Resveratrol triggers autophagy‐related apoptosis to inhibit the progression of colorectal cancer via inhibition of FOXQ1

Colorectal cancer (CRC) is a significant health problem with elevated mortality rates, prompting intense exploration of its complex molecular mechanisms and innovative therapeutic avenues. Resveratrol (RSV), recognised for its anticancer effects through SIRT1 activation, is a promising candidate for CRC treatment. This study focuses on elucidating RSV's role in CRC progression, particularly its effect on autophagy‐related apoptosis. Using bioinformatics, protein imprinting and immunohistochemistry, we established a direct correlation between FOXQ1 and adverse CRC prognosis. Comprehensive in vitro experiments confirmed RSV's ability to promote autophagy‐related apoptosis in CRC cells. Plasmids for SIRT1 modulation were used to investigate underlying mechanisms. Molecular docking, glutathione‐S‐transferase pull‐down experiments and immunoprecipitation highlighted RSV's direct activation of SIRT1, resulting in the inhibition of FOXQ1 expression. Downstream interventions identified ATG16L as a crucial autophagic target. In vivo and in vitro studies validated RSV's potential for CRC therapy through the SIRT1/FOXQ1/ATG16L pathway. This study establishes RSV's capacity to enhance autophagy‐related cell apoptosis in CRC, positioning RSV as a prospective therapeutic agent for CRC within the SIRT1/FOXQ1/ATG16L pathway.

Cancer poses a significant global public health challenge, ranking as the second leading cause of mortality.As of early 2023, colorectal cancer (CRC) has emerged as the third most prevalent malignancy in male and female populations (Siegel et al., 2023).CRC incidence exhibits substantial geographic variations, with high rates reported in developed countries (Patel et al., 2022).CRC risk factors include age, family history, high-processed meat, low-fibre diet, obesity, smoking and sedentary lifestyle.Timely detection and treatment are pivotal for enhancing CRC patient prognosis (Dekker et al., 2019).CRC, affecting the colon or rectum, which are part of the large intestine, usually originates as small polyps that may progress into cancer over time (Patel et al., 2022).As comprehensive CRC management research expands, a predominant therapeutic approach involves combining chemotherapy and targeted therapy, particularly for postoperative patients and palliative treatment (Weng et al., 2022).Despite advancements in combination therapy, managing advanced CRC remains challenging, because of its unfavourable prognosis and median overall survival of 25-30 months (Muro et al., 2019;Yoshino et al., 2018).Hence, the search for innovative synergistic therapeutic approaches has become a key focus in CRC research.In particular, monomeric compounds from traditional Chinese medicine, extracted from natural botanical sources, are widely recognised for their potential in adjunctive therapy against malignant tumours (Bilgin et al., 2023;Esmeeta et al., 2022;Li et al., 2008).Monomeric compounds demonstrate anticancer effects in CRC by inducing ferroptosis (Brockmueller, Girisa, Motallebi, et al., 2023;Zhang et al., 2022), apoptosis (Castrillon-Lopez et al., 2022), suppression of stemness (Liao et al., 2023;Miyazaki et al., 2023) and modulation of antioxidant activities (Roshani et al., 2022;Shakibaei et al., 2015).However, the intricate molecular mechanisms that underscore their efficacy demands further in-depth exploration.
Resveratrol (RSV), a natural polyphenolic compound, is found in plants such as grapes, berries, peanuts and various Chinese medicines (Ren et al., 2021).With minimal toxicity and few side effects, RSV is an attractive candidate for cancer treatment (Shaito et al., 2020).Some of the most well-documented mechanisms are as follows: ①Inhibition of cancer cell growth: RSV has been shown to inhibit the growth of cancer cells by inducing cell cycle arrest and apoptosis (programmed cell death).It regulates the expression of various genes and proteins involved in these processes (Ren et al., 2021;Roshani et al., 2022).②Anti-inflammatory and antioxidant effects: RSV exhibits anti-inflammatory and antioxidant effects, mitigating DNA damage and inhibiting the formation of cancerous cells (Meng et al., 2021).③Inhibition of angiogenesis (Jiang et al., 2023): RSV inhibits angiogenesis, starving tumours of essential nutrients by preventing the formation of new blood vessels.④Activation of the immune system (Malaguarnera, 2019;Nadile et al., 2022): RSV activates the immune system, enhancing the body's ability to recognise and eliminate cancer cells.⑤ Chemo-sensitising impact (Brockmueller, Girisa, Kunnumakkara, & Shakibaei, 2023;Khaleel et al., 2016): In CRC adjuvant chemotherapy, RSV enhances drug efficacy, thereby delaying resistance, ensuring prolonged effectiveness and minimising side effects.As a result of its potential health benefits, RSV is gaining attention as a dietary supplement for cancer prevention and treatment (Breuss et al., 2019).Moreover, it activates silent information regulator 1 (SIRT1), a key regulator of autophagy linked to cancer progression (Pignet et al., 2021;Vernousfaderani et al., 2021).The diverse mechanisms underlying RSV's actions in cancer prevention and treatment position it as a promising therapeutic agent.However, further research is essential to comprehensively elucidate its clinical applications, optimal dosage and delivery methods.
FOXQ1 is an extensively studied factor in cancer research; it belongs to the forkhead family of transcription factors (Yang et al., 2022).Its abnormal expression is intricately linked to the onset and progression of diverse cancers.Overexpression of FOXQ1 is associated with malignancy (Wei et al., 2019), driving transformations, cell proliferation and metastasis in tumours (Mitchell et al., 2022).
FOXQ1 exerts regulatory control over crucial cellular processes, including cell cycle modulation; increased proliferation; and involvement in apoptosis, migration and invasion (Wu, Zheng, et al., 2023).
Within the tumour microenvironment, FOXQ1 potentially affects cancer development by influencing angiogenesis and immune evasion (Wei et al., 2019).Notably, FOXQ1 is implicated in the regulation of tumour stem cell characteristics and drug resistance (Wu, Zheng, et al., 2023), offering promising avenues for therapeutic exploration.A comprehensive understanding of FOXQ1's molecular mechanisms and interaction networks is crucial for unravelling the intricacies of cancer development and identifying novel therapeutic strategies.
This study aimed to elucidate the potential therapeutic effects of RSV in CRC, specifically focusing on its role in activating the SIRT1/ FOXQ1/ATG16L signalling pathway.By evaluating RSV's influence on essential cellular processes, including proliferation, apoptosis and autophagy within CRC cells, we sought to address existing research limitations.The investigation also aimed to comprehensively explore the inhibitory effects of RSV on CRC progression and understand the intricate signalling cascades involved.The findings are expected to enhance our understanding of RSV's therapeutic prospects in CRC treatment, potentially driving innovative advancements in cancer therapeutics.

| Bioinformatics analysis
Bioinformatics analysis on FOXQ1 in tumours was achieved through the Oncomine database (https://www.oncomine.com).Differential expression of the FOXQ1 gene across various tumour types was statistically analysed by examining its correlation with clinical parameters.
Pathway and functional enrichment analyses were carried out to uncover the biological functions and pathway regulations involving FOXQ1 in tumours, laying a solid bioinformatics foundation for further research.In addition, a comprehensive investigation of differential gene expression patterns in patients with CRC was conducted using the GEO dataset (https://www.ncbi.nlm.nih.gov/geo).We aimed to explore the potential overexpression of FOXQ1, specifically in CRC intestinal tissue.Furthermore, an extensive analysis of differential gene expression in tumour tissues from the TCGA database (https:// portal.gdc.cancer.gov/) was performed.This work involved non-paired pan-cancer analysis and paired differential analyses to reveal distinct gene expression patterns associated with tumorigenesis.

| Reagents and cells
In this study, CRC cells (SW480, LOVO, SW480-FOXQ1sh, SW480-FOXQ1 over, LOVO-FOXQ1 and LOVO-FOXQ1 over) were sourced from the Shanghai Institute of Life Sciences, Chinese Academy of Sciences.The cells underwent rigorous quality assessments.
Cell seeding involved the meticulous execution of isolating cells, transferring them into culture dishes with a growth medium and ensuring appropriate cell density through dissociation and counting.
Culture conditions, including temperature, humidity, gas composition and coating of culture vessels, were meticulously controlled at 37 C, 5% carbon dioxide and high humidity in a CO 2 incubator.Cell passaging, critical for sustaining cell proliferation and survival, was performed based on specific density or growth states, involving detachment and reseeding to maintain cell health for subsequent experiments.

| Human intestinal tissue collection and patient information sorting
All participants in this study were newly diagnosed with CRC through  Other drugs were administered through intraperitoneal injection.

| Subcutaneous tumour formation in nude mice
SW480-derived cancer cells were used for tumour transplantation.These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillinstreptomycin in a humidified incubator at 37 C with 5% CO 2 .Prior to implantation, SW480 cell viability was assessed using the trypan blue exclusion method.The cells were suspended in sterile phosphatebuffered saline (PBS) at a concentration of 1 Â 10 7 cells/mL.Using a sterile syringe with a 27-gauge needle, 100 μL of the cell suspension was subcutaneously injected into each mouse's flank region.The injection site was monitored daily for tumour formation.Tumour growth was measured with callipers, and tumour volume was calculated using the following formula: tumour volume (mm 3 ) = (length Â width 2 )/2.Once tumours reached a certain size (e.g., 100-200 mm 3 ), mice were randomly divided into different treatment groups.Drug administration or control treatment followed the experimental design.
Throughout the experiment, mice were monitored daily for signs of distress, changes in body weight and overall health status.

| Azoxymethane/dextran sodium sulphateinduced colorectal cancer model
In the initial phase of model construction, mice received a single injection of azoxymethane (AOM) to induce intestinal mutations.AOM, dissolved in saline, was injected into the peritoneal cavity of mice by using a sterile needle and syringe.Subsequently, in the second phase of model construction, mice were exposed to a 4% dextran sodium sulphate (DSS) solution to induce colonic inflammation and injury.The DSS solution was administered to the mice through drinking water for a specified duration.The cycles of AOM and DSS treatments were repeated, with treatment duration and dosage determined by the experimental design.This cyclic AOM/DSS treatment mimicked the developmental process of CRC, encompassing inflammation, ulceration and tumour formation.Throughout the experiment, mice were monitored daily for changes in body weight, stool characteristics and overall health status.The severity of colonic inflammation and the timing of tumour formation were observed and documented.

| Haematoxylin and eosin (HE) staining
Tissue sections underwent HE staining following standard procedures outlined in the science journal.Initially, samples were fixed in formalin, embedded in paraffin and thinly sliced.The sections were deparaffinised, rehydrated and immersed in sequential staining solutions.Haematoxylin, an alkaline dye, imparted a blue hue to cell nuclei, whereas eosin, an acidic dye, provided a red colouration to the cytoplasm and extracellular matrix.Following staining, sections underwent dehydration, rinsing and mounting for microscopic examination.Specialised software facilitated image acquisition and subsequent analysis.By comparing stained sections with control normal tissue samples and others, a comprehensive evaluation of tissue structure, cellular morphology and pathological alterations was conducted.

| Immunohistochemistry (IHC) experiment
The IHC experiment adhered to a standardised protocol.Tissue samples were initially fixed in formalin, embedded in paraffin and thinly sectioned.Sections underwent deparaffinisation and rehydration, followed by antigen retrieval using appropriate methods.To inhibit endogenous peroxidase activity and minimise nonspecific binding, we treated sections with hydrogen peroxide and serum blocking solution, respectively.The primary antibody specific to the target antigen was then applied and incubated overnight at the recommended temperature.After washing, sections were incubated with a secondary antibody conjugated with horseradish peroxidase (HRP) or fluorescent dyes.In cases of HRP-conjugated antibodies, a chromogenic substrate such as diaminobenzidine (DAB) was used, generating a brown precipitate at the target antigen site.Counterstaining with haematoxylin was conducted to visualise cellular morphology.
Following dehydration, sections were mounted and observed under a microscope.Image acquisition and analysis employed specialised software.Staining specificity was validated by omitting the primary antibody or substituting it with an isotype-matched negative control antibody.

| Western blot (WB)
The WB technique is a robust method for detecting and characterising proteins in biological specimens.Beginning with sample preparation, cell or tissue lysis liberates target proteins.SDS-PAGE then segregates proteins by size for membrane transfer.Blocking agents mitigate nonspecific binding, ensuring selective identification of the target protein.
After binding, the primary antibody undergoes washing, followed by a

| Quantitative real-time polymerase chain reaction (qRT-PCR)
The experimental workflow involves several key steps.Initially, highquality and pure RNA is extracted from the desired sample, such as cells or tissues, using appropriate methods.Subsequently, reverse transcription converts the RNA into complementary DNA (cDNA) using reverse transcriptase enzyme and specific or random primers.
The resulting cDNA serves as the template for qRT-PCR, amplifying the target gene with primers and fluorescent probes.Fluorescence signal increases during qPCR with accumulating PCR products.Realtime fluorescence detection instruments monitor these changes, determining the target gene's expression level based on the threshold cycle (Ct) value.Data analysis involves calculating relative expression levels, normalising to reference genes and conducting statistical analysis.qRT-PCR results offer insights into expression differences of target genes in various samples.Additional techniques like WB or functional assays can be employed to validate findings.Detailed primer sequences for all genes in the study are provided in Table 1.

| Clone formation assay
CRC cells were cultured to achieve optimal density in a culture medium.The experimental group underwent RSV treatment at a specific concentration, whereas the control group experienced identical conditions without RSV.Subsequently, cells were reseeded into culture dishes with agar and a nutrient-rich medium.Following incubation under controlled conditions, colonies were formed.After a designated period, colonies were fixed, stained and quantified.A comprehensive assessment of RSV's impact on CRC cell clone formation was made by comparing the quantity and distinctive features of colonies between the experimental and control groups.

| Methyl thiazolyl tetrazolium (MTT) assay
In CRC cell culture plates post-RSV intervention, MTT was added to the medium, forming purple formazan crystals through reduction by viable cells.These crystals created a purple intermediate in live cells.
After dissolving the samples, they were transferred to a microplate, and absorbance was measured using an immunoassay or spectrophotometer.The MTT formazan in viable cells absorbs light at a specific wavelength, with absorbance directly proportional to viable cell numbers.Comparing absorbance values between the experimental and control groups allows an evaluation of the tested substances' effects on cell proliferation and viability.High absorbance indicates good cell growth, whereas low values may suggest cell inhibition or apoptosis.

| Apoptosis detection
Flow cytometry for cell apoptosis detection involves key steps.Cells are collected, washed and resuspended in a buffer solution.Subsequently, annexin V, which binds to exposed phosphatidylserine on apoptotic cells, and propidium iodide (PI), which distinguishes apoptosis stages, are used for staining.After incubation, a flow cytometer analyses the stained cells, detecting emitted fluorescence.Specialised software processes the data to quantify the percentage of apoptotic cells based on annexin V and PI staining.This technique offers a reliable, quantitative method for studying cell apoptosis, providing insights into cellular responses and the effects of treatments or stimuli.

| Cell plasmid transfection experiment
The cell transfection experiment involves key steps.Initially, target cells are cultured and prepared in a suitable medium.Plasmid DNA, containing the gene of interest, is mixed with a transfection reagent to form complexes absorbed by the cells.After adding the complexes to the cells and incubating for a specific period, internalisation and expression of the transfected gene occur.Post-incubation, cells are washed to remove any remaining untransfected DNA or reagent.Subsequently, cells are cultured under suitable conditions for expression and propagation of the transfected gene.Finally, the gene's expression can be assessed using techniques such as fluorescence microscopy, WB or qPCR.This experiment enables researchers to introduce and express specific genes in target cells, facilitating the study of gene function, protein expression and cellular responses to genetic manipulations.

| Cell lentivirus transfection experiment
The lentiviral vector transduction experiment involves crucial steps.
Initially, a lentiviral vector carrying the gene of interest is generated by co-transfecting host cells with packaging plasmids, producing lentiviral particles.These particles, containing the gene of interest, are harvested from the transfected cell supernatant.Target cells are seeded and incubated with lentiviral particles in the presence of a transduction enhancer, allowing internalisation.Lentiviral particles deliver the gene into the host cells' genome.Post-transduction, cells are selected or sorted based on a selectable marker in the lentiviral vector to retain only those carrying the gene of interest.Transduced cells are cultured to allow gene expression for subsequent analysis.This method facilitates stable introduction and long-term expression of genes in target cells, proving valuable for functional studies and gene therapy research.

| Molecular docking
Molecular docking, a computational chemistry method, predicts interactions and binding modes between molecules.The process involves obtaining and optimising the 3D structures of the ligand and receptor, T A B L E 1 The primer sequences of genes in real-time quantitative polymerase chain reaction experiment.
followed by a spatial search for potential binding sites and conformations.Evaluation employs scoring functions to rank binding modes based on interaction forces and complementarity.Additional analysis calculates binding free energy and predicts affinity.Experimental validation ensures the accuracy and biological significance of predicted binding modes.

| Glutathione-S-transferase pull-down experiment
The GST pull-down experiment, a method for studying protein- This experiment provides direct insights into protein interactions, aiding the study of protein function, signalling pathways and disease mechanisms.

| Immunoprecipitation (IP)
IP, also known as co-immunoprecipitation (co-IP), involves the selective purification of a target protein and its associated interacting partners using specific antibodies.The process typically consists of several steps.Firstly, the target protein and its interacting partners are extracted from cells or tissues using lysis buffers.Secondly, the protein lysate is incubated with antibodies that specifically recognise the target protein.These antibodies can be either monoclonal or polyclonal, depending on the experimental requirements.During incubation, the antibodies bind to the target protein, forming an antibody-protein complex.Protein A or protein G beads are added to the mixture to capture the antibody-protein complex.These beads have a high affinity for the Fc region of antibodies and can effectively isolate the antibody-protein complex from the rest of the lysate.After incubation and gentle washing to remove nonspecifically bound proteins, the antibody-protein complex is eluted from the beads and subjected to further analysis.
Subsequent analysis can include techniques such as SDS-PAGE, immunoblotting or mass spectrometry to identify and characterise the interacting partners.IP allows for the study of protein-protein interactions, protein modifications and protein complex composition, providing valuable insights into cellular processes and signalling pathways. 2.17

| Statistical analysis
The data were subjected to rigorous statistical analysis using Graphpad Prism software version 8.2.Descriptive statistics are reported as means ± standard deviation (SD).Group comparisons for variables following a normal distribution were assessed using one-way ANOVA, followed by post hoc analysis using the least significant difference (LSD) method for pairwise mean separations.A significance level of p < 0.05 was employed to determine statistical significance.
T A B L E 3 Relationship between Hepatocyte Nuclear Factor 3 Forkhead Homolog 1 (FOXQ1) expression and clinicopathological features of colonic adenocarcinoma (COAD) patients in database.

| Resveratrol inhibits CRC cell proliferation
Following the elucidation of FOXQ1's role in patients with CRC, we aimed to uncover novel therapeutic targets and agents for CRC treatment by exploring upstream regulators capable of inhibiting FOXQ1.
Previous investigations have unveiled the remarkable anticancer properties of RSV, derived from natural sources, against CRC (Figure 3a).
Nevertheless, the precise molecular mechanisms underlying its efficacy necessitate in-depth exploration.Consequently, this study preliminarily determined the potential of RSV in attenuating CRC progression through FOXQ1 inhibition.On the basis of preliminary investigations, by utilising a concentration of 100 μΜ, RSV was administered to SW480 and LOVO cell lines for 48 h (Figure 3b).concentrations (<35 μM), significantly impeding tumour cell viability.
However, as the concentration surpassed the threshold of 35 μM, a remarkable reversal in the inhibitory effect of RSV on cell viability was observed upon autophagy inhibition, eventually reaching a state of saturation beyond 80 μM (Figure 3e).These findings confirmed the pivotal role of autophagy in promoting apoptotic demise of tumour cells under high-concentration conditions.Moreover, to provide a comprehensive morphological perspective, we substantiated the inhibitory effect of RSV on CRC cell proliferation through robust clonogenic assays (Figure 3f).Collectively, the study highlights RSV's remarkable potential in restraining CRC cell proliferation by orchestrating intricate autophagy regulatory networks.

| RSV induces autophagy-associated apoptosis in CRC cells
As previously mentioned, the inhibition of CRC progression by RSV is (Figure 5c).Furthermore, corroborating the results at the genetic level, qPCR analysis provided supplementary evidence (Figure 5d).These preliminary observations offer a glimpse into the putative role of FOXQ1 as a downstream effector of SIRT1 activation triggered by RSV, necessitating further investigations to unravel its direct targeting potential.

| FOXQ1 as a direct downstream target of SIRT1 in CRC
Through tandem affinity purification/mass spectrometry, the downstream proteins that interacted with SIRT1 were rapidly identified.
Table 5 reveals that p85 was among the potential candidate proteins that interacted with LZTS2, with a peptide bond count of 8.This finding implied a potential interaction between SIRT1 and FOXQ1.Subsequently, molecular docking simulations of SIRT1 and FOXQ1 proteins were conducted using state-of-the-art molecular docking techniques.
The protein-protein interactions were rigorously evaluated and scored, revealing the presence of multiple energetically favourable chemical bonding interactions between SIRT1 and FOXQ1.These WB analysis revealed that GST-FOXQ1 was able to bind to SFB-SIRT1, whereas GST-only did not bind to SFB-FOXQ1, indicating a direct interaction between FOXQ1 and SIRT1 (Figure 7d).The aforementioned investigation provides empirical support for the direct interaction between SIRT1 and FOXQ1, firmly establishing FOXQ1 as a downstream effector of SIRT1.

| ATG16L is a critical autophagy target involved in RSV-mediated intervention of FOXQ1
The aforementioned findings proved the direct involvement of SIRT1 in modulating FOXQ1.However, the precise mechanisms by which FOXQ1 regulates autophagy remain elusive.Researchers hypothesised that FOXQ1 may exert its influence on autophagy through downstream effector molecules.To explore this hypothesis, we assessed the mRNA expression levels of autophagy-related molecules in SW480 cells overexpressing FOXQ1 via stable transfection with a lentivirus.Through this approach, a preliminary screening identified ATG16L as a significantly differentially expressed candidate (p < 0.001) upon FOXQ1 overexpression (Figure 8a), strongly suggesting its potential role as a downstream target of FOXQ1 in the regulation of autophagy.Subsequently, we employed luciferase reporter gene experiments to provide further evidence that FOXQ1, functioning as a transcription factor, governed the regulatory promoter region of the 3 0 -UTR of the pivotal autophagy molecule, ATG16L (Figure 8b).

| DISCUSSION
The process of autophagy is known to contribute to cancer progression, enabling cancer cells to endure challenging conditions such as nutrient deprivation (Zhao et al., 2022).SIRT1, a member of the sirtuin family, participates in cellular processes related to autophagy-induced apoptosis (Luo et al., 2019).FOXQ1, a transcription factor, regulates various cellular functions, including cell proliferation (Hong et al., 2020), differentiation (Xiang et al., 2020) and apoptosis (Zhuang et al., 2020).Another key player is ATG16L, which is a protein involved in the autophagy process, and it is responsible for the degradation of damaged cellular components (Laine-Menéndez et al., 2022).The present study builds upon prior evidence demonstrating the significant activation of SIRT1 by RSV and other interventions (Huang et al., 2021;Shu et al., 2020;Wang et al., 2020Wang et al., , 2022)).
Furthermore, emerging research suggested that RSV exerts its inhibitory effects on CRC through the activation of pathways associated with autophagy and apoptosis (Wu, Xiong, et al., 2023).However, a dearth of targeted investigations into the intricate mechanisms underlying these effects remains, with most studies primarily focusing on the assessment of established canonical autophagy and apoptosis pathways (Behroozaghdam et al., 2022;El-Readi et al., 2019;Ferraresi et al., 2021).
RSV safety has been extensively examined in preclinical and clinical settings (Amintas et al., 2023).Animal studies show minimal toxicity, and human clinical trials suggest general tolerance with few reported adverse events (Amintas et al., 2023;Liao et al., 2023).At appropriate doses, RSV is considered safe, yet caution is advised against excessive or prolonged use.Despite positive safety evidence, consulting healthcare professionals, especially for those with underlying conditions or taking other medications, is recommended before RSV supplementation (Liao et al., 2023;Wu, Xiong, et al., 2023).
Ongoing research is vital for a comprehensive understanding of RSV safety, particularly in long-term use or specific patient populations.
In this study, on the basis of previously validated phenotypic observations, we employed a comprehensive approach encompassing bioinformatics analysis, molecular docking, CO-IP, GST-pull down and pathology and had undergone no prior treatment except surgery, following the signing of informed consent.Participants were exclusively selected from the Department of Gastrointestinal Surgery at Union Hospital, affiliated with Tongji Medical College, Huazhong University of Science and Technology.The research adhered to The Code of Ethics of the World Medical Association (Declaration of Helsinki) for human experiments.Approval for all studies was obtained from the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (Ethics approval number: UHCT220831).
Experimental animals, obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd., comprised female BALB/c nude mice and C57BL/6 mice aged 6-8 weeks.They were housed in a specific pathogen-free (SPF) facility at the Experimental Animal Center of Tongji Medical College, Huazhong University of Science and Technology, with controlled temperature, humidity and a 12-h light/dark cycle.Animal experiments strictly adhered to the animal ethics guidelines of the Institutional Animal Care and Use Committee (IACUC) of the US National Institutes of Health (NIH), in accordance with ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines and EU Directive 2010/63/EU for animal experiments (ethics approval number: HKTJCT108634).RSV (Aladdin, CAS No. 501-36-0) was initially dissolved in dimethylsulfoxide (DMSO) and then diluted in physiological saline to create an oral gavage suspension (50 and 80 μmol/L).
secondary antibody introduction, conjugated with an enzyme or fluorescent dye for signal amplification.Signal detection employs chemiluminescence or fluorescence-based methods.Chemiluminescent signals produce luminescence captured by x-ray film or imaging systems, whereas fluorescent signals emit light at specific wavelengths, enabling visualisation through fluorescence imaging technologies.The resulting protein bands or signals are analysed to elucidate the presence, abundance and interactions related to the target protein.Quantitative analysis involves comparing band intensities with molecular weight markers or using loading controls.WB provides valuable insights into various biological processes and diseases by revealing information about protein expression, modifications and intermolecular associations.
protein interactions, utilises GST fused proteins as bait to selectively purify and analyse target proteins.The procedure involves cloning the target protein's coding sequence into a GST fusion expression vector, expressing it in host cells and obtaining the cell lysate containing the GST fusion protein.The GST fusion protein is incubated with glutathione-agarose resin, binding selectively via the GST-glutathione interaction.This resin acts as an affinity resin, purifying the GST fusion protein.The target protein sample is then incubated with the pre-bound GST fusion protein resin, facilitating a specific interaction.After washing to remove nonspecifically bound proteins, analysis techniques such as SDS-PAGE and immunoblotting reveal the GST fusion protein and bound target protein.
| Dual-luciferase reporter assay (DLRA)DLRA is a powerful experimental technique used to investigate gene expression and regulatory mechanisms.It involves the use of two different luciferase reporter genes, firefly luciferase (FLuc) and Renilla luciferase (Rluc), to monitor the activity of a promoter or the effect of specific regulatory elements.The Fluc reporter gene is typically under the control of the promoter region of interest, whereas the Rluc reporter gene serves as an internal control to normalise for transfection efficiency.The experimental workflow generally consists of the following steps.Firstly, the promoter region of interest is cloned upstream of the Fluc reporter gene in a plasmid vector.The Rluc reporter gene is also present in the same plasmid vector, driven by a constitutive promoter.Secondly, the plasmid vector is transfected into the target cells via a suitable transfection method.After a specific incubation period, the cells are lysed, and the luciferase activities are measured using specific luciferase assay reagents.Fluc and Rluc emit different wavelengths of light when their respective substrates are added, allowing for the simultaneous quantification of both luciferase activities using a luminometer or a plate reader.Fluc activity represents the promoter activity or regulatory effect, whereas Rluc activity serves as an internal control for normalisation purposes.By calculating the ratio of Fluc to Rluc activity, the specific effects of the promoter or regulatory elements can be determined.DLRA provides a sensitive and reliable method to study gene expression and regulatory mechanisms, enabling researchers to gain insights into transcriptional regulation, promoter activity and the effects of various factors on gene expression.

FOXQ1
as a molecular marker associated with tumorigenesis in diverse cancer types.Our investigation was centred on assessing the expression pattern of FOXQ1 in CRC, with particular emphasis on the prevalent adenocarcinoma subtype.We conducted a comprehensive analysis utilising datasets from the GEO database (https://www.ncbi.nlm.nih.gov/geo),where we compared CRC tissues with their corresponding adjacent normal tissues.Our examination revealed a remarkable upregulation of FOXQ1 expression within the tumour tissues, as evidenced by the findings in Figure1b.These observations closely corroborated the consistently elevated FOXQ1 expression patterns identified in CRC, as documented in the comprehensive Oncomine database.The results were validated through comprehensive pan-cancer analysis (Figure1c) and tumour-specific paired analysis (Figure1d) in the TCGA database (https://portal.gdc.cancer.gov/).These supplementary analyses not only serve as additional confirmation for our findings but also suggest the robustness and universality of the correlation between FOXQ1 expression and cancer across diverse malignancies.They further indicate the significance of FOXQ1 as a key molecular determinant in the context of CRC.The investigators gathered tumour and normal tissues from 10 patients diagnosed with CRC to conduct molecular biology validation.Immunohistochemical analysis and subsequent statistical analysis demonstrated a pronounced elevation of FOXQ1 expression in tumour tissues, whereas its expression was comparatively low in normal colorectal tissues (Figure 1e,g).Furthermore, by contrasting the protein levels of FOXQ1 between normal intestinal tissues and CRC tissues, F I G U R E 1 Elevated expression of Hepatocyte Nuclear Factor 3 Forkhead Homolog 1 (FOXQ1) in colorectal cancer (CRC).(a) FOXQ1 is overexpressed in several types of cancers (Oncomine database; cancer vs. normal: overexpression [red colour] and downexpression [blue colour]).(b) FOXQ1 is overexpressed in CRC (GEO database, cancer vs. normal: tumour tissue [pink colour] and normal tissue [blue colour]; overexpression [red colour] and downexpression [green colour]).(c) Pan-cancer analysis showed that FOXQ1 is highly expressed in CRC.(d) FOXQ1 expression was analysed in tumour and normal tissues (TCGA database, overexpression [red colour] and downexpression [blue colour]).(e, g).Immunohistochemical analysis and statistical analysis of FOXQ1 expression in human tissue.(f, h).Protein expression and statistical analysis of FOXQ1 in human tumour and normal tissues.the researchers observed concordant outcomes (Figure 1f,h).These results establish the heightened expression of FOXQ1 as a plausible molecular signature in CRC.Upon establishing the specific expression pattern of FOXQ1 in CRC, we analysed its potential as a prognostic biomarker for patients with CRC.We utilised the Kaplan-Meier Plotter database by employing an automated selection of optimal cut-off values to stratify patients.Subsequently, the prognostic significance of FOXQ1 in patients with CRC (n = 477) was comprehensively assessed.Remarkably, heightened FOXQ1 expression was found to be significantly associated with inferior outcomes across multiple parameters, including overall survival (OS, HR = 0.7654 [0.54-1.36],p = 0.0384), progression-free interval (PFS; HR = 0.7654 [0.57-1.47],p = 0.0103) and disease-specific survival (DSS; HR = 0.7654 [0.387-1.49],p = 0.0428; Figure 2a-c).To evaluate the influence of FOXQ1 expression on the survival probability of patients within specific tumour subtypes, we conducted survival analyses based on the corresponding TNM staging.The findings demonstrated that heightened FOXQ1 expression exhibited the most prominent influence on patient prognosis in the context of T3 stage tumours (HR = 1.61, 95% CI: 1.01-2.57,p = 0.047), N1 stage tumours (HR = 4.80, 95% CI: 1.77-13.07,p = 0.002) and M1 stage tumours (HR = 0.59, 95% CI: 0.28-T A B L E 2 The expression of Hepatocyte Nuclear Factor 3 Forkhead Homolog 1 (FOXQ1) in colonic adenocarcinoma (COAD) in Ocomine database.

F
I G U R E 2 High expression of Hepatocyte Nuclear Factor 3 Forkhead Homolog 1 (FOXQ1) predicts poor prognosis of colorectal cancer (CRC).(a-c) Graphs generated from the Kaplan-Meier Plotter database show the prognostic values of FOXQ1 in patients with CRC.Overall survival (OS), progression-free interval (PFS) and disease-specific survival (DSS).(d-f) Influence of FOXQ1 expression on the survival probability of patients within specific tumour subtypes (TNM).
Subsequent morphological observations and initial statistical analysis revealed a significant inhibition of RSV on the proliferation of CRC cells.To determine the optimal concentration and duration of RSV for inhibiting CRC cells, we conducted MTT assays to assess cell viability.The results revealed a dose-and time-dependent reduction in cell viability upon RSV treatment.High concentrations and long durations of RSV treatment led to a significant decrease in cell viability in CRC cells.Compared with the control group, low concentrations (<25 μM) or short durations (<12 h) of RSV treatment promoted cell viability in SW480 and LOVO cells.However, when RSV concentration exceeded 150 μM or treatment duration surpassed 48 h, the inhibitory trend on cell viability weakened, potentially due to the activation of cell death mechanisms associated with high RSV concentrations.Thus, the researchers identified the most pronounced inhibition of CRC cells by RSV at 50 and 80 μM with a treatment duration of approximately 48 h (Figure 3c,d).Previous research findings revealed that RSV exhibits potent induction of cellular autophagy.The underlying regulatory mechanisms of autophagy were determined as follows.Building upon the foundation of 120 μM RSV treatment, the researchers employed the autophagy inhibitor 3-MA to selectively modulate autophagic processes in two distinct cell lines, assessing cell viability through MTT assays.Intriguingly, the results unveiled a synergistic effect between autophagy inhibition and RSV treatment at low F I G U R E 3 Resveratrol (RSV) inhibits colorectal cancer cell proliferation.(a) 3D structural diagram of RSV (grey: carbon atoms, red: hydroxy oxygen atoms; PubChem-https://pubchem.ncbi.nlm.nih.gov/).(b) Cellular distribution map after 48 h of culture was observed under an optical microscope.(c) Cell viability was assessed by MTT assay, with the colours representing different time points: brown for 12 h, red for 24 h, green for 48 h and blue-purple for 72 h.Various concentrations of RSV (25, 50, 100, 150, and 200 μM) were used to intervene and evaluate cell viability.(d) Effects of different concentrations of RSV on the cell viability of LOVO and SW480 cells were compared at 12, 24, 48, and 72 h.(e) Effect of different concentrations of RSV on the cell viability of LOVO and SW480 cells was compared after the application of an autophagy inhibitor.(f) Tumour cell colony formation assay.The data are presented as mean ± SD from three independent experiments.*p < 0.05; **p < 0.01; ***p < 0.001.
closely associated with the activation of autophagy.Furthermore, cellular apoptosis, a prevalent form of cell death, plays a pivotal role in the anti-tumour activity of natural extracts.Consequently, the researchers selected two concentrations of RSV, namely, 50 and 80 μM, to assess apoptosis through flow cytometry.The findings demonstrated a significant induction of apoptosis in CRC cells by RSV, with the high concentration of 80 μM exhibiting a high apoptotic rate.Remarkably, the autophagy inhibitor 3-MA effectively reversed the apoptotic process induced by RSV (Figure4a).The aforementioned results demonstrated a consistent pattern across both cell lineages.Subsequently, the two cell lines underwent molecular biology-level examinations by the researchers.Primarily, protein-level analyses were conducted.Compared with the DMSO control group, the RSV intervention groups (50 and 80 μM) exhibited significant upregulation of the pro-apoptotic protein Bax expression (p < 0.001), concurrent with significant downregulation of the anti-apoptotic protein Bcl2 expression (p < 0.001).Furthermore, they demonstrated substantial upregulation of the expression of the autophagy-related proteins Beclin1 and LC3-II (p < 0.001).By contrast, the 3-MA intervention group exhibited pronounced inhibition of apoptosis, as demonstrated in the accompanying bar graph.Collectively, these findings substantiated the role of RSV in enhancing the protein expression of apoptosis-related molecules associated with autophagy (Figure4b).Continuing the investigation, we conducted gene-level analyses using qPCR to assess the expression profiles of the aforementioned pro-apoptotic gene Bax, anti-apoptotic gene Bcl2, as well as proautophagy genes Beclin1 and LC3-II.The results revealed the potent induction of autophagy-related apoptotic genes by RSV (Figure4c), thereby furnishing additional evidence to support our findings.3.4 | RSV modulates FOXQ1 expression through SIRT1 activationRSV, acknowledged as a potent activator of SIRT1, has garnered substantial recognition for its role in suppressing the progression of CRC based on previous investigations.Considering the observed high expression of FOXQ1 in CRC tissues, we postulated that RSV can modulate FOXQ1 expression by activating SIRT1, thereby impeding the advancement of CRC.To validate this conjecture, we implemented a graded RSV treatment approach with three distinct concentrations (20, 50, and 80 μM) in SW480.The findings demonstrated a discernible dose-dependent upregulation of SIRT1 protein expression induced by escalating RSV concentrations, concomitant with a gradual attenuation of FOXQ1 protein expression (Figure5a).Through genelevel analysis using qPCR, the findings regarding SIRT1 and FOXQ1 demonstrated the ability of RSV to enhance the expression of SIRT1 mRNA while suppressing FOXQ1 expression.The precise mechanism underlying the observed decline in FOXQ1 inhibition rate following treatment with 50 μM RSV requires in-depth investigation (Figure5b).Autophagy plays a pivotal role in the therapeutic efficacy of RSV in CRC, thereby rendering the assessment of autophagyrelated biomarkers as a reliable reflection of RSV's intervention.To elucidate the molecular mechanisms underlying RSV's intervention in CRC, we employed an intricate lentiviral transfection system to establish SW480 cell lines overexpressing SIRT1 and FOXQ1 knockout, which were subsequently subjected to RSV intervention.The downstream molecular cascades modulated by RSV intervention were probed via WB analysis of autophagy-inducing molecules (Beclin1 and LC3-II) and autophagic substrates (p62).The findings unveiled a synergistic induction of autophagy in CRC by RSV and SIRT1 overexpression, which was further amplified upon FOXQ1 suppression Previous studies have established the role of RSV in promoting autophagy-associated apoptosis in CRC, which is closely associated with the activation of SIRT1 and its downstream target FOXQ1.Consequently, the researchers postulated that RSV's activation of SIRT1/ FOXQ1 cascade facilitates the occurrence of autophagy-associated apoptosis in CRC.To investigate this hypothesis, we engineered SIRT1 overexpression plasmids, SIRT1 mutant plasmids and FOXQ1 overexpression plasmids (Figure 6a).Transfection of the SIRT1 overexpression plasmids and SIRT1 mutant plasmids, followed by RSV intervention, revealed that RSV activated SIRT1 and suppressed FOXQ1 expression.Notably, the inhibitory effect of RSV on FOXQ1 was completely abolished upon SIRT1 knockout.These results proved the role of SIRT1 in mediating the influence of RSV on FOXQ1 (Figure 6b).Subsequently, the investigators assessed protein markers associated with autophagy-mediated apoptosis.Treatment with RSV F I G U R E 4 Resveratrol (RSV) induces autophagy-associated apoptosis in colorectal cancer cells.(a) Detection of RSV-induced apoptosis using flow cytometry.(b) Western blot analysis of RSV-induced autophagy-related apoptosis.(c) Quantitative polymerase chain reaction assessment of RSV-induced autophagy-related apoptosis.RSV: RSV, 3-MA: autophagy inhibitor.The data are presented as mean ± SD from three independent experiments.*p < 0.05; **p < 0.01; ***p < 0.001.and transfection with the SIRT1 overexpression plasmid effectively stimulated the expression of SIRT1.Simultaneously, they facilitated the upregulation of autophagy and apoptosis-associated proteins (including Bax, Beclin1, LC3-II and cleaved caspase3) but suppressed the expression of Bcl2, p62 and caspase-3.Remarkably, the complete abrogation of the aforementioned autophagy-related apoptotic effects was observed upon SIRT1 knockout (Figure 6c).Similarly, comparable outcomes were observed at the genetic level through qPCR analysis, as depicted in Figure 6d.These results highlight the indispensable role of SIRT1 modulation in mediating the regulatory effects of RSV on autophagy-related apoptosis in CRC.SIRT1 and FOXQ1 display a discernible regulatory interplay in RSV-mediated F I G U R E 5 Resveratrol (RSV) modulates FOXQ1 expression through SIRT1 activation.(a) Expression profiles of SIRT1/FOXQ1 following intervention with RSV at concentrations of 20, 50, and 80 μM were examined by Western blot analysis.The corresponding histograms depicting grayscale values are presented on the right.(b) A scatter plot was generated to illustrate the expression levels of SIRT1/FOXQ1 after RSV intervention, as quantified by qPCR.(c) Western blot analysis was employed to evaluate the expression patterns of SIRT1/FOXQ1, as well as the autophagy-related proteins Beclin1, LC3-II and p62, and elucidate the downstream molecular mechanisms of RSV intervention, including SIRT1 overexpression and FOXQ1 knockout.The grayscale value histograms are displayed on the right.(d) A scatter plot was utilised to depict the expression profiles of SIRT1/FOXQ1, along with the autophagy-related genes Beclin1, LC3-II and p62, following RSV intervention, as determined by qPCR analysis.The data are presented as mean ± SD from three independent experiments.*p < 0.05; **p < 0.01; ***p < 0.001.F I G U R E 6 Legend on next page.ZHOU ET AL.intervention of CRC.However, the precise mechanistic linkage by which SIRT1 governs the autophagy-related apoptotic processes through FOXQ1 modulation necessitates further elucidation.We conducted FOXQ1 overexpression experiments to investigate the occurrence of autophagy-related apoptosis following SIRT1 overexpression in CRC.Remarkably, the data (Figure6e) revealed the capacity of FOXQ1 overexpression to counteract the incidence of SIRT1-induced autophagy-related apoptosis, thereby fostering the progression of CRC.These findings underscore the ability of RSV to curtail FOXQ1 expression via SIRT1 activation, thereby fostering autophagy-related apoptosis in CRC.Nonetheless, the precise existence of a direct regulatory nexus between SIRT1 and FOXQ1 warrants further analysis.
findings strongly support the theoretical possibility of their interaction, suggesting a robust potential for functional interplay between SIRT1 and FOXQ1 (Figure7a).To further substantiate this hypothesis, we constructed SFB-SIRT1 and Myc-FOXQ1 plasmids and separately transfected them into 293 T cells, generating the SFB-SIRT1 + Vector, Myc-FOXQ1 + Vector and SFB-SIRT1 + Myc-FOXQ1 groups.Utilising forward and reverse CO-IP assays, we confirmed an interaction between SFB-SIRT1 and Myc-FOXQ1 (Figure7b,c).To further validate the direct interaction between SIRT1 and FOXQ1, we performed GST pull-down experiments.The bacteria-expressed proteins GST-only and GST-FOXQ1 were purified, whereas SFB-SIRT1 was transfected into 293 T cells, and protein extracts were prepared.
other cutting-edge methodologies to elucidate the oncogenic role of the transcription factor FOXQ1. Intriguingly, our findings revealed a pronounced inhibitory influence of SIRT1 on FOXQ1, thereby reinforcing the pivotal involvement of the SIRT1/FOXQ1/ATG16L pathway in the regulatory cascade orchestrated by RSV.This study confirmed that the SIRT1/FOXQ1/ATG16L pathway is a molecular pathway that has been implicated in CRC autophagy related apoptosis.RSV exerts its anti-tumour effects on CRC by activating the SIRT1/FOXQ1/ ATG16L signalling pathway, thereby facilitating autophagy-associated apoptosis and suppressing tumour growth.This study identified a discrepancy in the findings, where RSV, at concentrations below 25 μM and exposure durations less than F I G U R E 9 Enhancing colorectal cancer autophagy-associated cell apoptosis through in vivo investigation of resveratrol via the NADdependent deacetylase sirtuin-1 (SIRT1)/Hepatocyte Nuclear Factor 3 Forkhead Homolog 1 (FOXQ1)/autophagy-related 16 like 1 (ATG16L) pathway.(a) Subcutaneous tumour mouse weight changes.(b) Subcutaneous tumour mouse survival analysis.(c) Subcutaneous tumour gross and statistical results.(d) Western blot analysis of SIRT1/FOXQ1 expression in subcutaneous tumour tissues.(e) Quantitative polymerase chain reaction analysis of SIRT1/FOXQ1 expression in subcutaneous tumour tissues.(f) Azoxymethane/dextran sodium sulphate-induced in situ tumour mouse weight changes.(g) AOM/DSS-induced in situ tumour mouse survival analysis.(h) AOM/DSS-induced in situ tumour gross and statistical results.(i) Western blot analysis of SIRT1/FOXQ1/ATG16L pathway and autophagy-related apoptotic molecules in AOM/DSS-induced in situ tumour mouse intestinal tissues.(j) Mechanism diagram.The mitochondria-related content of the diagram has been widely confirmed.The data are presented as mean ± SD from three independent experiments.*p < 0.05; **p < 0.01; ***p < 0.001.