Role of a Novel Heparanase Inhibitor on the Balance between Apoptosis and Autophagy in U87 Human Glioblastoma Cells

Background: Heparanase (HPSE) is an endo-β-glucuronidase that cleaves heparan sulfate side chains, leading to the disassembly of the extracellular matrix, facilitating cell invasion and metastasis dissemination. In this research, we investigated the role of a new HPSE inhibitor, RDS 3337, in the regulation of the autophagic process and the balance between apoptosis and autophagy in U87 glioblastoma cells. Methods: After treatment with RDS 3337, cell lysates were analyzed for autophagy and apoptosis-related proteins by Western blot. Results: We observed, firstly, that LC3II expression increased in U87 cells incubated with RDS 3337, together with a significant increase of p62/SQSTM1 levels, indicating that RDS 3337 could act through the inhibition of autophagic-lysosomal flux of LC3-II, thereby leading to accumulation of lipidated LC3-II form. Conversely, the suppression of autophagic flux could activate apoptosis mechanisms, as revealed by the activation of caspase 3, the increased level of cleaved Parp1, and DNA fragmentation. Conclusions: These findings support the notion that HPSE promotes autophagy, providing evidence that RDS 3337 blocks autophagic flux. It indicates a role for HPSE inhibitors in the balance between apoptosis and autophagy in U87 human glioblastoma cells, suggesting a potential role for this new class of compounds in the control of tumor growth progression.


Introduction
The endo-β-glucuronidase heparanase (HPSE) is the primary enzyme that cleaves heparan sulfate (HS) side chains linked to proteoglycan core proteins, leading to the disassembly of the extracellular matrix (ECM). This event is therefore involved in the main biological phenomena associated with tissue remodeling and cell invasion, including inflammation, angiogenesis, and metastasis [1][2][3][4].
HPSE is synthesized in the endoplasmic reticulum as a precursor of 68 kDa, which, in the Golgi, is then processed in proHPSE (65 kDa). After secretion in the extracellular space as a latent 65-kDa enzyme, HPSE rapidly interacts with membrane heparan sulfate proteoglycans (HSPGs), such as syndecans [5,6] for being endocytosed and processed into a highly active 50-kDa enzyme [7]. HPSE has been shown to reside primarily within endocytic vesicles, and it assumes a polar, perinuclear localization colocalizing with lysosomal markers [8]. Recent studies have identified cathepsin L, a lysosomal protease, responsible for the processing and activation of HPSE [9,10]. Activated HPSE may have different destinations in the cell; it can be secreted, included in autophagosomes, shuttled into the nucleus, or anchored on the surface of exosomes [11][12][13].
we investigated the role of the HPSE inhibitor RDS 3337 (7 g) [33] in the regulation of the autophagic process and the balance between apoptosis and autophagy in U87 human glioblastoma cells. nanomolar range (80 nM). It is worthy of note that, while RDS 3337 is less potent than roneparstat, one of the most potent inhibitors of HPSE described (that is, in clinical trials), the amount potentially needed for clinical use could be about half of that of roneparstat, due to their enormous difference in molecular weight. Of interest, it has been shown that this compound was able to reduce the in vitro invasive ability of human glioblastoma cell lines (U87), although the underlying mechanisms involved are not completely clear. In this research, we investigated the role of the HPSE inhibitor RDS 3337 (7 g) [33] in the regulation of the autophagic process and the balance between apoptosis and autophagy in U87 human glioblastoma cells.

Cell Cultures and Treatments
RPE-1 human non-cancer neuro-ectodermal cells (ATCC) were grown in DMEM-F12, and human SK-N-BE2 (ATCC) neuroblastoma cells were grown in an RPM1 1640 medium. Human glioblastoma U87 cells (ATCC, Manassas, VA, USA) were grown in Dulbecco's modified Eagle medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA). All culture media were supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humified 5% CO2 atmosphere. All experiments were carried out using cells split no more than seven times. For autophagy induction, RPE1 cells were stimulated under the condition of nutrient deprivation with Hanks' Balanced Salt Solution (HBSS, Sigma H9269) for 16 h at 37 °C. The optimal incubation time with HBSS was selected on the basis of preliminary experiments. To inhibit autophagic flux, 100 nM Bafilomycin A1 (Baf A1) (Sigma-Aldrich B1793) was added 2 h before lysis.

In Vitro Treatment with HPSE Inhibitors
Cells were treated with the benzazolyl derivative RDS 3337 endowed with potent anti-HPSE activity, as described previously [33]. The compound RDS 3337 was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) at the 10 mM stock solution and then used at the concentration of 80, 320, and 1280 nM. We considered vehicle cells without any treatment, with only culture medium plus DMSO used as a vehicle to dissolve the compound. HPSE activity was also evaluated, as reported in Supplementary Figure S1.

Trypan Blue Assay
As previously reported [33], Trypan Blue (Sigma-Aldrich) assay was used to evaluate the cell viability of both RPE-1 and U87 cells. Cells were seeded into cell culture plates at a concentration of 5 × 10 5 cells/mL and kept for 24 h at 37 °C with 5% CO2. Then, cells were treated with different concentrations of RDS 3337 (80, 320, 1280 nM) for an incubation time of 24, 48, or 72 h. Vehicle-treated cells or cells incubated with RDS 3337 were analyzed by Trypan Blue (Sigma-Aldrich) assay to assess cell viability. DMSO is the vehicle to dissolve the compound, and we consider cells with only DMSO as vehicle-treated cells. All experiments were carried out in quintuplicate.

Cell Cultures and Treatments
RPE-1 human non-cancer neuro-ectodermal cells (ATCC) were grown in DMEM-F12, and human SK-N-BE2 (ATCC) neuroblastoma cells were grown in an RPM1 1640 medium. Human glioblastoma U87 cells (ATCC, Manassas, VA, USA) were grown in Dulbecco's modified Eagle medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA). All culture media were supplemented with 10% fetal bovine serum (FBS) at 37 • C in a humified 5% CO 2 atmosphere. All experiments were carried out using cells split no more than seven times. For autophagy induction, RPE1 cells were stimulated under the condition of nutrient deprivation with Hanks' Balanced Salt Solution (HBSS, Sigma H9269) for 16 h at 37 • C. The optimal incubation time with HBSS was selected on the basis of preliminary experiments. To inhibit autophagic flux, 100 nM Bafilomycin A1 (Baf A1) (Sigma-Aldrich B1793) was added 2 h before lysis.

In Vitro Treatment with HPSE Inhibitors
Cells were treated with the benzazolyl derivative RDS 3337 endowed with potent anti-HPSE activity, as described previously [33]. The compound RDS 3337 was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) at the 10 mM stock solution and then used at the concentration of 80, 320, and 1280 nM. We considered vehicle cells without any treatment, with only culture medium plus DMSO used as a vehicle to dissolve the compound. HPSE activity was also evaluated, as reported in Supplementary Figure S1.

Trypan Blue Assay
As previously reported [33], Trypan Blue (Sigma-Aldrich) assay was used to evaluate the cell viability of both RPE-1 and U87 cells. Cells were seeded into cell culture plates at a concentration of 5 × 10 5 cells/mL and kept for 24 h at 37 • C with 5% CO 2 . Then, cells were treated with different concentrations of RDS 3337 (80, 320, 1280 nM) for an incubation time of 24, 48, or 72 h. Vehicle-treated cells or cells incubated with RDS 3337 were analyzed by Trypan Blue (Sigma-Aldrich) assay to assess cell viability. DMSO is the vehicle to dissolve the compound, and we consider cells with only DMSO as vehicle-treated cells. All experiments were carried out in quintuplicate.

BrdU Cell Proliferation Assay
Cell proliferation was analyzed by the BrdU/anti-BrdU method. Cells were seeded in 6-well plates (5 × 10 5 ) in the presence or absence of the test compound and incubated for 24, 48, or 72 h. Next, cells were labeled for 90 min with 10 µM of BrdU (Sigma-Aldrich) and washed twice with ice-cold PBS. Subsequently, cells were detached, and the cell pellet was resuspended in ice-cold PBS and fixed in ice-cold acetone/methanol (1:5, v:v) for 1 h at 4 • C. Cells were washed twice with PBS containing Tween 20 (0.5%), followed by incubation with 2 N HCl (Merck, Darmstadt, Germany) for 45 min at room temperature. The cells were then washed twice, and the pellet resuspended in 0.1 M Na 2 B 4 O 7 ; after washing in PBS/Tween, cells were incubated with FITC-conjugated anti-BrdU (Becton Dickinson Biosciences, San Jose, CA, USA) at RT for 30 min. After two washes in PBS/Tween, the cells were analyzed by a CytoFlex flow cytometer (Beckman Coulter, Coulter Brea, CA, USA).

Preparation of Cell Extracts
Cells untreated or treated with RDS 3337 for 18 or 72 h at 37 • C in 5% CO 2 in the presence or absence of bafilomycin A1 (Baf A1; 100 nM) were lysed in lysis buffer, containing 20 mM HEPES, pH 7.2; 1% Nonidet P-40, 10% glycerol, 50 mM NaF, 1 mM Na 3 VO 4 , and a protease inhibitors cocktail (Sigma-Aldrich). Proteins were recovered after centrifugation of lysates at 15,000× g for 15 min at 4 • C. Then, samples were resuspended in a lysis buffer, and whole-cell extracts were obtained, as reported above. Protein contents were determined by Bradford assay.

Autophagy Evaluation by Flow Cytometry
RPE-1 cells were starved with HBSS for 16 h, or treated with 320 nM RDS 3337 for 72 h in the presence or absence of bafilomycin A1 (Baf A1; 100 nM). At the end of treatment, cells were analyzed by flow cytometry after single staining with a Cyto-ID detection kit (ENZ-51031-K200, Enzo Life Sciences, Exeter, UK). This assay was optimized for the evaluation of autophagy at the cellular level by flow cytometry using a 488 nm-excitable probe that becomes fluorescent in autophagic vesicles (autophagosomes) produced during autophagy. To detect p62/SQSTM1 levels, cells were analyzed by flow cytometry after fixation with 4% paraformaldehyde in PBS and permeabilization with 0.5% Triton X-100 in PBS for 5 min, with anti-p62/SQSTM1 (rabbit, Cell Signaling Technology) primary antibodies followed by anti-rabbit Alexa Fluor 488 (A11008, Invitrogen, Waltham, MA, USA). A representative experiment among 3 is shown. The bar graph reports the mean ± SD obtained in three independent experiments. U87 cells were seeded at a density of 5 × 10 5 cells/mL per well. After an incubation time of 24 h, 48 h, or 72 h with RDS 3337 at the concentration of 320 nM at 37 • C in 5% CO 2 , cells were collected, centrifuged, and resuspended in a fresh medium. Alternatively, cells were incubated with RDS 3337 for 24 h, 48 h, or 72 h and then with HBSS for 16 h. As a positive control for apoptosis, cells were treated with 1 µM staurosporine (STS) (Sigma-Aldrich) for 8 h at 37 • C in 5% CO 2 . After, they were washed with PBS, fixed in 70% ethanol in PBS for 1 h at 4 • C, washed twice with PBS, resuspended in 125 µL of PBS, 12.5 µL of 5 µg/mL RNase (Sigma-Aldrich), and then stained with 125 µL of 100 µg/mL PI (Sigma-Aldrich). Lastly, cells were incubated for 30 min in the dark at RT before analyzing their DNA content. The fluorescence was measured by a Cytoflex flow cytometer (Beckman Coulter Brea).

Statistical Analysis
All the statistical procedures were performed by GraphPad Prism Software Inc. (San Diego, CA, USA). All data were verified in at least 3 different experiments in duplicate and reported as mean ± standard deviation (SD). Normally distributed variables were summarized using the mean ± SD. The p-values for all graphs were generated using Student's t-test as indicated in the figure legends; * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001.

HPSE Inhibitor RDS 3337 Promotes LC3-II and p62 Accumulation in RPE-1 Cells
It is known that HPSE has been recently shown to play a role in the autophagy of cancer cells, leading to chemoresistance and tumor progression. Thus, in this study, we first examined HPSE inhibitor RDS 3337 effects on human non-cancer neuro-ectodermal cell line RPE-1, which represents a non-transformed alternative to cancer cell lines. With this aim, we used the HPSE inhibitor RDS 3337, a compound with high anti-HPSE activity, showing nanomolar potency, with an IC 50 value of 80 nM. First of all, we tested the cytotoxic effect of RDS 3337 under our experimental conditions, using both Trypan Blue and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2h-tetrazolium, and monosodium salt (WST-1) assays. RPE-1 cells were treated with RDS 3337 at different concentrations (80-1280 nM) and analyzed by both cell counting and cell viability, as shown in Figure 2A,B.
Since the highest concentration (1280 nM) showed a significantly higher cytotoxic effect, as revealed by Trypan Blue, as well as by WST-1 assay, we selected the dose of 320 nM. Similar findings were found using the BrdU assay ( Figure 2C). Next, to explore the effect of HPSE inhibitor RDS 3337 on the expression of the autophagic marker microtubule-associated protein1 light chain 3 (LC3), we employed immunoblotting analysis in RDS 3337-treated and untreated RPE-1 cells. As shown in Figure 2D, the analysis revealed a mild but significant increase of LC3 II after incubation with 320 nM RDS 3337 for 18 h or 72 h. Quantitative analysis confirmed these data ( Figure 2D, see histograms). This finding suggests an accumulation of autophagosomes [34]. As expected, when cells were stimulated with HBSS for 16 h at 37 • C for autophagy induction, the analysis revealed an increase of LC3 II levels together with a significant decrease Cells 2023, 12, 1891 6 of 14 of p62/SQSTM1 levels, as confirmed by densitometric analysis. This finding indicates the activation of the autophagic flux [34]. On the contrary, when HBSS pre-treated cells were successively exposed to RDS 3337 (18 h or 72 h), the increase of LC3 II was accompanied by a significant enhancement of p62/SQSTM1 levels ( Figure 2D, see histograms), indicating an impairment of the autophagic clearance. Indeed, p62/SQSTM1 is a selective autophagy receptor, which sequesters ubiquitinated proteins into autophagosome vesicles by interacting with LC3. Moreover, since p62 is a substrate for autophagic degradation, its degradation can represent a marker of autophagic clearance. Supplementary Figure S2. As expected, this methodological approach is less sensitive as compared to Western blot for this test.
In sum, these findings indicate that HPSE inhibitor RDS 3337 induces a significant increase of Cyto-ID staining, with a high level of p62/SQSTM1 in RPE-1 cells pre-treated with HBSS, suggestive of an autophagosomes accumulation and a consistent arrest of the autophagic flux.  were analyzed by Western blot, using rabbit anti-LC3 pAb or rabbit anti-SQSTM1 mAb. Anti-actin mAb was used as a loading control. A representative experiment among the three is shown. The bar graph on the right shows densitometric analysis. Results represent the mean ± SD from three independent experiments. * p < 0.05, ** p < 0.005, *** p < 0.001, **** p < 0.0001. No statistically significant differences were found between BafA1 and RDS 3337 18 h or 72 h + BafA1 samples. (E) Autophagy evaluation by flow cytometry in RPE-1 cells. Cells were starved with HBSS for 16 h, or treated with 320 nM RDS3337 for 72 h, and the autophagic flux was monitored in the presence or absence of 100 nM Baf A1. At the end of treatment, cells were analyzed by flow cytometry after single staining with a Cyto-ID autophagy detection kit. To detect p62/SQSTM1 levels, cells were analyzed by flow cytometry after fixation with 4% paraformaldehyde in PBS and permeabilization by 0.5% Triton X-100 in PBS for 5 min at room temperature, with anti-p62/SQSTM1, followed by anti-rabbit Alexa Fluor 488. The bar graph reports the mean ± SD obtained in three independent experiments. * p < 0.05 versus vehicle, ** p < 0.005 versus vehicle, *** p < 0.0001 versus vehicle, **** p < 0.0001 versus vehicle.
To discriminate between autophagy inducers and blockers, we blocked autophagy with bafilomycin A1 after treatment and evaluated the content of LC3B-II and p62/SQSTM1 by Western blot. Thus, as the control of autophagy flux, RPE-1 cells were treated with BafA1. As expected, cells treated with HBSS and successively with BafA1 displayed a significant accumulation of LC3-II together with a significant increase of p62/SQSTM1 in comparison with control cells (Figure 2D), indicating a block of the autophagic flux [34]. Of interest, also in samples incubated with BafA1 after RDS 3337 treatment for 18 h or 72 h, a significant accumulation of LC3-II, accompanied by a significant increase of p62/SQSTM1, was found ( Figure 2D). This finding indicates an effect of the compound in the block of the autophagic flux.
Western blot results obtained, as above, were also confirmed by flow cytometry, using Cyto-ID and anti-p62/SQSTM1 Ab. Following treatment with RDS 3337, the analysis revealed a significant increase in Cyto-ID staining, which indicates autophagosome formation, with a high level of p62/SQSTM1, indicating a blockage of the autophagic flux. These findings confirmed an accumulation of autophagosomes [34]. In parallel, cells stimulated with HBSS for 16 h at 37 • C showed an increase of Cyto-ID staining as compared to control cells, together with a significant decrease of p62/SQSTM1 levels, which indicated the activation of the autophagic flux. On the contrary, in cells pre-treated with HBSS and then with RDS 3337, an increase of Cyto-ID staining without degradation of p62/SQSTM1 was observed, indicating an impairment of the autophagic clearance. As expected, cells pre-treated with HBSS and successively with BafA1 displayed a significant increase of Cyto-ID staining, with a high level of p62/SQSTM1, indicating a block of the autophagic flux. Interestingly, again, in samples incubated with BafA1 after RDS 3337 treatment, a significant increase of Cyto-ID staining, suggestive of autophagosome accumulation, together with a high level of anti-p62/SQSTM1 staining, was found, indicating an effect of the HPSE inhibitor in the block of autophagic flux. In Figure 2E, the mean fluorescence intensities are reported; all the cytofluorimetric panels are shown in Supplementary Figure S2. As expected, this methodological approach is less sensitive as compared to Western blot for this test.
In sum, these findings indicate that HPSE inhibitor RDS 3337 induces a significant increase of Cyto-ID staining, with a high level of p62/SQSTM1 in RPE-1 cells pre-treated with HBSS, suggestive of an autophagosomes accumulation and a consistent arrest of the autophagic flux.

HPSE Inhibitor RDS 3337 Blocks Autophagic Flux in U87 Human Glioblastoma Cells
Since previous studies have demonstrated that HPSE is highly expressed in many cancer types and is localized within autophagosomes [17], in this current study, we investigated the potential mechanism of the HPSE inhibitor RDS 3337 in regulating autophagy in U87 human glioblastoma cells in which autophagy is markedly increased [35]. For this purpose, we preliminary tested the cytotoxic effect of RDS 3337 in U87 cells, using Trypan Blue ( Figure 3A), WST-1 assays ( Figure 3B), and BrdU assay ( Figure 3C). U87 glioblastoma  Figure 3A,B. Similar findings were found using the BrdU assay ( Figure 3C). Similarly to RPE-1, we selected the dose of 320 nM also for U87 cell experiments. The samples were analyzed for the evaluation of autophagic flux by Western blot, using rabbit anti-LC3 pAb or rabbit anti-SQSTM1 mAb. The loading control was evaluated using anti-actin mAb. A representative experiment among the three is shown. The bar graph on the right shows densitometric analysis. Results represent the mean ± SD from three independent experiments * p < 0.05, ** p < 0.005, *** p < 0.001. No statistically significant differences were found between BafA1 and RDS 3337 18 h or 72 h + BafA1 samples.

RDS 3337 Inhibitor Sensitizes U87 Human Glioblastoma Cells to Apoptosis
To assess whether the suppression of autophagic flux upon RDS 3337 treatment can activate cytotoxic mechanisms in U87 human glioblastoma cells, we investigated cell death rates following treatment with the HPSE inhibitor. We preliminary determined the activation of caspase 3, a key molecule for induction of caspase and poly (ADP-ribose)polymerase 1 (Parp1) protein by Western blot analysis in RDS 3337 treated cells. As shown in Figure 4A, RDS 3337 treatment induced a significant increase of the cleaved-caspase 3 in a time-dependent manner, as compared with control cells. Furthermore, we also observed that the level of cleaved Parp1 protein, the nuclear enzyme Parp1 engaged in DNA repair, increased with the time of incubation upon RDS 3337 treatment.
All results were also confirmed by using a neuroblastoma cell line, i.e., SK-N-BE2 neuroblastoma cells (Supplementary Figure S4). presence or absence of 100 nM Baf A1, were lysed in a lysis buffer. The samples were analyzed for the evaluation of autophagic flux by Western blot, using rabbit anti-LC3 pAb or rabbit anti-SQSTM1 mAb. The loading control was evaluated using anti-actin mAb. A representative experiment among the three is shown. The bar graph on the right shows densitometric analysis. Results represent the mean ± SD from three independent experiments * p < 0.05, ** p < 0.005, *** p < 0.001. No statistically significant differences were found between BafA1 and RDS 3337 18 h or 72 h + BafA1 samples.
Thus, we analyzed the effect of this compound on the autophagic pathway in U87 cells, which basically show quite a high level of LC3-II together with a low level of p62/SQSTM1 ( Figure 3D). With this aim, we evaluated the protein levels of the autophagy-related markers, including both LC3-II and p62/SQSTM1, in U87 cells after incubation with 320 nM RDS 3337 for 18 h or 72 h versus control cells (vehicle). As shown in Figure 3D, immunoblotting analysis of lipidated LC3-II and its quantification (histograms on the right) revealed a significant increase of LC3-II in U87 RDS 3337 treated cells compared to untreated ones (vehicle). Interestingly, the analysis of p62/SQSTM1, under the same experimental conditions, showed a significant increase with respect to untreated cells, suggesting a consistent arrest of its degradation. These findings suggest an accumulation of autophagosomes.
Next, to better clarify the action of RDS 3337 in the autophagic process in U87 glioblastoma cells, we verified the autophagic flux using 100 nM BafA1 for 2 h, which prevents lysosomal acidification and, as a consequence, accumulates LC3-II by inhibiting p62/SQSTM1 degradation. Thus, the levels of both lipidated LC3-II and p62/SQSTM1 were assessed by Western blot in RDS 3337-treated cells in combination with BafA1. As shown in Figure 3B, cells treated with RDS 3337 and successively with BafA1 displayed a consistent and significant accumulation of both LC3-II and p62/SQSTM1 as compared to control cells or with RDS 3337-treated cells, indicating a block of the autophagic flux.
All results were also confirmed by using a neuroblastoma cell line, i.e., SK-N-BE2 neuroblastoma cells (Supplementary Figure S3).
Altogether, these data suggest the ability of HPSE inhibitor RDS3 3337 to induce both LC3-II and p62/SQSTM1 increase, suggestive of an autophagosome accumulation and a consistent arrest in the autophagic flux.

RDS 3337 Inhibitor Sensitizes U87 Human Glioblastoma Cells to Apoptosis
To assess whether the suppression of autophagic flux upon RDS 3337 treatment can activate cytotoxic mechanisms in U87 human glioblastoma cells, we investigated cell death rates following treatment with the HPSE inhibitor. We preliminary determined the activation of caspase 3, a key molecule for induction of caspase and poly (ADP-ribose)polymerase 1 (Parp1) protein by Western blot analysis in RDS 3337 treated cells. As shown in Figure 4A, RDS 3337 treatment induced a significant increase of the cleaved-caspase 3 in a time-dependent manner, as compared with control cells. Furthermore, we also observed that the level of cleaved Parp1 protein, the nuclear enzyme Parp1 engaged in DNA repair, increased with the time of incubation upon RDS 3337 treatment.   All results were also confirmed by using a neuroblastoma cell line, i.e., SK-N-BE2 neuroblastoma cells (Supplementary Figure S4).
Data obtained on the proapoptotic activity of RDS 3337 were confirmed by cytofluorimetric analysis using PI staining. These analyses showed a significant time-dependent increase in the sub-G1 phase, which identifies DNA fragmentation, when cells were treated with 320 nM RDS 3337 for 24, 48, or 72 h. Interestingly, after treatment with RDS 3337 for the indicated times (24, 48,

Discussion
Results of the present research support the notion that HPSE promotes autophagy, providing evidence that the RDS 3337 HPSE inhibitor blocks autophagic flux.
HPSE, which traditionally functions extracellularly by cleavage of heparan sulfate and promoting the remodeling of the extracellular matrix (ECM), has long been associated with an increase in tumor metastasis and angiogenesis [36]. However, the recent literature strongly supports a non-enzymatic activity of HPSE in the regulation of intracellular signaling pathways [37]. In this regard, a variety of biological functions and key signaling molecules have been investigated, including cell proliferation, mobility, angiogenesis, and activation of β1 integrin, HIF-2α, Flk-1, and/or AKT signaling [38][39][40][41]. New discoveries have led to the consideration of a possible role for HPSE in modulating autophagy, mainly in the context of tumor growth and chemoresistance, although such a function still remains to be elucidated [12]. A higher increase of autophagy was observed following HPSE overexpression in tumor-derived cells, with an enhancement of tumor growth and chemoresistance. This agrees with a strong pre-clinical and clinical correlation between HPSE expression and the progression of these malignancies [42,43]. Accordingly, studies in HPSE-deficient or transgenic mice established its contributions to autophagy [12].
Enrichment of HPSE in lysosomes suggests that the enzyme may control the normal physiology of these organelles [12,28]. Since it is well known that double-membrane vesicles, called autophagosomes, are fused with lysosomes during autophagy, it cannot be ruled out that lysosomal HPSE may play a role in the completion of autophagy. This emerges from the observation that HPSE is localized within autophagosomes in association with LC3-II in SIHN-013 laryngeal carcinoma cells overexpressing HPSE [12].
More recently, Yang et al. showed that either active or enzymatically inactive HPSE enhanced both autophagosome formation and the expression of related genes in gastric cancer cells [44]. In particular, the presence of non-enzymatic HPSE was able to upregulate both LC3-II protein expression and the level of LAMP2, a lysosomal membrane protein.
In the present study, we analyzed the effect of RDS 3337 HPSE inhibitor on human non-cancer neuro-ectodermal cell line RPE-1 with low levels of autophagy and on U87 human glioblastoma cells, which are endowed with increased levels of autophagy. With this aim, the transit of LC3-II through the autophagic pathway was examined by Western blot analysis. As a rule, autophagic flux is deduced on the basis of LC3-II turnover in both the presence and absence of lysosomal or vacuolar degradation [45]. Here, our findings point out an increase of LC3-II amount both in RPE-1 cells and, more evidently, in U87 human glioblastoma cells following HPSE inhibitor RDS 3337 treatment, suggestive of an autophagosomes accumulation and a consistent arrest in the autophagic flux, as confirmed by cell treatment with BafA1.
Consistently, the RDS 3337 treatment might compromise the pro-autophagy function of the intracellular HPSE, considering the ability of the inhibitor to easily cross the cellular plasma membrane. Up to the present, there are still some important issues unresolved about the role of HPSE inhibitors in affecting autophagic pathways and what are the underlying mechanisms involved. Autophagy related to HPSE appears to involve the mechanistic target of rapamycin complex 1 (MTORC1), an autophagy-suppressive regulator that integrates growth factor, nutrient, and energy signals; inhibition of mTOR1 leads to autophagy induction [46]. Recently, a direct correlation between HPSE overexpression and mTOR1 activity has also been proposed. In fact, in HPSE overexpressing SIHN-013 laryngeal carcinoma cells, mTOR1 activity was downregulated, and this condition positively stimulated the autophagic process [12].
Stress-activated autophagy is known to favor the survival of tumor cells, mostly when apoptosis is defective, protecting them from anticancer therapy, including chemotherapy or radiotherapy, and facilitating multi-drug-resistance development [47]. In this regard, it has been shown that autophagy made MCF-7 cells resistant to apoptosis induced by epirubicin, one of the most effective drugs against breast cancer. On the other hand, inhibition of autophagy by the downregulation of Beclin-1, a crucial upstream protein of autophagy, increased the epirubicin sensitivity of MCF-7 cells by accelerating caspase-9 activity and intrinsic apoptosis [48].
Here, we assessed whether alteration in autophagic activity upon RDS 3337 treatment in U87 human glioblastoma cells could lead to the induction of apoptosis. The relationship between autophagy and apoptosis is complex; similar stimuli can induce either autophagy or apoptosis, and the two phenomena may involve signal transduction pathways, which in turn are dependent on the type of cell nature and duration of stimulus, and stress. In many cases, when the apoptotic response is started, autophagy stops to function, partially because of the caspase-mediated cleavage of essential autophagy proteins [49]; thus, the cascade of caspase-activation associated with apoptosis shuts off the autophagic machinery [50].
Based on these considerations, we investigated the cell death rates by the evaluation of caspase 3 activation. Our results showed that RDS 3337 treatment induced a significant increase, in a time-dependent manner, of the cleaved-caspase 3 as compared with control cells. Furthermore, we also observed that activation of caspase 3 was associated with an increase of both cleaved Parp1 protein, the nuclear enzyme engaged in DNA repair, as well as the sub-G1 phase, which identifies DNA fragmentation of apoptotic cells.
Taken together, the downregulation of the autophagic process may sensitize cells to anticancer drugs, since the deregulation of signal pathways leading to autophagy plays a crucial role in producing critical pathophysiological consequences in numerous cellular mechanisms, including the process of tumorigenesis [51]. Our evidence on apoptosis activation may suggest a role for HPSE inhibitor, making its use particularly advantageous for therapeutic applications, where the progression of tumor growth can be controlled by acting in a balance between apoptosis and autophagy and regulating the autophagic process, without a cytotoxic mechanism affecting cell viability.

Conclusions
Thus, our findings contribute to further knowledge of autophagy regulation by HPSE, supporting the view that the use of clinically applicable autophagy inhibitors may be one of the important strategies for the control of tumor growth progression.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data underlying this article will be shared on reasonable request to the corresponding author.