KYP-2047, an Inhibitor of Prolyl-Oligopeptidase, Reduces GlioBlastoma Proliferation through Angiogenesis and Apoptosis Modulation

Simple Summary Glioblastoma (GB) is the most aggressive brain tumor characterized by necrosis, excessive proliferation, and invasiveness. Despite relevant progress in conventional treatments, the survival rate for patients with GB remains low. The present study investigated the potential effect of KYP-2047, an inhibitor of the prolyl-oligopeptidase (POP or PREP), in an in vivo U87-xenograft model and in an in vitro study on human GB cells. This study demonstrated the abilities of KYP-2047 to counteract and reduce GB progression through angiogenesis and apoptosis modulation. Abstract Glioblastoma (GB) is the most aggressive tumor of the central nervous system (CNS), characterized by excessive proliferation, necrosis and invasiveness. The survival rate for patients with GB still remains low. Angiogenesis and apoptosis play a key role in the development of GB. Thus, the modulation of angiogenesis and apoptosis processes represent a possible strategy to counteract GB progression. This study aimed to investigate the potential effect of KYP-2047, an inhibitor of the prolyl-oligopeptidase (POP), known to modulate angiogenesis, in an in vivo U87-xenograft model and in an in vitro study on human GB cells. Our results showed that KYP-2047 at doses of 2.5 mg/kg and 5 mg/kg was able to reduce tumor burden in the xenograft-model. Moreover, KYP-2047 significantly reduced vascular endothelial-growth-factor (VEGF), angiopoietins (Ang) and endothelial-nitric-oxide synthase (eNOS) expression. In vitro study revealed that KYP-2047 at different concentrations reduced GB cells’ viability. Additionally, KYP-2047 at the concentrations of 50 µM and 100 µM was able to increase the pro-apoptotic protein Bax, p53 and caspase-3 expression whereas Bcl-2 expression was reduced. Thus, KYP-2047 could represent a potential therapeutic treatment to counteract or reduce GB progression, thanks its abilities to modulate angiogenesis and apoptosis pathways.


Introduction
Gliomas are the main neoplastic diseases affecting the central nervous system (CNS) [1]. Among gliomas, glioblastoma (GB) is the most common primary malignant tumor of CNS, with an incidence of about 3-4 cases per 100,000 people per year [2]. GB is classified by the World Health Organization (WHO) as grade IV astrocytoma, characterized by poorly Furthermore, the control group + KYP-2047 1 mg/kg was only subjected to histological evaluation, mean tumor burden and mean tumor weight, because it did not induce any beneficial effect; therefore, we decided to continue analyzing only KYP-2047 2.5 mg/kg and 5 mg/kg groups.

Histological Evaluation
Histological evaluation was performed as previously described by Esposito et al. [16]. Tumor samples were fixed with 10% neutral formalin, embedded in paraffin, and sectioned at 7 µm. Sections were deparaffinized with xylene and stained with hematoxylin and eosin. The slides were analyzed by a pathologist blinded to the treatment groups. All sections were analyzed using an Axiovision microscope (Zeiss, Milan, Italy).

Caspase-3 Activity Measurement
Caspase-3 activity in tumor lysate was measured using a colorimetric Assay Kit (cat#ab39401, Abcam, Cambridge, UK) as suggested by manufacturer's instruction.

RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR)
Total RNA of tumor samples was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA isolation was performed as previously described by Weinert et al. [18]. First-strand cDNA obtained from RNA samples was stored at −80 • C until use.

Enzyme-Linked Immunosorbent Assay (ELISA) Kit
An enzyme-linked immunosorbent assay (ELISA) kit was performed to evaluate PREP expression in serum of each mice using Mouse PREP ELISA kit (cat#Q9QUR6 RayBiotech, Peachtree Corners, GA, USA) as suggested by manufacturer's instructions. The serum of each animal was collected and measured by ELISA kit once a week. Immunofluorescence staining was performed as previously described by Campolo et al. [20]. Tumor samples were collected and processed for immunofluorescence staining. Tissue sections of 7 µm were incubated with the following primary antibody anti-CD34 at 37 • C overnight (1:100; Santa Cruz Biotechnology, Dallas, TX, USA; sc-74499). Then, tissue sections were washed with PBS and incubated with secondary antibody anti-mouse Alexa Fluor-488 antibody (1:1000 v/v, Molecular Probes, Altrincham, UK) for 1 h at 37 • C. For nuclear staining, 4 ,6 -diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt, Germany) (2 µg/mL) in PBS was added. Sections were observed and photographed at 40× magnification using a Leica DM2000 microscope. The experiments were repeated three times to verify the data. For western blot analysis and immunofluorescence assay on U-87, A-172 and U-138 cells, we decided to continue to analyze only KYP-2047 at the concentrations of 50 µM and 100 µM because represented the most cytotoxic concentrations revealed by MTT assay.

Cell Viability Assay
Cell viability assay on U-87, U-138 and A-172 cells were performed using a mitochondriadependent dye for live cells (tetrazolium dye; MTT) to formazan [20]. GB cells were pre-treated with increasing concentrations of KYP-2047 for 24 h. After 24 h, cells were incubated at 37 • C with MTT (0.2 mg/mL) for 1 h. The medium was removed, and the cells lysed with dimethyl sulfoxide (DMSO) (100 µL). The extent of reduction in MTT to formazan was quantified by measurement of optical density (OD) at 550 nm with a microplate rider.

Western Blot Analysis
Western blot analysis on U-87, A-172 and U-138 cell lysates was performed as previously described by Campolo et al. [20]. Human GB cells were washed with ice-cold PBS harvested and resuspended in Tris-HCl 20 mM pH 7.5, NaF 10 mM, 150 µL NaCl, 1% Nonidet P-40 and protease inhibitor cocktail (Roche). After 40 min, cell lysates were centrifuged at 16,000× g for 15 min at 4 • C. Protein concentration was estimated by the Bio-Rad protein assay using bovine serum albumin as standard. Samples were then heated at 95 • C for 5 min and equal amounts of protein separated on a 10-15% SDS-PAGE gel and transferred to a PVDF membrane (Immobilon-P). The membranes were incubated overnight at 4 • C with primary antibodies: anti-Bax (1:500; Santa Cruz Biotechnology, Dallas, TX, USA; sc-7480); anti-Bcl2 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA; sc-7382); anti-p53 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA; sc-126). To ascertain that blots were loaded with equal amounts of protein lysate, they were also incubated with the antibody β-actin for cytosolic fraction (1:500; Santa Cruz Biotechnology; Dallas, TX, USA. sc-8432) and lamin A/C for nuclear fraction (1:500; Santa Cruz Biotechnology; Dallas, TX, USA, sc-376248). Signals were detected with enhanced chemiluminescence (ECL) detection system reagent according to the manufacturer's instructions (Thermo Fisher, Waltham, MA, USA). The relative expression of the protein bands was quantified by densitometry with BIORAD ChemiDocTMXRS + software.

Immunofluorescence Assay for Transforming Growth Factor-β (TGF-β) and Caspase-3
Immunofluorescence assay was performed on U-87, A-172 and U-138 cells as previously described by Donaldson [21]. GB cells on glass cover slips were rinsed briefly in phosphate-buffered saline (PBS:0.15 M NaCl, 10 mM Na 2 HPO 4 , pH 7.4), permeabilized in 0.2% Triton X-100/PBS and blocked with 10% goat serum. The cells were stained overnight (O/N) at 4 • C with primary antibodies: anti-transforming growth factor-β (TGFβ, 1:50, Santa Cruz Biotechnology, Dallas, TX, USA; sc-130348) and anti-caspase-3 (1:50, Santa Cruz Biotechnology, Dallas, TX, USA; sc-56053). At the end of the incubation with the primary antibody, the sections were abundantly washed with PBS and incubated with a secondary antibody anti-mouse Alexa Fluor-488 antibody (1:1000 v/v Molecular Probes, UK) for 1 h at 37 • C. Sections were washed in PBS and for nuclear staining 4 ,6 -diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt; Germany) 2 µg/mL in PBS was added. Sections were observed and photographed at 40× magnification using a Leica DM2000 microscope (Leica, Axio Vision, Feldbach, Schweiz). All images were digitalized at a resolution of 8 bits into an array of 2560 × 1920 pixels. Optical sections of fluorescence specimens were obtained using a HeNe laser (543 nm), an ultraviolet (UV) laser (361-365 nm), and an argon laser (458 nm) at a 1 min, 2 s scanning speed with up to eight averages; 1.5 µm sections were obtained using a pinhole of 250. Contrast and brightness were established by examining the most brightly labeled pixels and applying settings that allowed clear visualization of structural details while keeping the highest pixel intensities close to 250.

Materials
KYP-2047 and all other chemicals were obtained by Sigma-Aldrich (Milan, Italy). All stock solutions were prepared in non-pyrogenic saline (0.9% NaCl, Baxter, Milan, Italy).

Statistical Analysis
All values are expressed as mean ± standard error of the mean (SEM) of "n" observations. Each analysis was performed three times with three samples replicates for each one. The results were analyzed by one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant.

Effect of KYP-2047 on Tumor Growth
The histological analysis of the control group ( Figure 1A) showed a significant subcutaneous tumor mass, associated to an increase in necrosis and neutrophil infiltration; while the treatment with KYP-2047 at doses of 2.5 mg/kg and 5 mg/kg showed a reduction in tumor sections as well as neutrophil infiltration ( Figure 1C,D), much more than KYP-2047 at the dose of 1 mg/kg ( Figure 1B). Furthermore, we observed a marked reduction of mean tumor burden, tumor volume and tumor weight following KYP-2047 treatment at doses of 2.5 mg/kg and 5 mg/kg, much more than KYP-2047 1 mg/kg ( Figure 1E-G). Moreover, to better understand if the expression levels of PREP changed during the course of treatment in the tumors, we decided to verify the expression of PREP during the treatment with KYP-2047 by ELISA kit. The results showed that KYP-2047 at doses of 2.5 mg/kg and 5 mg/kg was able to reduce significantly PREP levels particularly from day 14 ( Figure 1H). During the course of treatment, no important change in animals' weight was seen ( Figure 1I).
All values are expressed as mean ± standard error of the mean (SEM) of "n" observations. Each analysis was performed three times with three samples replicates for each one. The results were analyzed by one-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test for multiple comparisons. A p-value of less than 0.05 was considered significant.

Effect of KYP-2047 on Tumor Growth
The histological analysis of the control group ( Figure 1A) showed a significant subcutaneous tumor mass, associated to an increase in necrosis and neutrophil infiltration; while the treatment with KYP-2047 at doses of 2.5 mg/kg and 5 mg/kg showed a reduction in tumor sections as well as neutrophil infiltration ( Figure 1C,D), much more than KYP-2047 at the dose of 1 mg/kg ( Figure 1B). Furthermore, we observed a marked reduction of mean tumor burden, tumor volume and tumor weight following KYP-2047 treatment at doses of 2.5 mg/kg and 5 mg/kg, much more than KYP-2047 1 mg/kg ( Figure 1E-G). Moreover, to better understand if the expression levels of PREP changed during the course of treatment in the tumors, we decided to verify the expression of PREP during the treatment with KYP-2047 by ELISA kit. The results showed that KYP-2047 at doses of 2.5 mg/kg and 5 mg/kg was able to reduce significantly PREP levels particularly from day 14 ( Figure 1H). During the course of treatment, no important change in animals' weight was seen ( Figure  1I).

Effect of KYP-2047 on Angiogenesis
Angiogenesis is an essential process for tumor growth [22]. GB is characterized by a deregulation of angiogenic growth factors as VEGF and eNOS expression, which play a key role in maintaining vascular homeostasis and vessel integrity [22][23][24]. Therefore, in this study we decided to investigate by immunohistochemical staining the levels of VEGF and eNOS. Our results demonstrated a significant increase of VEGF and eNOS levels in the control group (Figures 2A and 3A respectively); however, the treatment with KYP-2047 at doses of 2.5 mg/kg and 5 mg/kg significantly reduced their expression ( Figure 2B,C, see immunohistochemistry score Figure 2D; Figure 3B,C, see immunohistochemistry score Figure 3D respectively) in a dose-dependent manner. These results were confirmed also by Western blot analysis and RT-qPCR, showing a significantly reduction of VEGF and eNOS expression in the groups treated with KYP-2047 at doses of 2.5 mg/kg and 5 mg/kg compared to control group ( Figure 2M, see densitometric analysis Figure 2M1,N and Figure 3E, see densitometric analysis Figure 3E1,F).
Additionally, we evaluated the expression of CD34, a transmembrane glycoprotein involved in the process of newly-forming tumour vessels [25] by immunohistochemistry and immunofluorescence analysis. In this context, our results showed a significant reduction of CD34 expression in the groups treated with KYP-2047 at doses of 2.5 mg/kg and 5 mg/kg compared to control group (   Studies on angiogenesis have emphasized the importance of others angiogenic factors involved in tumor growth such as angiopoietins, in particular angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2), currently proposed as biomarkers of GB [26,27]. Therefore, we detected Ang1 and Ang2 expression by Western blot analysis on tumor samples. Our results showed a significantly decrease of Ang1 and Ang2 levels following KYP-2047 treatment at doses of 2.5 mg/kg and 5 mg/kg compared to control group ( Figure 4A, see densitometric analysis 4A1; Figure 4B, see densitometric analysis 4B1) in a dose-dependent manner.
Furthermore, we investigated the role of Ki-67, a nuclear protein associated with tumor proliferation and progression [28,29]. As shown in the Figure 4C, the blot revealed a marked expression of Ki-67 in the control group whereas the treatment with KYP-2047 at doses of 2.5 mg/kg and 5 mg/kg significantly reduced its expression (see densitometric analysis 4C1). Moreover, Ki-67 was evaluated also by immunohistochemistry assay confirming the results obtained as showed in the Figure 4D-F (see immunohistochemistry score 4G). Studies on angiogenesis have emphasized the importance of others angiogenic factors involved in tumor growth such as angiopoietins, in particular angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2), currently proposed as biomarkers of GB [26,27]. Therefore, we detected Ang1 and Ang2 expression by Western blot analysis on tumor samples. Our results showed a significantly decrease of Ang1 and Ang2 levels following KYP-2047 treatment at doses of 2.5 mg/kg and 5 mg/kg compared to control group ( Figure 4A, see densitometric analysis 4A1; Figure 4B, see densitometric analysis 4B1) in a dose-dependent manner.
Furthermore, we investigated the role of Ki-67, a nuclear protein associated with tumor proliferation and progression [28,29]. As shown in the Figure 4C, the blot revealed a marked expression of Ki-67 in the control group whereas the treatment with KYP-2047 at doses of 2.5 mg/kg and 5 mg/kg significantly reduced its expression (see densitometric analysis 4C1). Moreover, Ki-67 was evaluated also by immunohistochemistry assay confirming the results obtained as showed in the Figure 4D-F (see immunohistochemistry score 4G).

Effect of KYP-2047 on Apoptosis Pathway
Considering the key role of apoptosis in GB progression [30], we evaluated the proapoptotic Bax, and anti-apoptotic Bcl2 protein by western blot analysis on tumor samples. The results showed that KYP-2047 was able to increase Bax expression and reduce Bcl2 expression ( Figure 5A; see densitometric analysis 5A1; Figure 5B, see densitometric analysis 5B1). Moreover, the ability of KYP-2047 to modulate Bcl2 expression was confirmed by immunohistochemistry as shown in Figure 5C-E (see immunohistochemistry score Figure 5F). Furthermore, we detected caspase-3 levels by immunohistochemistry and by a colorimetric assay kit on tumor samples, showing that KYP2047 at doses of 2.5 and 5 mg/kg significantly increased caspase-3 activity compared to the control group ( Figure  6A-C; see immunohistochemistry score Figure 6D,E

Effect of KYP-2047 on Apoptosis Pathway
Considering the key role of apoptosis in GB progression [30], we evaluated the proapoptotic Bax, and anti-apoptotic Bcl2 protein by western blot analysis on tumor samples. The results showed that KYP-2047 was able to increase Bax expression and reduce Bcl2 expression ( Figure 5A; see densitometric analysis 5A1; Figure 5B, see densitometric analysis 5B1). Moreover, the ability of KYP-2047 to modulate Bcl2 expression was confirmed by immunohistochemistry as shown in Figure 5C-E (see immunohistochemistry score Figure 5F). Furthermore, we detected caspase-3 levels by immunohistochemistry and by a colorimetric assay kit on tumor samples, showing that KYP2047 at doses of 2.5 and 5 mg/kg significantly increased caspase-3 activity compared to the control group ( Figure

Effect of KYP-2047 on Apoptosis Pathway
Apoptosis plays a key role in the development of cancer including GB [31]. Deregulation of apoptotic process is a relevant hallmark of a tumor [31], responsible not only for its progression but also for tumor resistance to therapies [32]. Therefore, we investigated the effect of KYP-2047 on the apoptosis pathway in U-87, A-172 and U-138 cell lysates evaluating the pro-apoptotic Bax, tumor suppressor p53 and anti-apoptotic Bcl2 protein by Western blot analysis (The original Western blot can be found in Figure S5). Our results revealed an increase of Bax and p53 levels following KYP-2047 treatment in U87 cell lysates performed for 24 h at the concentrations of 50 µM and 100 µM compared to control group ( Figure 8A, see densitometric analysis 8A1; Figure 8B, see densitometric analysis 8B1, respectively); while Bcl2 expression was significantly reduced following KYP-2047 treatment compared to control group ( Figure 8C; see densitometric analysis 8C1) in a concentration-dependent manner. The same results appear for A-172 and U-138 cell lysates, confirming an increase of pro-apoptotic Bax and p53 expression following KYP-2047 treatment compared to control group ( Figure S1A, see densitometric analysis 1A1; Figure S1B, see densitometric analysis 1B1, respectively) ( Figure S2A, see densitometric analysis 2A1; Figure S2B, see densitometric analysis 2B1, respectively) and a decrease of anti-apoptotic

Effect of KYP-2047 on Apoptosis Pathway
Apoptosis plays a key role in the development of cancer including GB [31]. Deregulation of apoptotic process is a relevant hallmark of a tumor [31], responsible not only for its progression but also for tumor resistance to therapies [32]. Therefore, we investigated the effect of KYP-2047 on the apoptosis pathway in U-87, A-172 and U-138 cell lysates evaluating the pro-apoptotic Bax, tumor suppressor p53 and anti-apoptotic Bcl2 protein by Western blot analysis (The original Western blot can be found in Figure S5). Our results revealed an increase of Bax and p53 levels following KYP-2047 treatment in U87 cell lysates performed for 24 h at the concentrations of 50 µM and 100 µM compared to control group ( Figure 8A, see densitometric analysis 8A1; Figure 8B, see densitometric analysis 8B1, respectively); while Bcl2 expression was significantly reduced following KYP-2047 treatment compared to control group ( Figure 8C; see densitometric analysis 8C1) in a concentration-dependent manner. The same results appear for A-172 and U-138 cell lysates, confirming an increase of pro-apoptotic Bax and p53 expression following KYP-2047 treatment compared to control group ( Figure S1A, see densitometric analysis 1A1; Figure S1B, see densitometric analysis 1B1, respectively) ( Figure S2A, see densitometric analysis 2A1; Figure S2B, see densitometric analysis 2B1, respectively) and a decrease of anti-apoptotic Bcl2 protein expression ( Figure S1C; see densitometric analysis 1C) ( Figure S2C; see densitometric analysis 2C1).

Effect of KYP-2047 on TGF-β and Caspase-3 Expression by Immunofluorescence Assay
Current studies have focused on the role of TGF-β in the tumor microenvironment suggesting that it plays a key role for GB progression [33,34]. Therefore, we investigated In addition to the regulation of the cell cycle and differentiation, TGF-β is able to induce apoptosis [35] promoting the activation of pro-apoptotic caspase-3, a member of the cysteine-aspartic acid protease family [36]. Thus, in this study we detected caspase-3 expression by immunofluorescence assay in all three GB cell lines. The results obtained showed an increase of caspase-3 expression following KYP-2047 treatment at the concentrations of 50 µM and 100 µM compared to the control group in U-87 cells ( Figure 9E

Effect of KYP-2047 on TGF-β and Caspase-3 Expression by Immunofluorescence Assay
Current studies have focused on the role of TGF-β in the tumor microenvironment suggesting that it plays a key role for GB progression [33,34]. Therefore, we investigated In addition to the regulation of the cell cycle and differentiation, TGF-β is able to induce apoptosis [35] promoting the activation of pro-apoptotic caspase-3, a member of the cysteine-aspartic acid protease family [36]. Thus, in this study we detected caspase-3 expression by immunofluorescence assay in all three GB cell lines. The results obtained showed an increase of caspase-3 expression following KYP-2047 treatment at the concentrations of 50 µM and 100 µM compared to the control group in U-87 cells ( Figure 9E

Discussion
Glioblastoma (GB) is the most common and aggressive primary brain tumor in adults [37]. GB arise from glial cells but can also develop from astrocytic or neural stem/progenitor cells [38]. GB can be classified into primary and secondary subtypes, based on preexisting lesion [38]. The primary GB subtype develops rapidly de novo in elderly patients without clinical or histologic evidence, whereas the secondary subtype develops from evolution of low-grade astrocytic tumours over the course of 4-5 years [38]. In the last decade, many studies have focused on the role of genetic mutations which contribute to GB initiation as TP53 and isocitrate dehydrogenase (IDH) mutations [37,39]. GB is characterized by a high degree of invasiveness, cell proliferation, angiogenesis and apoptosis alteration [2]. Despite scientific advances, the survival rate for patients with GB remains low and additional therapies are needed [12]. Previous studies have demonstrated that angiogenesis and apoptosis play a key role in GB pathology promoting cell survival and proliferation [10,31]. Therefore, the modulation of angiogenesis and apoptosis processes represent a valid strategy to counteract or reduce GB progression. KYP-2047 (4-phenylbutanoyl-L-prolyl-2(S)-cyanopyrrolidine) was developed as a highly specific and potent POP (or PREP) inhibitor, a serine protease involved in the angiogenesis process [40]. Recent studies revealed that KYP-2047 was able to modulate not only angiogenesis [11] but also cell cycle and differentiation [12,13]. Therefore, in this study we investigated the potential effect of KYP-2047 on angiogenesis and apoptosis pathways in an in vivo U87-xenograft model and in vitro study on the human GB cell line.
Firstly, we evaluated the ability of KYP-2047 to inhibit tumor growth in the xenograft model. Our results showed a high-grade necrosis and neutrophil infiltration in the control group, while KYP-2047 at higher doses significantly reduced subcutaneous tumor mass

Discussion
Glioblastoma (GB) is the most common and aggressive primary brain tumor in adults [37]. GB arise from glial cells but can also develop from astrocytic or neural stem/progenitor cells [38]. GB can be classified into primary and secondary subtypes, based on pre-existing lesion [38]. The primary GB subtype develops rapidly de novo in elderly patients without clinical or histologic evidence, whereas the secondary subtype develops from evolution of low-grade astrocytic tumours over the course of 4-5 years [38]. In the last decade, many studies have focused on the role of genetic mutations which contribute to GB initiation as TP53 and isocitrate dehydrogenase (IDH) mutations [37,39]. GB is characterized by a high degree of invasiveness, cell proliferation, angiogenesis and apoptosis alteration [2]. Despite scientific advances, the survival rate for patients with GB remains low and additional therapies are needed [12]. Previous studies have demonstrated that angiogenesis and apoptosis play a key role in GB pathology promoting cell survival and proliferation [10,31]. Therefore, the modulation of angiogenesis and apoptosis processes represent a valid strategy to counteract or reduce GB progression. KYP-2047 (4-phenylbutanoyl-L-prolyl-2(S)-cyanopyrrolidine) was developed as a highly specific and potent POP (or PREP) inhibitor, a serine protease involved in the angiogenesis process [40]. Recent studies revealed that KYP-2047 was able to modulate not only angiogenesis [11] but also cell cycle and differentiation [12,13]. Therefore, in this study we investigated the potential effect of KYP-2047 on angiogenesis and apoptosis pathways in an in vivo U87-xenograft model and in vitro study on the human GB cell line.
Firstly, we evaluated the ability of KYP-2047 to inhibit tumor growth in the xenograft model. Our results showed a high-grade necrosis and neutrophil infiltration in the control group, while KYP-2047 at higher doses significantly reduced subcutaneous tumor mass as well as neutrophil infiltration. Moreover, KYP-2047 significantly decreased mean tumor burden and tumor weight at higher doses, without encountering important weight differences.
Interestingly, treatment with KYP-2047 was able to reduce. PREP levels in serum of animals, particularly from day 14.
GB is one of the most highly angiogenic solid tumor [41]. Its tumor vasculature is both structurally and functionally abnormal, characterized by a dense network of vessels tortuous with increased diameter and thickened basement membranes [41]. Thus, angiogenesis is considered as a pathologic hallmark of GB, leading to VEGF activation, an angiogenic growth factor that promotes glioblastoma proliferation and CD34 activation, a transmembrane glycoprotein involved in the process of newly-forming tumour vessels [25,42]. Therefore, in this study we investigated VEGF and CD34 expression, showing that KYP-2047 at higher doses was able to reduce their expression significantly compared to the control group. Moreover, we investigated the role of eNOS, a relevant endothelial enzyme that modulates vascular homeostasis and vessel integrity [24]. In this context, our results showed that the control group was characterized by an increase of eNOS expression, whereas KYP-2047 significantly reduced eNOS expression.
The formation of new blood vessels is an essential process for GB growth [22]. In addition to VEGF, this process requires the involvement of other angiogenic factors as the angiopoietins, in particular angiopoietin 1 (Ang1) and angiopoietin 2 (Ang2) which have similar functions [26,43]. Previous studies revealed that Ang1 and Ang2 regulate vascular development and remodelling, promoting tumor growth [43,44]. Therefore, we decided to investigate the expression of Ang1 and Ang2 in GB; our results showed that the control group was characterized by an increase of Ang1 and Ang2 expression, while the treatment with KYP-2047 was able to significantly reduce their expression, inhibiting GB proliferation.
An increased vascularization provides to the tumor cells more oxygen and nutrients, promoting metastatic spread and cell proliferation [22]. In this context, Mastronardi et al. evaluated the correlation between angiogenesis and proliferation processes, through Ki-67 evaluation, a nuclear protein that regulates the cell cycle and differentiation [45]. Ki-67 is considered a relevant marker of tumor proliferation in GB [45,46]. Thus, we decided to evaluate Ki-67 expression, demonstrating that the control group was characterized by an increase of Ki-67 level, while KYP-2047 treatment was able to significantly reduce its expression.
Moreover, considering the key role of apoptosis in GB progression [3], we decided to investigate Bax, Bcl2 and caspase-3 expression in the U87-xenograft model, showing that KYP-2047 at doses of 2.5 mg/kg and 5 mg/kg was able to increase pro-apoptotic Bax and caspase-3 expression while Bcl2 expression was significantly reduced following KYP-2047 treatment.
To confirm the promising results obtained by an in vivo U87-xenograft model, we decided to conduct an in vitro model of GB on U-87, A-172 and U-138 cell lines. Firstly, we evaluated the cytotoxicity of KYP-2047 at different concentrations on U-87, A-172 and U-138 GB cells, demonstrating that KYP-2047 was able to significantly reduce cell viability in all three GB cell lines in a concentration-dependent manner.
Previous studies revealed that also apoptosis plays a key role in the development of GB [31,47]. It has been demonstrated that a down-regulation of apoptosis is associated with tumor survival [31,47]. Therefore, in this study we decided to evaluate the effect of KYP-2047 on the apoptosis pathway by evaluating protein levels of pro-apoptotic Bax, p53 and anti-apoptotic Bcl2 on U-87, A-172 and U-138 cell lysates. Our results revealed that KYP-2047 reduced Bcl2 expression, while Bax and p53 expression were significantly increased following KYP-2047 treatment in a concentration-dependent manner in all three GB cell lines, confirming apoptosis modulation.
Tumor proliferation is associated with an increase of TGF-β expression [48]. TGF-β regulates cell differentiation and apoptosis, promoting caspase-3 activation, a key regulator in apoptotic pathway [36]. Thus, we investigated the expression of TGF-β and caspase-3 in the in vitro model [49].
The results showed an increase of TGF-β expression in the control group while KYP-2047 treatment significantly reduced its expression in U-87, A-172 and U-138 cell lines. In addition, a marked increase of pro-apoptotic caspase-3 expression was revealed following KYP-2047 treatment, highlighting the ability of KYP-2047 to modulate apoptosis in all three GB cell lines.
Thus, the results obtained in an in vivo xenograft model and in an in vitro study on human GB cell lines revealed that KYP-2047 was able to reduce GB progression and growth by modulating angiogenesis and apoptosis pathways. Therefore, KYP-2047 could be considered as an alternative therapeutic strategy to counteract or reduce GB progression.

Conclusions
The data obtained revealed the ability of KYP-2047 to modulate angiogenesis and apoptosis pathways in an in vivo xenograft model and in an in vitro model of GB, reducing tumor progression. Therefore, on the basis of these results, KYP-2047 could represent an available strategy for the treatment of GB.
Author Contributions: S.A.S. prepared the manuscript; S.A.S., G.C., and A.A. performed experiments; S.F., L.C., and S.S. carried out formal analysis; I.P., E.E., and S.C. planned the experiments and critically revised the manuscript. M.C. supervised the research and revised the manuscript. All authors have read and agreed to the published version of the manuscript.