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Received Date : 20-Aug-2014 Revised Date : 19-Oct-2014 Accepted Date : 15-Dec-2014 Article type : Research Article
Binding and anticancer properties of plumbagin with human serum albumin Yi Gou1Ψ, Yao Zhang1Ψ, Jinxu Qi1, Linlin Kong1, Zuping Zhou2, Shichu Liang2*, Feng Yang1*, Hong Liang2*
1
State Key Laboratory Cultivation Base for the Chemistry and Molecular
Engineering of Medicinal Resources, Ministry of Science and Technology of China, Guangxi Normal University, Guilin, Guangxi, China 2
Key Laboratory of Ecology of Rare an Endangered species and Environmental
Protection, Ministry of education of the People's Republic of China, Guangxi Normal University, Guilin, Guangxi, China
*To whom correspondence should be addressed: Shichu Liang, Feng Yang, Hong Liang Address: 15 Yucai Road, Guilin, Guangxi, China. Zip code: 541004 Email: [email protected]; [email protected], [email protected] Phone/Fax: 86-773-212-0958
Ψ
Yi Gou and Yao Zhang pay the same contribution to the article.
Keywords: human serum albumin, plumbagin, drug carrier, protein complex structure, anticancer activity. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12501 This article is protected by copyright. All rights reserved.
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Abstract Plumbagin (PBG) has received extensive attention as a promising anticancer drug. Therefore, we investigated the binding and anticancer properties of PBG with human serum albumin (HSA). Fluorescence results demonstrated that PBG interacts with HSA, although its binding affinity may be affected to various extents by different compounds. The HSA-PBG complex structure revealed that PBG binds to the hydrophobic cavity in the IIA subdomain of HSA through hydrogen bonding and hydrophobic interactions. The PBG-HSA complex enhances cytotoxicity by 2- to 3-fold particularly in cancer cells but has no effect on normal cells in vitro. Compared with the unbound drug, the HSA-PBG complex promotes HeLa cell apoptosis and has a stronger capacity for cell cycle arrest at the G2/M phase of HeLa cells. In conclusion, this study contributes to the rational design and development of PBG-based drugs and a drug-HSA delivery system.
Introduction To date, anticancer drugs are still a double-edged sword given their severe side effects during cancer treatment (1). Therefore, less toxic but more effective and target-specific anticancer agents have been extensively sought and developed coupled with the exploitation of drug This article is protected by copyright. All rights reserved.
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delivery systems to improve their targeting and decrease their side effects (2–5). Human serum albumin (HSA) is the most abundant protein in plasma. HSA has attracted great interest given its ability to bind a remarkable variety of drugs, impacting their delivery and efficacy and altering their pharmacokinetic and pharmacodynamic properties (6–8). Furthermore, the interaction of a potential drug with HSA is one of the important factors taken into consideration in drug development (9, 10). HSA is a promising drug delivery system due to its unique biochemical and pharmacological characteristics, which ultimately leads to higher efficacy of treatment and reduced side effects due to the enhanced permeability and retention effect of macromolecules on tumours (11–15). HSA and its complexes contain three structurally similar α-helical domains (I–III). Each domain consists of A and B subdomains, which contain six and four α-helices, respectively (16, 17). Despite it being well known that the majority of ligands that bind to HSA are hydrophobic and anionic heavy metals, a complete understanding of HSA’s binding abilities remain largely unknown due to its multiple binding sites (18–22). Therefore, while it has been particularly difficult to assess the interactions between different ligands and HSA, this is critical for the understanding of how HSA behaves in vivo (23–25). Thus, to evaluate the This article is protected by copyright. All rights reserved.
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physiologically-relevant interactions between HSA and its ligands, the HSA biding sites and their relative affinities for various ligands must be determined (26, 27). Plumbagin (PBG, Figure 1A) is extracted from the roots of the plumbago species, which has been ascribed with remarkable medicinal properties. PBG exhibits highly potent biological activities, including antioxidant, anti-inflammatory, anticancer, antibacterial and antifungal activities (28). Recent studies have indicated that PBG effectively induces apoptosis and causes cell cycle arrest (29, 30). Further, it has been suggested that designing ‘hybrid drug molecules’ composed of PBG with other appropriate anticancer agents may lead to the generation of novel and potent anticancer drugs with pleiotropic action against human cancers. Although PBG as a lead drug has attracted considerable attention, the binding mechanism and anticancer activity of the HSA-PBG complex remain to be elucidated. Therefore, this study investigates the binding and anticancer properties of PBG with an HSA carrier. The results of this study may provide forward-looking data on the rational design and development of PBG-inspired anticancer drugs and HSA-based drug delivery systems.
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Materials and methods Plumbagin, shikimic acid, salicylic acid, cinnamic acid, rhein, luteolin, HSA, fetal bovine serum, RNA polymerase, acridine orange/ethidium bromide
(AO/EB),
Hoechst
3-(4,5-dimathylthiazol-2-yl)-2,5-diphenyltetrazolium
33258, bromide
and (MTT)
were purchased from Sigma (USA). JC-1 and Annexin V-FITC/PI assay kits were purchased from Beyotime Biotech and Jiamay Biotech, respectively. Human hepatoma cell lines BEL7404, human cervical cancer cell lines HeLa, human gastric cancer cell lines MGC-803, human lung carcinoma cell lines A549, and human hepatocyte cell lines HL-7702 were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were maintained in a humid atmosphere containing 5% CO2 at 37°C in GIBCO DMEM medium supplemented with 100 units mL−1 penicillin, 100 µg mL–1 streptomycin, and 10% fetal bovine serum. X-ray Crystallography To investigate binding mechanism of PBG to HSA, we solved structure of PBG and HSA complex. We in brief defined the procedure of getting crystal. Fatty acid (FA) free HSA was purified by removing HSA dimers and multimers as published (12). HSA and PBG complexes were prepared by mixing 100 µL HSA (100 mg/mL), 380 µL FA (2.5 mM) and This article is protected by copyright. All rights reserved.
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150 µL PBG (10 mM) together overnight, then were concentrated to 100mg/ml with a Millipore spin filter (10,000 dalton cutoff ). The crystallization was carried out by sitting drop vapor diffusion at room temperature. Equal volume of the HSA complex was mixed with the reservoir solution, consisting of 28–32% (w/v) polyethylene glycol 3350, 50 mM potassium phosphate (pH 7.5), 6% glycerol, 4% DMSO. Crystals were directly picked from drop solution and then frozen in liquid nitrogen. X-ray diffraction data were collected under cryo-conditions (110 K) using Shanghai Synchrotron Radiation Facility. The HSA complex data was integrated and scaled with HKL2000, and processed in space group P1. The structure of HSA complex was resolved by molecular replacement with the program AMORE using a modified model of the HSA–MYR structure (PDB code 1BJ5) that was stripped of its ligands. This model was initially refined using a rigid body protocol in CNS and then subjected to cycles of positional and B-factor refinement before the calculation of initial Fo-Fc and 3Fo-2Fc maps. These maps were used to guide the position of the ligands, and to make manual adjustments to the protein model prior to further cycles of refinement. The Figures depicting the crystal structure were prepared with PyMOL (31). Data collection details and unit cell parameters are provided in Table 1.
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Determining binding parameters of PBG to HSA The binding constant can be calculated from the modified Stern-Volmer plot according to the following equation (32, 33)： Log((F0－F)/F) = logK + n×log(Q) (1) where F and F0 are the fluorescence intensities of protein in the presence and absence of the quencher, respectively; n is the number of binding sites; K is the binding constant and Q is quencher concentration. When the temperature change is not very large, the enthalpy change (ΔH0) of a system can be regarded as a constant (33). Under these conditions, both the enthalpy change (ΔH0) and entropy change (ΔS0) can be evaluated from the van’t Hoff equation: lnK = – ΔH0/RT + ΔS0/R (2) where R is the gas constant. The enthalpy change (ΔH0) is calculated from the slope of the van’t Hoff plot, while the entropy change (ΔS0) is calculated from the intercept. The free energy change (ΔG0) is then estimated from the following equation: ΔG0 = ΔH0 – TΔS0 (3) All fluorescence emission spectroscopy were recorded from 300 to 450 nm (excited at 280 nm) at 298 K. HSA solution concentrations in quenching experiments were 1.0×10−6 M. Both excitation and emission slit widths were 5 nm. The range of drug concentration is from 0 to 10×10−6. Furthermore, to investigate the impact of other drugs on binding This article is protected by copyright. All rights reserved.
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constants of HSA for PBG, HSA were incubated with other drugs overnight prior to taking our measurements. Cytotoxicity evaluation (MTT assay) The in vitro cytotoxicity of drugs against cells was determined in growth inhibition assays (34). 100 μL of cell suspensions at a density of 5 × 104 cells/mL was seeded in triplicate in 96-well plates and incubated for 24 h at 37°C in 5% CO2. Then graded concentrations of complexes was added to the wells in 10 µL of FBS free culture medium and the plates were incubated in a 5% CO2 humidifying atmosphere for 48 h. The MTT solution (10 mL, 5 mg/mL) was added into each well and the cells were incubated further for 4 h at 37℃. Then 100 µL DMSO was used to dissolve the formed formazan crystals and the absorbance was recorded at 570 nm and 630 nm using an Enzyme-linked Immunosorbent Assay (ELISA) reader. The sensitivity of cells to drugs treatment was obtained in terms of IC50 by the Bliss method (n = 5). Apoptosis evaluation Acridine orange/ethidium bromide (AO/EB) double staining was used to detect the apoptosis of PBG as described previously (35). HeLa cells were respectively treated with same concentration of PBG and HSA–PBG at a fixed incubation time of 24 h. Cells were washed twice with PBS and fixed with 4% paraformaldehyde. Then, the cells were This article is protected by copyright. All rights reserved.
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stained with AO/EB staining solution for 10 min. After washing twice with PBS, the cells of morphological observation were obtained under a reflected fluorescence microscope (Nikon MF30 LED, Japan). Cellular apoptosis was evaluated through annexin V-FITC apoptosis detection kit. Briefly, HeLa cells of exponential growth were in 6-well plates. Cells were collected after drugs treatment for 24 h, and washed twice with PBS, then resuspended in 100 µL 1×binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). Cells were transferred to a 1.5 mL eppendorf tube, and then 5 µL of annexin V-FITC and 5 µL of propidium iodide were added. Cells were incubated for 1h at room temperature in the dark. After 400 µL 1×binding buffer was added into each tube, the stained cells were analyzed by flow cytometry (FACScan, Bection Dickinson, San Jose, CA). The rate of cell apoptosis was analyzed. Cell cycle analysis Cell cycle analysis was carried out by flow cytometry (36). Briefly, 5 × 105 HeLa cells were treated with 20 µM PBG and HSA-PBG at 37℃ for 48 h, respectively. Cells were harvested in cold PBS, centrifuged, resuspended and fixed in 70% EtOH overnight at -20℃. After a further washing steps with cold PBS, cells were digested by RNase A (100 µg/mL) at 37℃ for 30 min, and stained with PI (50 mg/mL) in the dark at This article is protected by copyright. All rights reserved.
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room temperate for 30 min. The distribution of the cell cycle was analyzed by flow cytometry.
Results and Discussion Structural basis of PBG binding to HSA To determine the binding mode and binding site of PBG to HSA, the HSA-PBG complex structure was resolved. Electron density maps clearly revealed that a PBG molecule is located at the IIA subdomain of HSA (Figure 1B). The overall structure of the PBG, HSA and fatty acid (FA) complex is ‘heart-shaped’; six FA molecules and one PBG molecule are asymmetrically distributed throughout the HSA structure. One FA molecule is located at the IB subdomain, one at the interface between the IA and IIA subdomains, one in the interface between the IIA and IIB subdomains, two in the IIIA subdomain and one in the IIIB subdomain (Figure 1C). The large hydrophobic binding pocket in the IIA subdomain of HSA is delimited by residues, including Trp150, Glu153, Ser192, Gln196, Cys245, Lys195, Phe211, Leu203, Ala210, Ala215, Arg218, Lys199, Trp214, Leu219, Phe223, Leu238, His242, Arg257, Leu260, Ile264, Ser287, Ile290 and Ala291. The structure of the HSA-PA-PBG complex showed that one PBG molecule occupies only a small part of the large
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hydrophobic cavity of the IIA subdomain formed by Arg218, Arg222, Lys199, Trp214, Leu219, Phe223, Leu238, His242, Arg257, Leu260, Ile264, Ser287, Ile290 and Ala291 (Figures 1D and 1E). Among these residues, Arg218 and Arg222 project out of the binding pocket to form the pocket opening; the pocket’s top and bottom consists of Ala291 and Leu238 residues, respectively (Figure 1D). The hydrophobic part of PBG is surrounded within Leu219, Phe223, Leu238, His242, Arg257, Leu260, Ile264, Ser287, Ile290 and Ala291, mainly undergoing hydrophobic interactions with the surrounding side chains. The range of the closest distance between the hydrophobic group and the surrounding HSA side chains is approximately 3.0 to 5.0 Å (Table 2). The HSA-PA-PBG structure revealed that the hydroxyl group of PBG undergoes hydrogen bonding with His-242 (3.24 Å) and the acetyl oxygen atom of PBG undergoes hydrogen bonding with Lys-199 (3.26 Å) and His-242 (3.12 Å) (Figure 1D). Thermodynamic binding properties of PBG to HSA The intrinsic fluorescence properties of HSA are attributable to the single tryptophan residue at amino acid residue 214 in the IIA subdomain. Thus, compounds that bind to the IIA subdomain would diminish the fluorescence emission of HSA. This phenomenon has been used to study the nature of the interaction of drugs with HAS (37, 38). Figure 2A shows that an increased PBG concentration leads to increased quenching of the This article is protected by copyright. All rights reserved.
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HSA fluorescence signal, suggesting an interaction between PBG and HSA. The binding constants of the IIA subdomain of HSA for PBG were estimated based on Eq. (1), (Figure 2B and Table 3). Meanwhile, the thermodynamic parameters of PBG binding to HSA were calculated by using Eq. (2) and (3). Based on the binding constants of PBG to HSA obtained at three different temperatures (298, 310, 320 K), the thermodynamic parameters were obtained from the linear van’t Hoff plot (Figure 2C and Table 3). The data in Table 3 shows that the binding process of PBG to HSA at different temperatures is spontaneous because the Gibbs free energy (ΔG0) is negative. Additionally, ΔH0 and ΔS0 are also negative, indicating that both hydrogen bonds and van der Waals forces play a major role in the reaction between PBG and HSA (33,37,39), which fit well with the HSA-PBG structural results. The presence of other compounds could alter PBG binding to the IIA subdomain of HSA (Table 3). We found that PBG binding decreases significantly in the presence of rhein (RH) as observed by fluorescence quenching. In the presence of luteolin, the binding affinity of PBG decreased by approximately 2.6-fold compared to that of HSA-PBG. Shikimic acid, salicylic acid (SA) and cinnamic acid (CA) led to appreciable reductions in PBG binding affinity (Table 3). These results not only show that various compounds can inhibit PBG binding to the IIA This article is protected by copyright. All rights reserved.
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subdomain of HSA, but also suggest that these compounds may occupy the same binding site as PBG on HSA. Superimposition of HSA-FA-SA, HSA-FA-CA, HSA-FA-RH and HSA-FA-PBG structures shows that SA, CA, RH and PBG share common binding sites in the IIA subdomain (Figure 3) (20,40,41). The binding affinity of HSA for PBG decreases to a different extent in the presence of various compounds due the differences in their interaction forces with HSA. Obviously, the extent of reduction in the binding affinity of PBG to HSA has a direct relation with the competing drugs’ binding affinities. Anti-cancer properties of HSA-PBG To evaluate the influence of HSA on anticancer activity of PBG in vitro, MTT assays were performed. In comparison with the naked compounds, the cytotoxic activity of the HSA-PBG complex on cancer cells increased by approximately 2- to 3-fold without any effect on normal cells. HSA itself has no cytotoxic activity to cancer cells or normal cells (Table 5). HeLa cells were used to assess whether HSA affected the apoptosis and cell cycle arrest induced by PBG. HeLa cells were chosen because the HSA-PBG complex has high cytotoxic activity in HeLa cells relative to other cancer cell lines. HeLa cells were stained with acridine orange/ethidium bromide, revealing that the amount of apoptosis induced by the HSA-PBG complex is higher than that induced by the naked This article is protected by copyright. All rights reserved.
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compound (Figure 4). Results of the Annexin V-FITC/PI staining showed that the percentages of apoptosis in HeLa cells were 13.1% for PBG and 28.4% for HSA-PBG, indicating that HSA-PBG can promote apoptosis in cancer cells (Figure 5). Furthermore, the percentage of cells in the G2/M phase when treated with PBG (21.22%) increased to 30.73% when treated with HSA-PBG. In contrast, 8.47% of the control cells (with no drug treatment) were in the G2/M phase. These results suggest that PBG may cause an accumulation of cells in the G2/M phase of the cell cycle by delaying or inhibiting cell cycle progression at this phase. Obviously, HSA improves the capacity of PBG for cell cycle accumulation at the G2/M phase. According to our results, HSA has a significant influence on the anticancer properties of PBG, improving its targeting and efficiency to some degree due to the selective accumulation of drug-bound HSA in tumour cells and not in normal cells (42, 43). An ideal drug carrier should improve drug efficiency and control its release rate at the target site. The binding affinity of the drug to the carrier exerts an influence on its release rate (5). Thus, the binding affinity of active compounds to HSA can be regulated by optimizing the drugs or modifying certain amino acids based on the HSA complex structures (44, 45). Utilizing HSA as a drug delivery system has many beneficial characteristics. HSA is a non-toxic and non-antigenic endogenous protein that can carry different hydrophobic and hydrophilic drugs through the This article is protected by copyright. All rights reserved.
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circulatory system (45, 46). Remarkably, HSA possesses several amino acid residues, including Lys, Asp, Glu and Cys. These residues are conveniently able to form complexes with targeting molecules such as antibodies and various overexpressed receptors (48–50). In terms of biomedical applications, HSA has an immense potential in the field of drug delivery.
Conclusions Our study investigated the binding parameters and anticancer activities of HSA as a PBG carrier and resolved the X-ray structure of HSA-PAL-PBG complex. Using the structural and experimental data regarding PBG with has allows not only the rational development of PBG-inspired drugs, but also the utilization of HSA as a carrier to selectively deliver drugs to cancer cells and ultimately decrease the side effects associated with current anticancer treatments.
Acknowledgments We are grateful to staff of Shanghai Synchrotron Radiation Facility for their technical assistance in data collection. This work was supported by the Natural Science Foundation of China (31060121, 31460232, 21431001 and 21171043), Natural Science Foundation of Guangxi (2012GXNSFCB053001,
2013GXNSFGA019010
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and
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2014GXNSFDA118016), Technology division of Guilin (20130403-1) and Guangxi ‘Bagui’ scholar program to ZP Zhou.
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Figure Legends:
Figure 1 (A) Chemical structure of PBG. (B) Experimental sigmaA weighted 2Fo-Fc electron density map of PBG. (C) The overall structure of PBG with HSA (D) Structural binding environment of PBG to IIA subdomain of HSA (E) Binding cavity of PBG to IIA subdomain. Carbon atoms of ligands molecule are shown in grey; oxygen in red; nitrogen in blue; hydrogen bond is shown in dash lines. Figure 2 (A) The emission spectrum of HSA (1×10-6 M) in the presence of increasing amounts of PBG at 298 K. (B) Scatchard plots of fluorescence titration of PBG with HSA (C) Van’t Hoff plot for the interaction of HSA and PBG at 298 K, pH 7.4. Figure 3 Superposition of IIA subdomain of HSA–FA–SA, HSA–FA–CA, HSA–FA–RH and HSA–FA–PBG. SA molecule (carbon = purple); CA molecule (carbon = green); RH molecule (carbon = yellow) and PBG molecule (carbon = grey), Nitrogen = blue; oxygen = red. Hydrogen bonds/salt bond are shown in dash lines. Figure 4 Representative images of HeLa cells morphology of AO/EB double staining treated by (A) Control (B) PBG (C) HSA-PBG.
[PBG] = [HSA-PBG] = 25 µM.
Figure 5 The apoptosis detection on Annexin V/propidium iodide assay of HeLa cells treated by (A) Control (B) PBG (C) HSA-PBG. [PBG] = [HSA-PBG] = 25 µM. Figure 6 Effect of PBG/HSA-PBG on cell cycle progression in HeLa cells. (A) Control (B) PBG (C) HSA-PBG. [PBG] = [HSA-PBG] = 20 µM.
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Table 1 X-ray data collection and model refinement statistics for the crystal structures of HSA-FA-PBG complex HSA complex
HSA-FA-PBG
Data collection Space group
P1
Cell parameters, a,b,c (Å)
94.87, 95.73,38.44
Cell parameters,
75.37,89.98,78.30
Resolution range (Å)
40-2.3
Data redundancy
4.0
Completeness (%)
98% (99.6%)
I/σa
14.6 (4.8)
Rmerge (%)
b
6.9% (23.7%)
Model refinement Rmodel (%)c d
22.56%
Rfree (%)
29.01%
R.m.s. deviation from ideal bond lengths
0.007 Å
R.m.s. deviation from ideal angles (°)
1.112
a Values for the outermost resolution shell are given in parentheses. b Rmerge=100×ΣhΣj| Ihj-Ih|/ΣhΣj Ihj where Ih is the weighted mean intensity of the symmetry-related refrections Ihj. c Rmodel=100×Σhkl|Fobs -Fcalc|/ΣhklFobs where Fobs and Fcalc are the observed and calculated structure factors, respectively. d Rfree is the Rmodel calculated using a randomly selected 5% sample of reflection data omitted from the refinement.
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Table 2 Hydrogen bond and Vander Waals interactions between PBG and HSA Group/atom
Hyrogen bond (Å)
Van der Waals interactions (Distances of the closest approach, Å)
OH
His242 (3.24)
O (2)
Lys199 (3.26), His242 (3.12)
Trp214(6.16), Leu238(3.64), Leu219(3.28), Phe223(4.41), Ile264(3.98), Ile290(3.13), Ala291(3.96), Leu238(3.35), Val241(4.62), Leu260(3.83), Arg257(4.28), Arg222(4.28), Arg218(5.21)
Table 3: Effect of other compounds on binding constants (K) of HSA for PBG, λex = 280 nm, T = 298 K, pH 7.4
Mol ratio of drug to HSA (1:1)
K (× 104/M)
n
ΔG(kJ/mol)
Salicylic acid
4.05 ± 0.07
1.11
–26.28
Cinnamic acid
3.09 ± 0.02
1.03
–25.61
Shikimic acid
2.61 ± 0.02
0.97
–25.20
Luteolin
1.82 ± 0.01
0.89
–24.30
Rhein
0.64 ± 0.02
0.78
–22.20
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Table 4: Binding and thermodynamic characteristics of the PGB–HSA system.
T(K)
K0 (×104/M)
n
298
4.71 ± 0.04
1.06
310
2.49 ± 0.05
1.03
320
1.53 ± 0.02
1.04
ΔH (kJ· mol-1 )
ΔG (kJ·mol-1 )
–44.89 ± 0.04
–26.18 ± 0.04
ΔS (J· mol-1K-1)
–26.79 ± 0.04 –60.33 ± 0.04
–25.58 ± 0.04
Table 5 IC50 (µM) of PBG and HSA-PBG to cancer cells and normal cell (HL-7702).
PBG and HSA
cell growth inhibition, IC50 ± SD (μM) HeLa
BEL 7404
A549
MGC-803
HL-7702
HSA
>100
>100
>100
>100
>100
PBG
21.2 ± 1.2
>50
23.5 ± 0.9
27.5 ± 1.4
16.3 ± 1.9
PBG-HSA
6.9 ± 0.3
22.3 ± 0.4
10.2 ± 0.8
10.5 ± 0.3
33.2 ± 2.1
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Received Date : 20-Aug-2014 Revised Date : 19-Oct-2014 Accepted Date : 15-Dec-2014 Article type : Research Article
Binding and anticancer properties of plumbagin with human serum albumin Yi Gou1Ψ, Yao Zhang1Ψ, Jinxu Qi1, Linlin Kong1, Zuping Zhou2, Shichu Liang2*, Feng Yang1*, Hong Liang2*
1
State Key Laboratory Cultivation Base for the Chemistry and Molecular
Engineering of Medicinal Resources, Ministry of Science and Technology of China, Guangxi Normal University, Guilin, Guangxi, China 2
Key Laboratory of Ecology of Rare an Endangered species and Environmental
Protection, Ministry of education of the People's Republic of China, Guangxi Normal University, Guilin, Guangxi, China
*To whom correspondence should be addressed: Shichu Liang, Feng Yang, Hong Liang Address: 15 Yucai Road, Guilin, Guangxi, China. Zip code: 541004 Email: [email protected]; [email protected], [email protected] Phone/Fax: 86-773-212-0958
Ψ
Yi Gou and Yao Zhang pay the same contribution to the article.
Keywords: human serum albumin, plumbagin, drug carrier, protein complex structure, anticancer activity. This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12501 This article is protected by copyright. All rights reserved.
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Abstract Plumbagin (PBG) has received extensive attention as a promising anticancer drug. Therefore, we investigated the binding and anticancer properties of PBG with human serum albumin (HSA). Fluorescence results demonstrated that PBG interacts with HSA, although its binding affinity may be affected to various extents by different compounds. The HSA-PBG complex structure revealed that PBG binds to the hydrophobic cavity in the IIA subdomain of HSA through hydrogen bonding and hydrophobic interactions. The PBG-HSA complex enhances cytotoxicity by 2- to 3-fold particularly in cancer cells but has no effect on normal cells in vitro. Compared with the unbound drug, the HSA-PBG complex promotes HeLa cell apoptosis and has a stronger capacity for cell cycle arrest at the G2/M phase of HeLa cells. In conclusion, this study contributes to the rational design and development of PBG-based drugs and a drug-HSA delivery system.
Introduction To date, anticancer drugs are still a double-edged sword given their severe side effects during cancer treatment (1). Therefore, less toxic but more effective and target-specific anticancer agents have been extensively sought and developed coupled with the exploitation of drug This article is protected by copyright. All rights reserved.
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delivery systems to improve their targeting and decrease their side effects (2–5). Human serum albumin (HSA) is the most abundant protein in plasma. HSA has attracted great interest given its ability to bind a remarkable variety of drugs, impacting their delivery and efficacy and altering their pharmacokinetic and pharmacodynamic properties (6–8). Furthermore, the interaction of a potential drug with HSA is one of the important factors taken into consideration in drug development (9, 10). HSA is a promising drug delivery system due to its unique biochemical and pharmacological characteristics, which ultimately leads to higher efficacy of treatment and reduced side effects due to the enhanced permeability and retention effect of macromolecules on tumours (11–15). HSA and its complexes contain three structurally similar α-helical domains (I–III). Each domain consists of A and B subdomains, which contain six and four α-helices, respectively (16, 17). Despite it being well known that the majority of ligands that bind to HSA are hydrophobic and anionic heavy metals, a complete understanding of HSA’s binding abilities remain largely unknown due to its multiple binding sites (18–22). Therefore, while it has been particularly difficult to assess the interactions between different ligands and HSA, this is critical for the understanding of how HSA behaves in vivo (23–25). Thus, to evaluate the This article is protected by copyright. All rights reserved.
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physiologically-relevant interactions between HSA and its ligands, the HSA biding sites and their relative affinities for various ligands must be determined (26, 27). Plumbagin (PBG, Figure 1A) is extracted from the roots of the plumbago species, which has been ascribed with remarkable medicinal properties. PBG exhibits highly potent biological activities, including antioxidant, anti-inflammatory, anticancer, antibacterial and antifungal activities (28). Recent studies have indicated that PBG effectively induces apoptosis and causes cell cycle arrest (29, 30). Further, it has been suggested that designing ‘hybrid drug molecules’ composed of PBG with other appropriate anticancer agents may lead to the generation of novel and potent anticancer drugs with pleiotropic action against human cancers. Although PBG as a lead drug has attracted considerable attention, the binding mechanism and anticancer activity of the HSA-PBG complex remain to be elucidated. Therefore, this study investigates the binding and anticancer properties of PBG with an HSA carrier. The results of this study may provide forward-looking data on the rational design and development of PBG-inspired anticancer drugs and HSA-based drug delivery systems.
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Materials and methods Plumbagin, shikimic acid, salicylic acid, cinnamic acid, rhein, luteolin, HSA, fetal bovine serum, RNA polymerase, acridine orange/ethidium bromide
(AO/EB),
Hoechst
3-(4,5-dimathylthiazol-2-yl)-2,5-diphenyltetrazolium
33258, bromide
and (MTT)
were purchased from Sigma (USA). JC-1 and Annexin V-FITC/PI assay kits were purchased from Beyotime Biotech and Jiamay Biotech, respectively. Human hepatoma cell lines BEL7404, human cervical cancer cell lines HeLa, human gastric cancer cell lines MGC-803, human lung carcinoma cell lines A549, and human hepatocyte cell lines HL-7702 were obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were maintained in a humid atmosphere containing 5% CO2 at 37°C in GIBCO DMEM medium supplemented with 100 units mL−1 penicillin, 100 µg mL–1 streptomycin, and 10% fetal bovine serum. X-ray Crystallography To investigate binding mechanism of PBG to HSA, we solved structure of PBG and HSA complex. We in brief defined the procedure of getting crystal. Fatty acid (FA) free HSA was purified by removing HSA dimers and multimers as published (12). HSA and PBG complexes were prepared by mixing 100 µL HSA (100 mg/mL), 380 µL FA (2.5 mM) and This article is protected by copyright. All rights reserved.
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150 µL PBG (10 mM) together overnight, then were concentrated to 100mg/ml with a Millipore spin filter (10,000 dalton cutoff ). The crystallization was carried out by sitting drop vapor diffusion at room temperature. Equal volume of the HSA complex was mixed with the reservoir solution, consisting of 28–32% (w/v) polyethylene glycol 3350, 50 mM potassium phosphate (pH 7.5), 6% glycerol, 4% DMSO. Crystals were directly picked from drop solution and then frozen in liquid nitrogen. X-ray diffraction data were collected under cryo-conditions (110 K) using Shanghai Synchrotron Radiation Facility. The HSA complex data was integrated and scaled with HKL2000, and processed in space group P1. The structure of HSA complex was resolved by molecular replacement with the program AMORE using a modified model of the HSA–MYR structure (PDB code 1BJ5) that was stripped of its ligands. This model was initially refined using a rigid body protocol in CNS and then subjected to cycles of positional and B-factor refinement before the calculation of initial Fo-Fc and 3Fo-2Fc maps. These maps were used to guide the position of the ligands, and to make manual adjustments to the protein model prior to further cycles of refinement. The Figures depicting the crystal structure were prepared with PyMOL (31). Data collection details and unit cell parameters are provided in Table 1.
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Determining binding parameters of PBG to HSA The binding constant can be calculated from the modified Stern-Volmer plot according to the following equation (32, 33)： Log((F0－F)/F) = logK + n×log(Q) (1) where F and F0 are the fluorescence intensities of protein in the presence and absence of the quencher, respectively; n is the number of binding sites; K is the binding constant and Q is quencher concentration. When the temperature change is not very large, the enthalpy change (ΔH0) of a system can be regarded as a constant (33). Under these conditions, both the enthalpy change (ΔH0) and entropy change (ΔS0) can be evaluated from the van’t Hoff equation: lnK = – ΔH0/RT + ΔS0/R (2) where R is the gas constant. The enthalpy change (ΔH0) is calculated from the slope of the van’t Hoff plot, while the entropy change (ΔS0) is calculated from the intercept. The free energy change (ΔG0) is then estimated from the following equation: ΔG0 = ΔH0 – TΔS0 (3) All fluorescence emission spectroscopy were recorded from 300 to 450 nm (excited at 280 nm) at 298 K. HSA solution concentrations in quenching experiments were 1.0×10−6 M. Both excitation and emission slit widths were 5 nm. The range of drug concentration is from 0 to 10×10−6. Furthermore, to investigate the impact of other drugs on binding This article is protected by copyright. All rights reserved.
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constants of HSA for PBG, HSA were incubated with other drugs overnight prior to taking our measurements. Cytotoxicity evaluation (MTT assay) The in vitro cytotoxicity of drugs against cells was determined in growth inhibition assays (34). 100 μL of cell suspensions at a density of 5 × 104 cells/mL was seeded in triplicate in 96-well plates and incubated for 24 h at 37°C in 5% CO2. Then graded concentrations of complexes was added to the wells in 10 µL of FBS free culture medium and the plates were incubated in a 5% CO2 humidifying atmosphere for 48 h. The MTT solution (10 mL, 5 mg/mL) was added into each well and the cells were incubated further for 4 h at 37℃. Then 100 µL DMSO was used to dissolve the formed formazan crystals and the absorbance was recorded at 570 nm and 630 nm using an Enzyme-linked Immunosorbent Assay (ELISA) reader. The sensitivity of cells to drugs treatment was obtained in terms of IC50 by the Bliss method (n = 5). Apoptosis evaluation Acridine orange/ethidium bromide (AO/EB) double staining was used to detect the apoptosis of PBG as described previously (35). HeLa cells were respectively treated with same concentration of PBG and HSA–PBG at a fixed incubation time of 24 h. Cells were washed twice with PBS and fixed with 4% paraformaldehyde. Then, the cells were This article is protected by copyright. All rights reserved.
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stained with AO/EB staining solution for 10 min. After washing twice with PBS, the cells of morphological observation were obtained under a reflected fluorescence microscope (Nikon MF30 LED, Japan). Cellular apoptosis was evaluated through annexin V-FITC apoptosis detection kit. Briefly, HeLa cells of exponential growth were in 6-well plates. Cells were collected after drugs treatment for 24 h, and washed twice with PBS, then resuspended in 100 µL 1×binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). Cells were transferred to a 1.5 mL eppendorf tube, and then 5 µL of annexin V-FITC and 5 µL of propidium iodide were added. Cells were incubated for 1h at room temperature in the dark. After 400 µL 1×binding buffer was added into each tube, the stained cells were analyzed by flow cytometry (FACScan, Bection Dickinson, San Jose, CA). The rate of cell apoptosis was analyzed. Cell cycle analysis Cell cycle analysis was carried out by flow cytometry (36). Briefly, 5 × 105 HeLa cells were treated with 20 µM PBG and HSA-PBG at 37℃ for 48 h, respectively. Cells were harvested in cold PBS, centrifuged, resuspended and fixed in 70% EtOH overnight at -20℃. After a further washing steps with cold PBS, cells were digested by RNase A (100 µg/mL) at 37℃ for 30 min, and stained with PI (50 mg/mL) in the dark at This article is protected by copyright. All rights reserved.
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room temperate for 30 min. The distribution of the cell cycle was analyzed by flow cytometry.
Results and Discussion Structural basis of PBG binding to HSA To determine the binding mode and binding site of PBG to HSA, the HSA-PBG complex structure was resolved. Electron density maps clearly revealed that a PBG molecule is located at the IIA subdomain of HSA (Figure 1B). The overall structure of the PBG, HSA and fatty acid (FA) complex is ‘heart-shaped’; six FA molecules and one PBG molecule are asymmetrically distributed throughout the HSA structure. One FA molecule is located at the IB subdomain, one at the interface between the IA and IIA subdomains, one in the interface between the IIA and IIB subdomains, two in the IIIA subdomain and one in the IIIB subdomain (Figure 1C). The large hydrophobic binding pocket in the IIA subdomain of HSA is delimited by residues, including Trp150, Glu153, Ser192, Gln196, Cys245, Lys195, Phe211, Leu203, Ala210, Ala215, Arg218, Lys199, Trp214, Leu219, Phe223, Leu238, His242, Arg257, Leu260, Ile264, Ser287, Ile290 and Ala291. The structure of the HSA-PA-PBG complex showed that one PBG molecule occupies only a small part of the large
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hydrophobic cavity of the IIA subdomain formed by Arg218, Arg222, Lys199, Trp214, Leu219, Phe223, Leu238, His242, Arg257, Leu260, Ile264, Ser287, Ile290 and Ala291 (Figures 1D and 1E). Among these residues, Arg218 and Arg222 project out of the binding pocket to form the pocket opening; the pocket’s top and bottom consists of Ala291 and Leu238 residues, respectively (Figure 1D). The hydrophobic part of PBG is surrounded within Leu219, Phe223, Leu238, His242, Arg257, Leu260, Ile264, Ser287, Ile290 and Ala291, mainly undergoing hydrophobic interactions with the surrounding side chains. The range of the closest distance between the hydrophobic group and the surrounding HSA side chains is approximately 3.0 to 5.0 Å (Table 2). The HSA-PA-PBG structure revealed that the hydroxyl group of PBG undergoes hydrogen bonding with His-242 (3.24 Å) and the acetyl oxygen atom of PBG undergoes hydrogen bonding with Lys-199 (3.26 Å) and His-242 (3.12 Å) (Figure 1D). Thermodynamic binding properties of PBG to HSA The intrinsic fluorescence properties of HSA are attributable to the single tryptophan residue at amino acid residue 214 in the IIA subdomain. Thus, compounds that bind to the IIA subdomain would diminish the fluorescence emission of HSA. This phenomenon has been used to study the nature of the interaction of drugs with HAS (37, 38). Figure 2A shows that an increased PBG concentration leads to increased quenching of the This article is protected by copyright. All rights reserved.
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HSA fluorescence signal, suggesting an interaction between PBG and HSA. The binding constants of the IIA subdomain of HSA for PBG were estimated based on Eq. (1), (Figure 2B and Table 3). Meanwhile, the thermodynamic parameters of PBG binding to HSA were calculated by using Eq. (2) and (3). Based on the binding constants of PBG to HSA obtained at three different temperatures (298, 310, 320 K), the thermodynamic parameters were obtained from the linear van’t Hoff plot (Figure 2C and Table 3). The data in Table 3 shows that the binding process of PBG to HSA at different temperatures is spontaneous because the Gibbs free energy (ΔG0) is negative. Additionally, ΔH0 and ΔS0 are also negative, indicating that both hydrogen bonds and van der Waals forces play a major role in the reaction between PBG and HSA (33,37,39), which fit well with the HSA-PBG structural results. The presence of other compounds could alter PBG binding to the IIA subdomain of HSA (Table 3). We found that PBG binding decreases significantly in the presence of rhein (RH) as observed by fluorescence quenching. In the presence of luteolin, the binding affinity of PBG decreased by approximately 2.6-fold compared to that of HSA-PBG. Shikimic acid, salicylic acid (SA) and cinnamic acid (CA) led to appreciable reductions in PBG binding affinity (Table 3). These results not only show that various compounds can inhibit PBG binding to the IIA This article is protected by copyright. All rights reserved.
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subdomain of HSA, but also suggest that these compounds may occupy the same binding site as PBG on HSA. Superimposition of HSA-FA-SA, HSA-FA-CA, HSA-FA-RH and HSA-FA-PBG structures shows that SA, CA, RH and PBG share common binding sites in the IIA subdomain (Figure 3) (20,40,41). The binding affinity of HSA for PBG decreases to a different extent in the presence of various compounds due the differences in their interaction forces with HSA. Obviously, the extent of reduction in the binding affinity of PBG to HSA has a direct relation with the competing drugs’ binding affinities. Anti-cancer properties of HSA-PBG To evaluate the influence of HSA on anticancer activity of PBG in vitro, MTT assays were performed. In comparison with the naked compounds, the cytotoxic activity of the HSA-PBG complex on cancer cells increased by approximately 2- to 3-fold without any effect on normal cells. HSA itself has no cytotoxic activity to cancer cells or normal cells (Table 5). HeLa cells were used to assess whether HSA affected the apoptosis and cell cycle arrest induced by PBG. HeLa cells were chosen because the HSA-PBG complex has high cytotoxic activity in HeLa cells relative to other cancer cell lines. HeLa cells were stained with acridine orange/ethidium bromide, revealing that the amount of apoptosis induced by the HSA-PBG complex is higher than that induced by the naked This article is protected by copyright. All rights reserved.
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compound (Figure 4). Results of the Annexin V-FITC/PI staining showed that the percentages of apoptosis in HeLa cells were 13.1% for PBG and 28.4% for HSA-PBG, indicating that HSA-PBG can promote apoptosis in cancer cells (Figure 5). Furthermore, the percentage of cells in the G2/M phase when treated with PBG (21.22%) increased to 30.73% when treated with HSA-PBG. In contrast, 8.47% of the control cells (with no drug treatment) were in the G2/M phase. These results suggest that PBG may cause an accumulation of cells in the G2/M phase of the cell cycle by delaying or inhibiting cell cycle progression at this phase. Obviously, HSA improves the capacity of PBG for cell cycle accumulation at the G2/M phase. According to our results, HSA has a significant influence on the anticancer properties of PBG, improving its targeting and efficiency to some degree due to the selective accumulation of drug-bound HSA in tumour cells and not in normal cells (42, 43). An ideal drug carrier should improve drug efficiency and control its release rate at the target site. The binding affinity of the drug to the carrier exerts an influence on its release rate (5). Thus, the binding affinity of active compounds to HSA can be regulated by optimizing the drugs or modifying certain amino acids based on the HSA complex structures (44, 45). Utilizing HSA as a drug delivery system has many beneficial characteristics. HSA is a non-toxic and non-antigenic endogenous protein that can carry different hydrophobic and hydrophilic drugs through the This article is protected by copyright. All rights reserved.
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circulatory system (45, 46). Remarkably, HSA possesses several amino acid residues, including Lys, Asp, Glu and Cys. These residues are conveniently able to form complexes with targeting molecules such as antibodies and various overexpressed receptors (48–50). In terms of biomedical applications, HSA has an immense potential in the field of drug delivery.
Conclusions Our study investigated the binding parameters and anticancer activities of HSA as a PBG carrier and resolved the X-ray structure of HSA-PAL-PBG complex. Using the structural and experimental data regarding PBG with has allows not only the rational development of PBG-inspired drugs, but also the utilization of HSA as a carrier to selectively deliver drugs to cancer cells and ultimately decrease the side effects associated with current anticancer treatments.
Acknowledgments We are grateful to staff of Shanghai Synchrotron Radiation Facility for their technical assistance in data collection. This work was supported by the Natural Science Foundation of China (31060121, 31460232, 21431001 and 21171043), Natural Science Foundation of Guangxi (2012GXNSFCB053001,
2013GXNSFGA019010
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and
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2014GXNSFDA118016), Technology division of Guilin (20130403-1) and Guangxi ‘Bagui’ scholar program to ZP Zhou.
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Figure Legends:
Figure 1 (A) Chemical structure of PBG. (B) Experimental sigmaA weighted 2Fo-Fc electron density map of PBG. (C) The overall structure of PBG with HSA (D) Structural binding environment of PBG to IIA subdomain of HSA (E) Binding cavity of PBG to IIA subdomain. Carbon atoms of ligands molecule are shown in grey; oxygen in red; nitrogen in blue; hydrogen bond is shown in dash lines. Figure 2 (A) The emission spectrum of HSA (1×10-6 M) in the presence of increasing amounts of PBG at 298 K. (B) Scatchard plots of fluorescence titration of PBG with HSA (C) Van’t Hoff plot for the interaction of HSA and PBG at 298 K, pH 7.4. Figure 3 Superposition of IIA subdomain of HSA–FA–SA, HSA–FA–CA, HSA–FA–RH and HSA–FA–PBG. SA molecule (carbon = purple); CA molecule (carbon = green); RH molecule (carbon = yellow) and PBG molecule (carbon = grey), Nitrogen = blue; oxygen = red. Hydrogen bonds/salt bond are shown in dash lines. Figure 4 Representative images of HeLa cells morphology of AO/EB double staining treated by (A) Control (B) PBG (C) HSA-PBG.
[PBG] = [HSA-PBG] = 25 µM.
Figure 5 The apoptosis detection on Annexin V/propidium iodide assay of HeLa cells treated by (A) Control (B) PBG (C) HSA-PBG. [PBG] = [HSA-PBG] = 25 µM. Figure 6 Effect of PBG/HSA-PBG on cell cycle progression in HeLa cells. (A) Control (B) PBG (C) HSA-PBG. [PBG] = [HSA-PBG] = 20 µM.
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Table 1 X-ray data collection and model refinement statistics for the crystal structures of HSA-FA-PBG complex HSA complex
HSA-FA-PBG
Data collection Space group
P1
Cell parameters, a,b,c (Å)
94.87, 95.73,38.44
Cell parameters,
75.37,89.98,78.30
Resolution range (Å)
40-2.3
Data redundancy
4.0
Completeness (%)
98% (99.6%)
I/σa
14.6 (4.8)
Rmerge (%)
b
6.9% (23.7%)
Model refinement Rmodel (%)c d
22.56%
Rfree (%)
29.01%
R.m.s. deviation from ideal bond lengths
0.007 Å
R.m.s. deviation from ideal angles (°)
1.112
a Values for the outermost resolution shell are given in parentheses. b Rmerge=100×ΣhΣj| Ihj-Ih|/ΣhΣj Ihj where Ih is the weighted mean intensity of the symmetry-related refrections Ihj. c Rmodel=100×Σhkl|Fobs -Fcalc|/ΣhklFobs where Fobs and Fcalc are the observed and calculated structure factors, respectively. d Rfree is the Rmodel calculated using a randomly selected 5% sample of reflection data omitted from the refinement.
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Table 2 Hydrogen bond and Vander Waals interactions between PBG and HSA Group/atom
Hyrogen bond (Å)
Van der Waals interactions (Distances of the closest approach, Å)
OH
His242 (3.24)
O (2)
Lys199 (3.26), His242 (3.12)
Trp214(6.16), Leu238(3.64), Leu219(3.28), Phe223(4.41), Ile264(3.98), Ile290(3.13), Ala291(3.96), Leu238(3.35), Val241(4.62), Leu260(3.83), Arg257(4.28), Arg222(4.28), Arg218(5.21)
Table 3: Effect of other compounds on binding constants (K) of HSA for PBG, λex = 280 nm, T = 298 K, pH 7.4
Mol ratio of drug to HSA (1:1)
K (× 104/M)
n
ΔG(kJ/mol)
Salicylic acid
4.05 ± 0.07
1.11
–26.28
Cinnamic acid
3.09 ± 0.02
1.03
–25.61
Shikimic acid
2.61 ± 0.02
0.97
–25.20
Luteolin
1.82 ± 0.01
0.89
–24.30
Rhein
0.64 ± 0.02
0.78
–22.20
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Table 4: Binding and thermodynamic characteristics of the PGB–HSA system.
T(K)
K0 (×104/M)
n
298
4.71 ± 0.04
1.06
310
2.49 ± 0.05
1.03
320
1.53 ± 0.02
1.04
ΔH (kJ· mol-1 )
ΔG (kJ·mol-1 )
–44.89 ± 0.04
–26.18 ± 0.04
ΔS (J· mol-1K-1)
–26.79 ± 0.04 –60.33 ± 0.04
–25.58 ± 0.04
Table 5 IC50 (µM) of PBG and HSA-PBG to cancer cells and normal cell (HL-7702).
PBG and HSA
cell growth inhibition, IC50 ± SD (μM) HeLa
BEL 7404
A549
MGC-803
HL-7702
HSA
>100
>100
>100
>100
>100
PBG
21.2 ± 1.2
>50
23.5 ± 0.9
27.5 ± 1.4
16.3 ± 1.9
PBG-HSA
6.9 ± 0.3
22.3 ± 0.4
10.2 ± 0.8
10.5 ± 0.3
33.2 ± 2.1
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