Protein kinase C inhibitor anchored BRD4 PROTAC PEGylated nanoliposomes for the treatment of vemurafenib-resistant melanoma
Abstract
Limited treatment options and development of resistance to targeted therapy within few months pose significant challenges in the treatment of BRAF-mutated malignant melanoma. Moreover, extensive angiogenesis and vas- culogenic mimicry promote the rapid progression of disease. The purpose of this study was to develop a protein kinase C inhibitor anchored BRD4 PROTAC (ARV) loaded PEGylated nanoliposomes (LARPC). Palmitoyl-DL- carnitine chloride (PC) was used as a protein kinase C inhibitor to provide a cationic surface charge to LARPC. The formulation was characterized for particle size, zeta potential, drug release and various cell culture assays using HUVEC and vemurafenib resistant melanoma cells. The particle size of LARPC was found to be 105.25 ± 2.76 nm with a zeta potential of +26.6 ± 6.25 mV. Inhibition of angiogenesis was demonstrated by ARV and LARPC using human umbilical vein endothelial cells (HUVEC)-based matrigel basement membrane model. Additionally, LARPC demonstrated very low IC50 with promising inhibition of vasculogenic mimicry channel formation, cell migration as well as colony formation in vemurafenib-resistant melanoma cell lines. Hence, the outcome of this combination therapy indicated the suitability of LARPC as a potential and novel approach for eradicating vemurafenib-resistant melanoma.
1. Introduction
Melanoma is a malignant tumor formed from melanocytes. Mela- noma is more likely to spread and hence it is one of the most aggressive and deadliest skin cancer [1]. Due to the poor overall survival (OS) and the resistance problem of conventional chemotherapy, there were limited treatment options before the discovery of BRAFi [2]. Discovery of BRAF mutation (around 50% of melanoma patients) and BRAFi such as vemurafenib and dabrafenib based on mitogen-activated protein ki- nase (MAPK) pathway are major breakthroughs in the field of unre- sectable or metastatic BRAF mutant melanoma treatment [3]. Even though BRAFi showed promising clinical outcome and prolonged the OS, development of resistance within a few months of treatment is inevitable [4]. A combination of BRAFi and MEKi showed a remarkable tumor response rate and improved patient survival but off-target toXicity and cross-resistance limit the clinical potential of this approach [5–8]. Therefore, there is a need to identify novel or alternative cancer drugs and develope appropriate delivery system for the treatment of melanoma.
Bromodomain and extra-terminal domain (BET) proteins are epige- netic readers that can control gene expression. BET inhibitors (BETi) have gained a lot of interest in targeting the acetyl-lysine-binding property of BET proteins due to its encouraging anticancer efficacy in various types of cancer at a preclinical level [9–11]. Segura et al., 2013 reported that BRD4 protein, one of protein in BET family, is overex- pressed in primary and metastatic melanoma, and therefore exerted remarkable anti-melanoma effect after the treatment with BET inhibitor [12]. BET inhibitor, JQ, demonstrated a synergistic effect with BRAF inhibitor vemurafenib in vitro and in vivo models of BRAF-mutant mel- anoma and mitigated vemurafenib-mediated drug resistance in mela- noma [13–15]. However, lack of specificity and restoration of pre-existing transcription level limit the potential use of JQ1 [16,17]. Novel BET protein PROTAC (PROteolysis-Targeting Chimera) ARV-825 (ARV) was developed by researchers, which selectively degrades BRD4 protein by hijacking the E3 ubiquitin ligase cereblon instead of mere inhibiting it, resulting in quick and prolonged degradation of BRD4 protein [18]. Additionally, ARV-825 was also investigated as a novel therapeutic approach in vemurafenib-resistant melanoma, which showed very promising results in our previous work [19].
Along with the development of resistance in melanoma, BRAFi treatment was found to paradoXically activate the MAPK pathway in tumor macrophages to produce vascular endothelial growth factor (VEGF) and resulted in melanoma cell proliferation, survival, and metastasis based on VEGF-dependent angiogenesis [20]. Furthermore, vasculogenic mimicry was observed in the microcirculation in mela- noma cells in vitro and in vivo [21]. These highly patterned blood channels are generated by melanoma cells and are physiologically connected as tumor-lined networks, which are independent of angio- genesis and facilitate tumor growth by providing nutrients [22]. Considering the highly vascular nature of melanoma, angiogenesis in- hibitor should be amenably used. Nevertheless, inefficient delivery of anti-angiogenetic agents limits its therapeutic application [23,24]. For instance, AXitinib by Pfizer failed in melanoma clinical trials due to its off-target side effects. Besides, an angiogenesis inhibitor may promote metastasis [25,26]. Hence, new targeting strategies to inhibit angio- genesis needs further attempt for investigation.
Protein kinase C (PKC) family is a serine/threonine protein kinase, which includes multiple isozymes with various functions on signaling pathways in both melanocytes and melanoma. PKC isoforms expressed in malignant melanoma play important role in melanogenesis, regula- tion of cell growth, and metastasis-related properties [27,28]. It was reported that PKC activation especially, PKC isoform β2, is a predomi- nant regulator for VEGF induced angiogenesis, proliferation, and tumor development [29,30]. It was also found to be absent in melanoma cell lines, which is the major differences between normal and malignant cells and it was considered to be the reason for the growth of melanoma [31, 32]. Moreover, overexpression of PKC isoform α was found in murine B16 melanoma and exerted inhibition effect on cell growth [33–35]. Also, PKCα was found to promote angiogenic activity and induces VEGF of human endothelial cell, which in turn stimulates it to release in a sustain manner by an autocrine positive feedback loop [36]. PKC in- hibitor is suggested to be combined with chemotherapy as new thera- peutics against high-grade malignant gliomas [37]. Therefore, a PKC inhibitor would provide a promising anti-melanoma effect. In the pre- sent study, Palmitoyl-DL-carnitine chloride (PC) was selected as a PKC inhibitor to combine with PROTAC molecule – ARV825 for the investi- gation of anti-angiogenic and anti-vasculogenic mimicry effect on vemurafenib-resistant melanoma.
The novel PROTAC molecule, ARV, was reported as a CYP3A4 sub- strate with extremely poor water solubility [19]. Thus, it is very chal- lenging to develop an oral formulation based on the physicochemical properties of ARV and PC. In cancer therapy, conventional chemo-therapeutics always lead to severe side effects due to the poor targeting to tumor site. Stealth liposomes with PEGylation are exten- sively used as a nanocarrier to deliver anti-cancer drugs, which avoid detection by the immune system – reticuloendothelial system (RES) and thus prolong the circulation in the blood. Moreover, because of the enhanced permeability and retention (EPR) effect, which is known as EPR-mediated passive tumor targeting, nanoliposome tend to accumu- late into tumor tissue due to poor lymphatic system and leaky vascula- ture and thus improve the efficacy of drug delivery [38]. Liposomes are therefore considered as the most suitable formulation to deliver both ARV and PC, since it can incorporate ARV into the lipid bilayer as well as provide a safe and effective way for chemo-drug delivery. Surface characteristics of liposome also play a key role in tumor-specific delivery of anti-cancer molecules. Previous studies have reported that cationic liposomes can accumulate more in tumor vessels and thus improve intratumorally delivery of anti-cancer molecules [39,40]. Additionally, acidic pH of solid tumor is due to lactate secretion from anaerobic glycolysis [41]. Negatively-charge of neovasculature, tumor cells and extracellular matriX are contributed by glycocalyX, flipped phosphati- dylserine and interstitial hyaluronic acid, respectively [42–44]. There- fore, positively charged particles are able to facilitate cell penetration and endosomal escape due to electrostatic interaction with negative charged vascular endothelial cells (vessel wall charge density 0.05C/m2) [45,46]. Taken together, the purpose of the present study is to develop a protein kinase C inhibitor anchored BRD4 PROTAC (ARV) loaded PEGylated nanoliposomes (LARPC) and to investigate the anti- cancer activity of LARPC in vemurafenib-resistant melanoma.
2. Materials and methods
2.1. Materials
ARV was purchased from ChemieTek (Indianapolis, IN, USA), Pal- mitoyl-DL-carnitine chloride was obtained from Chemcruz (Dallas, TX, USA) and Vemurafenib was purchased from LC Laboratories (Woburn, MA, USA), 1,2-Dioleoyl-sn-glycero-3 phosphocholine (DOPC) was pur- chased from Cordenpharma (Liestal, Switzerland), PE 18:0/18:0- PEG2000 was obtained from Lipoid (Ludwigshafen, Germany), Choles- terol and Chloroform were purchased from Sigma-Aldrich (MO, USA), Fetal Bovine Serum (FBS) was procured from Atlanta Biologics (Oak- wood, GA, USA), Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Thermo Fisher Scientific Inc. (Waltham. MA, USA), Penicillin-Streptomycin-Amphotericin B (PSA) was purchased from MP Biomedicals, LLC (Solon, Ohio, USA). High performance liquid chro- matography (HPLC) grade water, acetonitrile and other solvents of analytical grade were procured from Fisher Scientific (Hampton, NH, USA).
2.2. Cell lines and culture conditions
Melanoma cell lines (A375 and SK-MEL-28) were purchased from ATCC (Manassas, USA) and were cultured in DMEM, supplemented with 10% fetal bovine serum (FBS), 10 mg/mL antibiotics (PSA) at 37 ◦C with 5% CO2 in humidified air. Cell concentrations in the culture were adjusted to allow for exponential growth. Vemurafenib-resistant mela- noma cell lines (A375R and SK-MEL-28 R) were developed by the method described before and experiments were carried out with A375R and SK-MEL-28 R [19]. Briefly, vemurafenib was added to A375 and SKK-Mel-28 up to 20 passages Vemurafenib was withdrawn once resis- tance against Vemurafenib was developed. The vemurafenib resistance was confirmed prior to studies.Human umbilical vein endothelial cells (HUVECs) were purchased from ATCC (Manassas, USA) were seeded and grown in T-25 tissue culture flasks in Endothelial Cell Growth Basal Medium (EBM-2 Me- dium) with the EGM-2 BulletKits (Lonza). HUVECs were incubated at 37 ◦C and 5% CO2. EXperiments were performed with cells between passages 5 to 8.
2.3. HPLC analysis
Chromatographic separation of ARV was achieved using Waters e2695 separation module with 2998 PDA (Photodiode array) detector and Hypersil ODS C18 column (250 mm 4.6 mm, 5 μm). Acetonitrile: Phosphate buffer pH 3.5 (60:40) was used as the mobile phase at a flow rate of 1 mL/min and an injection volume of 10 μL. The output signal was monitored by Empower 3 software. ARV was detected at 247 nm with the retention time of ~6.9 ± 0.2 min.
2.4. In vitro cytotoxicity test
CytotoXicity of ARV, PC and LARPC was evaluated in A375R, and SK- MEL-28 R using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates at a density of 5000 cells/well and allowed to grow for 24 h before treatment. Drugs and formulation were diluted in cell culture medium at different con- centrations for treatment. After 48 h treatment, cell viability was determined using MTT assay. Briefly, MTT dye was dissolved at a final concentration of 5 mg/mL in PBS. Cells were incubated with 20 μL of 5 mg/mL MTT solution in each well for 3 h at 37 ◦C, 5% CO2. MTT- formazan crystals were dissolved by adding 100 μL dimethyl sulfoXide (DMSO) to each well. The quantity of MTT-formazan was determined by measuring the absorbance at 570 nm.
2.5. Preparation of liposome
LARPC was prepared by modified hydration method described pre- viously [47]. Briefly, ARV, DOPC, cholesterol and DSPE-PEG2000 were dissolved in chloroform. The chloroform solution was then added dropwise to parenteral-grade mannitol (200 μm) with constant stirring at 45 ◦C and left overnight for evaporation of chloroform. The resultant powder was then dispersed in water containing PC at 55 ◦C followed by probe sonication (30% amplitude) for 2 min. The optimized molar ratio of ARV: DOPC: cholesterol: DSPE-PEG2000: PC was 1:72:24: 3:9. ARV liposomes were made with the same composition without PC.
2.6. Characterization of liposomes
The average size, size distribution by intensity, zeta potential, and polydispersity index (PDI) were measured using a dynamic light scattering (DLS) particle size analyzer (Malvern Zetasizer Nano ZS, UK). Samples were analyzed using disposable cuvettes at 25 ◦C having a scattering angle of 173◦. All the experiments were carried out in triplicates.
2.7. Stability study
Total drug content was measured from freshly prepared liposomes at day 0. Samples were collected at different time points, centrifuged at 5000 rpm for 10min to separate any precipitated ARV. The supernatant was diluted with acetonitrile and the concentration of ARV was analyzed by HPLC. Drug content was plotted at different time points.
2.8. In vitro release study
Drug release of ARV from LARPC was analyzed using the dialysis bag method. Before use, dialysis bags (Spectra/Por® 7) were soaked in Milli- Q water at room temperature overnight to remove the preservative, followed by rinsing thoroughly in Milli-Q water prior to its use. The dialysis sac filled with LARPC was immersed in beaker with 150 mL of PBS (pH 7.4) including 0.5% w/v TPGS and was maintained at 37 ◦C ensuring constant stirring. The samples were withdrawn from the release medium at various time points. The concentration of ARV in the release media was evaluated by HPLC.
2.9. Anti-angiogenesis assay
BME (basement membrane extract: reconstituted protein matriX comprised of laminin, collagen IV, entactin, and heparin sulphate pro- teoglycan) was precoated in a 96 well plate. Human umbilical vein endothelial cells (HUVECs) were incubated with calcine after washing with phosphate-buffered saline (PBS). The cells were then diluted with endothelial growth medium in the presence of 40 nM ARV, 360 nM PC and LARPC (contains the same concentration of ARV and PC as single drug treatment) and seeded into the wells at a density of 3 104/well. Docetaxel was used at 800 nM as a positive control. The plate was incubated for 10 h in a 37 ◦C incubator containing 5% CO2. Endothelial tube formation was visualized under a fluorescence microscope. Tube formation was quantified as the number of branch points and tube length using ImageJ software.
2.10. Vasculogenic mimicry
A375 and A375R cells suspension at 1 × 105/ml were incubated with ARV (200 nM), PC (1.8 μM) and LARPC (ARV 200 nM and PC 1.8 μM) for 5 min and then seeded at a density of 1 × 104/well in a 96 well plate precoated with BME. After 24 h incubation at 37 ◦C with 5% CO2, images were taken using an EVOS light microscope at 10 . Tube formation was quantified as the number of junctions formed.
2.11. Migration assay
Vemurafenib-resistant cell lines (A375R and SK-MEL-28 R) were seeded in a 96-well plate at a density of 5000 cells/well, and incubated at 37 ◦C and 5% CO2 overnight. Once the cells in each well reached 90–95% confluency, a uniform scratch was made in the center of the well. Cells were treated with ARV (5 μM), PC (45 μM) and LARPC (contains 5 μM ARV and 45 μM PC) till the gap of the control group was bridged. Then the cells were fiXed with 3.7% formaldehyde and stained with 0.5% w/v crystal violet followed by washing with PBS and air-dried for 48 h. Percent bridging of migration area was analyzed by calculating the area of the scratch gap using ImageJ software.
2.12. Clonogenic assay
The procedure for the clonogenic assay was carried out according to the procedure described previously [48]. Cells at an exponentially growing stage were harvested and plated in siX-well microplates at a density of 1000 cells/well for A375R and SK-MEL-28 R cells. Cells were allowed to adhere overnight. Then the cells were treated with 40 nM ARV, 360 nM PC and LARPC (contains 40 nM ARV and 360 nM PC) before the population doubling time. The treatments were withdrawn and replaced with the fresh media after 24 h and cells were maintained at 37 ◦C with 5% CO2 for 5 days. Thereafter, the medium was removed from the wells and cells were rinsed with PBS. Colonies were fiXed with glutaraldehyde (6.0% v/v) followed by 0.5% crystal violet staining of clones for 30 min. The plate was then washed with water and was air-dried. Colonies were counted (the colony is defined to consist of at least 50 cells) on the following day. Plating efficiency (PE) and survival fraction (SF) was calculated by the following equations: PE = number of colony formed/number of cell seeded * 100% (1) SF = PE of treasted sample/PE of control * 100%.
2.13. Statistics
Statistics were carried out using GraphPad Prism7 Software (La Jolla, CA, USA). Statistical analysis was performed by one-way ANOVA. P- value lower than 0.05 was considered a statistically significant differ- ence between groups.
3. Results
3.1. In vitro cytotoxicity test
In vitro cytotoXicity of ARV, PC and LARPC were evaluated in vemurafenib-resistant cell lines (A375R and SK-MEL-28 R). As shown in Table 1, the IC50 of individual drug and formulation are similar in both vemurafenib-resistant cell lines. ARV exhibited much lower IC50 compared to PC. IC50 value of nanoliposome LARPC showed further reduction in A375R whereas found to be similar in SK-MEL-28 R in comparison with ARV.
Fig. 1. Characterization of LARPC (a) DLS particle size distribution of LARPC (b) Zeta potential of LARPC. (c) Effect of serum on average particle size of LARPC. LARPC were incubated with water, FBS free DMEM and 5% FBS DMEM. Data was plotted as mean ± S.D. (n = 3). S.D. = Standard deviation.
Fig. 2. In Vitro Angiogenesis assay (a) HUVEC cells with treatment of ARV (40 nM), PC (360 nM) and LARPC (with 40 nM ARV and 360 nM PC). Untreated HUVEC was used as control and docetaxel treated was used as positive control. (b) Number of branch points HUVEC after treatment with PC, ARV and LARPC. Graph shows significant inhibi- tion of endothelial tube formation by PC, ARV and LARPC compared to the control; (c) Tube length showed significantly inhibited by ARV and LARPC compared to the control. Data was plotted as mean ± S.D. (n = 3). (*p < 0.05, **p < 0.01).
3.2. Characterization and stability of liposomes
Particle size and zeta potential are important aspects in terms of drug delivery system which may affect permeability, uptake and stability of nanoparticles. As shown in Fig. 1a, the hydrodynamic diameter of LARPC was 105.25 ± 2.76 nm with PDI less than 0.25. The zeta potential was found to be +26.6 ± 6.25 mV which is attributed to carnitine on the surface (Fig. 1b). Whereas, the particle size of the ARV liposomes without PC was more than 130 nm with a PDI of over 0.30 and zeta potential of 46.4 12.8. Further, we also investigated the impact of serum on LARPC. There was no difference in particle size of LARPC was observed on incubation of LARPC liposomes in FBS free DMEM and 5% FBS DMEM for 2 h and 24 h (Fig. 1c). The LARPC were stable for particle size and zeta potential for one month at 4 ◦C storage. In vitro release study revealed that there was no ARV release detected up to 24 h. Based on limits of analytical detection we can assume that less than 1% of ARV was released in 24 h. Thus, ARV is strongly confined to liposomal bilayer and will not leak/release ARV in the systemic circulation.
Fig. 3. Evaluating the effect of ARV formulation on A375 and A375R vasculogenic mimicry (a) Vasculogenic mimicry images of cells treated with ARV (200 nM), PC (1.8 μM) and LARPC. Number of branching points after treated with ARV, PC and LARPC treatment in (b) A375 and (c) A375R. Data were expressed as mean ± S.D. LARPC shows significantly less number of branching points compared to other treatment group. (*p < 0.05, **p < 0.01).
3.3. Angiogenesis assay
Primary HUVECs are the most widely used in-vitro model to inves- tigate the anti-angiogenesis effect of drugs. The result of the HUVEC capillary tube formation assay revealed that ARV, PC and LARPC have strong anti-angiogenic activity. ARV and LARPC showed an anti- angiogenic effect at nanomolar concentration. As low as 4 nM of ARV and 36 nM of PC showed stronger inhibition of tubular formation after 10 h incubation (Fig. 2). Moreover, the number of branch points was significantly inhibited by ARV, PC and LARPC. The tube length was also significantly decreased by LARPC group compared to ARV and PC alone. Since there was no branching point observed in docetaxel - positive control group, it was not represented in the graph.
3.4. Vasculogenic mimicry (VM)
The concept of vasculogenic mimicry was introduced by Maniotis et al. which describes the formation of de novo formation of extracellular matriX (ECM)-rich and vasculogenic-like networks found in aggressive tumor cells in 3- dimensional matrices (3D) culture, which is indepen- dent of angiogenesis [21]. This VM tube formation is another mode to deliver nutrients to the tumor tissue besides angiogenesis, which pro- motes tumor progression and metastasis. The VM structures in the control group were observed in both A375 and A375R on the matriX gel. Wheras, PC and ARV showed drastic inhibition of VM channel formation, and LARPC was found to further inhibit the VM channel formation in A375 and A375R as shown in Fig. 3. Treatment with ARV and PC significantly decrease the branching points while the combina- tion of ARV with PC in LARPC further enhanced the inhibition of VM channel formation. Moreover, reduction in the number of branching points by ARV and LARPC was more in resistant cells compared to parent cells.
3.5. Migration assay
Migration assay is also known as scratch-wound assay, which is a two-dimensional method to evaluates the migration ability of cells after exposure to the chemo-treatment. The vemurafenib-resistant melanoma cell lines were exposed to the treatments for 48 h. As shown in Fig. 4, ARV and LARPC treated groups showed significant inhibition of the scratch bridging compared to the control group in both A375R and SK- MEL-28 cell lines and the LARPC exhibited more inhibition in terms of migration ability compared to single-drug treatment.
3.6. Clonogenic assay
Clonogenic assay is a method to determine cell reproductive death after treatment. The number of colonies was drastically reduced by the exposure of LARPC and ARV compared to control as well as the PC treated group in both A375R and SK-MEL-28 R (Fig. 5). PC showed no reduction in colonies compared to the control group. Moreover, there was no difference in the number of colonies in the ARV and LARPC groups. Further, plating efficiency (PE) was 65% for A375R and 72% for SK-MEL-28 R. The survival fraction (SF) of ARV was much lower compared to other treatment groups as shown in Table 2.
Fig. 4. In-vitro migration assay. (a) Microscopic images with crystal violet in A375R and SK-MEL-28 R after treatments. Percentage of migration inhibition in (b) A375R and (c) SK-MEL-28 R cell lines. LARPC (contains 5 μM ARV and 45 μM PC) showed significantly inhibition of bridging compared to control and treatment group (ARV 5 μM, 45 μM PC). (**p < 0.01, ***p < 0.001, ****p < 0.0001).
4. Discussion
Functionalized liposomes co-delivering anticancer drugs to target multiple pathways have previously succeeded in overcoming drug- resistant tumors [49–51]. Nano-liposomal formulations provide the possibility to incorporate chemo-drugs with different chemical proper- ties as well as increase anti-tumoral efficacy while minimizing side ef- fects on normal tissues. Moreover, PEGylated liposomes possess the advantage of avoiding mononuclear phagocyte system uptake and thus prolong the accumulation in blood circulation. Poor aqueous solubility of PROTAC class of molecules warrants a biocompatible delivery system for its efficient parenteral administration. The aim of this research is to develop protein kinase C inhibitor and BRD4 PROTAC co-loaded nano- liposomes and to test for its cytotoXicity, angiogenic and vasculogenic mimicry in vemurafenib-resistant melanoma.LARPC were successfully developed with a particle size of 105.25 2.76 nm, which would tend to accumulate in tumor tissue due to EPR effect [38]. Since ARV is a very hydrophobic molecules, development of physically stable liposomes was very challenging. The incorporation of protein kinase C inhibitor - palmitoyl carnitine (PC) in lipid bilayer resulted in reduction in particle size with improving the PDI. Surface active property of PC – lipophilic palmitoyl chain and hydrophilic carnitine head group could be responsible for particle size reduction compared to PC free liposomes. The stability study of LARPC showed that drug content remained the same at room temperature for a month, which demonstrated the role of the PC as a stabilizer. Due to its lipo- philic nature, the palmitoyl chain of PC and ARV both are expected to reside within lipid bilayers. A substantial change of zeta potential from 46.4 in PC free liposomes to 26.6 clearly indicated that carnitine head has occupied space on the liposomal surface. Positively charged LARPC would preferentially be taken up by tumor cells and facilitate its permeation into cells due to the negatively-charged neovasculature and acidic extracellular pH.
The cytotoXicity study of LARPC in vemurafenib-resistant cell lines showed lower IC50 in the comparison with single molecules especially PC, which means the cytotoXicity of LARPC is mainly due to ARV while PC did not show any antagonistic effect when combined with ARV. The very low IC50 of LARPC suggests the potency of the drug as well as its potential use for the vemurafenib-resistant melanoma treatment. Pre- viously, Sonja et al., reported the IC50 of palmitoyl carnitine in HepG2 cell lines to be 76 μM, which means protein kinase C inhibition does not have potent cytotoXicity but can be used as a cytotoXic agent at higher concentration [52]. The release study was carried out to predict the release behavior of ARV in vivo. Since the release of LARPC of ARV is negligible, we can conclude that ARV will not have burst release in plasma, but it will most likely release from LARPC after internalization into the tumor. Therefore, the side effects of the anti-cancer drugs could be minimized in the blood circulation.
Neovascularization plays an essential role in tumor growth and malignant melanoma has been known as an angiogenic tumor type with new blood vessel formation in the tumor progression process [53]. Angiogenesis is known as the growth of new blood vessels from a pre-existing vasculature and angiogenesis remodels the vascular network during tumor development, which provide the tumor with nutrition and oXygen for its growth and metastasis [54]. However, the response rate of patients varies with anti-angiogenic therapies, which limited the use of anti-angiogenic agents. Hence, it is crucial to look for new molecules that can effectively inhibit angiogenesis. Up to the pre- sent time, primary human umbilical vein endothelial cells (HUVECs) are the most widely used model for tube formation and for evaluating the angiogenic activity of endothelial cells after treatment using Matrigel in vitro [55]. Targeting BET bromodomain using BET inhibitor JQ1 has been shown to inhibit angiogenesis as a novel therapeutic method for the treatment of angiogenesis-related cancers [56,57]. The suppression of angiogenesis may be due to the loss of BRD4 in the regions of genes that participate in angiogenesis [58]. In our study, ARV – a BRD4 protein degrader, has a significant anti-angiogenic effect even at very low con- centrations. PC did not show cytotoXicity in melanoma cells but showed marked inhibition of HUVEC branching point and tube length. Thus, protein kinase C inhibition could be more helpful in inhibiting the growth of new blood vessels compared to tumor cells. Further, the combination of PC with ARV in the LARPC showed significantly higher inhibition of tube length but not in the number of branch points.
Fig. 5. Effect of PC, ARV and LARPC treatment on the colony forming ability of A375R and SK-MEL-28 R cell lines. (a) Colonies images stained with crystal violet after treatments followed by 5 days incubation with DMEM in A375R and SK-MEL-28 R. Cells are treated with 40 nM ARV, 360 nM PC and LARPC (contains 40 nM ARV and 360 nM PC) for 24 h. (b) Number of colonies with ARV, PC and LARPC treatment and control in A375R and SK-MEL-28 R.
Number of colonies with LARPC and ARV treatment were significantly reduced compared to control and PC treatment group (**p < 0.01).
A distinct characteristic of solid melanoma tumor is the ability of melanoma cells to form a blood vessel-like structure which is known as vasculogenic mimicry (VM). Such de novo vascular networks are observed in aggressive melanoma and often responsible for the poor clinical results [59]. This tumor cell-generated patterned channels play an important role in tumor development, invasion, and metastasis in- dependent of tumor angiogenesis. It was reported that the metastasis of melanoma is due to the overexpression of c-Myc, which promotes vas- culogenic mimicry via the c-Myc/snail/Bax signaling pathway [60]. c-Myc is proven to be a very challenging target due to its structure and therefore, targeting the BET bromodomain could be a therapeutic strategy for targeting c-Myc [61]. There is no previous report demon- strating the potential of BET degradation in inhibiting vasculogenic mimicry of melanoma cells. Both ARV and PC showed significant inhi- bition of the branching points, which may be due to the targeting effect of c-Myc and the role of PKC inhibitor. Interestingly, inhibition of the branching points by LARPC is more significant in A375R than A375, which indicates that combination is more efficient against vemurafenib resistant melanoma cells than parent melanoma cells.
Metastasis is detected in the late stage of advanced cancers and it is rendered that cell invasion starts from single colonial growth tumor cell or collective cell migration [62]. In the process of carcinoma metastasis, tumor cells initially go through the basement membrane and invade to the extracellular matriX (ECM), then migrate to the blood and lymphatic circulation and finally get embedded in the stromal ECM [63]. Thus, it is crucial to understand the proliferation characteristics of the tumor cells and the inhibiting potential of the treatment molecules. Clonogenic assay or colony formation assay is an in vitro cell survival-based assay that determines the ability of a cell to form a colony and indefinite di- vision. It is widely used to study cell reproductive death after treatment with ionizing radiation and the effects of radiation on cells, but it can also be used to determine the chemosensitivity of chemotherapy agents [48]. We found ARV and LARPC attenuated the ability of melanoma cells to form colonies while PC alone did not show any effect. Our result is in accordance with the previous work which states that ARV-825 has potent anti-clonogenic activity even compared to OTX015 and JQ1 in cholangiocarcinoma cells [64]. Downregulation of c-MYC was also shown to decrease the clonogenic potential in melanoma cell lines [65]. This reduction of clonogenic activity may be due to MYC-driven epige- netic reprogramming [66]. The migration study results suggested that the capability to grow and proliferate was significantly reduced after the treatment with ARV and LARPC. PC also showed inhibition of migration to some extent, but not as significant as LARPC or ARV, which implied that role of PC in the LARPC is mainly target towards angiogenesis and vasculogenic mimicry while the potential of inhibition of clonogenicity and migration are compromised. This also confirmed the crucial need for combining two molecules with different targeting mechanisms.
5. Conclusion
In conclusion, we provided a novel chemotherapeutic combination to treat vemurafenib-resistant melanoma in the present study. Protein kinase C inhibitor anchored BRD4 PROTAC (ARV) PEGylated nano- liposomes (LARPC) were successfully developed by the modified hy- dration method. This is the very first report on the preparation of parenteral liposomal formulation of the PROTAC class of molecules. Incorporation of PC not only enhanced anti-angiogenesis and anti- vasculogenic mimicry effects, but also enhanced the stability of LARPC and reduced the particle size. Hence, this work suggested that LARPC could be an approach ACBI1 for the treatment of vemurafenib-resistant melanoma.