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1.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(4): 1008-1012, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31418349

RESUMO

OBJECTIVE: To investigate the role of nucleophosmin (NPM) in the proliferation of chronic myeloid leukemia cells (K562 cells) and its mechanism by RNAi technology. METHODS: shRNA was used to inhibit the expression of NPM. The expression of NPM gene was detected by real-time quantitative PCR. The effect of inhibiting NPM gene on cell proliferation was detected by MTS assay. Change of cell cycle was detected by flow cytometry. Western blot was used to detect the expression of cell cycle-related proteins. RESULTS: The shRNA lentiviral vector targeting at NPM gene was successfully constructed and used to transfect the K562 cells. The results showed that compared with the control groups, suppression of NPM gene expression in K562 cells could inhibit the cell proliferation and decrease the cell colony formation. Moreover, interference of NPM gene could prolong G0/G1 phase and arrest cell cycle, which may be related to the down-regulation of NPM gene expression and activation of p21 protein expression, thereby inhibited the formation of CDK2/ Cyclin E complex. CONCLUSION: Down-regulation of NPM gene expression in K562 cells can induce cell cycle arrest and inhibit cell proliferation.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Apoptose , Proliferação de Células , Técnicas de Silenciamento de Genes , Humanos , Células K562 , Proteínas Nucleares
2.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(4): 1064-1070, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31418358

RESUMO

OBJECTIVE: To construct a K562 and adriamycin-resistant K562 (KAR) cell line with stably down-regulation of NCL (nucleolin) expression, and to investigate the effect of NCL down-regulation on the drug resistance in K562 and KAR cells. METHODS: K562 and KAR cells were infected with lentivirus, and stably transfected cell clones were obtained by puromycin screening. The cell proliferation was detected by MTS assay, the cell apoptosis was detected by flow cytometry, and the expression level of drug resistance related genes was detected by real-time PCR. RESULTS: The K562 and KAR cells with stable down-regulation of NCL were successfully constructed. Compared with the control group, the proliferation of K562 and KAR cells with down-regulating NCL expression decreased significantly (P <0.05), the apoptosis of cells increased significantly (P <0.05), and cell resistance to adriamycin was down-regulated. CONCLUSION: Inhibition of NCL expression may increase the sensitivity of cells to adriamycin, which may be related with the promotion of apoptosis of K562 and KAR cells.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Leucemia Mielogênica Crônica BCR-ABL Positiva , Apoptose , Doxorrubicina , Humanos , Células K562 , Fosfoproteínas , Proteínas de Ligação a RNA
3.
Fitoterapia ; 137: 104264, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31299275

RESUMO

Five undescribed triterpene-type saponins, parkibicolorosides A-E, a cassane-type diterpene, and a known trimethoxy benzene glucoside were isolated from the roots of Parkia bicolor A. Chev. Their structures were elucidated by different spectroscopic methods including 1D- and 2D-NMR experiments as well as HR-ESI-MS analysis. Their cytotoxic activity against the chronic myeloid leukemia (K562) cell line was evaluated. The monosaccharides saponins exhibited a moderate antiproliferative activity with IC50 ranging from 48.49 ±â€¯0.16 to 81.66 ±â€¯0.17 µM.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Fabaceae/química , Raízes de Plantas/química , Saponinas/farmacologia , Triterpenos/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Costa do Marfim , Humanos , Células K562 , Estrutura Molecular , Compostos Fitoquímicos/isolamento & purificação , Compostos Fitoquímicos/farmacologia , Saponinas/isolamento & purificação , Triterpenos/isolamento & purificação
4.
Chem Biodivers ; 16(8): e1900325, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31290253

RESUMO

Chronic myeloid leukemia (CML) is a lethal malignancy, and the progress toward long-term survival has stagnated in recent decades. Pristimerin, a quinone methide triterpenoid isolated from the Celastraceae and Hippocrateaceae families, is well-known to exert potential anticancer activities. In this study, we investigated the effects and the mechanisms of action on CML. We found that pristimerin inhibited cell proliferation of K562 CML cells by causing G1 phase arrest. Furthermore, we demonstrated that pristimerin triggered autophagy and apoptosis. Intriguingly, pristimerin-induced cell death was restored by an autophagy inhibitor, suggesting that autophagy is cross-linked with pristimerin-induced apoptosis. Further studies revealed that pristimerin could produce excessive reactive oxygen species (ROS), which then induce JNK activation. These findings provide clear evidence that pristimerin might be clinical benefit to patients with CML.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Triterpenos/farmacologia , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Células K562 , Espécies Reativas de Oxigênio/metabolismo , Triterpenos/química
5.
Cancer Invest ; 37(6): 242-252, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31296070

RESUMO

Drug resistance to TKIs and the existance of CML leukemia stem cells is an urgent problem. In this study, we demonstrate that quinacrine (QC) induces apoptosis in BCR-ABL positive CML and acute lymphoblastic leukemia (ALL) cells. Interestingly, QC inhibits the colony formation of primary CD34+ progenitor/stem leukemia cells from CML patients. QC targets RNA polymerase I, which produces ribosomal (r)RNA, involving in protein translation process. Also, QC treatment prolongs CML-like mice survival and inhibits K562 tumor growth in vivo. In conclusion, we demonstrate that QC depletes BCR-ABL protein and suppresses Ph-positive leukemia cells in vitro and in vivo.


Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Quinacrina/uso terapêutico , Animais , Antígenos CD34/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico
6.
Zhongguo Zhong Yao Za Zhi ; 44(12): 2532-2537, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31359720

RESUMO

According to drug design flattening principle,a series of novel indole podophyllotoxin derivatives which were introduced different indole substituents in C-4 position on the basis of podophyllotoxin nucleus were synthesized with the starting material podophyllotoxin and 1 H-indole-5-carboxylic acid. Its anti-tumor activity in vitro was tested in order to screen for high-efficiency and low-toxic compounds. Six target compounds were synthesized,and were confirmed by~1 H-NMR,~(13)C-NMR,HR-ESI-MS and melting point determination analysis. All these target compounds were not reported by previous literature. Using etoposide as positive control drug,all the target compounds were screened for cytotoxicity against He La cells,K562 cells and K562/A02 cell in vitro by MTT method. The antitumor activity screening results showed that compounds 4 b,4 e,4 f exhibited higher inhibitory rate against He La cells and K562 cells than those of control drug VP-16. This route has the advantages on simple operation and reasonable design,provides some practical reference value for the further development on the structure modification of podophyllotoxin and study on anti-tumor activity.


Assuntos
Antineoplásicos/farmacologia , Indóis/farmacologia , Podofilotoxina/farmacologia , Antineoplásicos/síntese química , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Indóis/síntese química , Células K562 , Podofilotoxina/síntese química , Relação Estrutura-Atividade
7.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(3): 685-691, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31204917

RESUMO

OBJECTIVE: To investigate the effect of Bmi-1 gene silence on the proliferation ability of K562 cells in vitro and in vivo, and to explore the relation of molecular mechanism between proliferation ability of K562 cells in vitro and in vivo with PTEN/pAKT signaling pathway. METHODS: The Bmi-1 small interference RNA (siRNA) sequences were transfected into K562 cells for decreasing Bmi-1 expression. The effect of Bmi-1 siRNA on the proliferation of K562 cells in vitro and in vivo was detected by MTT method and colony-forming test, the effect of Bmi-1 siRNA on the tumorogenicity of K562 cells was observed by subcutaneous inoculation of K562 cells, LY294002 and Bpv treated K562 cells in nude mice, the expression of Bmi-1, PTEN and pAKT proteins were detected by Western blot. RESULTS: The Bmi-1 siRNA could inhibit the proliferation activity, colony-forming and tumor-forming abilities of K562 cells. After the silence of Bmi-1 gene, the PTEN expression in Bmi-1 gene-silenced group was significantly enhanced. While the pAKT expression in Bmi-1 gene-silenced group was significantly reduced; after the K562 cells were treated with LY294002 (an inhibitor of pAKT), the pAKT expression colony-forming and tumor forming abilities were reduced in comparison with untreated K562 cells; after the K562-S1 cells were treated with Bpv (an inhibitor of PTEN), the PTEN expression decreased, while the pAKT expression, colony forming and tumor-forming abilities were restored. CONCLUSION: The Bmi-1 gene possibly involves in regulation of K562 proliferation in vivo and in vitro, the effect of PTEN/pAKT signaling pathway maybe one of molecular mechanisms mediating this regulation.


Assuntos
Apoptose , Leucemia , Animais , Proliferação de Células , Humanos , Células K562 , Camundongos , Camundongos Nus , PTEN Fosfo-Hidrolase , Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas c-akt , RNA Interferente Pequeno , Transdução de Sinais
8.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(3): 708-716, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31204920

RESUMO

OBJECTIVE: To investigate the effect of stably down-regulating the FMI expression of K562 cells on the sensitivity of K562 cells to Imatinib (IM) and its possible mechanism. METHODS: Western-blot was used to detect the expression of FMI protein in K562 cells and peripheral blood mononuclear cells from the patients with chronic myelogenous leukemia, chronic myeloid blast crisis and healthy volunteers. The specific interference sequences targeting at the human FMI gene were designed and ligated into the lentiviral vector LV3; the three plasmid system-packaged lentivirus particles were used to transfect K562 cells to screen K562 cells that stably down-regulated FMI. CCK-8 assay and flow cytometry were used to determine effect of IM on cell proliferation and apoptosis. The transcription level of FMI and Fz8 in leukemia cells was detected by fluorescent quantitative PCR. The protein expression levels of FMI, Fz8, NFAT1, BCR-ABL and ß-catenin in leukemia cells were detected by Western-blot. RESULTS: The expression of FMI protein could be detected in peripheral blood mononuclear cells of the patients with CML-BC and K562 cells, the FMI expression could not be detected in all the patients with CML-CP and healthy volunteers. The recombinant lentiviral vector LV3/FMI had been successfully constructed the lentivirus was packaged, and the K562 cells stably down-regulating the FMI protein were screened. After stable down-regulation of FMI expression in K562 cells, the proliferation rate of leukemia cells decreased and the apoptosis rate was increased under the same drug concentration. Both the transcription and protein expression levels of Fz8 decreased. The NFAT1 total protein level increased, as well as the nuclear translocation of protein was enhanced. There was no significant change in the expression level of BCR-ABL fusion protein. The expression level of ß-catenin protein decreased. CONCLUSION: After the stable down-regulation of FMI expression, the sensitivity of K562 cells to IM and apoptosis of cells increase, which are performed possibly by inhibiting the FMI-Fz8 signaling pathway and activating the Ca2+-NFAT and Wnt/ß-catenin signaling pathway.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucócitos Mononucleares , Apoptose , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl , Humanos , Mesilato de Imatinib , Células K562
9.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(3): 763-768, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31204929

RESUMO

OBJECTIVE: To investigate the regulatory effect of miR-543 on proliferation and apoptosis of human leukemia cell line K562 and its mechanism. METHODS: 46 CML patients treated in our hospital from 2018.2-2018.9 was selected and enrolled in CML group, and 30 healthy persons were selected and enrolled in control group at the same time. After Lipofectamine 2000 liposome was used to transfer the miR-543 mimic and negative control (Scramble mimic) to K562 cells, CCK8 assay was used to detect the effect of miR-543 on proliferation of K562 cells; Luciferase reporter assay was used to report the targeting relationship of miR-543 to Wnt gene. Flow cytometry was used to detect the effect of miR-543 on apoptosis of K562 cells; Western blot method was used to detect the Wnt, ß-catenin, BCL-2, c-MYC and BAX expression level. RESULTS: The level of miRNA-543 in CML patients was significantly lower than that in healthy controls (P<0.05). The expression level of miR-543 in mimic group was significantly higher than that in blank control group (P<0.05). The Wnt gene expression level in mimic group was significantly lower than that in blank control group (P<0.05). The expression of luciferase of Wnt wild plasmid was decreased by miR-543 mimic (P<0.05), and the luciferase activity of Wnt mutant plasmid was not inhibited by miR-543 mimic after mutation of binding site (P<0.05). There was no significant difference in the proliferation ability of K562 cells between the blank control group and the negative control (Scramble mimic) group (P>0.05). The proliferation level of K562 cells in mimic group was significantly lower than that in blank control group and negative control (Scramble mimic) group (P<0.05). Apoptotic level of K562 cells in miR-543 mimic group was significantly higher than that in blank control group and negative control (Scramble mimic) group (P<0.05). Western blot assay showed that Wnt, ß-catenin, BCL-2 and c-MYC protein levels in miR-543 mimic group were significantly lower than those in blank control group and negative control (Scramble mimic) group (P<0.05); BAX protein level in miR-543 mimic group was higher than that in blank control group and negative control (Scramble mimic) group (P<0.05). CONCLUSION: miR-543 can target Wnt protein to inhibit the activity of Wnt signaling pathway, thereby inhibiting the proliferation of leukemia cells and increasing the level of apoptosis.


Assuntos
Leucemia , MicroRNAs/genética , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Células K562
10.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(3): 785-789, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31204932

RESUMO

OBJECTIVE: To explore the reversal effect of pioglitazone (PIO) on multidrug resistance in K562/ADR cells and its mechanism. METHODS: The proliferation inhibition rate, half inhibition concentration (IC50) and drug-resistance reversal multipe were detected and the curve of proliferation inhibition rate was drawn by MTT assay, the transcription of PPARγ, CYP2C8 and CYP2J2 genes was detected by RT-PCR; the expression of PPARγ, CYP2C8 and CYP2J2 proteins was detected by Western blot. RESULTS: The IC50 of PIO on K562 and K562/ADR cells for 60 h was 326.7 µmol/L and 349.1 µmol/L respectively. The reversal multiple of 30 µmol/L PIO on ADR-resistance of K562/ADR cells was 6.4. After treatment of K562/ADR cells with PIO, the transcription of CYP2C8 and CYP2J2 and the protein expression of CYP2C8 and CYP2J2 significantly decreased, the transcription of PPARγ gene and the expression of PPARγ protein were not changed. CONCLUSIONS: Pioglitazone can reverse the adriamycin-resistance in K562/ADR cells that is closely related to the decrease of protein expression of CYP2C8 and CYP2J2. Pioglitazone is an effective multidrug resistance reversal agent for tumors.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Doxorrubicina , Resistência a Múltiplos Medicamentos , Humanos , Células K562 , Pioglitazona
11.
Eur J Med Chem ; 178: 232-242, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31185413

RESUMO

As a continuation to our research, a series of novel Bcr-Abl inhibitors incorporated with 6-phenyl-1H-indazol-3-amine as hinge binding moiety (HBM) were developed based on confirmation analysis. Biological results indicated that these compounds exhibited an enhanced inhibition against Bcr-AblWT and Bcr-AblT315I in kinases assays, along with improved anti-proliferative activities in K562 cell assays. In particular, compound Y9 displayed comparable potency with that of imatinib. It potently inhibited Bcr-AblWT and Bcr-AblT315I kinases with IC50 of 0.043 µM and 0.17 µM, respectively. Furthermore, compound Y9 inhibited the proliferation of K562 and K562R cells with IC50 of 1.65 µM and 5.42 µM, respectively. Therefore, 6-phenyl-1H-indazol-3amine as HBM, combined with flexible linker, is a successful strategy contribute to research on T315I mutant resistance, and compound Y9 could be served as a starting point for further optimization.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Indazóis/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Benzamidas/síntese química , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacologia , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Desenho de Drogas , Proteínas de Fusão bcr-abl/química , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Mesilato de Imatinib/farmacologia , Indazóis/síntese química , Indazóis/química , Indazóis/metabolismo , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Simulação de Acoplamento Molecular , Mutação , Piperazinas/síntese química , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/farmacologia , Maleabilidade , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo
12.
Ann Hematol ; 98(9): 2045-2052, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31243572

RESUMO

Thalassemia has a high prevalence in Thailand. Oxidative damage to erythroid cells is known to be one of the major etiologies in thalassemia pathophysiology. Oxidative stress status of thalassemia is potentiated by the heme, nonheme iron, and free iron resulting from imbalanced globin synthesis. In addition, levels of antioxidant proteins are reduced in α-thalassemia and ß-thalassemia erythrocytes. However, the primary molecular mechanism for this phenotype remains unknown. Our study showed a high expression of miR-144 in ß- and α-thalassemia. An increased miR-144 expression leads to decreased expression of nuclear factor erythroid 2-related factor 2 (NRF2) target, especially in α-thalassemia. In α-thalassemia, miR-144 and NRF2 target are associated with glutathione level and anemia severity. To study the effect of miR-144 expression, the gain-loss of miR-144 expression was performed by miR inhibitor and mimic transfection in the erythroblastic cell line. This study reveals that miR-144 expression was upregulated, whereas NRF2 expression and glutathione levels were decreased in comparison with the untreated condition after miR mimic transfection, while the reduction of miR-144 expression contributed to the increased NRF2 expression and glutathione level compared with the untreated condition after miR inhibitor transfection. Moreover, miR-144 overexpression leads to significantly increased sensitivity to oxidative stress at indicated concentrations of hydrogen peroxide (H2O2) and rescued by miR-144 inhibitor. Taken together, our findings suggest that dysregulation of miR-144 may play a role in the reduced ability of erythrocyte to deal with oxidative stress and increased RBC hemolysis susceptibility especially in thalassemia.


Assuntos
Eritrócitos/metabolismo , MicroRNAs/biossíntese , Fator 2 Relacionado a NF-E2/biossíntese , Estresse Oxidativo , Regulação para Cima , Talassemia alfa/metabolismo , Talassemia beta/metabolismo , Eritrócitos/patologia , Feminino , Glutationa/biossíntese , Glutationa/genética , Hemólise , Humanos , Peróxido de Hidrogênio/metabolismo , Células K562 , Masculino , MicroRNAs/genética , Fator 2 Relacionado a NF-E2/genética , Talassemia alfa/genética , Talassemia alfa/patologia , Talassemia beta/genética , Talassemia beta/patologia
13.
Planta Med ; 85(13): 1088-1097, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31216579

RESUMO

As part of our search for new cytotoxic and antimicrobial natural products from endolichenic fungi, 19 compounds including 1 new 10-member lactone (2: ), 1 new polyacetylene glycoside (3: ), 1 new brasilane-type sesquiterpenoid glycoside (4: ), and 2 isobenzofuran-1(3H)-one derivatives (5: and 6: ) were isolated from the solid culture of the endolichenic fungus Hypoxylon fuscum. Their structures were unambiguously elucidated by NMR spectroscopic data, MS, ECD (electronic circular dichroism) calculation, and chemical methods. The cytotoxic effects on K562, SW480, and HEPG2 cell lines and the antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Candida albicans were assessed. Compounds 1, 2: , and 5: exhibited moderate cytotoxicity against K562, SW480, and HEPG2 cell lines while compounds 1, 9: , and 11: displayed weak antibacterial activity against S. aureus.


Assuntos
Citotoxinas/isolamento & purificação , Xylariales/metabolismo , Anti-Infecciosos/isolamento & purificação , Anti-Infecciosos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Dicroísmo Circular , Citotoxinas/farmacologia , Escherichia coli/efeitos dos fármacos , Células Hep G2/efeitos dos fármacos , Humanos , Células K562/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Staphylococcus aureus/efeitos dos fármacos , Xylariales/química
14.
Drug Des Devel Ther ; 13: 1107-1115, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31040647

RESUMO

Background: With the development of drug delivery, novel tools and technological approaches have captured the attention of researchers in recent years. Several target drug delivery systems (DDSs) including nanoparticles (NPs) have been developed as an important strategy to deliver classical medicine. Objective: The objective of this study was to evaluate the application of novel daunorubicin (DNR)-loaded poly(lactic-co-glycolic acid)-poly-l-lysine-polyethylene glycol-transferrin (Tf) nanoparticles (DNR-loaded NPs) in hematologic malignancies in vitro and in vivo. Materials and methods: DNR-loaded NPs were prepared by the modified double-emulsion solvent evaporation/diffusion method, and its microscopic form was observed under scanning electron microscope. Intracellular distribution of DNR was directly detected by fluorescence microscopy. After establishment of a tumor xenograft model by injecting K562 cells into the left leg of nude mice, the therapeutic effect of the DNR-loaded NPs on the growth of tumors was measured by calculating the tumor size, and the relative expression of Caspase-3 protein was detected by immunohistochemical staining. Furthermore, intracellular concentration of DNR and the extent of cell apoptosis in primary leukemia cells were quantified by flow cytometry. Results: DNR-loaded NPs had a spherical shape of about 180 nm in diameter. DNR-loaded NP group showed a significant enhancement of cellular uptake in K562 cells compared with DNR group. Tumor inhibition rate was higher in DNR-loaded NP group in comparison with DNR group, and the relative expression of Caspase-3 protein was upregulated in DNR-loaded NP group compared with DNR group. Furthermore, DNR-loaded NPs obviously increased intracellular concentration of DNR in primary leukemia cells compared with DNR group, but there was no significant difference in primary cell apoptosis between the two groups. These findings suggest that the novel NP DDS can enhance the performance of conventional antitumor drugs and may be suitable for further application in the treatment of hematologic malignancies.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Daunorrubicina/farmacologia , Neoplasias Hematológicas/tratamento farmacológico , Nanopartículas/química , Poliésteres/química , Polilisina/análogos & derivados , Transferrina/química , Animais , Antibióticos Antineoplásicos/química , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Daunorrubicina/química , Ensaios de Seleção de Medicamentos Antitumorais , Neoplasias Hematológicas/patologia , Humanos , Células K562 , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Polilisina/química , Células Tumorais Cultivadas
15.
BMC Bioinformatics ; 20(1): 292, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142264

RESUMO

BACKGROUND: Although several studies have provided insights into the role of long non-coding RNAs (lncRNAs), the majority of them have unknown function. Recent evidence has shown the importance of both lncRNAs and chromatin interactions in transcriptional regulation. Although network-based methods, mainly exploiting gene-lncRNA co-expression, have been applied to characterize lncRNA of unknown function by means of 'guilt-by-association', no strategy exists so far which identifies mRNA-lncRNA functional modules based on the 3D chromatin interaction graph. RESULTS: To better understand the function of chromatin interactions in the context of lncRNA-mediated gene regulation, we have developed a multi-step graph analysis approach to examine the RNA polymerase II ChIA-PET chromatin interaction network in the K562 human cell line. We have annotated the network with gene and lncRNA coordinates, and chromatin states from the ENCODE project. We used centrality measures, as well as an adaptation of our previously developed Markov State Models (MSM) clustering method, to gain a better understanding of lncRNAs in transcriptional regulation. The novelty of our approach resides in the detection of fuzzy regulatory modules based on network properties and their optimization based on co-expression analysis between genes and gene-lncRNA pairs. This results in our method returning more bona fide regulatory modules than other state-of-the art approaches for clustering on graphs. CONCLUSIONS: Interestingly, we find that lncRNA network hubs tend to be significantly enriched in evolutionary conserved lncRNAs and enhancer-like functions. We validated regulatory functions for well known lncRNAs, such as MALAT1 and the enhancer-like lncRNA FALEC. In addition, by investigating the modular structure of bigger components we mine putative regulatory functions for uncharacterized lncRNAs.


Assuntos
Cromatina/metabolismo , Redes Reguladoras de Genes , RNA Longo não Codificante/genética , Análise de Sequência de RNA/métodos , Algoritmos , Regulação da Expressão Gênica , Humanos , Células K562 , RNA Mensageiro/genética
16.
Int J Mol Sci ; 20(9)2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31071955

RESUMO

Since imatinib (Glivec or Gleevec) has been used to target the BCR-ABL fusion protein, chronic myeloid leukemia (CML) has become a manageable chronic disease with long-term survival. However, 15%-20% of CML patients ultimately develop resistance to imatinib and then progress to an accelerated phase and eventually to a blast crisis, limiting treatment options and resulting in a poor survival rate. Thus, we investigated whether histone deacetylase inhibitors (HDACis) could be used as a potential anticancer therapy for imatinib-resistant CML (IR-CML) patients. By applying a noninvasive apoptosis detection sensor (NIADS), we found that panobinostat significantly enhanced cell apoptosis in K562 cells. A further investigation showed that panobinostat induced apoptosis in both K562 and imatinib-resistant K562 (IR-K562) cells mainly via H3 and H4 histone acetylation, whereas panobinostat targeted cancer stem cells (CSCs) in IR-K562 cells. Using CRISPR/Cas9 genomic editing, we found that HDAC1 and HDAC2 knockout cells significantly induced cell apoptosis, indicating that the regulation of HDAC1 and HDAC2 is extremely important in maintaining K562 cell survival. All information in this study indicates that regulating HDAC activity provides therapeutic benefits against CML and IR-CML in the clinic.


Assuntos
Proteínas de Fusão bcr-abl/genética , Histona Desacetilase 1/genética , Histona Desacetilase 2/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Acetilação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Sistemas CRISPR-Cas/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Técnicas de Inativação de Genes , Inibidores de Histona Desacetilases/farmacologia , Humanos , Mesilato de Imatinib/efeitos adversos , Mesilato de Imatinib/farmacologia , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Panobinostat/farmacologia
17.
Ann Hematol ; 98(8): 1905-1918, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31104089

RESUMO

Efficient and safe delivery of siRNA in vivo is the biggest roadblock to clinical translation of RNA interference (RNAi)-based therapeutics. To date, lipid nanoparticles (LNPs) have shown efficient delivery of siRNA to the liver; however, delivery to other organs, especially hematopoietic tissues still remains a challenge. We developed DLin-MC3-DMA lipid-based LNP-siRNA formulations for systemic delivery against a driver oncogene to target human chronic myeloid leukemia (CML) cells in vivo. A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with siRNA molecules targeting the BCR-ABL fusion oncogene found in CML. We show a highly efficient and non-toxic delivery of siRNA in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of a leukemic model. By targeting the BCR-ABL fusion oncogene, we show a reduction of leukemic burden in our myeloid leukemia mouse model and demonstrate reduced disease burden in mice treated with LNP-BCR-ABL siRNA as compared with LNP-CTRL siRNA. Our study provides proof-of-principle that fusion oncogene specific RNAi therapeutics can be exploited against leukemic cells and promise novel treatment options for leukemia patients.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/farmacologia , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Expressão Gênica , Marcação de Genes/métodos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Lipídeos/administração & dosagem , Lipídeos/química , Camundongos , Camundongos Nus , Nanopartículas/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacocinética , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Cancer Sci ; 110(8): 2421-2430, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31145521

RESUMO

Although the targeted tyrosine kinase inhibitor imatinib mesylate (IM) has achieved significant responses against CML in the clinical setting, a small proportion of patients fail to respond to IM treatment and their disease continues to progress, indicating resistance to IM therapy. As a secreted extracellular matrix protein, cysteine-rich protein 61 (Cyr61) plays an important role in the resistance of solid tumors to chemotherapy, but its role in CML is unclear. In the present study, we observed that Cyr61 levels were upregulated in the plasma and bone marrow (BM) of patients with CML as well as in K562 cells. This upregulation of Cyr61 significantly decreased IM-induced cellular apoptosis of K562 cells through nuclear factor kappa B/B-cell lymphoma 2 pathways. Inhibition of Cyr61 restored the chemosensitivity of K562 cells to IM both in vitro and in vivo. Thus, our results showed for the first time that Cyr61 plays an important role in regulating the chemosensitivity of CML cells to IM, suggesting that selectively targeting Cyr61 directly or its relevant effector pathways may provide potential value in improving the clinical response of patients with CML to IM treatment.


Assuntos
Proteína Rica em Cisteína 61/metabolismo , Mesilato de Imatinib/farmacologia , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Inibidores de Proteínas Quinases/farmacologia , Regulação para Cima/efeitos dos fármacos
19.
Nat Commun ; 10(1): 2233, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110232

RESUMO

The recently developed single-cell CRISPR screening techniques, independently termed Perturb-Seq, CRISP-seq, or CROP-seq, combine pooled CRISPR screening with single-cell RNA-seq to investigate functional CRISPR screening in a single-cell granularity. Here, we present MUSIC, an integrated pipeline for model-based understanding of single-cell CRISPR screening data. Comprehensive tests applied to all the publicly available data revealed that MUSIC accurately quantifies and prioritizes the individual gene perturbation effect on cell phenotypes with tolerance for the substantial noise that exists in such data analysis. MUSIC facilitates the single-cell CRISPR screening from three perspectives, i.e., prioritizing the gene perturbation effect as an overall perturbation effect, in a functional topic-specific way, and quantifying the relationships between different perturbations. In summary, MUSIC provides an effective and applicable solution to elucidate perturbation function and biologic circuits by a model-based quantitative analysis of single-cell-based CRISPR screening data.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Modelos Genéticos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Biologia Computacional/métodos , Conjuntos de Dados como Assunto , Estudos de Viabilidade , Perfilação da Expressão Gênica/métodos , Técnicas de Silenciamento de Genes , Humanos , Células Jurkat , Células K562 , RNA Guia/genética
20.
Nat Commun ; 10(1): 2212, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101808

RESUMO

In mammalian cells, double-stranded DNA breaks (DSBs) are preferentially repaired through end-joining processes that generally lead to mixtures of insertions and deletions (indels) or other rearrangements at the cleavage site. In the presence of homologous DNA, homology-directed repair (HDR) can generate specific mutations, albeit typically with modest efficiency and a low ratio of HDR products:indels. Here, we develop hRad51 mutants fused to Cas9(D10A) nickase (RDN) that mediate HDR while minimizing indels. We use RDN to install disease-associated point mutations in HEK293T cells with comparable or better efficiency than Cas9 nuclease and a 2.7-to-53-fold higher ratio of desired HDR product:undesired byproducts. Across five different human cell types, RDN variants generally result in higher HDR:indel ratios and lower off-target activity than Cas9 nuclease, although HDR efficiencies remain strongly site- and cell type-dependent. RDN variants provide precision editing options in cell types amenable to HDR, especially when byproducts of DSBs must be minimized.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Engenharia Genética/métodos , Rad51 Recombinase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reparo de DNA por Recombinação , Proteína 9 Associada à CRISPR/genética , Quebras de DNA de Cadeia Dupla , Edição de Genes/métodos , Células HEK293 , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas , Células K562 , Rad51 Recombinase/genética , Proteínas Recombinantes de Fusão/genética , Transfecção/métodos
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