RESUMO
Ponatinib is used for advanced treatment of chronic myeloid leukemia (CML), although low doses to prevent side effects do not suppress survival pathways and eradicate leukemia stem cells (LSCs). We evaluated the potential of ponatinib and PI3K/mTOR dual-inhibitor VS-5584 combination (PoVS) therapy to increase the anti-leukemic effects of ponatinib and investigated the underlying mechanisms at the molecular level. We measured the cytotoxicities of ponatinib, VS-5584, and PoVS (CCK-8 assay), and used the median-effect equation for combination analyses. We investigated the effects of inhibitory concentrations on apoptosis, cell viability and cell-cycle regulation (flow cytometry), protein levels (ELISA, Western blot), transcriptional activities (dual-luciferase reporter assay), gene expressions (qRT-PCR). VS-5584 exerted selective cytotoxic effects against CML and LSC cell lines. VS-5584 inhibited the PI3K/Akt/mTOR pathway, resulting in reduced cell viability, slightly induced caspase-independent apoptosis, prominent G0/G1 cell-cycle blockade that is not a consequence of quiescence. Normal hematopoietic stem cell line was the least affected. Moreover, ponatinib and VS-5584 mediated synergistic anti-leukemic effects on leukemic cells. VS-5584 reduced the ponatinib dose required to target leukemic cells. PoVS treatment inhibited PI3K/Akt/mTOR pathway more consistently than either of the two agents alone through reducing p-Akt, p-mTOR, p-S6K, p-PRAS40, p-S6. The subsequent downstream effects were an increase in C/EBP transcriptional activity and decreases in activities of E2F/DP1, Myc/Max, CREB, STAT3, NFκB, AP-1, Elk-1/SRF. Transcriptional regulation resulted in alterations in the expression levels of target mRNAs. Our results highlight PoVS can be a promising treatment strategy for eliminating CML cells and LSCs selectively, with the reduced ponatinib doses.
Assuntos
Antineoplásicos/farmacologia , Imidazóis/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Purinas/farmacologia , Piridazinas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Janus Quinases/genética , Janus Quinases/metabolismo , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/efeitos dos fármacosRESUMO
Circular RNAs (circRNAs) have been demonstrated to be biomarkers in human cancers. CircRNAs are majorly recognized in the transcript formation of eukaryotic genes. Research has also revealed that circRNAs regulate gene expression at the transcriptional, post-transcriptional, and translational levels. Notably, there have been studies demonstrating that they contribute to the improvement of various diseases, including cancer. In this regard, they have potential applications in the diagnosis and treatment of cancer. In circRNA studies of blood fluids, plasma circRNAs have been identified as biomarkers in human cancers. For instance, the acute myeloid leukemia-associated hsa_circ_0004277 has been reported to be a biomarker in targeted treatments. This links with circRNAs being highly expressed in hematopoietic tissue; in haematopoiesis, the cell states are controlled by the main regulators and the complex circuits of the RNA family. In particular, circRNA serve a role in cellular processes including self-renewal, apoptosis and proliferation. In the current review, the aim was to explain the role of the defined pathogenic circRNAs derived from leukemia fusion genes and of hsa_circ_0004277 in leukemia cells.
RESUMO
BCR-ABL tyrosine kinase inhibitors (TKIs) are selective therapies for the patients with Chronic Myeloid Leukemia (CML). Imatinib and ponatinib have remarkable long-term efficacy on a major molecular response. Although TKI related induction of cytotoxicity and apoptosis have been clearly investigated in molecular levels, their comparative effect on autophagy and miRNome are largely unknown. This study aimed to investigate the involvement of alterations of miRNA expressions in CML progression, and how imatinib and ponatinib affect this process, by comparing CML, imatinib-resistant CML and leukemia stem cells (LSC). Cytotoxicity analysis was conducted by WST-1, apoptosis was evaluated by AnnexinV, autophagy was analyzed by Tb/GFP TR-FRET LC3B assay and changes in miRNomes were evaluated with microarray method. Ponatinib showed higher cytotoxicity and apoptosis at far fewer concentrations than imatinib. Both imatinib and ponatinib was able to trigger autophagy in imatinib-resistant K562ima3 cell line but not in LSC. We pointed that imatinib and ponatinib caused significant miRNA profile alterations, especially in the expressions of miR-214-pre, miR-218, miR-19a-5p, miR-19b-1-5p, miR-27b-pre, miR-23b-pre, miR-320e, miR-200a-pre, miR-508-3p, miR-33-pre and miR-766. This study is the first comparative miRNome analysis of CML, resistant CML and LSCs following the imatinib or ponatinib treatment and may guide to identify new markers for diagnosis, follow-up of the disease and to develop novel therapeutic strategies if supported by preclinical studies.