Farnesiferol C Induces Apoptosis in Chronic Myelogenous Leukemia Cells as an Imatinib Sensitizer via Caspase Activation and HDAC (Histone Deacetylase) Inactivation

Herein the underlying apoptotic mechanism of Farnesiferol C (FC) derived from Ferula assafoetida was elucidated in chronic myelogenous leukemia (CML) K562 and KBM5 cells. FC showed significant cytotoxicity in K562 and KBM5 cells, more so than in U937 and UL-60 acute myeloid leukemia (AML) cells. Cleaved PARP and caspase 9/3 attenuated the expression of Bcl2 and induced G1 arrest in K562 and KBM5 cells. Also, FC effectively abrogated the expression of cell cycle related proteins, such as: Cyclin D1, Cyclin E, Cyclin B1 in K562, and KBM5 cells, but caspase 3 inhibitor Z-DEVD-FMK rescued the cleavages of caspase 3 and PARP induced by FC in K562 cells. Of note, FC decreased histone deacetylase 1 (HDAC1) and HDAC2, and enhanced histone H3 acetylation K18 (Ac-H3K18) in K562 and KBM5 cells. Furthermore, combination of FC and Imatinib enhanced the apoptotic effect of Imatinib as a potent Imatinib sensitizer in K562 cells. Overall, our findings provide scientific evidence that inactivation of HDAC and caspase activation mediate FC induced apoptosis in CML cells.


Introduction
Among bone marrow or blood cancers, leukemia with the abnormal proliferation feature of white blood cells is generally classified into myelogenous leukemia and lymphoblastic leukemia [1]. Chronic myeloid leukemia (CML) is a myeloproliferative disorder of transformed hematopoietic stem cells by the Philadelphia chromosome or the Philadelphia translocation (Ph) of chromosomes 9 and 22 with constitutively activated Bcr-abl tyrosine kinase [2]. Although various chemotherapeutical agents have been developed such as Imatinib mesylate (Gleevec ® , Novartis), Busulfan (Myleran ® , Busilvex), or Hydroxyurea (Hydrea ® , Bristol-Myers Squibb Pharmaceuticals) for the treatment of CML, their therapeutic efficacies have been limited due to their side effects, such as lethargy, fluid retention, thrombocytopenia, nausea, and diarrhea [3,4].
Recently, anticancer agents from natural products such as decursin [5], tanshinone IIA [6], and curcumin [7] are attractive in CML cells due to their lesser toxicity and potent synergy with conventional anticancer drugs.
Histone acetylation is regarded as a dynamic process regulated by the antagonistic actions of two large families of enzymes, such as histone deacetylases (HDACs) and histone acetyltransferases (HATs) [8]. Histone deacetylases (HDACs) function to remove acetyl group from an N-acetyl lysine amino acid on a histone. Usually HDACs are overexpressed in several cancers and are known to be involved in cell survival, inflammation, proliferation, angiogenesis, and immunity [9]. Hence, HDAC inhibitors have also been considered potent agents for adjuvant therapy and cancer treatment [10].
Farnesiferol C (FC) derived from Ferula asafoetida species is a polycyclic aromatic compound containing a 1-benzopyran moiety with a ketone group at the C2 carbon atom. Though FC is known to have antileishmanial [11], antiangiogenic [12], and apoptotic effects [13][14][15][16], to date its underlying antitumor mechanisms still remain unclear in CML cells. Hence, in the current study, apoptotic mechanism of FC and its potential as an Imanitib sensitizer for combination therapy were evaluated in K562 and KBM5 CML cells.

FC (Farnesiferol C) Induces Significant Cytotoxicity in K562 and KBM5 Cells.
To confirm the cytotoxicity of FC ( Figure 1A), a cell viability assay was conducted in K562, KBM5, U937, and HL-60 cells by an MTT assay. Here, FC significantly reduced the viability of K562 and KBM5 cells (CML) in a concentration dependent fashion, better than in U937 and HL-60 AML cells ( Figure 1B). (HATs) [8]. Histone deacetylases (HDACs) function to remove acetyl group from an N-acetyl lysine amino acid on a histone. Usually HDACs are overexpressed in several cancers and are known to be involved in cell survival, inflammation, proliferation, angiogenesis, and immunity [9]. Hence, HDAC inhibitors have also been considered potent agents for adjuvant therapy and cancer treatment [10]. Farnesiferol C (FC) derived from Ferula asafoetida species is a polycyclic aromatic compound containing a 1-benzopyran moiety with a ketone group at the C2 carbon atom. Though FC is known to have antileishmanial [11], antiangiogenic [12], and apoptotic effects [13][14][15][16], to date its underlying antitumor mechanisms still remain unclear in CML cells. Hence, in the current study, apoptotic mechanism of FC and its potential as an Imanitib sensitizer for combination therapy were evaluated in K562 and KBM5 CML cells.

FC Regulates Apoptosis Related Proteins and Induces G1 Arrest in CML Cells.
To examine whether the cytotoxic effect of FC is associated with apoptosis, the effect of FC on apoptosis related genes was evaluated in K562 or KBM5 cells. FC induced the cleavages of PARP, caspase-9, and caspase-3, and decreased the expression of Bcl-2 in K562 and KBM5 cells (Figure 2A,B). Additionally, as shown in Figure 2C, FC increased sub-G1 population in K562 cells. Conversely, caspase 3 inhibitor Z-DEVD-FMK rescued cleavages of caspase 3 and PARP in K562 cells ( Figure 2D).
To examine whether the cytotoxic effect of FC is associated with apoptosis, the effect of FC on apoptosis related genes was evaluated in K562 or KBM5 cells. FC induced the cleavages of PARP, caspase-9, and caspase-3, and decreased the expression of Bcl-2 in K562 and KBM5 cells (Figure 2A, 2B). Additionally, as shown in Figure 2C, FC increased sub-G1 population in K562 cells. Conversely, caspase 3 inhibitor Z-DEVD-FMK rescued cleavages of caspase 3 and PARP in K562 cells ( Figure 2D).

FC Regulates Cell Cycle Related Proteins
It is well known that FC induces cell cycle arrest in breast cancer cells [17]. To investigate whether FC regulates cell cycle proteins, Western blotting was performed in K562 and KBM5 cells. As shown in Figure 3, FC inhibited the expression of cyclin D1, cyclin E, and cyclin B1.

FC Regulates Cell Cycle Related Proteins
It is well known that FC induces cell cycle arrest in breast cancer cells [17]. To investigate whether FC regulates cell cycle proteins, Western blotting was performed in K562 and KBM5 cells. As shown in Figure 3, FC inhibited the expression of cyclin D1, cyclin E, and cyclin B1.

The Role of HDAC1 in FC Induced Apoptosis in K562 Cells.
Next, the role of HDAC1 was examined in FC induced apoptosis. The effect of HDAC1 overexpression was tested on pro-PARP expression in K562 cells in the presence or absence of FC. As shown in Figure 5, overexpression of HDAC1 weakly reduced the apoptotic ability of FC to attenuate pro-PARP expression compared to the FC alone control, which may be due to transfection efficiency in K562 suspension cells.

The Role of HDAC1 in FC Induced Apoptosis in K562 Cells.
Next, the role of HDAC1 was examined in FC induced apoptosis. The effect of HDAC1 overexpression was tested on pro-PARP expression in K562 cells in the presence or absence of FC. As shown in Figure 5, overexpression of HDAC1 weakly reduced the apoptotic ability of FC to attenuate pro-PARP expression compared to the FC alone control, which may be due to transfection efficiency in K562 suspension cells.

FC Sensitizes K562 Cells to Imatinib Induced Apoptosis.
To confirm the potential of FC as an Imatinib sensitizer, the combinatorial effect of FC and Imatinib was evaluated in K562 cells. As shown in Figure 6A, cotreatment of FC and Imatinib significantly reduced viability of K562 cells compared to Imatinib alone control ( Figure 6A). Similarly, cotreatment of FC and Imatinib attenuated the expression of HDAC1, upregulated Ac-H3K18, and cleaved PARP in K562 cells ( Figure 6B).

FC Sensitizes K562 Cells to Imatinib Induced Apoptosis.
To confirm the potential of FC as an Imatinib sensitizer, the combinatorial effect of FC and Imatinib was evaluated in K562 cells. As shown in Figure 6A, cotreatment of FC and Imatinib significantly reduced viability of K562 cells compared to Imatinib alone control ( Figure 6A). Similarly, cotreatment of FC and Imatinib attenuated the expression of HDAC1, upregulated Ac-H3K18, and cleaved PARP in K562 cells ( Figure 6B).

The Role of HDAC1 in FC Induced Apoptosis in K562 Cells.
Next, the role of HDAC1 was examined in FC induced apoptosis. The effect of HDAC1 overexpression was tested on pro-PARP expression in K562 cells in the presence or absence of FC. As shown in Figure 5, overexpression of HDAC1 weakly reduced the apoptotic ability of FC to attenuate pro-PARP expression compared to the FC alone control, which may be due to transfection efficiency in K562 suspension cells.

FC Sensitizes K562 Cells to Imatinib Induced Apoptosis.
To confirm the potential of FC as an Imatinib sensitizer, the combinatorial effect of FC and Imatinib was evaluated in K562 cells. As shown in Figure 6A, cotreatment of FC and Imatinib significantly reduced viability of K562 cells compared to Imatinib alone control ( Figure 6A). Similarly, cotreatment of FC and Imatinib attenuated the expression of HDAC1, upregulated Ac-H3K18, and cleaved PARP in K562 cells ( Figure 6B).

Discussion
In the current project, the underlying apoptotic mechanism of FC and its potential of Imanitib sensitizer for combinatorial therapy were explored in CML cells. Herein, FC exerted significant cytotoxicity in K562 and KBM5 CML cells better than in U937 and HL-60 AML cells, implying FC may be more susceptible to CML cells with less blast cells than AML cells with more blast cells. It also suggests the better antitumor effect of FC in CML cells, since AML cells have more than 20% of blast cells for proliferation and differentiation than CML cells. In addition, FC was a known nontoxic in normal mesenchymal stem cells [16].
Generally, apoptosis is induced via intrinsic (mitochondrial) or extrinsic (cell death) dependent pathway with features of chromatin condensation, nuclear fragmentation, blebbing, cell shrinkage, and global mRNA decay [19,20]. Here, FC induced cleavages of PARP and caspase 9/3, attenuated the expression of Bcl2, one of antiapoptotic proteins [21] in K562 and KBM5 cells, and conversely caspase 3 inhibitor Z-DEVD-FMK rescued cleavages of PARP and caspase 3 induced by FC in K562 cells, indicating apoptotic effect FC via mitochondrial dependent apoptotic pathway.
It is well documented that cell cycle consists of four distinguishable phases such as G1, S (synthesis), G2 (interphase), and M (mitosis) phases for cell division and duplication, and inhibition of cell cycle is considered a potent strategy for cancer therapy [22,23]. Here FC induced G1 arrest in K562 cells and effectively inhibited the expression of Cyclin D1, Cyclin E, Cyclin B1 that are related to G1-S phase transition [24], demonstrating G1 arrest effect of FC.
Emerging evidences reveal that histone acetylation modulated by histone deacetylases (HDACs) and histone acetyltransferases (HATs) is critically involved in cancer progression [10,25,26]. Here FC decreased histone deacetylase 1 (HDAC1) and HDAC2, and induced acetylation of H3 K9 , H3 K18 , and H4 K8 in K562 and KBM5 cells, implying the critical role of HDAC inhibition in FC induced apoptosis in CML cells. Similarly, FC was known to induce apoptosis via modulation of c-Myc and in non-small-cell lung cancers [13] and also exerted antitumor and antiangiogenic effects by multiple targets of VEGFR1 or VEGFR2 signaling [12].
Though Imatinib has been extensively used for treatment of CML for years, recently combination therapy with low dose of Imatinib is attractive to reduce side effects due to its toxicity in normal cells [27,28]. In the same line, FC sensitized K562 cells to Imatinib induced apoptosis by inhibition of HDAC1, activation of Ac-H3K18, and PARP cleavage in K562 cells, strongly indicating combinatorial therapy potential of FC with Imatinib.
In summary, FC showed significant cytotoxicity, cleaved PARP, and caspase 9/3, attenuated the expression of Bcl2, Cyclin D1, Cyclin E, Cyclin B1, and induced G1 arrest in K562 and/or KBM5 cells. Also, caspase 3 inhibitor Z-DEVD-FMK rescued cleavages of caspase 3 and PARP induced by FC in K562 cells. Furthermore, FC decreased HDAC1 and HDAC2, enhanced Ac-H3 K18 in K562 cells, and sensitized K562 cells to Imatinib induced apoptosis. Taken together, our findings provide insight that inactivation of HDAC and caspase3 activation are critically involved in FC induced apoptosis in CML cells (Figure 7).

Discussion
In the current project, the underlying apoptotic mechanism of FC and its potential of Imanitib sensitizer for combinatorial therapy were explored in CML cells. Herein, FC exerted significant cytotoxicity in K562 and KBM5 CML cells better than in U937 and HL-60 AML cells, implying FC may be more susceptible to CML cells with less blast cells than AML cells with more blast cells. It also suggests the better antitumor effect of FC in CML cells, since AML cells have more than 20% of blast cells for proliferation and differentiation than CML cells. In addition, FC was a known nontoxic in normal mesenchymal stem cells [16].
Generally, apoptosis is induced via intrinsic (mitochondrial) or extrinsic (cell death) dependent pathway with features of chromatin condensation, nuclear fragmentation, blebbing, cell shrinkage, and global mRNA decay [19,20]. Here, FC induced cleavages of PARP and caspase 9/3, attenuated the expression of Bcl2, one of antiapoptotic proteins [21] in K562 and KBM5 cells, and conversely caspase 3 inhibitor Z-DEVD-FMK rescued cleavages of PARP and caspase 3 induced by FC in K562 cells, indicating apoptotic effect FC via mitochondrial dependent apoptotic pathway.
It is well documented that cell cycle consists of four distinguishable phases such as G1, S (synthesis), G2 (interphase), and M (mitosis) phases for cell division and duplication, and inhibition of cell cycle is considered a potent strategy for cancer therapy [22,23]. Here FC induced G1 arrest in K562 cells and effectively inhibited the expression of Cyclin D1, Cyclin E, Cyclin B1 that are related to G1-S phase transition [24], demonstrating G1 arrest effect of FC.
Emerging evidences reveal that histone acetylation modulated by histone deacetylases (HDACs) and histone acetyltransferases (HATs) is critically involved in cancer progression [

Cell Cycle Analysis
Based on cell cycle method shown in Yun et al.'s paper [29], cell cycle analysis was conducted in K562 cells treated with or without FC by propidium iodide (PI) staining. Cell cycle distributions were calculated by FACS Calibur (Becton Dickinson, Franklin Lakes, NJ, USA) by using the Cell Quest program (Becton Dickinson).

Western Blotting
K562 or KBM5 cells were exposed to FC and/or Imanitib for 24 h and subjected to Western blotting based on method shown in Lee et al.'s [12]. The protein samples were separated and transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies of HDAC1, HDAC2, Ac-H3 K9 , Ac-H3 K18 , and Ac-H4 K8 , cleaved PARP, PARP, caspase-9, caspase 3, and finally, incubated with HRP-conjugated secondary antibody (1:2000). The expression was visualized by using ECL Western blotting detection reagent (GE Healthcare, Amersham, UK).

Transfection Assay
K562 cells were transfected with control vector or HDAC1 plasmid with Lipofectamine 2000 and Interferin™ transfection reagent (Polyplus-transfection Inc., New York, NY, USA). The mixtures of HDAC1 plasmid and transfection reagent were together incubated for 10 min and then the cells were incubated at 37 • C for 36 h before exposure to FC (16 µM for 24 h).

Statistical Analysis
The data stand for means ± SD from at least three independent experiments. Student's t-test was used for two group comparison and the one-way analysis of variance (ANOVA), followed by a Turkey post-hoc test for multi-group comparison by using GraphPad Prism software (Version 5.0, California, CA, USA). The statistically significance was accepted, only when the difference p-value between groups was less than 0.05.