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Back to Journal »International Journal of Nanomedicine» Volume 11

Author Archibald M, Pritchard T, Nehoff H, Rosengren RJ, Greish K, Taurin S

Published on January 8, 2016, 2016 volume: 11 pages, pages 179-201

DOI https://doi.org/10.2147/IJN.S97286

Single anonymous peer review

Editor approved for publication: Dr. Thomas J Webster

Monica Archibald, 1 Tara Pritchard, 1 Hayley Nehoff, 1 Rhonda J Rosengren, 1 Khaled Greish, 1,2 Sebastien Taurin 1 1 Department of Pharmacology and Toxicology, University of Otago, Dunedin, New Zealand; 2 Aljawhara Center for Molecular Medicine, Arabian Gulf University, Manama, Kingdom of Bahrain Abstract: Due to the lack of effective treatments, castration-resistant prostate cancer (CRPC) is still incurable. Several tyrosine kinases are involved in the development and growth of CRPC, so targeting these kinases may provide alternative therapeutic strategies. We identified a combination of two tyrosine kinase inhibitors (TKIs), sorafenib and nilotinib, as the most cytotoxic drugs. In addition, in order to improve their bioavailability and reduce their metabolism, we encapsulated sorafenib and nilotinib into styrene-co-maleic acid micelles. Characterized the charge, size and release rate of micelles. We evaluated the effects of the combination on cytotoxicity, cell cycle, apoptosis, protein expression, tumor sphere integrity, migration and invasion. The micelles have an average diameter of 100 nm, are neutrally charged, and exhibit a high degree of stability. Compared with free TKI, micellar TKI promotes greater cytotoxicity, reduces cell proliferation, and increases cell apoptosis. In addition, the combination reduced the expression and activity of several tyrosine kinases more effectively than single treatment, and reduced the tumor spheroid integrity and metastatic potential of CRPC cell lines more effectively. This combination increases the therapeutic potential and proves the relevance of targeted combination therapy and CRPC therapy. In addition, the efficacy of encapsulated drugs provides a basis for in vivo preclinical testing. Keywords: sorafenib, nilotinib, castration-resistant prostate cancer, tyrosine kinase inhibitor, nanomedicine

Prostate cancer is the most common non-skin cancer diagnosed in men in developed countries, and it remains the leading cause of cancer-related deaths in men worldwide. 1 The standard treatment for localized prostate cancer includes surgery and radiotherapy. Although this treatment can be cured, in 20%–30% of patients, the tumor will eventually spread beyond the boundaries of the prostate. 2 If progress occurs, androgen deprivation therapy (ADT) after radiotherapy has been shown to improve the overall survival of patients. 3, 4 Early studies by Huggins and Hodges5 showed that chemical castration can cause tumor regression. Currently, ADT includes luteinizing hormone releasing hormone agonists and androgen receptor (AR) antagonists. 6 Approximately 80% of patients have a clinical response to ADT, in which tumor regression is initially associated with a decrease in serum prostate-specific antigen (PSA), a clinical biomarker. 7 Despite ADT, as the tumor progresses to a more aggressive phenotype and eventually spreads to distant sites, the serum level of PSA will increase. 8 Castration resistance is a hallmark of prostate cancer, with a 5-year relative survival rate of approximately 25%–33%. 9 In recent years, several treatments have been approved for the treatment of castration-resistant prostate cancer (CRPC), which can extend overall survival by up to 35%. 10-13 Although the current treatment strategy is that for some patients, no medicine can be cured, so it is necessary to develop alternative or complementary therapies.

Several contributors have been proposed to explain the recurrence of prostate cancer, its evolution to metastatic CRPC (mCRPC), and acquired resistance to anticancer treatments. The AR expression status was initially overlooked because prostate cancer appears to be growing despite low circulating androgen levels. However, recent research has determined that AR is the main enabler of CRPC. The amplification of AR gene copy number, the existence of splicing mutations, the recognition of gain-of-function point mutations, the altered expression of AR co-regulatory factors, and even the autocrine androgens produced by prostate tumor cells have been identified as causing the development of mCRPC. 14,15 In addition, the activation of AR may also be due to dysregulation of signal pathways involving receptor tyrosine kinases (RTKs) (such as epidermal growth factor receptor (EGFR) or c-Met) or non-RTKs (such as SRC) 16,17 EGFR overexpression is observed in more than 40% of CRPC patients, but the use of specific EGFR inhibitors in the clinic has failed to provide survival benefits. 18 Other mechanisms that are independent of AR status and involve tyrosine kinases also contribute to the development of mCRPC, including alternative and illegal pathways that rely on the local overexpression of growth factors or related receptors, onco activation genes, and tumor suppressor genes inhibition. 19

In a preliminary study, we selected eight tyrosine kinase inhibitors (TKIs) to target proteins related to the development and progression of mCRP (as listed in Table 1), and evaluated their effects individually or in combination. Cytotoxicity. The combination of sorafenib and nilotinib proved to be most effective against CRPC cells in vitro. Sorafenib targets a variety of tyrosine and serine/threonine kinases, including the vascular endothelial growth factor receptor (VEGFR) family, platelet-derived growth factor receptor (PDGFR)-β, stem cell factor receptor (KIT) , RET tyrosine kinase, Flt-3 kinase, B-RAF and C-RAF. 20 Sorafenib has been approved by the US Food and Drug Administration (FDA) for the treatment of hepatocellular carcinoma, renal cell carcinoma and thyroid cancer. 21-23 The anti-cancer effect of sorafenib on hepatocellular carcinoma has been evaluated for the treatment of CRPC in phase II clinical trials. 24 Nilotinib is a TKI that targets Bcr-Abl and other kinases, including PDGFR-α, KIT, discoid domain receptor and colony stimulating factor receptor-1.25 Nilotinib is currently approved by the FDA For the treatment of imatinib-resistant chronic myeloid leukemia. 26 The anti-cancer effect of nilotinib in CRPC has only been confirmed in one study, with mixed results. 27

Table 1 Target expression of TKIs and their proteins in CRPC Abbreviations: VEGFR, vascular endothelial growth factor receptor; PDGFR, platelet-derived growth factor receptor; FLT3, Fms-like tyrosine kinase 3 receptor; ALK, anaplastic lymphoid Oncokinase; ND, undefined; DDR, discoid domain receptor; CSF1R, colony stimulating factor 1 receptor; FGFR, fibroblast growth factor receptor; EGFR, epidermal growth factor receptor; JAK, Janus kinase; c -KIT, stem cell factor receptor; RET, neurotrophic factor receptor derived from glial cell line.

Sorafenib and Nilotinib have poor solubility in aqueous environments. 28,29 In addition, the use of sorafenib in elderly patients is discouraged due to signs of cardiotoxicity. 30 Encapsulation of sorafenib and nilotinib into nanoparticles can improve the solubility and stability of the drug, minimize side effects, and make the drug in the tumor site through enhanced permeability and retention (EPR) effect build up. 31 In this study, we prepared poly(styrene-co-maleic acid [SMA]) micelles loaded with sorafenib and nilotinib. We characterized micelles, including their load, size, charge, and release rate, and evaluated their anticancer effects in vitro using two CRPC cell lines.

With an average Mn ~1,700, N-(3-dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride (EDAC), penicillin, streptomycin and sulforhodamine B (SRB) Propylene-terminated poly(styrene-co-maleic anhydride) was obtained from Sigma-Aldrich Ltd. (St. Louis, Missouri, USA). Sorafenib (Sor), Nilotinib (Nilo), Crizotinib (Crizo), PD173074 (PD), Pazopanib (Pazo), Sunitinib (Suni), Lapatinib ( Lap), Tofacitinib (Tofa), and Semedinib (Selu) were purchased from LC Laboratories (Woburn, Massachusetts, USA).

The CRPC cell lines PC3 and LNCaP were obtained from the American Type Culture Collection (Manassas, Virginia, USA). Maintain the cells in a complete growth medium composed of Dulbecco's Modified Eagle medium/Ham's F12, supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomyces Vegetarian and 2.2 g/L sodium bicarbonate. For all procedures, cells were harvested using TrypLE Express (Thermo Fisher Scientific, Waltham, MA, USA) and kept in a humidified atmosphere at 37°C and 5% CO2. The study was conducted in accordance with the principles of the Declaration of Helsinki.

Preparation of SMA-Sor and SMA-Nilo micelles

The preparation methods of SMA-Sor and SMA-Nilo micelles are as described above. 32 In short, adjust the pH of the hydrolyzed SMA solution to 5; dissolve sorafenib or nilotinib in the smallest volume of dimethyl sulfoxide (DMSO) and add to the SMA solution. Then water-soluble EDAC was added to the mixture and stirred at pH 5 for 20 minutes. The solution was then adjusted to pH 11 with 0.1N NaOH and stirred for 30 minutes to form micelles. Readjust the pH of the clear solution to pH 7.4 with 0.1 N HCl. The clarified micellar suspension was ultrafiltered four times using a laboratory-scale ultrafiltration system equipped with a Pellicon XL filter 10 kDa (Merck Millipore, Billerica, MA, USA). The concentrated micellar solution is lyophilized to obtain the final SMA-Sor or SMA-Nilo powder.

Load SMA-Sor and SMA-Nilo

The standard curves of sorafenib and nilotinib were prepared in DMSO and measured at 270 nm. The drug content of SMA-Sor and SMA-Nilo micelles was determined by dissolving the micelles (1 mg/mL) in DMSO and measuring the absorbance at 270 nm to compare with their respective standard curves. The loading is expressed as the weight percentage of sorafenib or nilotinib in the final micelles compared with the total weight of the recovered SMA-Sor or SMA-Nilo, respectively. We determined that the loads of SMA-Sor and SMA-Nilo are 16.8% and 16.4% w/w, respectively.

Size and zeta potential of SMA-Sor and SMA-Nilo micelles

SMA-Sor and SMA-Nilo micelles (8 mg/mL) were dissolved in sodium bicarbonate (0.1 M, pH 7.4) or water to determine the average intensity-weighted diameter and zeta potential, respectively. All measurements of size distribution and zeta potential were performed using the Malvern ZEN3600 Zetasizer Nano series (Malvern Instruments Inc., Westborough, MA, USA). The measured values ​​were taken from three independent experiments and performed in triplicate.

Release profile of sorafenib and nilotinib

The release of sorafenib and nilotinib from the micellar construct was assessed using a dialysis method. SMA-Sor and SMA-Nilo micelles were dissolved in phosphate buffered saline (PBS) at a concentration of 1 mg/mL and placed in a dialysis bag (2 mL) with a molecular weight cut-off of 12 kDa. The micellar resuspension was dialyzed with 20 mL of PBS, and the PBS was adjusted to pH 5.5, 6.8, or 7.4. Within 96 hours, remove an aliquot of the solution outside the dialysis bag and measure the absorbance at 270 nm. The quantification of the release percentage is determined by the ratio of the absorbance of the solution outside the bag at a specified time divided by the absorbance in the bag at t=0. All experiments were performed in triplicate. The percentage release is reported as the mean ± standard error.

Cytotoxicity of Sorafenib, Nilotinib, SMA-Sor and SMA-Nilo

Inoculate PC3 (4×103 cells/well) and LNCaP (8×103 cells/well) cells in a 96-well plate, and incubate at 37°C and 5% CO2 for 24 hours, then use from 0 to 15 μM free sorafenib, free nilotinib, SMA-Sor or SMA-Nilo. The cells were incubated for 72 hours and fixed with trichloroacetic acid. The number of cells was determined using the SRB assay. 33 Use Graphpad Prism™ software (GraphPad Software, Inc., San Diego, California, USA). The measured values ​​are taken from three independent experiments performed in triplicate. The preliminary study used various TKIs at a concentration of 2.5 μM and evaluated the cytotoxicity as a single or combination therapy in PC3 cells, as described previously. For time course studies, cells were treated with 2.5 μM without sorafenib or micelles, 3 μM without nilotinib or micelles, and DMSO or SMA for 12, 24, 36, 48, and 72 hours. At the specified time, the cells are processed as described above. All experiments were performed in triplicate and repeated three times independently.

Inoculate PC3 (8×104 cells/well) and LNCaP (3×105 cells/well) cells in a six-well plate in 1.5 mL of complete growth medium. Cells were treated with 2.5 μM free or micellar sorafenib, 3 μM free or micellar nilotinib, DMSO or SMA for 48 hours. As mentioned earlier, the cell cycle distribution was assessed using propidium iodide staining. 34 Use the FACScalibur flow cytometer (BD Biosciences, San Jose, CA, USA) to analyze the samples, and analyze the cell ratios in G0/G1-, S- and G2 to determine the /M phase using CellQuest Pro software (BD Biosciences). All experiments were performed in triplicate and repeated three times independently.

Inoculate PC3 (8×104 cells/well) and LNCaP (3×105 cells/well) cells in a six-well plate in 1.5 mL of complete growth medium. Cells were treated with 2.5 μM free or micellar sorafenib, 3 μM free or micellar nilotinib, DMSO or SMA for 48 hours. As mentioned earlier, annexin-V-FLUOS/propidium iodide staining was used to assess apoptosis. 34 Use the FACScalibur flow cytometer to analyze the samples, and use the CellQuest Pro software (BD Biosciences) to determine the proportion of apoptotic cells. All experiments were performed in triplicate and repeated three times independently.

PC3 (1×106) and LNCaP (1.5×106) cells were seeded in a 10 cm Petri dish in 10 ml of complete growth medium and incubated for 36 hours. Cells were treated with 2.5 μM free or micellar sorafenib, 3 μM free or micellar nilotinib, DMSO or SMA for 48 hours. Cells containing Tris-HCl 50 mM (pH 8), sodium chloride 150 mM (NaCl), Triton X-100 1%, sodium dodecyl sulfate (SDS) 1%, sodium fluoride 1 mM, orthovanadic acid Sodium 200 μM, and protease inhibitors (leupeptin 1 μg/mL, aprotinin 1 μg/mL, phenylmethylsulfonyl fluoride 1 mM). The insoluble matter of the lysate was removed by centrifugation at 20,000×g for 10 minutes, boiled in Laemmli buffer, and subjected to polyacrylamide gel electrophoresis, and analyzed by Western blot using a polyvinylidene fluoride membrane. The antibodies used are as follows: anti-VEGFR-2 (D5B1, Cell Signaling Technology; Beverly, MA, USA); anti-phosphorylated focal adhesion kinase (FAK) (D20B1, cell signaling); anti-FAK (D2R2E, cell signaling); anti-PDGFR (D1E1E, Cell Signaling), anti-phospho-EGFR (tyr1148) (Cell Signaling), anti-EGFR (D38B1, Cell Signaling), anti-phospho-AKT (193H2, Cell Signaling), anti-AKT (Cell Signaling), anti-phospho -SRC (D49G4, Cell Signaling), anti-SRC (32G6, Cell Signaling) and β-tubulin (2-28-3, Sigma-Aldrich Ltd). The secondary horseradish peroxidase (HRP) conjugated antibody is from Merck Millipore, which can be detected with SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific).

Immunofluorescence was performed as previously described. 34,35 LNCaP cells (2.5×104 cells/well) were seeded on a glass slide and incubated for 36 hours, and then treated with 2.5 μM free or micellar sorafenib, 3 μM free or micellar nilotinib, DMSO, or SMA 48 hours. The cells were washed twice with ice-cold PBS, fixed with 4% paraformaldehyde in PBS at room temperature for 15 minutes, washed again with PBS, permeabilized in 0.2% Tween-20 PBS for 15 minutes, and then incubated with 1% cattle Serum albumin (BSA) in PBS for 1 hour. The cells were then incubated with anti-AR antibody (D6F11 XP, Cell Signaling Technology; 5 μg/mL in PBS/BSA, as previously described) at 4°C overnight, washed four times with PBS, and then incubated with Dylight 594 goat. Rabbit IgG antibody (Vector Laboratories, Burlingame, CA, USA; 10 μg/mL PBS/BSA, as described above) was kept at room temperature for 1 hour. The slides were washed again with PBS four times, and the coverslips were mounted with Gel/Mount aqueous mounting medium (Fisher, Pittsburgh, PA, USA). The image was taken with a Nikon Eclipse Ni-E upright epi-fluorescence microscope (Nikon Corporation, Tokyo, Japan).

Tumor spheroids and cell viability measured by acid phosphatase

The tumor spheroids were prepared as described by Friedrich et al. 36 In short, transfer PC3 (4×103 cells) and LNCaP (8×103 cells/well) cells to a 96-well plate pre-coated with agarose (1.5% w/v). Cell incubation 4 Day, then treated with 2.5 μM free sorafenib or SMA-Sor at an equivalent sorafenib concentration, 3 μM free nilotinib or SMA-Nilo, DMSO or SMA at an equivalent nilotinib concentration 15 sky. The medium and treatment are updated every 4 days. At the end of the treatment period, photographs were taken and cell viability was assessed by the acid phosphatase assay as previously described. 36 In short, the tumor spheroids are collected, washed in PBS, and in acid phosphatase buffer (0.1 M sodium acetate, 0.1% Triton X-100 and p-nitrophenyl phosphate [2 mg/mL]) in 37 Leave it at °C for 90 minutes. Stop the reaction with NaOH (1 N) and quantify at 405 nm on a microplate reader. The results are expressed as a percentage of the control. Three independent experiments were performed in six replicates.

The in vitro cell scratch test was used to measure the migration of PC3 cells. After the cells grown in the six-well plate reach 90% confluence, they are scratched with a 10 μL pipette tip and then extensively washed with serum-free medium to remove cell debris. Then add free or micellar sorafenib (2.5 μM) or free or micellar nilotinib (3 μM) or control (SMA or DMSO). Before taking pictures, allow the cells to migrate to the scratched area for up to 20 hours at 37°C and 5% CO2. The experiment was performed in triplicate and repeated three times independently.

PC3 cells (4×104) were seeded into Boyden cells (8 μm wells; In Vitro) coated with Geltrex (Life Technologies) and treated with free or micellar sorafenib (2.5 μM) or free or micellar nilotinib Technologies, Auckland, New Zealand) (3 μM) or control (SMA or DMSO). Fetal bovine serum (5%) is used as a chemoattractant in the lower chamber containing complete growth medium. After 20 hours, the filter was fixed in methanol and stained with Diff-Quick staining solution. Count the cells in each well under an inverted microscope at 200 times magnification. Invasion was expressed as the percentage of cells that passed through the basement membrane layer to the number of cells counted in the control wells without basement membrane. Data were collected from three independent experiments and performed in triplicate. The student's t-test was used to count and analyze the migrating cells.

MMP-9 activity measurement and MMP-9 and isthmin-1 secretion

PC3 cells (1×106) were seeded in a 10 cm petri dish in 10 ml of complete growth medium and incubated for 36 hours. The cells were washed with PBS, then incubated in serum-free medium and treated with 2.5 μM free or micellar sorafenib, 3 μM free or micellar nilotinib, DMSO or SMA for 48 hours. After treatment, the medium was collected, centrifuged to remove cell debris, and freeze-dried for 12 hours. Rehydrate the sample and mix with the loading buffer (0.4 M Tris, pH 6.8, 5% SDS, 20% glycerol, 0.03% bromophenol blue). For the zymogram, load the sample on a 10% SDS-polyacrylamide gel containing 1 mg/mL gelatin. After electrophoresis, incubate the gel in a refolding solution (2.5% Triton-X-100 (w/v)) for 30 minutes at room temperature, and then incubate the gel in a developing buffer containing 50 mM Tris, pH 7.5 at 37°C for 24 Hours, 200 mM NaCl, 4 mM calcium chloride (CaCl2) and 0.02% NP40. The gel was then stained with Coomassie Blue R250, and the unstained area indicated gelatin lysis. The gel is simply rinsed and scanned. For the secretion of MMP-9 and isthmin-1 (ISM1), load the sample on a 10% SDS-polyacrylamide gel and use anti-MMP-9 (D6O3H, Cell Signaling) and anti-isthmin-1 antibodies (Biorbyt, San Francisco, California, USA).

For Figures 1, 4, 5, 8, and 9, and Supplementary Figures 1 and 2, use Student's t-test or one-way analysis of variance (ANOVA) to compare the groups. Figures 2 and 3 use two-way analysis of variance. In all cases, the analysis of variance was combined with the Student-Newman-Keuls post-hoc test. For all analyses, P<0.05 is the minimum requirement for statistically significant differences.

Figure 1 The effect of single and combined TKI on PC3 cell viability. Note: The cells are processed for 72 hours and fixed at the specified time point, and the number of cells is determined by the sulforhodamine B assay. Data are expressed as mean ± SEM (n=3). * P<0.05 compared with control, ** P<0.05 compared with all other treatments. Abbreviations: SEM, standard error of the mean; TKI, tyrosine kinase inhibitor.

Figure 2 Release rate of Sorafenib and Nilotinib from SMA-Sor (A) and SMA-Nilo (B) at pH 5.5, 6.8 and 7.4. Note: The release of sorafenib and nilotinib was evaluated using dialysis and compared with sorafenib and nilotinib in a dialysis bag at t=0 hours. The released substances are evaluated over a period of 4 days. Data are expressed as mean ± SEM (n=3) (for pH 5.5 vs. pH 6.8, *P<0.05 and **pH 5.5 vs. pH 7.4). Abbreviations: SMA, poly(styrene-co-maleic acid) acid; SEM, standard error of the mean.

Figure 3 Time-dependent cytotoxicity of sorafenib, nilotinib, SMA-Sor, SMA-Nilo and their combinations on the proliferation of PC3 (A) and LNCaP (B) cells. Note: Cells were treated with free or micellar sorafenib 2.5 μM and/or nilotinib 3 μM for 72 hours. At the designated time points, the cells were fixed and sulforhodamine B was used to determine the number of cells. Data are expressed as mean ± SEM (n=3). * P<0.05 compared with the control, starting from 36 hours, ** P<0.05 compared with a single treatment, starting from 36 hours, *** P<0.05 compared with other treatments, starting from 36 hours. Abbreviations: SEM, standard error of the mean; SMA, poly(styrene-co-maleic acid) acid.

The combination of sorafenib and nilotinib reduces the cell viability of PC3 cells

In order to determine the best combination of TKIs that are cytotoxic to CRPC cells, PC3 cells were treated with 2.5 μM TKI as a single treatment or a combined treatment for 72 hours. The combination of sorafenib and nilotinib reduced cell viability by 80% after 72 hours, which is statistically significant compared to all other treatments (Figure 3). Therefore, this combination was further studied.

Synthesis and characterization of SMA-Sor and SMA-Nilo

SMA is an amphiphilic block copolymer that forms a self-assembled micelle structure in an aqueous solution. We have generated SMA-Sor and SMA-Nilo micelles with a theoretical loading of 20%, encapsulation efficiency between 85% and 88%, and loading efficiencies of 16.8% and 16%, respectively, depending on the weight ratio of the micelles . The drug exceeds the total weight of SMA micelles (Table 2). Encapsulation of sorafenib and nilotinib into SMA micelles increased their solubility in aqueous solutions by 1,357 times (0.00171–2.32 mg/mL) and 1,313 times (0.00201–2.65 mg/mL), respectively. For SMA-Sor and SMA-Nilo, the average micelle diameters measured by dynamic light scattering were 109±28.9 and 113±35.2 nm, respectively (Table 2). The charge of SMA-Sor and SMA-Nilo is close to neutral (Table 2). When administered systemically, the physicochemical characteristics of the size and charge of the two micelles can predict improved biodistribution and circulating half-life. The size of micelles larger than 7 nm will escape kidney filtration, 37 and the size and neutral charge of the micelles will reduce their possible interaction with the reticuloendothelial system (RES). 37

Table 2 The characteristics of sorafenib and nilotinib micelles

Drug release rate from micelles

As shown in Figure 1, the release profiles of SMA-Sor and SMA-Nilo micelles were stable at physiological pH 7.4, and the release of sorafenib and nilotinib were lower than 3.4% and 3.3% respectively in the first 12 hours ( figure 1). The release of the two drugs lasted more than 96 hours, and 9.7% and 10.7% of sorafenib and nilotinib were released from the micelles (Figure 1). This release rate indicates that the micelles are stable at physiological pH. At a pH of 6.8, which is equivalent to the pH around the tumor tissue, the release of sorafenib and nilotinib after 96 hours was 10.1% and 11.7%, respectively (Figure 1). The low release in the tumor microenvironment may promote their internalization by tumor cells through endocytosis, thereby synergistically acting on targeted tumor cells. The release at pH 5.5 (equivalent to the pH of the inner body cavity) is relatively faster than at pH 6.8 and 7.4. At pH 5.5, 12% SMA-encapsulated sorafenib and 18.8% SMA-encapsulated nilotinib were released after 96 hours (Figure 1). The acidic environment promotes the conformational transition of SMA, 38 allowing the encapsulated drug to be released slowly.

Cytotoxicity of Sorafenib, SMA-Sor, Nilotinib and SMA-Nilo

The cytotoxicity of Sorafenib, SMA-Sor, Nilotinib and SMA-Nilo micelles to LNCaP and PC3 cells was evaluated in vitro using the SRB assay within 72 hours. As listed in Table 3, the concentration of SMA-Sor that reduces the number of cells by 50% (IC50) is almost the same as the free sorafenib in LNCaP cells. In PC3 cells, the encapsulation of sorafenib slightly increased its cytotoxicity, and reduced the IC50 of sorafenib and SMA-Sor from 4.08 μM to 3.29 μM, respectively, a reduction of nearly 20%. Encapsulation of Nilotinib increased its cytotoxicity in LNCaP cells by nearly 46%, with IC50 values ​​of 2.98–1.59 μM, and an increase of 22% in PC3 cells. The IC50 values ​​of Nilotinib and SMA-Nilo Respectively 3.97 and 3.08 μM (Table 3)). The same cell line was used to further evaluate the effects of sorafenib and nilotinib and their micellar formulations in a 72-hour period. Cells were treated with 2.5 μM free or micellar sorafenib, 3 μM free or micellar nilotinib; DMSO (0.034%) or SMA (1.55 mg/mL) were used as controls. In PC3 cells, Sorafenib treatment reduced the cell number by 23%, while SMA-Sor reduced it by 27.2% (Figure 2A). A similar trend was observed after nilotinib and SMA-Nilo treatment, in which the number of cells was reduced by 13.1% and 27.2%, respectively (Figure 2A). The combination of sorafenib and nilotinib and their micellar preparations reduced the number of cells by 59.2% and 76.4%, respectively (Figure 2A). In LNCaP cells, Sorafenib and SMA-Sor reduced the cell numbers by 45% and 58.7%, respectively, while Nilotinib and SMA-Nilo treatment reduced the cell numbers by 41.3% and 45%, respectively (Figure 2B) . Free and micellar combination treatments reduced cell numbers by 76% and 82%, respectively (Figure 2B). These results indicate that the combination of sorafenib and nilotinib is more effective than single-drug therapy, and the encapsulation of the drug only further improves their cytotoxicity.

Table 3 IC50 values ​​of free sorafenib and nilotinib drugs and their micellar preparations in two CRPC cell lines PC3 and LNCaP cells. Abbreviations: CRPC, castration-resistant prostate cancer; IC50, half maximum inhibitory concentration ; SMA, poly(styrene-co-maleic acid) acid.

Sorafenib and Nilotinib treatment reduced the cell cycle progression of PC3 and LNCaP cells

PC3 and LNCaP cells were treated with free or micellar sorafenib (2.5 μM) and/or nilotinib (3 μM) for 48 hours, and the effect on cell cycle progression was measured by flow cytometry. In PC3 cells, Sorafenib and Nilotinib moderately increased the number of cells in the G0/G1 phase of the cell cycle by 3.7% and 4.1%, respectively (Figure 4A). A concomitant decrease in the S phase was observed, while the ratio of cells in the G2/M phase did not change (Figure 4A). Similar observations were made after treatment with SMA-Sor and SMA-Nilo, where the percentage of cells in the G0/G1 phase increased by 5.1% and 4.5%, respectively. However, the combination of sorafenib and nilotinib triggered the accumulation of cells in the G2/M phase, which increased from 25.6% to 33%, accompanied by a decrease in the proportion of cells in the S (-3.4%) and G0/G1 phase. −4%) (Figure 4A). In contrast, treatment with a combination of micelles does not promote G2/M accumulation in cells. The micelle combination increased the number of cells in the G0/G1 phase by 5%, while the number of cells in the S phase decreased (Figure 4A). In LNCaP cells, a similar pattern was observed, where the combination of sorafenib, nilotinib, SMA-Sor, SMA-Nilo and micelles increased the number of cells in the G0/G1 phase by 6.2%, 3.7%, 5.5 %, 5.7%, and 6.9%, respectively (Figure 4B). It was observed that the number of cells in S phase and G2/M phase decreased at the same time. The combination of sorafenib and nilotinib increased the number of LNCaP cells in the S (+3.7%) and G2/M (+4.2%) phases (Figure 4B).

Figure 4 The effects of sorafenib, nilotinib, SMA-Sor, SMA-Nilo and their combinations on cell cycle progression. Note: PC3 (A) and LNCaP cells (B) were treated with free or micellar sorafenib 2.5 μM and/or nilotinib 3 μM for 48 hours. SMA and DMSO were used as controls. Data are expressed as mean ± SEM (n=3). *Compared with the control, P<0.05. Abbreviations: SMA, poly(styrene-co-maleic acid) acid; DMSO, dimethyl sulfoxide; SEM, standard error of the mean.

The combination of free and micellar sorafenib and nilotinib enhances apoptosis

After 48 hours of incubation, annexin V and propidium iodide were used to determine the effects of sorafenib, nilotinib and their micellar preparations on cell apoptosis and analyzed by flow cytometry. As shown in Figure 5A, compared with the control, sorafenib and nilotinib increased apoptosis by 2.9 times and 2.3 times, respectively, and their combination increased the proportion of apoptotic cells in PC3 cells by 5.3 times (Figure 5A). SMA-Sor, SMA-Nilo, and their combinations follow the same pattern as observed with free drugs. The apoptosis of SMA-Sor, SMA-Nilo and micellar combinations increased 3.6, 3.5, and 6.9 times, respectively. In LNCaP cells, the drug has a more profound cytotoxic effect. Compared with the control, sorafenib, nilotinib and their combination increased apoptotic cells by 5.7, 3.5 and 8.6 times, respectively (Figure 5B). In addition, the micellar preparation of each drug slightly increased the number of apoptotic cells compared with the free drug. Compared with SMA, the combination of SMA-Sor, SMA-Nilo and micelles increased the number of apoptotic cells by 8.8, 6.1, and 12.2 times (Figure 5B). In general, the combination of the two micellar drugs increased the cytotoxicity to CRPC cell lines, which may be the result of inhibiting multiple signaling pathways.

Figure 5 The effect of sorafenib, nilotinib, SMA-Sor, SMA-Nilo and their combination on apoptosis PC3 (A) and LNCaP cells (B) with free or micellar sorafenib 2.5 μM and / Or Nilotinib 3 μM treatment for 48 hours. Note: SMA and DMSO are used as controls. Data are expressed as mean ± SEM (n=3). *P<0.05 compared with control, **P<0.05 compared with free drug and micellar therapy, and ***P<0.05 compared with free drug and combination therapy. Abbreviations: SMA, poly(styrene-co-maleic acid) acid; DMSO, dimethyl sulfoxide; SEM, standard error of the mean.

The combination reduces the expression of proteins necessary for cancer cell proliferation and survival

We evaluated the effects of free drug or micellar drug combination therapy on the expression of sorafenib and nilotinib protein targets, as well as signaling proteins that are essential for cell proliferation and apoptosis. After free or micellar combination treatment, the expression of VEGFR2 was not affected in PC3 cells (Figure 6A). In LNCaP cells, the expression of VEGFR2 was reduced by 46% and 81% after free and micellar combination treatments (Figure 6B and S1 [quantitative]). Both Sorafenib and Nilotinib are inhibitors of PDGFR; in PC3 cells, after combined free and micellar treatments, the combined treatment reduced expression by 65% ​​and 80%, respectively (Figure 6A and S1). In LNCaP cells, PDGFR expression was reduced by 96% and 82% after combined free and micellar treatments (Figure 6B). EGFR is a tyrosine kinase receptor, which is elevated in CRPC and helps tumor cell proliferation. Free and micellar combined treatments reduced the expression of EGFR in PC3 cells by 15% and 29% after combined treatment of free and micellar cells, and eliminated EGFR expression in LNCaP cells (Figure 6A, B and S1). FAK is a ubiquitous non-RTK, and it is also related to the progression of prostate cancer. 39 In PC3 cells, the combination of free and micelles reduced FAK expression by 76% and 80%, respectively (Figure 6A and S1). In LNCaP cells, the combination of free and micelles reduced FAK expression by 85% and 78%, respectively (Figure 6B and S1).

Figure 6 The effects of sorafenib, nilotinib, SMA-Sor, SMA-Nilo and their combination therapy on the expression of various tyrosine kinases and related proteins. Note: PC3 (A) and LNCaP cells (B) were treated with free or micellar sorafenib 2.5 μM and/or nilotinib 3 μM for 48 hours. SMA and DMSO were used as controls. The total lysate was analyzed by Western blot using antibodies against the indicated protein. The experiment was performed in triplicate. Abbreviations: VEGFR, vascular endothelial growth factor receptor; PDGFR, platelet-derived growth factor receptor; EGFR, epidermal growth factor receptor; FAK, focal adhesion kinase; AR, androgen receptor; SMA, poly(styrene-co- Maleic acid) acid; DMSO, dimethyl sulfoxide.

In addition, we evaluated the effect of the combination on downstream signaling proteins such as AKT and SRC. In PC3 cells, the combination therapy eliminated AKT phosphorylation, but overall AKT expression was only moderately affected (-10%). The micellar assembly also completely eliminated phosphorylation, while AKT expression was reduced by 40% (Figure 6A and S1). In LNCaP cells, the combination of free drug and micellar drug inhibited the phosphorylation of AKT. However, the total AKT expression was reduced by 88% after the combination treatment, and the micellar combination treatment eliminated it (Figure 6B and S1). After combined free and micellar treatments, the expression of total SRC was reduced by 57% and 69% in PC3 cells, but not in LNCaP cells (Figure 6A and B). After free or micellar combination treatment, the phosphorylation of SRC decreased in PC3 cells and disappeared in LNCaP cells (Figure 6A, B and S1). We also considered the effect of treatment on AR expression in LNCaP cells. LNCaP cells overexpress mutated AR and respond to low levels of androgens and other steroids. The combination of sorafenib and nilotinib free or micelles eliminated AR expression in LNCaP cells (Figure 6B and S1), but had no effect on AR expression in PC3 cells (Figure 6A and S1). Finally, we evaluated the effect of treatment on the activation of the classic apoptosis marker caspase-3. In PC3 cells, the increase in apoptosis is not related to the detectable level of active caspase-3. However, the combination of dissociation and micellar treatment of LNCaP cells increased the active caspase-3 tenfold (Figure 6B and S1).

Sorafenib, Nilotinib and their combination have different effects on the nuclear localization of AR

AR is a DNA-binding transcription factor that is essential for the expression of multiple genes in LNCaP cells. We evaluated the ability of sorafenib and nilotinib treatment to interfere with AR nuclear localization in LNCaP cells. In cells treated with carrier or SMA, AR is distributed throughout the cytoplasm and nucleus. After treatment with sorafenib, AR changes and is basically located in the cytoplasm (Figure 7). A similar effect was observed after SMA-Sor treatment, where SMA-Sor promoted the co-localization of AR and plasma membrane (Figure 7). However, free nilotinib failed to change the nuclear localization of AR, but reduced its overall expression (Figure 7). SMA-Nilo reduces the nuclear localization of AR, but increases its co-localization with the plasma membrane. The combined treatment of free and micelles eliminated the nuclear accumulation of AR and promoted its co-localization with the plasma membrane.

Figure 7 The effect of sorafenib, nilotinib, SMA-Sor, SMA-Nilo and their combination therapy on AR expression and localization in LNCaP cells. Note: The cells were treated and incubated for 48 hours as instructed, and then examined by immunofluorescence microscopy with anti-AR antibody. The scale bar represents 10 μm. Abbreviations: SMA, poly(styrene-co-maleic acid) acid; AR, androgen receptor.

The combination of sorafenib and nilotinib reduces tumor spheroid viability

We used PC3 and LNCaP tumor spheroid models to further evaluate the effects of free and micellar sorafenib, nilotinib and their combination therapy on cell viability. The three-dimensional culture model simulates certain aspects of tumor tissue in the body and better summarizes the response of cells to drugs. As shown in Figures 8A and C, after free sorafenib, nilotinib, and combination treatment, the cell viability of PC3 spheres measured by acid phosphatase activity was reduced by 34%, 18%, and 54%, respectively (Figure 8B ). After treatment with SMA-Sor, SMA-Nilo and the combination, the encapsulation of the drugs increased their cytotoxicity and reduced cell viability by 47%, 29%, and 66% (Figure 8B). In addition, the combination of micelles changed the interactions between cells, thereby changing the organization of the tumor spheroids (Figure 8A). LNCaP tumor spheroids (Figure 8C) follow a similar pattern. Sorafenib and combination therapy reduced acid phosphatase activity by 47% and 69%, respectively (Figure 8D). However, nilotinib treatment does not affect the integrity of the tumor spheroids. Compared with free drug treatment, micellar preparations were more effective, and acid phosphatase activity was reduced by 69.5%, 19%, and 79% after SMA-Sor, SMA-Nilo and combination treatments, respectively (Figure 8D).

Figure 8 The morphology and viability of PC3 and LNCaP tumor spheroids treated with free or micellar sorafenib 2.5 μM and/or nilotinib 3 μM for 15 days. Note: Representative pictures of PC3 (A) and LNCaP (C) tumor spheroids were taken after the indicated treatment. Tumor spheroid activity was measured by the acid phosphatase activity of PC3 (B) and LNCaP (D) tumor spheroids. *P<0.05 compared with control, **P<0.05 compared with free drug and micellar therapy, and ***P<0.05 compared with free drug and combination therapy. The scale bar represents 100 μm. Abbreviation: SMA, poly(styrene-co-maleic) acid.

Combination reduces the metastatic potential of PC3 cells

As shown in Figure 6, the combination therapy reduces the expression and/or activity of proteins necessary for cell migration and invasion, such as EGFR, FAK, SRC, and AKT. Therefore, we evaluated the effects of single treatments and their combinations on the metastatic potential of CRPC cells. First, we use the wound healing test to determine the effect on cell migration. PC3 cells are grown on a petri dish until they reach confluence, and then a pipette tip is used to stroke the cell monolayer. The wound healing was evaluated 20 hours after the use of sorafenib, nilotinib, or combination therapy. As shown in Figure 9A, nilotinib treatment moderately reduced wound closure, while sorafenib and combination therapy were more effective. The micellar TKI formulation eliminated the migration of PC3 cells alone and in combination (Figure 9A). In addition, we use Boyden chambers with or without geltrex coating to assess cell invasion. Sorafenib (2.5 μM), Nilotinib (3 μM), and combination treatment reduced invasion by 48%, 41%, and 68%, respectively (Figure 9B). Micellar sorafenib (2.5 μM), nilotinib (3 μM) and the combination slightly enhanced the anti-invasive effect, reducing it by 53%, 50%, and 72%, respectively (Figure 9B). These results indicate that the combination of sorafenib and nilotinib reduces migration and invasion, and this effect is further enhanced by encapsulating them in micelles. Matrix metalloproteinases (MMP) are essential for migration and invasion; we use zymography and western blotting to detect the activity and secretion of MMP-9 in cell culture media. Sorafenib reduced the activity and secretion of MMP-9 by 50%, while nilotinib had no effect (Figure 9C). However, this combination reduced MMP-9 activity by 69%. The combined treatment of SMA-Sor, SMA-Nilo and micelles increased the secretion and activity of MMP-9 by 62%, 33%, and 86%, respectively (Figure 9C and S2). The anti-angiogenic effect of sorafenib has been previously confirmed; here, we report that sorafenib and combination therapy trigger the secretion of ISM-1 (Figure 9C and S2), which is a kind of promoting endothelial cell apoptosis Secreted anti-angiogenic protein. 40 However, when the cells are exposed to the micellar formula.

Figure 9 Anti-metastatic potential of free and micellar sorafenib in combination with nilotinib. Note: The effects of sorafenib, nilotinib, SMA-Sor, SMA-Nilo and their combination therapy on inhibiting cell migration (A), invasion (B) and angiogenesis (C). Scratch the PC3 cell monolayer, treat it with free or micellar sorafenib 2.5 μM and/or nilotinib 3 μM, and incubate for 20 hours. Take representative photos at 0 hours and 20 hours (A). For cell invasion, PC3 cells were treated as previously shown. After 20 hours, the cells that migrated to the lower chamber were fixed and stained with Diff Quick. Take a representative picture and count the number of cells in each field of view. The bars represent the mean ± SEM of three independent experiments (B). As mentioned above, the conditioned medium was collected from the cell culture after 48 hours of culture, and the MMP-9 activity was analyzed by gelatin zymography and the expression of MMP-9 and ISM-1 was analyzed by Western blotting (C). The experiment was performed in triplicate (n=3). * P<0.05 compared with control, ** P<0.05 compared with free drug and combination therapy. The scale bar represents 100 μm. Abbreviations: SMA, poly(styrene-co-maleic acid) acid; SEM, standard error of the mean; MMP, matrix metalloproteinase; ISM-1, isthmin-1; t, time.

Despite the emergence of new therapies, CRPC remains an important health challenge. New therapies include chemotherapy drugs such as docetaxel and cabazitaxel, vaccines based on dendritic cells such as sipuleucel-T, bone-targeted radiopharmaceuticals such as radium 223, and compounds that interfere with androgen-stimulated tumor growth, such as AR antagonists (Enzalutamide)) or androgen synthesis inhibitor (abiraterone). It is recommended to continue androgen deprivation for mCRPC indefinitely. 41 AR amplification was observed in 30% of CRPC patients with recurrent tumors, but AR amplification was not observed in tissues taken from the same patient before ADT. 42 Therapies such as abiraterone acetate/prednisone, enzalutamide, radium 223 and docetaxel/prednisone improve patient survival and quality of life, while docetaxel/prednisone is moderately toxic . 41 For men with asymptomatic or mild symptoms, sipuleucel-T improves the survival rate of patients. 41 These new therapies have the ability to shorten the overall survival of patients from 6-10 months to 24 months for some therapies. 43,44 Despite these clinical improvements, it is clear that further advances in treatment are needed. Recent studies have shown that the progress of CRPC and the development of resistance to current treatments are related to the increase in tyrosine kinase signaling activity. 45 However, in contrast to other cancer types, somatic mutations or amplifications of tyrosine kinase genes are rarely found. CRPC patients. 45 The lack of mutations also indicates that tumors are not dependent on dominant signaling pathways and may promote the inter-tumor and intra-tumor heterogeneity of their response to specific inhibitors. In recent years, people have made progress in understanding the important role RTK plays in the development of prostate cancer, and therefore various TKIs have been evaluated in clinical trials (for a review, see Ojemuyiwa et al. 46). However, the results of using TKI as a single treatment have been disappointing. Therefore, a more promising strategy is to use them in combination with other TKIs or chemotherapy drugs. 46

We have shown that the combination of two TKIs, sorafenib and nilotinib, produces the greatest cytotoxicity to PC3 cells. However, both of these TKIs are highly hydrophobic, which greatly reduces their bioavailability in vivo. 47,48 The nanoparticles developed in this study have a similar size of about 100 nm and a near-neutral charge, and achieve about 16% of the SMA load-Sor and SMA-Nilo. The size and charge of micelles are important parameters for prolonging the cycle half-life and ideal biodistribution. Elimination of nanoparticles from the circulation is mainly mediated by the kidneys and RES. Nanoparticles with a hydrodynamic size greater than 5.5 nm escape renal clearance, 49 and nanoparticles of 100 nm or lower are unlikely to be recognized and eliminated by RES. 50 In addition, the near-neutral charge of micelles reduces their conditioning effect and recognizes RES, 51 helps to prolong blood circulation. Therefore, encapsulating these drugs in SMA micelles can increase their solubility, but also reduce metabolism, increase bioavailability, reduce toxicity to surrounding tissues, and promote accumulation in tumors.

For effective anti-cancer treatment, the stability of micelles is an important criterion. Micellar SMA-Sor and SMA-Nilo are very stable at physiological pH, which is measured by a low release rate of only 10% after 4 days. At a pH comparable to that observed near the tumor, the release rate is slightly higher, while at a lower pH of 5.5, micelles are less stable and release more encapsulated drugs. The stability of these micelles will promote their passive accumulation at the tumor site through the EPR effect. All currently approved nano-drugs mainly rely on the EPR effect for anti-cancer targeting. 37 The abnormal structure of tumor blood vessels with large windows and insufficient lymphatic drainage lead to the EPR effect. 52 The accumulation of nanoparticles in the tumor stroma will facilitate their internalization by tumor cells. We have previously demonstrated that the cellular uptake of SMA micelles is mediated through endocytosis that forms caveolin vesicles. 53 In addition, the stability and loading of the micelles indicate that the high levels of the two drugs will be released into the cytoplasm and allow the drugs to work together.

We found that sorafenib and nilotinib moderately reduced cell viability and increased apoptosis in PC3 cells, and were strong in androgen-sensitive LNCaP cells. The combination of the two drugs enhances the cytotoxic effect, and under all treatment conditions, the encapsulation of the drug will trigger a more profound cytotoxic effect. The cytotoxicity promoted by sorafenib or nilotinib treatment depends on their intracellular concentration. Sorafenib is transported to liver cells via organic cation transporters 1 and -3 and organic anion transport peptides. 54,55 These transporters are also expressed in CRPC tumors and cell lines (including PC3 and LNCaP cells). 56-59 However, the transporter nilotinib has not been identified, indicating passive transport through the cell membrane. 25 The efflux mechanism of sorafenib and nilotinib has been reported in a number of studies, involving breast cancer resistance protein (BCRP), P-glycoprotein (P-gp) and multidrug resistance protein (MRP). )-2 only applies to sorafenib. 60–63 Interestingly, although the basal expression of P-gp has not been reported in these cell lines, treatment with chemotherapeutic drugs may induce the expression of P-gp protein. 64 On the other hand, BRCP was shown to be expressed in both cell lines, while MRP-2 was only found in PC3 cells. 65,66 Despite the redundancy, one possible explanation for the increased cytotoxicity after combination therapy is that the target protein is a small TKI and a known inhibitor of these efflux pumps. Nilotinib has been identified as an inhibitor of P-gp and BCRP protein activity, while sorafenib can reduce the expression of P-gp protein. 67,68 The higher cytotoxicity observed after combination therapy may be the result of inhibition of P-gp protein. Outflow of medicine.

The uptake of drug-loaded micelles is mediated by endocytosis, where internalization is not rate limited. This, combined with the inhibition of the efflux mechanism, can promote a higher intracellular accumulation of drugs responsible for inhibiting multiple signaling pathways.

Sorafenib and Nilotinib are characterized by broad specificity and can inhibit the autophosphorylation of a variety of tyrosine kinases. 69,70 Sorafenib was originally developed to inhibit Raf/MEK1/2/ERK1/2 signaling pathway or its mutant (V600E)72 by inhibiting Raf-171 and B-Raf. However, PC3 and LNCaP cells are characterized by low expression levels of components of the Raf/MEK1/2/ERK1/2 protein cascade. 73 In addition, AKT identified in PC3 and LNCaP cells by mutation of PTEN protein also contributes to the high basal activity of AKT, which can inhibit the Raf/MEK1/2/ERK1/2 signal cascade. 73 These findings indicate that the effect of sorafenib treatment in these cell lines is caused by the inhibition of one or more RTKs. We evaluated the effects of free and micellar combinations on the expression of several RTK and non-RTKs, such as VEGFR-2, PDGFR, EGFR and FAK and their downstream effector kinases, including SRC, AKT and AR, especially in the midline of androgen-sensitive cells . These proteins are overexpressed or constitutively activated in CRPC, and promote tumor cell proliferation, survival and migration. In addition, if the cells are sensitive to androgen such as LNCaP cells or androgen resistant such as PC3 cells, the sensitivity to single-drug therapy is different. Various mechanisms can explain the difference in sensitivity, such as the number of transporters present on the cell surface, the abundance of RTK, and/or the expression of metabolism and efflux proteins. Overall, encapsulation in SMA enhanced the effect of a single drug, while the combined effect on RTK and non-RTK expression followed a similar pattern in PC3 and LNCaP cells. The combination of free and micellar sorafenib and nilotinib also eliminated AKT and SRC activity and reduced their expression. AKT and SRC are at the crossroads of several signaling pathways, including AR, and promote androgen-independent growth of CRPC. 74 The PTEN mutation found in LNCaP cells leads to increased AKT activity, which has been shown to work synergistically with AR. 75 In addition, the association between SRC and AR has also been shown to depend on EGFR activity. 76 In LNCaP cells, the maintenance of AR activity under androgen deprivation is the activation of multiple signaling pathways, including RTK. In our study, sorafenib and nilotinib slightly reduced AR expression; however, sorafenib reduced AR nuclear localization. The combination of these two drugs reduces AR expression and eliminates its nuclear localization. Treatment with micellar drugs eliminated AR nuclear localization. Interestingly, the expression profile of AR is similar to that of EGFR in LNCaP cells. We were able to visualize AR expression in PC3 cells; however, AR expression was not affected by any processing conditions. According to reports, PC3 cells are AR negative; however, some studies have demonstrated AR expression. 77,78 However, the expression of AR does not seem to be related to the activity of tyrosine kinase, because the inhibitory effect of single agent and combination therapy on SRC is a known modulator of AR activity in LNCaP cells, 79 failure affects its performance in PC3 cells Express. The role of AR in PC3 cells is still elusive. The decrease in the activity and expression of these proteins helps to activate the apoptosis and lysis of caspase-3 in LNCaP cells. However, under all treatment conditions, the increase in cytotoxicity and apoptosis observed in PC3 cells was not related to the detectable level of caspase-3. Using immunocytochemical methods, sorafenib was shown to induce caspase-3 lysis at higher concentrations in PC3 cells. 80 In human leukemia cells, sorafenib has been shown to promote endoplasmic reticulum stress, a process that is partly independent of the activation of caspase-3.81. In addition, we used a spheroid model of tumor PC3 and LNCaP cells to evaluate the cytotoxicity of free and micellar combination therapy. Tumor spheroids are usually used to verify the cytotoxicity of drugs in vitro, and then to evaluate their efficacy in preclinical animal studies. Tumor spheres mimic the complexity of cell interactions observed in vivo to a certain extent. 82 The combination of sorafenib and nilotinib reduced the size of PC3 and LNCaP spheres, and their activity was measured by reducing acid phosphatase activity. The encapsulation of the drug further enhanced this effect in the two cell models, demonstrating the superiority of the micellar system in providing greater cytotoxic concentrations. In addition, we evaluated the possible anti-metastatic effects of the combination of sorafenib and nilotinib. It has previously been demonstrated that treatment with sorafenib can inhibit the invasion of PC3 cells. 83 We demonstrated that free or micellar nilotinib and sorafenib can reduce the migration and invasion of PC3 cells. However, through zymography and western blot evaluation, only micellar nilotinib reduced the secretion of MMP-9. Combination therapy with free drugs or micellar drugs can reduce cell invasion and significantly reduce MMP-9 secretion, indicating that combination therapy has a higher potential in inhibiting metastasis. Sorafenib and Nilotinib are also characterized by their anti-angiogenic properties: Sorafenib is an effective anti-angiogenic agent by inhibiting VEGFR-2 and PDGFR, 84 while Nilotinib interferes with PDGFR signaling Conductive and has been shown to have anti-angiogenic activity. 85 In addition, we believe that different treatments affect the secretion of ISM-1. ISM-1 is an effective angiogenesis inhibitor that acts on endothelial cells through the receptor αvβ5 integrin and glucose regulatory protein 78 kDa (GRP78). 40 Interestingly, Sorafenib treatment and its combination with Nilotinib triggered the release of ISM-1 from PC3 cells. However, encapsulating the drug in micelles inhibited the secretion of ISM-1. These results indicate that endocytosis inhibits the effect of sorafenib on ISM-1 secretion.

In summary, we provide insights into the molecular response of the micelle combination of sorafenib and nilotinib in two CRPC cell models. In addition, we demonstrated the significantly enhanced cytotoxicity of multiple TKI combination therapy, which may provide a way to overcome single treatment and evade treatment.

The authors report no conflicts of interest in this work.

Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global cancer statistics, 2012. CA Cancer J Clin. 2015;65(2):87–108.

Kelly K, Yin JJ. Prostate cancer and metastasis initiating stem cells. Cell Resources 2008;18(5):528-537.

Bolla M, Van Tienhoven G, Warde P, etc. External beam radiation therapy with or without long-term androgen suppression for prostate cancer with a high risk of metastasis: 10-year results of a randomized EORTC study. Lancet tumor. 2010;11(11):1066-1073.

Brundage M, Sydes MR, Parulekar WR, etc. The effect of radiotherapy in androgen deprivation therapy for locally advanced prostate cancer: Long-term quality of life results from the NCIC CTG PR3/MRC PR07 randomized trial. J Clinical Oncology. 2015;33(19):2151-2157.

Hudgens C, Hodges resume. Prostate cancer research. I. The effect of castration, estrogen and androgen injection on serum phosphatase of prostate metastatic cancer. Cancer research. 1941; 1(4): 293-297.

PenningTM. Androgen biosynthesis in castration-resistant prostate cancer. Endocrine related cancers. 2014;21(4):T67–T78.

Greasley R, Khabazhaitajer M, Rosario DJ. Overview of the use of enzalutamide in the treatment of advanced castration-resistant prostate cancer. Cancer management research. 2015; 7: 153-164.

Pound CR, Partin AW, Eisenberger MA, Chan DW, Pearson JD, Walsh PC. Natural history of progression after radical prostatectomy with elevated PSA. Magazine. 1999;281(17):1591-1597.

Oh Brawley. Epidemiology of prostate cancer in the United States. World J Urol. 2012;30(2):195–200.

Fizazi K, Scher HI, Molina A, etc. Abiraterone acetate in the treatment of metastatic castration-resistant prostate cancer: final overall survival analysis of COU-AA-301 randomized, double-blind, placebo-controlled phase 3 study. Lancet tumor. 2012;13(10):983–992.

Scher HI, Fizazi K, Saad F, etc. The use of enzalutamide after chemotherapy improves the survival rate of prostate cancer. N Engl J Med. 2012;367(13):1187–1197.

Saad F, Chi KN, Finelli A, etc. 2015 CUA-CUOG Management Guidelines for Castration Resistant Prostate Cancer (CRPC). Can Urol Assoc J. 2015; 9(3–4):90–96.

Kantoff PW, Higano CS, Shore ND, etc. Sipuleucel-T immunotherapy is used for castration-resistant prostate cancer. N Engl J Med. 2010;363(5):411–422.

Ceraline J, Cruchant, MD, Erdmann E, etc. Activation of androgen receptor through point mutations in the hinge region: a new mechanism for androgen-independent growth in prostate cancer. International J Cancer. 2004;108(1):152-157.

Montgomery RB, Mostacher EA, Visella R, etc. Intratumoral androgen maintenance in metastatic prostate cancer: the mechanism of castration-resistant tumor growth. Cancer research. 2008;68(11):4447–4454.

Tu WH, Zhu C, Clark C, Christensen JG, Sun Z. The efficacy of c-Met inhibitors in the treatment of advanced prostate cancer. BMC cancer. 2010; 10:556.

Nabhan C, Parsons B, Touloukian EZ, Stadler WM. New methods and future directions for castration-resistant prostate cancer. Ann Angkor. 2011;22(9):1948-1957.

Canil CM, Moore MJ, Winquist E, etc. A randomized phase II study of two doses of gefitinib in the treatment of hormone-refractory prostate cancer: a trial in the clinical trial group of the National Cancer Institute of Canada. J Clinical Oncology. 2005;23(3):455–460.

Lee EC, Tenniswood M. Programmed cell death and survival pathways in prostate cancer cells. Arch Andro. 2004;50(1):27-32.

Adnane L, Trail PA, Taylor I, Wilhelm SM. Sorafenib (BAY 43-9006, Nexavar), a dual-acting inhibitor, targets the RAF/MEK/ERK pathway in tumor cells and the tyrosine kinase VEGFR/PDGFR in tumor vasculature. Method enzyme. 2006;407:597-612.

Llovet JM, Villanueva A, Lachenmayer A, Finn RS. Progress in targeted therapy for hepatocellular carcinoma in the genome era. Nat Rev Clin Oncol. 2015;12(7):408-424.

Ivy SP, Wick JY, Kaufman BM. Overview of small molecule inhibitors of VEGFR signaling. Nat Rev Clin Oncol. 2009;6(10):569–579.

Carneiro RM, Carneiro BA, Agulnik M, Kopp PA, Giles FJ. Targeted therapy for advanced differentiated thyroid cancer. Cancer Therapy Revised 2015;41(8):690–698.

Coast N, Mason M, Derek TM. New developments in castration-resistant prostate cancer. BJU International 2012; 109 (Supplement 6): 22-32.

Blay JY, von Mehren M. Nilotinib: A new type of selective tyrosine kinase inhibitor. Smart tumor. 2011; 38 (Supplement 1): S3–S9.

Giles FJ, Rosti G, Beris P, etc. As a first-line treatment for chronic myelogenous leukemia, nilotinib is superior to imatinib: ENESTnd study. Expert Rev Hematol. 2010; 3(6): 665–673.

Schneider M, Korzeniewski N, Merkle K, etc. The tyrosine kinase inhibitor Nilotinib has anti-tumor activity in prostate cancer cells, but it can upregulate the ERK survival signal-which has an impact on targeted therapy. Urol tumor. 2015;33(2):72.e1–72.e7.

Cao Hong, Wang Yao, He Xiao, et al. Co-delivery of sorafenib and curcumin through directed self-assembled nanoparticles can enhance the therapeutic effect of hepatocellular carcinoma. Moore Pharmaceuticals. 2015; 12(3): 922–931.

Hazarika M, Jiang X, Liu Qiang, etc. Tasigna is used for chronic and accelerated philadelphia chromosomes-positive chronic myeloid leukemias that are resistant or intolerant to imatinib. Clinical cancer research. 2008;14(17):5325–5331.

Sasine JP, Schiller GJ. Emerging strategies for high-risk and relapsed/refractory acute myeloid leukemia: new drugs and new methods currently in clinical trials. Blood Rev. 2015;29(1):1-9.

Maeda H, Nakamura H, Fang J. EPR effect of macromolecular drug delivery to solid tumors: improve tumor uptake, reduce systemic toxicity, and different tumor imaging in vivo. Adv Drug Deliv Rev. 2013; 65(1): 71–79.

Greish K, Sawa T, Fang J, Akaike T, Maeda H. SMA-doxorubicin, a new polymer micellar drug that effectively targets solid tumors. J control release. 2004;97(2):219-230.

Vichai V, Kirtikara K. Sulforhodamine B colorimetric method is used for cytotoxicity screening. National agreement. 2006;1(3):1112-1116.

Somers-Edgar TJ, Taurin S, Larsen L, Chandramouli A, Nelson MA, Rosengren RJ. The active mechanism of heterocyclic cyclohexanone curcumin derivatives in estrogen receptor-negative human breast cancer cell lines. Invest in new drugs. 2011;29(1):87-97.

Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO. Cyclic AMP-dependent protein kinase phosphorylation of β-catenin. J Biochemistry. 2006;281(15):9971–9976.

Friedrich J, Seidel C, Ebner R, Kunz-Schughart LA. Sphere-based drug screening: considerations and practical methods. National agreement. 2009;4(3):309–324.

Taurin S, Nehoff H, Greish K. Anti-cancer nanomedicine and tumor vascular permeability; where is the missing link? J control release. 2012;164(3):265-275.

Banerjee S, Sen K, Pal TK, Guha SK. Poly(styrene-co-maleic acid)-based pH-sensitive liposomes mediate the cytosolic delivery of drugs and are used to enhance cancer chemotherapy. Int J Pharm. 2012;436(1–2):786–797.

Parsons JT, Slack-Davis J, Tilghman R, Roberts WG. Focal adhesion kinase: Targeting the adhesion signaling pathway for therapeutic intervention. Clinical cancer research. 2008;14(3):627–632.

Xiang Wei, Ke Zhi, Zhang Y, et al. Isthmin is a new secretory angiogenesis inhibitor that can inhibit tumor growth in mice. J Cell Molecular Medicine. 2011;15(2):359–374.

Basch E, Loblaw DA, Oliver TK, etc. Systemic treatment of men with metastatic castration-resistant prostate cancer: American Society of Clinical Oncology and Ontario Cancer Care Clinical Practice Guidelines. J Clinical Oncology. 2014;32(30):3436–3448.

Visakorpi T, Hyytinen E, Koivisto P, etc. In vivo amplification of the androgen receptor gene and the progression of human prostate cancer. Nat Ginette. 1995; 9(4): 401-406.

El-Amm J, Aragon-Ching JB. The changing pattern of treatment for metastatic castration-resistant prostate cancer. The Adv Med Oncol. 2013;5(1):25-40.

Heidegger I, Massoner P, Eder IE, etc. A new treatment for castration-resistant prostate cancer. J Steroid Biochemistry and Molecular Biology. 2013; 138: 248-256.

Drake JM, Graham NA, Stoyanova T, etc. The oncogene-specific activation of the tyrosine kinase network during the progression of prostate cancer. Proc Natl Acad Sci US A. 2012;109(5):1643–1648.

Ojemuyiwa MA, Madan RA, Dahut WL. Tyrosine kinase inhibitors in the treatment of prostate cancer: the next step in clinical development. Expert opinion on emerging drugs. 2014;19(4):459–470.

Wang XQ, Fan JM, Liu YO, Zhao B, Jia ZR, Zhang Q. The bioavailability and pharmacokinetics of sorafenib suspension, nanoparticles, and nanomatrix for oral administration in rats. Int J Pharm. 2011;419(1–2):339–346.

Di Gion P, Kanefendt F, Lindauer A, etc. Clinical pharmacokinetics of tyrosine kinase inhibitors: focus on pyrimidine, pyridine and pyrrole. Clinical pharmacokinetics. 2011;50(9):551–603.

Choi HS, Liu W, Liu F, etc. Design considerations for tumor targeting nanoparticles. Nat Nanotechnology. 2010;5(1):42-47.

Davis ME, Chen Z, Shen DM. Nanoparticle therapy: an emerging cancer treatment. Nat Rev drug discovery. 2008; 7(9): 771–782.

Roser M, Fischer D, Kissel T. Surface-modified biodegradable albumin nano and microspheres. II: The effect of surface charge on phagocytosis and biodistribution of rats in vitro. Eur J Pharm Biopharm. 1998;46(3):255-263.

Noguchi Y, Wu J, Duncan R, etc. Early tumor accumulation of macromolecules: The clearance rate between tumor and normal tissue is very different. Jpn J Cancer Research. 1998;89(3):307-314.

Taurin S, Nehoff H, van Aswegen T, Rosengren RJ, Greish K. The new role of raloxifene nanomicelles in the treatment of castration-resistant prostate cancer. Biomedical Res Int. 2014; 2014: 323594.

Swift B, Nebot N, Lee JK, etc. Sorafenib Hepatobiliary Disposal: The mechanism by which the liver takes up and disposes of the produced metabolites. Drug metabolism treatment.​​​ 2013;41(6):1179–1186.

Namisaki T, Schaeffeler E, Fukui H, etc. Differential expression of drug uptake and efflux transporters in Japanese hepatocellular carcinoma patients. Drug metabolism treatment.​​​ 2014;42(12):2033-2040.

Gong S, Lu X, Xu Y, Swiderski CF, Jordan CT, Moscow JA. Identify OCT6 as a novel organic cation transporter preferentially expressed in hematopoietic cells and leukemia. Exp heme. 2002;30(10):1162–1169.

Arakawa H, Nakanishi T, Yanagahara C, etc. Enhanced expression of organic anion transport polypeptide (OATP) in androgen receptor-positive prostate cancer cells: the possible role of OATP1A2 in adaptive cell growth under androgen depletion conditions. Biochemical Pharmacology. 2012;84(8):1070-1077.

Joerger M, van Schaik RH, Becker ML, etc. Multidrug and toxin extrusion 1 and human organic cation transporter 1 polymorphisms in castration-resistant prostate cancer patients receiving metformin treatment (SAKK 08/09). Prostate cancer. Prostate disease. 2015;18(2):167–172.

Chen L, Hong C, Chen EC, etc. Genetic and epigenetic regulation of organic cation transporter 3, SLC22A3. Pharmacogenomics J. 2013; 13(2): 110–120.

Huang WC, Hsieh YL, Hung CM, et al. BCRP/ABCG2 inhibition makes hepatocellular carcinoma cells sensitive to sorafenib. Public Science Library One. 2013; 8(12): e83627.

Tang SC, de Vries N, Sparidans RW, Wagenaar E, Beijnen JH, Schinkel AH. The effect of gene dosage of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) on the plasma pharmacokinetics and brain accumulation of dasatinib, sorafenib and sunitinib. J Pharmacol Exp Ther. 2013;346(3):486–494.

Shibayama Y, Nakano K, Maeda H, etc. Multidrug resistance protein 2 suggests anticancer drug resistance to sorafenib. Biopharmaceutical bull. 2011;34(3):433–435.

Shukla S, Kouanda A, Silverton L, Tallele TT, Ambudkar SV. The three-dimensional quantitative structure-activity relationship method was used to model the pharmacophore of nilotinib as an ATP-binding cassette drug transporter and a BCR-ABL kinase inhibitor. Moore Pharmaceuticals. 2014;11(7):2313–2322.

Sanchez C, Mendoza P, Contreras HR, etc. The expression of multidrug resistance protein in prostate cancer is related to the sensitivity of cells to chemotherapy drugs. prostate. 2009;69(13):1448-1459.

Xie Y, Xu Ke, Linn DE, et al. 44-kDa Pim-1 kinase phosphorylates BCRP/ABCG2, thereby promoting its multimerization and drug resistance activity in human prostate cancer cells. J Biochemistry. 2008;283(6):3349-3356.

Cai C, Omwancha J, Hsieh CL, Shemshedini L. Androgen induces the expression of the multidrug resistance protein gene MRP4 in prostate cancer cells. Prostate cancer. Prostate disease. 2007;10(1):39–45.

Couture L, Nash JA, Turgeon J. ATP binding cassette transporter and its effect on drug disposal: a special observation on the heart. Pharmacology Revised 2006;58(2):244-258.

He Ming, Wei Mingjie. Reversal of multidrug resistance through tyrosine kinase inhibitors. Chinese Journal of Cancer. 2012;31(3):126-133.

Fletcher JA, Rubin BP. KIT mutation in GIST. Curr Opin Genet Dev. 2007;17(1):3-7.

Wilhelm SM, Adnane L, Newell P, Villanueva A, Llovet JM, Lynch M. Preclinical overview of Sorafenib. Sorafenib is a multi-kinase inhibitor targeting Raf and VEGF and PDGF receptor tyrosine kinase signaling. Moore cancer treatment. 2008;7(10):3129–3140.

Lyons JF, Wilhelm S, Hibner B, Bollag G. Discovery of new Raf kinase inhibitors. Endocrine related cancers. 2001;8(3):219-225.

William SM, Carter C, Don L, etc. BAY 43-9006 has a broad-spectrum oral anti-tumor activity, targeting RAF/MEK/ERK pathways and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer research. 2004;64(19):7099-7109.

McCubrey JA, Steelman LS, Chappell WH, etc. The role of Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Journal of Biochim Biophys. 2007;1773(8):1263–1284.

Perner S, Cronauer MV, Schrader AJ, Klocker H, Culig Z, Baniahmad A. The adaptive response of androgen receptor signaling in castration-resistant prostate cancer. Tumor target. 2015;6(34):35542–35555.

Xin L, Teitell MA, Lawson DA, Kwon A, Mellinghoff IK, Witte ON. Prostate cancer progresses through the synergy of AKT and androgen receptor genotypic and non-genotypic effects. Proc Natl Acad Sci US A. 2006;103(20):7789-7794.

Castoria G, Giovannelli P, Di Donato M, etc. Targeting the androgen receptor/Src complex can impair the aggressive phenotype of human fibrosarcoma cells. Public Science Library One. 2013; 8(10): e76899.

Alimirah F, Chen J, Basrawala Z, Xin H, Choubey D. DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: effects on androgen receptor function and regulation. FEBS let. 2006;580(9):2294-2300.

Tong da Y, Wu X, Sun H, Jin Y, Liu Z, Zhou F. Expression changes and regulation of AR and IGF-1 in PC3 prostate cancer cells treated with sex hormones and flutamide. Cancer Biology 2012;33(6):2151-2158.

Guo Zhi, Dai B, Jiang Tian, ​​etc. Regulate androgen receptor activity through tyrosine phosphorylation. cancer cell. 2006;10(4):309-319.

Huang R, Chen XQ, Huang Y, Chen N, Zeng H. Sorafenib, a multi-kinase inhibitor, induces caspase-dependent apoptosis in PC-3 prostate cancer cells. Asian J Androl. 2010; 12(4): 527-534.

Rahmani M, Davis EM, Crabtree TR, etc. The kinase inhibitor sorafenib induces cell death through a process involving the induction of endoplasmic reticulum stress. Moore Cell Biology. 2007;27(15):5499–5513.

Hirschhaeuser F, Menne H, Dittfeld C, West J, Mueller-Klieser W, Kunz-Schughart LA. Multicellular tumor spheres: an underrated tool catching up again. J Biotechnology. 2010;148(1):3-15.

Yu Ping, Ye Li, Wang Hua, et al. NSK-01105 is a new type of sorafenib derivative that inhibits the growth of human prostate tumors by inhibiting VEGFR2/EGFR-mediated angiogenesis. Public Science Library One. 2014; 9(12): e115041.

Yamamoto Y, De Velasco MA, Kura Y, etc. The in vivo response to sorafenib treatment was evaluated in a preclinical mouse model of PTEN-deficient prostate cancer. J Transl Med. 2015; 13:150.

Heine A, Held SA, Bringmann A, Holderried TA, Brossart P. Immunomodulatory effects of anti-angiogenic drugs. leukemia. 2011;25(6):899-905.

Mahon KL, Henshall SM, Sutherland RL, Horvath LG. The chemotherapy resistance pathway of castration-resistant prostate cancer. Endocrine related cancers. 2011; 18(4): R103–R123.

Robinson D, He F, Pretlow T, Kung HJ. Tyrosine kinase profile of prostate cancer. Proc Natl Acad Sci US A. 1996;93(12):5958-5962.

Brooks C, Sheu T, Bridges K, etc. Preclinical evaluation of sunitinib (a multi-tyrosine kinase inhibitor) as a radiosensitizer for human prostate cancer. Radiation tumor. 2012; 7:154.

Jackson MW, Roberts JS, Heckford SE, etc. The potential autocrine effect of vascular endothelial growth factor in prostate cancer. Cancer research. 2002;62(3):854–859.

Huber RM, Lucas JM, Gomez-Sarosi LA, etc. DNA damage induces the secretion of GDNF in the tumor microenvironment, which has a paracrine effect that promotes prostate cancer treatment resistance. Tumor target. 2015;6(4):2134–2147.

Heigener DF, Reck M. Crizotinib. Recent results of cancer research. 2014;201:197-205.

Drake JM, Graham NA, Lee JK, etc. Metastatic castration-resistant prostate cancer reveals intra-patient similarity and inter-patient heterogeneity of therapeutic kinase targets. Proc Natl Acad Sci US A. 2013;110(49):E4762–E4769.

Viticchiè G, Muller PA. c-Met and other cell surface molecules: interaction, activation, and functional consequences. biomedicine. 2015; 3(1): 46–70.

Kim IY, Zelner DJ, Lee C. TGF-β1 signaling in the human prostate cancer cell line LNCaP does not require the traditional transforming growth factor-β (TGF-β) receptor type I. Exp Cell Res. 1998; 241(1): 151-160.

Ergüven M, Muramatsu T, Bilir A. Midkine: From embryogenesis to pathogenesis and treatment. Springer: The Netherlands; 2012.

Patel D, Suthar MP, Patel V, Singh R. BCR ABL kinase inhibitors are used in cancer treatment. Int J Pharm Sci Drug Res. 2010; 2:80-90.

Simak R, Capodieci P, Cohen DW, etc. The expression of c-kit and kit-ligand in benign and malignant prostate tissues. Histopathology. 2000;15(2):365–374.

Shimada K, Nakamura M, Ishida E, etc. Prostate cancer antigen 1 promotes cell survival and invasion in human prostate cancer through discoidin receptor 1. Cancer science. 2008;99(1):39-45.

Ide H, Seligson DB, Memarzadeh S, etc. The expression of colony stimulating factor 1 receptor during prostate development and prostate cancer progression. Proc Natl Acad Sci US A. 2002;99(22):14404-14409.

Wiesner C, Nabha SM, Dos Santos EB, etc. C-kit and its ligand stem cell factor: potential contribution to bone metastasis of prostate cancer. Tumor formation. 2008;10(9):996–1003.

Yan Z, Jin S, Wei Z, et al. Discoid domain receptor 2 promotes prostate cancer bone metastasis by regulating parathyroid hormone-related protein. Journal of Biochim Biophys. 2014;1842(9):1350–1363.

Kwabi-Addo B, Ozen M, Ittmann M. The role of fibroblast growth factor and its receptor in prostate cancer. Endocrine related cancers. 2004;11(4):709–724.

Corn PG, Wang F, McKeehan WL, Navone N. Targets the fibroblast growth factor pathway in prostate cancer. Clinical cancer research. 2013;19(21):5856–5866.

Ozen M, Giri D, Ropiquet F, Mansukhani A, Ittmann M. The role of fibroblast growth factor receptor signaling in the survival of prostate cancer cells. J Natl Cancer Institute. 2001;93(23):1783-1790.

McCormack PL. Pazopanib: A review of its application in the treatment of advanced renal cell carcinoma. drug. 2014;74(10):1111–1125.

Kim S, Ding W, Zhang L, Tian W, Chen S. The clinical response of sunitinib as a multi-target tyrosine kinase inhibitor (TKI) in solid cancer: a review of clinical trials. Onco goals. 2014; 7: 719-728.

Whang YE, Armstrong AJ, Rathmell WK, etc. A phase II study of lapatinib (a dual inhibitor of EGFR and HER-2 tyrosine kinase) in the treatment of castration-resistant prostate cancer patients. Urol tumor. 2013;31(1):82–86.

Sridhar SS, Hotte SJ, Chin JL, etc. A multicenter phase II clinical trial of lapatinib (GW572016) in advanced prostate cancer without hormone therapy. It's J Clin Oncol. 2010;33(6):609-613.

Pignon JC, Koopmansch B, Nolens G, Delacroix L, Waltregny D, Winkler R. Androgen receptors control different levels of EGFR and ERBB2 gene expression in prostate cancer cell lines. Cancer research. 2009;69(7):2941–2949.

El Sheikh SS, Domin J, Abel P, Stamp G, Lalani EN. Phosphorylation of EGFR and ErbB2 are reliable predictors of prostate cancer cell response to EGF. Tumor formation. 2004; 6(6): 846-853.

Gu L, Talati P, Vogiatzi P, etc. The pharmacological inhibition of JAK1/2 by JAK1/2 inhibitor AZD1480 can effectively inhibit the formation of experimental prostate cancer metastasis induced by IL-6. Moore cancer treatment. 2014;13(5):1246–1258.

Meyer SC, Levine RL. Molecular pathway: The molecular basis of sensitivity and resistance to JAK kinase inhibitors. Clinical cancer research. 2014;20(8):2051-2059.

Weiss-Messer E, Merom O, Adi A, etc. Growth hormone (GH) receptors in prostate cancer: gene expression and characterization in human tissues and cell lines, GH signaling and androgen receptor regulation in LNCaP cells. Moore cell endocrine. 2004;220(1–2):109–123.

Jorvig JE, Chakraborty A. Zerumbone inhibits the growth of hormone-refractory prostate cancer cells and increases paclitaxel sensitivity by inhibiting the JAK2/STAT3 pathway. Anti-cancer drugs. 2015;26(2):160–166.

O'Neill BH, Goff LW, Kauh JS, etc. A phase II study of mitogen-activated protein kinase 1/2 inhibitor simetinib in patients with advanced hepatocellular carcinoma. J Clinical Oncology. 2011;29(17):2350-2356.

Figure S1 Quantification of Western blot. Note: The protein of interest is normalized to β-tubulin and expressed as a percentage of the control. *P<0.05 compared with control, **P<0.05 compared with free drug and micellar therapy, and ***P<0.05 compared with free drug and combination therapy. Abbreviations: VEGFR, vascular endothelial growth factor receptor; PDGFR, platelet-derived growth factor receptor; EGFR, epidermal growth factor receptor; FAK, focal adhesion kinase; AR, androgen receptor; SMA, poly(styrene-co- Maleic acid) acid.

Figure S2 Western blot and quantification of zymogram. Note: MMP-9 activity and MMP-9 and ISM-1 expression have been standardized as controls and expressed as percentages of controls. * P<0.05 compared with control, ** P<0.05 compared with free drug and micellar treatment. Abbreviations: SMA, poly(styrene-co-maleic acid) acid; MMP, matrix metalloproteinase; ISM-1, isthmin-1.

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