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  • Involvement of Adiponectin in the regulation of imatinib res

    2024-07-09

    Involvement of Adiponectin in the regulation of imatinib resistance in human CML cells has not been hitherto examined. Therefore, in this study, we first established imatinib-resistant K562 CML cells, and then evaluated the effect of Adiponectin in reversing imatinib resistance. The data presented here showed that Adiponectin was able to reverse K562 resistance to imatinib in vitro and in vivo. Additional data with molecular approaches suggested that the reversion of Adiponectin in imatinib resistance signals through AdipoR1 but not AdipoR2 to downregulate Bcr-Abl expression and effect in imatinib-resistant K562 CML cells.
    Materials and methods
    Results
    Discussion In this study, we first established imatinib-resistant K562 CML cells which referred to IMR-K562. Continuous exposure to stepwise increasing concentrations of imatinib resulted in the selection of sub-clones resistance to imatinib, which has been used in several studies to derive imatinib-resistant cell lines [15]. Our results showed that imatinib is ineffective in proliferation suppressing and apoptosis induction in IMR-K562 even at the high dose. Furthermore, mRNA expression level of AdipoR1 was strikingly increased in IMR-K562 cells compared with K562 cells, whereas AdipoR2 mRNA level was found to be unchanged, indicating a possible novel mechanism information about imatinib-mediated resistance. Adiponectin, a functional ligand of AdipoR1, suppressed cell proliferation and induced apoptosis [3], [20], [21]. Earlier studies reported that Adiponectin serum levels have been inversely associated with childhood acute myeloblastic leukemia. Moreover, a recent study revealed that AdipoR1 expression was significantly increased in imatinib treated CML patients [5]. Correspondently, our results also showed that AdipoR1 but not AdipoR2 was upregulated in imatinib-resistant K562 CML cells compared with parental-sensitive cells. Taken together, Adiponectin and its functional receptor AdipoR1 might WZ811 mg play a critical role in imatinib resistance. Therefore, we next assessed whether Adiponectin was involved in imatinib resistance. Interestingly, MTT assay and WZ811 mg analysis depicted that IMR-K562 cells resensitized to imatinib treatment in the presence of Adiponectin in a dose-dependent manner, that is to say, Adiponectin was able to reverse K562 CML cells resistance to imatinib. What’s more, the reversible effect of Adiponectin in imatinib resistance was abolished by silencing AdipoR1 but not AdipoR2, suggesting that AdipoR1 but not AdipoR2 exhibited a central role in Adiponectin signaling in reversing imatinib resistance. Resistance development is a multifactorial phenomenon in cells exposed to the kinase inhibitor imatinib [22]. Increasing evidences have since described overexpression of Bcr-Abl was the most frequent cause of resistance identified in cell lines which were engineered to develop resistance [15]. Most importantly, amplification of Bcr-Abl was reported in patients with acquired resistance [23], [24]. The amplification of the fusion gene can lead to increased Bcr-Abl activity thus overcoming the inhibiting function of imatinib. To investigate possible downstream mechanisms involved in imatinib resistance, the DNA sequence of the ATP-binding site of Bcr-Abl in IMR-K562 and K562 cells was examined by direct sequencing and MDR1 gene expression were analyzed using real-time PCR in these cells, however, neither mutations detected in Bcr-Abl in these cells nor overexpression of MDR1 in IMR-K562 was found (data not shown). The data presented here then showed that the mRNA expression level of Bcr-Abl was strikingly increased in IMR-K562 cells compared with K562 cells. Furthermore, Adiponectin dose dependently reduced Bcr-Abl expression in both mRNA and protein level. In the meanwhile, Adiponectin also attenuated phosphorylated CrkL which is Bcr-Abl substrate, indicating Adiponectin was able to downregulate Bcr-Abl expression but its tyrosine kinase activity. Additionally, these downregulation effect of Bcr-Abl and its tyrosine kinase activity in Adiponectin was abandoned by gene silence of AdipoR1 but not AdipoR2, which was consistent with data mentioned above, the reversible effect of Adiponectin in imatinib resistance was due to AdipoR1, not AdipoR2. In other words, the reversion of Adiponectin in imatinib resistance signals through AdipoR1 to downregulate Bcr-Abl expression and effect in IMR-K562 cells.