Treatment with IM is the standard of care for patients newly diagnosed with CML, while several second generation inhibitors, such as dasatinib and nilotinib, have become available with the promise of overcoming some of the mutations associated with acquired resistance in these patients [
18]. However, primary TKI therapy resistance and relapse due to the persistence of leukemic stem cells (LSCs) remain a major clinical problem. Moreover, TKI monotherapy is not curative. Recently, combination therapies such as the dual-targeting of the Bcr-Abl and JAK2 activities have been shown to lead to more effective disease eradication, particularly for CML patients at high risk for TKI resistance and disease progression [
19],[
20]. Natural products, such as oridonin, which has been shown to induce the apoptosis of t(8;21) acute myeloid leukemic (AML) cells and inhibit the activity of c-Kit (+) leukemia-initiating cells [
21],[
22], have been considered to play an important role in treating cancer and drug resistance, and they are promising and safe antitumor agents due to their natural origin. DHTMF is a polymethoxyflavone isolated from
Laggera pterodonta that is a traditional herbal medicine. In a previous study, DHTMF demonstrated antiproliferative activities against a number of tumor cell lines and induced the apoptosis of CNE cells
in vitro in a time- and dose-dependent manner while exhibiting low cytotoxicity in the two normal cell lines Vero and EVC304 [
17]. Therefore, in this study, we used DHTMF in IM-resistant K562 cells (K562R) to investigate its anti-tumor effect. According to this finding, it would be interesting to characterize the effects of DHTMF in anti-leukemia therapy, particularly for the drug resistance of refractory leukemia. In this study, we analyzed the effects of DHTMF in IM-resistant K562 cells (K562R) and found that DHTMF alone or in combination with IM could significantly inhibit proliferation in a time- and dose-dependent manner. As such, the inhibitory ratio was 36.36% when 5 μg/mL DHTMF was used alone for 24 h, while 5 μg/mL DHTMF combined with 1 μg/mL IM could increase the inhibitory activity to 46.63%. These results suggest that DHTMF indeed has inhibitory effects in K562R cells and at the same time improves the sensitivity of K562R cells to IM. By flow cytometry, we confirmed that the percentage of apoptotic cells was significantly increased when K562R cells were treated with 5 μg/mL DHTMF or 1 μmol/L IM +5 μg/mL DHTMF for 24 h. These findings indicate that DHTMF alone or in combination with IM inhibits cell proliferation by inducing apoptosis in K562R cells. There are two main apoptosis pathways: the extrinsic death receptor pathway and the intrinsic mitochondrial pathway [
23]. Many polymethoxyflavone induce apoptosis via the mitochondrial apoptosis pathway [
24]–[
26]. To further investigate the potential mechanisms of action involved using apoptosis-related analysis, we first observed changes in the mitochondrial membrane potential by flow cytometry and laser confocal microscopy. The results showed that 5 μg/mL DHTMF and 1 μmol/L IM +5 μg/mL DHTMF for 24 h significantly decrease the mitochondrial membrane potential. These results are consistent with the findings of previous studies in which DHTMF was shown to induce the apoptosis of CNE cells in vitro in a time- and dose-dependent manner [
17]. These changes suggest that DHTMF is likely to induce apoptosis via the mitochondrial pathway. Caspases are crucial players in the induction of apoptotic cell death. Caspase-9 is an initiator caspase that has been implicated in the mitochondria-dependent pathway [
27]. In this study, we demonstrated that DHTMF alone or in combination with IM induces the cleavage of caspase-9, caspase-3, caspase-7, and PARP in DHTMF-treated K562R cells. DHTMF also markedly increased the levels of cleaved caspases and PARP, indicating that the apoptosis mechanism in DHTMF-treated K562R cells might be mediated by activation of the mitochondrial apoptotic pathway and subsequently activate the caspase pathway. Further study is required to elucidate additional mechanisms underlying DHTMF function and determine the exact molecular mechanisms of action of DHTMF in CML cells with IM resistance.