Synergistic Lethality Effects of Apatinib and Homoharringtonine in Acute Myeloid Leukemia

Purpose The significance of vascular endothelial growth factor receptor (VEGFR)-2 in numerous solid tumors and acute myeloid leukemia (AML) has been demonstrated, but Apatinib remains largely unexplored. In this study, whether Apatinib combined with homoharringtonine (HHT) kills AML cell lines and its possible mechanisms have been explored. Methods AML cell lines were treated with Apatinib and HHT in different concentrations with control, Apatinib alone, HHT alone, and Apatinib combined with HHT. The changes of IC50 were measured by CCK8 assay, and apoptosis rate, cell cycle, and the mitochondrial membrane potential in each group were measured by flow cytometry. Finally, the possible cytotoxicity mechanism was analyzed by Western blotting. Results Our results noted that Apatinib combined with HHT remarkably inhibited cell proliferation, reduced the capacity of colony-forming, and induced apoptosis and cell cycle arrest in AML cells. Mechanistically, Apatinib and HHT play a role as a suppressor in the expression of VEGFR-2 and the downstream signaling cascades, such as the PI3K, MAPK, and STAT3 pathways. Conclusion Our preclinical data demonstrate that Apatinib combined with HHT exerts a better antileukemia effect than Apatinib alone by inhibiting the VEGFR-2 signaling pathway, suggesting the potential role of Apatinib and HHT in the treatment of AML. This study provides clinicians with innovative combination therapies and new therapeutic targets for the treatment of AML.


Introduction
Acute myeloid leukemia (AML) is derived from leukemia stem cells or progenitor cells. e American Cancer Society found that there are about 19, 940 new cases of AML per annum with 11,180 deaths [1]. Although it has significantly improved the molecular biology of this disease and over the last forty years treatment has changed, the outcome remains poor for most patients [2]. Cytogenetic abnormalities are closely associated with clinical features and therapeutic responses in AML [3]. erefore, there is a crying need for novel AML therapies, ranging from drugs targeting specific vascular endothelial growth factors (VEGF), and oncogenic proteins to immunotherapies. In the last few years, a large number of studies have shown that angiogenesis is involved not only in leukemogenesis but also in leukemia progression [4]. An increased angiogenesis in the bone marrow niche relates to acute myeloid leukemia progression and resistance to treatment [5][6][7][8][9]. In consideration of the important role of angiogenic activity in different kinds of tumor-like hematological malignancies, targeting vasculogenesis signaling has gained more and more attention as a new therapy to avoid cancer metastasis and resistance. erefore, antiangiogenic drugs or VEGFR inhibitors may provide a novel innovative approach for AML treatments [10][11][12].
Apatinib (also known as YN968D1), a small-molecule receptor tyrosine kinase inhibitor, can target VEGFR-2 selectively [13], which has been approved in China as a subsequent treatment for advanced gastric cancer [14]. Moreover, it has also been tested in phase II/III clinical trials of other cancers, including non-small-cell lung cancer and breast cancer [15]. In this case, we are intrigued to explore whether Apatinib can be used for AML treatment and its relevant mechanisms. Homoharringtonine (HHT) (also known as omacetaxine mepesuccinate), a classical antileukemia drug, has been applied for about forty years in China. HHT has a variety of antitumor effects, including AML [16] and CML [17]. e researchers found that HHT is a protein synthesis inhibitor affecting leukemic cells, which potentiates the therapeutic efficacy of anthracycline and cytarabine [18]. In China, HHT has been widely used in the treatment of AML for more than 30 years because of its good curative effect and low treatment cost [19]. Although HHT has been used for the treatment of a variety of tumors, the specific targets are still unknown [18,20]. In this paper, we studied whether HHT would strengthen the antileukemic effect when combined with Apatinib. Besides, we also studied the underlying mechanisms of the cooperative effect of both two drugs.

Materials and Methods of Drugs
Apatinib (Houston, TX, USA). HHT (Zhejiang Minsheng Pharmaceutical, Zhejiang, China) was dissolved in dimethyl sulfoxide (DMSO) at 1 mg/mL and stored at −20°C. HHT was diluted with a culture medium in subsequent experiments.

Cell Viability
Assay. AML cell lines (2 × 10 4 cells/well) were plated in 96-well plates and then treated with different concentrations of Apatinib and HHT for 24 h and 48 h. e cytotoxic effect was determined by cell counting kit-8 (CCK8; Dojindo, Japan) assay. IC 50 (half-maximal inhibitory concentration) values were determined using a microplate reader (BIO-TEK EPOCH, USA).

Apoptosis Assay.
To assess apoptosis, MV4-11, MOLM-13, OCI-AML2, and OCI-AML3 cells were cultured and presented with different doses of Apatinib or HHT alone or in combination for 24 h or 48 h then double-labeled with Annexin V/DAPI (eBioscience). However, the same concentration did not work in non-FLT3-ITD mutations in AML cell lines, for example, THP1. e stained cells were analyzed with a NovoCyte flow cytometer (ACEA Biosciences, Inc.) with NovoExpress software. Annexin Vpositive cells were defined as apoptotic cells.

In Vitro Clonogenicity
Assay. MV4-11, MOLM-13, OCI-AML2, and OCI-AML3 cells (2 × 10 5 /well) were used to test colony-forming abilities. AML cells were seeded in 24-well plates and then, treated with 20 µM Apatinib or 16 nM HHT alone or both molecules. After 24 h, cells were washed and then cultured in a complete methylcellulose medium at a cell density of 500 cells/well in 3.5 cm dishes for 10-14 days. e percentage of CFU was determined by counting colonies (≥50 cells). Data were presented as the mean ± SD of three independent experiments.
2.5. Cell Cycle Analysis. AML cells were treated for 24 h, then cells were fixed overnight with 75% ethanol, washed with PBS three times, and then incubated in buffer containing 50 μg/mL PI and 100 μg/mL RNase A for 30 min at room temperature. Cells were resuspended with DAPI saline solution and subjected to flow cytometry (NovoCyte ™ ).

Statistical Analysis.
We used GraphPad Prism software v7.0 to analyze the data. All experiments performed at least three independent experiments. Multigroup comparisons were using a one-way test of variance (ANOVA). Statistical analyses were presented using SPSS 20.0 software (La Jolla, CA).

e Cytotoxic Effect of Apatinib and HHT on FLT3-ITD
Mutations AML Cell Lines. We used the CCK8 assay to test the activity of Apatinib and HHT alone and in combination to verify the synergistic effect in inhibition of FLT3-ITD mutations AML cell viability. e concentrations of Apatinib and HHT are shown in Figures 1(a)-1(d). We found that AML cell lines treated with both Apatinib and HHT showed a much better inhibitory effect, especially in the FLT3-ITD group than those treated with each reagent alone in a time-dependent manner. However, Apatinib and HHT displayed no effect on the THP1 cell line without FLT3-ITD mutations in cell proliferation (Supplemental Figure 1). e IC 50 values for Apatinib and HHT were calculated using   GraphPad Prism software v7.0 in AML cells, respectively (Table 1). Together, these results indicate that Apatinib synergistically interacts with HHT to reduce the viability of AML cells.  Table 2). A CI value of less than 1.0 means a synergistic effect. In contrast, the combination of Apatinib and HHTdisplayed no effect on the THP1 cell line without FLT3-ITD mutations (Figure 2(d)). ese findings suggested that the combination of Apatinib and HHT might be a hopeful therapy for AML with FLT3-ITD mutation cells.

e Synergistic Effects of Apatinib Combined with HHTon the Formation of Colonies.
Next, we studied the effects of Apatinib, HHT, or the combination of these two drugs on the cell colony formation of FLT3-ITD mutation cells. MV4-11, MOLM-13, OCI-AML2, and OCI-AML3 cells were treated with different concentrations of Apatinib or HHT alone or in combination for 48 h. Neither Apatinib (20 μM) nor HHT (16 nM) alone diminished the colony formation abilities of FLT3-ITD mutations in AML cells. However, when Apatinib was combined with HHT, the colonyforming units significantly decreased (p < 0.001 vs. control, Apatinib alone, or HHT alone) (Figures 3(a)-3(d)).

Apatinib and HHT Induce Cell Cycle Arrest in FLT3-ITD
Mutations AML Cells. Cell cycle assays were carried out to investigate whether Apatinib combined with HHTaffects the cell cycle capacity in FLT3-ITD mutation cells. MV4-11 and MOLM-13 cells were treated with different concentrations of Apatinib alone or combined with HHT for 24 hours. As shown in Figures 4(a) and 4(b), cells treated with Apatinib at 20 μM or HHT at 16 nM, these concentrations did not affect G0/G1 or S phase cells notably. However, Apatinib and HHT exerted a prominent G0/G1 phase arrest and the S phase decreased. en, we used Western blots to explore the cyclerelevant proteins, such as cyclin A2, cyclin D1, and P21, and the results were consistent with the former (Figure 4(c)). Consistent with the cell cycle assays, cell cycle-relevant proteins of Apatinib plus HHT induced a reduction of cyclin D1 and a rising of P21. Also, we found cyclin A2 was increased, which was a relation to the fewer cycling cells in the S phase.

Apatinib Combined with HHT Dose-Dependent Manner
Reduces the Mitochondrial Membrane Potential. We used the JC-1 probe to test the mitochondrial membrane potential (MMP, ΔΨm) to validate the joint effects of Apatinib and HHT. As we expected, compared with each reagent alone, co-treatment with Apatinib and HHT remarkably reduced the MMP in MV4-11 and MOLM-13 cells after 24 h treatment (Figures 5(a) and 5(b)). Apatinib combined with HHT downregulates the VEGFR-2 and its downstream signaling pathways in AML cells. As we mentioned above, Apatinib was a selective target to the VEGFR-2 pathway. What surprises us was that when Apatinib was combined with HHT the VEGFR-2 expression was affected. Apatinib and HHT treatment markedly inhibited the downstream signals, PI3K and p-Akt, as well as antiapoptotic proteins like BCL-2 and MCL-1 ( Figure 5(c)). We can summarize the mechanism through a brief schematic mechanism as follows: on FLT3-ITD mutations AML cells Apatinib combined with HHT induced cell apoptosis by decreasing the mitochondrial membrane potential, inhibiting cell cycle, and regulating vascular endothelial growth factor as well as its downstream signaling pathways ( Figure 5(d)).

Discussion
e evidence is overwhelming that the poor prognosis and higher disease relapse rate of AML accompanied by FLT3-ITD mutations make FLT3-ITD a perfect therapeutic target in individualized treatment [21][22][23]. rough our experimental results, we found that on FLT3-ITD (+) AML cell lines, compared to the monotherapy group, Apatinib and HHT could significantly inhibit cell proliferation, promote cell apoptosis, and regulate Apatinib-relevant protein VEGFR-2. However, these results were not found on the FLT3-ITD (-) THP1 cell line. Hence, we proposed and proved for the first that the combination of Apatinib and HHT exerted a significant antileukemic action. Mechanistically, the combination of Apatinib and HHT synergistically decreases phosphorylated forms of VEGFR-2 protein and its downstream PI3K, BCL-2, Akt, and MCL-1, resulting in cell arresting at G1 and apoptosis. As we know, approximately 30% of AML with normal karyotype will have FLT3 (FMS-like tyrosine kinase 3) gene with mutations of internal tandem duplications (ITD) in the juxtamembrane domain [24]. Meanwhile, AML with FLT3-ITD mutations is concerned with poor overall survival (OS) and decreased disease-free survival (DFS) [25]. Although FLT3-mutant AML patients can be treated with FLT3 tyrosine kinase inhibitors (TKI), the relapse and rapid drug resistance limit its use [26]. Lately, several studies have found that HHT exerted a sensitive cytotoxic function on FLT3-ITD (+) AML cells [27].
e key role of angiogenesis is the process of forming blood vessels in the growth and maintenance of solid tumors. In the last few decades, a large number of studies show the involvement of angiogenesis in leukemogenesis as well as leukemia progression [28]. An increase in angiogenesis in the bone marrow niche is related to both acute lymphoblastic leukemia and acute myeloid leukemia [5][6][7][8][9]. erefore, targeting angiogenesis with antiangiogenic agents Journal of Oncology or VEGFR inhibitors is likely to be a new method for AML treatment. Apatinib is a novel small-molecule tyrosine kinase inhibitor, which inhibits the phosphorylation of vascular endothelial growth factor receptors selectively, with a binding affinity ten times that of sorafenib. Besides, compared with Apatinib sorafenib with only one-tenth of the anti-VEGFR-2 efficacy, it is insufficient for antitumor angiogenesis [10,14,29]. Its antitumor activity in all kinds of tumors has been proved in many studies [20,30,31]. Results of those clinical trials indicated the antitumor role of Apatinib across a large scale of advanced cancers, but the specific function of tumor angiogenesis in AML pathogenesis remains unknown. Furthermore, our research has found that Apatinib combined with a variety of antitumor drugs can improve the curative effect [32,33]. It was interesting to note that accompanied FLT3-ITD mutant AML cells presented a significant synergistic effect after being treated with Apatinib and HHT. Nevertheless, it makes no difference to the AML cell line, which was without FLT3-ITD mutant (Figure 2(d)). It has been well reported that the important role of PI3K signaling in the progression of all kinds of tumors, including leukemia [34,35]. e evasion of apoptosis was an important characteristic of cancer, which was caused by the activation of antiapoptotic molecules of the BCL-2 protein family [36][37][38]. Aberrant activation of BCL-2 members such as BCL-2 and MCL-1 were related to antiapoptosis and drug resistance in FLT3-ITD mutant AML [39][40][41].
VEGF is considered a target in leukemia treatment and a variety of strategies have been applied to downregulate or inhibit the VEGF signaling pathway. e new strategy which inhibits the VEGF signaling pathway could be able to block the autocrine VEGF pathway in AML cells or the typical vessel development by the vascular endothelial cells [42]. We and other researchers found that Apatinibinduced cytotoxicity was related to inhibition of the VEGFR-2 and PI3K/Akt pathways, and induction of mitochondrial membrane protein (MMP)-mediated apoptosis. To our surprise, Apatinib and HHT could significantly enhance this phenomenon. As everyone knows that FLT3-ITD mutations result in missing the autoinhibitory function for FLT3 kinase, which next leads to activation of its downstream signaling pathways, such as PI3K/Akt and JAK/STAT5 [43,44], our results indicated that Apatinib combined with HHT synergistically suppressed the growth and induced apoptosis of FLT3-ITD mutations AML cells by synergistically downregulating the expression of phosphorylated forms of VEGFR-2 and PI3K/Akt signaling pathways as well as affecting the expression level of cell cycle regulatory protein, upregulating the expressions of cyclin A2 and P21 and downregulating the expression of cyclin D1 in FLT3-ITD (+) AML cell lines. Nevertheless, mechanisms of combined application of Apatinib and HHT in regulating P21, cyclin A2, and cyclin D1 proteins remain unclear and need further study.

Conclusions
All in all, our research first reveals the synergistic antileukemic effect between Apatinib and HHT on FLT3-ITD mutant AML cells, likely through inhibiting VEGFR-2mediated signaling pathways, and suggests potential benefits and clinical application of Apatinib combined with HHT in the treatment of AML patients.
Data Availability e datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
is article does not contain any studies with human participants or animals performed by any of the authors.

Disclosure
A preprint has previously been published [45].

Conflicts of Interest
e authors declare that they have no potential conflicts of interest.

Authors' Contributions
YFS and YX performed in vitro experiments and animal studies. DDX, HFS, and YZ carried out the statistical analysis. XJY, JJ, and DWC designed the study. WZX wrote the manuscript. All authors read and approved the final manuscript. YFS, DDX, and YX contributed equally to this work. Journal of Oncology 11