OL Oncology Letters 1792-1074 1792-1082 D.A. Spandidos 10.3892/ol.2020.12040 OL-0-0-12040 Articles Sterically stabilized liposomes targeting P21 (RAC1) activated kinase-1 and secreted phospholipase A2 suppress prostate cancer growth and metastasis VermaArti 1 2 * Najahi-MissaouiWided 3 * CummingsBrian S. 3 4 SomanathPayaningal R. 1 2 5 Program in Clinical and Experimental Therapeutics, University of Georgia, Augusta, GA 30912, USA Charlie Norwood Veterans Affairs Medical Center, Augusta, GA 30904, USA Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30602, USA Interdisciplinary Toxicology Program, University of Georgia, Augusta, GA 30602, USA Department of Medicine and Cancer Center, Augusta University, Augusta, GA 30602, USA Correspondence to: Professor Payaningal R. Somanath, Program in Clinical and Experimental Therapeutics, University of Georgia, HM 102-College of Pharmacy, 914 New Bailie Street, Augusta, GA 30912, USA, E-mail: sshenoy@augusta.edu

Contributed equally

 11 2020 31 08 2020 20 5 179 08052020 22072020 Copyright: © Verma et al. 2020 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Metastatic prostate cancer (PCa) has a very high mortality rate in men, in Western countries and lacks reliable treatment. The advanced-stage PCa cells overexpress P21 (RAC1) activated kinase-1 (PAK1) and secreted phospholipase A2 (sPLA2) suggesting the potential utility of pharmacologically targeting these molecules to treat metastatic PCa. The small molecule, inhibitor targeting PAK1 activation-3 (IPA3) is a highly specific allosteric inhibitor of PAK1; however, it is metabolically unstable once in the plasma thus, limiting its utility as a chemotherapeutic agent. In the present study, the efficacy and specificity of IPA3 were combined with the stability and the sPLA2-targeted delivery method of two sterically stabilized liposomes [sterically stabilized long-circulating liposomes (SSL)-IPA3 and sPLA2 responsive liposomes (SPRL)-IPA3, respectively] to inhibit PCa growth and metastasis. It was found that twice-a-week administration of either SSL-IPA3 or SPRL-IPA3 for 3 weeks effectively suppressed the growth of PC-3 cell tumor xenografts implanted in athymic nude mice. Both drug formulations also inhibited the metastasis of intravenously administered murine RM1 PCa cells to the lungs of C57BL/6 mice. Whereas the twice-a-week administration of SSL-IPA3 significantly inhibited the spontaneous PCa metastasis to the lungs in Transgenic Adenocarcinoma of the Mouse Prostate mice, the administration of free IPA3 had no significant therapeutic benefit. The results present two novel IPA3 encapsulated liposomes to treat metastatic PCa.

 P21 (RAC1) activated kinase-1 secreted phospholipase A2 liposomes prostate cancer metastasis Introduction

Prostate cancer (PCa) ranks second for cancer-related mortality in men, with an expected 33,330 deaths estimated to occur in 2020 worldwide (1,2). Treatment options for the advanced metastatic PCa are limited due to the uncertainties in the molecular mechanisms and the serious side effects of chemotherapy. While the advent of novel screening methods, such as novel serum-based models like 4Kscore® and prostate health index (PHI) (3), and hormone ablation therapies have achieved a ~100% 5-year survival rate for patients with localized PCa, treating patients harboring metastatic PCa, who have a 5-year survival rate of 31%, remains a challenge (4,5). In-depth molecular characterization to detect novel druggable targets, identifying compounds with precise molecular targets and limited off-target effects, and developing novel strategies for drug delivery is critical in improving the 5-year survival rate in patients with metastatic PCa.

Our previous studies and those from other researchers have shown that P21 (RAC1) activated kinase-1 (PAK1) promotes PCa growth and metastasis (6–11) by facilitating cell proliferation, cell survival, motility, invasion, and epithelial-to-mesenchymal transition (11–14). Previous studies have also shown that the small molecule, an inhibitor targeting PAK1 activation-3 (IPA3) was found to be an effective allosteric inhibitor of PAK1, which decreases PCa tumor growth, and metastasis (12–14). However, despite the promising effect of IPA3 on PCa, the compound has limitations related to its pharmacokinetic properties. Specifically, IPA3 is metabolically unstable, therefore, daily administration is required to exert its anti-cancer effects (15), which is not feasible in a clinical setting. Therefore, this limitation was addressed by developing two distinct liposomal formulations of IPA3, one based on the classical sterically stabilized long-circulating liposomes (SSL) (16), and the other, that incorporates lipids, which are selectively targeted by secreted phospholipase A2 (sPLA2), an esterase overexpressed in several types of cancer, including PCa (17).

SSL-IPA3 are long-circulating liposomes designed for passive targeting by the enhanced permeability and retention effect (18–20). The base formulation of these SSLs is clinically used for the enhanced delivery of doxorubicin for the treatment of breast cancer (21). The mechanisms of increased efficiency for SSL stems, in part, from the presence of the polyethylene glycol (PEG) coating, which decreases SSL clearance by phagocytes in the reticuloendothelial system, therefore extending their systemic circulation time and alters the pharmacokinetics of the encapsulated drug (22). The efficacy of SSL for the treatment of cancer is further enhanced due to the leaky vasculature of tumors and the lack of a functional lymphatic system, which provides access for SSL to enter and accumulate in the tumors by the enhanced permeability and retention phenomenon (23–26). Besides, this also provides passive targeting of SSL to the tumors, as the intact vasculature in normal tissue limits the entry of SSL, decreasing off-target toxicity (27–29). sPLA2 responsive liposomes, or SPRL, are the base formulation of SSL with alterations that include an increase in the negatively charged glycerophospholipids (22). These alterations ensure that SPRL is more responsive to cancers, that overexpress SPLA2, including prostate, breast, gastric, lung, and colon cancers (30,31). sPLA2 cleave phospholipids at the sn-2 (a nucleophilic substitution in which the rate-determining step involves 2 components) bond of the glycerol backbone releasing fatty acid and lysophospholipids (31–35). Unlike other PLA2, sPLA2 has a strong affinity for negatively charged phospholipid head groups, in particular, phosphatidylserine, phosphatidylglycerol, and phosphatidylethanolamine (17,36). In PCa, sPLA2 overexpression was associated with poor clinical prognosis and 5-year survival rate, and the levels of sPLA2 in PCa tissues were reportedly 10–20-fold higher compared with that in normal tissue (31,37–39). Our previous studies demonstrated the increased ability of SPRL, which contained doxorubicin, to decrease PCa growth in a mouse xenograft model (19) and validated its use as a targeted drug delivery system (17,19). SPRL increased their affinity to bind to the cell surface-expressed sPLA2 on the tumor cells, due to the higher concentrations of phosphatidylethanolamine (PE) in their formulation (22,31,36,37).

Despite our previous success with SPRL, it has only been tested in non-metastatic cancer models and has only been formulated with doxorubicin, a drug that is not commonly used for the treatment of PCa (19). Therefore, in the present study, IPA3-encapsulated SSL and SPRL (SSL-IPA3 and SPRL-IPA3, respectively) were created and their efficacies in inhibiting the growth of PCa xenografts and PCa metastasis to the lungs was investigated. The results showed that both SSL and SPRL had increased efficacy compared with that in free IPA3, in the treatment of the tumors, which only required twice-weekly IP injections, as opposed to daily use.

 Materials and methods <sec> <title>Cell culture

The human PC-3 (CRL-1435) and murine RM-1 (CRL-3310) metastatic PCa cells were purchased from ATCC. Cells were grown in either DMEM high-glucose medium (for PC-3 cells) or RPMI 1640 medium (for RM-1 cells) (Hyclone; GE Healthcare Life Sciences), supplemented with 10% FBS (R&D Systems, Inc.), 100 U/ml penicillin, and 100 mg/ml streptomycin (Themo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO2. Cells were passaged when they were 80–90% confluent. All the other analytical reagents were purchased from Thermo Fisher Scientific, Inc. unless otherwise stated.

 Animals

All the animal procedures were performed according to the protocol approved by the Institutional Animal Care and Use Committee at the Charlie Norwood Veterans Affairs Medical Center, (GA, USA; protocol no. 19-04-114). The protocols were also in agreement with the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines (40). Briefly, animals were housed 2–4 mice per cage, in rooms maintained at 65–75°F (~18-23°C), 40–60% humidity, a 10/14-h light/dark cycle, and ad libitum access to food and water. Animals were handled as minimally as possible, with minimal noise levels to avoid any stress. Athymic nude mice (Harlan Laboratories, Inc.) were maintained in sterile cages (2 mice per cage) in a separate sterile room, with the provision of sterile food and water. Isoflurane (3–4% in oxygen) was used to anesthetize mice at the end of the experiment, before euthanasia by cervical dislocation. A total of 90 male 8–10 week-old mice weighing between 25–29 g were used for the tumor and metastasis experiments, with 6 to 11 mice per experiment. Mice were monitored every day for any potential infections or sickness and weighed every 2nd day to determine any weight loss beyond 20%. No animals died before the end of the experiment.

 Preparation of liposomal encapsulated IPA3

SSL-IPA3 liposomes were prepared as described in our previous study (16), using the thin lipid hydration method followed by freeze-thaw cycles and a high-pressure extrusion (19,22). Briefly, cholesterol (5 µmol/ml), phospholipids, including 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (9 µmol/m) and DSPE-PEG (1 µmol/ml) in chloroform, and IPA3 (4 µmol/ml) in ethanol were added into a round bottom flask. SPRL-IPA3 was also prepared similarly using 5 µmol/ml cholesterol, 8 µmol/ml DSPC, 1 µmol/ml 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[poly(ethylene glycol) 2000] (DSPE-PEG), 1 µmol/ml DSPE (17). The solvents were evaporated under vacuum in a water bath at 65°C using a rotary evaporator (Buchi Labortechnik AG). The formed thin film was then hydrated and suspended in PBS to achieve a final lipid concentration of 10 µmol/ml. The formulation then underwent five liquid nitrogen freeze-thaw cycles, above the phase transition temperature of the primary lipid, before passing five times through a Lipex Extruder (Northern Lipids, Inc.) at 65°C using double-stacked polycarbonate membranes (80-nm; Suez Water Technologies and Solutions). Excess unencapsulated IPA3 and lipids were eliminated using dialysis in 10% (w/v) sucrose for at least 20 h, with three changes of the dialysis media. Liposome suspensions were stored at 4°C, protected from light, and used within 24–48 h of preparation. Empty SPRL was also formulated and used as vehicle controls. Quantification of IPA3 was evaluated using methods previously described (16). The quality control used during the formulation of SSL and SPRL included the measurement of size, zeta potential, and drug encapsulation. The size was measured by both dynamic light scattering and by tandem electron microscopy. Liposomes that did not meet the minimum required characteristics of 1,000 µM IPA3 encapsulation, a size of 100 nm hydrodynamic diameter, a poly-dispersity index of <0.3, and charge of at least-20 mV zeta potential were not used for the experiments.

 Mouse genotyping

For the genotyping of Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mice (Jackson Laboratory), DNA was extracted from the ear punch of 10 to 21-day old litters. Tissues were incubated with 50 µl of alkaline lysis buffer containing 25 ml H2O, 62.5 µl of 10 N NaOH, and 10 µl of 0.5 M disodium EDTA at 95°C for 90 min, followed by neutralization with 50 µl buffer containing 24 ml H2O, 1 ml 1 M Tris-HCl (41). The TRAMP transgene (600 bp) was amplified using the following primer sequences: Forward 5′-GCGCTGCTGACTTTCTAAACATAAG-3′ and reverse, 5′-GAGCTCACGTTAAGTTTTGATGTGT-3′, with an annealing temperature of 55°C. GAPDH was measured as an internal positive control using the following primer seuqneces: Forward, 5′-CTAGGCCACAGAATTGAAAGATCT-3′ and reverse, 5′-GTAGGTGGAAATTCAGCATCATCC-3′. PCR was performed using the GoTaq® M712C green master mix (Promega Corporation) with the following thermocycling conditions: Step-1: 95°C for 3 min; Step-2: 94°C for 30 sec; Step-3: 60°C for 1 min; Step-4: 72°C for 1 min (step-2-4 repeated for 35 cycles); Step-5: 72°C for 2 min and Step-6: Hold at 4°C until further processing. The products were visualized using ethidium bromide containing agarose gel (2%) under UV light.

 Cell proliferation assay

Cell proliferation following IPA3 treatment was assessed using an MTT assay (Thermo Fisher Scientific, Inc.), as previously described (13). Briefly, cells were seeded into 48-well cell culture plates, at a density of 5×104 cells/ml per well and incubated at 37°C in a humidified incubator with 5% CO2 for 24 h. Cells were treated with either 10, 20, or 30 µM IPA3 (cat. no. 3622; Tocris BioScience) encapsulated in SSL and SPRL liposomes, empty SSL and SPRL or dimethyl sulfoxide (DMSO) (vehicle) as controls for 24 h. Following this, MTT reagent was added, at a final concentration of 0.25 mg/ml, and the plates were incubated at 37°C for 2 h. After incubation, non-reduced MTT and the medium were aspirated and MTT formazan crystals were dissolved using DMSO. Following an additional 15 min incubation, with constant shaking (2.8×10−3 × g using a Vari-Mix Platform Rocker (Thermo Fisher Scientific, Inc.), plates were read at 590 nm using a Biotek plate reader (Agilent Technologies, Inc.).

 In vivo prostate tumor xenograft assay

PC-3 cells were grown to 60–70% confluent in T75 flasks. Next, the cells were collected and suspended in sterile normal saline. Cell suspension (3×106 cells/100 µl) was subcutaneously injected into the right flank of 6 to 8-week-old male athymic nude mice (Harlan Laboratories, Inc.). All treatments (empty liposomes, SSL-IPA3, and SPRL-IPA3 (5 mg/kg) were started on day 3 from tumor implantation and were administered two times a week, by intraperitoneal (i.p.) injection. Tumor diameters were measured using digital calipers on days 7, 14, 19, and 21, and the tumor volume (mm3) was calculated by the modified ellipsoidal formula (tumor volume=½ [length × width2]). The average size of the tumors before treatment was 42 mm3. Mice were sacrificed on day 25 and tumors were dissected, weighed, and snap-frozen for further analysis. We included 12 mice in each group at the start of the experiment. However, one mouse from the empty liposome group, one from the SSL-IPA3 group and two mice from the SPRL-IPA3 group were later removed due to sickness.

 In vivo mouse lung colonization (metastasis) assay

PC-3 and RM-1 cells grown to 60–70% confluence in T75 flasks were washed once with 1X PBS, detached using trypsin, and re-suspended in 0.9% saline. A total volume of 150 µl cell suspension, containing 0.5×106 cells was injected through the tail-vein into 8-week-old C57BL/6 mice, as well as athymic nude mice. Animals in each group were injected (i.p.) with either 5 mg/kg SSL-IPA3 or SPRL-IPA3, or vehicle control (PBS or empty liposomes) twice weekly as previously described (18). Alternately, 27-week old TRAMP mice were injected with the vehicle (sterile PBS), free IPA3 twice a week, free IPA3 once daily, or SSL-IPA3 twice a week for 3 weeks. Mice weight was monitored every 3 days, up to day 21. On day 21, mice were euthanized, the lungs were collected and snap-frozen or directly fixed for hematoxylin and eosin (H&E) staining. The number of lung nodules was counted by three blinded reviewers (individuals within the laboratory) and the average of their scores was used for analysis. In the TRAMP mice study, 8 mice were included in each at the start of the experiment. However, one mouse from the twice-weekly free IPA3 group and two mice from the once per day free IPA3 group were removed due to sickness.

 Histological examination of the mouse lungs

Tissue sections were embedded in paraffin and 5 µm sections were cut for H&E staining. For staining, tissues were first dehydrated twice with 95% ethanol for 30 min each, followed by soaking in xylene for 1 h at 60–70°C. The sections were subsequently dipped in paraffin for 12 h. Tissue sections were stained with Harris' hematoxylin solution for 6 h at 60–70°C, rinsed in tap water and immersed in a destaining solution containing 10% acetic acid and 85% ethanol in water for 2 h and an additional 10 h at room temparature. Washing slides were soaked in a saturated lithium carbonate solution for 12 h and rinsed with tap water. Finally, sections were stained with eosin Y in ethanol for 48 h at room temparature. Imaging was performed using a brightfield Keyence BZ-X800 microscope (Keyence Corporation; magnification, ×4). Lung micrometastasis was analyzed using ImageJ software (version 1.48v; National Institutes of Health) (13). Briefly, the H&E images of the lung were converted to grayscale followed by splitting the image into RGB channels. The area of individual channels was measured and subtracted from the total lung area to determine the metastatic area.

 Statistical analysis

Data are presented as the mean ± SD. The ‘n’ value for each figure indicates the number of samples in each group. MTT assays were performed 6 times in 3 replicates. All the data were analyzed using parametric tests, the Student's unpaired t-test for comparing two groups or one-way ANOVA for comparing more than two groups, followed by Tukey's post hoc test (with pooled variance) and the GraphPad Prism v6.01 software (GraphPad, Software, Inc.) P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>SSL-IPA3 and SPRL-IPA3 suppress the growth of PC-3 cell tumor xenografts in athymic nude mice with similar efficacy

Our previous study found that SSL-IPA3 was more effective in decreasing PC-3 tumor xenograft growth compared with that for free IPA3 (16); however, the ability of SPRL-IPA3 to decrease tumor growth in xenograft models has never been investigated. Twice-weekly administration of SSL-IPA3 (5 mg/kg) inhibited the growth of PC-3 tumor xenografts compared with that in the control group (Fig. 1A-C). Furthermore, the same results were demonstrated with SPRL-IPA3, administrated at the same dose and schedule (Fig. 1A-C). The results were found to be significantly different for tumor volume (Fig. 1D) and weight (Fig. 1E). There was no significant difference between empty liposomes, SSL-IPA3, and SPRL-IPA3 treatment on the total body weight of mice after 25 days (Fig. 1F). The data revealed that SSL-IPA3 and SPRL-IPA3 are effective in inhibiting the growth of prostate tumor xenografts.

 SSL-IPA3 and SPRL-IPA3 inhibit murine RM-1 PCa cell proliferation in vitro

The lung metastasis model used in the present study required mouse PCa cells; however, to the best of our knowledge the effect of SSL- or SPRL-IPA3 to inhibit mouse prostate cell growth has not been previously investigated. Therefore, the effect of this formulation on murine prostate RM-1 metastatic PCa cell proliferation in vitro was performed using the MTT assay. It was found that 20 and 30 µM doses for both SSL- and SPRL-IPA3 decreased the number of cells compared with that in cells treated with 10 µM after 24 h (Fig. 2). There was a significant decrease in cell proliferation with 30 µM SRPL-IPA3 compared with that in cells treated with 20 µM SPRL-IPA3, but not between 20 and 30 µM SSL-IPA3. There was also a modest but significant reduction in RM-1 cell proliferation in cells treated with 30 µM SPRL-IPA3 compared with that in cells treated with 30 µM SSL-IPA3. These data suggested that both SSL- and SPRL-IPA3 have antiproliferative effects on RM-1 cells.

 SSL-IPA3 and SPRL-IPA3 inhibit RM-1 cell metastasis to the lungs of mice

To the best of our knowledge, liposomal encapsulated IPA3, in any formulation, has never been investigated for its ability to inhibit cancer metastasis. Therefore, the effect of twice-weekly administration of SSL-IPA3 and SPRL-IPA3 on lung metastasis in C57BL/6 mice, following their intravenous injection with RM-1 cells, was determined. Histological examination of the mouse lungs demonstrated a significant reduction in lung metastasis, as measured by the number of colonies and areas of lung metastasis indicated by red arrows with intravenous RM-1 cell injection compared with that in the vehicle group (Fig. 3A). Both SSL-IPA3 and SPRL-IPA3 (5 mg/kg) significantly reduced the number of lung nodules and metastatic areas in the mouse lungs compared with that in the empty liposomes or vehicle control (PBS) groups (Fig. 3B and C). SSL-IPA3 and SPRL-IPA3 were found to be equally effective in inhibiting PCa cell lung metastasis in mice.

 Twice-weekly administered SSL-IPA3 but not free IPA3 inhibited lung metastasis in TRAMP mice

To further investigate the ability of SSL- and SPRL-IPA3 to prevent metastasis the effect of twice-weekly injected liposomal IPA3 was compared with that in free IPA3, to inhibit spontaneous lung metastasis, that occurs in TRAMP mice. This mouse model closely resembles the pathogenesis of human PCa and is known to spontaneously develop metastasis at the age of 28–30 weeks (41). TRAMP mice, at 27 weeks of age were injected twice a week with a vehicle (sterile PBS) or free IPA3, or once daily with free IPA3, or twice a week with SSL-IPA3 for 3 weeks (until 30 weeks of age). SPRL-IPA3 was not investigated, as both SSL-IPA3 and SPRL-IPA3 were both efficacious at reducing tumor growth in the aforementioned experiments. Spontaneous lung metastasis in the TRAMP (control and treated) mice were examined using H&E staining of lung sections (Fig. 4A), following 3 weeks of treatments. Histological examination of the lung sections revealed lung metastatic nodules in vehicle-treated TRAMP mice. The area of metastatic nodules was markedly lower in TRAMP mice, administered daily with free IPA3. Notably, twice-weekly treatment with free IPA3 (5 mg/kg) in TRAMP mice did not inhibit PCa lung metastasis in TRAMP mice (Fig. 4A and B). As expected, SSL-IPA3 (5 mg/kg) administration, twice per week for 3 weeks significantly inhibited PCa lung metastasis in TRAMP mice (Fig. 4A and B). These results indicated that the administration of liposome-encapsulated IPA3 twice weekly for 3 weeks could significantly decrease lung metastasis in vivo and could be developed into a future therapeutic approach to treat metastatic PCa in humans.

 Discussion

Despite the FDA approval of three new drugs, abiraterone acetate, enzalutamide, and radium-223, for front-line use in men with metastatic castration-resistant PCa in the past 5 years, the life expectancy for patients with PCa, over the past decade has only been prolonged by one year (42,43). As a result, patients with metastatic PCa can succumb to the disease, which accounts for the second leading cause of cancer-associated death in men in the US as of 2020 (1,2). Thus, there is an urgent requirement for novel and effective therapeutic strategies for the treatment of metastatic PCa. The higher expression level of PAK1 protein has been found in human PCa tissues and PCa metastasized lung lesions in patients compared with that in the normal tissues from healthy controls (9). Our previous studies have demonstrated that PAK1 was essential for PCa growth, epithelial-to-mesenchymal transition and metastasis, and that the PAK1 allosteric inhibitor, IPA3, was a potential drug to treat metastatic PCa (6,8–10,12,13,16). These findings suggested that PAK1 could be a potential therapeutic target to prevent PCa growth and metastasis in humans.

Several PAK inhibitors have been studied for their anti-cancer efficacies in vitro and in vivo (10,14,15,44,45). Several PAK1 inhibitors, such as G-5555 (46), FL172 (47), PF-3758309 (48), and AZ13705339 (49) have demonstrated PAK1 activity suppression in vitro; however, these compounds also exhibited off-target effects on several other kinases, such as the Src family of kinases, Akt1, AMP Kinases, Cyclin-dependent kinase-7 and serum glucocorticoid kinase, due to their competition for the ATP-binding site, that is conserved in several kinases (44,45). Among these, PF-3758309 and PF-03758309 are specific inhibitors of PAK4, a group II PAK isoform (50), and a clinical trial on PF-03758309 for advanced solid tumors (NCT00932126) was terminated, due to the undesirable pharmacokinetic characteristics, such as unfavorable levels of the drug in the plasma and the lack of a dose-response association. Therefore, the allosteric PAK1 inhibitors, such as IPA3 (15) and NVS-PAK1-1 (51) were preferred for pharmacological interventions in cancer research. Our previous studies have demonstrated that IPA3, suppresses epithelial-to-mesenchymal transition, and micro-invasion of PCa cells in vitro and tumor growth in vivo (6,9). Daily administration of IPA3 also attenuated PCa-induced metastasis and bone remodeling in athymic nude mice (13). Unfortunately, IPA3 in its free form can be rapidly metabolized in the plasma and has a very short half-life (45), which limits its use as a therapeutic agent for cancer. Therefore, it is not feasible to treat patients with cancer in the clinic daily. To overcome this, two new liposome formulations were utilized, with distinct lipid composition to encapsulate IPA3 into nanoliposomes to serve as a reservoir for its slow release thus, increasing the half-life of the drug in the plasma. Due to the different lipid composition, while both SSL-IPA3 and SPRL-IPA3 have the benefit of high stability and long half-life once in the blood, SPRL-IPA3 has the specificity to respond to the highly expressed sPLA2 in the tumor microenvironment (12,17,36).

In the present study, loading IPA3 into nanoliposomes reduced the frequency of drug administration without compromising its efficacy. The efficacy of SSL-IPA3 or SPRL-IPA3, when administered twice a week, was comparable to the efficacy of daily administration of free IPA3 in preventing PCa lung metastasis in 2 different mouse models. A total of 30 µM SPRL-IPA3 exhibited slightly higher efficacy on reducing cell survival compared with that in cells treated with a similar dose of SSL-IPA3; however, the efficacies of either of IPA3-containing liposomes were similar even in inhibiting the growth of human PCa tumor xenografts implanted in immunocompromised mice. This frequency of drug administration was not effective with the free form of IPA3 to prevent PCa growth, as evidenced in our previous study (16). The advantage of liposomal IPA3 was also clear from the observation that twice a week administration of free IPA3 did not prevent PCa metastasis to the mouse lungs thus, presenting the liposomal-IPA3 approach as a reliable method to treat metastatic PCa.

Several laboratories have utilized nanoparticles for targeted drug delivery in various types of cancer, including breast, lung, colon, and gastric cancers, which is similar to our approach, where these nanoparticles were enriched in tumors thus, delivering small molecule inhibitors specifically at the tumor site (52–56). To the best of our knowledge, these data are the first to demonstrate the ability of liposomal formulations of IPA3 to inhibit the lung metastasis of PCa. This was observed in two different models of PCa metastasis, representing two different strains of mice, which supports the rigor of our findings. Since PAK1 hyperactivation has also been reported in other diseases, such as osteoarthritis (57), neurodevelopmental disorders (58), macrocephaly (59), intellectual disability (60), and kidney injury (61), liposomal IPA3 could have potential therapeutic applications for several non-cancer human diseases.

The results from the present study show that the efficacies of SSL-IPA3 and SPRL-IPA3 were very similar. The exact reason for this is currently unknown, and requires further investigation; however, it could be due to the specific advantage of each of these formulations; SSL was highly stable and long-acting, while SPRL had additional specificity in targeting the tumor tissues. Another possibility is that both liposomes release their contents before reaching the tumor tissue, but are still able to maintain a constant level of IPA3 in the plasma. Furthermore, a previous study demonstrated the increased efficacy of SPRL over SSL using doxorubicin as the payload (19). This suggested that the differential efficacy of these liposomes may be drug-dependent. It is also important to note that SPRL was designed and validated against human Group IIA sPLA2, as opposed to the mouse sPLA2 isoform. Thus, a potential difference in the activity of group IIA sPLA2 in mice compared with humans may have accounted for SPRL-IPA3 not having a superior efficacy over SSL-IPA3. Additional investigations are required to validate this hypothesis. However, these data still provide strong evidence that PCa growth and metastasis could be targeted effectively by the encapsulation of IPA3, in two different lipid-based nanoparticles. This treatment strategy would allow for the administration of the drug less frequently and improve drug efficacy while minimizing its side-effects.

 Acknowledgements

Not applicable.

 Funding

The work was primarily funded by the Department of Defense Prostate Cancer Research Program Idea Development Award (grant no. PC150431 GRANT11996600). Partial financial support was also provided by the National Heart Lung and Blood Institute (grant no. R01HL103952), National Center for Advancing Translational Sciences (grant no. UL1TR002378), Wilson Pharmacy Foundation (intramural), and Translational Research Initiative grant (intramural). This work has been accomplished using the resources and facilities at the VA Medical Center in Augusta, GA (USA).

 Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author upon reasonable request.

 Author's contributions

AV, WNM, BSC, and PRS contributed to the design and conception of the study and acquired, analyzed, and/or interpreted the data. AV, BSC, WNM, and PRS drafted the initial manuscript. All authors approved the final version of the manuscript.

 Ethics approval and consent to participate

Ethics approval was granted by the Institutional Animal Care and Use Committee at the Charlie Norwood Veterans Affairs Medical Center (Georgia, USA; approval no. ACORP #19-04-114).

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

 References American Cancer SocietyCancer Facts and Figures 2020AtlantaAmerican Cancer Society SiegelRLMillerKDJemalACancer statistics, 2020CA Cancer J Clin70730202010.3322/caac.2160131912902 TanGHNasonGAjibKWoonDTHerrera-CaceresJAlhunaidiOPerlisNSmarter screening for prostate cancerWorld J Urol37991999201910.1007/s00345-019-02719-530859272 SiegelRLMillerKDJemalACancer statistics, 2019CA Cancer J Clin69734201910.3322/caac.2155130620402 MalvezziMCarioliGBertuccioPBoffettaPLeviFLa VecchiaCNegriEEuropean cancer mortality predictions for the year 2018 with focus on colorectal cancerAnn Oncol2910161022201810.1093/annonc/mdy03329562308 Al-AzayzihAGaoFSomanathPRP21 activated kinase-1 mediates transforming growth factor β1-induced prostate cancer cell epithelial to mesenchymal transitionBiochim Biophys Acta185312291239201510.1016/j.bbamcr.2015.02.02325746720 Al-MaghrabiJEmamEGomaaWAl-QaydyDAl-MaghrabiBBuhmeidaAAbuzenadahAAl-QahtaniMAl-AhwalMOverexpression of PAK-1 is an independent predictor of disease recurrence in colorectal carcinomaInt J Clin Exp Pathol81589515902201526884861 GocAAbdallaMAl-AzayzihASomanathPRRac1 activation driven by 14-3-3ζ dimerization promotes prostate cancer cell-matrix interactions, motility and transendothelial migrationPLoS One7e40594201210.1371/journal.pone.004059422808202 GocAAl-AzayzihAAbdallaMAl-HuseinBKavuriSLeeJMosesKSomanathPRP21 activated kinase-1 (Pak1) promotes prostate tumor growth and microinvasion via inhibition of transforming growth factor β expression and enhanced matrix metalloproteinase 9 secretionJ Biol Chem28830253035201310.1074/jbc.M112.42477023258534 KichinaJVGocAAl-HuseinBSomanathPRKandelESPAK1 as a therapeutic targetExpert Opin Ther Targets14703725201010.1517/14728222.2010.49277920507214 ParkJKimJMParkJKHuangSKwakSYRyuKAKongGParkJKooBSAssociation of p21-activated kinase-1 activity with aggressive tumor behavior and poor prognosis of head and neck cancerHead Neck37953963201510.1002/hed.2369524634274 Najahi-MissaouiWQuachNDJenkinsADabkeISomanathPRCummingsBSEffect of P21-activated kinase 1 (PAK-1) inhibition on cancer cell growth, migration, and invasionPharmacol Res Perspect7e00518201910.1002/prp2.51831516713 VermaAArthamSAlwhaibiAAdilMSCummingsBSSomanathPRPAK1 inhibitor IPA-3 mitigates metastatic prostate cancer-induced bone remodelingBiochem Pharmacol177113943202010.1016/j.bcp.2020.11394332240651 OngCCGierkeSPittCSagollaMChengCKZhouWJubbAMStricklandLSchmidtMDuronSGSmall molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agentsBreast Cancer Res1759201510.1186/s13058-015-0564-525902869 DeaconSWBeeserAFukuiJARennefahrtUEMyersCChernoffJPetersonJRAn isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinaseChem Biol15322331200810.1016/j.chembiol.2008.03.00518420139 Al-AzayzihAMissaouiWNCummingsBSSomanathPRLiposome-mediated delivery of the p21 activated kinase-1 (PAK-1) inhibitor IPA-3 limits prostate tumor growth in vivoNanomedicine1212311239201610.1016/j.nano.2016.01.00326949163 QuachNDMockJNScholpaNEEggertMWPayréCLambeauGArnoldRDCummingsBSRole of the phospholipase A2 receptor in liposome drug delivery in prostate cancer cellsMol Pharm1134433451201410.1021/mp500174p25189995 AdaminaMBolliMAlboFCavazzaAZajacPPadovanESchumacherRReschnerAFederCMartiWREncapsulation into sterically stabilised liposomes enhances the immunogenicity of melanoma-associated Melan-A/MART-1 epitopesBr J Cancer90263269200410.1038/sj.bjc.660147314710238 MockJNCostynLJWildingSLArnoldRDCummingsBSEvidence for distinct mechanisms of uptake and antitumor activity of secretory phospholipase A2 responsive liposome in prostate cancerIntegr Biol (Camb)5172182201310.1039/c2ib20108a22890797 WoodleMCLasicDDSterically stabilized liposomesBiochim Biophys Acta1113171199199210.1016/0304-4157(92)90038-C1510996 ZhouRMazurchukRStraubingerRMAntivasculature effects of doxorubicin-containing liposomes in an intracranial rat brain tumor modelCancer Res6225612566200211980650 ZhuGMockJNAljuffaliICummingsBSArnoldRDSecretory phospholipase A2 responsive liposomesJ Pharm Sci10031463159201110.1002/jps.2253021455978 FenskeDBCullisPRLiposomal nanomedicinesExpert Opin Drug Deliv52544200810.1517/17425247.5.1.2518095927 SakakibaraTChenFAKidaHKuniedaKCuencaREMartinFJBankertRBDoxorubicin encapsulated in sterically stabilized liposomes is superior to free drug or drug-containing conventional liposomes at suppressing growth and metastases of human lung tumor xenograftsCancer Res563743374619968706018 MaruyamaKIntracellular targeting delivery of liposomal drugs to solid tumors based on EPR effectsAdv Drug Deliv Rev63161169201110.1016/j.addr.2010.09.00320869415 FangJNakamuraHMaedaHThe EPR effect: Unique features of tumor blood vessels for drug delivery, factors involved, and limitations and augmentation of the effectAdv Drug Deliv Rev63136151201110.1016/j.addr.2010.04.00920441782 DeshpandePPBiswasSTorchilinVPCurrent trends in the use of liposomes for tumor targetingNanomedicine (Lond)815091528201310.2217/nnm.13.11823914966 SawantRRTorchilinVPChallenges in development of targeted liposomal therapeuticsAAPS J14303315201210.1208/s12248-012-9330-022415612 ShulerTERiddleCDJrPottsDWPolymicrobic septic arthritis caused by Kingella kingae and enterococcusOrthopedics1325425619902308886 YamashitaSOgawaMSakamotoKAbeTArakawaHYamashitaJElevation of serum group II phospholipase A2 levels in patients with advanced cancerClin Chim Acta2289199199410.1016/0009-8981(94)90280-17988039 DongQPatelMScottKFGrahamGGRussellPJSvedPOncogenic action of phospholipase A2 in prostate cancerCancer Lett240916200610.1016/j.canlet.2005.08.01216182442 BelinskyGSRajanTVSariaEAGiardinaCRosenbergDWExpression of secretory phospholipase A2 in colon tumor cells potentiates tumor growthMol Carcinog46106116200710.1002/mc.2027117091473 WangMHaoFYWangJGXiaoWGroup IIa secretory phospholipase A2 (sPLA2IIa) and progression in patients with lung cancerEur Rev Med Pharmacol Sci1826482654201425317799 YamashitaSYamashitaJOgawaMOverexpression of group II phospholipase A2 in human breast cancer tissues is closely associated with their malignant potencyBr J Cancer6911661170199410.1038/bjc.1994.2298198986 ZhangCYuHXuHYangLExpression of secreted phospholipase A2-Group IIA correlates with prognosis of gastric adenocarcinomaOncol Lett1030503058201510.3892/ol.2015.373626722288 QuachNDArnoldRDCummingsBSSecretory phospholipase A2 enzymes as pharmacological targets for treatment of diseaseBiochem Pharmacol90338348201410.1016/j.bcp.2014.05.02224907600 DongZLiuYScottKFLevinLGaitondeKBrackenRBBurkeBZhaiQJWangJOleksowiczLLuSSecretory phospholipase A2-IIa is involved in prostate cancer progression and may potentially serve as a biomarker for prostate cancerCarcinogenesis3119481955201010.1093/carcin/bgq18820837598 JiangJNeubauerBLGraffJRChedidMThomasJERoehmNWZhangSEckertGJKochMOEbleJNChengLExpression of group IIA secretory phospholipase A2 is elevated in prostatic intraepithelial neoplasia and adenocarcinomaAm J Pathol160667671200210.1016/S0002-9440(10)64886-911839587 LuSDongZOverexpression of secretory phospholipase A2-IIa supports cancer stem cell phenotype via HER/ERBB-elicited signaling in lung and prostate cancer cellsInt J Oncol5021132122201710.3892/ijo.2017.396428440478 KilkennyCBrowneWCuthillICEmersonMAltmanDGNC3Rs Reporting Guidelines Working GroupAnimal research: Reporting in vivo experiments: The ARRIVE guidelinesBr J Pharmacol16015771579201010.1111/j.1476-5381.2010.00872.x20649561 GaoFAlwhaibiASabbineniHVermaAEldahshanWSomanathPRSuppression of Akt1-β-catenin pathway in advanced prostate cancer promotes TGFβ1-mediated epithelial to mesenchymal transition and metastasisCancer Lett402177189201710.1016/j.canlet.2017.05.02828602980 SonnenburgDWMorgansAKEmerging therapies in metastatic prostate cancerCurr Oncol Rep2046201810.1007/s11912-018-0692-z29644451 TeoMYRathkopfDEKantoffPTreatment of advanced prostate cancerAnnu Rev Med70479499201910.1146/annurev-med-051517-01194730691365 SemenovaGChernoffJTargeting PAK1Biochem Soc Trans457988201710.1042/BST2016013428202661 RudolphJCrawfordJJHoeflichKPWangWInhibitors of p21-activated kinases (PAKs)J Med Chem58111129201510.1021/jm501613q25415869 NdubakuCOCrawfordJJDrobnickJAliagasICampbellDDongPDornanLMDuronSEplerJGazzardLDesign of Selective PAK1 Inhibitor G-5555: Improving Properties by Employing an Unorthodox Low-pK a Polar MoietyACS Med Chem Lett612411246201510.1021/acsmedchemlett.5b0039826713112 MaksimoskaJFengLHarmsKYiCKissilJMarmorsteinRMeggersETargeting large kinase active site with rigid, bulky octahedral ruthenium complexesJ Am Chem Soc1301576415765200810.1021/ja805555a18973295 MurrayBWGuoCPirainoJWestwickJKZhangCLamerdinJDagostinoEKnightonDLoiCMZagerMSmall-molecule p21-activated kinase inhibitor PF-3758309 is a potent inhibitor of oncogenic signaling and tumor growthProc Natl Acad Sci USA10794469451201010.1073/pnas.091186310720439741 McCoullWHennessyEJBladesKChuaquiCDowlingJEFergusonADGoldbergFWHoweNJonesCRKemmittPDOptimization of highly kinase selective bis-anilino pyrimidine PAK1 InhibitorsACS Med Chem Lett711181123201610.1021/acsmedchemlett.6b0032227994749 ShaoYGNingKLiFGroup II p21-activated kinases as therapeutic targets in gastrointestinal cancerWorld J Gastroenterol2212241235201610.3748/wjg.v22.i3.122426811660 KarpovASAmiriPBellamacinaCBellanceMHBreitensteinWDanielDDenayRFabbroDFernandezCGalubaIOptimization of a dibenzodiazepine hit to a potent and selective allosteric PAK1 inhibitorACS Med Chem Lett6776781201510.1021/acsmedchemlett.5b0010226191365 HuQShangLWangMTuKHuMYuYXuMKongLGuoYZhangZCo-Delivery of paclitaxel and interleukin-12 regulating tumor microenvironment for cancer immunochemotherapyAdv Healthc Mater91901858202010.1002/adhm.201901858 UnalOAkkocYKocakMNalbatEDogan-EkiciAIYagci AcarHGozuacikDTreatment of breast cancer with autophagy inhibitory microRNAs carried by AGO2-conjugated nanoparticlesJ Nanobiotechnology1865202010.1186/s12951-020-00615-432345308 ZhaoHMuXZhangXYouQLung cancer inhibition by betulinic acid nanoparticles via adenosine 5′-Triphosphate (ATP)-binding cassette transporter G1 gene downregulationMed Sci Monit26e922092202032277808 AzimeeSRahmatiMFahimiHMoosaviMATiO2 nanoparticles enhance the chemotherapeutic effects of 5-fluorouracil in human AGS gastric cancer cells via autophagy blockadeLife Sci248117466202010.1016/j.lfs.2020.11746632101760 TanLHanSDingSXiaoWDingYQianLWangCGongWChitosan nanoparticle-based delivery of fused NKG2D-IL-21 gene suppresses colon cancer growth in miceInt J Nanomedicine1230953107201710.2147/IJN.S12803228450784 MaWWangXWangCGongMRenPUp-regulation of P21-activated kinase 1 in osteoarthritis chondrocytes is responsible for osteoarthritic cartilage destructionBiosci Rep40BSR20191017202010.1042/BSR2019101731868209 OhoriSMitsuhashiSBen-HaimRHeymanESengokuTOgataKMatsumotoNA novel PAK1 variant causative of neurodevelopmental disorder with postnatal macrocephalyJ Hum Genet65481485202010.1038/s10038-020-0728-832005903 HornSAuMBasel-SalmonLBayrak-ToydemirPChapinACohenLEltingMWGrahamJMGonzaga-JaureguiCKonenODe novo variants in PAK1 lead to intellectual disability with macrocephaly and seizuresBrain14233513359201910.1093/brain/awz26431504246 KernohanKDMcBrideAHartleyTRojasSKCare4Rare Canada ConsortiumDymentDABoycottKMDyackSp21 protein-activated kinase 1 is associated with severe regressive autism, and epilepsyClin Genet96449455201910.1111/cge.1361831392718 ZyndaERMaloyMHKandelESThe role of PAK1 in the sensitivity of kidney epithelial cells to ischemia-like conditionsCell Cycle18596604201910.1080/15384101.2019.157814930724698

SSL-IPA3 and SPRL IPA3 significantly inhibits the growth of PC-3 cell tumor xenografts in athymic nude mice. Images of athymic nude mice bearing PC-3 cell tumor xenografts (left) and extracted tumors (right) treated with (A) empty liposomes, (B) SSL-IPA3, and (C) SPRL-IPA3 (5 mg/kg). (D) Line graph showing the volume of PC-3 cell tumor xenografts on days 7, 14, 21 and 25, following implantation in athymic nude mice treated with empty liposomes, SSL-IPA3 and SPRL-IPA3 (5 mg/kg), respectively. Bar graph showing the (E) weight of the tumors and (F) body weight of PC-3 cell tumor xenograft athymic nude mice treated with empty liposomes, SSL-IPA3 and SPRL-IPA3 (5 mg/kg), respectively on day 25 post-tumor implantation. Data are presented as the mean ± SD. One-way ANOVA was used to compare when there were more than two groups. *P<0.001. i.p., intraperitoneal; SRPL, secreted phospholipase A2 responsive liposomes; SSL, sterically stabilized long-circulating liposomes; IPA3, P21 (RAC1) activated kinase-1. Empty liposomes (n=11), SSL-IPA3 group (n=11), SPRL-IPA3 group (n=10).

SSL-IPA3 and SPRL-IPA3 significantly inhibit RM-1 murine metastatic PCa cell proliferation in vitro. A bar graph representing a dose-dependent reduction in cell proliferation following 24 h treatment with SSL-IPA3 and SPRL-IPA3 (5 mg/kg) in RM-1 cells compared with that in cells treated the vehicle (DMSO) and respective empty liposome-treated controls (n=6). Data are presented as the mean + SD. One-way ANOVA was used to compare when there were more than two groups. *P<0.001 and **P<0.01. DMSO, dimethyl sulfoxide; SRPL, secreted phospholipase A2 responsive liposomes; SSL, sterically stabilized long-circulating liposomes; IPA3, P21 (RAC1) activated kinase-1.

SSL-IPA3 and SPRL-IPA3 significantly inhibit lung metastasis of intravenously administered RM-1 cells in C57BL/6 mice. (A) Representative H&E-stained lung section images showing a marked decrease in lung colonization of RM-1 cells, 21 days following injection with SSL-IPA3, and SPRL-IPA3 (5 mg/kg) compared with that in the control and empty SSL and SPRL liposome-treated groups (n=6). The red arrows indicate the areas of colonization. Bar graphs showing a significant reduction in the (B) number of nodules and (C) area of lung colonization on day 21 following treatment with SSL-IPA3 and SPRL-IPA3 (5 mg/kg) compared with that in the control and empty SSL and SPRL groups. (n=6). Data are presented as the mean ± SD. An unpaired Student's t-test was used to compare between two groups and one-way ANOVA was used to compare among all the groups. *P<0.05; **P<0.01. i.p., intraperitoneal; i.v., intravenous; SRPL, secreted phospholipase A2 responsive liposomes; SSL, sterically stabilized long-circulating liposomes; IPA3, P21 (RAC1) activated kinase-1; PBS, control group.

SSL-IPA3 inhibits lung metastasis in TRAMP mice. TRAMP mice, at 27 weeks of age were injected with vehicle, empty SSL, and SSL-IPA3 (5 mg/kg) twice weekly and compared with free IPA3 administered daily or twice weekly for 3 weeks, for lung colonization. (A) Images of H&E-stained TRAMP lung sections showing changes in lung metastasis between the control, free IPA3, and SSL-IPA3-treated groups for 3 weeks. The arrows indicate the area of metastasis. (B) Bar graph indicating reduced lung metastasis in daily administered free IPA3 and twice-weekly administered SSL-IPA3 groups compared with that in the control groups and twice-weekly administered free IPA3 group. Data are presented as the mean ± SD. An unpaired Student's t-test was used to compare between two groups and one-way ANOVA for more than two groups. **P<0.01; #P<0.001. TRAMP, Transgenic Adenocarcinoma of the Mouse Prostate; PBS, vehicle group; i.p., intraperitoneal; SRPL, secreted phospholipase A2 responsive liposomes; SSL, sterically stabilized long-circulating liposomes; IPA3, P21 (RAC1) activated kinase-1; Ctrl, control. Control, Empty SSL (n=8), twice-weekly administered free IPA3 (n=7), daily administered free IPA3 for 3 weeks (n=6), twice-weekly administered SSL-IPA3 for 3 weeks (n=8).