2-Methoxyestradiol

Co-administration of paclitaxel and 2-methoxyestradiol using folate-conjugated human serum albumin nanoparticles for improving drug resistance and antitumor efficacy

Xinyang Liua*, Taoqian Zhaoa, Yue Xua, Pengchao Huoa, Xia Xua, Zhenzhong Zhanga, Qingfeng Tianb and Nan Zhanga

ABSTRACT

The use of chemotherapeutic drug paclitaxel (PTX) for the treatment of tumors has several limitations, including multidrug resistance (MDR) and serious adverse reactions. This research aims to co-encapsulate PTX and the chemosensitizer 2-methoxyestradiol (2-ME) into folate-conjugated human serum albumin nanoparticles (FA-HSANPs) to reduce multiple drug resistance and improve antitumor efficiency. The results show PTX/2-ME@FA-HSANPs had uniform particle size (180 ± 12.31 nm) and high encapsulation efficacy. It also exhibited highly potent cytotoxicity and apoptosis-inducing activities in the G2/M phase of PTX-resistant EC109/Taxol cells. Moreover, PTX/2-ME@FA-HSANPs not only displayed better inhibition of tumor growth in S-180 tumor-bearing mice than PTX alone but also reduced pathological damage to normal tissues. In summary, PTX/2-ME@FA-HSANPs could be a promising vehicle for tumor therapy and reducing drug resistance. This research will also provide references for other MDR treatment.

KEYWORDS
Anticancer; paclitaxel;
2-methoxyestradiol; drug resistance; folate- conjugated human serum albumin; nanoparticles

Introduction

Paclitaxel (PTX), a natural alkaloid that extracted from the bark of yew has become one of the first-line anticancer drugs due to its excellent chemotherapeutic effect toward various types of can- cers, including ovarian, breast, non-small cell lung, stomach, and esophageal cancers (Wang et al. 2011). The potent anticancer effect of PTX is associated with its activity of promoting and stabi- lizing the microtubule assembly, and then, inducing mitotic arrest and eventually leading to the programmed cell death (Kingston and Snyder 2014). Current main marketed formulation of PTX in clinic is the solvent combination of Cremophor EL and dehydrated ethanol (50:50 (v/v)) (Huang et al. 2018) because of poor solubility of PTX (<0.01 mg/mL) (Hong et al. 2016). However, the PTX prod- ucts has serious limitation in clinic because many patients are allergic to Cremophor EL. In 2005, an innovative formulation of human albumin-bound PTX nanoparticles (NPs) (AbraxaneVR ) was approved by the US-FDA (Sofias et al. 2017). AbraxaneVR reduces the allergic reaction, and significantly increased the water solubil- ity of PTX (Miele et al. 2009). Moreover, human albumin-bound PTX nanoparticles (PTX@HSANPs) is able to passively target to the tumor tissue because of the nanoscale size (Maeda et al. 2000). Multidrug resistance (MDR) is another important issue for the failure of PTX to treat various types of cancers (Holohan et al. 2013; Iyer et al. 2013). The cancer patients are very sensitive to chemo- therapeutic agent in the early stage, but resistance may occur in the later period of chemotherapy. MDR is often caused by the overexpression of active efflux transporters which export the anticancer drugs out of the cancer cells, and then reduces the intracellular concentration of drugs. The active efflux transporters includes DNA methyltransferase1 (DNMT1), P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance- related protein 1 (MRP1) (Aller et al. 2009). Currently, many reports indicated the combination of a chemotherapeutic drug and chemo- sensitizer could improve the drug efficiency for the cancers drug resistance by inhibiting the drug efflux by P-gp (Chen et al. 2017; Zou et al. 2017), such as doxorubicin and curcumin (Wang et al. 2015), PTX and curcumin (Ganta and Amiji 2009), and PTX and quercetin (Kang et al. 2017). 2-methoxyestradiol (2-ME), a natural metabolite of estradiol (Hamel et al. 1996), could effectively over- come drug resistance in tumor cells. The co-administration of 2-ME with PTX has the better anticancer effect than PTX alone (Chauhan et al. 2002). Previous study showed the better effects of tumor inhibition by the combination of PTX and 2-ME (Han et al. 2005). To improve the poor water solubility, reduce the side effects and drug resistance of PTX, current research aimed to develop a folate-conjugated human serum albumin nanoparticles for PTX and 2-ME (PTX/2-ME@FA-HSANPs), which could simultaneously deliver both drugs into tumor cells and expect a good inhibition effect. Experiments Materials 2-ME (purity > 98.0%) was synthesized by Zhengzhou University (Zhengzhou, China). HSA and FA-HSA were purchased from Hefei Botai Biotechnology Co. Ltd. Fluorescein isothiocyanate (FITC) and dimethyl sulfoxide (DMSO) were purchased from Beijing Dingguo Biotech Co. Ltd. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazo- lium bromide (MTT), Dulbecco’s modified Eagle’s medium(DMEM), penicillin, and streptomycin were purchased from Gibco Invitrogen. Dialysis bags (MWCO = 12 000) were purchased from Spectrum Laboratories Inc. Ethanol and acetonitrile were of analytic grade. Methanol was of high-performance liquid chromatog- raphy (HPLC) grade.

Cell cultures

The human esophageal cancer cell line EC109 and its PTX-resist- ant cells (EC109/Taxol) were obtained from the Chinese Academy of Sciences Cell Bank. Both these two cells are overexpressed fol- ate receptor. The cells were cultured in normal DMEM with 10% FBS and 1% penicillin/streptomycin in 5% CO2 and 95% air at 37 ◦C in a humidified incubator.

BCA protein assay and western blotting

Bicinchoninic acid (BCA) protein-assay and western blotting were performed to evaluate the differences in expression levels of drug-resistant proteins in EC109 and EC109/Taxol cells. Briefly, EC109 and EC109/Taxol cells (2 × 105 cells/well in 6-well plates) were treated with 2-ME (2, 5, and 10 mM) and the positive control 5-Aza-2′-deoxycytidine (5-Aza-Dc, 10 mM). The harvested cells were washed twice with PBS buffer and incubated in radioimmunoprecipitation assay buffer with protease inhibitor phenyl methane sulfonyl fluoride in ice bath for 30 min. Insoluble debris was pre- cipitated by centrifugation (12 000 rpm, 15 min, 4 ◦C) and the supernatant was assayed for protein concentration by a BCA protein assay kit. Western blotting was performed according to standard protocols. The levels of DNMT1, P-gp, BCRP, and MRP1 were analyzed using the following antibodies, including human polyclonal anti-DNMT1, anti-P-gp, anti-BCRP, and anti-MRP1 (1:500). And it was detected by the electrochemiluminescence (ECL) western blotting detection reagents.

Nanoparticles preparation

PTX/2-ME@FA-HSANPs and PTX/2-ME@HSANPs were prepared by solvent evaporation method (Elzoghby et al. 2012). Briefly, 30 mg FA-HSA and HSA was first dissolved in 2 mL of water under the stirring with 600 rpm at room temperature, respectively. PTX (1 mg) and 2-ME (1 mg) were dissolved in 6 mL of ethanol. Then, the drug solution was dropped into the FA-HSA solution until complete mixture. Ninety microliter of 1% glutaraldehyde solution was added into the mixed solution and the crosslinking process was stirred for 12 h. Ethanol was removed at 45 ◦C by using a rotary evaporator. For purifying the nanoparticles, they were dissolved in PBS buffer and filter out the free drug by repeated ultrafiltration centrifuga- tion (1.6 × 104 g, 15 min, Beckman Coulter, USA). Finally, these nanoparticles were re-dispersed in purified water for fur- ther researches.

Characterization of tested nanoparticles

The particle size, zeta potential, and polydispersity index (PDI) of tested nanoparticles were measured by dynamic light scattering (Zetasizer Nano ZS-90, Malvern, UK). Transmission electron micro- scope (TEM; Tecnai G2 20, FEI) was used for the morphological examination. The concentrations of PTX and 2-ME were meas- ured by HPLC with the mobile phase (water: carbinol: aceto- nitrile, 41:23:36) at 227 nm wavelength. The encapsulated efficiency and drug loading were calculated using the following equations:

In vitro drug release from tested nanoparticles

First, the PTX/2-ME@FA-HSANPs (1 mL) and PTX/2-ME@HSANPs (1 mL) were placed in a dialysis membrane (MWCO = 10 000). Then, the sample was incubated at 37 ◦C in a mixed solution of 10% PBS (pH 7.4) and 1% SDS under stirring (100 rpm). The solu- tion was drawn at regular intervals. Finally, the amount of released drug from tested nanoparticles was measured by HPLC.

In vitro cytotoxicity

In vitro cytotoxicity of different formulations in EC109 and EC109/ Taxol cells was evaluated by MTT assay. First, EC109 and EC109/ Taxol cells were seeded at the density of 5 × 104 cells/well in 96- well plates. After 24 h incubation, the various concentrations of 2- ME, PTX, a mixture of PTX and 2-ME, PTX/2-ME@HSANPs and PTX/ 2-ME@FA-HSANPs, were dissolved in DMSO. The DMSO solutions were added into the medium with the concentration of 2% v/v.
The drug-containing medium was injected into the cell wells for another 48 h co-incubation. The cells with the blank medium only were used as control. Then, 50 lL of MTT solution (2 mg/mL) was added to each well and incubated for additional 4 h. After removal of themedium, 200 lL of DMSO was added and the fluorescence intensity was measured at 490 nm using a microplate reader (Thermo Fisher Scientific Varioskan Flash). The cellular inhibition rate and IC50 values were calculated by GraphPad prism 5.0 Software. Resistant index (RI) was also calculated using the following equation: RI = (IC50 of sample in EC109/Taxol cells)/(IC50 of sample in EC109 cells)

Cellular uptake

In this study, the fluorescence microscopy was used to evaluate the cellular uptake of FA-targeted and non-targeted NPs (Zhang et al. 2014). HSANPs and FA-HSANPs were labeled with FITC (green color) (Zhang et al. 2014). FITC solution at the concentra- tion of 1 mg/mL was slowly added to the HSANPs or FA-HSANPs solution. The solution was prepared by 7.56 g NaHCO3, 1.06 g Na2CO3, 7.36 g NaCl at 1 L water. Then, the solution was placed at 4 ◦C for 8 h in the dark. NH4Cl solution with 50 mM concentration was added to terminate the reaction and the solution was placed at 4 ◦C for 2 h. Finally, the crosslinking solution was dialyzed with four times. Next, EC109/Taxol cells were seeded in 6-well plates (2 × 105 cells/well) for 24 h. After the original culture medium was discarded, these cells were washed with PBS for three times. Then, the cells were treated with control, FITC, FITC-HSANPs, and FITC-FA-HSANPs, respectively. After 4-h incubation and the ori- ginal culture medium were discarded, these cells were washed with PBS for three times. Then, 70% ethanol was added to allow fixation for 30 min. The cells were observed by the fluorescence microscope (Nikon, Eclipse 80i). Finally, the cells were harvested and detected by FACS.

Cell apoptosis and cell cycle assay

First, the EC109/Taxol cells were seeded in 6-well plates (2 × 105 cells/well). After 24-h incubation, the culture medium was replaced with fresh medium containing PTX, 2-ME, the mixture of PTX and 2-ME, PTX/2-ME@HSANPs, and PTX/2-ME@FA-HSANPs, respectively. Then, the cells were incubated for 24 h with the untreated EC109/Taxol cells as control. After this, the cells were harvested, washed three times with cold PBS, and re-suspended in 500 mL of the binding buffer. To detect apoptosis and necrosis, the final step is that labeled cells with 5 mL of Annexin V-FITC (KeyGEN BioTECH, Nanjing) and 5 mL of propidium iodide, and fur- ther incubated for 15 min in the dark. For cell cycle assay, the cells were processed as the same protocol of apoptosis, except for the final step in which Cycle TEST PLUS DNA (KeyGEN BioTECH, Nanjing) reagent was added to the samples. Both apoptosis and cell cycle assays were performed on the BD LSRFortessa flow cytometer (Alam et al. 2013).

In vivo pharmacokinetics

All animal experiments were consistent with the guideline of ani- mal researches by Zhengzhou University. The female Sprague Dawley rats were injected intravenously with the mixture of PTX and 2-ME and the formulation (PTX/2-ME@FA-HSANPs) at the dose of PTX (12.5 mg/kg) and 2-ME (12.5 mg/kg), respectively. Due to the poor solubility of 2-ME and PTX in water, a 1:1(v/v) mixture of Cremophor EL (polyoxyethylene castor oil) and absolute etha- nol was applied as a co-solvent. Equal amounts 2-ME and PTX were put into the solvent of polyoxyethylene castor oil and abso- lute ethanol (v/v, 1:1). The mixture was, then, diluted in with the physiological saline at a ratio of 1:4 (v/v) for further applications. In addition, 0.5 mL of blood samples were collected in the hepari- nized tube from the orbital venous plexus at 0.125, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 12, and 24 h. These blood samples were immediately separated by centrifugation at 3000 rpm for 5 min. After that, those samples were extracted by methanol for the HPLC analysis with the mobile phase (water: carbinol: acetonitrile, 41:23:36) at 227 nm wavelength.

In vivo antitumor efficacy

To evaluate the in vivo antitumor efficacy of PTX/2-ME@FA- HSANPs, we started the pharmacodynamics assay. All animal experiments were consistent with the guideline of animal researches by Zhengzhou University. First, the Kunming (KM) mice (18 ± 2 g) were purchased from the Henan Laboratory Animal Center. Then, 2 × 106 S180 cells were subcutaneously injected into the left armpits of the mice to form a tumor model. The tumor was allowed to grow to the volume of 80–100 mm3 for fur- ther experiments.
The KM mice samples were divided into six groups equally as follows: saline, 2-ME, PTX, PTX + 2-ME, PTX/2-ME@HSANPs, PTX/2-ME@FA-HSANPs. Intravenous injections (12.5 mg/kg) were given once daily for 13 days. The tumor size and body weight were monitored every day. The tumor weight and relative tumor volume [V = (tumor length) × (tumor width)2/2] were recorded to determine antitumor efficacy. The biochemical examination was performed by Zhengzhou University Hospital, including the routine blood analysis and liver function tests. The biochemical tests were used as the indicators to evaluate the myelosuppressive effect and hepatotoxicity of PTX and 2-ME. At the end of the treatment, the mice were euthanized to collect the heart, liver, spleen, lung, kidney, and tumor. At last, the samples were fixed in 10% formalin for hematoxylin and eosin (H&E) staining.

Statistical analysis

In the results, all values were expressed as means ± SD (standard deviation). Student’s t-test or one-way analysis of variance (ANOVA) was applied to test for significance in the experiments. Statistical differences were considered significant according to the Graphpad statistics guide. (*p < 0.05, significant; **p < 0.01, very significant; ***p < 0.001, extremely significant; ****p < 0.0001, extremely significant). Results and discussion Reducing drug resistance of PTX in EC109/Taxol cells by 2-ME Due to the drug resistance of PTX, the mechanism of 2-ME in EC109/Taxol was first investigated. Previous studies showed that the expression of DNMT1, P-gp, BCRP, and MRP1 might be corre- lated with the PTX resistance in human esophageal cancer (Han et al. 2005; Davoren et al. 2007). As shown in Figure 1, our results showed that the expression of DNMT1, P-gp, BCRP, and MRP1 were all significantly higher in PTX-resistant esophageal cancer cells (EC109/Taxol cells) than that in EC109 cells (see Figure 1). To compare the effects of 2-ME with 5-Aza-dC on drug-resistance protein expression in EC109 and EC109/Taxol cells, the expression of DNMT1 and MRP1 were downregulated by 2-ME in EC109/ Taxol cells. Therefore, the combination of PTX and 2-ME is able to reduce PTX resistance and enhance the antitumor activity. However, both PTX and 2-ME have very poor water solubility. Thus, FA-HSANPs for both drugs were prepared to improve their water solubility, target the tumors, and achieve the more antitu- mor effect. Synthesis and characterization of tested nanoparticles To maximize the use of carrier materials, we packed two drugs as much as possible. The preparation process of tested nanoparticles was shown in Figure 2. The Table 1 listed important characteristic parameters of the test nanoparticles, such as particle sizes, zeta potential, EE, and DL. The particle size of PTX/2-ME@FA-HSANPs is 180 nm and PTX/2-ME@HSANPs is 176 nm. The zeta potential of both two tested nanoparticles is negative. Compare these data of these two nanoparticles; we can see that the characteristics of the two nanoparticles are similar, so it can be used in the subsequent comparative study. In vitro drug release of tested nanoparticles In vitro drug release profiles of tested nanoparticles were shown in Figure 3. About 20% drug molecules were released in the first 3 h from nanoparticles and the cumulative release of both drugs reached 80% in 24 h. Moreover, these nanoparticles formulation was able to release the drug with zero order, which was in agreement with previous study of albumin nanoparticles with sus- tained drug release ability. This sustained release profile could effectively reduce the frequency of administration and improve patient compliance (Li et al. 2011). In vitro cytotoxicity The cytotoxicity of different formulations was evaluated in EC109 and EC109/Taxol cells by MTT assay. The IC50 values and cell inhibition ratio were shown in Figure 4 and Table 2. In EC109/ Taxol cells, the cytotoxicity of PTX was significantly low, and the IC50 value of PTX was 45.83 lg/mL, which was higher than that in EC109 cells. Meanwhile, the IC50 values of PTX in PTX/2- ME@HSANPs and PTX/2-ME@FA-HSANPs were 1.13 and 2.16 lg/ mL, respectively. Both these two nanoparticles were less than that uptake into tumor cells than HSANPs formulation in Figure 5. The possible reason was that FA-HSANPs delivery system had active targeting effect to the high expression of FA-receptor on tumor cell surface. The RI was also calculated to evaluate MDR reversal effects by PTX + 2-ME and the FA-HSANPs. As shown in Table 1, the RI value of PTX + 2-ME was 2.21, which was 2.52-fold decrease than that of PTX (5.58). This result indicated that 2-ME could overcome MDR by reducing the expression of DNMT1, P-gp, BCRP and MRP1 in EC109/Taxol cells. Moreover, the RI values of PTX/2- ME@HSANPs and PTX/2-ME@FA-HSANPs were 1.84 and 1.20, respectively, which further demonstrated that the combination of PTX and 2-ME co-encapsulated in FA-HSANPs could significantly enhance the sensitivity of resistant cells and overcome MDR. Cellular uptake and endocytosis mechanism of PTX/2- ME@FA-HSANPs Two fluorescence microscopy methods were adopted to investi- gate the uptake differences of PTX/2-ME@FA-HSANPs in EC109 and EC109/Taxol cells both with overexpressed folate receptor. The internalization of the NPs with the FITC labeled into the cells was tracked through the colocalization of FITC signal with green fluorescence. The main principle of this method is to use the N = C=S group of FITC to chemically react with free -NH2 of protein, and then the fluorescence signal can be observed under the excitation of a specific wavelength source. As shown in Figure 5(A), the fluorescence intensity of FITC-FA-HSANPs in cells was the strongest than that of FITC and FITC-HSANPs. In addition, we can see the bar chart with the results of FACS from Figure 5(B). FITC- FA-HSANPs group had the most uptake ratio in both EC109 and EC109/Taxol. Therefore, the above results show that FA-HSANPs showed high affinity toward EC109 and EC109/Taxol cells through the FA receptor on the cell surface and had strong transmem- brane capacity (Ulbrich et al. 2011; Zhang et al. 2018). Apoptosis-promoting effects of PTX/2-ME@FA-HSANPs In this article, the Apoptosis Assay kit was used to detect the apoptosis-promoting effects of 2-ME, PTX, and other formulations. Previous studies reported that both PTX and 2-ME can induce cell apoptosis (Ganta and Amiji 2009; Zhang et al. 2014). As shown in Figure 6(A), on the one hand, we can see that the PTX induced 4.30% cell apoptosis, while the combination of PTX with 2-ME induced 16.83% apoptosis. This result showed that 2-ME and PTX demonstrated the synergic effects on the apoptosis by inhibiting NF-jB pathway (Cortes and Saura 2010; Nie et al. 2017). On the other hand, we can see that the PTX/2-ME@HSANPs and PTX/2- ME@FA-HSANPs induced 19.02% and 24.24% apoptosis in EC109/ Taxol cells, respectively. To sum up, the PTX/2-ME@FA-HSANPs achieved the best effects on cell apoptosis, which further demonstrated that our formulation exhibited enhanced antitumor effects and more intracellular uptake compared with the combination of PTX with 2-ME. Block in cell cycle by PTX/2-ME@FA-HSANPs The Figure 6(B) showed the cell cycle of EC109/Taxol cells by dif- ferent samples. The percentage of G2/M phase cells by the ‘PTX + 2-ME’ group was 26.88%, while that of ‘PTX’ group was 15.22%. The percentage of cells in the G2/M phase with PTX/2-ME@FA- HSANPs comes to 65.96%, which achieved remarkable rise. It revealed that FA-HSANPs were very efficient to block cell cycle and enhance cell apoptosis in cancer therapy. In vivo pharmacokinetics The mean plasma concentration-time profiles of PTX and 2-ME after intravenous administration of different formulations were shown in Figure 7, which reveals the significant differences between ‘PTX + 2-ME’ and PTX/2-ME@FA-HSANPs. Compared with PTX + 2-ME and PTX/2-ME@FA-HSANPs, the plasma concen- tration of PTX and 2-ME were significantly improved after intra- venous administration of PTX/2-ME@FA-HSANPs. The AUC from ROC curve, of 2-ME in PTX/2-ME@FA-HSANPs was approximately 2-times higher than in PTX +2-ME group. Similarly, PTX was about one- fold increase in comparison with that in PTX +2-ME group. It was reported that the AUC of Abraxane in rats and humans, were nearly twice than that of PTX alone (Ibrahim et al. 2002). These results further confirmed that PTX/2-ME@FA-HSANPs could obvi- ously extend the blood circulation time and improve thera- peutic efficiency. In vivo antitumor effects The antitumor efficacy of different formulations was tested in the mice bearing S180 tumor and the results were displayed in Figure 8. From the trends of different lines in Figure 8(B), we can see that the tumor volumes in mice with the saline treatment increased rapidly. Moreover, the PTX + 2ME treated group exhibited the considerable tumor inhibition than single drug and the saline-treated group. Also, the PTX/2-ME@FA-HSANPs exhibited the most significant tumor inhibition effect than other groups. In addition, the tumor weight after treatment in Figure 8(C), the weight was just 0.2 g of PTX/2-ME@FA-HSANPs group, which sug- gested that PTX/2-ME@FA-HSANPs exhibited excellent antitumor effects in vivo. In vivo safety The systemic side effects of formulations were also evaluated since high toxicity of PTX has been a major obstacle for its clinical use (Ibrahim et al. 2002). As shown in Figure 9(A), the body weight of saline-treated mice increased with the rapid growth of tumor, while the body weight of mice in 2-ME-treated group increased slightly, which indicated that 2-ME was relatively safe. However, the body weight reduced obviously after the treatment with PTX and PTX + 2-ME, which indicated the strong toxic effects of PTX. As for the mice treated by PTX/2-ME@HSANPs and PTX/2-ME@FA-HSANPs, the body weight increased steadily, which showed that our nanoparticles could effectively reduce the sys- temic side effects of PTX. The bone marrow suppression and liver function tests were performed as shown in Figure 9(B,C). In the biochemical test results, the values of HGB, WBC, Neu, and PLT in tumor-bearing mice by PTX/2-ME@FA-HSANPs were significantly higher than those of the PTX + 2-ME group, which revealed that PTX/2-ME@FA-HSANPs could reduce in vivo myelosuppression of ‘PTX + 2-ME’. Furthermore, the values of AST, ALT, and TBIL in the liver function biochemical examination treated by FA-HSANPs were lower than those of the PTX + 2-ME group, which showed that FA-HSANPs could remarkably alleviate the liver toxicity of PTX. The Figure 10 displayed the picture grids of H&E-stained organ tissue treated with different formulations. From Figure 10, it can be seen that the PTX treatment led to inflammation and necrosis in the liver, while it was not shown in targeted formula- tions treatment. 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