Autophagy Inhibition via Hydroxychloroquine or 3-Methyladenine Enhances Chemotherapy-Induced Apoptosis in Neuro-Blastoma and Glioblastoma

Neuroblastoma is the most common tumour in children under 1 year old, accounting for 12–15% of childhood cancer deaths. Although current treatments are relatively efficacious against this cancer, associated adverse effects could be detrimental to growth and development. In contrast, glioblastoma accounts for 52% of brain tumours and has an extremely poor prognosis. Current chemotherapeutics include temozolomide, which has numerous negative side-effects and a low-effective rate. Previous studies have shown the manipulation of autophagy to be a promising method for targeting cancers, including glioblastoma. We sought to determine the effects of autophagic alterations in combination with current chemotherapies in both neuroblastoma and glioblastoma. Supplementing cisplatin or temozolomide with autophagy activator rapamycin stabilized cancer cell mitochondria, despite having little effect on apoptosis or oxidative stress. Autophagy inhibition via 3-methyladenine or hydroxychloroquine alongside standard chemotherapies enhanced apoptosis and oxidative stress, with 3-methyladenine also disrupting mitochondrial health. Importantly, combining hydroxychloroquine with 0.5 µM cisplatin or 50 µg/mL temozolomide was as or more effective than 2 µM cisplatin or 100 µg/mL temozolomide alone. Analyzing these interesting results, a combined treatment of autophagy inhibitor with a standard chemotherapeutic agent could help to improve patient prognosis and reduce chemotherapy doses and their associated side-effects.


Introduction
Neuroblastoma (NB) is one of the most common embryonic tumors that begins from immature nerve cells, making it a tumor of the peripheral sympathetic nervous system. It is an extracranial solid tumor that exhibits unique features like early onset, representing around 8-10% of all childhood cancers, and a high tendency for spontaneous regression [1]. Commonly affecting children 5 years old and younger, it is one of the most prevalent pediatric cancers characterized by a poor prognosis resulting in 15% of all pediatric cancer fatalities [2]. In the past 30 years, the 5-year survival rate for NB patients has improved from 52% to 74%; however, approximately 55% of patients in the high-risk group experience relapse [3].
In contrast, glioblastoma (GBM) is one of the most common and aggressive malignant brain tumors diagnosed in adults, occurring in the spinal cord or brain. This cancer makes up about 16% of all primary brain and central nervous system tumors and 45.2% of malignant primary brain tumors [4,5]. Current treatments for GBM are highly ineffective Int. J. Mol. Sci. 2023, 24, 12052 2 of 34 resulting in a median survival of only 15 months [6]. The initial treatment is typically surgery, as this is the best and easiest way to remove the tumor barring prior metastasis. However, if the tumor has metastasized, multiple surgeries or a combination of other treatment methods may be required, including chemotherapies like cisplatin and temozolomide (TMZ), used for NB and GBM respectively, and radiation therapy [7][8][9]. Although radiation and chemotherapy drugs are an effective way to kill cancer and control further growth, they also cause intensive damage to surrounding healthy cells and tissues resulting in many unfavorable side-effects to the patient. According to the Canadian Cancer Society, these include low blood cell count, hearing problems, infections, alopecia, thinking and memory changes, inflamed mucous membranes, anemia, and an increased risk of developing a second cancer [10]. Though most side-effects are temporary, hearing issues, thinking and memory changes, inflamed membranes, and increased risk of secondary cancers can last for long periods of time or become permanent. This is of particular importance in young children battling with NB as severe damage could hinder their growth and development. The poor prognosis and significant side-effects associated with these cancers and chemotherapeutics highlight the vital need to improve current treatments. One potential method to improving patient outcome is through the manipulation of autophagy in conjunction with standard chemotherapeutic treatments.
Autophagy is a pro-survival, self-degradative process that plays a housekeeping role in the cell as it removes aggregated and misfolded proteins or dysfunctional cell components that could negatively impact cellular homeostasis [11]. Autophagy initiation occurs when the cell recognizes an aberrant protein or organelle that requires removal and results in the formation of a double membraned vesicle called a phagophore [12]. Next, nucleation involves the localization of autophagic proteins to the phagophore promoting the continuation of autophagy. Elongation results in phagophore growth eventually forming an autophagosome around the entire protein/organelle. The autophagosome fuses with an acidic lysosome forming an autophagolysosome where the lysosome degrades the autophagosome contents. Another critical cellular process, apoptosis (programmed cell death) is crucial for overall health as it ensures proper functioning and causes minimal damage to surrounding tissues [13]. Both autophagy and apoptosis are vital for optimal cellular functioning during encounters with internal or external stressors including uncontrolled cell growth, high levels of oxidative stress, and dysfunctional organelles.
A variety of autophagic modulators including rapamycin, 3-methyladenine (3-MA), and hydroxychloroquine (HCQ) may improve anti-cancer activity in combination with current chemotherapies in NB (SH-SY5Y) and GBM (U-87 Mg) cells. Rapamycin, an autophagy activator, works through the inhibition of mammalian target of rapamycin (mTOR) which results in the formation of a phagophore and subsequent autophagosome [14]. In contrast, 3-MA and HCQ are early and late-stage autophagy inhibitors respectively. Specifically, 3-MA inhibits class III phosphatidylinositol 3-kinase which mediates vesicle nucleation [15,16] whereas HCQ prevents vesicular fusion between the autophagosome and lysosome resulting in the build-up of autophagic vacuoles [17].
Due to the crucial nature of autophagy as a cell maintenance mechanism, alteration of this pathway can lead to a plethora of problems in the cell. In relation to cancer, defective autophagic machinery is a major causative effect involved in initial cancer tumorigenesis [18]. Conversely, autophagy in established tumors helps cancer cells withstand metabolic stressors and resist death induced by chemotherapy. Moreover, it allows cells to maintain viability in periods of stress which can lead to tumor dormancy, progression, and therapeutic resistance [19].
Although much work has been done on autophagy modulation in NB and GBM, the optimal strategies in which autophagy modulation combined with chemotherapy can be used as an anti-cancer treatment is not understood. Common chemotherapies cisplatin and TMZ have been shown to induce both dose-dependent apoptosis and autophagy in NB and GBM cells respectively [20,21]. Various groups have shown that supplementation of HCQ in NB or GBM cells sensitizes them to chemotherapy and increases apoptotic levels [20,22].
Another study demonstrated that 3-MA induced cell injury and decreased cell viability further supporting the idea that autophagy can act as a cytoprotective mechanism, and its inhibition may promote apoptosis in NB [23]. Alternatively, rapamycin had no significant effect on cell viability but did provide neuroprotection to NB SH-SY5Y cells against amyloidβ-induced toxicity, oxidative stress, and neurotoxicity. However, other studies have shown rapamycin's potential as a possible therapeutic agent to treat NB and GBM as it halts cell proliferation and induces cytotoxicity [24,25].
Since many anti-cancer drugs such as chemotherapeutic agents create cytotoxic stress in the cell, they activate autophagy which, if performed for an excessive period, could result in cancer cell death [18]. Conversely, it has been found that the inhibition of autophagy enhances anti-cancer drug induced cell death, suggesting a novel therapeutic method to treat cancer. Clearly, a knowledge gap exists about the role of autophagy in tumor survival or suppression in these different cancers. This paper aims to investigate whether autophagy modulation can be an effective treatment in combination with current chemotherapies along with further mechanistic elucidation. We hypothesize that the inhibition of autophagy alongside standard chemotherapy regimens will result in enhanced apoptotic levels and could be utilized to reduce chemotherapy doses in NB and adverse effects in children, as well as increasing the lifespan/quality of life for GBM patients.

Cisplatin-Induced Autophagy Is Reduced in SH-SY5Y Neuroblastoma Cells by 3-MA Supplementation
Autophagy induction has been shown to be crucial to cisplatin resistance in various cancers including non-small-cell lung cancer and ovarian cancer [26][27][28][29]. To determine whether autophagy could be influenced in NB, we measured autophagic vacuole levels in SH-SY5Y cells following treatment with cisplatin, autophagy activator rapamycin, autophagosome inhibitor 3-MA, and autophagosome-lysosome fusion inhibitor HCQ through monodansylcadaverine (MDC) staining and fluorescent microscopy (Figure 1), as well as Cyto-ID staining with image-based cytometry ( Figure 2). Immunofluorescent staining for lysosomal-associated membrane protein 1 (LAMP1) and autophagosome marker microtubule-associated proteins 1A/1B light chain 3B (LC3B) following 24-h treatments   Cisplatin induced autophagy compared to while combinations with rapamycin were comparable to cisplatin alone. 3-MA in combinat cisplatin reduced autophagy levels compared to cisplatin alone. Results were obtained using based cytometry to assess the percentage of cells with active autophagy compared to contro are expressed as mean ± SD from 3 independent experiments. Statistical calculations w formed using single factor analysis of variance. *** p < 0.001.

Autophagic Induction in U-87 Mg Glioblastoma Cells via TMZ Is Decreased by 3-MA or HCQ Supplementation
Some chemotherapy treatments induce autophagy as a pro-survival mechanism [30]. The objective was to evaluate the effect of known autophagy modulators alongside chemotherapy. We measured autophagic flux through Cyto-ID staining of autophagic vacuoles in U-87 Mg cells treated for 24 h with combinations of TMZ and autophagy regulators ( Figure 4). Both TMZ and rapamycin enhanced autophagy compared to control but showed comparable levels to TMZ alone when used in combination. 3-MA was able to inhibit TMZ-induced autophagy while HCQ treatment resulted in increased Cyto-ID staining, likely due to inhibited vesicular fusion. These results were confirmed at 48 h through LAMP1/LC3B immunofluorescence, as shown in Figure 5.

Autophagic Induction in U-87 Mg Glioblastoma Cells via TMZ Is Decreased by 3-MA or HCQ Supplementation
Some chemotherapy treatments induce autophagy as a pro-survival mechanism [30]. The objective was to evaluate the effect of known autophagy modulators alongside chemotherapy. We measured autophagic flux through Cyto-ID staining of autophagic vacuoles in U-87 Mg cells treated for 24 h with combinations of TMZ and autophagy regulators ( Figure 4). Both TMZ and rapamycin enhanced autophagy compared to control but showed comparable levels to TMZ alone when used in combination. 3-MA was able to inhibit TMZ-induced autophagy while HCQ treatment resulted in increased Cyto-ID staining, likely due to inhibited vesicular fusion. These results were confirmed at 48 h through LAMP1/LC3B immunofluorescence, as shown in Figure 5.    Nuclei were counterstained with Hoechst (blue). This experiment is designed to show qualitative manipulation of autophagy with fluorescence measurements using ImageJ software version 1.52. Micrographs were taken at 200× magnification. Scale bar = 100 µm.

Autophagy Inhibition via 3-MA or HCQ Enhances Chemotherapy-Induced Apoptosis in Neuroblastoma and Glioblastoma
Both autophagy activation and inhibition have been proposed as potential mechanisms for inducing cell death in NB [31,32] and GBM [33,34]. As a result, we sought to examine the effect of autophagy regulators rapamycin, 3-MA, and HCQ in combination with commonly used chemotherapies cisplatin and TMZ using in-vitro models of NB and GBM. This was done through fluorescent staining of SH-SY5Y ( Figure 6) and U-87 Mg ( Figure 7) for apoptotic markers annexin V (AV) and propidium iodide (PI). In agreement with its lack of autophagic influence in chemotherapy treated cells, rapamycin had little effect on apoptotic levels alone or in combination with cisplatin or TMZ. Treatment with either 3-MA or HCQ alone induced apoptosis compared to control in both NB ( Figure 6) and GBM ( Figure 7). When combined with cisplatin or TMZ, both autophagy inhibitors enhanced AV/PI staining, with HCQ resulting in considerably increased levels of apoptosis.    In combination with 50 µg/mL TMZ, rapamycin and 3-MA had minor influence while HCQ led to drastic autophagic vacuole build-up. 100 µg/mL TMZ did not induce autophagy but LC3B and LAMP1 fluorescence increased when rapamycin or HCQ was supplemented as well. Nuclei were counterstained with Hoechst (blue). This experiment is designed to show qualitative manipulation of autophagy with fluorescence measurements using ImageJ software version 1.52. Micrographs were taken at 200× magnification. Scale bar = 100 µm.

Autophagy Inhibition via 3-MA or HCQ Enhances Chemotherapy-Induced Apoptosis in Neuroblastoma and Glioblastoma
Both autophagy activation and inhibition have been proposed as potential mechanisms for inducing cell death in NB [31,32] and GBM [33,34]. As a result, we sought to examine the effect of autophagy regulators rapamycin, 3-MA, and HCQ in combination with commonly used chemotherapies cisplatin and TMZ using in-vitro models of NB and GBM. This was done through fluorescent staining of SH-SY5Y ( Figure 6) and U-87 Mg ( Figure 7) for apoptotic markers annexin V (AV) and propidium iodide (PI). In agreement with its lack of autophagic influence in chemotherapy treated cells, rapamycin had little effect on apoptotic levels alone or in combination with cisplatin or TMZ. Treatment with either 3-MA or HCQ alone induced apoptosis compared to control in both NB ( Figure 6) and GBM (Figure 7). When combined with cisplatin or TMZ, both autophagy inhibitors enhanced AV/PI staining, with HCQ resulting in considerably increased levels of apoptosis. In combination with 50 µg/mL TMZ, rapamycin and 3-MA had minor influence while HCQ led to drastic autophagic vacuole build-up. 100 µg/mL TMZ did not induce autophagy but LC3B and LAMP1 fluorescence increased when rapamycin or HCQ was supplemented as well. Nuclei were counterstained with Hoechst (blue). This experiment is designed to show qualitative manipulation of autophagy with fluorescence measurements using ImageJ software version 1.52. Micrographs were taken at 200× magnification. Scale bar = 100 µm.

Autophagy Inhibition Enhances Reactive Oxygen Species (ROS) Production in SH-SY5Y and U-87 Mg Cells
Due to the enhanced metabolic rates of cancerous cells, elevated levels of ROS are produced, and oxidative damage can be a therapeutic target [35]. As a result, we aimed to analyze the influence of autophagy regulators rapamycin, 3-MA, and HCQ on ROS levels in NB and GBM. We used 2 ,7 -dichlorodihydrofluorescein diacetate (H2DCFDA), which diffuses across the cell membrane and is cleaved by intracellular esterases where it can be converted to the fluorescent molecule 2 ,7 -dichlorofluorescein (DCF) if oxidized by ROS. This fluorescence was examined via microscopy following 24-h treatments with rapamycin, 3-MA, and HCQ on both NB ( Figure 8) and GBM ( Figure 9). Autophagy activator rapamycin showed little effect on DCF fluorescence in SH-SY5Y but drastically reduced levels in U-87 Mg cells. Autophagy inhibition via 3-MA or HCQ led to increased ROS production, particularly in SH-SY5Y cells (Figure 8). Results were supported through immunofluorescence with peroxidised lipid marker 4-hydroxynonenal (4-HNE). Minor differences were observed in SH-SY5Y whereby rapamycin reduced, and 3-MA increased fluorescent levels both alone and in combination with cisplatin ( Figure 10). In GBM, rapamycin decreased 4-HNE staining when used alone or in combination with TMZ, while 3-MA increased oxidative stress alone but not in combination with TMZ ( Figure 11). Furthermore, HCQ had little effect on U-87 Mg alone but drastically enhanced fluorescent levels when combined with 50 µg/mL.

SH-SY5Y AVPI Quantification
Annexin V Propidium Iodide  nofluorescence with peroxidised lipid marker 4-hydroxynonenal (4-HNE). Minor differences were observed in SH-SY5Y whereby rapamycin reduced, and 3-MA increased fluorescent levels both alone and in combination with cisplatin ( Figure 10). In GBM, rapamycin decreased 4-HNE staining when used alone or in combination with TMZ, while 3-MA increased oxidative stress alone but not in combination with TMZ ( Figure 11). Furthermore, HCQ had little effect on U-87 Mg alone but drastically enhanced fluorescent levels when combined with 50 µg/mL.  treatments with autophagy regulators rapamycin, 3-MA, and HCQ was performed. Rapamycin had no effect on DCF staining while autophagy inhibition by 3-MA or HCQ increased levels of ROS. This experiment is designed to show qualitative ROS levels with fluorescence measurements using ImageJ software version 1.52. Micrographs were taken at 200× magnification. Scale bar = 100 µm.

Rapamycin-Induced Autophagy Activation Increases Mitochondrial Stability While Autophagy Inhibition Reduces Mitochondrial Functionality
Mitochondrial vulnerabilities in cancer are another possible target for therapeutic regimens [36]. Our objective was to determine the status of functional mitochondria under various treatments using Tetramethylrhodamine, methyl ester (TMRM), a cell permeant dye that accumulates in active mitochondria with intact membrane potentials. Rapamycin was able to enhance TMRM staining in both cell lines alone and in combination with cisplatin or TMZ. Despite reducing active mitochondria staining alone, 3-MA had little effect in combination with either chemotherapy as did HCQ. However, it should be noted that TMRM staining increased with HCQ treatment alone in SH-SY5Y ( Figure 12) while it decreased in U-87 Mg (Figure 13).   HCQ had no effect on oxidative stress when used alone but drastically increased staining when combined with 50 µg/mL TMZ. Nuclei were counterstained with Hoechst (blue). This experiment is designed to qualitatively show levels of oxidative stress with fluorescence measurements using ImageJ software version 1.52. Micrographs were taken at 200× magnification. Scale bar = 100 µm.

Rapamycin-Induced Autophagy Activation Increases Mitochondrial Stability While Autophagy Inhibition Reduces Mitochondrial Functionality
Mitochondrial vulnerabilities in cancer are another possible target for therapeutic regimens [36]. Our objective was to determine the status of functional mitochondria under  and (b) fluorescent quantification following 24-h treatments with TMZ, rapamycin, 3-MA, and HCQ were conducted. Rapamycin treatment was able to reduce fluorescent levels and oxidative stress when used alone or in combination with TMZ. 3-MA increased 4-HNE staining alone but had little effect on ROS production when combined with TMZ. HCQ had no effect on oxidative stress when used alone but drastically +10]increased staining when combined with 50 µg/mL TMZ. Nuclei were counterstained with Hoechst (blue). This experiment is designed to qualitatively show levels of oxidative stress with fluorescence measurements using ImageJ software version 1.52. Micrographs were taken at 200× magnification. Scale bar = 100 µm.

Supplementation of Standard Chemotherapies with 3-MA or HCQ Has Little Effect on Apoptotic Levels in Normal Healthy Cells
After demonstrating the anti-neoplastic effects of these autophagy inhibitors, we sought to examine their effect on apoptotic levels in non-cancerous cells. First, Cyto-ID staining for autophagic vacuoles was performed on normal colon mucosal (NCM-460) cells to determine the regulatory capacity of rapamycin, 3-MA, and HCQ at the same doses used in cancer cells (Figure 14). Autophagy inhibitors 3-MA and HCQ had little effect alone while rapamycin and standard chemotherapy TMZ enhanced green fluorescence compared to control. A notable difference was observed when combining TMZ with 3-MA resulting in reduced fluorescent levels comparable to the control. Fluorescent markers AV and PI alongside fluorescent microscopy were used to assess cell death in NCM-460 cells as well as normal human skin fibroblasts (NHF2) in Figures 15 and 16 respectively. Apoptotic levels were slightly raised by 3-MA alone or combined with cisplatin in NCM-460, with little difference observed in combination with TMZ, despite reductions in autophagy levels. Interestingly, despite minor increases in apoptosis when used alone, HCQ reduced AV and PI staining when combined with standard chemotherapeutics. Autophagy overactivation via rapamycin supplementation resulted in slight increases to apoptotic levels when combined with standard chemotherapeutics in NCM-460 cells. The autophagy modulators had little to no effect on apoptosis-induction in NHF2 when used alone or alongside standard chemotherapeutics.

Supplementation of Standard Chemotherapies with 3-MA or HCQ Has Little Effect on Apoptotic Levels in Normal Healthy Cells
After demonstrating the anti-neoplastic effects of these autophagy inhibitors, sought to examine their effect on apoptotic levels in non-cancerous cells. First, Cyto staining for autophagic vacuoles was performed on normal colon mucosal (NCM-4 cells to determine the regulatory capacity of rapamycin, 3-MA, and HCQ at the sa doses used in cancer cells (Figure 14). Autophagy inhibitors 3-MA and HCQ had l effect alone while rapamycin and standard chemotherapy TMZ enhanced green fluo cence compared to control. A notable difference was observed when combining TMZ w 3-MA resulting in reduced fluorescent levels comparable to the control. Fluorescent m ers AV and PI alongside fluorescent microscopy were used to assess cell death in NC 460 cells as well as normal human skin fibroblasts (NHF2) in Figures 15 and 16 resp tively. Apoptotic levels were slightly raised by 3-MA alone or combined with cisplati NCM-460, with little difference observed in combination with TMZ, despite reduction autophagy levels. Interestingly, despite minor increases in apoptosis when used alo HCQ reduced AV and PI staining when combined with standard chemotherapeutics. tophagy overactivation via rapamycin supplementation resulted in slight increase apoptotic levels when combined with standard chemotherapeutics in NCM-460 cells. autophagy modulators had little to no effect on apoptosis-induction in NHF2 when u alone or alongside standard chemotherapeutics.

Discussion
In this report, we have provided new details as to how the activation of autophag in response to chemotherapy treatment is critical for cancer cell health. Prior research in autophagy modulation alongside standard chemotherapy has provided mixed resul with both the activation and inhibition of autophagy proposed as potential anti-canc treatments. This project aimed specifically to examine and compare the impact of earl and late-stage autophagy inhibition alongside standard chemotherapies using in-vit models of neuroblastoma and glioblastoma. Furthermore, the roles of oxidative stress an mitochondrial dysfunction in cancer following treatment with chemotherapy and autop agy modulators were examined in these cancers for the first time. By inhibiting autophag induction with early or late-stage inhibitors, apoptotic levels were selectively enhance compared to levels normally observed with chemotherapy treatment alone in NB an GBM. Combining 3-MA with standard chemotherapies cisplatin or TMZ inhibited a tophagy, destabilized mitochondria, enhanced ROS levels, and induced apoptosis. Lat stage autophagy inhibitor HCQ resulted in the build-up of autophagic vesicles while i creasing oxidative stress on the cancer cells, together leading to apoptosis. Though bo autophagy inhibitors led to apoptosis in cancer, late-stage autophagy inhibition was mo effective at enhancing chemotherapy-induced apoptosis in NB and GBM. Importantl this work showed for the first time that increased oxidative stress following autophag inhibition is a potential mechanism through which these autophagy modulators, bo early-and late-stage, can induce apoptosis in combination with chemotherapy in NB an GBM. Overall, these autophagic inhibitors are effective at targeting cancer cells throug multiple mechanisms in combination with standard chemotherapeutics while havin minimal effects on non-cancerous healthy cells.
First off, we confirmed that treatment of SH-SY5Y or U-87 Mg cells with commo chemotherapies, cisplatin or TMZ respectively, results in the induction of autophag [37,38]. Known to be critical for maintenance of cellular homeostasis through protein/o ganelle recycling, autophagy upregulation is likely serving as a pro-survival mechanis to deal with chemotherapy-associated cell stress [39]. Importantly, we demonstrated th effectiveness of the autophagosome inhibitor 3-MA in reducing autophagy levels in SH SY5Y following cisplatin treatment. A similar result was observed in U-87 Mg cells whe combined with TMZ. Furthermore, HCQ, a known inhibitor of lysosomal fusion, e hanced autophagic vacuole staining due to vesicular build-up. These results indicate th

Discussion
In this report, we have provided new details as to how the activation of autophagy in response to chemotherapy treatment is critical for cancer cell health. Prior research into autophagy modulation alongside standard chemotherapy has provided mixed results with both the activation and inhibition of autophagy proposed as potential anti-cancer treatments. This project aimed specifically to examine and compare the impact of earlyand late-stage autophagy inhibition alongside standard chemotherapies using in-vitro models of neuroblastoma and glioblastoma. Furthermore, the roles of oxidative stress and mitochondrial dysfunction in cancer following treatment with chemotherapy and autophagy modulators were examined in these cancers for the first time. By inhibiting autophagic induction with early or late-stage inhibitors, apoptotic levels were selectively enhanced compared to levels normally observed with chemotherapy treatment alone in NB and GBM. Combining 3-MA with standard chemotherapies cisplatin or TMZ inhibited autophagy, destabilized mitochondria, enhanced ROS levels, and induced apoptosis. Latestage autophagy inhibitor HCQ resulted in the build-up of autophagic vesicles while increasing oxidative stress on the cancer cells, together leading to apoptosis. Though both autophagy inhibitors led to apoptosis in cancer, late-stage autophagy inhibition was more effective at enhancing chemotherapy-induced apoptosis in NB and GBM. Importantly, this work showed for the first time that increased oxidative stress following autophagy inhibition is a potential mechanism through which these autophagy modulators, both earlyand late-stage, can induce apoptosis in combination with chemotherapy in NB and GBM. Overall, these autophagic inhibitors are effective at targeting cancer cells through multiple mechanisms in combination with standard chemotherapeutics while having minimal effects on non-cancerous healthy cells.
First off, we confirmed that treatment of SH-SY5Y or U-87 Mg cells with common chemotherapies, cisplatin or TMZ respectively, results in the induction of autophagy [37,38]. Known to be critical for maintenance of cellular homeostasis through protein/organelle recycling, autophagy upregulation is likely serving as a pro-survival mechanism to deal with chemotherapy-associated cell stress [39]. Importantly, we demonstrated the effectiveness of the autophagosome inhibitor 3-MA in reducing autophagy levels in SH-SY5Y following cisplatin treatment. A similar result was observed in U-87 Mg cells when combined with TMZ. Furthermore, HCQ, a known inhibitor of lysosomal fusion, enhanced autophagic vacuole staining due to vesicular build-up. These results indicate that both 3-MA and HCQ could be utilized to reduce chemotherapy-associated autophagic flux in both NB and GBM. On the other hand, although rapamycin-induced autophagy activation was observed, this was rendered virtually ineffective when combined with either cisplatin or TMZ. Figure 17 outlines the process of autophagy and the point of intervention for each of these autophagic modulators. both 3-MA and HCQ could be utilized to reduce chemotherapy-associated autophagic flux in both NB and GBM. On the other hand, although rapamycin-induced autophagy activation was observed, this was rendered virtually ineffective when combined with either cisplatin or TMZ. Figure 17 outlines the process of autophagy and the point of intervention for each of these autophagic modulators. Figure 17. General diagram outlining the steps of autophagy and the modulation by rapamycin, 3-MA, and HCQ. Initiation of autophagy involves the formation of the double-membraned phagophore in response to a cargo that needs degrading. Rapamycin targets this step through the inhibition of mTOR resulting in phagophore formation. Nucleation occurs when autophagic proteins localize to the growing phagophore. 3-MA can inhibit this process by blocking class III phosphatidyl 3-kinase, normally involved in vesicle nucleation. During elongation, the phagophore grows into an autophagosome which traps the cargo inside. Fusion involves the joining of the autophagosome with an acidic lysosome forming an autophagolysosome. The late-stage autophagy inhibitor HCQ prevents this fusion from occurring resulting in the build-up of these autophagic vacuoles. Finally, degradation results in the lysosomal breakdown of the desired cargo because of low pH.
Due to the complexity and knowledge gaps surrounding autophagy-apoptosis interactions, we sought to analyze the effect of these autophagy regulators in combination with cisplatin or TMZ on apoptosis. Debate continues as to whether reducing a cell's autophagic defense mechanism or inducing autophagy to cause cell death through lysosomal overactivation or immunogenic cell death is a more effective treatment option [40]. We demonstrated the induction of apoptosis following autophagy inhibition via 3-MA or HCQ treatment alone in both NB and GBM cell lines. As a result, we hypothesized that autophagic inhibition alongside standard chemotherapies could enhance apoptotic cell death. Indeed, a drastic uptick in apoptosis was observed among SH-SY5Y and U-87 Mg cells treated with cisplatin or TMZ respectively when they were combined with 3-MA or HCQ. Late-stage autophagy inhibition via HCQ appeared more effective than early-stage inhibitor 3-MA suggesting its potential use as an anti-cancer supplement. Autophagy upregulation via rapamycin supplementation had little effect on apoptotic levels when used alone or in combination with chemotherapies. As a result, the induction of autophagy seen with cisplatin treatment in NB or TMZ treatment in GBM appears to be serving as a prosurvival mechanism. Fortunately, through 3-MA or HCQ autophagic inhibition, levels of Figure 17. General diagram outlining the steps of autophagy and the modulation by rapamycin, 3-MA, and HCQ. Initiation of autophagy involves the formation of the double-membraned phagophore in response to a cargo that needs degrading. Rapamycin targets this step through the inhibition of mTOR resulting in phagophore formation. Nucleation occurs when autophagic proteins localize to the growing phagophore. 3-MA can inhibit this process by blocking class III phosphatidyl 3-kinase, normally involved in vesicle nucleation. During elongation, the phagophore grows into an autophagosome which traps the cargo inside. Fusion involves the joining of the autophagosome with an acidic lysosome forming an autophagolysosome. The late-stage autophagy inhibitor HCQ prevents this fusion from occurring resulting in the build-up of these autophagic vacuoles. Finally, degradation results in the lysosomal breakdown of the desired cargo because of low pH.
Due to the complexity and knowledge gaps surrounding autophagy-apoptosis interactions, we sought to analyze the effect of these autophagy regulators in combination with cisplatin or TMZ on apoptosis. Debate continues as to whether reducing a cell's autophagic defense mechanism or inducing autophagy to cause cell death through lysosomal overactivation or immunogenic cell death is a more effective treatment option [40]. We demonstrated the induction of apoptosis following autophagy inhibition via 3-MA or HCQ treatment alone in both NB and GBM cell lines. As a result, we hypothesized that autophagic inhibition alongside standard chemotherapies could enhance apoptotic cell death. Indeed, a drastic uptick in apoptosis was observed among SH-SY5Y and U-87 Mg cells treated with cisplatin or TMZ respectively when they were combined with 3-MA or HCQ. Late-stage autophagy inhibition via HCQ appeared more effective than early-stage inhibitor 3-MA suggesting its potential use as an anti-cancer supplement. Autophagy upregulation via rapamycin supplementation had little effect on apoptotic levels when used alone or in combination with chemotherapies. As a result, the induction of autophagy seen with cisplatin treatment in NB or TMZ treatment in GBM appears to be serving as a pro-survival mechanism. Fortunately, through 3-MA or HCQ autophagic inhibition, levels of apoptosis are radically increased in SH-SY5Y and U-87 Mg compared to cisplatin or TMZ treatment alone.
To determine whether the cell death enhancement is specific to cancerous cells, we analyzed the effect of the autophagy inhibitors 3-MA and HCQ on two normal cell lines, a healthy skin fibroblast model (NHF2) and healthy colon epithelial cells (NCM-460). Through live-cell fluorescent staining, we observed a slight increase in NCM-460 cell death when combining 3-MA with cisplatin, and a small decrease when combined with TMZ, despite a drastic reduction in autophagic levels. Little to no difference was observed in the NHF2 cells indicating that any change in apoptotic levels in non-cancerous cells is minimal with 3-MA treatment. Interestingly, the large increase in apoptotic levels observed in both cancer lines when adding HCQ to standard chemotherapy was not observed in either healthy cell line when combined with cisplatin or TMZ. This could be a result of reduced potency in normal healthy cells compared to cancerous tissue as a build-up of autophagic vacuoles was not seen with Cyto-ID staining in NCM-460 as it was in both SH-SY5Y and U-87 Mg cell lines. This selectivity of the HCQ-chemotherapy combination to cancerous cells, namely SH-SY5Y and U-87 Mg, compared to healthy cells, NHF2 and NCM-460, indicates that reduced doses of chemotherapy could be used to achieve the same cell death in cancerous tissues, while possibly reducing harmful side-effects.
Although autophagy inhibition itself could be detrimental to cancer cell survival, we aimed to determine whether other common mechanisms of targeting cancer were being exploited by these autophagy inhibitors. Due to the rapidly multiplying nature of cancerous cells and the elevated metabolic rates along with it, cancer cells typically produce elevated amounts of reactive oxygen species (ROS) compared to normal cells. This excessive oxidative stress is critical to both the formation and progression of cancer due to resulting genomic DNA mutations that it produces [41]. However, elevated levels of ROS can be detrimental, resulting in cancer cells becoming particularly susceptible to further oxidative stress due to their already high ROS production [35]. An associated increase in the production of ROS was observed with autophagy inhibition via 3-MA or HCQ, particularly in NB. Conversely, autophagy activation via rapamycin diminished ROS production in GBM while having little effect on NB. Using 4-HNE as a marker of peroxidised lipids, very little difference was observed with rapamycin or HCQ, whether alone or combined with cisplatin. 3-MA resulted in small increases in the number of peroxidised lipids both alone and in combination with cisplatin. In GBM however, rapamycin showed antioxidant capabilities alone and with TMZ, confirming the results from Figure 9. 3-MA resulted in some oxidative stress alone but had very minimal effect when combined with TMZ on U-87 Mg. Interestingly, HCQ, which had very little effect on ROS levels alone in GBM, led to increased oxidative stress when combined with 50 µg/mL TMZ.
Another unique metabolic feature of cancer cells is their preference for lactic fermentation rather than aerobic respiration [36,42]. Dubbed the Warburg Effect, this phenomenon results in an acidified cytoplasm and changes in the MMP, increasing mitochondrial vulnerability. We determined that rapamycin treatment increased the number of active mitochondria in both SH-SY5Y and U-87 Mg cells, likely due to its associated reductions in oxidative stress. When rapamycin induces autophagy, this could result in the clearing of aberrant mitochondria, reducing ROS levels often produced from defective mitochondria. A vicious cycle exists whereby ROS production will further damage other mitochondria which will then produce ROS themselves. As a result, it stands to reason that through autophagy induction, rapamycin can relieve oxidative stress within the cell while increasing the number of viable mitochondria. This trend continued in combination with chemotherapy where the addition of rapamycin was able to enhance TMRM staining and increase mitochondrial health in cancer cells. The inhibition of autophagy through 3-MA or HCQ resulted in reduced mitochondrial health in SH-SY5Y (only 3-MA) and U-87 Mg. However, when combined with chemotherapies cisplatin or TMZ, 3-MA appeared to reduce mitochondrial functionality at lower doses of chemo (0.5 µM cisplatin or 50 µg/mL TMZ) but not at higher doses (2 µM cisplatin or 100 µg/mL TMZ). Alternatively, autophagy inhibition via HCQ appeared to have little effect in combination with any dose of chemotherapy.
Throughout this study, we combined autophagy regulators rapamycin, 3-MA, and HCQ with commonly used chemotherapies cisplatin and TMZ to determine their impact on cellular health and survival. We found that rapamycin induced autophagy in a dose-dependent manner but had little effect when combined with autophagy-inducing chemotherapies. Alone, rapamycin served as an antioxidant while stabilizing mitochondria in both SH-SY5Y and U-87 Mg cells. Its antioxidative benefits were limited when combined with chemotherapy; however, mitochondrial stabilization was still observed. Alternatively, 3-MA reduced autophagy levels while HCQ treatment caused the buildup of autophagic vacuoles both alone and combined with chemotherapy. In addition to autophagy inhibition, 3-MA treatment led to disruptions in MMP while both 3-MA and HCQ induced ROS production. Essentially, autophagy inhibition combined with cisplatin or TMZ treatment in NB and GBM respectively resulted in a noticeable increase in apoptotic levels, particularly with late-stage inhibition. As a result, the combination of autophagy inhibitors with standard chemotherapies should be investigated further as a potential treatment for both neuroblastoma and glioblastoma. These combination treatments could improve patient prognosis/survival as well as quality of life, ameliorating some negative side-effects associated with chemotherapy due to reduced dosing. Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available.