Phenazine-1,6-diol selectively inhibited non-small cell lung cancer via DR5-mediated apoptosis

Phenazine-1,6-diol selectively inhibited non-small cell lung cancer via DR5-mediated apoptosis | 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 Phenazine-1,6-diol selectively inhibited non-small cell lung cancer via DR5-mediated apoptosis Peng Yue, Xiaoping Zhao, Jianan Jin, Langyu Yang, Tianrui Li, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8162519/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Non-small cell lung cancer (NSCLC) is a lethal malignancy with limited treatment options. Natural products, particularly from marine sources, represent a promising avenue for discovering novel anticancer agents with high selectivity. Purpose This study investigated the antitumor efficacy and mechanism of action of phenazine-1,6-diol, a marine-derived compound, against NSCLC, with a focus on DR5-mediated apoptosis. Methods The cytotoxicity of phenazine-1,6-diol was assessed in NSCLC and normal lung cell lines. Apoptosis was evaluated via flow cytometry and Western blot analysis. The essential role of DR5 was confirmed in DR5-knockout H460 cells. The impact on PI3K/AKT/mTOR signaling was analyzed. In vivo antitumor activity and safety were evaluated in a Lewis lung carcinoma xenograft model. Results Phenazine-1,6-diol selectively inhibited the proliferation of NSCLC cells while sparing normal lung epithelial cells. It induced G2/M cell cycle arrest, suppressed colony formation, and triggered caspase-dependent apoptosis, as evidenced by increased cleavage of PARP and caspase-3/8, upregulation of Bax, and downregulation of Bcl-2. Mechanistically, phenazine-1,6-diol upregulated DR5 expression. Genetic ablation of DR5 profoundly attenuated its cytotoxicity and apoptotic effects. The compound also inhibited the PI3K/AKT/mTOR pathway. In mouse xenografts, phenazine-1,6-diol (20 and 30 mg/kg) significantly suppressed tumor growth, prolonged survival, and enhanced tumor apoptosis without causing systemic toxicity. Conclusion Phenazine-1,6-diol is a potent and selective anti-NSCLC agent that acts through two mechanisms: the induction of DR5-mediated apoptosis and the suppression of the PI3K/AKT/mTOR survival pathway. It presents a promising candidate for further development as a targeted therapy for NSCLC. Non-small cell lung cancer death receptor 5 marine natural products PI3K/AKT/mTOR signaling pathway apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Worldwide, lung cancer is one of the deadliest types of cancer. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer diagnoses(Rodak et al., 2021 ). Despite improvements in early detection and therapies, patients diagnosed with advanced-stage NSCLC still have very low survival rates(Mamdani et al., 2022 ; M. Wang et al., 2021 ). Treatment options such as chemotherapy, targeted agents, and immunotherapy have extended lives in some cases, but their effectiveness is frequently undermined by the development of resistance, significant toxicity, and the tendency of tumors to metastasize(Hirsch et al., 2016 ; Li et al., 2023 ; Xiang et al., 2024 ). Therefore, the identification of new therapeutic molecules—especially those derived from natural sources—has become a major focus in NSCLC research(Huang et al., 2017 ; Wen et al., 2021 ; Yuan et al., 2019 ). One promising molecular target is death receptor 5 (DR5), which plays a pivotal role in the extrinsic pathway of programmed cell death(Kim et al., 2024 ). Upon DR5 activation, signaling cascades lead to caspase activation and apoptosis, making DR5 an attractive locus for anticancer strategies(Han and Roman, 2010 ). Recent reports in NSCLC indicate that the activation of DR5 can inhibit tumor growth and sensitize malignant cells to other treatments(Chen et al., 2016 ; Iksen et al., 2021 ; Quan et al., 2022 ). Given its central function in triggering apoptosis, DR5 may help overcome resistance mechanisms that limit current NSCLC therapies(Jin et al., 2022 ). The structural diversity and biological potential of natural compounds from marine sources, including their antitumor effects, have attracted increasing interest(Iksen et al., 2021 ; Montuori et al., 2022 ). Among these, phenazine derivatives are notable: many natural phenazines and their synthetic analogs display anticancer, antimicrobial, and anti-inflammatory activity(Matulja et al., 2022 ; Pasdaran et al., 2023 ; Peng et al., 2023 ). Phenazine-1,6-diol, a compound isolated from the marine sponge Dysidea avara and found in the Xisha Islands of China, has demonstrated antimicrobial and anti-inflammatory properties(Banik and Banerjee, 2022 ; Takahashi et al., 2022 ). However, its anticancer potential, particularly in NSCLC, remains underexplored. The objectives of the present study were threefold: first, to characterize the inhibitory effect of phenazine-1,6-diol on the growth of non-small cell lung cancer cells; second, to determine whether it induces programmed cell death through activation of the death receptor DR5; and third, to examine whether it modulates the PI3K-AKT-mTOR signaling cascade. In addition, we evaluated its antitumor efficacy in vivo in Lewis lung carcinoma xenografts in mice to determine its potential as a therapeutic agent. 2. Materials and methods 2.1 Reagents and Materials For the preparation of stock solutions, phenazine-1,6-diol (Bide Pharmatech Ltd., Shanghai, China) was thoroughly dissolved in DMSO. To promote apoptosis during our experimental procedures, we employed the pancaspase inhibitor Z-VAD-FMK (Selleck Chemicals, Houston, TX, USA). The entirety of our cell culture requirements—including but not limited to RPMI-1640 and DMEM, fetal bovine serum (FBS), and various antibiotics—were procured from Gibco (Carlsbad, CA, USA). The quantification of apoptotic cell populations was achieved via an Annexin V-FITC/PI staining kit (BD Biosciences, San Jose, CA, USA), with strict adherence to the manufacturer's provided guidelines. For our Western blot analyses, we assembled a comprehensive collection of primary antibodies specifically targeting Cyclin B1 and p21, both phosphorylated and total forms of AKT, as well as AMPK, mTOR, P70S6K, S6, PARP, Bcl-2, Bax, cleaved Caspase-3, and β-actin—all sourced from Cell Signaling Technology (Danvers, MA, USA). The corresponding secondary antibodies compatible with our Odyssey imaging system were obtained from LI-COR Biosciences (Lincoln, NE, USA). 2.2 Drug quality identification Following trypsin digestion, we assessed the purity of phenazine-1,6-diol via liquid chromatography‒mass spectrometry (LC‒MS). To prepare the sample, we dissolved 1 mg of the compound in 1 mL of methanol (MeOH), then took 0.1 mL of this mixture and diluted it to a final volume of 10 mL with additional MeOH. The analysis was performed by injecting a 1 µL aliquot into the system. We utilized an Agilent 1260 ultrahigh-performance liquid chromatography system equipped with a binary pump and standard autosampler for our analysis. Chromatographic separation was achieved with an EP-C18 column (3.0×50 mm, 3.5 µm). Our mobile phase consisted of methanol (Component A) and 0.1% formic acid in Milli-Q water (Component B). The gradient program began with a 10:90 A:B ratio, transitioned to 95:5 A:B at 2.00 minutes, maintained this composition until 3.00 minutes, returned to the initial conditions at 3.10 minutes, and held steady until the 4.00-minute mark was reached. Throughout the analysis, we maintained a constant flow rate of 1.5 mL/min and kept the column at ambient room temperature. Detection was accomplished with an Agilent 6120 mass spectrometer paired with a diode array detector (DAD), which simultaneously monitored UV absorbance at three wavelengths: 214, 254, and 280 nm. For ionization, we employed electrospray ionization (ESI) in positive mode, with parameters set at 250 °C for the drying gas, a flow rate of 12.0 L/min, and a nebulizer pressure of 35 psi. 2.3 Cell culture and drug treatment We procured the H1975, PC9, H1650, H460, H358, and BEAS-2B cell lines from the American Type Culture Collection (ATCC) facility located in Manassas, Virginia. Non-small cell lung cancer (NSCLC) cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum. On the other hand, the nonmalignant bronchial epithelial BEAS-2B line received specialized care, thriving in DMEM supplemented with bovine serum albumin (0.01 mg/mL), type I collagen (0.03 mg/mL), and fibronectin (0.01 mg/mL). All the cell cultures were cultured in a humidified incubator maintained at 37°C with an atmospheric concentration of 5% CO₂. 2.4 Cell viability assay The cells were seeded into 96-well plates and allowed to acclimate overnight. After confirming proper cell attachment, the cultures were exposed to varying concentrations of phenazine-1,6-diol for a 48-hour period, with DMSO serving as the vehicle control. Each well was subsequently supplemented with 10 µL of MTT reagent, and the plates were returned to the incubator for four additional hours to facilitate formazan crystal formation. The culture medium was then carefully removed, and 100 µL of DMSO was added to each well to dissolve the resulting formazan precipitates. Absorbance measurements were obtained using a Tecan microplate reader at 490 nm, with a reference wavelength of 570 nm. Cell viability percentages were calculated relative to those of the control group, and the IC₅₀ value for phenazine-1,6-diol was established through dose‒response analysis conducted with GraphPad Prism 6.0 software. 2.5 Colony formation assay I seeded H460 cells into six-well plates at approximately 500 cells per well and allowed them to acclimate overnight. The following day, the cells were treated with phenazine-1,6-diol at concentrations of 0, 5, 10, and 20 µM for a 48-hour period. Following this treatment, the drug-containing medium was carefully aspirated, and the cells were gently washed with PBS. Fresh compound-free medium was then added, which was refreshed every three days. The cultures were maintained for approximately ten days, providing sufficient time for visible colony formation. At that stage, the colonies were fixed with 4% paraformaldehyde, stained with crystal violet, thoroughly rinsed, and photographed. Finally, I counted the colonies in each treatment group to assess their clonogenic potential. 2.6 Western blot analysis After the cells were subjected to 48 hours of immersion in phenazin-1,6-diol, they were subsequently treated with 1× RIPA buffer supplemented with protease and phosphatase inhibitors. The mixture was then chilled for 15 minutes on ice. The cell extracts were carefully collected from the plates via a cell scraper and carefully transferred into microcentrifuge tubes. These tubes were then spun at a whirl of 13,000 rpm for a full 10 min, all while they were chilled to 4°C. Once the spin was over, the clear liquid above the sediment was carefully separated. To determine the protein concentration, we relied on the DC™ Protein Assay Kit from Bio-Rad, a reputable brand from the USA. To ensure consistency, we mixed each sample with the same amount of protein (30 µg) and 5× loading buffer. The mixture was then subjected to high heat and boiled between 95–100°C for 5 minutes to prepare it for analysis. Next, the protein samples were separated via SDS‒PAGE, which involves 8–12% polyacrylamide gels. After the proteins were separated by electrophoresis, they were transferred onto PVDF membranes from Millipore. These membranes were then treated with 5% nonfat dry milk to block any potential interference and left to sit for an hour at room temperature. After blocking, the membranes were treated with primary antibodies, diluted to a ratio of 1:1000, and then allowed to marinate overnight in a refrigerator at 4°C. The following day, after a series of rigorous washes, the membranes were exposed to fluorescently labeled secondary antibodies, which were diluted to 1:10,000. This process lasted for an hour at room temperature while the samples were kept in the dark to prevent unwanted light-induced reactions. Finally, the resulting protein bands were visualized via the AI800 imaging system from GE Healthcare, a tool that allows for crystal-clear images and precise quantification of the target protein expression levels. 2.7 Apoptosis analysis We plated H460 cells at a concentration of 100,000 cells per well in six-well plates and incubated them with a 48-hour window. Following this incubation period, we exposed the cells to phenazine-1,6-diol at various doses—specifically, 0, 5, 10, and 20 micromoles—for another 48 hours. Once the treatment concluded, we collected the cells and suspended them in 100 µL of Annexin V binding buffer. The resulting mixture was then combined with 1.5 microliters of Annexin V-FITC and 3 microliters of propidium iodide (100 micrograms per milliliter), followed by a 15-minute incubation at room temperature shielded from light. After the staining process, we added 300 microliters of binding buffer to each sample and passed it through a filter to eliminate any cell clumps. Finally, we performed flow cytometric analysis on a BD FACSAria III instrument from BD Biosciences and interpreted the data via FlowJo software. 2.8 Quantitative real-time PCR (qRT‒PCR) We harvested total RNA from our cell samples via TRIzol reagent (Invitrogen, USA) according to the manufacturer's recommended protocol. To convert this RNA into complementary DNA (cDNA), we employed the PrimeScript™ RT Reagent Kit (TaKaRa, Tokyo, Japan), following the supplier's guidelines. For quantitative real-time PCR analysis, we utilized FastStart Universal SYBR Green Master Mix (ROX) (Roche, Basel, Switzerland) on a CFX96™ Real-Time PCR Detection System paired with a C1000™ Thermal Cycler (Bio-Rad, Hercules, CA, USA). Our primer sequences were as follows: GAPDH (reference gene): Forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′, Reverse: 5′-GGCTGTTGTCATACTTCTCATGG-3′; DR5 (target gene): Forward: 5′-TCCTGCCTCTCCCTGTTCTC-3′, Reverse: 5′-GGTCGTTGTGAGCTTCTGTC-3′. These primers were custom-designed and synthesized by BGI Tech Solutions (Beijing, China). To ensure reliability, each qPCR was carried out in triplicate. Finally, we determined the relative expression levels of our target genes via the comparative threshold cycle (2^−ΔΔCt) method, with GAPDH serving as our internal control to normalize the data. 2.9 Stable transfection The DR5-knockout lentiviral plasmid was acquired from Miaoling Bioscience (Wuhan, Hubei, China). H460 cells were transduced with either the DR5-targeting lentivirus or a control (nontargeting) lentivirus. To establish an effective puromycin concentration for selection, a kill curve experiment was carried out. On the basis of that titration, puromycin dihydrochloride (Thermo Fisher Scientific, Pittsburgh, PA, USA) was applied at the optimal concentration to select successfully transduced cells. After selection, the surviving cell population was expanded and used in downstream assays, including Western blot and flow cytometry. 2.10 Animal experiments All animal research procedures strictly adhered to the ethical standards sanctioned by the Animal Ethical Committee at Macau University of Science and Technology (Protocol Number: AL003/DICV/SIS/2018; Approval Date: April 13, 2018). We employed male athymic nude mice, ranging from 6–8 weeks of age, which received subcutaneous injections in their right forelimbs containing 1 × 10⁶ LLC cells suspended within growth factor-reduced Matrigel. Once the tumor masses reached the 100 mm³ mark, the mice were randomly distributed across three distinct experimental arms. The vehicle control group received a cocktail of 5% DMSO, 40% PEG-400, or 55% saline. The second cohort was treated with phenazine-1,6-diol at a dosage of 20 mg/kg on an every-other-day schedule. The third group received phenazine-1,6-diol at 30 mg/kg, which was also administered every other day. This therapeutic protocol was maintained for three weeks. Every three days, we measured the tumor dimensions (length and width) via digital calipers and recorded the body weights. The tumor volumes were calculated via the following standard formula: (length × width²)/2. Throughout the study, the animals were housed under carefully controlled environmental conditions (with regulated temperature, humidity, and light cycles), and their well-being was monitored by dedicated facility staff. When the experimental period concluded, we euthanized the mice via cervical dislocation and harvested the tumors for subsequent analysis. 2.11 Histology and immunohistochemistry The paraffin-embedded tissue samples were thinly sliced to approximately 5 micrometers and then subjected to the standard hematoxylin and eosin staining protocol. For Ki-67 immunohistochemical analysis, tissue sections were incubated with a primary antibody diluted 1:400. The subsequent detection process employed the DAKO EnVision + system, which strictly followed the manufacturer's guidelines. A Leica light microscope was used for microscopic assessment, while image documentation was accomplished via a Leica DFC310 FX digital camera. Finally, the staining was quantified via Leica Application Suite software (version 4.4.0, Switzerland). 2.12 TUNEL staining Tissue samples embedded in paraffin were sectioned and fixed in freshly prepared 4% formaldehyde solution for ~ 40 min at ambient temperature. Following fixation, the sections were washed with phosphate-buffered saline (two times) and then subjected to permeabilization on ice with 0.1% Triton X-100 in PBS for 2 min to allow for reagent penetration. The TUNEL reaction mixture (FITC-labeled from Boster, Wuhan, China) was assembled as per the supplier’s protocol and applied to the sections, which were incubated at 37°C for 1 hour in darkness. After incubation, the slides were washed in PBS, and fluorescence microscopy was used to observe and document apoptotic (TUNEL-positive) cells. 2.13 Statistical analysis All the quantitative results are displayed as the mean values with standard deviations (SDs) from three separate experimental runs to capture both the average values and the spread in the measurements. We conducted all the statistical analyses via GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA). When comparing more than two groups, we employed one-way analysis of variance (ANOVA) and subsequently used Dunnett's post hoc test to identify differences from the control group. For two-group comparisons, we utilized an unpaired t test to determine statistical significance. The thresholds for significance were set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. 3. Results 3.1 Chemical characterization and cytotoxicity profile of phenazine-1,6-diol The chemical structure of phenazine-1,6-diol (also known as 1,6-dihydroxyphenazine), a marine-derived small molecule, is illustrated in Fig. 1 A. LC‒MS analysis confirmed its purity, with a dominant molecular ion peak matching the expected mass (Fig. 1 B, C), validating its suitability for biological evaluation. To evaluate whether phenazine-1,6-diol suppresses proliferation, several NSCLC cell lines (H460, H1975, H358, H1650, and PC9) and normal lung epithelial BEAS-2B cells were exposed to increasing doses of the compound. Notably, the viability of BEAS-2B cells decreased only slightly even at higher concentrations (Fig. 2A), suggesting that the compound is selectively toxic to cancerous cells. Phenazine-1,6-diol significantly inhibited NSCLC cells in a dose-dependent manner (Fig. 2B–F). Among all the tested cell lines (Fig. 2G), KRAS-mutated H460 cells were the most sensitive to phenazine-1,6-diol treatment. These results suggest that phenazine-1,6-diol preferentially targets lung cancer cells while sparing normal lung-derived cells, highlighting its potential as a selective therapeutic agent for NSCLC. 3.3 Phenazine-1,6-diol inhibited colony formation and induced cell cycle arrest To assess the long-term proliferative capacity of NSCLC cells following treatment, a colony formation assay was performed in H460 cells exposed to increasing concentrations of phenazine-1,6-diol. It significantly reduced the number and size of colonies, indicating a strong suppressive effect on the clonogenic potential of H460 cells (Fig. 3A and B). To further investigate the mechanism underlying this growth inhibition, we examined the expression of cell cycle regulatory proteins. After 48 hours of treatment, phenazine-1,6-diol led to notable downregulation of Cyclin B1 and upregulation of P21 expression (Fig. 3C), suggesting G2/M phase arrest. This alteration in cell cycle markers is consistent with the observed reduction in colony formation, demonstrating that phenazine-1,6-diol could induce cell cycle arrest and colony formation in NSCLC cells. 3.3 Phenazine-1,6-diol induced NSCLC cell death via PI3K-AKT signaling pathway-mediated energy metabolism dysregulation. The PI3K–AKT cascade is a central regulator of cell fate, controlling both survival and apoptotic responses. Suppression of this pathway is known to provoke metabolic stress and activate caspase-dependent apoptosis(Ghareghomi et al., 2023 ; Quan et al., 2022 ). Increasing evidence also indicates that selective interference with PI3K–AKT signaling can elicit synthetic lethality in malignant cells without damaging normal tissues(Heavey et al., 2014 ; Wu et al., 2019 ). To clarify how phenazine-1,6-diol influences this pathway, we examined the phosphorylation status of PI3K, AKT, and their downstream effectors, including mTOR, a pivotal regulator linked to AMPK signaling. As shown in Fig. 3D–F, 48 hours of treatment with phenazine-1,6-diol led to a marked reduction in the phosphorylation of PI3K and AKT, whereas total protein levels remained unchanged. In parallel, the phosphorylation of mTOR and its downstream targets—p-AMPK, p-P70S, and p-S6—was also diminished (Zhang and Jin, 2024 ; Zhao et al., 2015 ). Collectively, these results indicate that phenazine-1,6-diol promotes cell death at least partially through inhibition of the PI3K–AKT signaling axis(Lim and Ma, 2019 ). 3.4 Phenazine-1,6-diol induced apoptosis in NSCLC cells 3.5 Caspase inhibition attenuated phenazine-1,6-diol–induced apoptosis in H460 cells 3.6 DR5 is essential for phenazine-1,6-diol–induced apoptosis in H460 cells To determine whether DR5 is involved in phenazine-1,6-diol–induced apoptosis, we first assessed DR5 expression following treatment. Quantitative real-time PCR revealed significant upregulation of DR5 mRNA in H460 cells after 48 hours of phenazine-1,6-diol treatment (Fig. 5A). Consistently, Western blot analysis confirmed elevated DR5 protein expression compared with that in untreated controls (Fig. 5B), indicating that phenazine-1,6-diol activates DR5 at both the transcriptional and protein levels. To functionally validate the role of DR5, we constructed a DR5-knockout H460 cell line via lentiviral transduction. MTT assays revealed that the cytotoxic effect of phenazine-1,6-diol was significantly attenuated in DR5-deficient cells compared with that in wild-type H460 cells (Fig. 5C), suggesting that DR5 is critical for mediating phenazine-1,6-diol–induced cell death. Western blot analysis further demonstrated that phenazine-1,6-diol strongly induced the expression of cleaved PARP and cleaved caspase-3 in wild-type H460 cells, whereas these apoptotic markers were notably reduced in DR5-knockout cells (Fig. 5D). Moreover, phenazine-1,6-diol–treated H460 cells displayed classical apoptotic nuclear morphology, including chromatin condensation and nuclear fragmentation, as observed by flow cytometry analysis via Annexin V/PI staining (Fig. F and G) and DAPI staining via confocal microscopy (Fig. 5H). In contrast, untreated wild-type H460 cells, DR5-knockout cells, and DR5-knockout cells treated with phenazine-1,6-diol presented no significant nuclear changes, further suggesting that DR5 is required for phenazine-1,6-diol–induced apoptosis. Collectively, these results demonstrated that DR5 is an indispensable upstream regulator of phenazine-1,6-diol–mediated apoptotic signaling in NSCLC cells. Loss of DR5 significantly diminished both the expression of apoptotic markers and the morphological features of apoptosis, confirming its central role in the antitumor mechanism of this compound. 3.7 Phenazine-1,6-diol exhibited significant in vivo antitumor efficacy in a mouse xenograft model To evaluate the in vivo antitumor activity of phenazine-1,6-diol, a Lewis lung carcinoma (LLC) xenograft model was established in C57BL/6J mice. The animals were randomly divided into three groups: the control group and the phenazine-1,6-diol (20 and 30 mg/kg) group, with intraperitoneal administration every other day for 17 days (Fig. 6A). Compared with that in the control group, tumor growth in both treatment groups was significantly suppressed. As shown in Fig. 6B, tumor volume increased steadily in the control group, whereas both phenazine-1,6-diol–treated groups exhibited markedly reduced tumor progression, with the 30 mg/kg group showing the most pronounced effect. Importantly, body weight remained stable across all the groups throughout the treatment period (Fig. 6C), indicating that phenazine-1,6-diol did not induce significant systemic toxicity. Survival analysis revealed a substantial increase in the median survival time of the mice treated with phenazine-1,6-diol, particularly those in the 30 mg/kg group (Fig. 6H). Postmortem tumor samples from the treatment groups were clearly smaller than those from the control groups were (Fig. 6D and E), and statistical analysis confirmed significant differences in tumor size and weight between the 20 mg/kg group and the control group (p < 0.05), supporting the efficacy of the compounds in vivo . To evaluate potential immune or hematologic toxicity, the spleens were excised and analyzed morphologically and by weight (Fig. 6F and G). Together, these results demonstrated that phenazine-1,6-diol effectively suppressed tumor growth and prolonged survival in vivo , with no apparent adverse effects on body weight or immune organs, supporting its potential as a safe and efficacious candidate for lung cancer therapy. 3.8 Histopathological analysis confirmed the antiproliferative and proapoptotic effects of phenazine-1,6-diol in vivo To further validate the in vivo antitumor mechanism of phenazine-1,6-diol, histopathological examinations were performed on tumor tissues harvested from LLC xenograft mice after 17 days of treatment. The tumor sections were subjected to hematoxylin and eosin (H&E) staining, Ki-67 immunohistochemistry, and TUNEL assays to evaluate tissue morphology, proliferative activity, and apoptosis, respectively. Panoramic scans of H&E-stained tumor sections revealed that tumors from phenazine–1,6–diol–treated mice displayed extensive regions of necrosis and reduced cellular density compared with those in the control group, indicating tissue damage (Fig. 6I, top row). Ki-67 staining revealed a marked reduction in the proliferative index in the phenazine-1,6-diol treatment groups, suggesting that the compound effectively inhibited tumor cell proliferation in vivo . Conversely, TUNEL staining revealed a substantial increase in the number of apoptotic nuclei (Fig. 6I, middle and bottom rows). Quantitative analysis of Ki-67 and TUNEL staining (Fig. 6J) confirmed statistically significant differences between the treatment and control groups (p < 0.01), with a significant decrease in Ki–67–positive cells and a corresponding increase in TUNEL-positive cells following phenazine-1,6-diol administration. These histological results provide robust evidence that phenazine-1,6-diol exerts its in vivo antitumor effects by simultaneously inhibiting tumor cell proliferation and promoting apoptosis in tumor tissues. 3. Discussion In this study, we systematically evaluated the antitumor properties of phenazine-1,6-diol, a marine-derived phenazine compound, and demonstrated for the first time its potent antiproliferative and proapoptotic effects in NSCLC models both in vitro and in vivo . Our findings revealed that phenazine-1,6-diol selectively inhibited NSCLC cell viability while sparing normal lung epithelial and fibroblasts, induced apoptosis via the DR5-caspase axis, and modulated the PI3K/AKT/mTOR signaling cascade(Lim and Ma, 2019 ). Our data revealed that H460 cells are particularly sensitive to phenazine-1,6-diol, suggesting that subtype-specific vulnerability is potentially linked to the EGFR wild-type status or specific DR5 expression patterns(Liu et al., 2015 ). The selective cytotoxicity observed in BEAS-2B and CCD-19Lu cells highlights the therapeutic window of phenazine-1,6-diol, reducing concerns of nonspecific toxicity(W. Wang et al., 2021 ). Importantly, phenazine-1,6-diol significantly suppressed colony formation and induced G2/M cell cycle arrest, which was accompanied by downregulation of Cyclin B1 and upregulation of P21, which is consistent with classical mechanisms of proliferation inhibition. Interestingly, while phenazine-1,6-diol activated PI3K and AKT phosphorylation, it paradoxically suppressed downstream effectors such as mTOR, p70S6K, and S6. This uncoupling may reflect a feedback inhibition loop or crosstalk with apoptotic signaling cascades, as observed with other proapoptotic agents that disrupt metabolic signaling in cancer cells. Such signaling disruption is likely to create a pro-apoptotic intracellular environment, enhancing the efficacy of phenazine-1,6-diol. Mechanistically, our Western blot and flow cytometry analyses demonstrated that phenazine-1,6-diol activates both the extrinsic and intrinsic apoptotic pathways, as evidenced by increased expression of cleaved PARP, cleaved caspase-3, and caspase-8; Bax upregulation; and Bcl-2 downregulation. These effects were significantly attenuated by the pancaspase inhibitor Z-VAD-FMK, confirming the caspase dependence of phenazine-1,6-diol–induced apoptosis. A critical finding of this study is the identification of DR5 as a key upstream mediator of phenazine-1,6-diol activity. Both the qRT‒PCR and Western blot results revealed that DR5 was upregulated upon treatment. More importantly, DR5-knockout H460 cells presented markedly reduced apoptotic responses and cytotoxicity, highlighting DR5 as an essential effector of phenazine-1,6-diol–mediated cell death. Confocal imaging of DAPI-stained nuclei further confirmed that DR5 deficiency abolishes apoptotic morphological features. In vivo , phenazine-1,6-diol demonstrated significant antitumor efficacy in an LLC xenograft model without affecting body weight or spleen size, indicating favorable safety and tolerability. Tumor volume, weight, and survival improved, and histopathological analyses confirmed the suppression of proliferation (Ki-67) and induction of apoptosis (TUNEL), corroborating our in vitro results. Previous studies on marine-derived phenazine derivatives have reported antimicrobial and anti-inflammatory activities, but few studies have explored their antitumor mechanisms in depth(Lang et al., 2019 ; Nguyen, 2022 ). This study bridges that gap and positions phenazine-1,6-diol as a promising lead compound with dual regulatory potential over extrinsic apoptotic signaling and PI3K/AKT/mTOR-mediated survival pathways. Despite these promising findings, several limitations remain. First, the detailed molecular mechanisms of DR5 induction and PI3K/AKT uncoupling require further investigation. Second, the pharmacokinetics and bioavailability of phenazine-1,6-diol in vivo need to be systematically evaluated for clinical translation. Finally, future studies should explore the synergy of phenazine-1,6-diol with standard chemotherapeutics or targeted agents in NSCLC treatment(MacManus and Hegi-Johnson, 2022 ; Yang et al., 2022 ; Zhou et al., 2023 ). Conclusions In summary, this study provides comprehensive evidence that phenazine-1,6-diol has potent antitumor activity against NSCLC. Phenazine-1,6-diol selectively inhibited NSCLC cell proliferation, induced G2/M phase cell cycle arrest, and triggered apoptosis through a caspase-dependent pathway. Mechanistically, it activated DR5 and altered PI3K/AKT/mTOR signaling, leading to apoptotic execution via both intrinsic and extrinsic pathways. Importantly, DR5 was identified as an essential mediator of phenazine–1,6–diol–induced apoptosis, as its knockout significantly impaired cytotoxic and apoptotic responses. In vivo experiments further confirmed the ability of the compound to suppress tumor growth and prolong survival without evident systemic toxicity. Histological analyses confirmed its dual antiproliferative and proapoptotic effects in tumor tissues. These findings suggest that phenazine-1,6-diol is a promising candidate for the development of novel therapeutic agents targeting NSCLC. Future studies are warranted to optimize its pharmacological profile and explore its therapeutic synergy with existing anticancer agents. Abbreviations AKT: Protein kinase B ANOVA: Analysis of variance ATCC: American Type Culture Collection BSA: Bovine serum albumin cDNA: Complementary DNA DAD: Diode array detector DAPI: 4',6-diamidino-2-phenylindole DMEM: Dulbecco's Modified Eagle Medium DMSO: Dimethyl sulfoxide DR5: Death Receptor 5 EGFR: Epidermal growth factor receptor ESI: Electrospray ionization FBS: Fetal bovine serum FITC: Fluorescein isothiocyanate H&E: Hematoxylin and eosin IC₅₀: Half-maximal inhibitory concentration IHC: Immunohistochemistry LC‒MS: Liquid Chromatography‒Mass Spectrometry LLC: Lewis Lung Carcinoma mTOR: Mammalian target of rapamycin MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NSCLC: Non-Small Cell Lung Cancer PARP: Poly (ADP‒ribose) polymerase PBS: Phosphate-buffered saline PI: Propidium iodide PI3K: Phosphoinositide 3-kinase qRT‒PCR: Quantitative real-time polymerase chain reaction SD: Standard deviation TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling Declarations Declaration of competing interests All the authors have no financial or commercial conflicts of interest to disclose. Acknowledgments We are grateful to the State Key Laboratory of Quality Research in Chinese Medicine for providing laboratory and relevant personnel management and technical support, as well as to the Macau University of Science and Technology for their support of this research, which was also funded by the Macau Science Development Foundation. Authorship contribution statement Peng Yue directed and guided the project. Peng Yue, Xiaoping Zhao and Jianan Jin designed the experiments and drafted the manuscript. Peng Yue, Xiaoping Zhao, Langyu Yang, Tianrui Lee, Un Ian Leong，Ziyang Zhang and Jiayi Sunperformed the experiments and analyzed the results. Chitin Hon and Jiyou Kou reviewed the manuscript and made significant revisions. The final manuscript has been approved by all authors. References Banik BK, Banerjee B. Heterocyclic Anticancer Agents. Walter de Gruyter GmbH & Co KG; 2022. Chen M, Wang X, Zha D, Cai F, Zhang W, He Y, Huang Q, Zhuang H, Hua Z-C. 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All the data were analyzed as the means ± SDs (n=3, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Cells IC 50 for 48 h （ μM ） BEAS-2B 160.2 ±2.2 H460 17.76 ±1.16 H1975 20.33 ±2.21 H358 58.98 ±1.43 H1650 PC9 83.79 ±12.2 25.67 ±3.42 Additional Declarations No competing interests reported. Supplementary Files SUPPLEMENTARYMATERIALS.docx westernblotfigure.docx imageabstract.png Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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1","display":"","copyAsset":false,"role":"figure","size":254257,"visible":true,"origin":"","legend":"\u003cp\u003e(A-C) Chemical structure of phenazine-1,6-diol and determination of its purity viamass spectrometry.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8162519/v1/f8e142527692164ba8fb5ef2.jpg"},{"id":96430816,"identity":"f06f3eb6-2a18-414b-a765-75323c5ba530","added_by":"auto","created_at":"2025-11-21 03:55:43","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":262326,"visible":true,"origin":"","legend":"\u003cp\u003e(A) The cytotoxic effect of phenazine-1,6-diol on normal lung cell lines was determined via the MTT assay. (B-F) The cytotoxic effect of phenazine-1,6-diol on NSCLC cells was determined via theMTT assay. All the data were analyzed as the means ± SDs(n=3, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"Figure2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8162519/v1/c55c965734598c9022aff5ba.jpeg"},{"id":96430820,"identity":"c1f4cfc4-fdbd-4539-84d6-c88a06d34c23","added_by":"auto","created_at":"2025-11-21 03:55:43","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":462252,"visible":true,"origin":"","legend":"\u003cp\u003e(A-B) Colony formation ability and statistical results of H460 cells treated with phenazine-1,6-diol. (C) Western blot analysis was conducted to determine the effectsof 1,6-dihydroxyphenazine on the cell cycle markers Cyclin B1 and P21 in H460 cells after 48 h of phenazine-1,6-diol treatment. (D-F) Phenazine-1,6-diol activated the PI3K/AKT pathway. All the data are presented as the means ± SDs (n=3, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8162519/v1/42604ead89ef22fc206dc589.jpeg"},{"id":96454849,"identity":"85f5ca37-d254-464b-a2bf-8953f06ca4d7","added_by":"auto","created_at":"2025-11-21 10:03:11","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":496161,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Cleaved PARP, Bcl-2, Bax, cleaved caspase-3, and cleaved caspase-8 protein expression was evaluated by western blotting. (B-C) Flow cytometric analysis of apoptosis after treatment with different concentrations of phenazine-1,6-diol and the apoptosis-positive reference agent sanguinarine for 48 h. (D) MTT assay showing the viability of H460 cells treated with phenazine-1,6-diol (20 μM) for 48 h and coculturedwith or without the pancaspase inhibitor Z-VAD-FMK (50 μM). (E) Western blot analysis of PARP and cleaved PARP in H460 cells after 48 h of treatment with phenazine-1,6-diol or sanguinarine in the presence or absence of Z-VAD-FMK. GAPDH was used as the loading control. (F) Flow cytometry analysis of apoptosis via Annexin V-FITC/PI staining in H460 cells treated with phenazine-1,6-diol and the apoptosis-positive reference agent sanguinarine for 48 hours. Representative dot plots are shown. (G) Quantification of apoptotic cells from (F). The data are presented as the means ± SDs from three independent experiments. All the data are presented as the means ± SDs(n=3, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"Figure4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8162519/v1/57f0ed67bb7152ae481ded92.jpeg"},{"id":96430835,"identity":"239a5013-5a2e-43f0-b7f6-96fb2b8ff8e7","added_by":"auto","created_at":"2025-11-21 03:55:44","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":482541,"visible":true,"origin":"","legend":"\u003cp\u003eThe results of normal H460 cells and DR5-knockout H460 cells after treatment with phenazine-1,6-diol for 48 hours. (A) DR5 expression in H460 cells after phenazine-1,6-diol administration was verified through a qPCR assay. (B) DR5 protein expression at the protein level in H460 cells after phenazine-1,6-diol administration was determined via a Western blot analysis. (C) MTT assays were used to examine the survival rates of wild-type H460 cells and H460 cells lacking DR5 after treatment with phenazine-1,6-diol for 48 hours. (D) The expression levels of various apoptosis-related proteins in wild-type H460 cells and H460 DR5 knockout cells were examined via Western blot analysis following 48 hours of phenazine-1,6-diol treatment. (E) The apoptosis of wild-type H460 cells and H460 DR5 knockout cells after phenazine-1,6-diol treatment for 48 hours was examined via flow cytometry. (F) Flow cytometry analysis of apoptotic wild-type H460 cells and H460 DR5 knockout cells after phenazine-1,6-diol treatment for 48 hours. (G) The phenazine-1,6-diol-treated cell nuclei of wild-type H460 cells and H460 cells with DR5 knockout were examined via confocal microscopy after DAPI staining for 48 hours; blue indicates DAPI-stained cell nuclei.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8162519/v1/f1e15656d8c1ade8ff8dd049.jpg"},{"id":96430839,"identity":"0fb8945a-7916-444f-b3f9-bfe189e9be03","added_by":"auto","created_at":"2025-11-21 03:55:44","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":802210,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e animal experimental results for phenazine-1,6-diol. (A) Schematic illustration of the \u003cem\u003ein vivo\u003c/em\u003e experimental design. (B) Tumor growth curves over the 17-day treatment period. Tumor volumes were measured every other day and calculated as (length × width²)/2. (C) Body weight changes during the treatment period. (D) Representative images of excised tumors at the end of treatment. (E) Quantification of tumor weights and volumes at the endpoint. (F) Representative images of spleens collected after 17 days of treatment. (G) Spleen weight analysis at the endpoint. (H) Kaplan–Meier survival curves for mice in different treatment groups. (I) Representative histological and immunohistochemical images of tumor sections stained with hematoxylin and eosin (H\u0026amp;E), Ki67, and TUNEL. (J) Quantification of Ki67-positive cells (proliferation index) and TUNEL-positive cells (apoptotic index) from (I).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8162519/v1/cde9709e227fce64139b332c.jpg"},{"id":96455216,"identity":"2ccbb6d6-ef02-40aa-b453-77f12da87c32","added_by":"auto","created_at":"2025-11-21 10:03:47","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":238639,"visible":true,"origin":"","legend":"\u003cp\u003eThe proposed mechanism of the effect of phenazine–1,6–diol on NSCLC cells.\u003c/p\u003e","description":"","filename":"Figure7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8162519/v1/cf4031226ef3e1b916d3b85d.jpeg"},{"id":96711312,"identity":"25919dde-2e64-444c-a8db-77b33ffdd21d","added_by":"auto","created_at":"2025-11-25 10:11:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4051554,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8162519/v1/101e4fe9-eaaf-4957-add0-9ac236734a04.pdf"},{"id":96455392,"identity":"32286425-61b7-4f79-8b1f-7a35dcf13131","added_by":"auto","created_at":"2025-11-21 10:04:05","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1242070,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTARYMATERIALS.docx","url":"https://assets-eu.researchsquare.com/files/rs-8162519/v1/1eead34cea7ddc8db001d725.docx"},{"id":96430829,"identity":"e65bf416-36cf-4dfe-8d90-796ced753e82","added_by":"auto","created_at":"2025-11-21 03:55:43","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1552477,"visible":true,"origin":"","legend":"","description":"","filename":"westernblotfigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-8162519/v1/a7c726538dec5c4226eecfce.docx"},{"id":96455325,"identity":"bbd6eedf-d18e-4b40-8abb-8c57ad3bb060","added_by":"auto","created_at":"2025-11-21 10:03:58","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":964454,"visible":true,"origin":"","legend":"","description":"","filename":"imageabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-8162519/v1/df1f6b69c4afa63cee1be54b.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phenazine-1,6-diol selectively inhibited non-small cell lung cancer via DR5-mediated apoptosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWorldwide, lung cancer is one of the deadliest types of cancer. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer diagnoses(Rodak et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Despite improvements in early detection and therapies, patients diagnosed with advanced-stage NSCLC still have very low survival rates(Mamdani et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; M. Wang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Treatment options such as chemotherapy, targeted agents, and immunotherapy have extended lives in some cases, but their effectiveness is frequently undermined by the development of resistance, significant toxicity, and the tendency of tumors to metastasize(Hirsch et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xiang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Therefore, the identification of new therapeutic molecules\u0026mdash;especially those derived from natural sources\u0026mdash;has become a major focus in NSCLC research(Huang et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Wen et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yuan et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOne promising molecular target is death receptor 5 (DR5), which plays a pivotal role in the extrinsic pathway of programmed cell death(Kim et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Upon DR5 activation, signaling cascades lead to caspase activation and apoptosis, making DR5 an attractive locus for anticancer strategies(Han and Roman, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Recent reports in NSCLC indicate that the activation of DR5 can inhibit tumor growth and sensitize malignant cells to other treatments(Chen et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Iksen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Quan et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Given its central function in triggering apoptosis, DR5 may help overcome resistance mechanisms that limit current NSCLC therapies(Jin et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe structural diversity and biological potential of natural compounds from marine sources, including their antitumor effects, have attracted increasing interest(Iksen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Montuori et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Among these, phenazine derivatives are notable: many natural phenazines and their synthetic analogs display anticancer, antimicrobial, and anti-inflammatory activity(Matulja et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Pasdaran et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Peng et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Phenazine-1,6-diol, a compound isolated from the marine sponge \u003cem\u003eDysidea avara\u003c/em\u003e and found in the Xisha Islands of China, has demonstrated antimicrobial and anti-inflammatory properties(Banik and Banerjee, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Takahashi et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). However, its anticancer potential, particularly in NSCLC, remains underexplored.\u003c/p\u003e\u003cp\u003eThe objectives of the present study were threefold: first, to characterize the inhibitory effect of phenazine-1,6-diol on the growth of non-small cell lung cancer cells; second, to determine whether it induces programmed cell death through activation of the death receptor DR5; and third, to examine whether it modulates the PI3K-AKT-mTOR signaling cascade. In addition, we evaluated its antitumor efficacy \u003cem\u003ein vivo\u003c/em\u003e in Lewis lung carcinoma xenografts in mice to determine its potential as a therapeutic agent.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Reagents and Materials\u003c/h2\u003e\u003cp\u003eFor the preparation of stock solutions, phenazine-1,6-diol (Bide Pharmatech Ltd., Shanghai, China) was thoroughly dissolved in DMSO. To promote apoptosis during our experimental procedures, we employed the pancaspase inhibitor Z-VAD-FMK (Selleck Chemicals, Houston, TX, USA). The entirety of our cell culture requirements\u0026mdash;including but not limited to RPMI-1640 and DMEM, fetal bovine serum (FBS), and various antibiotics\u0026mdash;were procured from Gibco (Carlsbad, CA, USA). The quantification of apoptotic cell populations was achieved via an Annexin V-FITC/PI staining kit (BD Biosciences, San Jose, CA, USA), with strict adherence to the manufacturer's provided guidelines. For our Western blot analyses, we assembled a comprehensive collection of primary antibodies specifically targeting Cyclin B1 and p21, both phosphorylated and total forms of AKT, as well as AMPK, mTOR, P70S6K, S6, PARP, Bcl-2, Bax, cleaved Caspase-3, and β-actin\u0026mdash;all sourced from Cell Signaling Technology (Danvers, MA, USA). The corresponding secondary antibodies compatible with our Odyssey imaging system were obtained from LI-COR Biosciences (Lincoln, NE, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Drug quality identification\u003c/h2\u003e\u003cp\u003eFollowing trypsin digestion, we assessed the purity of phenazine-1,6-diol via liquid chromatography‒mass spectrometry (LC‒MS). To prepare the sample, we dissolved 1 mg of the compound in 1 mL of methanol (MeOH), then took 0.1 mL of this mixture and diluted it to a final volume of 10 mL with additional MeOH. The analysis was performed by injecting a 1 \u0026micro;L aliquot into the system. We utilized an Agilent 1260 ultrahigh-performance liquid chromatography system equipped with a binary pump and standard autosampler for our analysis. Chromatographic separation was achieved with an EP-C18 column (3.0\u0026times;50 mm, 3.5 \u0026micro;m). Our mobile phase consisted of methanol (Component A) and 0.1% formic acid in Milli-Q water (Component B). The gradient program began with a 10:90 A:B ratio, transitioned to 95:5 A:B at 2.00 minutes, maintained this composition until 3.00 minutes, returned to the initial conditions at 3.10 minutes, and held steady until the 4.00-minute mark was reached. Throughout the analysis, we maintained a constant flow rate of 1.5 mL/min and kept the column at ambient room temperature. Detection was accomplished with an Agilent 6120 mass spectrometer paired with a diode array detector (DAD), which simultaneously monitored UV absorbance at three wavelengths: 214, 254, and 280 nm. For ionization, we employed electrospray ionization (ESI) in positive mode, with parameters set at 250 \u0026deg;C for the drying gas, a flow rate of 12.0 L/min, and a nebulizer pressure of 35 psi.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Cell culture and drug treatment\u003c/h2\u003e\u003cp\u003eWe procured the H1975, PC9, H1650, H460, H358, and BEAS-2B cell lines from the American Type Culture Collection (ATCC) facility located in Manassas, Virginia. Non-small cell lung cancer (NSCLC) cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum. On the other hand, the nonmalignant bronchial epithelial BEAS-2B line received specialized care, thriving in DMEM supplemented with bovine serum albumin (0.01 mg/mL), type I collagen (0.03 mg/mL), and fibronectin (0.01 mg/mL). All the cell cultures were cultured in a humidified incubator maintained at 37\u0026deg;C with an atmospheric concentration of 5% CO₂.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Cell viability assay\u003c/h2\u003e\u003cp\u003eThe cells were seeded into 96-well plates and allowed to acclimate overnight. After confirming proper cell attachment, the cultures were exposed to varying concentrations of phenazine-1,6-diol for a 48-hour period, with DMSO serving as the vehicle control. Each well was subsequently supplemented with 10 \u0026micro;L of MTT reagent, and the plates were returned to the incubator for four additional hours to facilitate formazan crystal formation. The culture medium was then carefully removed, and 100 \u0026micro;L of DMSO was added to each well to dissolve the resulting formazan precipitates. Absorbance measurements were obtained using a Tecan microplate reader at 490 nm, with a reference wavelength of 570 nm. Cell viability percentages were calculated relative to those of the control group, and the IC₅₀ value for phenazine-1,6-diol was established through dose‒response analysis conducted with GraphPad Prism 6.0 software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Colony formation assay\u003c/h2\u003e\u003cp\u003eI seeded H460 cells into six-well plates at approximately 500 cells per well and allowed them to acclimate overnight. The following day, the cells were treated with phenazine-1,6-diol at concentrations of 0, 5, 10, and 20 \u0026micro;M for a 48-hour period. Following this treatment, the drug-containing medium was carefully aspirated, and the cells were gently washed with PBS. Fresh compound-free medium was then added, which was refreshed every three days. The cultures were maintained for approximately ten days, providing sufficient time for visible colony formation. At that stage, the colonies were fixed with 4% paraformaldehyde, stained with crystal violet, thoroughly rinsed, and photographed. Finally, I counted the colonies in each treatment group to assess their clonogenic potential.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Western blot analysis\u003c/h2\u003e\u003cp\u003eAfter the cells were subjected to 48 hours of immersion in phenazin-1,6-diol, they were subsequently treated with 1\u0026times; RIPA buffer supplemented with protease and phosphatase inhibitors. The mixture was then chilled for 15 minutes on ice. The cell extracts were carefully collected from the plates via a cell scraper and carefully transferred into microcentrifuge tubes. These tubes were then spun at a whirl of 13,000 rpm for a full 10 min, all while they were chilled to 4\u0026deg;C. Once the spin was over, the clear liquid above the sediment was carefully separated. To determine the protein concentration, we relied on the DC\u0026trade; Protein Assay Kit from Bio-Rad, a reputable brand from the USA. To ensure consistency, we mixed each sample with the same amount of protein (30 \u0026micro;g) and 5\u0026times; loading buffer. The mixture was then subjected to high heat and boiled between 95\u0026ndash;100\u0026deg;C for 5 minutes to prepare it for analysis. Next, the protein samples were separated via SDS‒PAGE, which involves 8\u0026ndash;12% polyacrylamide gels. After the proteins were separated by electrophoresis, they were transferred onto PVDF membranes from Millipore. These membranes were then treated with 5% nonfat dry milk to block any potential interference and left to sit for an hour at room temperature. After blocking, the membranes were treated with primary antibodies, diluted to a ratio of 1:1000, and then allowed to marinate overnight in a refrigerator at 4\u0026deg;C. The following day, after a series of rigorous washes, the membranes were exposed to fluorescently labeled secondary antibodies, which were diluted to 1:10,000. This process lasted for an hour at room temperature while the samples were kept in the dark to prevent unwanted light-induced reactions. Finally, the resulting protein bands were visualized via the AI800 imaging system from GE Healthcare, a tool that allows for crystal-clear images and precise quantification of the target protein expression levels.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Apoptosis analysis\u003c/h2\u003e\u003cp\u003eWe plated H460 cells at a concentration of 100,000 cells per well in six-well plates and incubated them with a 48-hour window. Following this incubation period, we exposed the cells to phenazine-1,6-diol at various doses\u0026mdash;specifically, 0, 5, 10, and 20 micromoles\u0026mdash;for another 48 hours. Once the treatment concluded, we collected the cells and suspended them in 100 \u0026micro;L of Annexin V binding buffer. The resulting mixture was then combined with 1.5 microliters of Annexin V-FITC and 3 microliters of propidium iodide (100 micrograms per milliliter), followed by a 15-minute incubation at room temperature shielded from light. After the staining process, we added 300 microliters of binding buffer to each sample and passed it through a filter to eliminate any cell clumps. Finally, we performed flow cytometric analysis on a BD FACSAria III instrument from BD Biosciences and interpreted the data via FlowJo software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Quantitative real-time PCR (qRT‒PCR)\u003c/h2\u003e\u003cp\u003eWe harvested total RNA from our cell samples via TRIzol reagent (Invitrogen, USA) according to the manufacturer's recommended protocol. To convert this RNA into complementary DNA (cDNA), we employed the PrimeScript\u0026trade; RT Reagent Kit (TaKaRa, Tokyo, Japan), following the supplier's guidelines. For quantitative real-time PCR analysis, we utilized FastStart Universal SYBR Green Master Mix (ROX) (Roche, Basel, Switzerland) on a CFX96\u0026trade; Real-Time PCR Detection System paired with a C1000\u0026trade; Thermal Cycler (Bio-Rad, Hercules, CA, USA). Our primer sequences were as follows:\u003c/p\u003e\u003cp\u003eGAPDH (reference gene): Forward: 5\u0026prime;-GGAGCGAGATCCCTCCAAAAT-3\u0026prime;,\u003c/p\u003e\u003cp\u003eReverse: 5\u0026prime;-GGCTGTTGTCATACTTCTCATGG-3\u0026prime;;\u003c/p\u003e\u003cp\u003eDR5 (target gene): Forward: 5\u0026prime;-TCCTGCCTCTCCCTGTTCTC-3\u0026prime;,\u003c/p\u003e\u003cp\u003eReverse: 5\u0026prime;-GGTCGTTGTGAGCTTCTGTC-3\u0026prime;.\u003c/p\u003e\u003cp\u003eThese primers were custom-designed and synthesized by BGI Tech Solutions (Beijing, China). To ensure reliability, each qPCR was carried out in triplicate. Finally, we determined the relative expression levels of our target genes via the comparative threshold cycle (2^\u0026minus;ΔΔCt) method, with GAPDH serving as our internal control to normalize the data.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Stable transfection\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe DR5-knockout lentiviral plasmid was acquired from Miaoling Bioscience (Wuhan, Hubei, China). H460 cells were transduced with either the DR5-targeting lentivirus or a control (nontargeting) lentivirus. To establish an effective puromycin concentration for selection, a kill curve experiment was carried out. On the basis of that titration, puromycin dihydrochloride (Thermo Fisher Scientific, Pittsburgh, PA, USA) was applied at the optimal concentration to select successfully transduced cells. After selection, the surviving cell population was expanded and used in downstream assays, including Western blot and flow cytometry.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Animal experiments\u003c/h2\u003e\u003cp\u003e All animal research procedures strictly adhered to the ethical standards sanctioned by the Animal Ethical Committee at Macau University of Science and Technology (Protocol Number: AL003/DICV/SIS/2018; Approval Date: April 13, 2018). We employed male athymic nude mice, ranging from 6\u0026ndash;8 weeks of age, which received subcutaneous injections in their right forelimbs containing 1 \u0026times; 10⁶ LLC cells suspended within growth factor-reduced Matrigel. Once the tumor masses reached the 100 mm\u0026sup3; mark, the mice were randomly distributed across three distinct experimental arms. The vehicle control group received a cocktail of 5% DMSO, 40% PEG-400, or 55% saline. The second cohort was treated with phenazine-1,6-diol at a dosage of 20 mg/kg on an every-other-day schedule. The third group received phenazine-1,6-diol at 30 mg/kg, which was also administered every other day. This therapeutic protocol was maintained for three weeks. Every three days, we measured the tumor dimensions (length and width) via digital calipers and recorded the body weights. The tumor volumes were calculated via the following standard formula: (length \u0026times; width\u0026sup2;)/2. Throughout the study, the animals were housed under carefully controlled environmental conditions (with regulated temperature, humidity, and light cycles), and their well-being was monitored by dedicated facility staff. When the experimental period concluded, we euthanized the mice via cervical dislocation and harvested the tumors for subsequent analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11 Histology and immunohistochemistry\u003c/h2\u003e\u003cp\u003eThe paraffin-embedded tissue samples were thinly sliced to approximately 5 micrometers and then subjected to the standard hematoxylin and eosin staining protocol. For Ki-67 immunohistochemical analysis, tissue sections were incubated with a primary antibody diluted 1:400. The subsequent detection process employed the DAKO EnVision\u0026thinsp;+\u0026thinsp;system, which strictly followed the manufacturer's guidelines. A Leica light microscope was used for microscopic assessment, while image documentation was accomplished via a Leica DFC310 FX digital camera. Finally, the staining was quantified via Leica Application Suite software (version 4.4.0, Switzerland).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12 TUNEL staining\u003c/h2\u003e\u003cp\u003eTissue samples embedded in paraffin were sectioned and fixed in freshly prepared 4% formaldehyde solution for ~\u0026thinsp;40 min at ambient temperature. Following fixation, the sections were washed with phosphate-buffered saline (two times) and then subjected to permeabilization on ice with 0.1% Triton X-100 in PBS for 2 min to allow for reagent penetration. The TUNEL reaction mixture (FITC-labeled from Boster, Wuhan, China) was assembled as per the supplier\u0026rsquo;s protocol and applied to the sections, which were incubated at 37\u0026deg;C for 1 hour in darkness. After incubation, the slides were washed in PBS, and fluorescence microscopy was used to observe and document apoptotic (TUNEL-positive) cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.13 Statistical analysis\u003c/h2\u003e\u003cp\u003eAll the quantitative results are displayed as the mean values with standard deviations (SDs) from three separate experimental runs to capture both the average values and the spread in the measurements. We conducted all the statistical analyses via GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA, USA). When comparing more than two groups, we employed one-way analysis of variance (ANOVA) and subsequently used Dunnett's post hoc test to identify differences from the control group. For two-group comparisons, we utilized an unpaired t test to determine statistical significance. The thresholds for significance were set at *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Chemical characterization and cytotoxicity profile of phenazine-1,6-diol\u003c/h2\u003e\u003cp\u003eThe chemical structure of phenazine-1,6-diol (also known as 1,6-dihydroxyphenazine), a marine-derived small molecule, is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. LC‒MS analysis confirmed its purity, with a dominant molecular ion peak matching the expected mass (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C), validating its suitability for biological evaluation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo evaluate whether phenazine-1,6-diol suppresses proliferation, several NSCLC cell lines (H460, H1975, H358, H1650, and PC9) and normal lung epithelial BEAS-2B cells were exposed to increasing doses of the compound. Notably, the viability of BEAS-2B cells decreased only slightly even at higher concentrations (Fig.\u0026nbsp;2A), suggesting that the compound is selectively toxic to cancerous cells. Phenazine-1,6-diol significantly inhibited NSCLC cells in a dose-dependent manner (Fig.\u0026nbsp;2B\u0026ndash;F). Among all the tested cell lines (Fig.\u0026nbsp;2G), KRAS-mutated H460 cells were the most sensitive to phenazine-1,6-diol treatment. These results suggest that phenazine-1,6-diol preferentially targets lung cancer cells while sparing normal lung-derived cells, highlighting its potential as a selective therapeutic agent for NSCLC.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Phenazine-1,6-diol inhibited colony formation and induced cell cycle arrest\u003c/h2\u003e\u003cp\u003eTo assess the long-term proliferative capacity of NSCLC cells following treatment, a colony formation assay was performed in H460 cells exposed to increasing concentrations of phenazine-1,6-diol. It significantly reduced the number and size of colonies, indicating a strong suppressive effect on the clonogenic potential of H460 cells (Fig.\u0026nbsp;3A and B). To further investigate the mechanism underlying this growth inhibition, we examined the expression of cell cycle regulatory proteins. After 48 hours of treatment, phenazine-1,6-diol led to notable downregulation of Cyclin B1 and upregulation of P21 expression (Fig.\u0026nbsp;3C), suggesting G2/M phase arrest. This alteration in cell cycle markers is consistent with the observed reduction in colony formation, demonstrating that phenazine-1,6-diol could induce cell cycle arrest and colony formation in NSCLC cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Phenazine-1,6-diol induced NSCLC cell death via PI3K-AKT signaling pathway-mediated energy metabolism dysregulation.\u003c/h2\u003e\u003cp\u003eThe PI3K\u0026ndash;AKT cascade is a central regulator of cell fate, controlling both survival and apoptotic responses. Suppression of this pathway is known to provoke metabolic stress and activate caspase-dependent apoptosis(Ghareghomi et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Quan et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Increasing evidence also indicates that selective interference with PI3K\u0026ndash;AKT signaling can elicit synthetic lethality in malignant cells without damaging normal tissues(Heavey et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Wu et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To clarify how phenazine-1,6-diol influences this pathway, we examined the phosphorylation status of PI3K, AKT, and their downstream effectors, including mTOR, a pivotal regulator linked to AMPK signaling. As shown in Fig.\u0026nbsp;3D\u0026ndash;F, 48 hours of treatment with phenazine-1,6-diol led to a marked reduction in the phosphorylation of PI3K and AKT, whereas total protein levels remained unchanged. In parallel, the phosphorylation of mTOR and its downstream targets\u0026mdash;p-AMPK, p-P70S, and p-S6\u0026mdash;was also diminished (Zhang and Jin, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Collectively, these results indicate that phenazine-1,6-diol promotes cell death at least partially through inhibition of the PI3K\u0026ndash;AKT signaling axis(Lim and Ma, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Phenazine-1,6-diol induced apoptosis in NSCLC cells\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Caspase inhibition attenuated phenazine-1,6-diol\u0026ndash;induced apoptosis in H460 cells\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.6 DR5 is essential for phenazine-1,6-diol\u0026ndash;induced apoptosis in H460 cells\u003c/h2\u003e\u003cp\u003eTo determine whether DR5 is involved in phenazine-1,6-diol\u0026ndash;induced apoptosis, we first assessed DR5 expression following treatment. Quantitative real-time PCR revealed significant upregulation of DR5 mRNA in H460 cells after 48 hours of phenazine-1,6-diol treatment (Fig.\u0026nbsp;5A). Consistently, Western blot analysis confirmed elevated DR5 protein expression compared with that in untreated controls (Fig.\u0026nbsp;5B), indicating that phenazine-1,6-diol activates DR5 at both the transcriptional and protein levels. To functionally validate the role of DR5, we constructed a DR5-knockout H460 cell line via lentiviral transduction. MTT assays revealed that the cytotoxic effect of phenazine-1,6-diol was significantly attenuated in DR5-deficient cells compared with that in wild-type H460 cells (Fig.\u0026nbsp;5C), suggesting that DR5 is critical for mediating phenazine-1,6-diol\u0026ndash;induced cell death. Western blot analysis further demonstrated that phenazine-1,6-diol strongly induced the expression of cleaved PARP and cleaved caspase-3 in wild-type H460 cells, whereas these apoptotic markers were notably reduced in DR5-knockout cells (Fig.\u0026nbsp;5D). Moreover, phenazine-1,6-diol\u0026ndash;treated H460 cells displayed classical apoptotic nuclear morphology, including chromatin condensation and nuclear fragmentation, as observed by flow cytometry analysis via Annexin V/PI staining (Fig. F and G) and DAPI staining via confocal microscopy (Fig.\u0026nbsp;5H). In contrast, untreated wild-type H460 cells, DR5-knockout cells, and DR5-knockout cells treated with phenazine-1,6-diol presented no significant nuclear changes, further suggesting that DR5 is required for phenazine-1,6-diol\u0026ndash;induced apoptosis. Collectively, these results demonstrated that DR5 is an indispensable upstream regulator of phenazine-1,6-diol\u0026ndash;mediated apoptotic signaling in NSCLC cells. Loss of DR5 significantly diminished both the expression of apoptotic markers and the morphological features of apoptosis, confirming its central role in the antitumor mechanism of this compound.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.7 Phenazine-1,6-diol exhibited significant \u003cem\u003ein vivo\u003c/em\u003e antitumor efficacy in a mouse xenograft model\u003c/h2\u003e\u003cp\u003eTo evaluate the \u003cem\u003ein vivo\u003c/em\u003e antitumor activity of phenazine-1,6-diol, a Lewis lung carcinoma (LLC) xenograft model was established in C57BL/6J mice. The animals were randomly divided into three groups: the control group and the phenazine-1,6-diol (20 and 30 mg/kg) group, with intraperitoneal administration every other day for 17 days (Fig.\u0026nbsp;6A). Compared with that in the control group, tumor growth in both treatment groups was significantly suppressed. As shown in Fig.\u0026nbsp;6B, tumor volume increased steadily in the control group, whereas both phenazine-1,6-diol\u0026ndash;treated groups exhibited markedly reduced tumor progression, with the 30 mg/kg group showing the most pronounced effect. Importantly, body weight remained stable across all the groups throughout the treatment period (Fig.\u0026nbsp;6C), indicating that phenazine-1,6-diol did not induce significant systemic toxicity. Survival analysis revealed a substantial increase in the median survival time of the mice treated with phenazine-1,6-diol, particularly those in the 30 mg/kg group (Fig.\u0026nbsp;6H). Postmortem tumor samples from the treatment groups were clearly smaller than those from the control groups were (Fig.\u0026nbsp;6D and E), and statistical analysis confirmed significant differences in tumor size and weight between the 20 mg/kg group and the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), supporting the efficacy of the compounds \u003cem\u003ein vivo\u003c/em\u003e. To evaluate potential immune or hematologic toxicity, the spleens were excised and analyzed morphologically and by weight (Fig.\u0026nbsp;6F and G). Together, these results demonstrated that phenazine-1,6-diol effectively suppressed tumor growth and prolonged survival \u003cem\u003ein vivo\u003c/em\u003e, with no apparent adverse effects on body weight or immune organs, supporting its potential as a safe and efficacious candidate for lung cancer therapy.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Histopathological analysis confirmed the antiproliferative and proapoptotic effects of phenazine-1,6-diol \u003cem\u003ein vivo\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo further validate the \u003cem\u003ein vivo\u003c/em\u003e antitumor mechanism of phenazine-1,6-diol, histopathological examinations were performed on tumor tissues harvested from LLC xenograft mice after 17 days of treatment. The tumor sections were subjected to hematoxylin and eosin (H\u0026amp;E) staining, Ki-67 immunohistochemistry, and TUNEL assays to evaluate tissue morphology, proliferative activity, and apoptosis, respectively. Panoramic scans of H\u0026amp;E-stained tumor sections revealed that tumors from phenazine\u0026ndash;1,6\u0026ndash;diol\u0026ndash;treated mice displayed extensive regions of necrosis and reduced cellular density compared with those in the control group, indicating tissue damage (Fig.\u0026nbsp;6I, top row). Ki-67 staining revealed a marked reduction in the proliferative index in the phenazine-1,6-diol treatment groups, suggesting that the compound effectively inhibited tumor cell proliferation \u003cem\u003ein vivo\u003c/em\u003e. Conversely, TUNEL staining revealed a substantial increase in the number of apoptotic nuclei (Fig.\u0026nbsp;6I, middle and bottom rows). Quantitative analysis of Ki-67 and TUNEL staining (Fig.\u0026nbsp;6J) confirmed statistically significant differences between the treatment and control groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with a significant decrease in Ki\u0026ndash;67\u0026ndash;positive cells and a corresponding increase in TUNEL-positive cells following phenazine-1,6-diol administration. These histological results provide robust evidence that phenazine-1,6-diol exerts its \u003cem\u003ein vivo\u003c/em\u003e antitumor effects by simultaneously inhibiting tumor cell proliferation and promoting apoptosis in tumor tissues.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eIn this study, we systematically evaluated the antitumor properties of phenazine-1,6-diol, a marine-derived phenazine compound, and demonstrated for the first time its potent antiproliferative and proapoptotic effects in NSCLC models both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Our findings revealed that phenazine-1,6-diol selectively inhibited NSCLC cell viability while sparing normal lung epithelial and fibroblasts, induced apoptosis via the DR5-caspase axis, and modulated the PI3K/AKT/mTOR signaling cascade(Lim and Ma, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Our data revealed that H460 cells are particularly sensitive to phenazine-1,6-diol, suggesting that subtype-specific vulnerability is potentially linked to the EGFR wild-type status or specific DR5 expression patterns(Liu et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The selective cytotoxicity observed in BEAS-2B and CCD-19Lu cells highlights the therapeutic window of phenazine-1,6-diol, reducing concerns of nonspecific toxicity(W. Wang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Importantly, phenazine-1,6-diol significantly suppressed colony formation and induced G2/M cell cycle arrest, which was accompanied by downregulation of Cyclin B1 and upregulation of P21, which is consistent with classical mechanisms of proliferation inhibition. Interestingly, while phenazine-1,6-diol activated PI3K and AKT phosphorylation, it paradoxically suppressed downstream effectors such as mTOR, p70S6K, and S6. This uncoupling may reflect a feedback inhibition loop or crosstalk with apoptotic signaling cascades, as observed with other proapoptotic agents that disrupt metabolic signaling in cancer cells. Such signaling disruption is likely to create a pro-apoptotic intracellular environment, enhancing the efficacy of phenazine-1,6-diol. Mechanistically, our Western blot and flow cytometry analyses demonstrated that phenazine-1,6-diol activates both the extrinsic and intrinsic apoptotic pathways, as evidenced by increased expression of cleaved PARP, cleaved caspase-3, and caspase-8; Bax upregulation; and Bcl-2 downregulation. These effects were significantly attenuated by the pancaspase inhibitor Z-VAD-FMK, confirming the caspase dependence of phenazine-1,6-diol\u0026ndash;induced apoptosis. A critical finding of this study is the identification of DR5 as a key upstream mediator of phenazine-1,6-diol activity. Both the qRT‒PCR and Western blot results revealed that DR5 was upregulated upon treatment. More importantly, DR5-knockout H460 cells presented markedly reduced apoptotic responses and cytotoxicity, highlighting DR5 as an essential effector of phenazine-1,6-diol\u0026ndash;mediated cell death. Confocal imaging of DAPI-stained nuclei further confirmed that DR5 deficiency abolishes apoptotic morphological features. \u003cem\u003eIn vivo\u003c/em\u003e, phenazine-1,6-diol demonstrated significant antitumor efficacy in an LLC xenograft model without affecting body weight or spleen size, indicating favorable safety and tolerability. Tumor volume, weight, and survival improved, and histopathological analyses confirmed the suppression of proliferation (Ki-67) and induction of apoptosis (TUNEL), corroborating our \u003cem\u003ein vitro\u003c/em\u003e results. Previous studies on marine-derived phenazine derivatives have reported antimicrobial and anti-inflammatory activities, but few studies have explored their antitumor mechanisms in depth(Lang et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nguyen, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This study bridges that gap and positions phenazine-1,6-diol as a promising lead compound with dual regulatory potential over extrinsic apoptotic signaling and PI3K/AKT/mTOR-mediated survival pathways. Despite these promising findings, several limitations remain. First, the detailed molecular mechanisms of DR5 induction and PI3K/AKT uncoupling require further investigation. Second, the pharmacokinetics and bioavailability of phenazine-1,6-diol \u003cem\u003ein vivo\u003c/em\u003e need to be systematically evaluated for clinical translation. Finally, future studies should explore the synergy of phenazine-1,6-diol with standard chemotherapeutics or targeted agents in NSCLC treatment(MacManus and Hegi-Johnson, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study provides comprehensive evidence that phenazine-1,6-diol has potent antitumor activity against NSCLC. Phenazine-1,6-diol selectively inhibited NSCLC cell proliferation, induced G2/M phase cell cycle arrest, and triggered apoptosis through a caspase-dependent pathway. Mechanistically, it activated DR5 and altered PI3K/AKT/mTOR signaling, leading to apoptotic execution via both intrinsic and extrinsic pathways. Importantly, DR5 was identified as an essential mediator of phenazine\u0026ndash;1,6\u0026ndash;diol\u0026ndash;induced apoptosis, as its knockout significantly impaired cytotoxic and apoptotic responses. \u003cem\u003eIn vivo\u003c/em\u003e experiments further confirmed the ability of the compound to suppress tumor growth and prolong survival without evident systemic toxicity. Histological analyses confirmed its dual antiproliferative and proapoptotic effects in tumor tissues. These findings suggest that phenazine-1,6-diol is a promising candidate for the development of novel therapeutic agents targeting NSCLC. Future studies are warranted to optimize its pharmacological profile and explore its therapeutic synergy with existing anticancer agents.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eAKT:\u003c/strong\u003e Protein kinase B\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eANOVA:\u003c/strong\u003e Analysis of variance\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eATCC:\u003c/strong\u003e American Type Culture Collection\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eBSA:\u003c/strong\u003e Bovine serum albumin\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003ecDNA:\u003c/strong\u003e Complementary DNA\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eDAD:\u003c/strong\u003e Diode array detector\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eDAPI:\u003c/strong\u003e 4',6-diamidino-2-phenylindole\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eDMEM:\u003c/strong\u003e Dulbecco's Modified Eagle Medium\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eDMSO:\u003c/strong\u003e Dimethyl sulfoxide\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eDR5:\u003c/strong\u003e Death Receptor 5\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eEGFR:\u003c/strong\u003e Epidermal growth factor receptor\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eESI:\u003c/strong\u003e Electrospray ionization\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eFBS:\u003c/strong\u003e Fetal bovine serum\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eFITC:\u003c/strong\u003e Fluorescein isothiocyanate\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eH\u0026amp;E:\u003c/strong\u003e Hematoxylin and eosin\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eIC₅₀:\u003c/strong\u003e Half-maximal inhibitory concentration\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eIHC:\u003c/strong\u003e Immunohistochemistry\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eLC‒MS:\u003c/strong\u003e Liquid Chromatography‒Mass Spectrometry\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eLLC:\u003c/strong\u003e Lewis Lung Carcinoma\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003emTOR:\u003c/strong\u003e Mammalian target of rapamycin\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eMTT:\u003c/strong\u003e 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eNSCLC:\u003c/strong\u003e Non-Small Cell Lung Cancer\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003ePARP:\u003c/strong\u003e Poly (ADP‒ribose) polymerase\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003ePBS:\u003c/strong\u003e Phosphate-buffered saline\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003ePI:\u003c/strong\u003e Propidium iodide\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003ePI3K:\u003c/strong\u003e Phosphoinositide 3-kinase\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eqRT‒PCR:\u003c/strong\u003e Quantitative real-time polymerase chain reaction\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eSD:\u003c/strong\u003e Standard deviation\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eTUNEL:\u003c/strong\u003e Terminal deoxynucleotidyl transferase dUTP nick end labeling\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Declarations","content":"\u003col\u003e\n \u003cli\u003eDeclaration of competing interests\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eAll the authors have no financial or commercial conflicts of interest to disclose.\u003c/p\u003e\n\u003col start=\"2\"\u003e\n \u003cli\u003eAcknowledgments\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eWe are grateful to the State Key Laboratory of Quality Research in Chinese Medicine for providing laboratory and relevant personnel management and technical support, as well as to the Macau University of Science and Technology for their support of this research, which was also funded by the Macau Science Development Foundation.\u003c/p\u003e\n\u003col start=\"3\"\u003e\n \u003cli\u003eAuthorship contribution statement\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003ePeng Yue directed and guided the project. Peng Yue, Xiaoping Zhao and Jianan Jin designed the experiments and drafted the manuscript. Peng Yue, Xiaoping Zhao, Langyu Yang, Tianrui Lee, Un Ian Leong，Ziyang Zhang\u0026nbsp;and Jiayi Sunperformed the experiments and analyzed the results. Chitin Hon and Jiyou Kou reviewed the manuscript and made significant revisions. The final manuscript has been approved by all authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBanik BK, Banerjee B. Heterocyclic Anticancer Agents. Walter de Gruyter GmbH \u0026amp; Co KG; 2022.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChen M, Wang X, Zha D, Cai F, Zhang W, He Y, Huang Q, Zhuang H, Hua Z-C. Apigenin potentiates TRAIL therapy of non-small cell lung cancer by upregulating DR4/DR5 expression in a p53-dependent manner. 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All the data were analyzed as the means \u0026plusmn; SDs (n=3, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001).\u003cbr\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 277px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eCells\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 277px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIC\u003csub\u003e50\u003c/sub\u003e for 48 h\u003c/strong\u003e\u003cstrong\u003e（\u003c/strong\u003e\u003cstrong\u003e\u0026mu;M\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 277px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eBEAS-2B\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 277px;\"\u003e\n \u003cp\u003e160.2\u0026nbsp;\u0026plusmn;2.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 277px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eH460\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 277px;\"\u003e\n \u003cp\u003e17.76\u0026nbsp;\u0026plusmn;1.16\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 277px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eH1975\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 277px;\"\u003e\n \u003cp\u003e20.33 \u0026plusmn;2.21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 277px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eH358\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 277px;\"\u003e\n \u003cp\u003e58.98\u0026nbsp;\u0026plusmn;1.43\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 277px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eH1650\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePC9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 277px;\"\u003e\n \u003cp\u003e83.79 \u0026plusmn;12.2\u003c/p\u003e\n \u003cp\u003e25.67 \u0026plusmn;3.42\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Non-small cell lung cancer, death receptor 5, marine natural products, PI3K/AKT/mTOR signaling pathway, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-8162519/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8162519/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eNon-small cell lung cancer (NSCLC) is a lethal malignancy with limited treatment options. Natural products, particularly from marine sources, represent a promising avenue for discovering novel anticancer agents with high selectivity.\u003c/p\u003e\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eThis study investigated the antitumor efficacy and mechanism of action of phenazine-1,6-diol, a marine-derived compound, against NSCLC, with a focus on DR5-mediated apoptosis.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThe cytotoxicity of phenazine-1,6-diol was assessed in NSCLC and normal lung cell lines. Apoptosis was evaluated via flow cytometry and Western blot analysis. The essential role of DR5 was confirmed in DR5-knockout H460 cells. The impact on PI3K/AKT/mTOR signaling was analyzed. \u003cem\u003eIn vivo\u003c/em\u003e antitumor activity and safety were evaluated in a Lewis lung carcinoma xenograft model.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003ePhenazine-1,6-diol selectively inhibited the proliferation of NSCLC cells while sparing normal lung epithelial cells. It induced G2/M cell cycle arrest, suppressed colony formation, and triggered caspase-dependent apoptosis, as evidenced by increased cleavage of PARP and caspase-3/8, upregulation of Bax, and downregulation of Bcl-2. Mechanistically, phenazine-1,6-diol upregulated DR5 expression. Genetic ablation of DR5 profoundly attenuated its cytotoxicity and apoptotic effects. The compound also inhibited the PI3K/AKT/mTOR pathway. In mouse xenografts, phenazine-1,6-diol (20 and 30 mg/kg) significantly suppressed tumor growth, prolonged survival, and enhanced tumor apoptosis without causing systemic toxicity.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003ePhenazine-1,6-diol is a potent and selective anti-NSCLC agent that acts through two mechanisms: the induction of DR5-mediated apoptosis and the suppression of the PI3K/AKT/mTOR survival pathway. It presents a promising candidate for further development as a targeted therapy for NSCLC.\u003c/p\u003e","manuscriptTitle":"Phenazine-1,6-diol selectively inhibited non-small cell lung cancer via DR5-mediated apoptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-21 03:55:38","doi":"10.21203/rs.3.rs-8162519/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6155a988-66e1-488e-97ac-7fea85d0f581","owner":[],"postedDate":"November 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-25T04:23:32+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-21 03:55:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8162519","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8162519","identity":"rs-8162519","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}