Yandim Melis Kartal, Ceylan Cagatay, Elmas Efe, Baran Yusuf
Department of Molecular Biology and Genetics, İzmir Institute of Technology, Urla, 35430, İzmir, Turkey.
Department of Food Engineering, İzmir Institute of Technology, Urla, 35430, İzmir, Turkey.
Tumour Biol. 2016 Feb;37(2):2365-78. doi: 10.1007/s13277-015-4015-9. Epub 2015 Sep 15.
Chronic myeloid leukemia (CML) is a type of hematological malignancy that is characterized by the generation of Philadelphia chromosome encoding BCR/ABL oncoprotein. Tyrosine kinase inhibitors (TKIs), imatinib, nilotinib, and dasatinib, are used for the frontline therapy of CML. Development of resistance against these TKIs in the patients bearing T315I mutation is a major obstacle in CML therapy. Ponatinib, the third-generation TKI, is novel drug that is effective even in CML patients with T315I mutation. The exact mechanism of ponatinib in CML has been still unknown. In this study, we aimed to determine the potential mechanisms and structural metabolic changes activated by ponatinib treatment in imatinib-sensitive K562 human CML cell lines and 3 μM-imatinib-resistant K562/IMA3 CML cell lines generated at our lab. Apoptotic and antiproliferative effects of ponatinib on imatinib-sensitive and 3 μM-imatinib-resistant K562/IMA3 CML cells were determined by proliferation and apoptosis assays. Additionally, the effects of ponatinib on macromolecules and lipid profiles were also analyzed using Fourier transform infrared spectroscopy (FTIR). Our results revealed that ponatinib inhibited cell proliferation and induced apoptosis as determined by loss of mitochondrial membrane potential, increased caspase-3 enzyme activity, and transfer of phosphatidylserine to the plasma membrane in both K562 and K562/IMA-3 cells. Furthermore, cell cycle analyses revealed that ponatinib arrested K562 and K562/IMA-3 cells at G1 phase. Moreover, ponatinib treatment created a more ordered nucleic acid structure in the resistant cells. Although the lipid to protein ratio increased in imatinib-sensitive K562 cells with a little decrease in the K562/IMA-3 cells, ponatinib treatment indicated significant changes in the lipid composition such as a significant increase in the cellular cholesterol amounts much more in the K562/IMA-3 cells than the sensitive counterparts. Unsaturation in lipids was higher in the resistant cells; however, increases in lipids without phosphate and the number of acyl chains were much higher in the K562 cells. Taken together, all these results showed powerful antiproliferative and apoptotic effects of ponatinib in both imatinib-sensitive and imatinib-resistant CML cells in a dose-dependent manner, and hence, the use of ponatinib for the treatment of TKI-resistant CML patients may be an effective treatment approach in the clinic. More importantly, these results showed that FTIR spectroscopy can detect drug-induced physiological changes in cancer drug resistance.
慢性髓性白血病（CML）是一种血液系统恶性肿瘤，其特征是产生编码BCR/ABL癌蛋白的费城染色体。酪氨酸激酶抑制剂（TKIs），如伊马替尼、尼洛替尼和达沙替尼，用于CML的一线治疗。在携带T315I突变的患者中对这些TKIs产生耐药性是CML治疗的主要障碍。第三代TKI波纳替尼是一种新药，即使在携带T315I突变的CML患者中也有效。波纳替尼在CML中的确切机制仍然未知。在本研究中，我们旨在确定波纳替尼处理在我们实验室产生的伊马替尼敏感的K562人CML细胞系和3μM伊马替尼耐药的K562/IMA3 CML细胞系中激活的潜在机制和结构代谢变化。通过增殖和凋亡测定确定波纳替尼对伊马替尼敏感和3μM伊马替尼耐药的K562/IMA3 CML细胞的凋亡和抗增殖作用。此外，还使用傅里叶变换红外光谱（FTIR）分析了波纳替尼对大分子和脂质谱的影响。我们的结果表明，波纳替尼抑制细胞增殖并诱导凋亡，这通过线粒体膜电位丧失、caspase-3酶活性增加以及K562和K562/IMA-3细胞中磷脂酰丝氨酸向质膜的转移来确定。此外，细胞周期分析表明波纳替尼使K562和K562/IMA-3细胞停滞在G1期。此外，波纳替尼处理在耐药细胞中产生了更有序的核酸结构。虽然在伊马替尼敏感的K562细胞中脂蛋白比增加，而在K562/IMA-3细胞中略有下降，但波纳替尼处理表明脂质组成有显著变化，例如K562/IMA-3细胞中细胞胆固醇含量比敏感细胞显著增加得多。耐药细胞中脂质的不饱和度更高；然而，K562细胞中无磷酸脂质的增加和酰基链的数量更高。综上所述，所有这些结果表明波纳替尼在伊马替尼敏感和伊马替尼耐药的CML细胞中均具有强大的抗增殖和凋亡作用，且呈剂量依赖性，因此，在临床中使用波纳替尼治疗TKI耐药的CML患者可能是一种有效的治疗方法。更重要的是，这些结果表明FTIR光谱可以检测癌症耐药性中药物诱导的生理变化。
