Pleiotropic Effects of 3-O-Decanoylquercetin on U373-MG Human Glioma Cell Line

TL;DR

A new quercetin derivative, Q-3-Dec, shows promise in fighting glioma brain tumors by targeting multiple survival pathways in cancer cells.

Contribution

The study identifies Q-3-Dec as a multi-target agent that simultaneously disrupts key survival signals and DNA repair mechanisms in glioma cells.

Findings

Q-3-Dec treatment caused significant morphological changes and mitochondrial dysfunction in U373-MG glioma cells.

Q-3-Dec reduced NF-κB and STAT3 signaling, along with anti-apoptotic proteins and MGMT expression.

Exposure to Q-3-Dec led to approximately 30% cell death in glioma cells after 48 hours.

Abstract

Gliomas are among the most challenging brain tumors to treat, owing to their marked heterogeneity and the aberrant signaling networks that sustain tumor growth and resistance to therapy. Quercetin, a dietary flavonoid widely found in fruit and vegetables, exhibits documented anticancer activity, prompting the development of optimized derivatives with improved biological potency. In earlier work, we synthesized and evaluated a series of quercetin derivatives and identified the acylated compound 3-O-decanoylquercetin (Q-3-Dec) as particularly effective in reducing glioma cell viability. In this study, we explored Q-3-Dec as a multi-target agent, which concomitantly impairs NF-κB/STAT3-dependent survival signaling, mitochondrial function, and O6-Methylguanine-DNA Methyltransferase (MGMT) expression, a DNA repair enzyme closely associated with chemoresistance, in glioma cells. In U373-MG glioma cells, treatment with 50 μM Q-3-Dec triggered pronounced, time-dependent morphological changes and an early loss of mitochondrial membrane potential after 3 h. With prolonged exposure, Q-3-Dec markedly decreased NF-κB and STAT3 phosphorylation and reduced the expression of the anti-apoptotic proteins Bcl-2 and survivin, alongside a significant decrease in MGMT levels. These combined effects culminated in a progressive increase in cell death, reaching approximately 30% after 48 h. Together, these findings position Q-3-Dec as a multi-node modulator of glioma survival, supporting its potential for further preclinical development to improve future therapeutic strategies against glioma.

Figures

Full text

1. Introduction

Gliomas are primary brain tumors derived from glial cells and classified by the World Health Organization (WHO) into four grades reflecting increasing malignancy. While low-grade gliomas (Grades I–II) are associated with relatively favorable outcomes, high-grade gliomas (Grades III–IV) remain among the most lethal human cancers, with a median survival of less than 18 months despite aggressive multimodal therapy [1,2]. Although surgery, chemotherapy, and radiotherapy remain the standard of care, their efficacy is limited. This is largely attributed to the highly invasive nature of glioma cells and the restricted permeability of the blood–brain barrier (BBB), which limits the effectiveness of many chemotherapeutic agents [3,4]. Consequently, there is a pressing need for novel therapeutic compounds capable of crossing the BBB and simultaneously targeting multiple molecular pathways essential for glioma survival. Quercetin represents one such candidate [5].

Quercetin is a naturally occurring dietary flavonoid abundant in fruit and vegetables and characterized by a polyphenolic structure that underlies its broad biological activity. Numerous studies have demonstrated that quercetin exerts anti-inflammatory, anti-proliferative, and pro-apoptotic effects across several tumor types, including gliomas [6,7,8]. These effects are mediated through the modulation of key signaling pathways involved in oxidative stress responses, cell survival, and apoptosis, consistent with its antioxidant and signaling-regulatory properties [9,10]. Importantly, flavonoids are increasingly recognized as multi-node/pleiotropic modulators of cancer cell survival rather than single-target inhibitors. Recent evidence, including studies on prunin, isorhamnetin, and other flavonoid derivatives, highlights their ability to concurrently suppress transcriptional survival programs while promoting mitochondrial dysfunction and intrinsic apoptosis in malignant cells [11,12]. Comparative analyses of flavonoid subclasses further position quercetin within a broader structure–activity framework, revealing both shared and subclass-specific anticancer mechanisms [13].

In gliomas, aberrant activation of NF-κB and STAT3 is a hallmark of tumor aggressiveness and therapy resistance. Quercetin has been shown to suppress NF-κB signaling, including inhibition of TNF-α–mediated activation [8], while STAT3 activation downstream of cytokines and growth factors promotes glioma cell proliferation, survival, and immune evasion [14,15,16,17,18]. Notably, NF-κB and STAT3 function cooperatively sustain oncogenic transcriptional programs, regulating genes involved in cell cycle progression (Cyclin D1, c-Myc) and apoptosis resistance, including Bcl-2 and survivin [14,19,20,21,22].

Survivin, a member of the inhibitor of apoptosis protein (IAP) family, plays a dual role in mitotic regulation and apoptosis inhibition through caspase suppression [22]. In normal physiological conditions, survivin is highly expressed during fetal development and in actively proliferating cells, but it is scarce or absent in adult/differentiated tissues, while it is highly expressed in most cancers, including gliomas, where it correlates with tumor aggressiveness and poor prognosis [23,24,25,26,27]. Previous studies demonstrated that quercetin can sensitize malignant glioma cells to TRAIL-induced apoptosis through survivin downregulation, primarily via proteasomal degradation and Akt-dependent signaling [28]. Importantly, resistance to this effect was associated with sustained survivin expression, underscoring survivin as a critical molecular determinant of quercetin responsiveness in glioma cells.

Beyond the parent compound, accumulating evidence indicates that chemical modification of quercetin, particularly acylation, profoundly enhances its biological potential. Acylation increases lipophilicity, membrane affinity, and intracellular uptake, thereby improving metabolic stability and subcellular targeting compared with non-acylated quercetin [29]. Multi-site acetylation of quercetin hydroxyl groups has been shown to enhance intracellular persistence and mitochondria-associated apoptotic signaling in cancer cells [30]. Consistently, acylated quercetin derivatives exhibit increased anti-proliferative activity and altered redox behavior. These effects are attributed to enhanced membrane partitioning and modified subcellular interactions [31]. Moreover, flavonoids, including quercetin, directly modulate mitochondrial membrane potential, reactive oxygen species (ROS) homeostasis, and apoptotic signaling, supporting a mechanistic link between lipophilicity-driven mitochondrial targeting and enhanced anticancer activity [32].

In this context, we previously synthesized and characterized a series of quercetin derivatives. We observed a negligible toxicity on U373-MG treated with Quercetin (25–100 μM) for 24 h; this evidence prompted us to test whether the acylation of Quercetin could yield active cytotoxic compounds. To this aim, the structure of Quercetin was modified by adding hydrophobic acyl functional groups of different lengths, ranging from C2 to C16, as 3-O-acyl esters of the molecule. We analyzed the effects of each acylated Quercetin derivative to select the ones that yielded the highest effects on glioma cell survival. Among these, 3*-O-*decanoylquercetin (Q-3-Dec) with the C10 chain was identified as a compound with selective cytotoxic activity against U373-MG glioma cells, while sparing non-malignant astroglial cells and human fibroblasts [33]. Q-3-Dec induced mitochondrial apoptosis, as evidenced by caspase-3 activation, suggesting an enhanced engagement of intrinsic death pathways.

Building on these findings, the present study aimed to elucidate the molecular mechanisms underlying the anticancer activity of Q-3-Dec in glioma cells. Specifically, we investigated its effects on key survival regulators, including phosphorylated NF-κB and STAT3, survivin, and Bcl-2, and the possible functional mitochondrial impairment. In addition, given the clinical relevance of chemoresistance in glioma, we examined the impact of Q-3-Dec on MGMT expression, a critical determinant of resistance to alkylating agents, in light of recent evidence indicating that quercetin can sensitize MGMT-positive glioblastoma cells to apoptosis [34,35].

Overall, this study positions Q-3-Dec as a novel, lipophilicity-enhanced quercetin derivative acting as a multi-node modulator of glioma survival signaling and mitochondrial function, highlighting its potential as a promising flavonoid-based candidate for further preclinical development in glioma therapy.

2. Results

2.1. Morphological Changes in U373-MG Treated with 3-O-Decanoylquercetin

U373-MG cells treated with 50 μM Q-3-Dec for different times (1, 3, 6, 12, and 24 h) displayed progressive time-dependent morphological alterations (Figure 1). After 3 h of treatment, we observed a reduction in cell adhesion and increased cell rounding (balling). After 6 h, an increase in intercellular spacing became visible. After 12 h, cells showed distinct signs of stress, and by 24 h, they exhibited a notably narrowed morphology. On the other hand, the untreated/control cells maintained a compact, confluent appearance throughout the entire observation period.

2.2. Analysis of Phospho-NF-κB, Survivin and Phospho-STAT3 Expression Levels in U373-MG Treated with 3-O-Decanoylquercetin

Given that the NF-κB pathway, known also as an onco-pathway, can modulate the expression of genes beneficial for tumor survival/proliferation, we analyzed the NF-κB expression and phosphorylation levels by Western blotting in control and Q-3-Dec-treated U373-MG cells. Analysis of the protein at its basal and phosphorylated levels revealed a significant reduction (* p = 0.037) versus control cultures of 53% after 12 and 24 h of Q-3-Dec treatment. The Western blotting analysis of the survivin expression levels in U373-MG revealed, after 12–24 h of treatment with Q-3-Dec, a significantly higher decrease (** p = 0.001–0.002) of about 50–53% versus control untreated cells. Actin was used as a housekeeping gene and also as an internal control (Figure 2A–C).

The observed concomitant increase in P-NF-κB and survivin at 3 h indicates a transient elevation of both proteins in glioma cells exposed to Q-3-Dec, followed by a marked reduction in their levels compared with control at later time points (Figure 2).

Since STAT3 is known to cooperate with NF-κB in promoting the persistent survival of cancer cells, we analyzed the phosphorylation status of STAT3 under our experimental conditions. As shown in Figure 3A, Western blotting analyses revealed that Q-3-Dec treatment of U373-MG cells led to a marked decrease in STAT3 phosphorylation, ranging from 45% to 69%. Furthermore, we extended the analysis of STAT3 phosphorylation to later time points in order to clarify its temporal regulation. Specifically, because an increase in P-STAT3 levels was observed at 24 h, we included the 48 h time point to determine whether this represented a sustained activation or a transient response followed by inhibition. The maximal reduction is observed at 48 h (Figure 3B).

The observed transient increase in P-STAT3 at 24 h is consistent with a short-lived activation of survival signaling in response to Q-3-Dec-induced stress (Figure 3).

2.3. Immunocytochemical Analyses of Phospho-NF-κB, Phosho-STAT3 and Survivin Expression Levels in U373-MG Treated with 3-O-Decanoylquercetin

Immunocytochemical analyses of phospho-NF-κB, phospho-STAT3, and survivin were conducted to quantify fluorescent immunoreactivity and to evaluate their subcellular localization. Figure 4A shows U373-MG under each experimental condition, including the corresponding inverted-phase micrographs and immunocytochemical images for each target protein, allowing precise identification of their cytoplasmic and/or nuclear distribution. These evaluations were essential for determining the localization of each protein and understanding how its expression and activation/phosphorylation are regulated in response to the treatments. The left column of the phase contrast micrographs highlights cellular morphology and its changes in response to the treatments. Looking at the experimental time points progression, signs of cellular distress and cell death became clearly evident as early as 3 h after Q-3-Dec exposure (Figure 4A left column).

Immunocytochemical analyses of phospho-NF-κB, phospho-STAT3, and survivin were performed by quantifying immunoreactive signals across multiple automatically detected regions of interest (ROIs) in several scanned fields. ROIs were subsequently classified into two groups exhibiting either high or low fluorescence intensity (Hi or Li, respectively), as shown in histograms of Figure 4B.

The analysis of P-NF-κB revealed that in the control condition, the phosphorylated protein was strongly expressed in the cell, both in the cytoplasm and the nucleus. At the 1 h time-point, treatment resulted in a marked reduction in immunoreactivity for P-NF-κB.

Treated cells showed a very faint signal at both cytoplasmic and nuclear levels. After 24 h, a cytoplasmic restoration of a weak fluorescent signal was evident in a limited number of cells (Figure 4A). While in the control condition, no significant differences were observed between the percentage of Hi versus Li ROI, the treatments from the 1 h time point to 24 h revealed a highly significant decrease in the Hi-ROIs compared to Li-ROIs (**** p < 0.0001 Hi vs. Li at 1, 3, 6, 24 h). Consistently, there was a highly significant increase in the percentage of Li-ROIs versus that observed in the control condition. These observations demonstrate that the levels of phosphorylated NF-κB of control/untreated cells were modified by the use of the Q-3-Dec, which was able to decrease them, diminishing the amount of activated transcription factor working at the promoter of survival genes.

The results obtained from the immunocytochemical analyses of P-STAT3 at the different experimental conditions were quite similar to those observed for P-NF-κB. In control cells, clear immunoreactivity was detected in both the cytoplasm and the nucleus, although with lower intensity than that seen for P-NF-κB. Q-3-Dec treatment yielded a progressive reduction in the high intensity ROIs between 1 and 6 h. While a significant decrease from high- to low-intensity fluorescence was observed within this time window (* p = 0.03 at 1 h; * p = 0.01 at 3 and 6 h) (Figure 4A,C). The immunocytochemical analysis of the survivin showed strong nuclear immunoreactivity under control conditions (Figure 4A). After Q-3-Dec treatment, a highly significant decrease (* p = 0.01–0.04; ** p = 0.001) in the Hi nuclear fluorescence signal was detected. The nuclear Hi fluorescence observed in the control conditions of 40.2% dropped down to about 24% after 1–3 h of treatment and further decreased to 13.87% after 6 h of treatment. Consequently, Li signals increased over the same time course (Figure 4D). Collectively, these results support the hypothesis that Q-3-Dec treatment in U373-MG cells affects both the expression and the subcellular localization of survivin, including a marked reduction in its nuclear fraction, which is typically associated with mitotic regulation. Moreover, immunofluorescence analyses indicate that the reduction in phospho–NF-κB, P-STAT3, and survivin expression is already evident after 1 h of treatment and becomes more pronounced from 3 to 6 h.

2.4. Analysis of Bcl-2 Expression Levels in U373-MG Treated with 3-O-Decanoylquercetin

Bcl-2, a key anti-apoptotic protein within the Bcl-2 family, is commonly overexpressed across multiple cancer types and plays a significant role in the chemoresistance exhibited by many cancer cells, particularly glioma cells. To this aim, we have analyzed in our experimental model, through Western blotting, the expression levels of Bcl-2 in U373-MG to evaluate the possible effects of Q-3-Dec. The results obtained demonstrated a significant reduction in Bcl-2 level expression after 1 h treatment (* p = 0.04 1 h Q-3-Dec treated cells versus control cells) (Figure 5). This early decrease could be interpreted as an acute response to stress-adaptation, but more experimental evidences are needed. After 24 and 48 h of treatment, a further significant reduction of about 55–70%, respectively, was observed (** p = 0.002 in 24 h treated cells versus control cells and *** p ≤ 0.0001 in 48 h treated cells versus control cells) (Figure 5).

2.5. Mito- and Cyto-Toxicity of 3-O-Decanoylquercetin on U373-MG

To evaluate the mito- and/or cytotoxic effects induced by Q-3-Dec, U373-MG glioma cells seeded on coverslips in 24-well plates were treated with 50 μM Q-3-Dec for 1, 3, 6, and 24 h. Confocal laser-scanning immunofluorescence analyses revealed mitochondrial impairment and detectable cytotoxicity beginning at 3 h of treatment. At this time point, treated cells showed clear signs of cytotoxicity. The red fluorescence, emitted by the MitoHealth stain, which accumulates in functional mitochondria of healthy cells, was intense in control cells (CTR/DMSO) (Figure 6A). In treated cells, this signal showed a slight decrease at 1 h, followed by a more pronounced reduction at 3 h and at subsequent time points, indicating a progressive significant loss of mitochondrial membrane potential (* p = 0.04 for 1 h MitoHealth vs. 24 h MitoHealth). Conversely, the Image-iT DEAD Green dye, which exhibits a high affinity for DNA only when the plasma membrane is compromised, forming highly fluorescent and stable dye–nucleic acid complexes, became strongly evident already at 3 h and further increased at 24 h (**** p > 0.0001 for 3, 6, and 24 h DEAD green vs. CTR/DMSO). At the latest time point, a very intense fluorescent signal was detected in the nuclei, confirming extensive cell damage. Together, these evidences clearly demonstrate that Q-3-Dec induces mitochondrial impairment, within a few hours of exposure, contributing to reduced cell vitality and ultimately leading to glioma cell death.

2.6. Analysis of O6-MethylGuanine-DNA MethylTransferase (MGMT) Expression Levels in U373-MG Treated with 3-O-Decanoylquercetin

Quercetin has recently been investigated as a modulator of epigenetic pathways in anticancer strategies [36]. It is well established that the addition of alkyl groups to specific DNA regions can induce damage in cancer cells, thereby triggering apoptosis. This anticancer effect can be blocked by the activity of the MGMT enzyme, which removes alkyl groups from Temozolomide (TMZ) damaged DNA, thus reducing the therapeutic efficacy of alkylating agents. In this study, we evaluated the MGMT expression levels in control and Q-3-Dec-treated U373-MG cells (Figure 7). The results obtained through Western blotting analysis (Figure 7A) showed a significant reduction in MGMT protein levels between 6 and 12 h of treatment (* p = 0.04 at 6 h and * p = 0.01 at 12 h treatment vs. CTR/DMSO) and a highly significant decrease after 24 h Q-3-Dec exposure (** p = 0.002 at 24 h vs. CTR/DMSO) (Figure 7B). The results obtained indicate that Q-3-Dec induced a marked reduction in MGMT protein expression.

2.7. Analysis of Glioma Cells Survival in U373-MG Treated with 3-O-Decanoylquercetin

Figure 8 shows the morphological evidence of distressed cells after 24–48 h of treatment with 50 μM Q-3-Dec. The images from control cells (with/without DMSO) have shown no differences in their morphology, while highly significant detrimental signs were evident already at 24 h, worsening at 48 h of treatment (*** p = 0.0005 for 24 h vs. 48 h Q-3-Dec treated cells; and **** p < 0.0001 at 48 h treatment vs. CTR/DMSO).

3. Discussion

In this study, we analyzed the molecular effects of the quercetin derivative Q-3-Dec in glioma cells, focusing on key survival pathways and cellular mechanisms that sustain tumor survival and therapy resistance. The chemical formulation of Q-3-Dec was designed to achieve specific, high-performance functions through a carefully engineered combination of components. The parent molecule Quercetin was modified by adding a C10-length acyl functional group to obtain a more hydrophobic compound able to interact with cell structures. Our findings demonstrate that Q-3-Dec exerts a pleiotropic anticancer activity by coordinately impairing mitochondrial function, pro-survival transcriptional signaling, apoptosis regulation, and DNA repair capacity.

It is known that aberrant activity of signal transduction pathways plays a crucial role in glioma development and progression; among these, NF-κB and STAT3 contribute to uncontrolled cell growth, evasion of apoptosis, increased angiogenesis, and enhanced invasiveness [14,15]. NF-κB functions via canonical and non-canonical pathways, with the canonical pathway primarily regulating cell survival and proliferation [37]. In glioma, NF-κB is often constitutively active, driving the transcription of genes that promote cell survival, proliferation, and resistance to apoptosis [38]. In this study, Q-3-Dec treatment induced a significant, time-dependent reduction in NF-κB phosphorylation (Figure 2) and markedly decreased its nuclear and cytoplasmic localization (Figure 4A,B), indicating effective suppression of NF-κB transcriptional activity. Consistent with this inhibition, the expression of downstream anti-apoptotic targets was also reduced. Noteworthy, immunocytochemical analyses revealed a marked reduction in nuclear survivin following Q-3-Dec exposure, closely paralleling NF-κB dephosphorylation (Figure 4A,D). survivin, a key component of the chromosomal passenger complex, is required for proper mitotic progression, chromosome alignment, and cytokinesis in the nucleus, while also inhibiting apoptosis in the cytoplasm [39,40]. In glioma and other tumor models, survivin depletion or mislocalization has been linked to mitotic defects and increased susceptibility to apoptosis [40], suggesting that its reduction may contribute to the observed anti-proliferative and pro-apoptotic effects. Q-3-Dec exposure also led to a rapid and significant reduction in Bcl-2 levels, detectable as early as 1 h and persisting through 24–48 h (Figure 5). This sustained downregulation further compromised mitochondrial protection, reinforcing the pro-apoptotic impact of the treatment. Rapid decreases in Bcl-2 protein levels have already been observed in response to various cellular stresses, such as oxidative and endoplasmic reticulum stress, where Bcl-2 is targeted for ubiquitin-proteasome–mediated degradation [41]. In light of these findings, it is plausible that the very early reduction in Bcl-2 after Q-3-Dec exposure may involve acute post-transcriptional regulation and/or accelerated protein turnover in response to endoplasmic reticulum stress degradation [41], although the precise mechanisms need to be further elucidated.

All these findings indicated that Q-3-Dec disrupts NF-κB -dependent pro-survival transcriptional programs, thereby impairing survivin-associated proliferative and anti-apoptotic functions, overcoming apoptosis-resistant phenotypes characteristic of aggressive gliomas. Due to the known interaction of NF-κB with STAT3 [42,43], we also examined STAT3 activation. The results obtained showed a rapid and sustained decrease in STAT3 phosphorylation after Q-3-Dec treatment (Figure 3). Importantly, both NF-κB and STAT3 showed an early, transient increase in phosphorylation at specific time points, which is more consistent with an acute stress-adaptation response rather than with a random fluctuation. This short-lived activation likely reflects a compensatory attempt of glioma cells to counteract Q-3-Dec-induced mitochondrial and signaling stress by temporarily boosting pro-survival transcriptional programs. However, this early adaptive phase is subsequently overridden by the progressive dephosphorylation of NF-κB and STAT3 and by the concurrent downregulation of survivin and Bcl-2, ultimately resulting in the collapse of survival signaling that normally supports glioma cell proliferation, metabolic adaptation, and resistance to stress [44,45]. Furthermore, mitochondria are central regulators of glioma biology, functioning not only as the primary source of cellular energy but also as key modulators of inflammatory signaling and programmed cell death. In glioma cells, oxidative phosphorylation is frequently reprogrammed to meet increased bioenergetic demands and to support survival under hypoxic conditions [46,47]. In parallel, alterations in mitochondrial membrane permeability contribute to apoptosis resistance by limiting cytochrome c release and downstream caspase activation [48]. Because mitochondrial integrity represents a critical signaling hub linking metabolic adaptation to apoptotic regulation, we investigated whether Q-3-Dec treatment disrupts mitochondrial function. Our results demonstrate that Q-3-Dec induces a loss of the mitochondrial membrane potential, detectable as early as 1 h, which becomes more pronounced by 3 h after treatment, indicating a substantial mitochondrial toxicity at early time points (Figure 6). Notably, this mitochondrial dysfunction appears to precede the maximal inhibition of NF-κB and STAT3 phosphorylation, as well as the downregulation of the pro-survival factors survivin and Bcl-2, which were observed at later stages. This temporal sequence supports the interpretation that mitochondrial impairment may act as an initiating stress event that drives, rather than merely accompanies, the subsequent collapse of pro-survival transcriptional networks and activation of the cell death cascade. Figure 9 shows the possible mechanistic model of Q-3-Dec-induced mitochondrial dysfunction and apoptotic signaling in U373-MG glioma cells.

In addition to these effects, Q-3-Dec significantly downregulated the expression of MGMT (Figure 7). The progressive reduction in MGMT protein levels observed between 6 and 24 h after treatment indicates a time-dependent suppression of MGMT expression in our model, potentially implying an attenuation of MGMT-mediated DNA repair capacity, although neither DNA repair activity nor upstream regulatory mechanisms were directly assessed in this study. Previous reports have shown that quercetin reduces MGMT expression in MGMT-positive glioblastoma cells by repressing MGMT transcription and promoting protein downregulation, thereby enhancing apoptotic responses to Temozolomide [35]. Our results, which show the MGMT decrease and sustained inhibition of NF-κB and STAT3 signaling after the treatment of glioma cells with Q-3-Dec, are consistent with a flavonoid-mediated modulation of MGMT at both transcriptional and post-transcriptional levels [49].

These findings provide a rationale for future studies evaluating whether Q-3-Dec can potentiate the efficacy of Temozolomide or other alkylating agents in glioma. Importantly, early mitochondrial dysfunction combined with reduced NF-κB- and STAT3-dependent transcription, downregulation of the anti-apoptotic factors Bcl-2 and survivin, and impairment of MGMT-mediated DNA repair collectively remove key molecular safeguards against apoptosis. This convergence of effects offers a mechanistic framework that explains both the timing and magnitude of glioma cell death observed at later time points (Figure 8) following Q-3-Dec exposure, supporting the notion that Q-3-Dec exerts its cytotoxic activity through the coordinated disruption of multiple pro-survival pathways rather than the inhibition of a single molecular target.

Overall, this study identifies 3*-O-*decanoylquercetin (Q-3-Dec) as a novel quercetin derivative that exerts anticancer activity through the coordinated and pleiotropic modulation of multiple, interconnected pathways underlying glioma aggressiveness and therapeutic resistance. By simultaneously targeting NF-κB- and STAT3-dependent signaling, downregulating key anti-apoptotic proteins such as survivin and Bcl-2, and inducing early mitochondrial dysfunction, Q-3-Dec functions as a multi-node regulator of glioma cell viability rather than a single-pathway inhibitor. Importantly, our findings provide mechanistic evidence linking acylation-driven physicochemical modification to mitochondrial engagement and disruption of pro-survival signaling, thereby extending current understanding of how structurally modified flavonoids may overcome intrinsic resistance mechanisms in glioma cells. Together, these results offer a mechanistic rationale and proof-of-concept for further preclinical investigation, including in vivo validation, assessment of blood–brain barrier penetration, and evaluation of combination strategies with standard therapies. Nonetheless, substantial additional work will be required before any clinical translation can be considered.

4. Materials and Methods

4.1. Glioma Cell Line Cultures

The U373-MG glioma cell line was kindly provided by Dr. G. Finocchiaro at the Carlo Besta Neurological National Institute in Milan (Italy). In this study, we used the U373-MG glioma cell line, which was also employed as a model system in our previous study [33]. Cells were maintained in RPMI 1640 medium (GIBCO, Life Technologies™, Grand Island, NY, USA) supplemented with 10% heat-inactivated Fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA), 50 U/mL penicillin, 0.05 mg/mL streptomycin, 1% non-essential amino acids, 2 mM GlutaMAX, and 1 mM sodium pyruvate. Cultures were initially seeded in 25 cm^2^ flasks at a density of 2 × 10^6^ cells and incubated at 37 °C in a humidified atmosphere containing 5% CO_2_. The medium was refreshed every 2–3 days. When cultures reached approximately 85–90% confluence, cells were transferred to 100 mm dishes, Lab-Tek II chamber slides (Thermo Scientific™, Thermo Fisher Scientific Inc., Waltham, MA, USA), coverslips in 24-well plates, or 96-well plates, depending on the subsequent analyses, and maintained under the same incubation conditions.

4.2. Treatment of U373-MG Glioma Cell Line Cultures

Q-3-Dec was synthesized and prepared as previously described [33]. The concentration of 50 µM Q-3-Dec used in this study was selected on the basis of our prior findings, which demonstrated that this dose effectively reduces glioma cell survival [33]. As 50 µM represents the lowest concentration capable of significantly impairing glioma cell viability, we elected to use this single concentration for the present mechanistic investigation.

U373-MG cultures were exposed for 1, 3, 6, 12, 24, and 48 h to 50 μM Q-3-Dec solubilized in DMSO, and/or DMSO having a final concentration of 0.01% v/v, which was the same used to solubilize the quercetin derivative; we used this as a control (CTR/DMSO) group.

4.3. Western Blotting Analysis

U373-MG seeded in 100 mm Ø dishes after the experimental treatments with or without 50 μM Q-3-D were harvested with a cell scraper (Costar^®^, Corning Life Sciences, Tewksbury, MA, USA) and cold 1× PBS. Cells were then pelleted and homogenized in 1× lysis buffer [33]. Protein concentration was determined by using the BCA method (BCA Protein Assay Kit-Pierce™, Thermo Scientific, Waltham, MA, USA). 50 µg of total proteins were electrophoresed through 4–15% precast SDS-PAGE gels (Bio-Rad Laboratories, Hercules, CA, USA). The obtained filters were incubated with the specific primary antibodies such as: NF-κB p65 mouse monoclonal (# sc-8008; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), P-NF-κB p65^(Ser468)^ rabbit polyclonal (#PA5-37721-Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA), STAT3 rabbit monoclonal (**#**4904-Cell Signaling Technology, Inc., Danvers, MA, USA), P-STAT3^(Tyr705)^ rabbit monoclonal (# 9145, Cell Signaling Technology, Inc., Danvers, MA, USA), survivin mouse monoclonal (# sc-17779, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Bcl-2 rabbit polyclonal (#2876, Cell Signaling Technology, Inc., Danvers, MA, USA), MGMT mouse monoclonal (# MAB16200, Merck Life Science/Millipore Sigma, Burlington, MA, USA), and actin mouse monoclonal (#sc-8432, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), (dilution 1:1000). Anti-rabbit and anti-mouse secondary antibodies linked to alkaline phosphatase (AP) were used. WesternBreeze Chemiluminescent Western blot Immunodetection Kit (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA) was used to reveal immunobands. The immuno signals were detected through the ChemiDoc Imaging System (Bio-Rad Laboratories Srl, Milan, Italy) and evaluated by densitometric analysis using the Image Lab software (Bio-Rad version number: 6.1).

4.4. Immunocytochemical Assay

U373-MG cells were seeded in Lab-Tek II Chamber-Slide Systems at the final density of 0.5 × 10^5^ cells/mL and incubated at 37 °C in 5% CO_2_–95% air. 80% confluent cells were treated with DMSO and/or 50 μM Q-3-Dec for 1, 3, 6, and 24 h. After the treatments, the cells were fixed for 15 min with 4% paraformaldehyde, washed with PBS, and cellular membranes were permeabilized with 5% normal goat serum (NGS) in PBS containing 0.1% Triton X-100 at room temperature for 45 min, to block non-specific sites. Subsequently, they were incubated overnight at 4 °C in a humidified chamber, with mouse monoclonal-anti-P-NF-κB p65 (# sc-166748, Santa Cruz Biotechnology, INC) (diluted in PBS 1:100), rabbit monoclonal anti-P-STAT-3 (# 9145, Cell Signaling Technology) (diluted in PBS 1:200) or mouse monoclonal anti-survivin (# sc-17779, Santa Cruz Biotechnology, INC) (diluted in PBS 1:100) mouse monoclonal antibodies. Thereafter, the cells were washed with PBS, and incubated for 2 h with a Cy3-conjugated goat anti-mouse IgG secondary antibody or Cy3-conjugated goat anti-rabbit IgG secondary antibody (1:500 in PBS) and the slides were mounted with a DAPI-containing mounting medium (PBS/glycerol 50:50). The slides were scanned with a Nikon Ti-Eclipse inverted confocal microscope through a Plan Fluor 20×/0.5 or Plan Apochromat lambda 60×/1.4 oil immersion lens objective (Nikon, Tokyo, Japan) and acquired with NIS-Elements AR v4.60 software (Nikon, Tokyo, Japan). TRITC fluorescence was quantified by measuring the mean intensity of the TRITC channel in multiple auto-detected regions of interest (ROIs) from at least three background-corrected scanned images for each time point. To check for non-specific staining in U373-MG, the primary antibody was omitted in two chambers of the control incubations; no stain was observed.

4.5. Mitochondrial Health Assay

Mitotoxicity and cytotoxicity levels in U373-MG glioma cells exposed to Q-3-Dec were detected by using the HCS Mitochondrial Health Kit Invitrogen™ (Thermo Fisher Scientific, Waltham, MA, USA). Cells were cultured onto coverslips in 24 multiwell plates and exposed to 50 µM Q-3-Dec for different times (1, 3, 6, and 24 h). Control cells only received the same amount of DMSO used to vehicle the compound tested. After the treatments, according to the manufacturer’s instructions, cells underwent incubation for 30 min with MitoHealth, which accumulates in mitochondria in live cells proportional to the mitochondrial membrane potential and functionality, and Image-iT DEAD Green stain, which can form highly fluorescent dye-DNA complexes when plasma membrane integrity is compromised. Soon after, U373-MG were washed with PBS, fixed with 4% paraformaldehyde (PFA), and counterstained with the blue-fluorescent nuclear dye 4′,6-diamidino-2-phenylindole (DAPI). Coverslips were scanned with a Nikon Ti-Eclipse inverted microscope through either a Plan Apochromat lambda 60×/1.4 oil immersion lens Nikon© (Nikon, Tokyo, Japan) and acquired with NIS-Elements AR v4.60 software Nikon© (Nikon, Tokyo, Japan). Each fluorescence was analyzed and quantified by the evaluation of the mean intensity (MIF) of every single channel from multiple regions of interest (ROI), normalized to the background by using the NIS-Elements AR (Advanced Research) software (version 4.60) (Nikon Instruments Inc., Melville, NY, USA), as previously described [50].

4.6. MTT Bioassay

Cell viability/toxicity was assessed by MTT Test on untreated (control), or Q-3-Dec treated U373-MG. Cells seeded in 96-multiwell plates, after the treatments, were exposed to 20 µL of MTT stock solution, 5 mg/mL PBS, in 200 µL medium per well [51]. After 2 h incubation, the medium from each well was removed and replaced with 100 µL of DMSO. The optical density from each well was measured through the microplate reader (Cary-50 MPR-Varian© - Varian Inc., Palo Alto, CA, USA) at λ of 570 nm. Results are reported as a percentage of the control, taken as 100%, to normalize the different values obtained.

4.7. Statistical Data Analysis

Each analysis was executed in triplicate for each experimental condition. Three or four independent experiments were conducted. Data obtained were statistically analyzed using One-Way analysis of variance (ANOVA), followed by a post hoc Holm–Sidak test, or by Tukey’s multiple comparisons test or Brown–Forsythe test to evaluate significant differences among experimental groups. Data were expressed as mean ± S.D. Statistical significance is reported in figure legends.