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The management of chronic myelogenous leukemia (CML) has seen significant progress with the introduction of tyrosine kinase inhibitors (TKIs), particularly Imatinib. However, a notable proportion of CML patients develop resistance to Imatinib, often due to the persistence of leukemia stem cells and resistance mechanisms independent of BCR::ABL1 This study investigates the roles of IL6R, IL7R, and MYC in Imatinib resistance by employing CRISPR/Cas9 for gene editing and the Non-Invasive Apoptosis Detection Sensor version 2 (NIADS v2) for apoptosis assessment. The results indicate that Imatinib-resistant K562 cells (K562-IR) predominantly express IL6R, IL7R, and MYC, with IL6R and MYC playing crucial roles in cell survival and sensitivity to Imatinib. Conversely, IL7R does not significantly impact cytotoxicity, either alone or in combination with Imatinib. Further genetic editing experiments confirm the protective functions of IL6R and MYC in K562-IR cells, suggesting their potential as therapeutic targets for overcoming Imatinib resistance in CML. This study contributes to understanding the mechanisms of Imatinib resistance in CML, proposing IL6R and MYC as pivotal targets for therapeutic strategies. Moreover, the utilization of NIADS v2 enhances our capability to analyze apoptosis and drug responses, contributing to a deeper understanding of CML pathogenesis and treatment options.
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Biomarcadores , Leucemia Mielógena Crónica BCR-ABL Positiva , Proteínas Proto-Oncogénicas c-myc , Receptores de Interleucina-6 , Humanos , Apoptosis , Resistencia a Antineoplásicos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Highly polymorphic BCR-ABL kinase domains have been reported to harbor more than a hundred mutations, and among these, 40-60% have been identified as influencers of imatinib mesylate (IM) resistance. The emergence of IM resistance poses a significant challenge in the management of Chronic Myeloid Leukemia (CML). M351T (rs121913457), E255K (rs387906517), and Y253H (rs121913461) are of particular clinical significance due to their association with high-level imatinib resistance. This study was conducted to investigate the potential role of three significant SNPs in CML progression due to IM resistance. During the study period from 2018 to 2022 (48 months), the blood samples from 219 Reverse transcriptase-PCR-confirmed CML patients following RNA extraction and cDNA preparation were subjected to M351T, E255K, and Y253H mutation analysis by PCR-RFLP. After agarose gel visualization, the samples were subjected to Sanger sequencing to confirm the nucleotide change at the polymorphic loci. The wild-type genotype of all three ABL1 SNPs under investigation exhibits a significant reduction in frequency among IM non-responders compared to the responder group. The CGT haplotype frequency exhibits a significant difference between IM responder (4.2%) and non-responder (11.8%) (p = 0.002 < 0.05). Further, CGC haplotype was observed solely among the imatinib non-responder patients with a frequency percentage of 3.3% (p = 0.004), whereas the said genotype was absent among the responder group. A reduced overall survival rate was observed with deviation from wild-type genotype (M351T loci (T > C) with 1.217 times, E255K (G > A) with 1.485 and Y253H (T > C) with 1.399 times increase in hazard ratio) thereby enhancing mortality risk due to disease progression. The significant increase in the frequency of M351T, E255K, and Y253H loci among the IM non-responder group indicated their probable association with the development of IM resistance among CML patients. A haplotype frequency distribution pattern analysis of ABL1 loci further identified the CGC haplotype as an independent predictor for IM resistance. As such the study highlights the importance of patient characteristics, genotype distribution, and haplotype frequency distribution in predicting the response to IM treatment and clinical outcomes of CML patients.
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【Objective】 To observe the effect of Selinexor (SEL) combined with Imatinib (IM) on the proliferation and apoptosis of Imatinib-resistant chronic myeloid leukemia K562/G01(KG) cells and explore the possible mechanisms. 【Methods】 K562 cells and KG cells were treated with SEL or IM respectively or in combination. Cells viability was examined by MTT assay. Apoptosis was assessed by flow cytometry. BCR-ABL mRNA was detected by RT-PCR. XPO1 was detected by Western blotting. 【Results】 IM and SEL both inhibited the proliferation of K562 cells and KG cells; half maximal inhibitory concentration (IC50) for 48 h was 0.16 μmol/L vs. 6.48 μmol/L for IM and 132.0 nmol/L vs. 275.9 nmol/L for SEL. Compared with SEL or IM alone, SEL combined with IM significantly inhibited the proliferation of KG cells (P<0.05), induced KG cells apoptosis (P<0.05), downregulated the levels of BCR-ABL mRNA (P<0.05), and inhibited the expressions of XPO1 in KG cells (P<0.05). 【Conclusion】 SEL combined with IM can synergistically inhibit the proliferation and induce apoptosis of KG cells, and then inhibit the expressions of BCR-ABL mRNA and XPO1 to exert an anti-leukemia effect.
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INTRODUCTION: Imatinib Mesylate is an authenticated drug that aids in the treatment of Chronic Myeloid Leukaemia and Philadelphia patients which is recognized as a BCR-ABL tyrosine kinase inhibitor. Indeed, DNA Methylation occupies a key role in the stability of chromosomes. OBJECTIVE: Changes in the methylation status of genes may impart to the advancement of Chronic Myeloid Leukaemia. The present investigation aims to assess the role of expression analysis and methylation status of DDIT3 and MGMT genes in imatinib-resistant and nonresistant cases. METHODS: The Imatinib resistance was screened through RFLP. In this case maximum number of patients were recorded in the chronic phase belonging to the age group 40-59 and the accelerated and blast phase is more common in elderly patients showing the progressive nature of the disease with age. Hemoglobin and platelet count are found to be higher in cases where WBC count was minimal. A history of long-term alcohol consumption is found to be associated with the progression of the disease. RESULTS: The maximum level of expression of the DDIT3 gene was recorded in the chronic phase regardless of upstream (67.8%) and downstream (57.9%) regulation. The highest MGMT expression regulation was also observed in the case of chronic phase in both upstream (78.9%) and downstream (44%) regulation. Further, the MGMT gene showed the highest methylation of 6.6% and DDIT3 showed 3.3% in CML cases. CONCLUSION: In the present study notable depletion of survivality was established in the Imatinib resistance patients manifesting genetic malfunction of BCR-ABL transcripts among the North East Indian inhabitants and advocating for the expansion of the disease.
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Leucemia Mielógena Crónica BCR-ABL Positiva , Pirimidinas , Humanos , Anciano , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Pirimidinas/efectos adversos , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Inhibidores de Proteínas Quinasas/farmacología , Progresión de la Enfermedad , Epigénesis Genética , Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl/genética , Factor de Transcripción CHOP/genética , Factor de Transcripción CHOP/uso terapéutico , Metilasas de Modificación del ADN/genética , Proteínas Supresoras de Tumor/genética , Enzimas Reparadoras del ADN/genéticaRESUMEN
OBJECTIVES: Chronic myeloid leukemia (CML) is an aggressive malignancy originating from hematopoietic stem cells. Imatinib (IM), the first-generation tyrosine kinase inhibitor, has greatly improved theliving quality of CML patients. However, owing to the recurrence and treatment failure coming from tyrosine kinase inhibitor (TKIs) resistance, some CML patients still bear poor prognosis. Therefore, we aimed to seek potential signaling pathways and specific biomarkers for imatinib resistance. METHODS: We performed mRNA and miRNA expression profiling in imatinib-sensitive K562 cells (IS-K562) and imatinib-resistant K562 cells (IR-K562). Differentially expressed genes (DEGs) were identified and pathway enrichment analyses were performed to explore the potential mechanism. The protein-protein interaction (PPI) network and miRNA-mRNA regulatory network were constructed to explore potential relationships among these genes. RT-qPCR, western blot and CCK8 were used for further experiments. RESULTS: A total of 623 DEGs and 61 differentially expressed miRNAs were identified. GO revealed that DEGs were mainly involved in cell adhesion, cell migration, differentiation, and inflammatory response. KEGG revealed that DEGs were typically enriched in the Rap1 signaling pathway, focal adhesion, proteoglycans and transcriptional misregulation in cancer, signaling pathways regulating pluripotency of stem cells and some immune-related pathways. The protein-protein interaction (PPI) network and miRNA-mRNA regulatory network revealed a web of diverse connections among genes. Finally, we proved that RHoGDI2 played a critical role in imatinib resistance. CONCLUSION: The dynamic interplay between genes and signaling pathways is associated with TKIs resistance and RHoGDI2 is identified as a biomarker in IR-K562.
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Leucemia Mielógena Crónica BCR-ABL Positiva , MicroARNs , Humanos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Inhibidor beta de Disociación del Nucleótido Guanina rho , Células K562 , MicroARNs/genética , Inhibidores de la Disociación del Nucleótido Guanina rho-Específico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Biomarcadores , Biología ComputacionalRESUMEN
Chronic myeloid leukemia (CML) results from BCR-ABL oncogene, which blocks CML cells differentiation and protects these cells from apoptosis. T315I mutated BCR-ABL is the main cause of the resistance mediated by imatinib and second generation BCR-ABL inhibitor. CML with the T315I mutation has been considered to have poor prognosis. Here, we determined the effect of Jiyuan oridonin A (JOA), an ent-kaurene diterpenoid compound, on the differentiation blockade in imatinib-sensitive, particularly, imatinib-resistant CML cells with BCR-ABL-T315I mutation by cell proliferation assay, apoptosis analysis, cell differentiation analysis, cell cycle analysis and colony formation assay. We also investigated the possible molecular mechanism by mRNA sequencing, qRT-PCR and Western blotting. We found that JOA at lower concentration significantly inhibited the proliferation of CML cells expressing mutant BCR-ABL (T315I mutation included) and wild-type BCR-ABL, which was due to that JOA induced the cell differentiation and the cell cycle arrest at G0/G1 phase. Interestingly, JOA possessed stronger anti-leukemia activity than its analogues such as OGP46 and Oridonin, which has been investigated extensively. Mechanistically, the cell differentiation mediated by JOA may be originated from the inhibition of BCR-ABL/c-MYC signaling in CML cells expressing wild-type BCR-ABL and BCR-ABL-T315I. JOA displayed the activity of inhibiting the BCR-ABL and promoted differentiation of not only imatinib -sensitive but also imatinib -resistant cells with BCR-ABL mutation, which could become a potent lead compound to overcome the imatinib -resistant induced by inhibitors of BCR-ABL tyrosine kinase in CML therapy.
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BACKGROUND: Exon 18 D842V, which is a point mutation from aspartic acid to valine at codon 842, is the most frequent mutation in Platelet-Derived Growth Factor Receptor alpha (PDGFRA)-mutated gastrointestinal stromal tumor (GIST). In the Japanese GIST guidelines, no standard systematic therapy is available for this type of GIST, which is refractory after recurrence. Recently, pimitespib (PIMI), a novel heat shock protein 90 (HSP90) inhibitor, was approved for the treatment of advanced GIST in a phase III study. This report presents a case of a long-term response to PIMI in GIST with PDGFRA D842V mutation. CASE PRESENTATION: A 55-year-old woman was diagnosed with primary GIST of the stomach and underwent partial gastrectomy. Eight years after the operation, recurrent GISTs were identified as multiple recurrent peritoneal GISTs in the upper right abdomen and pelvic cavity. We administered tyrosine kinase inhibitors, but they achieved poor effects. After failure of the standard treatment, PIMI was administered and achieved a partial response in the patient. The highest reduction rate was 32.7%. After PIMI failed, we performed multiplex gene panel testing, which revealed the PDGFRA D842V mutation. CONCLUSIONS: We report the first case of long-term response to PIMI in PDGFRA D842V mutant GIST. Pimitespib may be effective for treating GIST harboring this mutation by inhibiting HSP90.
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Despite the effectiveness of imatinib, most gastrointestinal stromal tumors (GISTs) develop resistance to the treatment, mainly due to the reactivation of KIT tyrosine kinase activity. Sunitinib, which inhibits the phosphorylation of KIT and vascular endothelial growth factor (VEGF) receptor, has been established as second-line therapy for GISTs. The recently-developed heat shock protein 90 (HSP90) inhibitor pimitespib (PIM; TAS-116) demonstrated clinical benefits in some clinical trials; however, the effects were limited. The aim of our study was therefore to clarify the effectiveness and mechanism of the combination of PIM with sunitinib for imatinib-resistant GISTs. We evaluated the efficacy and mechanism of the combination of PIM with sunitinib against imatinib-resistant GIST using imatinib-resistant GIST cell lines and murine xenograft models. In vitro analysis demonstrated that PIM and sunitinib combination therapy strongly inhibited growth and induced apoptosis in imatinib-resistant GIST cell lines by inhibiting KIT signaling and decreasing auto-phosphorylated KIT in the Golgi apparatus. In addition, PIM and sunitinib combination therapy enhanced antitumor responses in the murine xenograft models compared to individual therapies. Further analysis of the xenograft models showed that the combination therapy not only downregulated the KIT signaling pathway but also decreased the tumor microvessel density. Furthermore, we found that PIM suppressed VEGF expression in GIST cells by suppressing protein kinase D2 and hypoxia-inducible factor-1 alpha, which are both HSP90 client proteins. In conclusion, the combination of PIM and sunitinib is effective against imatinib-resistant GIST via the downregulation of KIT signaling and angiogenic signaling pathways.
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Antineoplásicos , Tumores del Estroma Gastrointestinal , Humanos , Animales , Ratones , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Sunitinib/farmacología , Sunitinib/uso terapéutico , Tumores del Estroma Gastrointestinal/patología , Factor A de Crecimiento Endotelial Vascular , Piperazinas/farmacología , Pirimidinas , Resistencia a Antineoplásicos , Antineoplásicos/uso terapéutico , Proteínas Proto-Oncogénicas c-kit/metabolismo , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
In patients with gastrointestinal stromal tumors (GIST), systemic treatment after disease progression on imatinib is challenging. Sunitinib and regorafenib are approved in the second- and third-line setting, respectively, with activity against certain secondary mutations with comparatively much lower response rates and survival increment compared to imatinib. All three of these drugs were serendipitously found to have activity in GIST, starting with imatinib, which was formulated for its ability to inhibit BCR-ABL in chronic myelogenous leukemia. Ripretinib is a drug that was specifically developed as a more potent KIT tyrosine kinase inhibitor (TKI), with broad-spectrum activity against the mutations encountered in GIST. Encouraging responses in early and later lines of treatment in the Phase 1 trial of ripretinib in GIST led to the rapid development of this novel drug. In a Phase 3 randomized clinical trial with cross-over, ripretinib demonstrated superior PFS and overall survival (OS) in 4th-line treatment and beyond compared to placebo. This established 150 mg once daily ripretinib as the standard of care in this setting. Ripretinib is generally well tolerated, with common adverse effects of hair loss, diarrhea, cramps, fatigue and nausea. The favorable safety profile and efficacy of ripretinib prompted its evaluation in a randomized phase 3 trial in the 2nd-line treatment setting. However, it did not result in a longer PFS duration than sunitinib. Although the efficacy of ripretinib in this unselected patient population was not significantly different from that of sunitinib, the tolerability profile was better. This review article aims to review the efficacy and tolerability profile of ripretinib, together with its role in the setting of unresectable or metastatic GIST.
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An 82-year-old woman underwent laparoscopic partial gastrectomy for fundal gastrointestinal stromal tumor (GIST) in a previous hospital. She visited our hospital for the treatment of recurrence, because two nodules, one each on the gastric suture line and in the peritoneum of the left diaphragm, were pointed out. Imatinib therapy was immediately initiated, and the size of both nodules decreased. However, the one on the gastric suture line showed regrowth after 4 years, which was eventually diagnosed as imatinib-resistant GIST. Hence, laparoscopic partial gastrectomy with peritoneal imatinib-sensitive nodule resection was subsequently performed. Pathologically, the nodule on the gastric suture line was confirmed as a GIST, whereas the other was diagnosed as a hyaline-degenerated GIST. Surgical intervention for imatinib-resistant GISTs has been reported to improve the outcomes of recurrent GISTs. Laparoscopic resection of localized imatinib-resistant GISTs could safely and less invasively be performed in this case; however, caution should be paid to the indication of the approach.
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Antineoplásicos , Tumores del Estroma Gastrointestinal , Laparoscopía , Neoplasias Gástricas , Anciano de 80 o más Años , Antineoplásicos/uso terapéutico , Femenino , Gastrectomía , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/patología , Tumores del Estroma Gastrointestinal/cirugía , Humanos , Mesilato de Imatinib/uso terapéutico , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/cirugía , Resultado del TratamientoRESUMEN
c-Kit overexpression and activating mutations, which are reported in various cancers, including gastrointestinal stromal tumor (GIST), small-cell lung cancer (SCLC), acute myeloid leukemia, acral melanoma, and systemic mastocytosis (SM), confer resistance to tyrosine kinase inhibitors (TKIs). To overcome TKI resistance, an anti-c-Kit antibody-drug conjugate was developed in this study to treat wild-type and mutant c-Kit-positive cancers. NN2101, a fully human IgG1, was conjugated to DM1, a microtubule inhibitor, through N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) (to give NN2101-DM1). The antitumor activity of NN2101-DM1 was evaluated in vitro and in vivo using various cancer cell lines. NN2101-DM1 exhibited potent growth-inhibitory activities against c-Kit-positive cancer cell lines. In a mouse xenograft model, NN2101-DM1 exhibited potent growth-inhibitory activities against imatinib-resistant GIST and SM cells. In addition, NN2101-DM1 exhibited a significantly higher anti-cancer effect than carboplatin/etoposide against SCLC cells where c-Kit does not mediate cancer pathogenesis. Furthermore, the combination of NN2101-DM1 with imatinib in imatinib-sensitive GIST cells induced complete remission compared with treatment with NN2101-DM1 or imatinib alone in mouse xenograft models. These results suggest that NN2101-DM1 is a potential therapeutic agent for wild-type and mutant c-Kit-positive cancers.
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Tumores del Estroma Gastrointestinal , Inmunoconjugados , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Animales , Línea Celular Tumoral , Proliferación Celular , Resistencia a Antineoplásicos/genética , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Humanos , Mesilato de Imatinib/farmacología , Mesilato de Imatinib/uso terapéutico , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Ratones , Mutación/genética , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Proteínas Tirosina Quinasas Receptoras/genéticaRESUMEN
Imatinib is a nontoxic tyrosine kinase inhibitor, used in the treatment of advanced renal cell carcinoma. However, some patients with renal cell carcinoma develop resistance to imatinib. Protein disulfide isomerase family 6 (PDIA6) was involved in the chemo-resistance of lung adenocarcinoma. In this study, the effect of PDIA6 on imatinib-resistance of renal cell carcinoma was investigated. First, PDIA6 was found to be up-regulated in the imatinib-resistant renal cell carcinoma tissues and cells. Functional assays showed that knockdown of PDIA6 sensitized imatinib-resistant renal cell carcinoma cells to imatinib through decreasing the half-maximal inhibitory concentration (IC50) of imatinib-resistant renal cell carcinoma cells. Secondly, cell proliferation of imatinib-resistant renal cell carcinoma cells was suppressed by PDIA6 silencing, and the apoptosis was promoted with reduced Bcl-2, enhanced Bax and cleaved caspase-3. Moreover, the interference of PDIA6 increased phosphorylation of H2A histone family member X (γH2AX), while decreased Rad51 and phosphorylated DNA-dependent protein kinase (DNA-PK) (p-DNA-PK) in imatinib-resistant renal cell carcinoma cells. Lastly, protein expression levels of Wnt3a and Frizzled1 (FZD1) in imatinib-resistant renal cell carcinoma cells were down-regulated by silencing of PDIA6. Over-expression of FZD1 attenuated PDIA6 silencing-induced increase in cell apoptosis and decrease in cell proliferation in imatinib-resistant renal cell carcinoma cells. In conclusion, knockdown of PDIA6 sensitized imatinib-resistant renal cell carcinoma cells into imatinib through inactivation of Wnt3a-FZD1 axis.
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Carcinoma de Células Renales/tratamiento farmacológico , Resistencia a Antineoplásicos , Receptores Frizzled/genética , Regulación Neoplásica de la Expresión Génica , Mesilato de Imatinib/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Proteína Disulfuro Isomerasas/metabolismo , Proteína Wnt3A/metabolismo , Adulto , Apoptosis/efectos de los fármacos , Apoptosis/genética , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Daño del ADN , Reparación del ADN/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Receptores Frizzled/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Mesilato de Imatinib/farmacología , Neoplasias Renales/genética , Neoplasias Renales/patología , Persona de Mediana Edad , Proteína Disulfuro Isomerasas/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
In patients with chronic myelogenous leukemia (CML), resistance to tyrosine kinase inhibitor (TKI) therapy, like imatinib, can cause death, progression to accelerated phase or blast crises, and the need for maintenance treatment. Icaritin is an active component of the genus Epimedium, a traditional Chinese herbal medicine. Icaritin has been shown to notably inhibit the growth of CML cells. To explore the potential mechanisms of inhibiting growth and inducing cell senescence in imatinib-resistant CML cells by icaritin, MTT assays were used to assess the cell viability. The apoptosis and cell cycle arrest were evaluated using flow cytometry. The SA-ß-Gal staining and the intracellular reactive oxygen species (ROS) production were measured using flow cytometry to detect the senescent cells. qRT-PCR was conducted to assess the expression of the cell cycle-associated proteins, and western blotting was used to analyze the expressions of the JAK2 and STAT3 phosphorylation proteins. The results showed that icaritin inhibited cell growth and induced cell senescence in imatinib-resistant CML cells, which is associated with the regulation of the JAK2/STAT3/P21 axis and accompanied by the accumulation of ROS. Our data suggest that icaritin is a promising therapeutic strategy for the treatment of imatinib-resistant patients with CML.
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BACKGROUND: Chronic myelogenous leukemia (CML) is one of the most common cancers in the world. Imatinib is one of the most effective therapeutic strategies to inhibit the BCR-ABL tyrosine Kinase in patients with CML, but resistance is increasingly encountered. MATERIAL AND METHODS: Microarray data GSE7114, GSE92624 and GSE97562 were downloaded and analyzed from Gene Expression Omnibus (GEO) to identify the candidate genes in the imatinib-resistant CML cells. The differentially expressed genes (DEGs) were appraised, and the protein-protein interaction (PPI) network was created by using STRING and Cytoscape. RESULTS: We screened a total of 217 DEGs, including 151 upregulated genes and 66 downregulated genes. The enriched functions and pathways of genes include insulin-like growth factor I binding, cysteine-type endopeptidase inhibitor activity involved in apoptotic process, cell adhesion, positive regulation of nitric oxide biosynthetic process and hematopoietic cell lineage. Nine hub genes were appraised and Gene Ontology enrichment analysis revealed that these genes are mainly enriched in cell cycle, peptidase inhibitor activity and cell division. Several genes such as BIRC5, CCNE2 and MCM4 were identified in survival analysis and these genes alteration are significantly associated with worse overall survival and disease-free survival. CONCLUSIONS: These genes have the potential to become surrogate markers for a clinical evaluation of imatinib-resistant CML patients. Our results provide potential target genes for diagnosis and treatment of imatinib-resistant CML patients.
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Antineoplásicos/uso terapéutico , Resistencia a Antineoplásicos , Mesilato de Imatinib/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Antineoplásicos/farmacología , Biología Computacional , Bases de Datos Genéticas , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Ontología de Genes , Redes Reguladoras de Genes/efectos de los fármacos , Humanos , Mesilato de Imatinib/farmacología , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacosRESUMEN
PURPOSE: Imatinib, a small-molecule tyrosine kinase inhibitor, has shown good clinical activity by inhibiting adenosine triphosphate (ATP) binding to the receptor. Unfortunately, majority of patients eventually develop drug resistance, which limits the long-term benefits of the tyrosine kinase inhibitors and poses a significant challenge in the clinical management of GIST. The aim of our study was to explore the feasibility of blocking KIT dimerisation upstream of the phosphorylation in imatinib-resistant GIST. METHOD: KITMAb was prepared using hybridoma technique. The biological function of KITMAb was examined in KIT-dimer-expressing cells constructed by transfecting with liposomes using enzyme linked immunosorbent assay (ELISA), immunohistochemistry, western blot, MTT, Annexin V/FITC, and flow cytometry assay, respectively. RESULTS: KIT-dimer was expressed in 293 cells transfected with c-kit mutated-type pcDNA3.1. Treatment of KIT-dimer-expressing cells with the KITMAb significantly decreased the expression of both KIT-dimer and other phosphorylated proteins of KIT downstream signalling pathway. Furthermore, KITMAb slowed down cell growth and reduced the proportion of cells in the proliferative phase (S + G2-M). Finally, we also found that KITMAb treatment accelerated cell apoptosis. These results indicate that KITMAb strongly inhibits KIT receptor dimerisation-mediated signalling pathway and cell growth responses in vitro. CONCLUSIONS: We demonstrate c-kit mutation-driven KIT auto-dimerisation prior to tyrosine kinase phosphorylation as same as the procedure in ligand-dependent signalling pathway and describe a monoclonal antibody, KITMAb, with strong affinity to the dimerisation domain of KIT that blocks the important step in both the KIT signalling pathways. Further, the results suggest that treatment with KITMAb may be potentially therapeutic in imatinib-resistant GIST.
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Anticuerpos Monoclonales/farmacología , Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica , Mutación , Neoplasias/patología , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-kit/metabolismo , Apoptosis , Biomarcadores de Tumor/genética , Ciclo Celular , Proliferación Celular , Humanos , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-kit/química , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/inmunología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract. However, up to 40-50% of GISTs develop secondary resistance after an average of 24 months of imatinib treatment. It has been reported that autophagy can promote the survival of GIST cells and induce drug resistance. Presently, the specific mechanism of autophagy in GISTs with imatinib resistance is not clear. MATERIALS AND METHODS: The cell-counting kit (CCK)-8 method and flow cytometry were used for in vitro drug sensitivity testing and autophagy level detection. Detection of the apoptosis level was by flow cytometry with the annexin V Kit. Western blotting was used to analyze the role of autophagy and apoptosis in GIST cells with CQ alone, imatinib alone, or in combination, and to analyze MAPK pathway expression. In vitro results were confirmed by in vivo experiments using the mice model. Hematoxylin and eosin and immunohistochemical staining were used to detect the pathological characteristics and immunophenotype of the transplanted tumor. Detection of KIT and PDGFRA gene mutations in the transplanted imatinib-resistant GIST was done by denaturing high performance liquid chromatography (DHPLC) and direct sequencing. ERK and KIT expression and regulation levels were detected by Western blotting. RESULTS: In vitro and vivo experiments, the autophagy level of imatinib-resistant cells was higher than that of normal cells; CQ combined with imatinib can promote apoptosis by blocking autophagy of imatinib-resistant cells. In the meanwhile, we found that the phosphorylation level of ERK may be related to autophagy. CONCLUSION: Our data suggest that autophagy through the MAPK/ERK pathway may play a pivotal role in imatinib-resistant GIST proliferation. Moreover, combining an autophagy inhibitor with imatinib may be a potential valuable strategy in overcoming acquired resistance in GIST patients.
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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.
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Proteínas de Fusión bcr-abl/genética , Histona Desacetilasa 1/genética , Histona Desacetilasa 2/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Acetilación/efectos de los fármacos , Apoptosis/efectos de los fármacos , Sistemas CRISPR-Cas/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Técnicas de Inactivación de Genes , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Mesilato de Imatinib/efectos adversos , Mesilato de Imatinib/farmacología , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Células Madre Neoplásicas/efectos de los fármacos , Panobinostat/farmacologíaRESUMEN
BACKGROUND: Nilotinib, a second-generation tyrosine kinase inhibitor (TKI), is approved for the treatment of patients with chronic myeloid leukemia (CML) in many countries, including Taiwan. Though a number of controlled clinical trials have demonstrated the safety and efficacy of nilotinib, studies assessing the safety and efficacy of nilotinib in routine clinical practice are limited. METHODS: The current study was an open-label, single-arm study conducted across 12 centers in Taiwan in adult patients with CML in chronic or accelerated phase with confirmed Ph+ chromosome (or BCR-ABL) and resistant or intolerant to one or more previous TKIs. The primary objective was to collect the long-term safety data in patients treated with nilotinib 400 mg, twice daily for up to 2 years. RESULTS: The study enrolled 85 patients with CML, including 76 in the chronic phase (CML-CP) and 9 in the accelerated phase (CML-AP). Overall, 1166 adverse events (AEs) were reported in 80 patients (94.1%), of which 70 AEs (6%) in 28 patients (32.9%) were serious and 336 AEs (28.8%) reported in 60 patients (70.6%) were drug-related. Common drug-related AEs were thrombocytopenia (21.2%), increased alanine aminotransferase (21.2%) and pruritus (17.7%). Of the 85 patients, 19 switched from imatinib due to intolerance - AEs were resolved in 16 of these 19 patients (84.2%). By 24 months, the cumulative rates of complete cytogenetic response (CCyR), major molecular response (MMR), MR4.0 (BCR-ABL1IS ⩽0.01%) and MR4.5 (BCR-ABL1IS ⩽0.0032%) were 75.3, 56.8, 16.2 and 7.4%, respectively. Patients with CML-CP at baseline had higher overall survival (OS) and progression-free survival (PFS) than those with CML-AP. CONCLUSION: This is the first study that demonstrated that nilotinib is effective and well-tolerated in patients resistant or intolerant to imatinib in the real-world setting in Taiwan, reflecting effective management of CML by physicians under routine clinical practice in Taiwan.
RESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Ancient mediterranean diet was characterized by consuming the spontaneous forms of Cynara cardunculus L. (CCL), commonly called artichoke. Cultivated and/or spontaneous forms of CC studies have demonstrated that methanol extract of CCL flower and/or cynaropicrin showed remarkable anti-proliferative activity in vitro models of leukocyte cancer cell. AIM OF THE STUDY: Chronic myeloid leukemia (CML) is associated with a reciprocal translocation of the long arms of chromosomes 9 and 22 generating the BCR/ABL fusion gene, translated in the p210BCR/ABL oncoprotein kinase. This chimeric protein is the target of a kinase inhibitor, imatinib, but the development of mutations in the ABL kinase domain resulting in drug resistance and several approaches to overcoming resistance have been study. In this concern, we investigated the effect of CCL extract on human K562 CML and K562 imatinib resistant (IMAR) cell proliferation and on p210BCR/ABL expression. MATERIALS AND METHODS: Chemical characterization of the CCL extracts was performed by GC/MS analysis and semipreparative RP-HPLC chromatography. Structural characterization of compounds was assessed by 1H-13C NMR and LC/MS analysis. The effects of CCL extracts on the proliferation of K562 CML human cell line and K562 IMAR were screened by MTT assay. The p210BCR/ABL mRNA and protein expressions were analyzed by qRT-PCR and Western blot techniques respectively. RESULTS: We demonstrate that CCL extract affect cell viability of both K562 CML human cell line and K562 IMAR. The biocomponents of CCL were chemical characterized and we identify cynaropicrin and its deacyl derivative having the capability to down-regulate the p210BCR/ABL oncoprotein. CONCLUSIONS: Our study suggests that the use of those molecules could represent a novel and promising strategy to potentiate the ability of imatinib or of its analogues to induce cancer growth arrest in CML and to delay or overcome the resistance of CML to chemotherapy.
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Antineoplásicos Fitogénicos/uso terapéutico , Cynara/química , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéutico , Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Quimioterapia Adyuvante , Resistencia a Antineoplásicos/efectos de los fármacos , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Humanos , Mesilato de Imatinib/farmacología , Células K562 , Lactonas/farmacología , Sesquiterpenos/farmacologíaRESUMEN
Heat shock protein 90 (Hsp90) contains amino (N)-terminal domain, carboxyl(C)-terminal domain, and middle domains, which activate Hsp90 chaperone function cooperatively in tumor cells. One terminal occupancy might influence another terminal binding with inhibitor. The Bcr-Abl kinase is one of the Hsp90 clients implicated in the pathogenesis of chronic myeloid leukemia (CML). Present studies demonstrate that double inhibition of the N- and C-terminal termini can disrupt Hsp90 chaperone function synergistically, but not antagonistically, in Bcr-Abl-positive human leukemia cells. Furthermore, both the N-terminal inhibitor 17-AAG and the C-terminal inhibitor cisplatin (CP) have the capacity to suppress progenitor cells; however, only CP is able to inhibit leukemia stem cells (LSCs) significantly, which implies that the combinational treatment is able to suppress human leukemia in different mature states.