO
OBJECTIVE: To explore the sensitivity of tumor cell lines with acquired resistance to gefitinib to several chemotherapeutic drugs and provide preclinical basis of available chemotherapy regimens after failure of molecular targeted therapy. METHODS: Human lung adenocarcinoma cell lines PC9 and PC9/G with acquired resistance to gefitinib were cultured in vitro. The sensitivity to chemotherapeutic drugs and inhibition rate of cell proliferation was determined by MTT assay. Effects of drugs on apoptosis and expression of P-170 were determined by flow cytometry. Difference of gene expression profile between PC9 and PC9/G cells was analyzed by DNA microarray. Western blot was used to test the expression of Akt, phospho-Akt and integrin beta1. RESULTS: The resistance index of PC9/G cells to cisplatin was about 5.4-fold compared with that of PC9 cells. LY294002 may significantly elevate the sensitivity of PC9/G cells to cisplatin (P < 0.05). PC9/G cells were more sensitive to docetaxel than PC9 cells. No significant difference of sensitivity to pemetrexed was found between these two cell lines. Expression level of P-170 in PC9/G cells was lower than that in PC9 cells. In PC9/G cells, the expression of integrin beta1 and DNA healing gene was high and expression of gene during mitosis was low. The level of expression of Akt, phospho-Akt and integrin beta1 in PC9/G cells was higher than that in PC9 cells. CONCLUSION: In PC9/G cells, a cell line with acquired resistance to gefitinib, over-expression of PI3K, integrin and DNA restoration gene and continuous activation of PI3K is found to be correlated with resistance to cisplatin. Docetaxel or pemetrexed is a more reasonable choice than cisplatin for treatment of NSCLC patients who failed to respond to EGFR-TKI.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/patologia , Quinazolinas/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Cromonas/farmacologia , Docetaxel , Gefitinibe , Glutamatos/farmacologia , Guanina/análogos & derivados , Guanina/farmacologia , Humanos , Integrina beta1/metabolismo , Neoplasias Pulmonares/metabolismo , Morfolinas/farmacologia , Pemetrexede , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/metabolismo , Taxoides/farmacologiaRESUMO
OBJECTIVE: To investigate the mutations in exon 19 of epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer from Chinese patients. METHODS: Genomic DNA was extracted from 72 lung cancer tissues. Then the exon 19 of EGFR gene was amplified by nested PCR and sequenced. RESULTS: In 13 tumor tissues, multi-nucleotide in-frame deletion mutations at the exon 19 of EGFR gene, had been detected. There were 4 mutation types. The mutation rate was 18.1%. The mutations were all heterozygous. There was association of the exon 19 mutation of EGFR gene with adenocarcinoma, female patients and non-smokers. CONCLUSION: There were multi-nucleotide in-frame deletion mutations in exon 19 of EGFR gene. Mutations of the exon 19 of EGFR gene were higher in female, non-smoking and adenocarcinoma patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Genes erbB-1/genética , Adenocarcinoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , Éxons/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da Polimerase , Fatores Sexuais , FumarRESUMO
Enhanced MRI (magnetic resonance imaging) plays a vital role in the early detection of tumor but with low specificity. Molecular imaging of angiogenesis could efficiently deliver contrast agents to the tumor site by specific targeted carriers. We designed and synthesized dual-targeted paramagnetic liposomes functionalized with two angiogenesis-targeting ligands, the αVß3 integrin-specific RGD (Arg-Gly-Asp) and the neuropilin-1 (NRP-1) receptor-specific ATWLPPR (Ala-Thr-Trp-Leu-Pro-Pro-Arg) (A7R). These liposomes were proved to be in the nanoparticle range and demonstrated to effectively encapsulate paramagnetic MRI contrast agents Gd-DTPA (gadolinium-diethylenetriamine pentaacetic acid). T1 relaxivity of various liposome formulations was lower than pure Gd-DTPA but with no statistically significant difference. In vitro cellular uptake and competitive inhibition assay showed the higher binding affinity of dual-targeted liposomes to HUVECs (human umbilical vein endothelial cells) and A549 cells compared with pure Gd-DTPA, non-targeted, and single-targeted liposomes, which was proved to be mediated by the binding of RGD/ανß3-integrin and A7R/NRP1. For MR imaging of mice bearing A549 cells in vivo, dual-targeted liposomes reached the highest SER (signal enhancement rate) value with a significant difference at all experimental time points. It was about threefold increase compared to pure Gd-DTPA and non-targeted liposomes and was 1.5-fold of single-targeted liposomes at 2 h post injection. The SER was lowered gradually and decreased only by 40% of the peak value in 6 h. Dual-targeted liposomes were likely to exert a synergistic effect and the specificity of delivering Gd-DTPA to the tumor site. Therefore, dual-ανß3-integrin-NRP1-targeting paramagnetic liposome with a RGD-ATWLPPR heterodimeric peptide might be a potent system for molecular imaging of tumor.
RESUMO
Drug resistance is one of the major causes in treatment failure of non-small cell lung cancer. Interactions between tumor cells and extracellular matrix have been shown to be one of the mechanisms. We investigated the expression of extracellular matrix proteins and the effects of collagen IV on cisplatin-induced apoptosis in cisplatin-sensitive and -resistance lung cancer cells. Extracellular matrix may provide protection against cisplatin-induced apoptosis of lung cancer cells. Integrin ß(1) signaling and PI3-Kinase pathway may be important survival pathways of drug-induced apoptosis in lung cancer cells, and activation of this pathway may contribute to the generation of drug resistance.
Assuntos
Adenocarcinoma/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/farmacologia , Colágeno Tipo IV/farmacologia , Resistencia a Medicamentos Antineoplásicos , Matriz Extracelular/fisiologia , Neoplasias Pulmonares/tratamento farmacológico , Adenocarcinoma/patologia , Apoptose/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular , Ciclo Celular , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 QuinaseRESUMO
The present study aimed to explore the role of integrin ß1 and the relevant signaling pathways in acquired gefitinib resistance in non-small cell lung cancer (NSCLC). The inhibitory effects of gefitinib, with or without LY294002, on cellular proliferation were evaluated by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide assay. Cell cycle progression and apoptosis were analyzed by flow cytometry, while western blotting was used to evaluate the expression of EGFR, phosphorylated (phospho)-EGFR, protein kinase B (Akt), phospho-Akt, extracellular signal-regulated kinase (Erk) and phospho-Erk. The gene expression profiles of PC9 and PC9/G cells were determined by DNA microarray. Integrin ß1 was knocked down in PC9/G cells by transiently transfected short interfering RNA (siRNA). A scrambled siRNA sequence was used as a control. Apoptosis of transfected cells was determined by Annexin V-phycoerythrin-Cy5/propidium iodide staining. Sequencing products were amplified by nested PCR. The resistant index of PC9/G cells to gefitinib was ~138- to 256-fold higher than that of PC9 cells, and this resistance was accompanied by significant increase in integrin ß1 expression in PC9/G cells. Knockdown of integrin ß1 with short hairpin RNA in PC9/G cells markedly inhibited proliferation and enhanced apoptosis in response to gefitinib, restoring the sensitivity of PC9/G cells gefitinib. Phosphoinositide 3-kinase (PI3K)/Akt activation was observed in PC9/G cells in the presence of gefitinib and the sensitivity of PC9/G cells to gefitinib was also able to be restored by PI3K/Akt pathway inhibitor LY294002. Finally, knockdown of integrin ß1 significantly reduced the levels of phospho-Akt. These findings suggest that integrin ß1 signaling via the PI3K/Akt pathway may be a significant mechanism underlying gefitinib resistance, and may potentially present an alternative therapeutic target for the treatment of NSCLC unresponsive to EGFR inhibitors.
RESUMO
BACKGROUND: Non-small cell lung cancer (NSCLC) presents the highest morbidity and mortality among malignant tumors worldwide. The overall effective rate of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is 30% to 40%, and PFS (progression-free sruvival) is 12 months. However, EGFR-TKI resistance is typical in clinical observations, and this phenomenon significantly affects tumor suppression. To overcome this resistance, a new prognostic factor associated with lung cancer drug resistance should be discovered. This study investigated the relationship between the inhibitor of differentiation 1 (ID1) and non-small cell lung cancer EGFR-TKI resistance in vivo and in vitro to determine any statistical significance and discuss the underlying mechanism. METHODS: Western blot and qRT-PCR were used to quantify the expression of ID1 in lung cancer. IHC was used to detect the expression of ID1 in pathological tissues (lung cancer tissues and adjacent tissues). MTT was used to detect cell proliferation, in which the cells were treated with gefitinib after being transfected by ID1 slow virus vector. Lung cancer cells were inoculated in nude mice until the tumor diameter grew to certain measurement. Gefitinib treatment was started, and the tumor volume was estimated. RESULTS: ID1 was highly expressed in NSCLC (P<0.05). Both ID1 expression and drug resistance of EGFR-TKI in NSCLC were positively correlated (P<0.05). The treatment group with gefitinib showed obviously less expression than the control group. CONCLUSIONS: ID1 is highly expressed in NSCLC. ID1 expression was positively related to drug resistance of EGFR-TKI in NSCLC. Gefitinib can be used to effectively treat NSCLC, and the mechanism may be associated with an increased level of STAT3 phosphorylation.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Proteína 1 Inibidora de Diferenciação/genética , Neoplasias Pulmonares/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Proteína 1 Inibidora de Diferenciação/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
BACKGROUND: At present, it has been known that cyclooxygenase-2 (COX-2) plays a crucial role in invasion, development and metastasis of non-small cell lung cancer (NSCLC). In order to explore whether the expression of COX-2 inhibits the occurrence and development of NSCLC, antisense vector of human COX-2 gene is transfected into COX-2 highly expressing NSCLC cell line H1299 and its effects on proliferation and sensitivity to cisplatin of H1299 are analysed. METHODS: H1299 cells were transfected with antisense vector of human COX-2 gene using LipoVecTM transfecting technique. Transfected cells were selected with Geneticin (G418). The COX-2 mRNA level was examined by using reverse polymerase chain reaction (RT-PCR). The COX-2 protein level was examined by Western Blot. The proliferative status and sensitivity to cisplatin of cells was measured by methabenzthiazuron (MTT) assay. RESULTS: RT-PCR showed a lower COX-2 mRNA level in transfected cells. The level of COX-2 protein was decreased apparently. The proliferative index of the transfected cells decreased significantly (P < 0.05). The IC50 value of cisplatin decreased remarkably in transfected cells (1.8mg/l) compared with that in H1299 cells without transfection (3.8mg/l) (P < 0.05). CONCLUSIONS: Transfection with antisense vector of human COX-2 gene can not only inhibit the proliferation of H1299 cells, but also increase the sensitivity to cisplatin of H1299 cells.