Background
Ovarian cancer is the leading cause of death from gynecologic malignancy [
1‐
3], and is a silent killer with stable incidence and poor prognosis due to the difficulties of early diagnosis [
4]. The treatment of ovarian cancer usually involves debulking surgery and chemotherapy [
5,
6]. Seed oil of Brucea javanica (BJO), a traditional herbal medicine, is extracted from the seeds of Brucea javanica [
7], and has been used clinically to treat various tumors, including ovarian cancer [
7‐
9]. The mechanisms of antitumor activity of BJO include inducing tumor apoptosis and reversing multidrug resistance [
10,
11]. However, its clinical use is limited due to non-specific distribution, low therapeutic index and systemic side effects [
8].
Considerable research has been carried out to overcome these defects, including develop improved dosage and drug delivery systems. Liposomes have been developed as an effective drug carrier due to their ability to deliver encapsulated drugs to specific target sites, provide sustained drug release and protect encapsulated agents [
12‐
14]. Furthermore, many targeting ligand-conjugated liposomes have been tested in recent years, including natural or synthetic ligands. Luteinizing hormone releasing hormone receptor (LHRHR) is overexpressed in approximately 70–80 % of ovarian cancer cells, however, its expression in most of the other visceral organs is negligible [
15‐
17]. Since natural LHRH is unstable in vivo, LHRH analogue (LHRHa) with improved bioactivity has been synthesized as an ovarian cancer seeking agent to target LHRHR [
18]. Previous animal studies have shown that an LHRHa targeted drug delivery system exhibits no pituitary toxicity, insignificant influence on the luteinizing hormone concentration, and negligible effect on the reproductive functions [
17]. In addition, LHRHa is a stable protein with well-defined reaction sites for conjugation, and has been shown to undergo receptor-mediated endocytosis, and transporting the ligand-receptor complex into the cells [
18,
19]. More recently, we have successfully synthesized an LHRHa conjugated and paclitaxel loaded microbubbles for ultrasound mediated chemotherapy that induced ovarian cancer apoptosis in vitro and in vivo [
20‐
23].
In this study, we coupled the LHRHa ligands with BJO-loaded liposomes (LHRHa-BJOLs) to target human ovarian cancer A2780/DDP cells that express the LHRHR. The BJO loading rate in liposomes was detected by an ultraviolet spectrophotometry, and the LHRHa in BJO loaded liposomes (BJOLs) was detected by immune colloidal gold technique and observed by transmission electron microscopy. The targeted uptaking of LHRHa-BJOLs to cancer cells was observed by fluorescence microscope and analyzed by flow cytometry. The anticancer effects of LHRHa-BJOLs were then tested in vitro and in vivo. Our experiment verifies the hypothesis that LHRHa-BJOLs will enhance the efficacy of BJO therapy. To the authors’ knowledge, no similar study has been reported elsewhere.
Methods
Cell lines and culture
Human ovarian cancer A2780/DDP cells (LHRHR positive [
20]) were kindly provided by Professor Zehua Wang from Wuhan Union Hospital (Wuhan, China). SKOV3 cells (LHRHR negative [
22]) were obtained from School of Life and Health Sciences, Chongqing Medical University (Chongqing, China). The cells were grown in RPMI-1640 medium (HyClone, Utah, USA) at 37 °C under a humidified atmosphere of 5 % CO
2, and supplemented with 10 % fetal bovine serum (Tianhang Biotechnology Co., Ltd., China) and 1 % penicillin-streptomycin.
Animal model preparation
Female BALB/c-nu/nu nude mice (4–5 weeks old; body weight, 18–20 g) were purchased from the Animal Centre of Chongqing Medical University and housed in laminar flow rooms under constant temperature (22 ± 2 °C), humidity and specific pathogen-free conditions. Tumors were established by subcutaneous injection of A2780/DDP cells. A2780/DDP cells were suspended in serum-free RPMI-1640 medium to reach a cell density of 4 × 107/ml and injected subcutaneously into the left flanks of the mice at a dose of 0.2 ml, tumors were inspected by observation and palpation. All animal experiments were approved ethically and scientifically by the Chongqing Medical University in accordance with the Practice Guidelines for Laboratory Animals of China.
Preparation of LHRHa-BJOLs
LHRHa-BJOLs were prepared by the film-dispersion and biotin-streptavidin linkage methods. Briefly, the mixtures of Soybean phosphatidylcholine (Lipoid Co., Ltd., Germany), Cholesterol (Sigma Aldrich Co., Ltd., St. Louis, USA), 1,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[biotinyl (polyethyleneglycol) (2000)] (DSPE-PEG2000-Biotin, Avanti Polar Lipids Co., Ltd., Alabaster, USA) and BJO (Yaoda Pharmaceutical Co., Ltd., Shenyang, China) (194:50:6:75, w/w) were fully dissolved in sufficient chloroform and methanol (5:1, v/v) in a round bottom flask. The organic solvent was removed by rotary evaporation (37 °C, 100 r/min, 5Kpa, 30 to 40 min) and the resulting film was dried by storing in a vacuum overnight. After hydrating the film in 3 ml phosphate buffered saline (PBS, PH 7.4, 290 mosm, Zhongshanjinqiao Biotechnology Co., Ltd., Beijing, China), the solution was sonicated for 8 min at 4 °C in an ultrasonic water bath. The suspensions were then filtered by 0.45 μm and 0.22 μm microfiltration membrane in sequence, and the BJOLs were obtained. Streptavidin (SA, SA: DSPE-PEG2000-Biotin = 1:8, molar ratio) was added to the BJOLs solution and incubated at 4 °C with gentle stirring for 30 min. An excessive amount of the biotinylated LHRHa (Beijing Zhongkeyaguang Biotechnology Co., Ltd., Beijing, China) was then added into the solution and gentle stirred at 4 °C for another 30 min to form the LHRHa-BJOLs. Finally, Sephadex G-50 column (Pharmacia Biotech Inc., New Jersey, USA) was used to separate LHRHa-BJOLs from the free streptavidin and LHRHa peptide. Coumarin-6 loaded liposomes (C6Ls) and LHRHa-modified C6Ls (LHRHa-C6Ls), being used as fluorescently labelled probes, were prepared as described above by substituting the BJO with coumarin-6 (Sigma Aldrich Co., Ltd., St. Louis, USA).
Characterization of LHRHa-BJOLs
The particle size and zeta potential were determined using dynamic light scattering with the Zetasizer Nano ZS90 (Malvern Instrument Ltd., Worcestershire, UK), samples were diluted appropriately with double distilled water for the measurements. The content of BJO in liposomes was measured by ultraviolet spectrophotometry. LHRHa-BJOLs suspensions (1 ml) were eluted by PBS in Sephadex G-50 column, and then the opalescence part of the eluate was collected. The amount of BJO in the suspensions before and after passing over the Sephadex G-50 column (Wtotal and Wfree) were measured. The encapsulation efficiency (EE) of BJO in liposomes was calculated with the formula: EE = (Wtotal - Wfree)/Wtotal × 100 %.
Stability of LHRHa-BJOLs
LHRHa-BJOLs were stored at 4 and 25 °C in the dark for 3, 6, 9, 12, 15 and 30 days, respectively. The percolation rate (PR) of BJO in different storage conditions was calculated by the following equation: PR = (EE0 - EE)/EE0 × 100 %, where the EE0 and EE were the encapsulation efficiency of BJO before and after storage, respectively.
Measurement of LHRHa on the surface of LHRHa-BJOLs
The LHRHa on the BJOLs’ surface was detected by immune colloidal gold technique and observed by transmission electron microscopy (TEM, H-7500, JEOL, Japan). Briefly, the rabbit anti-human LHRH monoclonal antibody (Chemicon International, Inc., USA) was mixed with the LHRHa-BJOLs and BJOLs, respectively, followed by an incubation at 4 °C for overnight. After passed over the Sephadex G-50 column, the suspensions were incubated with goat anti-rabbit IgG labeled with 15 nm immunogold nanoparticles (Bioss Biotechnology Co., Ltd., Beijing, China) at room temperature for 90 min, and then washed with PBS on a Sephadex G-50 column and spread over a carbon-coated copper grid, negatively stained with 1 % phosphotungstic acid and allowed to dry. The morphology of the LHRHa-BJOLs was then observed using TEM.
In vitro study of the targeted binding affinity
The intracellular uptake of the LHRHa-Liposomes (LHRHa-Lip) was observed by laser scanning confocal microscope (LSCM, Leica, Heidelberg, Germany) and coumarin-6 was used as the fluorescent probe. Briefly, A2780/DDP and SKOV3 cells were seeded in 6-well plate in triplicate at a density of 4 × 105/well and incubated for 24 h, respectively. The medium was then changed with fresh culture medium containing either LHRHa-C6Ls or C6Ls at a coumarin-6 concentration of 2 μg/ml. After incubated for 1.5 h at 37 °C and washed three times with pre-cooling PBS to remove unbound liposomes, the cells were fixed in 4 % paraformaldehyde at 4 °C for 15 min and washed with PBS. After that, the cells were stained with DAPI-containing reagent for 5 min and washed with PBS to remove the free DAPI. The uptaking of LHRHa-C6Ls or C6Ls in the cancer cells were observed by LSCM.
The intracellular uptake of LHRHa-Lip was also quantitatively analyzed by flow cytometry (FCM, FACS, BD Biosciences, San Jose, CA, USA). Briefly, A2780/DDP and SKOV3 cells were seeded in 6-well plate in triplicate at a density of 4 × 105/well and incubated. When 80 % confluence was reached, the cells were washed with PBS, and the LHRHa-C6Ls or C6Ls (containing 2ug/ml coumarin-6) was added, respectively. After 1.5 h incubation, the cells were washed three times with PBS, harvested with trypsin, centrifuged, and resuspended at a concentration of 1 × 106/ml in culture medium. The mean fluorescent intensity (MFI) of the cells were analyzed by FCM.
In vitro evaluation of antitumor efficiency
A2780/DDP cells were seeded in 6-well plate (5 × 105 cells/well) in triplicate and incubated for 24 h to allow cell adhesion. After that, the cells were equally divided into the following four treatment groups: PBS, BJOE, BJOLs and LHRHa-BJOLs groups. For BJOE, BJOLs and LHRHa-BJOLs groups, BJO was administered at a dose of 0.4 mg/ml.
Cell viability assay
After different treatments, the cells were incubated for a further 24, 48 and 72 h and washed in PBS for three times. The number of viable cells in each treatment group relative to PBS group was evaluated using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (Sigma Aldrich Co., Ltd., St. Louis, USA). Briefly, at these designated time points, 20 μl MTT (5.0 mg/ml) was added and the cells were incubated for another 4 h. Then, the medium was removed, 150 μl dimethylsulfoxide was added and mixed on a vortex mixer for 10 min. Absorbance (A) at 490 nm was recorded using the microplate reader (BIO-RADModle 550, Bio-Rad Ltd., USA). The cell inhibitory rate (IR) was calculated by the following equation: IR (%) = [1 - (Atreated/Acontrol)] × 100 %.
Apoptosis assay
Twenty-four hours after different treatments, the cell apoptosis was assessed using an Annexin V-FITC/PI apoptosis detection kit (Beyotime Biotechnology Co., Ltd., Jiangsu, China) as described by the manufacturer’s instructions. Briefly, A2780/DDP cells treated with various treatments were harvested with trypsin, washed twice with PBS, resuspended in Annexin-V binding buffer and incubated in the dark for 15 min with 5 μl AnnexinV-FITC and 10 μl PI. The apoptosis was evaluated by FCM, and the apoptosis rate of each group was calculated using Cell Quest software.
JC-1 mitochondrial membrane potential (MMP) assay
Twenty-four hours after different treatments, the cell MMP (Δψm) was assessed using a JC-1 MMP assay kit (Beyotime Biotechnology Co., Ltd., Jiangsu, China) according to the manufacturer's instructions. Briefly, A2780/DDP cells treated with various treatments were collected and washed with PBS, and incubated with 0.5 ml JC-1 dye in the dark for 20 min at 37 °C. The cells were then washed twice with JC-1 buffer and then analyzed by FCM.
Detection of apoptosis by Hoechst 33258 fluorescent staining
Twenty-four hours after different treatments, A2780/DDP cells were washed with PBS, fixed with paraformaldehyde (Sigma, St. Louis, MO, USA) at room temperature for 15 min and washed with PBS, then stained in Hoechst 33258 dye (4 g/ml, Beyotime Institute of Biotechnology, China) in dark for 5 min at 37 °C. After been washed with PBS for three times, the cells were observed under fluorescence microscopy. Apoptotic cells were defined with the changes of nuclear morphology. Normal nuclei showed diffusely and homogeneously low-intensity fluorescent, apoptotic nuclei were hyperchromatic and compact at condensed or granular state.
Bcl-2, Bax and caspase-3 activity
In order to further understand the apoptosis-inducing effect of LHRHa-BJOLs in ovarian tumor cells, apoptosis-related proteins, including bcl-2, bax and caspase 3, were evaluated in A2780/DDP cells at 24 h after different treatments by western blot analysis. The cells in each group were lyzed in a lysis buffer (50 mM Tris–Cl, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1 % NP40, 0.25 % Na-deoxycholate, and 1 μg/ml of aprotinin, leupeptin and pepstatin). Equal amounts of protein (30 μg/sample) were separated electrophoretically by 15 % SDS-PAGE and blotted onto a polyvinylidene difluoride membrane, which was then blocked with PBS containing 5 % non-fat dried milk for at least 1 h. The blots were incubated at 4 °C overnight with a primary antibody against bcl-2 (Rabbit anti-human bcl-2 antibody, CST, Inc., USA), bax (Rabbit anti-human bax antibody, EPI, Inc., USA) and caspase 3 (Rabbit anti-human caspase-3, Santa, Inc., USA) respectively and followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 30 min in a blocking buffer at room temperature. After further washing, the blots were revealed by enhanced chemiluminescence (Pierce ECL detection kit) and exposed to X-ray film (Eastman-Kodak, Rochester, NY, USA). Equal loading was confirmed by β-actin detection. Band optical density (OD) was analyzed using a Labworks 4.6 UVP-image capture and analysis software package. The analysis results were expressed in the format of mean ± standard deviation (SD) as the ratio percentage of the protein of interest OD versus the β-actin OD.
In vivo evaluation of antitumor efficiency
The anticancer effect of LHRHa-Lip was demonstrated in an ovarian cancer xenografts model which were established as described above. Thirty-two ovarian cancer-bearing mice were randomly divided into the following four treatment groups (seven mice per group): PBS (control), BJOE, BJOLs and LHRHa-BJOLs groups. For each treatment groups, an equivalent BJO dose of 180 mg/kg was injected into the mouse caudal veins on days 15, 18, 21, 24 and 27 after tumor inoculation, respectively. Tumor diameter was measured by a dial caliper once a week until the 42th day after tumor inoculation. The subcutaneous tumor volume was estimated by the following formula: V (mm
3) = (length × width
2)/2 [
24]. Three mice in each group were sacrificed 24 h after the last treatment and the tumor tissues were harvested for further protein analysis. The rest of the mice (five in each group) were monitored daily for the signs of the reduced physical activity and the progression of the tumor. The survive time of each mouse was recorded.
Detection of Bcl-2, Bax and caspase-3 expression after in vivo treatment
The bcl-2, bax and caspase 3 protein expression of the tumor were characterized by western blot analysis. Briefly, samples in each treatment group were homogenized and centrifuged at 12000 g for 30 min. The supernatant was collected and the protein concentration of the lysate was determined by a Bradford protein assay (Bio-Rad, Hercules, CA, USA). For western blot analysis, equal amounts of protein were loaded for SDS-PAGE. The protein was then transferred to a nitrocellulose membrane, blocked with PBST (PBS with 0.1 % Tween-20) solution containing 5 % non-fat milk, and incubated overnight at 4 °C with a primary antibody against bcl-2, bax and caspase 3 (polyclonal, 1:1000, Abcam, UK),respectively. The antibody was detected by a horseradish peroxidase-conjugated secondary antibody (1:5000) after 1-hour incubation and developed with an ECL detection kit. Equal loading was confirmed by β-actin detection. Band optical density (OD) was analyzed using a Labworks 4.6 UVP-image capture and analysis software package. The analysis results were expressed in the format of mean ± standard deviation (SD) as the ratio percentage of the protein of interest OD versus the β-actin OD.
Statistical analysis
Data were analyzed using the Statistical Package for Social Sciences (SPSS) software for Windows, version 22.0 (SPSS Inc., Chicago, USA). One-way analysis of variance (ANOVA) for multiple-group analysis, unpaired student’s t-test was used for between two-group comparison, and log-rank test was used for the data of lifetime comparison. The data were expressed as mean value ± standard deviation \( \left(\overline{x}\pm s\right) \), a p value of less than 0.05 was considered statistically significant.
Discussion
Brucea javanica oil (BJO) is a complex mixture of fatty acids and its derivatives. It’s main component are oleic acid and linoleic acid [
9], and has been used to treat various tumors for many years in China [
26‐
29]. However, its non-specific distribution and low therapeutic index are two main reasons for poor prognosis in tumor therapy. Liposomes is regarded as a promising drug carrier with good biocompatibility, biodegradability and low cytotoxicity for cancer therapeutics [
30], and it is also beneficial for the parenteral delivery of insoluble drugs because it’s better stability in plasma [
12,
13]. LHRHa peptide has been used to target the corresponding LHRHR over-expressed in the plasma membrane of ovarian cancer [
15,
16,
31]. and we also have successfully coated microbubbles (MBs) with LHRHa to enhance the binding affinity with ovarian cancer cells [
20‐
22]. Therefore, we hypothesize that new LHRHa-targeted and BJO-loaded liposomes (LHRHa-BJOLs) will facilitate drug deposition at the tumor site for the enhanced therapeutic outcome. The results obtained in this in vitro and in vivo study are positive and in agreement with our study design, rendering the proposed LHRHa-BJOLs is a promising and novel targeting therapy strategy for the treatment of ovarian cancer.
In the present study, LHRHa-BJOLs have been successfully developed, and the colloidal gold immunoassay test had verified that LHRHa peptide was indeed conjugated on the surface of the liposomes. Particle size, zeta potential and encapsulation efficiency are the properties that influence the biopharmaceutical characteristics and stability of a liposomes. The smaller liposomes can more easily evade the reticuloendothelial system (RES) and prolong the time in circulation [
7,
32,
33]. It has been reported that particle size of the drug carrier should be bigger than 10 nm to escape the first-pass elimination by the kidney [
34‐
36], while with particle size less than 200 nm could increase drug accumulation in the tumor via “enhanced permeability and retention (EPR)” effect [
25,
37‐
40]. Meanwhile, the particle size also makes an effect on their interactions with the target cells [
40]. Therefore, the LHRHa-BJOLs synthesized in this study had an optimal size (155.1 ± 14.5 nm) for the tumor targeting by the EPR effect. The zeta-potential is a widely accepted parameter to represent the surface charge of the liposomes, and could influence on both its colloidal stability in suspension and its interaction with cells [
41]. For LHRHa-BJOLs synthesized in this study, the zeta potentials was - (24.1 ± 0.54) mV, demonstrating that the negatively charged DSPE-PEG
2000 was successfully inserted into the outer monolayer of the vesicles [
40]. Furthermore, the zeta potential was within the range from −20 mV to −30 mV, considering to be stable in aqueous environment without flocculation [
42]. Additionally, the encapsulation efficiency was (93.6 ± 1.04) % before conjugation, and declined to (92.2 ± 1.59) % after conjugation, similar to that reported by Cui et al. [
10]. The slight drug loss might be due to the release of BJO from the liposomes in the conjugation process, during which repeated and prolonged stirring was applied [
41].
To demonstrate the specific cell binding and internalization of LHRHa-Lip, LHRHR positive A2780/DDP cells were chosen as target cells, while LHRHR negative SKOV3 cells were applied as negative control, and the non-targeted liposomes was also prepared and used as the control. LSCM analysis revealed that the fluorescence intensity in A2780/DDP cells plus LHRHa-C6Ls group was stronger than that in A2780/DDP cells plus C6Ls and SKOV3 cells plus LHRHa-C6Ls groups, this is associated with the LHRHR targeting ability of LHRHa, and indicated that LHRHa conjugation does promote the entry of the liposomes into the LHRHR overexpressed cells by receptor-mediated endocytosis [
43]. FCM data further demonstrated that LHRHa-C6Ls resulted in significantly higher cellular uptake by A2780/DDP cells. All these results further suggested that LHRHa-Lip could enhance the specific cell binding and cellular uptake in A2780/DDP cells due to the mediating of LHRHa, and depending on the LHRHR expression level on cell surface as well.
BJOE, BJOLs and LHRHa-BJOLs were found to inhibit A2780/DDP cells proliferation and induce cell apoptosis in some extent, and the LHRHa-BJOLs exhibited the strongest inhibitory and apoptosis effect, this was consistent with the results of cellular uptake discussed above. Furthermore, the in vivo study showed that the median survival time of the animal in LHRHa-BJOLs group was significantly longer than that in BJOLs and BJOE groups at comparable dose of BJO. This may be a consequence of the targeted deposit of LHRHa-BJOLs in the tumor tissue. The mechanisms of this superior ovarian cancer targeting ability of LHRHa-Lip has not been fully understood yet. We believe that this mechanisms might be associated with at least the following contributing factors. First of all, LHRHa acted as an anchor to actively hold the liposomes specifically to the tumor site and promote the receptor-mediated endocytosis. Second, the DSPE-PEG2000 anchored to the surface of LHRHa-Lip are able to avoid rapid uptake by the RES, thus prolonging the circulation time and resulting in a higher accumulation of the liposomes in the tumor vasculature [
44]. Third, the optimal particle size of the LHRHa-Lip plays an important role in the increased accumulation by the EPR effect as discussed above. Forth, LHRHa-Lip improve the pharmacokinetic profile of BJO due to the PEGylated materials [
45,
46], which ultimately results in a higher accumulation in tumors. The results might further indicated that both active and passive tumor targeting mechanisms were involved in LHRHa-Lip accumulation within tumor, in which active targeting was achieved by LHRHa conjugation to liposomes and passive targeting was attributed to the EPR effect of liposomes [
47].
To further investigate the mechanisms underlying the enhanced tumor-inhibition activity of LHRHa-BJOLs, tumor apoptosis was also evaluated by western blot analysis. Activation of bax and caspase 3, inhibition of bcl-2 expression, and decline of MMP are crucial findings, the LHRHa-BJOLs group exhibited significantly more changes than the other groups both in vitro and in vivo. We speculated that the following possible mechanisms might be involved in the enhanced antitumor efficacy of LHRHa-BJOLs. Firstly, LHRHa modification and smaller particle size enhanced the accumulation of BJO in tumor cells which was discussed above, and this is the prerequisite for the BJO to function. Secondly, LHRHa-BJOLs upregulated bax, an important pro-apoptotic protein in mitochondrial pathway [
48], and downregulated bcl-2, one member of the anti-apoptotic bcl-2 family proteins and critical determinants of mitochondrial-dependent caspase activation [
49]. As a result, the mitochondrial-initiated apoptosis was promoted, and this was also confirmed by the reduced MMP of A2780/DDP cells, which is the result of a mitochondrial leak by opening the permeability transition pores [
44]. Thirdly, LHRHa-BJOLs upregulated caspase 3, an important promoters of the death receptor-mediated apoptosis [
50,
51], and resulted in activation of the death receptor pathway. Taken together, it could be proposed that LHRHa-BJOLs induced apoptosis in A2780/DDP cells in vitro or in ovarian tumor tissue in vivo via simultaneous activation of the death receptor pathway and the mitochondrial pathway.
Therefore, the LHRHa-BJOLs exhibited a promising ovarian cancer cells targeting capability and enhanced therapeutic efficiency both in vitro and in vivo experiments. However, this study also has some limitations. Firstly, BJO is a complex mixture of fatty acids and its derivatives, and its main activity components are oleic acid and linoleic acid with content of 63.3 and 21.2 % [
10], whether the anti-tumor effect of BJO in ovarian cancer was result from the existence of oleic acid or linoleic acid still needed to be further verified. Secondly, we fabricated the targeted liposomes via avidin–biotin interactions, although this method has been demonstrated successfully in animal models, its clinical feasibility is challenged by the unwanted immunogenicity. To overcome this limitation, one possible technical approach is to design avidin in the less immunogenic forms, and the other approach is to use other covalent and non-covalent binding strategies to synthesize tumor targeting liposomes. Further research and validation efforts are required before these technical advances can be implemented in a clinical setting. Finally, no complete recovery was observed for any treated mice in this study, because we chose to initiate the treatment on the 15
th day after tumor cell inoculation when the tumors were readily palpable and stop the treatment on the 27
th day after inoculation. In order to further improve the outcome, we will consider the optimal therapeutic strategies in the future, such as earlier initiation of the treatment, increase of dose and prolongation of treatment.
Acknowledgements
The authors are grateful to Dr. Zehua Wang (Department of Obstetrics and Gynecology, Tongji Medical College, Wuhan Union hospital Huazhong University of Science and Technology, Wuhan, China) for the kind supply of A2780/DDP cells, and Dr. Zhibiao Wang (Director of National Engineering Research Center of Ultrasound Medicine, Chongqing Medical University, Chongqing, China) for the generous support of the experimental facilities.