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BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous ailment in both biological and clinical concepts. Numerous efforts have been devoted to discover natural compounds for combating cancer, which showed great potential in cancer management. Methylsulfonylmethane (MSM), an organosulfur dietary supplement, is utilized for improving various clinical conditions, particularly osteoarthritis. MSM can exert antitumor activity in a wide range of cancers. OBJECTIVES: The molecular mechanisms of action underlying antileukemic activity of MSM remain unclear. In this regard, we aimed to investigate the anticancer properties of MSM on human AML cell lines (U937 and HL60) with focus on underlying cell death mechanism. METHODS: Anticancer activity of the MSM was examined employing MTT assay, Annexin V-PE/7AAD staining, caspase3/7 activity test, and real-time qPCR. Both cell lines were treated with different concentrations (50-400 mM) of MSM for 24 h. Pretreatment of the cells with a caspase inhibitor (i.e., Z-VAD-fmk) was performed for the assessment of apoptosis induction. RESULTS: The results of MTT assay revealed that in both cell lines, the MSM markedly reduced cell viability in comparison to the control cells. Additionally, findings of Annexin V-7AAD staining revealed that MSM induced apoptosis and activated caspase 3/7 in both cell lines markedly. Real-time quantitative PCR results also supported the induction of apoptosis in AML cells. MSM altered the expression levels of various apoptotic genes (BAX, BAD, and BIM). CONCLUSION: Overall, our results indicated that MSM could induce apoptosis in AML cell lines in a dose-dependent manner, which therefore could be utilized as an antileukemic agent.
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Chemotherapy resistance is a major obstacle in cancer therapy, and identifying novel druggable targets to reverse this phenomenon is essential. The exosome-mediated transmittance of drug resistance has been shown in various cancer models including ovarian and prostate cancer models. In this study, we aimed to investigate the role of exosomal miRNA transfer in chronic myeloid leukemia drug resistance. For this purpose, firstly exosomes were isolated from imatinib sensitive (K562S) and resistant (K562R) chronic myeloid leukemia (CML) cells and named as Sexo and Rexo, respectively. Then, miRNA microarray was used to compare miRNA profiles of K562S, K562R, Sexo, Rexo, and Rexo-treated K562S cells. According to our results, miR-125b-5p and miR-99a-5p exhibited increased expression in resistant cells, their exosomes, and Rexo-treated sensitive cells compared to their sensitive counterparts. On the other hand, miR-210-3p and miR-193b-3p were determined to be the two miRNAs which exhibited decreased expression profile in resistant cells and their exosomes compared to their sensitive counterparts. Gene targets, signaling pathways, and enrichment analysis were performed for these miRNAs by TargetScan, KEGG, and DAVID. Potential interactions between gene candidates at the protein level were analyzed via STRING and Cytoscape software. Our findings revealed CCR5, GRK2, EDN1, ARRB1, P2RY2, LAMC2, PAK3, PAK4, and GIT2 as novel gene targets that may play roles in exosomal imatinib resistance transfer as well as mTOR, STAT3, MCL1, LAMC1, and KRAS which are already linked to imatinib resistance. MDR1 mRNA exhibited higher expression in Rexo compared to Sexo as well as in K562S cells treated with Rexo compared to K562S cells which may suggest exosomal transfer of MDR1 mRNA.
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In this study, we aimed to analyze the effects of first and second-generation Bcr-Abl tyrosine kinase inhibitors, imatinib and nilotinib on LPS/IFN gamma activated RAW 264.7 macrophages. Our data revealed that imatinib was less effective on nitrite levels and more toxic on macrophages compared to nilotinib. Therefore, we further analysed the effect of nilotinib on various inflammatory markers including iNOS, COX-2, NFkB, IL-6, p-ERK, p-p38 and p-JNK in LPS/IFN gamma activated RAW264.7 macrophages. Spectrophotometric viability test and Griess assay,western blot, RT-PCR and luciferase reporter assays were used to analyze the biological activity of nilotinib. Our findings revealed that nilotinib decreases nitrite levels, iNOS mRNA, iNOS and p-p38 protein expressions significantly whereas induces IL-6 mRNA and p-JNK protein expressions at particular doses. We did not find significant effect of nilotinib on COX-2, p-ERK and nuclear p65 proteins and NFkB transcriptional activity. In addition, the binding mode of nilotinib to iNOS protein was predicted by molecular docking. According to the docking analyses, nilotinib exhibited hydrophobic interactions between MET349, ALA191, VAL346, PHE363, TYR367, MET368, CYS194, TRP366 residues at the binding pocket and the molecule as well as van der Waals interactions at specific residues. In conclusion, our results reveal that, in addition to its anticancer activity, nilotinib can exhibit immune modulatory effects on macrophages through its effects on iNOS, IL-6, p-p38 and p-JNK.
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Lipopolissacarídeos , Nitritos , Mesilato de Imatinib/farmacologia , Lipopolissacarídeos/farmacologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Nitritos/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Simulação de Acoplamento Molecular , Macrófagos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Pirimidinas/toxicidade , RNA Mensageiro/metabolismoRESUMO
Imatinib (IMA) and nilotinib are the first and second generations of BCR-ABL tyrosine kinase inhibitors, which widely applied in chronic myeloid leukemia (CML) treatment. Here we aimed to provide new targets for CML treatment by transcriptome analysis. Microarray data GSE19567 was downloaded and analyzed from Gene Expression Omnibus (GEO) to identify common genes, which are downregulated or upregulated in K562-imatinib and K562-nilotinib treated cells. The differentially expressed genes (DEGs) were assessed, and STRING and Cytoscape were used to create the protein-protein interaction (PPI) network. In imatinib and nilotinib treated groups' comparison, there were common 626 upregulated and 268 downregulated genes, which were differentially expressed. The GO analysis represented the enrichment of DEGs in iron ion binding, protein tyrosine kinase activity, transcription factor activity, ATP binding, sequence-specific DNA binding, cytokine activity, the mitochondrion, sequence-specific DNA binding, plasma membrane and cell-cell adherens junction. KEGG pathway analysis revealed that downregulated DEGs were associated with pathways including microRNAs in cancer and PI3K-Akt signaling pathway. Furthermore, upregulated DEGs were involved in hematopoietic cell lineage, lysosome and chemical carcinogenesis. Among the upregulated genes, MYH9, MYH14, MYL10, MYL7, MYL5, RXRA, CYP1A1, FECH, AKR1C3, ALAD, CAT, CITED2, CPT1A, CYP3A5, CYP3A7, FABP1, HBD, HMBS and PPOX genes were found as hub genes. Moreover, 20 downregulated genes, YARS, AARS, SARS, GARS, CARS, IARS, RRP79, CEBPB, RRP12, UTP14A, PNO1, CCND1, DDX10, MYC, WDR43, CEBPG, DDIT3, VEGFA, PIM1 and TRIB3 were identified as hub genes. These genes have the potential to become target genes for diagnosis and therapy of CML patients.
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Biologia Computacional , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva , Pirimidinas , Humanos , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Biologia Computacional/métodos , Pirimidinas/farmacologia , Mapas de Interação de Proteínas , Perfilação da Expressão Gênica , Antineoplásicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células K562 , Redes Reguladoras de Genes/efeitos dos fármacosRESUMO
Imatinib (IMA) is a tyrosine kinase inhibitor (TKI) introduced for the chronic myeloid leukemia (CML) therapy. Emergence of IMA resistance leads to the relapse and failure in CML therapy. Benzimidazole is a heterocyclic organic compound which is widely investigated for the development of anticancer drugs. In this study, we aimed to explore the anticancer effects of some 2-[4-(1H-benzimidazol-1-yl) phenyl]-1H-benzimidazole derivatives on K562S (IMA-sensitive) and K562R (IMA-resistant) cells. To analyze the cytotoxic and apoptotic effects of the compounds, K562S, K562R, and L929 cells were exposed to increasing concentrations of the derivatives. Cytotoxic effects of compounds on cell viability were analyzed with MTT assay. Apoptosis induction, caspase3/7 activity were investigated with flow cytometry and BAX, BIM, and BAD genes expression levels were analyzed with qRT-PCR. Rhodamine123 (Rho-123) staining assays were carried out to evaluate the effect of compounds on P-glycoprotein (P-gp) activity. The hit compounds were screened using molecular docking, and the binding preference of each compounds to BCR-ABL protein was evaluated. Our results indicated that compounds triggered cytotoxicity, caspase3/7 activation in K562S and K562R cells. Rho-123 staining showed that compounds inhibited P-gp activity in K562R cells. Overall, our results reveal some benzimidazole derivatives as potential anticancer agents to overcome IMA resistance in CML.
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Antineoplásicos , Resistencia a Medicamentos Antineoplásicos , Humanos , Mesilato de Imatinib/farmacologia , Simulação de Acoplamento Molecular , Células K562 , Antineoplásicos/farmacologia , Apoptose , Proteínas de Fusão bcr-abl/genética , Inibidores de Proteínas Quinases/farmacologia , Benzimidazóis/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATPRESUMO
PURPOSE: Chronic myeloid leukemia (CML) is a hematological malignancy characterized by the presence of BCR-ABL protein. Imatinib (IMA) is considered as the first line therapy in management of CML which particularly targets the BCR-ABL tyrosine kinase protein. However, emergence of resistance to IMA hinders its clinical efficiency. Hence, identifying novel targets for therapeutic approaches in CML treatment is of great importance. Here, we characterize a new subpopulation of highly adherent IMA-resistant CML cells that express stemness and adhesion markers compared to naive counterparts. MATERIALS AND METHODS: We performed several experimental assays including FISH, flow cytometry, and gene expression assays. Additionally, bioinformatics analysis was performed by normalized web-available microarray data (GSE120932) to revalidate and introduce probable biomarkers. Protein-protein interactions (PPI) network was analyzed by the STRING database employing Cytoscape v3.8.2. RESULTS: Our findings demonstrated that constant exposure to 5 âµM IMA led to development of the adherent phenotype (K562R-adh). FISH and BCR-ABL expression analysis indicated that K562R-adh cells were derived from the original cells (K562R). In order to determine the role of various genes involved in epithelial-mesenchymal transition (EMT) and stem cell characterization, up/down-regulation of various genes including cancer stem cell (CSC), adhesion and cell surface markers and integrins were observed which was similar to the findings of the GSE120932 dataset. CONCLUSION: Treating CML patients with tyrosine kinase inhibitors (TKIs) as well as targeting adhesion molecules deemed to be effective approaches in prevention of IMA resistance emergence which in turn may provide promising effects in the clinical management of CML patients.
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Leucemia Mielogênica Crônica BCR-ABL Positiva , Inibidores de Proteínas Quinases , Humanos , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Células K562 , Apoptose , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Proteínas de Fusão bcr-abl/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , FenótipoRESUMO
Chronic myeloid leukemia (CML) is a malignant hematopoietic stem cell disease resulting in the fusion of BCR and ABL genes and characterized by the presence of the reciprocal translocation t(9;22)(q34;q11). BCR-ABL, a product of the BCR-ABL fusion gene, is a structurally active tyrosine kinase and plays an important role in CML disease pathogenesis. Imatinib mesylate (IMA) is a strong and selective BCR-ABL tyrosine kinase inhibitor. Although IMA therapy is an effective treatment, patients may develop resistance to IMA therapy over time. This study investigated the possible genetic resistance mechanisms in patients developing resistance to IMA. We did DNA sequencing in order to detect BCR-ABL mutations, which are responsible for IMA resistance. Moreover, we analyzed the mRNA expression levels of genes responsible for apoptosis, such as BCL-2, P53, and other genes (SCD-1, PTEN). In a group of CML patients resistant to IMA, when compared with IMA-sensitive CML patients, a decrease in SCD-1 gene expression levels and an increase in BCL-2 gene expression levels was observed. In this case, the SCD-1 gene was thought to act as a tumor suppressor. The present study aimed to investigate the mechanisms involved in IMA resistance in CML patients and determine new targets that can be beneficial in choosing the effective treatment. Finally, the study suggests that the SCD-1 and BCL-2 genes may be mechanisms responsible for resistance.
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Lepidium sativum L. is a common herb distributed worldwide, used as a food ingredient and therapeutic agent in traditional medicine for treating health-related disorders. L. sativum and its extracts have been described to possess numerous biological activities including antimicrobial, antidiabetic, antioxidant, antidiarrheal, anticancer, and numerous health-promoting effects in in vivo and in vitro studies. The purpose of this review is to summarize the findings describing important biological functions and therapeutic effects of L. sativum in various cell lines and animal models. In this review, the English-language articles were gathered from electronic databases including Web of Science, PubMed and Google Scholar with no time limit applied to any database. The search terms used in this review include, "Lepidium sativum L." and/or "chemical composition", "health benefits", "antimicrobial", "antioxidant", "anticancer", "diuretic", "nephro-protection", "antidiarrheal", "antidiabetic", "anti-asthmatic", "neuroprotection", "metabolic", "bone fracture", and "reproductive performance". Additional and eligible studies were collected from reference lists of appropriate articles. The information presented will be helpful to attract more interest toward medicinal plants by defining and developing novel clinical applications and new drug formulations in the future. Pre-clinical studies showed that L. sativum possesses potent health-promoting effects involving various molecular mechanisms. Taken all together, data suggested that identified herbal plants such as L. sativum, can be exploited as nutritional and therapeutic agents to combat various ailments. Despite much research in this field, further comprehensive in vitro/in vivo studies and clinical trials are needed to identify the mechanisms underlying the biological and therapeutic activities of L. sativum.
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INTRODUCTION: Chronic Myeloid Leukemia (CML) is a hematological disease which is characterized by the presence of BCR-ABL fusion protein. Imatinib (IMA), a tyrosine kinase inhibitor of BCR-ABL, is used as a frontline treatment.Although IMA aids in killing a majority of leukemia cells, it may not kill CML stem cells which are the primary roots of disease and therapy resistance. Recently, antimicrobial drugs have been gaining attention because of their selective targeting of cancer cells. Therefore, we now ask if combinational therapy of IMA with a targeted antimicrobial drug Azithromycin (AZT) can enhance the treatment efficiency in IMA resistant CML. METHODS: K562S (IMA sensitive) and K562R (IMA resistant) cells were treated with increasing concentrations of AZT to determine its effects on cell proliferation and apoptosis. Cell viability, apoptosis, caspase3/7 activity and P-glycoprotein (Pgp) function were investigated with spectrophotometric MTT assay and flow cytometric Annexin V staining, caspase 3/7 activity, and Rhodamine123 staining assays respectively. The expression levels of pro-apoptotic (BAX, BAD and BIM), anti- apoptotic (BCL-XL and BCL-2) and drug transporter (MDR-1 and MRP-1) genes were assessed with qRT-PCR. RESULTS: AZT treatment alone inhibited cell viability, induced apoptosis and enhanced caspase 3/7 activity in both K562S and high MDR-1 (Pgp) expressing K562R cells. Moreover, combination of AZT/IMA suppressed cell viability, induced apoptosis and caspase3/7 activity more effectively and significantly compared to K562R cells treated with only IMA or AZT. Furthermore, AZT and AZT/IMA combination decreased Pgp function in K562R cells in comparison with their controls. Based on qRT-PCR data, single AZT and combined AZT/IMA treatment also induced BAX/BCL-2 ratio significantly in both K562S and K562R cells. CONCLUSION: Single AZT and AZT/IMA combinational treatment can be proposed as a promising and effective treatment strategy for CML. One of the mechanisms underlying the potent anticancer effect of combined AZT/IMA could be its ability to inhibit Pgp function and increase intracellular accumulation of IMA which leads to the induction of apoptosis in K562R cells.
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Antineoplásicos/farmacologia , Azitromicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Mesilato de Imatinib/farmacologiaRESUMO
Chronic Myeloid Leukemia (CML) is a clonal hematopoietic malignancy characterized by the formation of BCR-ABL fusion protein. Imatinib (IMA) is a BCR-ABL tyrosine kinase inhibitor (TKI), which exhibited a high rate of response for newly diagnosed CML patients. Emergence of IMA resistance considered as a major challenge in CML therapy. Recent studies reported the anti-cancer effect of natural extracts such as 6-Shogaol (6-SG) which is extracted from ginger and the mechanisms involved in targeting of cancer cells. In the present study, we aimed to explore the potential anticancer effect of 6-SG on K562S (Imatinib sensitive) and K562R (Imatinib resistant) cells. K562S and K562R cells were incubated with increasing concentrations of 6-SG (5 µM- 50 µM) to determine its cytotoxic and apoptotic effects. Cell viability and apoptosis were investigated with spectrophotometric MTT assay and flow cytometric Annexin V staining, respectively. The mRNA expression levels of apoptotic related genes (BAX and BCL-2) and drug transporter (MDR-1 and MRP-1) genes were evaluated with qRT-PCR. According to our results, 6-SG treatment inhibited cell viability, induced apoptosis in both K562S and K562R cells. Based on our RT-PCR results, 6-SG enhanced pro-apoptotic BAX gene and decreased anti-apoptotic BCL-2 gene expression levels significantly in both treated K562S and K562R cells. Furthermore, 6-SG increased MDR-1 mRNA expression level in K562S and K562R cells in comparison with their control counterparts. Whereas, 6-SG decrease MRP-1 mRNA expression level in K562S cells significantly. It is the first study that reveals the apoptotic effect of 6-SG in CML cell line and IMA resistance. Therefore, 6-SG treatment can be suggested as a promising strategy for CML therapy.
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Catecóis/farmacologia , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteína X Associada a bcl-2/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Cancer is a globally challenging health problem threatening mankind. Despite therapeutic advances in dealing with this malignancy, heterogeneous response and resistance to chemotherapeutic agents remain the hallmarks of cancer therapy. On the other hand, the involvement of the microbiota in affecting human health is well defined. An ever-growing body of evidence implicates the potential link between the microbiome and the efficacy of cancer therapies. Gut microbiota can modulate the metabolism of drugs in a number of ways. The presence of bacteria within the tumor environment can also impact the responses to cancer therapies; changing the chemical structure of chemotherapeutic drugs, affecting their activity, and local concentration. However, the underlying mechanisms by which gut and tumor microbial communities affect the response to cancer therapy are poorly understood and deciphering these mechanisms is of paramount importance. This review provides an overview of how gut and tumor microbiota might affect the efficacy of chemotherapy, radiotherapy, and immunotherapy and alleviate the adverse side effects of these therapies for the development of personalized and effective anticancer therapy.
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Antineoplásicos/uso terapêutico , Microbioma Gastrointestinal , Imunoterapia , Neoplasias/microbiologia , Neoplasias/terapia , Radioterapia , Humanos , Neoplasias/imunologia , Resultado do TratamentoRESUMO
Intercellular communications play a major role in tissue homeostasis. In pathologies such as cancer, cellular interactions within the tumor microenvironment (TME) contribute to tumor progression and resistance to therapy. Tunneling nanotubes (TNTs) are newly discovered long-range intercellular connections that allow the exchange between cells of various cargos, ranging from ions to whole organelles such as mitochondria. TNT-transferred mitochondria were shown to change the metabolism and functional properties of recipient cells as reported for both normal and cancer cells. Metabolic plasticity is now considered a hallmark of cancer as it notably plays a pivotal role in drug resistance. The acquisition of cancer drug resistance was also associated to TNT-mediated mitochondria transfer, a finding that relates to the role of mitochondria as a hub for many metabolic pathways. In this review, we first give a brief overview of the various mechanisms of drug resistance and of the cellular communication means at play in the TME, with a special focus on the recently discovered TNTs. We further describe recent studies highlighting the role of the TNT-transferred mitochondria in acquired cancer cell drug resistance. We also present how changes in metabolic pathways, including glycolysis, pentose phosphate and lipid metabolism, are linked to cancer cell resistance to therapy. Finally, we provide examples of novel therapeutic strategies targeting mitochondria and cell metabolism as a way to circumvent cancer cell drug resistance.
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Resistencia a Medicamentos Antineoplásicos , Mitocôndrias/metabolismo , Nanotubos , Neoplasias/metabolismo , Microambiente Tumoral , Animais , Humanos , Mitocôndrias/patologia , Neoplasias/patologiaRESUMO
OBJECTIVE: Chronic myleoid leukemia (CML) is a myeloproliferative disorder characterized with the constitutive activation of Bcr-Abl tyrosine kinase which is a target for imatinib, the first line treatment option for CML. Constitutive activation of NFκB and ß-catenin signaling promotes cellular proliferation and survival and resistance to Imatinib therapy in CML. Akirin-2 is a nuclear protein which is required for NFκB dependent gene expression as a cofactor and has been linked to Wnt/beta-catenin pathway. The purpose of this study is to examine Akirin-2, NFκB and ß-catenin in imatinib resistance of CML and to test if any direct physical protein-protein interaction exists between NFkB and both ß-catenin and Akirin-2. METHODS: RT-PCR and western blot were performed to determine Akirin-2, NFκB-p65 and ß-catenin gene and protein expressions, Co-immunoprecipitation and chromatin immunoprecipitation analysis were carried out to detect the direct physical interactions and binding of NFκB-p65 and ß-catenin proteins to MDR1 promoter region, respectively. RESULTS: ß-catenin and NFκB-p65 proteins bound to DNA promoter regions of MDR1 in imatinib-sensitive and resistant CML cells, whereas any direct protein-protein interaction could not be found between NFκB-p65 and Akirin-2 or ß-catenin proteins. Nuclear ß-catenin and NFκB-p65 levels increased in imatinib resistance. Moreover, increased Akirin-2 protein accumulation in the nucleus was shown for the first time in imatinib resistant CML cells. DISCUSSION: We show for the first time that Akirin-2 can be a novel biomarker in imatinib resistance. Targeting Akirin-2, NFκB and ß-catenin genes may provide an opportunity to overcome imatinib resistance in CML.
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Biomarcadores Tumorais , Proteínas de Ligação a DNA , Resistencia a Medicamentos Antineoplásicos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva , Proteínas de Neoplasias , Fator de Transcrição RelA , Fatores de Transcrição , beta Catenina , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fator de Transcrição RelA/biossíntese , Fator de Transcrição RelA/genética , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , beta Catenina/biossíntese , beta Catenina/genéticaRESUMO
Chronic myeloid leukemia (CML) is a hematopoietic malignancy characterized by the t(9; 22) and the related oncogene, BCR-ABL. Tyrosine kinase activity of fusion protein BCR-ABL is the main cause of CML. Even if imatinib is used as a tyrosine kinase inhibitor (TKI) for CML therapy, drug resistance may occur in patients and the clinical failure of imatinib treatment in resistant patients had resulted with the use of another alternative TKIs. BCR-ABL dependent and independent molecular mechanisms have crucial roles in drug resistance. To reveal the underlying molecular mechanisms which play significant roles in imatinib resistance in CML, we established K562 imatinib-resistant cell line (K562r5) which was continuously exposed to (5µM) imatinib to investigate molecular mechanisms which play significant roles in drug resistance. First of all, we analyzed T315I, M351T, F315L and F359C/L/V mutations with DNA sequencing as a BCR-ABL dependent mechanism in our cell lines. Moreover, we investigated BCR-ABL independent mechanisms such as apoptosis, autophagy, drug transport and DNA repair which affect drug resistance in these cell lines. In vitro cell viability was determined by MTT assay. DNA sequencing analysis was performed to detect BCR-ABL mutations. The apoptotic effect of imatinib on CML cell lines was tested by flow cytometric Annexin V-PE staining and caspase activation assays. Apoptotic, autophagic, drug transporter and DNA repair genes expression levels were determined by RT-PCR. The conventional cytogenetic analysis was performed on K562s and K562r cells. Our results indicate that inhibition of apoptosis, induction of autophagy, overexpression of efflux gene MDR1 and down-regulation of influx gene OCT1 play crucial roles in the progression of imatinib resistance.
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Antineoplásicos/farmacologia , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Mesilato de Imatinib/farmacologia , Células K562/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Transporte Biológico/efeitos dos fármacos , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Caspases/metabolismo , Análise Mutacional de DNA , DNA de Neoplasias/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática/efeitos dos fármacos , Proteínas de Fusão bcr-abl/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células K562/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação de Sentido Incorreto , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Mutação PuntualRESUMO
BACKGROUND: The tyrosine kinase inhibitor, imatinib, used as a first line treatment in Chronic Myeloid Leukemia (CML) patients, may lead to resistance and failure to therapy. Novel combinations of imatinib with other drugs is a strategy to improve treatment efficiency. OBJECTIVE: In this study, the antileukemic and apoptotic effects of a benzamide derivative XT5 and benzoxazole derivative XT2B and their combination with imatinib were investigated in imatinib-sensitive (K562S) and imatinib-resistant (K562R) CML cells. METHODS: In vitro cytotoxicity was determined by MTT assay. Then, apoptotic effect of XT5 on CML cell lines was tested by Annexin V flow cytometry, caspase activation and RT-PCR. Docking calculation was performed using AutoDock Vina in PyMOL environment using AutoDock/Vina plugin for PyMOL. RESULTS: According to our MTT assay data, XT5 indicated significant antiproliferative effect on cell lines, therefore we investigated apoptotic effects of XT5. Treatment of K562 cell lines with a combination of XT5 and imatinib-XT5 increased cytotoxicity, the Annexin V binding and caspase 3/7 activation. In addition to apoptosis assays, we observed an increase in the expression levels of the pro-apoptotic (BAX, BAD and BIM) genes in XT5 treated K562R and K562S cells. Molecular modelling experiments showed that XT5 showed hydrogenbonding interactions with important amino acids of BCR-ABL kinase receptor; however XT2B did not show any hydrogen bond interaction. CONCLUSION: Our results indicate that XT5 could be a potential candidate to be used as a new anticancer drug and XT5 combination with imatinib as an alternate treatment strategy for overcoming imatinib resistance.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Simulação de Acoplamento Molecular , Antineoplásicos/síntese química , Antineoplásicos/química , Benzamidas/síntese química , Benzamidas/química , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células K562 , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Retinoids which are vitamin A (Retinol) derivatives have been suggested to mediate the inhibition of cancer cell growth and apoptosis. It has been reported that all trans retinoic acid (ATRA) exhibited suppressive effects on different types of leukemia including chronic myelogenous leukemia. OBJECTIVE: In the present study, we aim to find out the effects of 6 synthetic N-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydronaphthalene-2-yl)-carboxamide derivatives (compound 6-12) on cell viability and apoptotic pathways in K562 human chronic myelogenous leukemia cell line. METHODS: Cell viability and apoptosis were examined by spectrophotometric thiazolyl blue tetrazolium bromide (MTT) and caspase-3 assay, western blot, RT-PCR and flow cytometry. RESULTS: Our results indicated that compound 6 (5-(1,2-Dithiolan-3-yl)-N-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydronaphthalen-2-yl)pentanamide), 8 (4-(3,4-Dimethoxyphenyl)-N-(3,5,5,8,8-pentamethyl-5,6,7,8- tetrahydronaphthalen-2-yl)butanamide) and 11 (E-3-(4-Hydroxy-3-methoxyphenyl)-N-(3,5,5,8,8-pentamethyl- 5,6,7,8-tetrahydronaphthalen-2-yl)acrylamide) exhibited apoptotic effects in K562 human chronic myelogenous leukemia cell line and induced caspase 3, PARP cleavage, Bax/Bcl-2 ratio, Bad and Bim gene expressions. CONCLUSION: Some retinoid derivatives tested in this study induced apoptosis of K562 cells which suggest that these compounds may serve as potential agents in the treatment of chronic myelogenous leukemia.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Tetra-Hidronaftalenos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Estrutura Molecular , Relação Estrutura-Atividade , Tetra-Hidronaftalenos/síntese química , Tetra-Hidronaftalenos/química , Células Tumorais CultivadasRESUMO
AIMS: The etiology of infertility is still unknown in almost half of all male infertility patients. In sperm, DNA condensation differs from somatic and female gamete cells, with the protamine (PRM) gene and its transcription factor, Y-box binding protein 2 (YBX2), playing key roles in making the structure more compact. Protamine polymorphisms have been studied in different populations, but various results have been acquired. MATERIALS AND METHODS: In our study, we examined, for the first time in a Turkish population, the association between protamine gene alleles (PRM1 c.-190C>A, PRM1 c.197G>T, and PRM2 c.248C>T), and YBX2 (c.187T>C and c.1095 + 16A>G) and male infertility. This was accomplished using polymerase chain reaction-restriction fragment length polymorphism analyses of 100 infertile and 100 fertile Turkish men. Sperm DNA fragmentation analysis was performed using the Comet technique. RESULTS: We found that the AA and CA genotypes of the PRM1 c.-190C>A polymorphism had a significant association with infertility (p < 0.001) and the AA genotype was also highly significantly associated with high sperm DNA damage (p < 0.001). CONCLUSION: This study demonstrates that the PRM1 c.-190C>A polymorphism is associated with sperm DNA fragmentation, which may impact male infertility in the Turkish population. Further research with larger groups and in various other study populations will be required to clarify the impact of protamine and YBX2 gene polymorphisms on male infertility.
Assuntos
Infertilidade Masculina/genética , Protaminas/genética , Proteínas de Ligação a RNA/genética , Adulto , Alelos , Frequência do Gene , Genótipo , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Protaminas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Espermatozoides/fisiologiaRESUMO
Methylsulfonylmethane (MSM) is an organic sulfur-containing compound which has been used as a dietary supplement for osteoarthritis. MSM has been shown to reduce oxidative stress and inflammation, as well as exhibit apoptotic or anti-apoptotic effects depending on the cell type or activating stimuli. However, there are still a lot of unknowns about the mechanisms of actions of MSM. In this study, MSM was tested on colon cancer cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay and flow cytometric analysis revealed that MSM inhibited cell viability and increased apoptotic markers in both HCT-116 p53 +/+ and HCT-116 p53 -/- colon cancer cells. Increased poly (ADP-ribose) polymerase (PARP) fragmentation and caspase-3 activity by MSM also supported these findings. MSM also modulated the expression of various apoptosis-related genes and proteins. Moreover, MSM was found to increase c-Jun N-terminal kinases (JNK) phosphorylation in both cell lines, dose-dependently. In conclusion, our results show for the first time that MSM induces apoptosis in HCT-116 colon cancer cells regardless of their p53 status. Since p53 is defective in >50% of tumors, the ability of MSM to induce apoptosis independently of p53 may offer an advantage in anti-tumor therapy. Moreover, the remarkable effect of MSM on Bim, an apoptotic protein, also suggests its potential use as a novel chemotherapeutic agent for Bim-targeted anti-cancer therapies.