Abstract

Introduction: The monolayer cell culture model is a popular model for screening anti-tumor activity of plant extracts. However, almost the extracts selected for screening in this model have failed in subsequent animal models. Therefore, there is only about 5 % of candidates from the original thousands of drugs that are screened which ultimately reach clinical trial. This study aimed to compare the differences in anti-tumor activity of 34 plant extracts against breast cancer cells in 2 models of monolayer cell culture (2D) and in three-dimensional (3D) cell culture.


Methods: Four breast cancer cell lines (MCF-7, CD44+CD24- MCF-7, VN9, and CD44+CD24- VN9) were used to generate the 2D and 3D models (the 3D model was developed by culturing breast cancer cells in matrigel). The extracts were got from the plant extract library that prepared in the previous study. The anti-tumor activity was evaluated via half inhibitory concentrations( IC50 values).


Results: Of the 34 extracts, E12, E7, E5 and E6 of them had an effect on MCF-7, CD44+CD24- MCF-7, VN9 and CD44+CD24- VN9 cells, respectively. The results indicated 10 potentially strong candidates for future drug development targeting hypoxic areas in breast cancer.


Conclusion: The 3D culture model exhibited higher resistance to extracts than the 2D culture model. The CD44+CD24- cell population of both VN9 and MCF-7 cell lines showed higher drug resistance than the original cell lines (VN9 and MCF-7).


 

Keywords: breast cancer drug screening natural extract CD44 CD24-


Introduction

For the discovery of new drugs, screening of natural compounds that target the proliferation of cancer cells is important1. For libraries with hundreds to thousands of extracts, they need to be screened with high-performance screening methods. Such methods allow the screening of many compounds at different concentrations at the same time on each target cell or the combination of compounds- with uniformity and high accuracy3, 2.

Screening of extracts on cancer cell models in 2-dimensional (2D) monolayer culture is limited because the monolayer model lacks the tumor cell characteristics of physiological tumors in the body4. Meanwhile, screening done on a cancer model in 3-dimensional (3D) culture may be better for studying drug effects since the 3D culture model is more similar to the in vivo animal models (and possibly clinical trials); the 3D model more closely reflects characteristics of in vivo tumors, such as differentiation, tumor microenvironment, and distribution of hypoxia in certain populations7, 6, 5. Many methods have been developed to create 3D cells like tumors in the body; these methods include use of U-shaped bottom well , the hanging drop, and cell growth in bio-matrix8, 6.

The method of using a U-shaped bottom well is heavily used in 3D cell model studies. However, one downside is that not all cell types can develop into 3D cell mass by this method9. For hanging drop culture, the advantage is that gravity is used to precipitate the cells together and thereby stimulate the cells to stick together into 3D spheres10. This method has a disadvantage of using very gentle manipulations and is difficult to develop if screened at high throughput automation. Meanwhile, the method of using biological substrates (like Matrigel) offers great potential for the development of 3D cell model13, 12, 11. Matrigel is usually stored in frozen form, at a concentration of 10-15 mg/mL; it is thawed at 4 °C and gelated in a temperature range of 24-37 °C for 30 minutes. Matrigel promotes the differentiation of different cell lines (e.g. prostate, salivary gland, mammary epithelium, pancreas, Schwann cells, intestinal cells, and bone cells), of primary cell lines (e.g. sertoli cells, blood cells, cartilage cells, epithelial cells, endometrial cells, and fallopian epithelial cells), and even tissue explants (e.g. neural crest, immature follicles, and zygote)14.

This study used matrigel to create a 3D cell model of breast cancer for the purpose of screening natural compounds that inhibit the growth of breast cancer cell . Modeling of 2D and 3D monolayer cancer cells was carried out in parallel (simultaneously) with the same evaluation agents, including Alarma Blue. The IC50 (half maximal inhibitory concentration) values were compared between 2D and 3D cancer cell models to evaluate and select the extract which showed different effects in these two models.

This study used 34 natural plant extracts and two control drugs (Doxorubicin and Tiparazamine) on 4 cell lines (MCF-7, CD44+CD24- MCF-7, VN9, and CD44+CD24- VN9 cells).

Methods

Cell lines

MCF-7 cell line was obtained from ATCC (Manassas, VA). VN9 cell line was obtained from the Stem Cell Institute, University of Science, VNU-HCM. MCF-7 and VN9 cells were cultured in DMEM/F12 (Sigma-Aldrich, St Louis, MO), 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO), 1% antibiotic-antimycotic (Sigma-Aldrich, St Louis, MO). The CD44+CD24- cells were sorted from VN9 cells (and termed CD44+CD24- VN9) or from MCF-7 (and termed CD44+CD24- MCF-7) by magnetic-activated cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany), and then expanded in M171 medium (Thermo Fisher Scientific, Waltham, MA) with MEGS Suplement (Thermo Fisher Scientific, Waltham, MA) for maintenance of stemness. The CD44+CD24- populations corresponded to the cancer stem cell (CSC) populations.

Table 1.

List of 34 natural extracts used in this study

Code of extract Plant (solvent) Code of extract Plant (solvent)
E4 Buchanania Latifolia – (CH3OH) E26 Anisoptera costata – (CH3OH)
E7 M. Camptosperma – (CH3OH) E27 Anisoptera costata – (CH3OH)
E8 D. Dyeri – (CH3OH) E28 Willughbeia cochinchinensis – (CH3OH)
E9 H. recopei – (CH3OH) E30 Streblus ilicifolius – (CH3OH)
E10 H. recopei – (CH3OH) E31 B. pandurate – (CH3OH)
E11 S. thorelii – (CH3OH) E32 Paramignya trimera – (CH3OH)
E12 S. thorelii – (CH3OH) E35 Mangifera mekongiensis – (CH3OH)
E13 D. turbinatus – (CH3OH) E36 Embelia ribes – (CH3OH)
E14 D. turbinatus – (CH3OH) E37 Willughbeia cochinchinensis – (C4H8O2)
E15 D. costatus – (CH3OH) E38 Artocarpus heterophyllus – (C4H8O2)
E16 D. costatus –(CH3OH) E39 Mangifera mekongiensis – (C4H8O2)
E17 Hopea odorata – (CH3OH) E40 Taxus wallichiana – (CH2Cl2)
E19 Vatica odorata ­– (CH3OH) E41 Caesalpinia sappan – (CH2Cl2)
E20 Vatica odorata – (CH3OH) E42 Trigona minor – (Hexan)
E21 Dipterocarpus alatus – (CH3OH) E43 B. pandurate - (Chloroform)
E22 Shorea roxburghii – (CH3OH) E45 Swintonia floribunda – (CH3OH)
E25 K. laurifolia – (CH3OH) E46 Mangifera reba Pierre 1897 – (CH3OH)

Chemicals

In the research study, the library of the 34 extract (Table 1 ), which were coded with ‘E’ as the initial label (i.e. E1-E34), were obtained from the Division of Medicinal Chemistry, Faculty of Chemistry, University of Science, Vietnam National University Ho Chi Minh City, Vietnam. Doxorubicin hydrochloride and tirapazamine were purchased from Sigma-Aldrich.

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Figure 1 . The 3D cell culture method using matrigel. The matrigel and the cells wered seed with density of 1000 cells/well. The matrigel was established on the edge of the well after 30 mins in 37 o C which has crescent shape. After 5 days in progress, the drug testing was process in 48 hours.

Cell culture in monolayer ( 2D ) and three-dimensional ( 3D ) culture

For 2D models, single cells (MCF-7, CD44+CD24- MCF-7, VN9 or CD44+CD24- VN-9) were harvested and seeded in 96-well plates at a final density of 1000 cells per well, and grown for 5 days. Fresh medium was replenished every two days. Cancer cells were cultured in DMEM/F12, 10% FBS (Sigma-Aldirch), and 1% antibiotic-antimycotic (Sigma-Aldrich). CD44+CD24- cancer cells were cultured in M171 medium (Thermo Fisher Scientific ) with MEGS supplement (Thermo Fisher Scientific).

For the 3D model, 5 µL of 1000 single cells was mixed with 5 µL of matrigel (Sigma-Aldrich) on ice and placed on the edge of the well. The plate was incubated at 37 oC in 10 minutes for gel polymerization, and then 100 µL of pre-warmed medium was added on top of the gel. The pre-warmed medium was a requisite for manipulation of 3D culture to avoid melting the gel (Figure 1 ).

Table 2.

The IC 50 of doxorubicin and tirapazamin on cell lines

Cell lines Models IC50 DOX (ng/mL) IC50 TPZ (µg/mL)
VN9 2D 1476 292
3D 1868 128
CD44+CD24- VN9 2D 98.52 315.2
3D 1711 105.4
MCF-7 2D 1674 159.4
3D 2354 68.14
CD44+CD24- MCF-7 2D 278.3 174.9
3D 3131 147

Cell viability assay and IC50 determination

After 5 days of culture, the cells and organoids were treated for 48 hours with the respective 34 extracts at the following concentrations: 31.25 µg/ml, 62.5 µg/ml, 125 µg/ml, 250 µg/ml, 500 µg/ml, or 1000 µg/ml. The concentrations of doxorubicin evaluated were: 62.5 nM, 125 nM, 250 nM, 500 nM, and 2000 nM; the concentrations of tirapazamine evaluated were: 15.625 µM, 31.25 µM, 62.5 µM, 125 µM, 250 µM, and 500 µM. Then, Alarma Blue (Sigma-Aldrich) was added to the wells at a final concentration of 10 µg/mL and incubated in the dark for 1 hour. The fluorescence intensity was read using an DTX880 system (Beckman Coulter, Brea, CA) at excitation wavelength of 535 nm, emission wavelength of 595 nm, and integration time of 500 µs. The data were normalized to control values (untreated wells) and IC50 values were calculated with GraphPad Prism 7 (GraphPad Software, Inc., La Jolla, CA).

Statistical analysis

All experiments were performed in triplicate. Statistical significance was set at P<0.05. Data were analyzed by GraphPad Prism 7.

Results

IC50 values of extracts are different on MCF-7 2D and 3D models

The IC50 results of doxorubicin and tirapazamine showed that both 2D and 3D models were successfully established for anti-tumor activity evaluation (Table 2). The IC50 results of the 34 plant extracts on MCF-7 breast cancer cells in 2D and 3D models are summarized in Table 3.

Table 3.

The IC 50 values of 34 extracts on MCF-7 breast cancer cell line

Extracts IC50 values (µg/mL) Extracts IC50 values (µg/mL)
2D model 3D model 2D model 3D model
E4 187.5 383.7 E25 597.4 870.8
E7 248.2 332.8 E27 165.3 242.5
E8 478.7 533.1 E28 299.7 673.3
E9 701.4 653.5 E30 1476 794
E10 310 154.7 E31 257.3 308.5
E11 342.9 198.3 E32 235.2 225.1
E12 303.5 160.4 E35 4450 615.9
E13 1779 1061 E36 2187 575.5
E14 348.6 593 E37 326.1 308.8
E15 1106 639.8 E38 368.1 692.2
E16 316.8 361.9 E39 345.6 270.6
E17 159.4 232.4 E40 70 1419
E18 112.4 230 E41 526.2 2063
E19 489.5 621.8 E42 499.7 359.6
E20 86.42 168.4 E43 306 620.4
E21 57.67 71.97 E45 155 361.9
E22 83.58 87.92 E46 135.4 387.6

Table 4.

The IC 50 values of 34 extracts on CD44 + CD24 - MCF-7 breast cancer cell line

Extracts IC50 values (µg/mL) Extracts IC50 values (µg/mL)
2D model 3D model 2D model 3D model
E4 66.2 360.3 E25 173.6 587.8
E7 69.42 307.7 E27 80.45 214.1
E8 103.2 937.8 E28 134.9 481.6
E9 153 887.8 E30 508.4 935.9
E10 50.48 162.2 EE31 85.04 253.5
E11 58.14 217.5 E32 56.65 223
E12 61.95 162 E35 73.95 624.6
E13 262.4 1243 E36 507 1215
E14 74.52 375.2 E37 103.5 322.7
E15 258.3 699.7 E38 229.8 980.4
E16 70.91 146.4 E39 62.65 386.6
E17 20.31 56.97 E40 274.5 1648
E18 145 39.89 E41 303.3 1257
E19 31.88 227.4 E42 102.3 293.9
E20 35.6 89.56 E43 60.97 449.6
E21 26.71 252.9 E45 32.16 295.5
E22 809.5 110.4 E46 71.98 459.6

There were 12/34 extracts which showed effects on both 2D and 3D culture models. These 12 extracts were: E4, E10, E11, E12, E17, E18, E20, E21, E22, E27, E45, and E46. However, most of the extracts predominantly had effects on the 2D model. In fact, 27 extracts on the 3D models were correlated with increased resistance by the cancer cells as compared to the resistance on the 2D models. Specifically, there were 7 extracts that had an IC50 values in the 3D model which were lower than in the 2D culture model. The 7 extracts were: E10, E12, E15, E30, E35, E36, and E42 (Figure 2). Thus, they are potential candidates for further use in the 3D culture model of MCF-7 breast cancer.

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Figure 2 . Comparing the IC 50 values of 34 extracts, doxorubicin and tirapazamine on MCF-7 breast cancer cell line . Scale 1 : Red corresponds to sensitivity, green corresponds to high resistance. Scale 2 : Black corresponds to, ratio of 2D/3D concentration is greater than 1. Gray white corresponds to, ratio of 2D/3D concentration is less than 1. Abbreviations : TPZ : tirapazamine, 2D : mononuclear cell culture, 3D : three-dimensional cell culture model, IC 50 : half inhibitory concentration

Table 5.

Summary of hit extracts on each cell types and models

Cells 2D model 3D model The extracts more sensitive on 3D than 2D
MCF-7 E20, E21, E22, E40 - E10, E12, E15, E30, E35, E36, E42
CD44+CD24-MCF-7 E4, E7, E10, E11, E12, E14, E16, E17, E19, E20, E21, E27, E31, E32, E35, E39, E43, E45, E46 E17, E18, E20 E26, E22
VN9 - - E15, E18, E22, E30
CD44+CD24-VN9 E4, E7, E10, E11, E12, E14, E16, E17, E19, E20, E21, E31, E32, E35, E39, E45 E7, E21 E18, E22

The results of hit extracts on CD44+CD24- MCF-7 in 2D and 3D models

There were 7/34 extracts that had effects on both 2D and 3D culture models. These 7 extracts were: E7, E10, E12, E17, E18, E19, E21, and E45. However, the majority of the extracts predominantly showed effects on the 2D model (Table 5 ). As seen in Table 5, cells grown in the 3D model showed more resistance to the effects of the 32 extracts than the cells grown in the 2D model. In particular, there were 2 extracts which had IC50 values in the 3D model that were lower than the values in the 2D model; those 2 extracts were E26 and E22 (Figure 3). Therefore, they are potential candidates for further research in the 3D culture model of the MCF-7 breast cancer stem cell (CSC). Comparing with the results on the MCF-7 cell line, it was observed that the CD44+CD24- sub-population of MCF-7 cells has a much higher resistance to the same extracts tested.

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Figure 3 . Comparing the IC 50 values of 34 extracts, Dox and TPZ on CD44 + CD24 - MCF-7 breast cancer cell line . Scale 1 : Red corresponds to sensitivity, green corresponds to high resistance. Scale 2 : Black corresponds to ratio of 2D/3D concentration is greater than 1. Gray white corresponds to ratio of 2D/3D concentration is less than 1. Abbreviations : Dox : doxorubicin, TPZ : tirapazamine, 2D : mononuclear cell culture, 3D : three-dimensional cell culture model

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Figure 4 . Comparing the IC 50 values of 34 extracts, Dox and TPZ on VN9 breast cancer cell line . Scale 1 : Red corresponds to sensitivity, green corresponds to high resistance. Scale 2 : Black corresponds to ratio of 2D/3D concentration is greater than 1. Gray white corresponds to ratio of 2D/3D concentration is less than 1. Abbreviations : Dox : doxorubicin, TPZ : tirapazamine, 2D : mononuclear cell culture, 3D : three-dimensional cell culture model

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Figure 5 . Comparing the IC 50 values of 34 extracts, Dox and TPZ on CD44 + CD24 - VN9 breast cancer cell line . Scale 1 : Red corresponds to sensitivity, green corresponds to high resistance. Scale 2 : Black corresponds to ratio of 2D/3D concentration is greater than 1. Gray white corresponds to ratio of 2D/3D concentration is less than 1. Abbreviations : Dox : doxorubicin, TPZ : tirapazamine, 2D : mononuclear cell culture, 3D : three-dimensional cell culture model

Table 6.

The IC 50 values of 34 extracts on VN9 breast cancer cell line

Extracts IC50 values (µg/mL) Extracts IC50 values (µg/mL)
2D model 3D model 2D model 3D model
E4 238.9 518.2 E25 4681 722.9
E7 345.6 297 E27 497.8 345.1
E8 1287 588.2 E28 1806 613.5
E9 916.8 535.7 E30 4976 1568
E10 712.6 270.9 E31 293.5 463.9
E11 559.2 686.4 E32 403.6 347
E12 635.9 496.2 E35 5799 3004
E13 7756 2706 E36 7437 3605
E14 531.4 638.3 E37 559.3 977.5
E15 5744 1088 E38 3158 2260
E16 377.7 987.1 E39 697.3 847.6
E17 211 431 E40 267.2 1212
E18 2055 430.6 E41 1083 871.1
E19 357.5 654 E42 2136 963.8
E20 103.8 122.6 E43 247.5 504.1
E21 304.2 146.1 E45 196.1 262
E22 2964 324.1 E46 1080 417.3

The results of hit extracts on VN9 cultured in 2D and 3D models

There were 5/34 extracts which showed effects on both 2D and 3D culture models. The 5 extracts were: E4, E7, E20, E21, and E45. However, most of the extracts had predominant effects on the 2D models (Table 6). As Table 6 demonstrates, 29 extracts on the 3D models were correlated with increased resistance by the cancer cells, as compared to their resistance on the 2D models. In particular, 5 extracts had IC50 values in the 3D model that were lower than the values in the 2D model. The 5 extracts were: E15, E18, E22, E25 and E30 (Figure 4). Therefore, these are potential candidates for further studies in the 3D culture model of VN9 breast cancer.

Table 7.

The IC 50 values of 34 extracts on CD44 + CD24 - VN9 breast cancer cell line

Extracts IC50 values (µg/mL) Extracts IC50 values (µg/mL)
2D model 3D model 2D model 3D model
E4 38.15 116.8 E25 292.7 2456
E7 69.24 74.54 E27 114.4 402.6
E8 112.8 255.3 E28 108.6 793.3
E9 118 249.6 E30 915.5 1218
E10 74.13 104 E31 67.14 480.1
E11 59.78 475 E32 76.9 479.4
E12 73.57 252.2 E35 99.37 738.9
E13 426.4 1107 E36 364.7 1855
E14 68.61 442.8 E37 119.1 333.4
E15 236 1296 E38 160.5 1022
E16 56.12 229.6 E39 73.18 362.4
E17 27.7 162.5 E40 137.5 992.6
E18 340.2 144.8 E41 372.8 790.6
E19 47.05 685.8 E42 146.6 515
E20 38.34 315.4 E43 126.1 301.5
E21 30.58 342.5 E45 39.07 90.3
E22 777.9 92.74 E46 90.46 193

R esults of hit extracts on CD44+CD24- VN9 cultured in 2D and 3D

There were 6/34 extracts affected both the 2D and 3D culture models: E4, E7, E10, E12, E18, and E45. Most of the extracts, however, mainly affected the 2D models (Table 7). As shown in Table 7, 32 extracts on the 3D models were correlated with increased resistance by the cancer cells, as compared to their resistance on the 2D models. In particular, there were 2 extracts which had IC50 values in the 3D model that were lower those in the 2D culture model; these extracts were E18 and E22 (Figure 5). Thus, they are potential candidates for further studies in the 3D VN9 breast CSC model. Comparison of screening results of VN9 with CD44+CD24- phenotype versus the original VN9 demonstrated that the CSC cell line (CD44+CD24- VN9) was more resistant to the extracts in the 3D culture model. Therefore, in this study, the number of extracts tested that showed an effect on this cell line was 2, indicating that VN9 CSC can carry more resistant characteristics than normal cells.

Discussion

The use of bio-matrix substrates (such as matrigel) to create 3D culture models is very convenient for drug screening. Use of a gel forming method- that contains the cells on the side of the culture well in a 96-well plate- facilitates easy manipulation without disrupting the gel structure or limiting cell growth in the form of single layer in the center of the well. This method also allows the creation of a 3D cell mass with a size of 100 μm within 5 days of culture. The drug test is conducted in 48 hours such that the entire drug testing procedure can be summarized in 7 days. In order to minimize errors when comparing 2D and 3D models, we conducted all experiments with both models in parallel. For both 2D and 3D models, the threshold of extracting effect was lower than 200 µg/mL.

A number of published studies have show that 3D breast cell culture better reflect the histological, biological, and molecular features of primary tumors than the same cells cultured using traditional 2D techniques15. In a study by Imamura et al., on a 3D breast cancer model, the breast cell mass was found to have the presence of a hypoxic cell population7; it is for this reason that the cell mass becomes sensitive to tiparazamine. In our study, we show that 10 extracts have the same effect as tiparazamine on breast cancer cells, and that they might be suitable candidates for hypoxia-targeted drug development (Table 5). Furthermore, in their study, Imamura and colleagues also showed that expression of Ki-67 was less in 3D breast cancer cell mass than in 2D, suggesting that the greater G0-dormant subpopulation was responsible for drug resistance in 3D culture.

Many studies have show that the breast cancer cell population with phenotype CD44+CD24- possesses higher tolerability to chemotherapy, hormone therapy, and radiotherapy21, 20, 19, 18, 17, 16. Thus, for the drug screening in our study, these 4 breast cancer cell lines were suitable for our evaluations: MCF-7, CD44+CD24- MCF-7, VN9, CD44+CD24- VN9. Morever, a promising outcome from out study is the identification of 10 extracts which have a more sensitive effect on the 3D culture model than the 2D culture model. These 10 extracts include: E10, E12, E15, E18, E22, E26, E30, E35, E36, and E42. These could be suitable candidates for the next steps towards developing drugs that target the hypoxic region in breast cancer. Therapies targeting cancer cells in areas of hypoxia and studies to discern mechanisms have garnered increase interest for cancer treatment. Hypoxia-related mechanisms such as overexpression of hypoxia-inducible factor (HIF) are also important avenues of research. Inhibiting HIF activity and changing the molecules involved in HIF offer hope for identifying molecular target to inhibit tumor growth or even completely halt growth22. HIF-1 also induces an increase in adenosine 2B receptor expression, thereby promoting the enrichment of breast cancer stem cells by activating protein kinase C-δ23.

Therefore, in the study herein, it was shown that the use of a 3D model of breast cancer cell culture for drug screening reflects a huge difference in drug resistance and drug sensitivity when compared to the 2D culture model. The matrigel 3D culture model is significant for screening compounds related to hypoxia-based therapy for breast cancer.

Conclusion

Medium-throughput screening on breast cancer cell models MCF-7, CD44+CD24- MCF-7, VN9, and CD44+CD24- VN9, in 2D and 3D culture, with 34 extracts showed that resistance to these extracts occurred when cancer cells were cultured in 3D. Resistance to extracts also manifested in the CD44+CD24- cell populations (i.e. CSC populations). There were 12/34 and 7/34 extracts which affected MCF-7 and CD44 +CD24- MCF-7 cells, respectively. For the Vietnamese breast cancer cell line (VN9), there were 5/34 and 6/34 extracts which affected the VN9 and CD44+CD24- VN9 cells, respectively. Overall, our study results indicated 10 potential candidates for future drug development targeting hypoxia in breast cancer.

Abbreviations

Dox: Doxorubicin

HIF: Hypoxia-Inducible Factor

TPZ: Tirapazamine

VN9: Vietnamse breast cancer cell line #9

Conflict of interest

The authors declare that there is no conflict of interests regarding the publication of this article.

Author contributions

Nhan Phan designed the project and carried out the experiments. Khuong Pham contributed to feasibility experiments. Mai Nguyen provided the extract. Nhan Phan analyzed the data and wrote the paper with contributions from all authors. Phuc Pham, Kiet Truong and Ngoc Phan suggested the idea, corrected the scientific matters, english wording and review all paper.

Acknowledgments

This work was supported by the Vietnam National University, Ho Chi Minh City, Vietnam, under grant number A2015-18-01.

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