Synergistic antitumor and radiosensitizing effects of α -sulfoquinovosyl-acylpropanediol (SQAP) via PI3K/Akt inhibition and DNA repair impairment in glioblastoma

Synergistic antitumor and radiosensitizing effects of α -sulfoquinovosyl-acylpropanediol (SQAP) via PI3K/Akt inhibition and DNA repair impairment in glioblastoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synergistic antitumor and radiosensitizing effects of α -sulfoquinovosyl-acylpropanediol (SQAP) via PI3K/Akt inhibition and DNA repair impairment in glioblastoma Takayuki Nishiwaki, Urara Kudo, Shinsuke Nakamura, Yoshiki Kuse, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6896488/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Aug, 2025 Read the published version in Journal of Neuro-Oncology → Version 1 posted 11 You are reading this latest preprint version Abstract Purpose Radiotherapy remains a key treatment modality for glioblastoma (GBM), but therapeutic resistance and radiation-induced toxicity severely limit its efficacy. Therefore, the development of novel, safe, and effective radiosensitizers is urgently needed. α-sulfoquinovosylacylpropandiol (SQAP), a marine-derived compound, has demonstrated potent radiosensitizing effects in cancer cells by improving tumor oxygenation and interfering with DNA repair. However, its impact on GBM has not yet been investigated. This study aimed to evaluate the biological effects of SQAP on GBM cells and to assess its potential as a radiosensitizer for future clinical application. Methods In vitro analyses—including cell viability, colony formation, immunoblotting, quantitative reverse transcription polymerase chain reaction, immunocytochemistry, and cell death/proliferation assays—were conducted to examine SQAP's mechanisms of action. In vivo efficacy and safety were evaluated using a murine intracranial glioma model. Results SQAP inhibited GBM cell proliferation while sparing normal astrocytes. In combination with radiotherapy SQAP significantly reduced colony formation and enhanced cell death without affecting mitosis. SQAP decreased PI3K/Akt phosphorylation and modulated expression of downstream apoptotic and cell cycle-related proteins. Additionally, SQAP suppressed HIF-1α and VEGF expression. Although SQAP alone did not cause DNA damage, it delayed radiotherapy-induced DNA repair, as shown by prolonged γH2AX expression and reduced 53BP1 nuclear expression. Conclusion SQAP exerts both antitumor and radiosensitizing effects in GBM models by inhibiting PI3K/Akt signaling, suppressing hypoxia-related pathways, and impairing DNA repair. These findings support its potential as a promising adjunctive agent in GBM therapy. Glioblastoma radiotherapy radiosensitizer DNA repair Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Radiation therapy is an integral component of the standard treatment for glioblastoma (GBM) owing to its high therapeutic efficacy combined with minimal invasiveness [ 1 ]. However, the high radiation doses used (~ 60 Gy) pose significant challenges, including treatment-related complications including radiation necrosis and difficulties with re-irradiation at recurrence. [ 2 , 3 ]. A promising strategy to overcome these limitations is the use of agents that selectively enhance the effects of radiation on tumor cells. Sulfoquinovosylacylpropandiol (SQAP) is a derivative developed based on sulfoquinovosylacylglycerol (SQAG), a natural marine product found in the red alga, Chondracanthus tenellus . It exhibits a potent radiosensitizing effect, with a radiation enhancement ratio of > 3.0 [ 4 – 6 ]. Furthermore, SQAP specifically binds to surface antigens on cancer cells, and its glycolipid structure allows it to be rapidly internalized and metabolized in normal cells, thereby exhibiting no cytotoxicity [ 7 , 8 ]. The radiosensitizing effects of SQAP are mediated by multiple mechanisms: a transient enhancement of tumor cell blood flow, which increases the local oxygen partial pressure [ 9 , 10 ], and the inhibition of DNA repair processes— specifically, by suppressing PARP-mediated repair of single-strand breaks (SSB) and by inhibiting double-strand break (DSB) repair via both homologous recombination (HR) and non-homologous end joining (NHEJ) [ 11 ]. In Japan, SQAP has been used to treat nasal adenocarcinoma in animals since September 2023 and remains the only available radiosensitizing agent. Preclinical studies have demonstrated radiosensitizing effects in human prostate and pancreatic cancer cells[ 12 , 13 ]; however, to date, no studies have evaluated this activity in GBM. Therefore, we aimed to investigate in detail the effects of SQAP on GBM cells and its potential radiosensitizing properties. Materials and Methods Cell lines and culture conditions Three human cells lines, U87MG, U251MG, and T98G, and one murine cell line, GL261, were used. The source of purchase is listed in Online Resource 1. All cell lines were seeded and cultured as described previously [ 14 ]. For normoxia experiments, cells were cultured at 37°C in 5% CO 2 , and for hypoxia experiments, cells were maintained under consistent 1% O 2 hypoxic condition [ 15 ]. Regents Temozolomide (TMZ) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and was dissolved in dimethyl sulfoxide (FUJIFILM Wako Pure Chemical Corporation). SQAP was purchased from Malignant Tumor Treatment Technologies (Osaka, Japan) and was dissolved in DMEM. Cell viability assay Cell viability was determined using the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) as previously described [ 16 ]. The absorbance of each well was measured at 450 nm, with a reference wavelength of 660 nm, using a Varioskan Flash 2.4 microplate reader (Thermo Fisher Scientific) 48 h after drug administration. Colony formation assay U87MG cells were seeded at 1.0×10 3 cells/well in six-well plates containing DMEM supplemented with 10% FBS. After 24 h, the medium was replaced, and SQAP treatment was administered 30 min prior to irradiation with an X-ray machine (MX-160Labo; mediXtec, Chiba, Japan). Thereafter, irradiation and drug treatments were performed every 3 days. After 14 days of treatment, cells were fixed with 100% methanol for 15 min and stained with 0.5% crystal violet for 30 min. Colonies comprising > 100 cells were counted using an All-in-One Fluorescence Microscope (BZ-X710; Keyence, Osaka, Japan). Immunoblotting U87MG cells were cultured in 12-well plates at a density of 2.5×10 4 cells/well in DMEM supplemented with 10% FBS. After a 24-h incubation, the medium was replaced with fresh DMEM containing 10% FBS, followed by the addition of 150 µM SQAP for 6, 12, 24, or 48 h. Immunoblotting procedures were detailed previously [ 17 ]. The primary antibodies were are provided in Online Resource 1 ; all primary antibodies were diluted 1:1000. The membranes were incubated with the following secondary antibodies: goat anti-rabbit IgG (32460, Thermo Fisher Scientific, Massachusetts, USA) or goat anti-mouse IgG (32430, Thermo Fisher Scientific). Protein bands were detected using Immuno Star LD (FUJIFILM Wako Pure Chemical Corporation). Band intensities were quantified using an Amersham Imager 680 (Cytiva, Tokyo, Japan). β-actin was used as a loading control to confirm equal protein loading. Additionally, phosphorylation signals were normalized to the total protein content of the corresponding target. Quantitative reverse transcription polymerase chain reaction To evaluate the effect of SQAP on the mRNA expression of BAX, Bcl-2, p21, cyclin D1, and VEGF, we conducted quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. U87MG-cells were cultured at a density of 2.5×10⁴ cells/well in culture medium supplemented with 10% FBS. After a 24-h incubation, the medium was replaced with fresh DMEM containing 10% FBS, followed by the addition of 150 µM SQAP for 3, 6, 12, or 24 h. Total RNA was extracted using the NucleoSpin® RNA II kit (Takara, Shiga, Japan), and RNA concentrations were measured using a biospectrometer (Eppendorf, Hamburg, Germany). Complementary DNA (cDNA) was synthesized from the isolated RNA via reverse transcription using the PrimeScript™ RT reagent kit (Perfect Real Time; Takara, Shiga, Japan). qRT-PCR was performed using TB Green® Fast qPCR Mix (Takara) on a TP800 Thermal Cycler Dice Real-Time System (Takara), following the manufacturer’s instructions. Primer sequences for BAX, Bcl-2, p21, cyclin D1, VEGF, and β-actin are provided in Online Resource 1. β-actin was used as an internal control. PCR cycling conditions followed the manufacturer’s protocol: 40 cycles of amplification, with each cycle comprising 5 s at 95°C and 30 s at 60°C. Gene expression was normalized to β-actin and is presented as relative expression values. Cell death assay U87MG cells were seeded in 96-well plates at a density of 5×10³ cells per well in DMEM supplemented with 10% FBS and incubated for 24 h. The culture medium was replaced with fresh DMEM containing 10% FBS, and SQAP was added at final concentrations of 30 or 150 µM. After 30 min of incubation, cells were irradiated with 4 Gy with an X-ray machine (MX-160Labo) and subsequently incubated for an additional 48 h. Cell death was assessed using Hoechst 33342 (Invitrogen) and propidium iodide (PI, Invitrogen) staining. Hoechst 33342 and PI were added to the culture medium at final concentrations of 8.1 and 1.5 µM, respectively, and incubated for 15 min. Stained cells were imaged using a Lionheart™ FX Automated Microscope (BioTek, Winooski, Vermont, USA). The percentage of PI-positive cells was determined by distinguishing fluorescence signals from Hoechst 33342 and PI. The cell death rate was calculated as follows: Cell death rate = PI-positive cells/Hoechst 33342-positive cells×100 (%). Bromodeoxyuridine (BrdU) cell proliferation assay U87MG cells were seeded in 96-well plates at a density of 5×10³ cells/well in DMEM supplemented with 10% FBS and incubated for 24 h. The culture medium was then replaced with fresh DMEM containing 10% FBS, and SQAP was added at final concentrations of 30 or 150 µM. After 30 min of incubation, cells were irradiated with 4 Gy with the X-ray machine (MX-160Labo) and subsequently incubated for an additional 48 h. The culture medium was replaced with fresh DMEM containing 10% FBS, followed by treatment with 10 µM BrdU for 3 h. Immunocytochemistry was performed according to the manufacturer's protocol using an anti-BrdU antibody (ab6326, Abcam, 1:500). Immunocytochemistry U87MG cells were seeded at a density of 1.0×10⁵ cells/well on four-well glass slides in DMEM supplemented with 10% FBS and incubated for 24 h. The culture medium was then replaced with fresh DMEM containing 10% FBS, and SQAP was added at final concentrations of 30 or 150 µM. After 30 min, cells were irradiated with 4 Gy with an X-ray machine (MX-160Labo) and subsequently incubated for 1, 3, 6, or 12 h. Following incubation, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at 37°C. After blocking with 10% horse serum (Vector Labs, Burlingame, CA, USA) at room temperature for 30 min, the slides were incubated overnight at 4°C with either rabbit anti-phospho-H2A.X (Ser139, 20E3) (9718S, Cell Signaling, 1:300) or rabbit anti-53BP1 (E7N5D) (88439S, Cell Signaling, 1:300). Next, cells were washed with PBS and incubated with Alexa Fluor® 647 goat anti-rabbit IgG secondary antibody (1:500, Thermo Fisher Scientific) for 1 h at room temperature. Cell nuclei were counterstained with Hoechst 33342, and fluorescence images were captured using the FLUOVIEW FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan). For quantification of pH2AX and 53BP1 foci, at least 100 cells/well were analyzed. Cells were categorized based on the number of foci as follows: strongly positive (≥ 11 foci), positive (1–10 foci), and negative (0 foci). The percentage of cells in each category was then calculated. Foci quantification was performed independently by two blinded observers, and the average values were used for analysis. Animals The Animal Experiment Committee of Gifu University, Japan (Ethics No.AG-P-N-20240062), reviewed and approved all animal procedures. The experiments were conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Eight-week-old C57BL/6J male mice were purchased from The Jackson Laboratory Japan, Inc. (Yokohama, Kanagawa, Japan). The mice were housed at 24 ± 2°C under a 12-h light–dark cycle with ad libitum access to food (CE-2, CLEA Japan, Tokyo, Japan) and water. On day 0, GL261 cells (1.0×10⁵ in 2 µL PBS) were stereotactically injected into the left striatum [ 18 ]. The mice were housed in groups of five per cage and randomly assigned to four experimental groups: vehicle (n = 10), SQAP-only (n = 5), RT-only (n = 8), and SQAP + RT (n = 7). In the SQAP-only and SQAP + RT groups, SQAP (4 mg/kg) dissolved in saline was administered via tail vein injection for 4 consecutive days, starting on day 6 post-GL261 cell injection. The vehicle group received an equivalent volume of saline (5 mL/kg). The SQAP dosage was determined based on the manufacturer’s instructions. In the RT-only and SQAP + RT groups, non-anesthetized 4 Gy per fraction irradiation with X-ray machine (MX-160Labo) was performed on days 7 and 8, 30 min after either SQAP or saline injection. On day 14, the mice were anesthetized and transcardially perfused with 4% paraformaldehyde (Wako Pure Chemicals, Osaka, Japan). The brains were dissected, post-fixed in 4% paraformaldehyde, embedded in paraffin (Leica Biosystems, Wetzlar, Germany), sectioned at 5-µm thickness, and stained with hematoxylin–eosin (HE). The maximum sectional tumor areas and tumor volumes were assessed using an All-in-One Fluorescence Microscope (BZ-X710; Keyence). Fifteen sections per mouse were analyzed, with two images obtained per section. Tumor volume was calculated using the formula: V = 4​πr 3 /3. This experiment was conducted in a non-blinded manner. Statistical analyses Data are presented as mean ± standard error of the mean (SEM). Statistical analyses were conducted using a one-way or two-way analysis of variance (ANOVA) followed by Dunnett’s test and Tukey’s test when comparing continuous values of two or more groups, and Kruskal-Wallis test for the ordinal variables of two or more group comparisons. These statistics were performed using Graph-Pad Prism v.10 (GraphPad Software, Boston, MA, USA) software. P < 0.05 was considered statistically significant. Results See Fig. 1 a for schematic summary of the key findings. SQAP suppressed GBM cell proliferation SQAP exhibited a concentration-dependent suppression of cell viability, regardless of the cell line used (Fig. 1 b–d). In contrast, the viability of astrocytes remained unchanged 48 h after SQAP administration at concentrations up to 150 µM. Similar results obtained under hypoxic conditions and for T98G cells are shown in Online Resource 2. Effect of SQAP combined with RT on GBM cell proliferation A colony formation assay was performed to evaluate the effect of combining RT with SQAP on GBM cell proliferation. Radiation and drug treatments were administered four times in each group, with total radiation doses of 8 and 16 Gy. In the no-irradiation group, similar to the CCK-8 assay, colony formation was not significantly reduced at SQAP concentrations up to 50 µM. However, in the 8-Gy group, colony numbers were significantly reduced at even 30 µM SQAP compared to the control group. A similar trend was observed in the 16-Gy group; however, the number of colonies in this group was already low even in the absence of SQAP, resulting in no significant differences (Fig. 1 e,f). Combination of SQAP and RT sensitized to the cell death while not affecting mitotic inhibition No significant increase in dead cells was observed up to 30 µM SQAP, and only a slight increase was observed following 4 Gy irradiation. Conversely, 150 µM SQAP not only markedly increased the number of dead cells but also significantly enhanced cell death in the 4 Gy group compared with that of the 0 Gy group (64.63 vs. 77.31, p < 0.01) (Fig. 1 g,h). Meanwhile, the BrdU assay demonstrated a concentration-dependent decrease in BrdU-incorporated cells with SQAP treatment; however, irradiation had no effect, as no significant differences were observed between the irradiated and non-irradiated groups (Fig. 1 i,j). SQAP decreased the expression of PI3K and Akt Immunoblotting was used to reveal the mechanisms of the anti-tumor effects of SQAP. The phosphorylation of Akt decreased over time over 24 h and the phosphorylation of PI3K decreased over time over 48 h (Fig. 2 a,b). The same test performed under hypoxia showed similar results (Online Resource 3). SQAP affected the expression of proteins downstream of the PI3K/Akt signaling pathway To elucidate the effects of SQAP on downstream components of the PI3K/Akt signaling pathway involved in apoptosis, we examined both the mRNA and protein expression of BAX (Bcl-2-associated X protein, an apoptosis-inducing protein) and Bcl-2 (B-cell lymphoma-2, an apoptosis-inhibiting protein). SQAP treatment increased BAX mRNA levels while decreasing those of Bcl-2, resulting in an elevated apoptosis-promoting BAX/Bcl-2 ratio over time (Fig. 2 c). A similar trend was observed at the protein expression level and under hypoxic conditions; however, the differences were not statistically significant (Online Resource 4). Next, we investigated the effects of SQAP on PI3K/Akt downstream components related to the cell cycle, including cyclin D1 and cyclin B1 (cell cycle-associated proteins) and p21 (a cyclin-dependent kinase inhibitor). SQAP treatment reduced cyclin B1 (Fig. 2 d) and p21 mRNA expression (Fig. 2 e) but did not influence cyclin D1 mRNA levels (Online Resource 5 showing results under hypoxic conditions). Furthermore, given previous reports on SQAP activity in other cancers, we evaluated its impact on HIF-1α and its downstream target VEGF. SQAP treatment induced a decrease in HIF-1α expression and a reduction in VEGF mRNA levels (Fig. 2 f,g). SQAP inhibited RT-induced DNA damage repair and enhanced nuclear γH2AX expression To determine whether SQAP enhanced radiosensitivity, we analyzed changes in expression of proteins involved in DNA repair and DNA damage following SQAP treatment. Compared with the control group, PARP expression significantly decreased over 48 h in the SQAP-treated groups (Fig. 3 a). Next, we examined the nuclear expression of γH2AX, a marker of DNA damage, and established inclusion and exclusion criteria for quantitative analysis (Fig. 3 b,d). Although the strongly positive rate increased with TMZ alone, no such increase was observed with SQAP alone (Fig. 3 c), suggesting that SQAP itself does not induce DNA damage. In the stacked bar graph, the bars represent the percentage of cells categorized as negative, positive, and strongly positive from the bottom up, with higher percentages at the top indicating greater DNA damage (Fig. 3 e). Three h after irradiation, γH2AX expression increased in a concentration-dependent manner. In the 150 µM SQAP group, the strongly positive rate increased, whereas the negative rate decreased (Fig. 3 e). By 12 h post-irradiation, the proportion of strongly positive cells no longer differed between groups. However, the number of negative cells remained significantly lower in the SQAP-treated group than in the control group, suggesting delayed DNA repair (Fig. 3 e). Similarly, on comparing nuclear 53BP-1 expression (Fig. 4 a), the strongly positive rate significantly decreased, and the negative rate increased in the 150 µM SQAP-treated group 3 h after irradiation (Fig. 4 b). A similar trend was observed at 30 µM. By 12 h post-irradiation, the strongly positive rate was nearly absent in both groups, but the negative rate was significantly lower in the 150 µM SQAP-treated group (Fig. 4 b). SQAP treatment and RT inhibited GBM growth in mice models The protocol for drug administration and irradiation is illustrated in Fig. 5 a. The mean maximum cross-sectional area and volume were more significantly reduced in the SQAP + RT group than in the RT alone group, when compared with that of the vehicle-treated group. Although the difference between the RT alone and the RT + SQAP combination did not reach statistical significance, it is noteworthy that tumor growth was markedly suppressed in nearly all cases in the combination group (Fig. 5 b,c). Discussion In this study, we investigated whether SQAP exerts radiosensitizing and antitumor effects on GBM cells using in vitro and in vivo experiments. Our findings demonstrated that SQAP directly exerts antitumor effects by inhibiting the PI3K/Akt signaling pathway. Furthermore, at lower drug concentrations, SQAP exhibited a radiosensitizing effect by suppressing DNA repair. To the best of our knowledge, this is the first study to demonstrate the antitumor and radiosensitizing effects of SQAP on GBM cells. SQAP derives from the natural compounds sulfoquinovosylmonoacylglycerol and SQAG, first described by Sahara et al. and Ohta et al. [ 5 , 19 ]. These compounds were initially developed as DNA polymerase inhibitors as they exhibited weak anticancer effect [ 20 , 21 ]. Subsequently, they were found to possess strong radiosensitizing effects [ 6 ]. Currently, SQAP has been successfully applied as a radiosensitizer for advanced adenocarcinoma in the nasal cavity in dogs. This study was conducted to explore the possibility of future clinical applications of human GBM. We first evaluated the standalone effects of SQAP on GBM cells. Although SQAP exhibited only modest anticancer activity during its preclinical development, our CCK-8 and colony formation assays under non-irradiated conditions (0 Gy) revealed that SQAP significantly reduced the proliferative capacity of GBM cells. Notably, in lung cancer cells (A549) with highly activated Akt, SQAP suppresses focal adhesion kinase (FAK) phosphorylation, leading to subsequent inhibition of Akt phosphorylation, reduced VEGF expression, and anti-angiogenic effects [ 22 ]. Although FAK was not investigated in our study, we observed suppression of phosphorylated Akt and its upstream regulator phosphorylated PI3K in GBM cells by SQAP. The PI3K/Akt signaling pathway, which is constitutively activated in GBM, represents a promising therapeutic target in this malignancy [ 23 , 24 ]. Akt, a central kinase in the PI3K/Akt pathway, plays critical roles in suppressing apoptosis and promoting cell cycle progression [ 25 ]. Akt indirectly inhibits BAX and enhances Bcl-2 to block apoptosis. Specifically, Akt phosphorylates FOXO transcription factors, leading to their cytoplasmic sequestration and subsequent suppression of BAX transcription [ 26 ]. Additionally, Akt stabilizes Bcl-2 expression by activating MDM2, which degrades p53 [ 27 , 28 ]. In the cell cycle, Akt promotes G1/S transition by phosphorylating and inactivating GSK-3β, thereby preventing the degradation of cyclin D1 [ 29 ]. Furthermore, Akt stabilizes cyclin B1, a key regulator of the G2/M phase, by inhibiting GSK-3β-mediated phosphorylation and subsequent proteasomal degradation of cyclin B1 [ 30 , 31 ]. Akt also suppresses CDK inhibitors p21 and p27, enabling uncontrolled cell cycle progression [ 32 ]. Although not all downstream signaling components of the PI3K/Akt signaling pathway were fully characterized, we observed critical molecular changes consistent with pathway modulation: (1) increased BAX/Bcl-2 ratio (pro-apoptotic shift), (2) upregulation of p21 (a CDK inhibitor), and (3) downregulation of cyclin B1 (a G2/M phase regulator). As collectively suggested by the cell death assay and BrdU test, SQAP induces apoptosis through mitochondrial dysfunction and impairs cell cycle progression at both G1/S and G2/M checkpoints, ultimately leading to reduced proliferative capacity in GBM cells. Beyond regulating cell death and proliferation, the Akt signaling pathway critically drives radioresistance by orchestrating DNA damage repair mechanisms. Upon radiation-induced DNA damage, Akt activation facilitates the recruitment and stabilization of key DNA repair proteins—including DNA-PKcs and 53BP1—at DSB sites. This enhances both HR and NHEJ, thereby reducing radiation-induced cytotoxicity [ 33 ]. Concurrently, Akt-mediated phosphorylation stabilizes the MDM2 oncoprotein, which suppresses p53-dependent apoptosis, further amplifying radioresistance [ 27 ]. Notably, pharmacological inhibition of Akt isoforms (e.g., MK-2206, Akt inhibitor IV) potentiates radiosensitivity in GBM cells, underscoring its therapeutic relevance [ 34 , 35 ]. Furthermore, SQAP inhibition of HIF-1α, VEGF [ 22 ], and von Hippel-Lindau protein (pVHL) [ 10 ] promotes vascular normalization and induces transient improvement in oxygenation, which may contribute to radiosensitizing effects [ 9 ]. In our study, we observed significant downregulation of HIF-1α and VEGF expression, suggesting that SQAP may modulate angiogenesis in GBM by attenuating hypoxic and pro-angiogenic signaling pathways. Although intratumoral oxygenation status and angiogenesis were not directly assessed in this study, the observed suppression of HIF-1α and VEGF suggests that these pathways—in parallel with Akt inhibition—may contribute to SQAP-mediated radiosensitization. The effect of SQAP on radiation-induced DNA damage was assessed using γH2AX, a phosphorylated histone H2AX variant that serves as a critical biomarker for DSBs induced by ionizing radiation. Its rapid formation at DSB sites facilitates the recruitment of DNA repair machinery [ 36 ], and the persistence of γH2AX foci correlates with unrepaired damage, making it a valuable tool for quantifying radiation-induced DNA damage and predicting therapeutic efficacy in cancer treatment. Depending on the cell type, γH2AX foci typically peak around 0.5–2 h after irradiation. As DNA repair progresses, most DSBs are repaired, and the number of foci returns to baseline levels by approximately 24 h [ 37 , 38 ]. In our study, the number of γH2AX foci increased with the addition of SQAP at 3 h after irradiation, coinciding with the peak of foci formation. At 6 and 12 h, the proportion of γH2AX-negative cells decreased after SQAP treatment, suggesting delayed DNA repair. Furthermore, this repair inhibition was observed even at concentrations as low as 30 µM, indicating that SQAP effectively inhibits DNA repair at low doses. 53BP-1 is a key protein involved in the repair of DNA DSBs. It is recruited to sites of damage through recognition of γH2AX-labeled chromatin and plays a critical role in facilitating the recruitment of downstream DNA repair factors in the NHEJ pathway. Notably, 53BP1 foci are absent once DNA repair is complete[ 39 , 40 ]. In the present study, 53BP-1 positivity decreased in an SQAP concentration-dependent manner during the early phase (1–3 h), when γH2AX expression was elevated. Thus, SQAP may inhibit the initial mobilization of 53BP1 to sites of DNA damage. In contrast, at 12 h, 53BP-1 positivity increased in a concentration-dependent manner following SQAP treatment. This likely reflects the fact that, in control cells, DNA repair had already been completed, and 53BP-1 foci had disappeared. However, on exposure to SQAP, DNA repair was delayed, and 53BP-1 likely remained active at this time point due to the ongoing repair of persistent DNA damage. Finally, to evaluate the in vivo efficacy and effects on brain lesions, we conducted a combination treatment study using radiotherapy and SQAP in a murine model. Although group sizes were limited due to model stability and the technical difficulty of tail vein injections, the group receiving combined RT and SQAP treatment showed a significant reduction in both maximum tumor area and tumor volume compared to the vehicle-treated group. Furthermore, although the difference was not significant, the RT-alone group exhibited only partial tumor shrinkage, whereas the combination group demonstrated consistent and pronounced tumor growth inhibition across all cases. Thus, SQAP may be useful as a radiosensitizer not only for intranasal adenocarcinoma, for which it is currently used clinically, but also for intracerebral tumors. However, the brain penetration of SQAP has not been evaluated, which warrants further research. To our knowledge, this study is the first to report the antitumor and radiosensitizing effects of SQAP in glioma. SQAP inhibits PI3K/Akt signaling, which plays a key role in DNA repair and cell proliferation. By downregulating HIF-1α and VEGF expression, SQAP may also normalize tumor vasculature, thereby indirectly enhancing radiosensitivity. Furthermore, inhibition of PI3K is associated with reduced resistance to temozolomide [ 24 ], suggesting that SQAP may potentiate the therapeutic effects of both radiotherapy and chemotherapy. Although SQAP shows promising potential as an adjunctive treatment for glioma, further research is needed to fully elucidate its safety profile, brain permeability, and underlying mechanisms of action. Conclusion This study is the first to demonstrate that SQAP exerts antitumor and radiosensitizing effects in both glioma cells and mouse models. SQAP enhances radiosensitivity through inhibition of the PI3K/Akt signaling pathway, suppression of HIF-1α and VEGF expression, and modulation of DNA repair dynamics, including attenuation of 53BP1 recruitment. In addition, a direct antitumor effect of SQAP via the induction of apoptosis was also observed. These findings highlight SQAP as a promising adjunctive agent for glioma treatment. However, further investigations are needed to elucidate the brain permeability, long-term safety, and detailed mechanisms of action in humans. Declarations Acknowledgements We would like to thank Editage (www.editage.com) for English language editing. Funding The authors did not receive support from any organization for the submitted work. Competing interests The authors have no conflicts of interest to declare that are relevant to the content of this article. Author contributions TN; Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation and Role/Writing—original draft. UK; Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation and Writing—review & editing. SN; Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Resources, Software, Validation, Supervision and Writing—review & editing. YK; Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Resources, Software, Validation, Supervision and Writing—review & editing. YH; Conceptualization, Investigation, Resources, Validation and Writing—review & editing. YF; Conceptualization, Investigation, Resources, Validation and Writing—review & editing. KS; Conceptualization, Investigation, Resources, Validation and Writing—review & editing. TY; Conceptualization, Investigation, Resources, Validation and Writing—review & editing. HT; Conceptualization, Investigation, Resources, Validation and Writing—review & editing. YE; Conceptualization, Validation, Supervision and Writing—review & editing. HH; Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Resources, Software, Validation, Supervision and Writing—review & editing. NN; Conceptualization, Validation, Supervision and Writing—review & editing. TI; Conceptualization, Validation, Supervision and Writing—review & editing. Masamitsu Shimazawa; Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Resources, Software, Validation, Supervision and Writing—review & editing. The first draft of the manuscript was written by TN, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Conceptualization; TN, UK,SN, YK, YH, FY, KS, TY, HT, YE, HH, NN, TI, MS, Data curation; TN, UK, SN, YK, HH, MS, Formal analysis; TN, UK, SN, YK, HH, MS, Investigation; TN, UK, YH, YF, KS, TY, HT, Methodology; TN, UK, SN, YK, HH, MS, Project administration; SN, YK, HH, MS, Resourse; TN, UK,SN, YK, YH, FY, KS, TY, HT, YE, HH, NN, TI, MS, Software; TN, SN, YK, HH, MS, Supervision; SN, YK, YE, HH, TI, MS, Validation; TN, UK,SN, YK, YH, FY, KS, TY, HT, YE, HH, NN, TI, MS. Roles/Writing—original draft; TN, Writing—review & editing; TN, UK,SN, YK, YH, FY, KS, TY, HT, YE, HH, NN, TI, MS. The first draft of the manuscript was written by TN, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request Ethics approval The Animal Experiment Committee of Gifu University, Japan (Ethics No.AG-P-N-20240062), reviewed and approved all animal procedures. The experiments were conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. Consent to participate Not applicable. Consent to publish Not applicable. References Stupp R, Hegi ME, Gilbert MR, Chakravarti A (2007) Chemoradiotherapy in malignant glioma: standard of care and future directions. J Clin Oncol 25: 4127-4136 doi:10.1200/jco.2007.11.8554 Minniti G, Niyazi M, Alongi F, Navarria P, Belka C (2021) Current status and recent advances in reirradiation of glioblastoma. Radiat Oncol 16: 36 doi:10.1186/s13014-021-01767-9 Siu A, Wind JJ, Iorgulescu JB, Chan TA, Yamada Y, Sherman JH (2012) Radiation necrosis following treatment of high grade glioma--a review of the literature and current understanding. Acta Neurochir (Wien) 154: 191-201; discussion 201 doi:10.1007/s00701-011-1228-6 Sakimoto I, Ohta K, Yamazaki T, Ohtani S, Sahara H, Sugawara F, Sakaguchi K, Miura M (2006) Alpha-sulfoquinovosylmonoacylglycerol is a novel potent radiosensitizer targeting tumor angiogenesis. Cancer Res 66: 2287-2295 doi:10.1158/0008-5472.Can-05-2209 Sahara H, Ishikawa M, Takahashi N, Ohtani S, Sato N, Gasa S, Akino T, Kikuchi K (1997) In vivo anti-tumour effect of 3'-sulphonoquinovosyl 1'-monoacylglyceride isolated from sea urchin (Strongylocentrotus intermedius) intestine. Br J Cancer 75: 324-332 doi:10.1038/bjc.1997.54 Ohta K, Murata H, Mori Y, Ishima M, Sugawara F, Sakaguchi K, Miura M (2010) Remodeling of the tumor microenvironment by combined treatment with a novel radiosensitizer, {alpha}-sulfoquinovosylmonoacylglycerol ({alpha}-SQMG) and X-irradiation. Anticancer Res 30: 4397-4404 Ruike T, Kanai Y, Iwabata K, Matsumoto Y, Murata H, Ishima M, Ohta K, Oshige M, Katsura S, Kuramochi K, Kamisuki S, Sahara H, Miura M, Sugawara F, Sakaguchi K (2019) Distribution and metabolism of (14)C-sulfoquinovosylacylpropanediol ((14)C-SQAP) after a single intravenous administration in tumor-bearing mice. Xenobiotica 49: 346-362 doi:10.1080/00498254.2018.1448949 Ohta K, Mizushina Y, Yamazaki T, Hanashima S, Sugawara F, Sakaguchi K (2001) Specific interaction between an oligosaccharide on the tumor cell surface and the novel antitumor agents, sulfoquinovosylacylglycerols. Biochem Biophys Res Commun 288: 893-900 doi:10.1006/bbrc.2001.5852 Takakusagi Y, Naz S, Takakusagi K, Ishima M, Murata H, Ohta K, Miura M, Sugawara F, Sakaguchi K, Kishimoto S, Munasinghe JP, Mitchell JB, Krishna MC (2018) A Multimodal Molecular Imaging Study Evaluates Pharmacological Alteration of the Tumor Microenvironment to Improve Radiation Response. Cancer Res 78: 6828-6837 doi:10.1158/0008-5472.Can-18-1654 Iwamoto H, Nakamura T, Koga H, Izaguirre-Carbonell J, Kamisuki S, Sugawara F, Abe M, Iwabata K, Ikezono Y, Sakaue T, Masuda A, Yano H, Ohta K, Nakano M, Shimose S, Shirono T, Torimura T (2015) Inhibition of hypoxia-inducible factor via upregulation of von Hippel-Lindau protein induces "angiogenic switch off" in a hepatoma mouse model. Mol Ther Oncolytics 2: 15020 doi:10.1038/mto.2015.20 Maeda J, Shellenberger KD, Kurihara W, Haga T, Kato TA (2023) Sulfoquinovosyl acylpropanediol (SQAP): Inhibition of poly(ADP-ribose) metabolism and enhanced cytotoxicity in homologous recombination repair-deficient Chinese hamster-derived cells. Mutat Res Genet Toxicol Environ Mutagen 892: 503703 doi:10.1016/j.mrgentox.2023.503703 Sawada Y, Omoto K, Kohei N, Sakaguchi K, Miura M, Tanabe K (2015) Sulfoquinovosylacylpropanediol is a novel potent radiosensitizer in prostate cancer. Int J Urol 22: 590-595 doi:10.1111/iju.12753 Takakusagi Y, Sugyo A, Tsuji AB, Sudo H, Yasunaga M, Matsumura Y, Sugawara F, Sakaguchi K, Higashi T (2022) The natural sulfoglycolipid derivative SQAP improves the therapeutic efficacy of tissue factor-targeted radioimmunotherapy in the stroma-rich pancreatic cancer model BxPC-3. Transl Oncol 15: 101285 doi:10.1016/j.tranon.2021.101285 Varricchio A, Khan S, Price ZK, Davis RA, Ramesh SA, Yool AJ (2023) Pharmacological Inhibition of Membrane Signaling Mechanisms Reduces the Invasiveness of U87-MG and U251-MG Glioblastoma Cells In Vitro. Cancers (Basel) 15 doi:10.3390/cancers15041027 Liu J, Gao L, Zhan N, Xu P, Yang J, Yuan F, Xu Y, Cai Q, Geng R, Chen Q (2020) Hypoxia induced ferritin light chain (FTL) promoted epithelia mesenchymal transition and chemoresistance of glioma. J Exp Clin Cancer Res 39: 137 doi:10.1186/s13046-020-01641-8 Yamada T, Tsuji S, Nakamura S, Egashira Y, Shimazawa M, Nakayama N, Yano H, Iwama T, Hara H (2021) Riluzole enhances the antitumor effects of temozolomide via suppression of MGMT expression in glioblastoma. J Neurosurg 134: 701-710 doi:10.3171/2019.12.Jns192682 Tsuji S, Nakamura S, Shoda K, Yamada T, Shimazawa M, Nakayama N, Iwama T, Hara H (2022) NMDA receptor signaling induces the chemoresistance of temozolomide via upregulation of MGMT expression in glioblastoma cells. J Neurooncol 160: 375-388 doi:10.1007/s11060-022-04154-w Yamada T, Tsuji S, Nakamura S, Egashira Y, Shimazawa M, Nakayama N, Yano H, Iwama T, Hara H (2020) Riluzole enhances the antitumor effects of temozolomide via suppression of MGMT expression in glioblastoma. J Neurosurg 134: 701-710 doi:10.3171/2019.12.JNS192682 Ohta K, Mizushina Y, Hirata N, Takemura M, Sugawara F, Matsukage A, Yoshida S, Sakaguchi K (1998) Sulfoquinovosyldiacylglycerol, KM043, a new potent inhibitor of eukaryotic DNA polymerases and HIV-reverse transcriptase type 1 from a marine red alga, Gigartina tenella. Chem Pharm Bull (Tokyo) 46: 684-686 doi:10.1248/cpb.46.684 Hanashima S, Mizushina Y, Ohta K, Yamazaki T, Sugawara F, Sakaguchi K (2000) Structure-activity relationship of a novel group of mammalian DNA polymerase inhibitors, synthetic sulfoquinovosylacylglycerols. Jpn J Cancer Res 91: 1073-1083 doi:10.1111/j.1349-7006.2000.tb00887.x Sahara H, Hanashima S, Yamazaki T, Takahashi S, Sugawara F, Ohtani S, Ishikawa M, Mizushina Y, Ohta K, Shimozawa K, Gasa S, Jimbow K, Sakaguchi K, Sato N, Takahashi N (2002) Anti-tumor effect of chemically synthesized sulfolipids based on sea urchin's natural sulfonoquinovosylmonoacylglycerols. Jpn J Cancer Res 93: 85-92 doi:10.1111/j.1349-7006.2002.tb01204.x Izaguirre-Carbonell J, Kawakubo H, Murata H, Tanabe A, Takeuchi T, Kusayanagi T, Tsukuda S, Hirakawa T, Iwabata K, Kanai Y, Ohta K, Miura M, Sakaguchi K, Matsunaga S, Sahara H, Kamisuki S, Sugawara F (2015) Novel anticancer agent, SQAP, binds to focal adhesion kinase and modulates its activity. Sci Rep 5: 15136 doi:10.1038/srep15136 Verdugo E, Puerto I, Medina M (2022) An update on the molecular biology of glioblastoma, with clinical implications and progress in its treatment. Cancer Commun (Lond) 42: 1083-1111 doi:10.1002/cac2.12361 Li X, Wu C, Chen N, Gu H, Yen A, Cao L, Wang E, Wang L (2016) PI3K/Akt/mTOR signaling pathway and targeted therapy for glioblastoma. Oncotarget 7: 33440-33450 doi:10.18632/oncotarget.7961 Liu R, Chen Y, Liu G, Li C, Song Y, Cao Z, Li W, Hu J, Lu C, Liu Y (2020) PI3K/AKT pathway as a key link modulates the multidrug resistance of cancers. Cell Death Dis 11: 797 doi:10.1038/s41419-020-02998-6 Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96: 857-868 doi:10.1016/s0092-8674(00)80595-4 Mayo LD, Donner DB (2001) A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc Natl Acad Sci U S A 98: 11598-11603 doi:10.1073/pnas.181181198 Zhou BP, Liao Y, Xia W, Zou Y, Spohn B, Hung MC (2001) HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nat Cell Biol 3: 973-982 doi:10.1038/ncb1101-973 Diehl JA, Cheng M, Roussel MF, Sherr CJ (1998) Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 12: 3499-3511 doi:10.1101/gad.12.22.3499 Katayama K, Fujita N, Tsuruo T (2005) Akt/protein kinase B-dependent phosphorylation and inactivation of WEE1Hu promote cell cycle progression at G2/M transition. Mol Cell Biol 25: 5725-5737 doi:10.1128/mcb.25.13.5725-5737.2005 Porter LA, Donoghue DJ (2003) Cyclin B1 and CDK1: nuclear localization and upstream regulators. Prog Cell Cycle Res 5: 335-347 Liang J, Slingerland JM (2003) Multiple roles of the PI3K/PKB (Akt) pathway in cell cycle progression. Cell Cycle 2: 339-345 Toulany M, Kehlbach R, Florczak U, Sak A, Wang S, Chen J, Lobrich M, Rodemann HP (2008) Targeting of AKT1 enhances radiation toxicity of human tumor cells by inhibiting DNA-PKcs-dependent DNA double-strand break repair. Mol Cancer Ther 7: 1772-1781 doi:10.1158/1535-7163.Mct-07-2200 Fujiwara K, Iwado E, Mills GB, Sawaya R, Kondo S, Kondo Y (2007) Akt inhibitor shows anticancer and radiosensitizing effects in malignant glioma cells by inducing autophagy. Int J Oncol 31: 753-760 Chautard E, Loubeau G, Tchirkov A, Chassagne J, Vermot-Desroches C, Morel L, Verrelle P (2010) Akt signaling pathway: a target for radiosensitizing human malignant glioma. Neuro Oncol 12: 434-443 doi:10.1093/neuonc/nop059 Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273: 5858-5868 doi:10.1074/jbc.273.10.5858 Tu WZ, Li B, Huang B, Wang Y, Liu XD, Guan H, Zhang SM, Tang Y, Rang WQ, Zhou PK (2013) γH2AX foci formation in the absence of DNA damage: mitotic H2AX phosphorylation is mediated by the DNA-PKcs/CHK2 pathway. FEBS Lett 587: 3437-3443 doi:10.1016/j.febslet.2013.08.028 Burdak-Rothkamm S, Short SC, Folkard M, Rothkamm K, Prise KM (2007) ATR-dependent radiation-induced gamma H2AX foci in bystander primary human astrocytes and glioma cells. Oncogene 26: 993-1002 doi:10.1038/sj.onc.1209863 Bártová E, Legartová S, Dundr M, Suchánková J (2019) A role of the 53BP1 protein in genome protection: structural and functional characteristics of 53BP1-dependent DNA repair. Aging (Albany NY) 11: 2488-2511 doi:10.18632/aging.101917 Fradet-Turcotte A, Canny MD, Escribano-Díaz C, Orthwein A, Leung CC, Huang H, Landry MC, Kitevski-LeBlanc J, Noordermeer SM, Sicheri F, Durocher D (2013) 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark. Nature 499: 50-54 doi:10.1038/nature12318 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6896488","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":472671427,"identity":"208ec973-db84-48cc-bd4d-0e795fd42892","order_by":0,"name":"Takayuki Nishiwaki","email":"","orcid":"","institution":"Gifu Pharmaceutical University","correspondingAuthor":false,"prefix":"","firstName":"Takayuki","middleName":"","lastName":"Nishiwaki","suffix":""},{"id":472671428,"identity":"e813c599-3b7b-431d-b9a3-234a4eee329d","order_by":1,"name":"Urara Kudo","email":"","orcid":"","institution":"Gifu Pharmaceutical 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04:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6896488/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6896488/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11060-025-05194-8","type":"published","date":"2025-08-11T15:58:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":84889474,"identity":"28a4b842-ef35-4147-8b55-976d8252cf06","added_by":"auto","created_at":"2025-06-18 12:27:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7055418,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Study overview. The figure illustrates the radiation-induced DNA repair mechanisms affected by SQAP, as well as the components of the PI3K/Akt signaling pathway investigated in this study. The dotted enclosure represents pathways presumed to be involved but not directly examined in the present study. \u003cstrong\u003eb\u003c/strong\u003e Cell viability of U87MG and U251MG cells treated with 10–150 µM SQAP for 48 h under normoxia. Data are shown as mean±SEM (n=6). ***p\u0026lt;0.001, ****p\u0026lt;0.0001 vs. Control group (Dunnett’s test) \u003cstrong\u003ec\u003c/strong\u003e Graph showing cell viability of astrocyte cells treated with 30–150 µM SQAP for 48 h. Data are shown as mean±SEM (n=6). \u003cstrong\u003ed\u003c/strong\u003e Graph showing cell viability of GL261cells treated with 10–150 µM SQAP for 48 h. Data are shown as mean±SEM (n=6). **p\u0026lt;0.01, ****p\u0026lt;0.0001 vs. Control group (Dunnett’s test) \u003cstrong\u003ee \u003c/strong\u003eRepresentative colony formation assay. U87 colonies were subjected to four sessions of SQAP treatment and irradiation (2 Gy or 4 Gy), administered every 3 days. Sampling was conducted 14 days after initial seeding. The scale bar is 10 mm. \u003cstrong\u003ef\u003c/strong\u003e Quantitative analysis of colony formation. The number of U87 colonies in controls with 0 Gy irradiation were set to 100%. Values are expressed as mean±SEM (n=3). **p\u0026lt;0.01, ***p\u0026lt;0.001,****p\u0026lt;0.0001 vs. Control group of same irradiation dose (Dunnett’s test) \u003cstrong\u003eg\u003c/strong\u003e Representative images of the cell death assay. PI is Propidium iodide. The scale bar is 100 µm. \u003cstrong\u003eh\u003c/strong\u003e Ratio of cell death observed in U87 cells treated with 30 or 150 µM SQAP with or without 4G irradiation. SQAP added 30 min before irradiation, and cell death assays were performed 48 h later. Data are shown as mean±SEM (n=6). *p\u0026lt;0.05, ****p\u0026lt;0.0001 vs. each group (one-way ANOVA followed by Tukey’s test). \u003cstrong\u003ei\u003c/strong\u003e Representative images of the BrdU assay. The scale bar is 100 µm. \u003cstrong\u003ej\u003c/strong\u003e Graph showing the BrdU incorporation ratio in U87 cells treated with 30 or 150 µM SQAP with 4 Gy irradiation or without irradiation. SQAP added 30 min before irradiation, and the BrdU assays were performed 24 h later. Data are shown as mean±SEM (n=6). ***p\u0026lt;0.001, ****p\u0026lt;0.0001 vs. each group (one-way ANOVA followed by Tukey’s test).\u003c/p\u003e","description":"","filename":"Fig.1.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6896488/v1/d5b23176f4bb6b228675d531.jpg"},{"id":84888753,"identity":"97c08fb5-6755-45dd-ad0e-7b2059baf743","added_by":"auto","created_at":"2025-06-18 12:19:06","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7264416,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in the expression of proteins in human glioblastoma cells. \u003cstrong\u003ea\u003c/strong\u003e Representative images of immunoblots. \u003cstrong\u003eb\u003c/strong\u003e Graphs showing the time course of expression of p-Akt (phospho-Akt), t-Akt (total-Akt), the phosphorylated to total Akt ratio, p-PI3K (phopho-PI3K), t-PI3K (total-PI3K), and the phosphorylated to total PI3K ratio in U87 cells treated with 150 µM SQAP under normoxia. Data are shown as mean±SEM (n=6). *p\u0026lt; 0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 , ****p\u0026lt;0.0001 vs. Control group (Dunnett’s test). \u003cstrong\u003ec \u003c/strong\u003eGraphs showing the results of quantitative reverse transcription PCR (qRT-PCR) analysis of U87 treated with 150 μM SQAP under normoxia. Each column shows the mRNA levels of BAX and Bcl-2 at different time points (3, 6, 12, and 24 h). Data are shown as mean±SEM (n=5). *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001 vs. Control groups (Dunnett’s test). \u003cstrong\u003ed, f \u003c/strong\u003eRepresentative images of immunoblots and graphs showing the time course of expression of cyclin B1 and HIF-1α in U87 cells treated with 150 µM SQAP under normoxia (and hypoxia for HIF-1α). Data are shown as mean±SEM (n=6). **p\u0026lt;0.01, ****p\u0026lt;0.0001, vs. Control groups. (Dunnett’s test) \u003cstrong\u003ee, g\u003c/strong\u003e Graphs showing qRT-PCR analysis in U87 treated with 150 μM SQAP under normoxia (and hypoxia for VEGF). Each column showing the mRNA levels of p21 and VEGF at different time points (3, 6, 12, and 24 h). Data are shown as mean±SEM (n=5). *p\u0026lt;0.05, **p\u0026lt;0.01, vs. Control groups. (Dunnett’s test)\u003c/p\u003e","description":"","filename":"Fig.2.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6896488/v1/f49391389e57d27b1ef5cec6.jpg"},{"id":84888748,"identity":"f454a566-b42f-42a8-84c1-da9d890b0c8d","added_by":"auto","created_at":"2025-06-18 12:19:06","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6429388,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Representative images of immunoblots and graphs showing the time course of expression of PARP in U87 cells treated with 150 µM SQAP under normoxia. Data are shown as mean±SEM (n=6). **p\u0026lt;0.01 vs. Control group (Dunnett’s test).\u003cstrong\u003e b \u003c/strong\u003eRepresentative image showing the exclusion criteria, foci count, and category criteria for evaluation of γH2AX and 53BP-1 immunofluorescence staining.\u003cstrong\u003e c \u003c/strong\u003eGraph showing strong γH2AX positivity rate of U87MG cells treated with 30 and 150 μM SQAP and 300 μM temozolomide (TMZ) for 6 h. \u003cstrong\u003ed \u003c/strong\u003eRepresentative images of immunofluorescence staining of γH2AX. The scale bar is 10 µm. \u003cstrong\u003ee\u003c/strong\u003e Stacked bar graph showing the percentage of cells categorized as negative, positive, and strongly positive of γH2AX foci from the bottom up. *p\u0026lt;0.05 and **p\u0026lt;0.01 vs. each group (Kruskal-Wallis test).\u003c/p\u003e","description":"","filename":"Fig.3.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6896488/v1/ba667439890208bb08a03e99.jpg"},{"id":84888750,"identity":"505726f2-f2ca-4f31-b394-6893e7a1d614","added_by":"auto","created_at":"2025-06-18 12:19:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5423694,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eRepresentative images of immunofluorescence staining of 53BP-1. The scale bar is 10 µm. \u003cstrong\u003eb\u003c/strong\u003e Stacked bar graph showing the percentage of 53BP-1 foci in cells categorized as negative, positive, and strongly positive. *p\u0026lt;0.05, **p\u0026lt;0.01 vs. each group (Kruskal-Wallis test).\u003c/p\u003e","description":"","filename":"Fig.4.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6896488/v1/659990d575fcbaa19193097f.jpg"},{"id":84888752,"identity":"51cdd5fd-b8bc-4817-a88e-0b42285e4ce1","added_by":"auto","created_at":"2025-06-18 12:19:06","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2930029,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Scheme showing the time point of administration of SQAP and radiation exposure to murine models. \u003cstrong\u003eb\u003c/strong\u003e Photomicrographs showing the HE-stained murine brain coronal sections following treatment of each group. Original magnification ×4. Scale bar=1 mm. The dotted lines indicate the tumor margins. \u003cstrong\u003ec\u003c/strong\u003e Bar graphs representing the mean maximum cross-sectional areas and mean tumor volumes of each group. Each column and bar represent the mean±SEM. *p\u0026lt;0.05 and **p\u0026lt;0.01 vs. each group (one-way ANOVA followed by Tukey’s test).\u003c/p\u003e","description":"","filename":"Fig.5.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6896488/v1/6abbac32ea5e84d1c04a9f93.jpg"},{"id":89310720,"identity":"b371b909-380a-452f-9a20-6dc6f4b325e9","added_by":"auto","created_at":"2025-08-18 16:10:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":30063118,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6896488/v1/dc7fda5e-9ec0-4b45-8ac3-6d96c2920b81.pdf"},{"id":84888747,"identity":"f17a6655-51db-465b-ace1-8322f1a3694d","added_by":"auto","created_at":"2025-06-18 12:19:06","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":34537,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6896488/v1/1e82cf230e19837058fc5389.docx"},{"id":84888745,"identity":"2c7d44bd-7e25-40d9-9e33-133f2c31cc0d","added_by":"auto","created_at":"2025-06-18 12:19:06","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":177278,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6896488/v1/4de30709a35176f84e32c4c8.pdf"},{"id":84889475,"identity":"fbe33073-f3b3-45cd-96c6-72f52c48b735","added_by":"auto","created_at":"2025-06-18 12:27:06","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":357478,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6896488/v1/e81a1af361663c355f6ccf15.pdf"},{"id":84889476,"identity":"815b5ebc-77d0-4e19-8b25-c1fd27d3365e","added_by":"auto","created_at":"2025-06-18 12:27:06","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":427786,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6896488/v1/8e332d74aa3e29078df1707b.pdf"},{"id":84889917,"identity":"65e8b218-945e-4d01-9dd4-c46c6b7fcf0a","added_by":"auto","created_at":"2025-06-18 12:35:07","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":260692,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineResource5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6896488/v1/44050e14aff682cb1ab7bfc5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic antitumor and radiosensitizing effects of α -sulfoquinovosyl-acylpropanediol (SQAP) via PI3K/Akt inhibition and DNA repair impairment in glioblastoma","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRadiation therapy is an integral component of the standard treatment for glioblastoma (GBM) owing to its high therapeutic efficacy combined with minimal invasiveness [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, the high radiation doses used (~\u0026thinsp;60 Gy) pose significant challenges, including treatment-related complications including radiation necrosis and difficulties with re-irradiation at recurrence. [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A promising strategy to overcome these limitations is the use of agents that selectively enhance the effects of radiation on tumor cells.\u003c/p\u003e \u003cp\u003eSulfoquinovosylacylpropandiol (SQAP) is a derivative developed based on sulfoquinovosylacylglycerol (SQAG), a natural marine product found in the red alga, \u003cem\u003eChondracanthus tenellus\u003c/em\u003e. It exhibits a potent radiosensitizing effect, with a radiation enhancement ratio of \u0026gt;\u0026thinsp;3.0 [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Furthermore, SQAP specifically binds to surface antigens on cancer cells, and its glycolipid structure allows it to be rapidly internalized and metabolized in normal cells, thereby exhibiting no cytotoxicity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The radiosensitizing effects of SQAP are mediated by multiple mechanisms: a transient enhancement of tumor cell blood flow, which increases the local oxygen partial pressure [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and the inhibition of DNA repair processes\u0026mdash; specifically, by suppressing PARP-mediated repair of single-strand breaks (SSB) and by inhibiting double-strand break (DSB) repair via both homologous recombination (HR) and non-homologous end joining (NHEJ) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn Japan, SQAP has been used to treat nasal adenocarcinoma in animals since September 2023 and remains the only available radiosensitizing agent. Preclinical studies have demonstrated radiosensitizing effects in human prostate and pancreatic cancer cells[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; however, to date, no studies have evaluated this activity in GBM. Therefore, we aimed to investigate in detail the effects of SQAP on GBM cells and its potential radiosensitizing properties.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell lines and culture conditions\u003c/h2\u003e \u003cp\u003eThree human cells lines, U87MG, U251MG, and T98G, and one murine cell line, GL261, were used. The source of purchase is listed in Online Resource 1. All cell lines were seeded and cultured as described previously [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For normoxia experiments, cells were cultured at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e, and for hypoxia experiments, cells were maintained under consistent 1% O\u003csub\u003e2\u003c/sub\u003e hypoxic condition [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRegents\u003c/h3\u003e\n\u003cp\u003eTemozolomide (TMZ) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and was dissolved in dimethyl sulfoxide (FUJIFILM Wako Pure Chemical Corporation). SQAP was purchased from Malignant Tumor Treatment Technologies (Osaka, Japan) and was dissolved in DMEM.\u003c/p\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eCell viability was determined using the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) as previously described [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The absorbance of each well was measured at 450 nm, with a reference wavelength of 660 nm, using a Varioskan Flash 2.4 microplate reader (Thermo Fisher Scientific) 48 h after drug administration.\u003c/p\u003e\n\u003ch3\u003eColony formation assay\u003c/h3\u003e\n\u003cp\u003eU87MG cells were seeded at 1.0\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well in six-well plates containing DMEM supplemented with 10% FBS. After 24 h, the medium was replaced, and SQAP treatment was administered 30 min prior to irradiation with an X-ray machine (MX-160Labo; mediXtec, Chiba, Japan). Thereafter, irradiation and drug treatments were performed every 3 days. After 14 days of treatment, cells were fixed with 100% methanol for 15 min and stained with 0.5% crystal violet for 30 min. Colonies comprising\u0026thinsp;\u0026gt;\u0026thinsp;100 cells were counted using an All-in-One Fluorescence Microscope (BZ-X710; Keyence, Osaka, Japan).\u003c/p\u003e\n\u003ch3\u003eImmunoblotting\u003c/h3\u003e\n\u003cp\u003eU87MG cells were cultured in 12-well plates at a density of 2.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well in DMEM supplemented with 10% FBS. After a 24-h incubation, the medium was replaced with fresh DMEM containing 10% FBS, followed by the addition of 150 \u0026micro;M SQAP for 6, 12, 24, or 48 h. Immunoblotting procedures were detailed previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The primary antibodies were are provided in Online Resource 1 ; all primary antibodies were diluted 1:1000. The membranes were incubated with the following secondary antibodies: goat anti-rabbit IgG (32460, Thermo Fisher Scientific, Massachusetts, USA) or goat anti-mouse IgG (32430, Thermo Fisher Scientific). Protein bands were detected using Immuno Star LD (FUJIFILM Wako Pure Chemical Corporation). Band intensities were quantified using an Amersham Imager 680 (Cytiva, Tokyo, Japan). β-actin was used as a loading control to confirm equal protein loading. Additionally, phosphorylation signals were normalized to the total protein content of the corresponding target.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative reverse transcription polymerase chain reaction\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of SQAP on the mRNA expression of BAX, Bcl-2, p21, cyclin D1, and VEGF, we conducted quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. U87MG-cells were cultured at a density of 2.5\u0026times;10⁴ cells/well in culture medium supplemented with 10% FBS. After a 24-h incubation, the medium was replaced with fresh DMEM containing 10% FBS, followed by the addition of 150 \u0026micro;M SQAP for 3, 6, 12, or 24 h. Total RNA was extracted using the NucleoSpin\u0026reg; RNA II kit (Takara, Shiga, Japan), and RNA concentrations were measured using a biospectrometer (Eppendorf, Hamburg, Germany).\u003c/p\u003e \u003cp\u003eComplementary DNA (cDNA) was synthesized from the isolated RNA via reverse transcription using the PrimeScript\u0026trade; RT reagent kit (Perfect Real Time; Takara, Shiga, Japan). qRT-PCR was performed using TB Green\u0026reg; Fast qPCR Mix (Takara) on a TP800 Thermal Cycler Dice Real-Time System (Takara), following the manufacturer\u0026rsquo;s instructions. Primer sequences for BAX, Bcl-2, p21, cyclin D1, VEGF, and β-actin are provided in Online Resource 1. β-actin was used as an internal control. PCR cycling conditions followed the manufacturer\u0026rsquo;s protocol: 40 cycles of amplification, with each cycle comprising 5 s at 95\u0026deg;C and 30 s at 60\u0026deg;C. Gene expression was normalized to β-actin and is presented as relative expression values.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell death assay\u003c/h3\u003e\n\u003cp\u003eU87MG cells were seeded in 96-well plates at a density of 5\u0026times;10\u0026sup3; cells per well in DMEM supplemented with 10% FBS and incubated for 24 h. The culture medium was replaced with fresh DMEM containing 10% FBS, and SQAP was added at final concentrations of 30 or 150 \u0026micro;M. After 30 min of incubation, cells were irradiated with 4 Gy with an X-ray machine (MX-160Labo) and subsequently incubated for an additional 48 h. Cell death was assessed using Hoechst 33342 (Invitrogen) and propidium iodide (PI, Invitrogen) staining. Hoechst 33342 and PI were added to the culture medium at final concentrations of 8.1 and 1.5 \u0026micro;M, respectively, and incubated for 15 min. Stained cells were imaged using a Lionheart\u0026trade; FX Automated Microscope (BioTek, Winooski, Vermont, USA). The percentage of PI-positive cells was determined by distinguishing fluorescence signals from Hoechst 33342 and PI. The cell death rate was calculated as follows: Cell death rate\u0026thinsp;=\u0026thinsp;PI-positive cells/Hoechst 33342-positive cells\u0026times;100 (%).\u003c/p\u003e\n\u003ch3\u003eBromodeoxyuridine (BrdU) cell proliferation assay\u003c/h3\u003e\n\u003cp\u003eU87MG cells were seeded in 96-well plates at a density of 5\u0026times;10\u0026sup3; cells/well in DMEM supplemented with 10% FBS and incubated for 24 h. The culture medium was then replaced with fresh DMEM containing 10% FBS, and SQAP was added at final concentrations of 30 or 150 \u0026micro;M. After 30 min of incubation, cells were irradiated with 4 Gy with the X-ray machine (MX-160Labo) and subsequently incubated for an additional 48 h. The culture medium was replaced with fresh DMEM containing 10% FBS, followed by treatment with 10 \u0026micro;M BrdU for 3 h. Immunocytochemistry was performed according to the manufacturer's protocol using an anti-BrdU antibody (ab6326, Abcam, 1:500).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eImmunocytochemistry\u003c/h2\u003e \u003cp\u003eU87MG cells were seeded at a density of 1.0\u0026times;10⁵ cells/well on four-well glass slides in DMEM supplemented with 10% FBS and incubated for 24 h. The culture medium was then replaced with fresh DMEM containing 10% FBS, and SQAP was added at final concentrations of 30 or 150 \u0026micro;M. After 30 min, cells were irradiated with 4 Gy with an X-ray machine (MX-160Labo) and subsequently incubated for 1, 3, 6, or 12 h. Following incubation, cells were washed with PBS and fixed with 4% paraformaldehyde for 20 min at 37\u0026deg;C. After blocking with 10% horse serum (Vector Labs, Burlingame, CA, USA) at room temperature for 30 min, the slides were incubated overnight at 4\u0026deg;C with either rabbit anti-phospho-H2A.X (Ser139, 20E3) (9718S, Cell Signaling, 1:300) or rabbit anti-53BP1 (E7N5D) (88439S, Cell Signaling, 1:300). Next, cells were washed with PBS and incubated with Alexa Fluor\u0026reg; 647 goat anti-rabbit IgG secondary antibody (1:500, Thermo Fisher Scientific) for 1 h at room temperature. Cell nuclei were counterstained with Hoechst 33342, and fluorescence images were captured using the FLUOVIEW FV3000 confocal laser scanning microscope (Olympus, Tokyo, Japan). For quantification of pH2AX and 53BP1 foci, at least 100 cells/well were analyzed. Cells were categorized based on the number of foci as follows: strongly positive (\u0026ge;\u0026thinsp;11 foci), positive (1\u0026ndash;10 foci), and negative (0 foci). The percentage of cells in each category was then calculated. Foci quantification was performed independently by two blinded observers, and the average values were used for analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e The Animal Experiment Committee of Gifu University, Japan (Ethics No.AG-P-N-20240062), reviewed and approved all animal procedures. The experiments were conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.\u003c/p\u003e \u003cp\u003eEight-week-old C57BL/6J male mice were purchased from The Jackson Laboratory Japan, Inc. (Yokohama, Kanagawa, Japan). The mice were housed at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under a 12-h light\u0026ndash;dark cycle with ad libitum access to food (CE-2, CLEA Japan, Tokyo, Japan) and water. On day 0, GL261 cells (1.0\u0026times;10⁵ in 2 \u0026micro;L PBS) were stereotactically injected into the left striatum [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The mice were housed in groups of five per cage and randomly assigned to four experimental groups: vehicle (n\u0026thinsp;=\u0026thinsp;10), SQAP-only (n\u0026thinsp;=\u0026thinsp;5), RT-only (n\u0026thinsp;=\u0026thinsp;8), and SQAP\u0026thinsp;+\u0026thinsp;RT (n\u0026thinsp;=\u0026thinsp;7). In the SQAP-only and SQAP\u0026thinsp;+\u0026thinsp;RT groups, SQAP (4 mg/kg) dissolved in saline was administered via tail vein injection for 4 consecutive days, starting on day 6 post-GL261 cell injection. The vehicle group received an equivalent volume of saline (5 mL/kg). The SQAP dosage was determined based on the manufacturer\u0026rsquo;s instructions. In the RT-only and SQAP\u0026thinsp;+\u0026thinsp;RT groups, non-anesthetized 4 Gy per fraction irradiation with X-ray machine (MX-160Labo) was performed on days 7 and 8, 30 min after either SQAP or saline injection. On day 14, the mice were anesthetized and transcardially perfused with 4% paraformaldehyde (Wako Pure Chemicals, Osaka, Japan). The brains were dissected, post-fixed in 4% paraformaldehyde, embedded in paraffin (Leica Biosystems, Wetzlar, Germany), sectioned at 5-\u0026micro;m thickness, and stained with hematoxylin\u0026ndash;eosin (HE). The maximum sectional tumor areas and tumor volumes were assessed using an All-in-One Fluorescence Microscope (BZ-X710; Keyence). Fifteen sections per mouse were analyzed, with two images obtained per section. Tumor volume was calculated using the formula: V\u0026thinsp;=\u0026thinsp;4​πr\u003csup\u003e3\u003c/sup\u003e/3. This experiment was conducted in a non-blinded manner.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Statistical analyses were conducted using a one-way or two-way analysis of variance (ANOVA) followed by Dunnett\u0026rsquo;s test and Tukey\u0026rsquo;s test when comparing continuous values of two or more groups, and Kruskal-Wallis test for the ordinal variables of two or more group comparisons. These statistics were performed using Graph-Pad Prism v.10 (GraphPad Software, Boston, MA, USA) software. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eSee Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea for schematic summary of the key findings.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSQAP suppressed GBM cell proliferation\u003c/h2\u003e \u003cp\u003eSQAP exhibited a concentration-dependent suppression of cell viability, regardless of the cell line used (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u0026ndash;d). In contrast, the viability of astrocytes remained unchanged 48 h after SQAP administration at concentrations up to 150 \u0026micro;M. Similar results obtained under hypoxic conditions and for T98G cells are shown in Online Resource 2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffect of SQAP combined with RT on GBM cell proliferation\u003c/h2\u003e \u003cp\u003eA colony formation assay was performed to evaluate the effect of combining RT with SQAP on GBM cell proliferation. Radiation and drug treatments were administered four times in each group, with total radiation doses of 8 and 16 Gy. In the no-irradiation group, similar to the CCK-8 assay, colony formation was not significantly reduced at SQAP concentrations up to 50 \u0026micro;M. However, in the 8-Gy group, colony numbers were significantly reduced at even 30 \u0026micro;M SQAP compared to the control group. A similar trend was observed in the 16-Gy group; however, the number of colonies in this group was already low even in the absence of SQAP, resulting in no significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee,f).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCombination of SQAP and RT sensitized to the cell death while not affecting mitotic inhibition\u003c/h2\u003e \u003cp\u003eNo significant increase in dead cells was observed up to 30 \u0026micro;M SQAP, and only a slight increase was observed following 4 Gy irradiation. Conversely, 150 \u0026micro;M SQAP not only markedly increased the number of dead cells but also significantly enhanced cell death in the 4 Gy group compared with that of the 0 Gy group (64.63 vs. 77.31, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg,h). Meanwhile, the BrdU assay demonstrated a concentration-dependent decrease in BrdU-incorporated cells with SQAP treatment; however, irradiation had no effect, as no significant differences were observed between the irradiated and non-irradiated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei,j).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSQAP decreased the expression of PI3K and Akt\u003c/h2\u003e \u003cp\u003eImmunoblotting was used to reveal the mechanisms of the anti-tumor effects of SQAP. The phosphorylation of Akt decreased over time over 24 h and the phosphorylation of PI3K decreased over time over 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea,b). The same test performed under hypoxia showed similar results (Online Resource 3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eSQAP affected the expression of proteins downstream of the PI3K/Akt signaling pathway\u003c/h2\u003e \u003cp\u003eTo elucidate the effects of SQAP on downstream components of the PI3K/Akt signaling pathway involved in apoptosis, we examined both the mRNA and protein expression of BAX (Bcl-2-associated X protein, an apoptosis-inducing protein) and Bcl-2 (B-cell lymphoma-2, an apoptosis-inhibiting protein). SQAP treatment increased BAX mRNA levels while decreasing those of Bcl-2, resulting in an elevated apoptosis-promoting BAX/Bcl-2 ratio over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). A similar trend was observed at the protein expression level and under hypoxic conditions; however, the differences were not statistically significant (Online Resource 4).\u003c/p\u003e \u003cp\u003eNext, we investigated the effects of SQAP on PI3K/Akt downstream components related to the cell cycle, including cyclin D1 and cyclin B1 (cell cycle-associated proteins) and p21 (a cyclin-dependent kinase inhibitor). SQAP treatment reduced cyclin B1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed) and p21 mRNA expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee) but did not influence cyclin D1 mRNA levels (Online Resource 5 showing results under hypoxic conditions). Furthermore, given previous reports on SQAP activity in other cancers, we evaluated its impact on HIF-1α and its downstream target VEGF. SQAP treatment induced a decrease in HIF-1α expression and a reduction in VEGF mRNA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef,g).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eSQAP inhibited RT-induced DNA damage repair and enhanced nuclear γH2AX expression\u003c/h2\u003e \u003cp\u003eTo determine whether SQAP enhanced radiosensitivity, we analyzed changes in expression of proteins involved in DNA repair and DNA damage following SQAP treatment. Compared with the control group, PARP expression significantly decreased over 48 h in the SQAP-treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eNext, we examined the nuclear expression of γH2AX, a marker of DNA damage, and established inclusion and exclusion criteria for quantitative analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb,d). Although the strongly positive rate increased with TMZ alone, no such increase was observed with SQAP alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), suggesting that SQAP itself does not induce DNA damage. In the stacked bar graph, the bars represent the percentage of cells categorized as negative, positive, and strongly positive from the bottom up, with higher percentages at the top indicating greater DNA damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). Three h after irradiation, γH2AX expression increased in a concentration-dependent manner. In the 150 \u0026micro;M SQAP group, the strongly positive rate increased, whereas the negative rate decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee). By 12 h post-irradiation, the proportion of strongly positive cells no longer differed between groups. However, the number of negative cells remained significantly lower in the SQAP-treated group than in the control group, suggesting delayed DNA repair (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSimilarly, on comparing nuclear 53BP-1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), the strongly positive rate significantly decreased, and the negative rate increased in the 150 \u0026micro;M SQAP-treated group 3 h after irradiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). A similar trend was observed at 30 \u0026micro;M. By 12 h post-irradiation, the strongly positive rate was nearly absent in both groups, but the negative rate was significantly lower in the 150 \u0026micro;M SQAP-treated group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eSQAP treatment and RT inhibited GBM growth in mice models\u003c/h2\u003e \u003cp\u003eThe protocol for drug administration and irradiation is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea. The mean maximum cross-sectional area and volume were more significantly reduced in the SQAP\u0026thinsp;+\u0026thinsp;RT group than in the RT alone group, when compared with that of the vehicle-treated group. Although the difference between the RT alone and the RT\u0026thinsp;+\u0026thinsp;SQAP combination did not reach statistical significance, it is noteworthy that tumor growth was markedly suppressed in nearly all cases in the combination group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb,c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated whether SQAP exerts radiosensitizing and antitumor effects on GBM cells using \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments. Our findings demonstrated that SQAP directly exerts antitumor effects by inhibiting the PI3K/Akt signaling pathway. Furthermore, at lower drug concentrations, SQAP exhibited a radiosensitizing effect by suppressing DNA repair. To the best of our knowledge, this is the first study to demonstrate the antitumor and radiosensitizing effects of SQAP on GBM cells.\u003c/p\u003e \u003cp\u003eSQAP derives from the natural compounds sulfoquinovosylmonoacylglycerol and SQAG, first described by Sahara et al. and Ohta et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These compounds were initially developed as DNA polymerase inhibitors as they exhibited weak anticancer effect [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Subsequently, they were found to possess strong radiosensitizing effects [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Currently, SQAP has been successfully applied as a radiosensitizer for advanced adenocarcinoma in the nasal cavity in dogs. This study was conducted to explore the possibility of future clinical applications of human GBM.\u003c/p\u003e \u003cp\u003eWe first evaluated the standalone effects of SQAP on GBM cells. Although SQAP exhibited only modest anticancer activity during its preclinical development, our CCK-8 and colony formation assays under non-irradiated conditions (0 Gy) revealed that SQAP significantly reduced the proliferative capacity of GBM cells. Notably, in lung cancer cells (A549) with highly activated Akt, SQAP suppresses focal adhesion kinase (FAK) phosphorylation, leading to subsequent inhibition of Akt phosphorylation, reduced VEGF expression, and anti-angiogenic effects [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Although FAK was not investigated in our study, we observed suppression of phosphorylated Akt and its upstream regulator phosphorylated PI3K in GBM cells by SQAP. The PI3K/Akt signaling pathway, which is constitutively activated in GBM, represents a promising therapeutic target in this malignancy [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Akt, a central kinase in the PI3K/Akt pathway, plays critical roles in suppressing apoptosis and promoting cell cycle progression [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Akt indirectly inhibits BAX and enhances Bcl-2 to block apoptosis. Specifically, Akt phosphorylates FOXO transcription factors, leading to their cytoplasmic sequestration and subsequent suppression of BAX transcription [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, Akt stabilizes Bcl-2 expression by activating MDM2, which degrades p53 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In the cell cycle, Akt promotes G1/S transition by phosphorylating and inactivating GSK-3β, thereby preventing the degradation of cyclin D1 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Furthermore, Akt stabilizes cyclin B1, a key regulator of the G2/M phase, by inhibiting GSK-3β-mediated phosphorylation and subsequent proteasomal degradation of cyclin B1 [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Akt also suppresses CDK inhibitors p21 and p27, enabling uncontrolled cell cycle progression [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough not all downstream signaling components of the PI3K/Akt signaling pathway were fully characterized, we observed critical molecular changes consistent with pathway modulation: (1) increased BAX/Bcl-2 ratio (pro-apoptotic shift), (2) upregulation of p21 (a CDK inhibitor), and (3) downregulation of cyclin B1 (a G2/M phase regulator). As collectively suggested by the cell death assay and BrdU test, SQAP induces apoptosis through mitochondrial dysfunction and impairs cell cycle progression at both G1/S and G2/M checkpoints, ultimately leading to reduced proliferative capacity in GBM cells.\u003c/p\u003e \u003cp\u003eBeyond regulating cell death and proliferation, the Akt signaling pathway critically drives radioresistance by orchestrating DNA damage repair mechanisms. Upon radiation-induced DNA damage, Akt activation facilitates the recruitment and stabilization of key DNA repair proteins\u0026mdash;including DNA-PKcs and 53BP1\u0026mdash;at DSB sites. This enhances both HR and NHEJ, thereby reducing radiation-induced cytotoxicity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Concurrently, Akt-mediated phosphorylation stabilizes the MDM2 oncoprotein, which suppresses p53-dependent apoptosis, further amplifying radioresistance [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Notably, pharmacological inhibition of Akt isoforms (e.g., MK-2206, Akt inhibitor IV) potentiates radiosensitivity in GBM cells, underscoring its therapeutic relevance [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, SQAP inhibition of HIF-1α, VEGF [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and von Hippel-Lindau protein (pVHL) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] promotes vascular normalization and induces transient improvement in oxygenation, which may contribute to radiosensitizing effects [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In our study, we observed significant downregulation of HIF-1α and VEGF expression, suggesting that SQAP may modulate angiogenesis in GBM by attenuating hypoxic and pro-angiogenic signaling pathways. Although intratumoral oxygenation status and angiogenesis were not directly assessed in this study, the observed suppression of HIF-1α and VEGF suggests that these pathways\u0026mdash;in parallel with Akt inhibition\u0026mdash;may contribute to SQAP-mediated radiosensitization.\u003c/p\u003e \u003cp\u003eThe effect of SQAP on radiation-induced DNA damage was assessed using γH2AX, a phosphorylated histone H2AX variant that serves as a critical biomarker for DSBs induced by ionizing radiation. Its rapid formation at DSB sites facilitates the recruitment of DNA repair machinery [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], and the persistence of γH2AX foci correlates with unrepaired damage, making it a valuable tool for quantifying radiation-induced DNA damage and predicting therapeutic efficacy in cancer treatment. Depending on the cell type, γH2AX foci typically peak around 0.5\u0026ndash;2 h after irradiation. As DNA repair progresses, most DSBs are repaired, and the number of foci returns to baseline levels by approximately 24 h [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In our study, the number of γH2AX foci increased with the addition of SQAP at 3 h after irradiation, coinciding with the peak of foci formation. At 6 and 12 h, the proportion of γH2AX-negative cells decreased after SQAP treatment, suggesting delayed DNA repair. Furthermore, this repair inhibition was observed even at concentrations as low as 30 \u0026micro;M, indicating that SQAP effectively inhibits DNA repair at low doses.\u003c/p\u003e \u003cp\u003e53BP-1 is a key protein involved in the repair of DNA DSBs. It is recruited to sites of damage through recognition of γH2AX-labeled chromatin and plays a critical role in facilitating the recruitment of downstream DNA repair factors in the NHEJ pathway. Notably, 53BP1 foci are absent once DNA repair is complete[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In the present study, 53BP-1 positivity decreased in an SQAP concentration-dependent manner during the early phase (1\u0026ndash;3 h), when γH2AX expression was elevated. Thus, SQAP may inhibit the initial mobilization of 53BP1 to sites of DNA damage. In contrast, at 12 h, 53BP-1 positivity increased in a concentration-dependent manner following SQAP treatment. This likely reflects the fact that, in control cells, DNA repair had already been completed, and 53BP-1 foci had disappeared. However, on exposure to SQAP, DNA repair was delayed, and 53BP-1 likely remained active at this time point due to the ongoing repair of persistent DNA damage.\u003c/p\u003e \u003cp\u003eFinally, to evaluate the \u003cem\u003ein vivo\u003c/em\u003e efficacy and effects on brain lesions, we conducted a combination treatment study using radiotherapy and SQAP in a murine model. Although group sizes were limited due to model stability and the technical difficulty of tail vein injections, the group receiving combined RT and SQAP treatment showed a significant reduction in both maximum tumor area and tumor volume compared to the vehicle-treated group. Furthermore, although the difference was not significant, the RT-alone group exhibited only partial tumor shrinkage, whereas the combination group demonstrated consistent and pronounced tumor growth inhibition across all cases. Thus, SQAP may be useful as a radiosensitizer not only for intranasal adenocarcinoma, for which it is currently used clinically, but also for intracerebral tumors. However, the brain penetration of SQAP has not been evaluated, which warrants further research.\u003c/p\u003e \u003cp\u003eTo our knowledge, this study is the first to report the antitumor and radiosensitizing effects of SQAP in glioma. SQAP inhibits PI3K/Akt signaling, which plays a key role in DNA repair and cell proliferation. By downregulating HIF-1α and VEGF expression, SQAP may also normalize tumor vasculature, thereby indirectly enhancing radiosensitivity. Furthermore, inhibition of PI3K is associated with reduced resistance to temozolomide [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], suggesting that SQAP may potentiate the therapeutic effects of both radiotherapy and chemotherapy. Although SQAP shows promising potential as an adjunctive treatment for glioma, further research is needed to fully elucidate its safety profile, brain permeability, and underlying mechanisms of action.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study is the first to demonstrate that SQAP exerts antitumor and radiosensitizing effects in both glioma cells and mouse models. SQAP enhances radiosensitivity through inhibition of the PI3K/Akt signaling pathway, suppression of HIF-1α and VEGF expression, and modulation of DNA repair dynamics, including attenuation of 53BP1 recruitment. In addition, a direct antitumor effect of SQAP via the induction of apoptosis was also observed. These findings highlight SQAP as a promising adjunctive agent for glioma treatment. However, further investigations are needed to elucidate the brain permeability, long-term safety, and detailed mechanisms of action in humans.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Editage (www.editage.com) for English language editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors did not receive support from any organization for the submitted work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTN; Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Resources, Software, Validation and Role/Writing\u0026mdash;original draft. UK; Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation and Writing\u0026mdash;review \u0026amp; editing. SN; Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Resources, Software, Validation, Supervision and Writing\u0026mdash;review \u0026amp; editing. YK; Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Resources, Software, Validation, Supervision and Writing\u0026mdash;review \u0026amp; editing. \u0026nbsp;YH; Conceptualization, Investigation, Resources, Validation and Writing\u0026mdash;review \u0026amp; editing. YF; Conceptualization, Investigation, Resources, Validation and Writing\u0026mdash;review \u0026amp; editing. KS; Conceptualization, Investigation, Resources, Validation and Writing\u0026mdash;review \u0026amp; editing. TY; Conceptualization, Investigation, Resources, Validation and Writing\u0026mdash;review \u0026amp; editing. HT; Conceptualization, Investigation, Resources, Validation and Writing\u0026mdash;review \u0026amp; editing. YE; Conceptualization, Validation, Supervision and Writing\u0026mdash;review \u0026amp; editing. HH; Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Resources, Software, Validation, Supervision and Writing\u0026mdash;review \u0026amp; editing. NN; Conceptualization, Validation, Supervision and Writing\u0026mdash;review \u0026amp; editing. TI; Conceptualization, Validation, Supervision and Writing\u0026mdash;review \u0026amp; editing. Masamitsu Shimazawa; Conceptualization, Data curation, Formal analysis, Methodology, Project administration, Resources, Software, Validation, Supervision and Writing\u0026mdash;review \u0026amp; editing. The first draft of the manuscript was written by TN, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Conceptualization; TN, UK,SN, YK, YH, FY, KS, TY, HT, YE, HH, NN, TI, MS, Data curation; TN, UK, SN, YK, HH, MS, Formal analysis; TN, UK, SN, YK, HH, MS, Investigation; TN, UK, YH, YF, KS, TY, HT, Methodology; TN, UK, SN, YK, HH, MS, Project administration; SN, YK, HH, MS, Resourse; TN, UK,SN, YK, YH, FY, KS, TY, HT, YE, HH, NN, TI, MS, Software; TN, SN, YK, HH, MS, Supervision; SN, YK, YE, HH, TI, MS, Validation; TN, UK,SN, YK, YH, FY, KS, TY, HT, YE, HH, NN, TI, MS. Roles/Writing\u0026mdash;original draft; TN, Writing\u0026mdash;review \u0026amp; editing; TN, UK,SN, YK, YH, FY, KS, TY, HT, YE, HH, NN, TI, MS. The first draft of the manuscript was written by TN, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Animal Experiment Committee of Gifu University, Japan (Ethics No.AG-P-N-20240062),\u0026nbsp;reviewed and approved all animal procedures. The experiments were conducted in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eStupp R, Hegi ME, Gilbert MR, Chakravarti A (2007) Chemoradiotherapy in malignant glioma: standard of care and future directions. J Clin Oncol 25: 4127-4136 doi:10.1200/jco.2007.11.8554\u003c/li\u003e\n\u003cli\u003eMinniti G, Niyazi M, Alongi F, Navarria P, Belka C (2021) Current status and recent advances in reirradiation of glioblastoma. Radiat Oncol 16: 36 doi:10.1186/s13014-021-01767-9\u003c/li\u003e\n\u003cli\u003eSiu A, Wind JJ, Iorgulescu JB, Chan TA, Yamada Y, Sherman JH (2012) Radiation necrosis following treatment of high grade glioma--a review of the literature and current understanding. Acta Neurochir (Wien) 154: 191-201; discussion 201 doi:10.1007/s00701-011-1228-6\u003c/li\u003e\n\u003cli\u003eSakimoto I, Ohta K, Yamazaki T, Ohtani S, Sahara H, Sugawara F, Sakaguchi K, Miura M (2006) Alpha-sulfoquinovosylmonoacylglycerol is a novel potent radiosensitizer targeting tumor angiogenesis. Cancer Res 66: 2287-2295 doi:10.1158/0008-5472.Can-05-2209\u003c/li\u003e\n\u003cli\u003eSahara H, Ishikawa M, Takahashi N, Ohtani S, Sato N, Gasa S, Akino T, Kikuchi K (1997) In vivo anti-tumour effect of 3\u0026apos;-sulphonoquinovosyl 1\u0026apos;-monoacylglyceride isolated from sea urchin (Strongylocentrotus intermedius) intestine. Br J Cancer 75: 324-332 doi:10.1038/bjc.1997.54\u003c/li\u003e\n\u003cli\u003eOhta K, Murata H, Mori Y, Ishima M, Sugawara F, Sakaguchi K, Miura M (2010) Remodeling of the tumor microenvironment by combined treatment with a novel radiosensitizer, {alpha}-sulfoquinovosylmonoacylglycerol ({alpha}-SQMG) and X-irradiation. Anticancer Res 30: 4397-4404\u003c/li\u003e\n\u003cli\u003eRuike T, Kanai Y, Iwabata K, Matsumoto Y, Murata H, Ishima M, Ohta K, Oshige M, Katsura S, Kuramochi K, Kamisuki S, Sahara H, Miura M, Sugawara F, Sakaguchi K (2019) Distribution and metabolism of (14)C-sulfoquinovosylacylpropanediol ((14)C-SQAP) after a single intravenous administration in tumor-bearing mice. Xenobiotica 49: 346-362 doi:10.1080/00498254.2018.1448949\u003c/li\u003e\n\u003cli\u003eOhta K, Mizushina Y, Yamazaki T, Hanashima S, Sugawara F, Sakaguchi K (2001) Specific interaction between an oligosaccharide on the tumor cell surface and the novel antitumor agents, sulfoquinovosylacylglycerols. Biochem Biophys Res Commun 288: 893-900 doi:10.1006/bbrc.2001.5852\u003c/li\u003e\n\u003cli\u003eTakakusagi Y, Naz S, Takakusagi K, Ishima M, Murata H, Ohta K, Miura M, Sugawara F, Sakaguchi K, Kishimoto S, Munasinghe JP, Mitchell JB, Krishna MC (2018) A Multimodal Molecular Imaging Study Evaluates Pharmacological Alteration of the Tumor Microenvironment to Improve Radiation Response. Cancer Res 78: 6828-6837 doi:10.1158/0008-5472.Can-18-1654\u003c/li\u003e\n\u003cli\u003eIwamoto H, Nakamura T, Koga H, Izaguirre-Carbonell J, Kamisuki S, Sugawara F, Abe M, Iwabata K, Ikezono Y, Sakaue T, Masuda A, Yano H, Ohta K, Nakano M, Shimose S, Shirono T, Torimura T (2015) Inhibition of hypoxia-inducible factor via upregulation of von Hippel-Lindau protein induces \u0026quot;angiogenic switch off\u0026quot; in a hepatoma mouse model. Mol Ther Oncolytics 2: 15020 doi:10.1038/mto.2015.20\u003c/li\u003e\n\u003cli\u003eMaeda J, Shellenberger KD, Kurihara W, Haga T, Kato TA (2023) Sulfoquinovosyl acylpropanediol (SQAP): Inhibition of poly(ADP-ribose) metabolism and enhanced cytotoxicity in homologous recombination repair-deficient Chinese hamster-derived cells. Mutat Res Genet Toxicol Environ Mutagen 892: 503703 doi:10.1016/j.mrgentox.2023.503703\u003c/li\u003e\n\u003cli\u003eSawada Y, Omoto K, Kohei N, Sakaguchi K, Miura M, Tanabe K (2015) Sulfoquinovosylacylpropanediol is a novel potent radiosensitizer in prostate cancer. Int J Urol 22: 590-595 doi:10.1111/iju.12753\u003c/li\u003e\n\u003cli\u003eTakakusagi Y, Sugyo A, Tsuji AB, Sudo H, Yasunaga M, Matsumura Y, Sugawara F, Sakaguchi K, Higashi T (2022) The natural sulfoglycolipid derivative SQAP improves the therapeutic efficacy of tissue factor-targeted radioimmunotherapy in the stroma-rich pancreatic cancer model BxPC-3. Transl Oncol 15: 101285 doi:10.1016/j.tranon.2021.101285\u003c/li\u003e\n\u003cli\u003eVarricchio A, Khan S, Price ZK, Davis RA, Ramesh SA, Yool AJ (2023) Pharmacological Inhibition of Membrane Signaling Mechanisms Reduces the Invasiveness of U87-MG and U251-MG Glioblastoma Cells In Vitro. Cancers (Basel) 15 doi:10.3390/cancers15041027\u003c/li\u003e\n\u003cli\u003eLiu J, Gao L, Zhan N, Xu P, Yang J, Yuan F, Xu Y, Cai Q, Geng R, Chen Q (2020) Hypoxia induced ferritin light chain (FTL) promoted epithelia mesenchymal transition and chemoresistance of glioma. J Exp Clin Cancer Res 39: 137 doi:10.1186/s13046-020-01641-8\u003c/li\u003e\n\u003cli\u003eYamada T, Tsuji S, Nakamura S, Egashira Y, Shimazawa M, Nakayama N, Yano H, Iwama T, Hara H (2021) Riluzole enhances the antitumor effects of temozolomide via suppression of MGMT expression in glioblastoma. J Neurosurg 134: 701-710 doi:10.3171/2019.12.Jns192682\u003c/li\u003e\n\u003cli\u003eTsuji S, Nakamura S, Shoda K, Yamada T, Shimazawa M, Nakayama N, Iwama T, Hara H (2022) NMDA receptor signaling induces the chemoresistance of temozolomide via upregulation of MGMT expression in glioblastoma cells. J Neurooncol 160: 375-388 doi:10.1007/s11060-022-04154-w\u003c/li\u003e\n\u003cli\u003eYamada T, Tsuji S, Nakamura S, Egashira Y, Shimazawa M, Nakayama N, Yano H, Iwama T, Hara H (2020) Riluzole enhances the antitumor effects of temozolomide via suppression of MGMT expression in glioblastoma. J Neurosurg 134: 701-710 doi:10.3171/2019.12.JNS192682\u003c/li\u003e\n\u003cli\u003eOhta K, Mizushina Y, Hirata N, Takemura M, Sugawara F, Matsukage A, Yoshida S, Sakaguchi K (1998) Sulfoquinovosyldiacylglycerol, KM043, a new potent inhibitor of eukaryotic DNA polymerases and HIV-reverse transcriptase type 1 from a marine red alga, Gigartina tenella. Chem Pharm Bull (Tokyo) 46: 684-686 doi:10.1248/cpb.46.684\u003c/li\u003e\n\u003cli\u003eHanashima S, Mizushina Y, Ohta K, Yamazaki T, Sugawara F, Sakaguchi K (2000) Structure-activity relationship of a novel group of mammalian DNA polymerase inhibitors, synthetic sulfoquinovosylacylglycerols. Jpn J Cancer Res 91: 1073-1083 doi:10.1111/j.1349-7006.2000.tb00887.x\u003c/li\u003e\n\u003cli\u003eSahara H, Hanashima S, Yamazaki T, Takahashi S, Sugawara F, Ohtani S, Ishikawa M, Mizushina Y, Ohta K, Shimozawa K, Gasa S, Jimbow K, Sakaguchi K, Sato N, Takahashi N (2002) Anti-tumor effect of chemically synthesized sulfolipids based on sea urchin\u0026apos;s natural sulfonoquinovosylmonoacylglycerols. Jpn J Cancer Res 93: 85-92 doi:10.1111/j.1349-7006.2002.tb01204.x\u003c/li\u003e\n\u003cli\u003eIzaguirre-Carbonell J, Kawakubo H, Murata H, Tanabe A, Takeuchi T, Kusayanagi T, Tsukuda S, Hirakawa T, Iwabata K, Kanai Y, Ohta K, Miura M, Sakaguchi K, Matsunaga S, Sahara H, Kamisuki S, Sugawara F (2015) Novel anticancer agent, SQAP, binds to focal adhesion kinase and modulates its activity. Sci Rep 5: 15136 doi:10.1038/srep15136\u003c/li\u003e\n\u003cli\u003eVerdugo E, Puerto I, Medina M (2022) An update on the molecular biology of glioblastoma, with clinical implications and progress in its treatment. Cancer Commun (Lond) 42: 1083-1111 doi:10.1002/cac2.12361\u003c/li\u003e\n\u003cli\u003eLi X, Wu C, Chen N, Gu H, Yen A, Cao L, Wang E, Wang L (2016) PI3K/Akt/mTOR signaling pathway and targeted therapy for glioblastoma. Oncotarget 7: 33440-33450 doi:10.18632/oncotarget.7961\u003c/li\u003e\n\u003cli\u003eLiu R, Chen Y, Liu G, Li C, Song Y, Cao Z, Li W, Hu J, Lu C, Liu Y (2020) PI3K/AKT pathway as a key link modulates the multidrug resistance of cancers. Cell Death Dis 11: 797 doi:10.1038/s41419-020-02998-6\u003c/li\u003e\n\u003cli\u003eBrunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME (1999) Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96: 857-868 doi:10.1016/s0092-8674(00)80595-4\u003c/li\u003e\n\u003cli\u003eMayo LD, Donner DB (2001) A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc Natl Acad Sci U S A 98: 11598-11603 doi:10.1073/pnas.181181198\u003c/li\u003e\n\u003cli\u003eZhou BP, Liao Y, Xia W, Zou Y, Spohn B, Hung MC (2001) HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nat Cell Biol 3: 973-982 doi:10.1038/ncb1101-973\u003c/li\u003e\n\u003cli\u003eDiehl JA, Cheng M, Roussel MF, Sherr CJ (1998) Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 12: 3499-3511 doi:10.1101/gad.12.22.3499\u003c/li\u003e\n\u003cli\u003eKatayama K, Fujita N, Tsuruo T (2005) Akt/protein kinase B-dependent phosphorylation and inactivation of WEE1Hu promote cell cycle progression at G2/M transition. Mol Cell Biol 25: 5725-5737 doi:10.1128/mcb.25.13.5725-5737.2005\u003c/li\u003e\n\u003cli\u003ePorter LA, Donoghue DJ (2003) Cyclin B1 and CDK1: nuclear localization and upstream regulators. Prog Cell Cycle Res 5: 335-347\u003c/li\u003e\n\u003cli\u003eLiang J, Slingerland JM (2003) Multiple roles of the PI3K/PKB (Akt) pathway in cell cycle progression. Cell Cycle 2: 339-345\u003c/li\u003e\n\u003cli\u003eToulany M, Kehlbach R, Florczak U, Sak A, Wang S, Chen J, Lobrich M, Rodemann HP (2008) Targeting of AKT1 enhances radiation toxicity of human tumor cells by inhibiting DNA-PKcs-dependent DNA double-strand break repair. Mol Cancer Ther 7: 1772-1781 doi:10.1158/1535-7163.Mct-07-2200\u003c/li\u003e\n\u003cli\u003eFujiwara K, Iwado E, Mills GB, Sawaya R, Kondo S, Kondo Y (2007) Akt inhibitor shows anticancer and radiosensitizing effects in malignant glioma cells by inducing autophagy. Int J Oncol 31: 753-760\u003c/li\u003e\n\u003cli\u003eChautard E, Loubeau G, Tchirkov A, Chassagne J, Vermot-Desroches C, Morel L, Verrelle P (2010) Akt signaling pathway: a target for radiosensitizing human malignant glioma. Neuro Oncol 12: 434-443 doi:10.1093/neuonc/nop059\u003c/li\u003e\n\u003cli\u003eRogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM (1998) DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273: 5858-5868 doi:10.1074/jbc.273.10.5858\u003c/li\u003e\n\u003cli\u003eTu WZ, Li B, Huang B, Wang Y, Liu XD, Guan H, Zhang SM, Tang Y, Rang WQ, Zhou PK (2013) \u0026gamma;H2AX foci formation in the absence of DNA damage: mitotic H2AX phosphorylation is mediated by the DNA-PKcs/CHK2 pathway. FEBS Lett 587: 3437-3443 doi:10.1016/j.febslet.2013.08.028\u003c/li\u003e\n\u003cli\u003eBurdak-Rothkamm S, Short SC, Folkard M, Rothkamm K, Prise KM (2007) ATR-dependent radiation-induced gamma H2AX foci in bystander primary human astrocytes and glioma cells. Oncogene 26: 993-1002 doi:10.1038/sj.onc.1209863\u003c/li\u003e\n\u003cli\u003eB\u0026aacute;rtov\u0026aacute; E, Legartov\u0026aacute; S, Dundr M, Such\u0026aacute;nkov\u0026aacute; J (2019) A role of the 53BP1 protein in genome protection: structural and functional characteristics of 53BP1-dependent DNA repair. Aging (Albany NY) 11: 2488-2511 doi:10.18632/aging.101917\u003c/li\u003e\n\u003cli\u003eFradet-Turcotte A, Canny MD, Escribano-D\u0026iacute;az C, Orthwein A, Leung CC, Huang H, Landry MC, Kitevski-LeBlanc J, Noordermeer SM, Sicheri F, Durocher D (2013) 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark. Nature 499: 50-54 doi:10.1038/nature12318\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuro-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"neon","sideBox":"Learn more about [Journal of Neuro-Oncology](https://www.springer.com/journal/11060)","snPcode":"11060","submissionUrl":"https://submission.nature.com/new-submission/11060/3","title":"Journal of Neuro-Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Glioblastoma, radiotherapy, radiosensitizer, DNA repair","lastPublishedDoi":"10.21203/rs.3.rs-6896488/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6896488/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eRadiotherapy remains a key treatment modality for glioblastoma (GBM), but therapeutic resistance and radiation-induced toxicity severely limit its efficacy. Therefore, the development of novel, safe, and effective radiosensitizers is urgently needed. α-sulfoquinovosylacylpropandiol (SQAP), a marine-derived compound, has demonstrated potent radiosensitizing effects in cancer cells by improving tumor oxygenation and interfering with DNA repair. However, its impact on GBM has not yet been investigated. This study aimed to evaluate the biological effects of SQAP on GBM cells and to assess its potential as a radiosensitizer for future clinical application.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn vitro analyses\u0026mdash;including cell viability, colony formation, immunoblotting, quantitative reverse transcription polymerase chain reaction, immunocytochemistry, and cell death/proliferation assays\u0026mdash;were conducted to examine SQAP's mechanisms of action. In vivo efficacy and safety were evaluated using a murine intracranial glioma model.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSQAP inhibited GBM cell proliferation while sparing normal astrocytes. In combination with radiotherapy SQAP significantly reduced colony formation and enhanced cell death without affecting mitosis. SQAP decreased PI3K/Akt phosphorylation and modulated expression of downstream apoptotic and cell cycle-related proteins. Additionally, SQAP suppressed HIF-1α and VEGF expression. Although SQAP alone did not cause DNA damage, it delayed radiotherapy-induced DNA repair, as shown by prolonged γH2AX expression and reduced 53BP1 nuclear expression.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eSQAP exerts both antitumor and radiosensitizing effects in GBM models by inhibiting PI3K/Akt signaling, suppressing hypoxia-related pathways, and impairing DNA repair. These findings support its potential as a promising adjunctive agent in GBM therapy.\u003c/p\u003e","manuscriptTitle":"Synergistic antitumor and radiosensitizing effects of α -sulfoquinovosyl-acylpropanediol (SQAP) via PI3K/Akt inhibition and DNA repair impairment in glioblastoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-18 12:19:01","doi":"10.21203/rs.3.rs-6896488/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-03T10:42:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-02T20:38:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-29T03:01:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-26T21:30:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"242035733373453667292774346250668148458","date":"2025-06-24T10:28:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4989769006562176944991825472932074578","date":"2025-06-20T16:19:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"104031924062534826673382833344147646848","date":"2025-06-17T16:14:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-06-16T13:47:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-16T13:42:10+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-16T13:40:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Neuro-Oncology","date":"2025-06-15T04:48:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-neuro-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"neon","sideBox":"Learn more about [Journal of Neuro-Oncology](https://www.springer.com/journal/11060)","snPcode":"11060","submissionUrl":"https://submission.nature.com/new-submission/11060/3","title":"Journal of Neuro-Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"657118ce-d58c-4a20-a7dc-1992ed4a5e7b","owner":[],"postedDate":"June 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-08-18T16:06:48+00:00","versionOfRecord":{"articleIdentity":"rs-6896488","link":"https://doi.org/10.1007/s11060-025-05194-8","journal":{"identity":"journal-of-neuro-oncology","isVorOnly":false,"title":"Journal of Neuro-Oncology"},"publishedOn":"2025-08-11 15:58:10","publishedOnDateReadable":"August 11th, 2025"},"versionCreatedAt":"2025-06-18 12:19:01","video":"","vorDoi":"10.1007/s11060-025-05194-8","vorDoiUrl":"https://doi.org/10.1007/s11060-025-05194-8","workflowStages":[]},"version":"v1","identity":"rs-6896488","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6896488","identity":"rs-6896488","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}