Assessment of Thiosemicarbazone-Containing Compounds as Potential Antileukemia Agents against P-gp Overexpressing Drug Resistant K562/A02 Cells

Article abstract of DOI:10.1002/cbdv.202000775

P-Glycoprotein (P-gp) overexpression is considered to be the leading cause of multidrug resistance (MDR) and failure of chemotherapy for leukemia. In this study, seventeen thiosemicarbazone-containing compounds were prepared and evaluated as potential ant

Full text of DOI:10.1002/cbdv.202000775

Title: Assessment of thiosemicarbazone-containing compounds as
potential anti-leukemia agents against P-gp overexpressing drug
resistant K562/A02 cells
Authors: Xiaoke Gu, Mingyu Guan, Chunyu Jiang, Qinghua Song, Xin
Li, Nan Sun, Jing Chen, and Jingying Qiu
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To be cited as: Chem. Biodiversity 10.1002/cbdv.202000775
Link to VoR: https://doi.org/10.1002/cbdv.202000775

10.1002/cbdv.202000775
Chemistry & Biodiversity
Chem. Biodiversity
Assessment of thiosemicarbazone-containing compounds as potential anti-
leukemia agents against P-gp overexpressing drug resistant K562/A02 cells
Xiaoke Gu*a, 1 , Mingyu Guana, 1, Chunyu Jianga, Qinghua Songa, Xin Lia, Nan Suna, Jing Chena, Jingying Qiu* a
a Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou 221004, People’s Republic of China, email:
gu_xk@xzhmu.edu.cn; jingyqiu@xzhmu.edu.cn
P‑glycoprotein (P-gp) overexpression is considered to be the leading cause of multidrug resistance (MDR) and failure of chemotherapy for leukemia. In
this study, seventeen thiosemicarbazone-containing compounds were prepared and evaluated as potential anti-leukemia agents against drug resistant
K562/A02 cell overexpressing P-gp. Among them, (E)-N-hydroxy-6-(2-(3-nitrobenzylidene)hydrazine-1-carbothioamido)hexanamide could significantly
inhibit K562/A02 cells proliferation with an IC50 value of 0.96 μM. Interestingly, (E)-N-hydroxy-6-(2-(3-nitrobenzylidene)hydrazine-1-
carbothioamido)hexanamide could dose-dependently increase ROS levels of drug resistant K562/A02 cells, thus displaying a potential collateral sensitivity
(CS)-inducing effect and selectively killing K562/A02 cells. Furthermore, (E)-N-hydroxy-6-(2-(3-nitrobenzylidene)hydrazine-1-carbothioamido)hexanamide
possessed potent inhibitory effect on HDAC1 and HDAC6, and could promote K562/A02 cells apoptosis via dose-dependently increasing Bax expression,
reducing Bcl-2 protein level, and inducing the cleavage of PARP and caspase3. These present findings suggest that (E)-N-hydroxy-6-(2-(3-
nitrobenzylidene)hydrazine-1-carbothioamido)hexanamide might be a promising lead to discover novel anti-leukemia agents against P-gp overexpressing
leukemic cells.
Keywords: Multidrug resistance; P-gp; Thiosemicarbazones; Collateral sensitivity.
Introduction
Multidrug resistance (MDR) is commonly considered as one of the major obstacle to successful chemotherapy for leukemia.[1, 2] Molecularly, the
occurrence of MDR is closely related to the ATP-binding cassette (ABC) transporters overexpression, which could efflux various kinds of anti-cancer drugs
out of cancer cells, thus resulting in the drug insensitivity. Among the ABC transporter members, P-glycoprotein (P-gp) encoded by MDR1 gene, is the
mostly studied and significant one.[3, 4] Therefore, discovery of agents that could overcome P-gp-mediated drug resistance is of great interest.[5]
Nowadays, three generations of inhibitors/modulators have been designed to overcome P-gp induced drug resistance, however, until now, there is no
single inhibitor available in clinic mainly because of the high inherent toxicity, poor target specificity, pharmacokinetic interaction, and/or insignificant
efficacy.[6, 7] Accordingly, alternative strategies are urgently needed to conquer P-gp-medicated MDR. Notably, resistant cancer cells overexpressing P-gp
may be hypersensitive to drugs with particular structure, a phenomenon termed collateral sensitivity (CS).[8] As more compounds were confirmed to have
a preferential toxicity towards resistant cancer cells, CS conveyed by P-gp represents a novel and promising strategy to overcome P-gp-mediated MDR.[9,
10]
Recently, plenty of studies showed that compounds with a thiosemicarbazone moiety, such as Bp44mT and Dp44mT (Figure 1), exhibited preferential
toxicities against resistant cancer cells compared to the nonresistant counterparts.[10-12] More importantly, thiosemicarbazones possessed diverse biological
activities (ie. anticancer, antivirus) and several agents with a thiosemicarbazone moiety have been evaluated in clinical trials against malignancies including
leukemia.[13] Keeping these in mind and considering that histone deacetylases (HDACs) play a significant role in leukemogenesis, and HDACs inhibitors
were reported as promising agents for the treatment of leukemia,[14-16] we speculated that hybridizing the thiosemicarbazone moiety with the HDACs
inhibitor pharmacophore might result in compounds with more effective anti-leukemia activity against resistant leukemic cells overexpressing P-gp. Thus,
seventeen thiosemicarbazone-containing compounds (3a-q, Figure 1) with a similar pharmacophore of HDACs inhibior vorinostat (SAHA) were synthesized,
and their anti-proliferation activities against P-gp overexpressing K562/A02 cells, and the preliminary mechanisms were subsequently evaluated in this
paper.1
1 1These authors contributed equally to this work.
1
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Figure 1. Structures of Bp44mT, Dp44mT and 3a-q
Results and Discussion
Chemistry
Synthetic procedure of compounds 3a-q was depicted in Scheme 1. Briefly, benzaldehyde or benzaldehyde derivatives were condensed with
thiosemicarbazide to obtain intermediates 1a-m, which were subsequently reacted with ethyl 6-bromohexanoate or ethyl 4-bromobutyrate to give
intermediates 2a-q, respectively. The crude products were finally aminolized with hydroxylamine hydrochloride to provide the target compounds 3a-q,
respectively.
Scheme 1. Synthesis of compounds 3a-q. Reagent and condition: a) Thiosemicarbazide, MeOH, 100 °C, 8 h; b) NaH, 1,4-Dioxane, Ethyl 6-bromohexanoate/Ethyl 4-
bromobutyrate, reflux, 10 h; c) EtOH, NH2OH.HCl, KOH, -10 °C, 4 h.
Biological evaluation
Inhibitory effect on K562/A02 cells proliferation
To investigate whether the target compounds could overcome P-gp-mediated drug resistance, we initially determined their anti-proliferation effect
against P-gp-overexpressed leukemic K562/A02 cell by MTT assay (Table 2). HDACs inhibitor SAHA, anti-cancer drug Adriamycin (ADR) and Dp44mT were
used as the positive controls and lead control, respectively. Moreover, we also examined their inhibitory effect on the proliferation of parental sensitive
leukemic K562 cells to investigate the ability of the target compounds to induce collateral sensitivity (CS) against P-gp. According to literature methods,[17,
18] CS can be quantitatively assessed by calculating the relative resistance (RR) index, which means the ration of IC50 values of a compound against resistant
and corresponding parental sensitive cells.
Table 1.Effect of compounds 3a-q on K562/A02 and K562 cell proliferation
IC50 (μM)[a]
IC50 (μM)[a]
Compound
K562
3.87 ± 0.37
K562/A02
4.05 ± 0.41
RR[b]
Compound
K562
0.92 ± 0.08
K562/A02
4.55 ± 0.47
RR[b]
3a
3b
3c
3d
3e
3f
1.05
0.95
0.95
1.77
3k
3l
4.95
4.55
4.81 ± 0.44
5.72 ± 0.61
4.34 ± 0.36
4.57 ± 0.51
5.42 ± 0.43
7.66 ± 0.75
4.63 ± 0.43
1.12 ± 0.11
4.16 ± 0.27
0.96± 0.089
2.51 ± 0.18
6.72 ± 0.62
2.15 ± 0.18
> 10
9.79 ± 0.95
> 10
[c]
3m
-
3n
> 10
> 10
-
0.86
-
4.90 ± 0.52
2.42 ± 0.26
4.23 ± 0.39
2.13 ± 0.23
2.63± 0.20
6.35 ± 0.59
0.95
0.46
0.98
0.45
0.95
1.06
3o
7.12 ± 0.83
> 10
6.09 ± 0.56
> 10
3p
3g
3h
3i
3q
> 10
> 10
-
SAHA
Dp44mT
ADR
1.35 ± 0.12
3.43 ± 0.40
0.83 ± 0.095
3.13 ± 0.35
3.02 ± 0.29
23.42 ± 4.73
2.32
0.88
28.21
3j
2
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[a]
[b]
Triplicate experiments were performed. Data are means ± SD. Relative resistance (RR) = IC50 (resistant cells)/IC50(parental cells). RR < 1 means compounds showed
preferential toxicity against MDR cell than parental sensitive cell. RR > 1 indicates cancer cell is resistant. When a compound displaying a RR ≤ 0.5, it is generally defined as a
CS-promoting agent. [c] Not determined.
As shown in Table 1, the RR values of ADR and SAHA was 28.21 and 2.32, respectively, indicating that K562/A02 cell was resistant to ADR and SAHA.
While, the RR values of the most active compounds were below 1, suggesting that these target compounds displayed selective anti-proliferation effect on
P-gp overexpressing K562/A02 cells. In addition, Dp44mT displayed a RR value of 0.88 under our experimental conditions, indicating that Dp44mT does
not display obvious preferential toxicities against K562/Ao2 cells. Notably, compounds 3f and 3h display more potent anti-proliferation activity against
K562/A02 cells (IC50 values: 1.12 and 0.96 μM, respectively) than positive controls SAHA (IC50 value: 3.13 μM), ADR (IC50 value: 23.42 μM) and lead control
Dp44mT (IC50 value: 3.02 μM). Interestingly, the RR values of compounds 3f and 3h was 0.46 and 0.45, respectively, indicating that both of them might
exhibit a potential ability to induce CS against drug resistant K562/A02 cells.
Effect of compound 3h on ROS levels in resistant K562/A02 cells
As mentioned above, one of the leading cause of MDR is the overexpression of P-gp, and CS is regarded as a novel and promising strategy to overcome
P-gp-mediated MDR. To clarify the potential mechanism of 3h, we firstly determined whether compound 3h could affect the expression and/or function of
P-gp, and found that 3h could not affect the efflux function as well as the expression of P-gp (data not shown). Considering that one of the mechanisms
underlying CS is the elevated production of ROS in drug resistant cell that overexpresses ABC transporters,[19, 20] we subsequently measured the effect of
3h on the ROS level in drug resistant K562/A02 cells as well as the parental sensitive K562 cells using DCFHA-DA assay.[21]
As shown in Figure 2, compound 3h could obviously increase the intracellular ROS in K562/A02 cells (Fig. 2A, blue bar) at a dose depended manner. And
the ROS levels were relatively higher than that in parental sensitive K562 cells (Fig. 2A, red bar). As expected, ROS scavenger Nacetyl-cysteine (NAC) could
remarkably diminish the levels of ROS in compound 3h-treated K562/A02 cells. In addition, data showed that the well-known P-gp inhibitor tariquidar (Tar)
could significantly reduce the levels of ROS in P-gp overexpressing K562/A02 cells incubated with 3h, but have little effect on sensitive K562 cells, indicating
that the increasing effect of 3h on the levels of ROS in K562/A02 cells was P-gp-dependent. Notably, non-toxic NAC could dramatically attenuate the anti-
proliferation effect compound 3h on K562/A02 cells (Fig. 2B), indicating that the cytotoxicity and CS-promoting effect of compound 3h on P-gp
overexpressing K562/A02 cells might rely on the increased production of the intracellular ROS, at least partly.
Figure 2. Effects of 3h on K562/A02 (blue bar) and K562 (red bar) ROS levels and viability. (A) Reactive oxygen species (ROS) level in K562/A02 cells (blue bar) and K562 cells
(red bar) were measured using the DCFHA-DA assay. **P < 0.01 vs. control group (blue bar). ##P < 0.01 vs. the 3h (3.0 μM) group in K562/A02 cells (blue bar). (B) K562/A02 cells
were incubated in the absence or presence of 3h (3.0 μM) for 24 h, the cell viability was then determined by MTT assay. In the case of inhibition, NAC (10 mM) or Tar (5.0 μM)
and 3h (3.0 μM) were incubated with K562/A02 cells together. **P < 0.01 vs. the 3h (3.0 μM) group.
Effect of compound 3h on K562/A02 cells apoptosis
Subsequently, Annexin V/PI assay was employed to investigate whether compound 3h could lead to enhanced cell apoptosis, a major mechanism of anti-
proliferation.[22, 23] Data showed that (Fig. 3), after the 48 h treatment of K562/A02 cells with 3h (1.0 or 3.0 μM), the percentage of early and late apoptotic
cells were significantly increased to 64.94% and 72.84%, respectively, indicating that compound 3h possessed an apoptosis-inducing effect on K562/A02
cells.
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Figure 3. Compound 3h induced the K562/A02 cells apoptosis (A) After 48 h treatment of K562/A02 cells with or without 3h (1.0 or 3.0 μM), cells were collected and stained using
Annexin V/PI. Early and late (Annexin-V+/PI−) apoptotic K562/A02 cells were analyzed by flow cytometry; (B) Percentage of cell distribution.
Effect of 3h on HDACs and the expression of apoptosis-related proteins in K562/A02 cells
HDACs play significant roles during the leukemogenesis.[14-16] In order to investigate the effect of the active compound 3h on HDACs activity, the
fluorescent-based HDAC biochemical activity were subsequently determined accordingly our previous method.[24] Thiosemicarbazone-containing
compound Dp44mT and SAHA were used as lead and positive controls, respectively.
As summarized in Table 2, Dp44mT showed little inhibitory activities on HDACs. While compound 3h exhibited potent inhibitory effect on HDAC1 and
HDAC6 activities with IC50 values even lower than those of classical HDACs inhibitor SAHA (28.2 nM and 24.3 nM vs. 37.7 nM and 33.1 nM, respectively).
However, 3h could not inhibit the activity of HDAC8, indicating that 3h might exhibit a selective inhibitory effect on HDACs activities.
Table 2. Inhibitory effect of target compound 3h on HDAC1, 6 and 8 activity.
IC50 (nM) [a]
Compounds
3h
HDAC1
28.2 ± 2.1
37.7 ± 3.7
> 5000
HDAC6
24.3 ± 3.2
33.1 ± 2.9
> 5000
HDAC8
> 5000
> 5000
> 5000
SAHA
Dp44mT
[a] Three independent experiments were performed. Values are mean ± SD.
To further confirm the HDACs inhibitory effect and apoptosis induction effect of the active compound 3h on drug resistant K562/A02 cells, we determined
the expression of Ac-H3, Ac-H4 (well-known substrates of HDAC1, 2 and 3),[25] and several key apoptotic proteins, including Bcl-2, Bax, and cleavage state
of caspase3 and PARP (apoptosis markers) in K562/A02 cells treated with 3h by western blot detection.
As depicted in Figure 4, 3h could dose-dependent increased Ac-H3, Ac-H4, and pro-apoptotic Bax proteins expression while reducing the expression of
anti-apoptotic Bcl-2. In addition, compared to control group, 3h could more obviously induce the cleavage of caspase3 and PARP. Together, these data
confirmed that 3h exhibited potential HDACs inhibitory activity and apoptosis-inducing effect against K562/A02 cells.
Figure 4. 3h affected the expression of Ac-H3, Ac-H4 and apoptosis-related proteins. (A) Apoptosis-related proteins and Ac-H3, Ac-H4 were determined by western blotting
assay. After 48 h treatment of K562/A02 cells with or without 3h (1.0 or 3.0 μM), cells were harvested and lysed to detect related proteins expression. (B) Density ratios of Ac-
H3, Ac-H4, apoptosis-related proteins to GADPH.
Structure-Activity Relationship
Based on the data in Table 1, the structure-activity relationships (SAR) of the target compounds on K562/A02 cells proliferation were preliminarily drawn
as follows: the length of the carbon chain linked to the thiosemicarbazone and hydroxamic acid moiety could significantly affect the anti-proliferation
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activity. Compound with a five-carbon chain showed more potent anti-proliferation activity than compound with a three-carbon chain (3e vs.3m; 3f vs.3n;
3i vs.3o; 3l vs.3p). In addition, compound with a meta substituent on the benzene ring exhibited more potent anti-proliferation activity than compound
with an ortho or para substitution (3f vs.3a; 3g vs.3b, 3j; 3h vs.3i). Notably, the type of meta substituent could also obviously affect the anti-proliferation
activity. Among them, nitro substituent was the best, followed by chlorine, and the trifluoromethyl and methyl groups were the worst (3h, 3f vs.3c, 3d).
Conclusions
In summary, seventeen thiosemicarbazone-containing compounds were prepared, and subsequently their anti-leukemia effect on K562/A02 cells were
determined. Among them, 3h possessed significant anti-proliferation activity against K562/A02 cells with an IC50 value of 0.96 μM. Interestingly, compound
3h exhibited a potential CS-inducing effect by increasing the level of intracellular ROS in K562/A02 cells, thus displaying a preferential toxicity against
K562/A02 cells. In addition, 3h could potently inhibit HDAC1 and HDAC6, and induce K562/A02 cells apoptosis by dose-dependently affecting the
expression of apoptosis-related proteins Bax and Bcl-2, and inducing the cleavage of caspase3 and PARP. Considering the potent anti-proliferation activity,
CS and apoptosis-inducing effects, and inhibitory effects on HDAC1 and HDAC6, compound 3h might be a promising lead to discover new anti-cancer
agents towards leukemic cells overexpressing P-gp.
Experimental Section
Chemical analysis
Column chromatography (thin layer chromatography or 200-300 mesh silica gel 60) were used to purify synthesized compounds. Melting point of each
compound was determined using an apparatus of model YRT-3 (uncorrected). Chemical structure of each target compound was confirmed by NMR (400
1
MHz for H and 100 MHz for 13C, JEOL), MS (Agilent 6530 QTOF and Agilent 6460 spectrometer), and IR (Shimadzu, FTIR-8400S) routinely. Chemical
structure analysis was performed in Xuzhou Medical University of Public Experimental Research Center. All the target compounds 3a-q were new
compounds. High-performance liquid chromatography (Agilent 1260 HPLC instrument, equipped with 150*4.6 mm Daicel CHIRALCEL OD-RH column and
UV detection at 254 nm) was used to measure the purity of each target compound. The mobile phase consistent of a binary gradient of MeOH (A) and H2O
with 0.1% formic acid (B). The flow rate was 0.8 mL/min.
Synthesis of target compounds 3a-q
Benzaldehyde (500 mg) or various substituted benzaldehyde and thiosemicarbazide (10 eq) was dissolved in methanol (100 mL). After 8 h stirring under
nitrogen (N2, gas) at 100 °C, the solution was filtered to obtain crude products 1a-m, respectively, which were then dissolved in 50 mL Dioxane. After 5 min
stirring at 0 °C, 1 eq NaH was added portionwise (keep the solution at 0 °C).Then 2 eq ethyl 6-bromohexanoate or ethyl 4-bromobutyrate was added to the
reaction solution. After 10 h stirring at 120 °C under nitrogen, the mixture was cooled down to room temperature. Then water was added to the mixture,
and dispersed with ethyl acetate. Organic phase was dried with anhydrous Na2SO4, and removed by evaporation to afford the corresponding intermediates
2a-q, respectively. The crude products 2a-q was dissolved in 50 mL ethanol. After cooling to -10 °C, hydroxylamine hydrochloride (20 eq) and potassium
hydroxide (21 eq) were added to the cooled solution, and stirred for another 4 h. Finally, 10 % HCl was added to the solution (neutral pH), which was
extracted with ethyl acetate, and dried over anhydrous Na2SO4. After removal of the solvent, crude products were finally purified using column
chromatography to give the target compounds 3a-q, respectively. CH2Cl2-MeOH (50:1-30:1, v/v) was selected as the eluent. The spectroscopic data of
compounds 3a-q were shown in the supporting information.
Cytotoxicity assay
[26,
K562 and K562/A02 cells (KeyGen Co. Ltd., Nanjing, China) were cultured as described previously
27]. 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay was used to determine the anti-proliferation activity of compounds 3a-q [28]. Briefly, 1 × 104 K562 or K562/A02
cells were seeded in 96-well culture plates and cultured for 24 h to maintain the cells in exponential growth phase. Then, cells were incubated for another
72 h with or without the test compounds at indicated concentrations, respectively. Then, MTT (final concentration of each well: 5 mg/mL) was added into
each well at 4 h before the test. The insoluble formazan was collected and dissolved in dimethylsulfoxide (DMSO, 0.5%), and absorbance was detected at
490 nm using a microplate enzyme-linked immunosorbent assay reader. The inhibition rate of cell growth (%) was calculated as [A490 (mean negative
control group)-A490 (mean compound treatment group)/(A490 (mean negative control group)-A490 (mean blank control group)] × 100%. In case of
inhibition, cells were incubated with Nacetyl-cysteine (NAC, 10 mM) and the test compounds.
Determination of ROS levels in K562 or K562/A02 cells
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After 12 h incubation of K562 or K562/A02 cells with 3h (1.0 or 3.0 μM) or control (DMSO), cells (1 × 106 cells/mL) were collected, and DCFHA-DA (3 mM)
was used to stain the cells in the dark at 37 °C for 20 min. After wash by PBS, FITC fluorescent intensity was subsequently determined via FACS Calibur
flowcytometer (Becton-Dickinson). ROS level in cells treated with 3h was normalized to the relative control. In case of inhibition, cells were incubated with
Nacetyl-cysteine (NAC, 10 mM) or Tar (5.0 μM) and the test compound 3h (3.0 μM).
Effect on HDAC1, 6 and 8 activity
HDAC1, 6 and 8 activity were determined using the HDAC fluorimetric activity assay kit (Signalchem Inc.) according to manufacturer's instruction. Briefly,
HDAC1, 6 or 8 enzyme solution was incubated with compound 3h at indicated concentrations in the presence of 20 μM Boc-Lys (Ac)-AMC (HDAC substrate)
at 37 °C for 30 min. Then the reaction was stopped by adding the lysine developer. Finally, the fluorescence intensities were recorded in a SpectraMax M5
microtiter plate reader (emission: 460 nm; excitation: 360 nm).
Analysis of Cells Apoptosis
An apoptosis assay kit was used to perform Annexin V staining (KeyGen, Nanjing, China). Briefly, after 48 h incubation of K562/A02 cells with or without
3h (1.0 or 3.0 μM), cells were washed with PBS, and reincubated with annexin V binding buffer (500 μL). Subsequently, after the harvesting and washing,
cells were stained in the darkness for 15 min with5 μL propidium iodide and annexin V-FITC. Finally, a FACS Calibur flow cytometer was served to determine
the percentage of apoptotic cells (Becton-Dickinson, San Jose, USA).
Effect of 3h on Ac-H3, Ac-H4 and apoptotic proteins expression
After separation of K562/A02 cells lysate with SDS-PAGE (40 μg protein/lane), each protein was transferred to the PVDF membrane via electroblotting.
Subsequently, each blot was blocked with milk (5%) and probed with antibody against acetyl-histone H3/4, Bcl-2, Bcl-xl, cleaved caspase-3 and PARP
(Sigma-Aldrich), respectively. After washing with TBST, each blot was incubated for 2 h with secondary antibodies. Finally, each blot was detected and
imaged with ECL and G:BOX chemiXR5 digital imaging system, respectively.
Supplementary Material
Supporting information for this article is available on line.
Acknowledgements
Thanks to the grants from the Jiangsu Provincial 333 High-level Talents Cultivation Project (BRA201927), Six Talent Peaks Project in Jiangsu Province
(2017-YY-039), Natural Science Foundation of Jiangsu Province (BK20171179 and BK20181151), Postgraduate Research & Practice Innovation Program of
Jiangsu Province (KYCX20_2506) and Jiangsu Training Programs of Innovation and Entrepreneurship for Undergraduates (201810313083X).
Author Contribution Statement
Xiaoke Gu and Mingyu Guan conducted the synthesis experiments, characterized the compounds, and prepared the first draft. Chunyu Jiang, Qinghua
Song, Xin Li, Nan Sun and Jing Chen performed biological tests and analyzed the data. Xiaoke Gu and Jingying Qiu designed and supervised the project and
wrote the article. The final revision of the manuscript was revised by all the authors.
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Twitter Text
Compound 3h showed potent anti-leukemia effect on P-gp overexpressing resistant K562/A02 cells by increasing ROS level, regulating
apoptosis-related proteins expression and inhibiting HDAC1/6 activity.
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