Mechanism of resveratrol affecting biological functions of breast cancer through glycolysis pathway

Mechanism of resveratrol affecting biological functions of breast cancer through glycolysis pathway | 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 Mechanism of resveratrol affecting biological functions of breast cancer through glycolysis pathway Yu Gao, Yaoyao Wang, Baodi Wang, Qunying Hu, Jirui Jiang, Bo Feng, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5210318/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 Phosphoglycerate kinase 1 (PGK1) plays a crucial role in the glycolytic pathway and its overexpression has a negative impact on tumor development and prognosis. Resveratrol, a natural polyphenolic compound, has gained significant attention in recent years due to its anti-inflammatory, antioxidant, and anti-tumor properties. However, the mechanism by which resveratrol inhibits breast cancer growth, invasion, and metastasis through the PGK1 glycolytic pathway is still not fully understood. Therefore, this study aimed to investigate the inhibitory effects of resveratrol on breast cancer cell proliferation and invasive migration, as well as its ability to promote apoptosis in vitro. Additionally, the study examined the inhibitory effects of resveratrol on the growth of mouse breast cancer graft tumors in vivo. Methods We analyzed the expression levels of glycolytic enzymes in different breast tissues and their correlation with the prognosis of breast cancer patients through GEPIA and The Human Protein Atlas database. Wound healing assay, Transwell migration and invasion assay were used to study the effect of resveratrol on the biological functions of breast cancer. RT-qPCR and Western blot methods were used to explore the potential molecular mechanism of resveratrol inhibiting the development of breast cancer through the PGK1 glycolysis pathway. In vivo experiments explore the biological mechanisms behind PGK1-mediated breast cancer proliferation and invasion. Results High expression of PGK1 is significantly associated with poor prognosis in breast cancer patients, and resveratrol induces breast cancer cell apoptosis in a dose-dependent manner, inhibiting PK, PGK1, LDH enzyme activity and ATP content. Resveratrol inhibits PGK1 expression and its related c-Myc/PI3K/AKT signaling pathway. Western blot experiments show that resveratrol affects the biological functions of breast cancer by inhibiting PCNA, MMP2 and MMP9 proteins and activating BAX/Bcl-2 and Caspase3/7 proteins. In addition, in vivo experiments show that resveratrol significantly inhibits the growth of transplanted tumors in breast cancer mice and inhibits breast cancer tumor proliferation, invasion and metastasis by downregulating PGK1 expression. Conclusion Resveratrol demonstrates inhibitory effects on breast cancer cell proliferation, invasive and migration, as well as induction of apoptosis in vitro. It also inhibits the growth of mouse breast cancer transplantation tumors in vivo and exhibits oncostatic effects both in vitro and in vivo. Moreover, resveratrol reduces the expression of PGK1 in breast cancer BT-549 cells by regulating the transcription factors c-Myc. This regulation leads to the blockage of cellular glycolysis pathways, ultimately inhibiting the malignant biological behavior of breast cancer cells. resveratrol breast cancer glycolysis invasion apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Breast cancer is a prevalent malignant disease among women, surpassing lung cancer in 2020 with a high incidence of 2.26 million cases and becoming the most common cancer worldwide. Surprisingly, the incidence rate of breast cancer among Chinese women of childbearing age accounts for 25% and represents 10% of all malignant tumors, This rate shows an increasing trend every year [ 1 ]. The etiology and pathogenesis of breast cancer have not yet been fully understood, and current treatment options primarily include chemotherapy, radiotherapy, and surgery. However, once tumor metastasis occurs, the chances of survival are significantly reduced. Despite advancements in treatment that have reduced patient mortality, breast cancer remains the leading cause of death among women. Resveratrol is a natural polyphenolic compound that can be found in various sources including blueberries, mulberries, Polygonum multiflorum, and cassia seeds [ 2 ]. It possesses numerous pharmacological activities such as anti-inflammatory, antioxidant, anti-tumor, metabolism regulation, and immune system modulation. Resveratrol has been shown to inhibit the growth of various types of tumors including colon cancer, pancreatic cancer, ovarian cancer, and breast cancer [ 3 ]. The anti-tumor effect of resveratrol has gained significant attention in research. However, the complete understanding of its action mechanism is still lacking and requires further investigation. Studies have shown that resveratrol can inhibit the formation and metastasis of abnormal crypt lesions by influencing cancer cell growth, apoptosis, and angiogenesis [ 4 ]. It exerts its anti-tumor activity by directly targeting relevant factors. These findings serve as a valuable reference for the development of new anti-tumor drugs and demonstrate significant inhibitory effects on various types of tumors [ 5 ]. Additionally, resveratrol block the cell cycle by activating the oncogenes p53 and suppressor gene PTEN, while also down-regulating PI3K. Ultimately, this leads to the induction of apoptosis in cancer cells [ 6 ]. Therefore, the potential of resveratrol as a cancer preventive agent in humans has been widely recognized. PGK1 is an essential enzyme in glycolysis, serving as both a metabolic enzyme and a protein kinase. It plays a crucial role in tumor growth, invasion, and metastasis by phosphorylating key substrates. As a metabolic enzyme, the primary function of PGK1 is to participate in the glycolysis reaction. This involves transferring the high-energy phosphate group from its substrate to ADP, generating ATP. This process is vital for sustaining cellular energy production, particularly in hypoxic conditions. PGK1 is a crucial molecular target in tumor therapy and has gained significant attention in recent years [ 7 ]. Recent studies have demonstrated that PGK1 plays a role in promoting breast cancer cell growth and lactate production, making it a potential target gene for breast cancer treatment [ 8 ]. Moreover, downregulation of PGK1 has been shown to significantly inhibit the invasive ability of breast cancer cells, reverse the process of epithelial-mesenchymal transition, and enhance the Warburg effect [ 9 ]. PGK1 is highly expressed in metastatic and invasive ductal breast cancer cells. It is significantly correlated with advanced tumor stage, suggesting its close association with breast cancer onset, progression, and metastasis [ 10 ]. Moreover, PGK1 overexpression is linked to poorer prognosis in breast cancer patients. Detecting PGK1 expression before chemotherapy can help predict the sensitivity of chemotherapeutic drugs, benefiting patients who are less responsive to drugs by avoiding unnecessary drug toxicity. In conclusion, reducing PGK1 expression offers a promising strategy to inhibit tumor growth in the future. Therefore, PGK1 can serve as an important biomarker for targeted tumor therapy. Materials and methods Reagents Resveratrol (≥ 99%, HPLC) was obtained from the National Institutes for Food and Drug Contro (Beijing, China). RPMI 1640 medium was purchased from Biosharp (Anhui, China). HK, PK and LDH activity assay reagents were purchased from Solarbio Company (Beijing, China). PGK1 ELISA reagents were purchased from Jianglai Biological (Shanghai, China). Muse Caspase-3/7 Kit was purchased from Luminex Company (Shanghai, China). Enhanced ATP detection reagent was purchased from Beyotime Biotechnology (Shanghai, China). PGK1, c-Myc, BAX and MMP2/9 antibodies were purchased from Cell Signaling Technology (Boston, USA). Antibodies to Caspase-3 and Caspase-7 were purchased from Wanlei Biotechnology (Shenyang, China). Correlation between PGK1 expression in breast cancer and patient prognosis analyzed by GEPIA and The Human Protein Atlas GEPIA ( http://gepia.cancer-pku.cn ), the Gene Expression Profiling Interactive Analysis, is able to predict PGK1 expression in breast cancer samples, in order to explore the expression level of PGK1 in breast cancer and its relationship with patients' prognostic survival. The Human Protein Atlas ( http://www.proteinatlas.org ) by analyzing the correlation between the expression level of human gene mRNA in tumor tissue and the prognostic data of cancer patients, the immunohistochemical (IHC) map of cancer tissue and the relationship between the expression of specific genes and the occurrence and development of specific tumors were obtained. Molecular docking predicts the binding energy of resveratrol and PGK1 Molecular docking of resveratrol with PGK1 was performed by AutoDock software to detect the docking binding energy. The chemical structure of the ligand was downloaded from the ZINC database ( http://zinc.docking.org/ ), and the Auto Dock Tools tool was used to set up a Grid Box centered on the ligand, and good docking active sites were obtained in the Autogrid module. Cell culture Human breast cancer cell BT-549 is from the Shanghai Cell Bank of the Chinese Academy of Medical Sciences. It is regularly cultured in RPMI-1640 medium containing 10% fetal bovine serum, in which 57.5 µL/100 mL insulin was added, and cultured at 37℃ with 5% CO 2 for 1 ~ 3 days for passage treatment. Cell proliferation assay The MTT assay was employed to investigate the impact of varying concentrations of resveratrol on the proliferation of BT-549 breast cancer cells. Briefly, resveratrol final concentrations of 2, 4, 8, 16, 32, 64 and 128 µg/mL were used in sterile 96-well plates inoculated with breast cancer BT-549 cells (6 × 10 3 cells/well) for 24 h. For the experiment, we utilized 180 µL of serum-free cell culture medium and added 20 µL of a 5 mg/mL MTT solution (Beyotime, China) to continue incubation for 4 h. Afterwards, 150 µL of DMSO (Sigma-Aldrich, USA) was added to each well, and the crystals were dissolved by shaking on a shaker for 10 min. The absorbance value was measured at 570 nm using a enzyme-labelling measuring, and the cell half inhibition rate (IC 50 ) was calculated. Determination of activity of glycolytic enzymes and ATP content Detects HK, PK, LDH and PGK1 enzyme activities The activities of HK and PK enzymes were measured using a UV spectrophotometer assay, while the LDH enzyme activity was measured using the enzyme-labelling measuring instrument. Firstly, breast cancer BT-549 1×10 4 cells were taken for the experiment, and the cells were lysed by adding appropriate amount of cell extract, centrifuged at 4℃, 10000 rpm for 15 min, and the supernatant was taken for the assay. Secondly, for HK and PK enzyme activity assay, mix the cell supernatant and the assay reagent according to the instructions, detect the absorbance value at 340 nm by UV spectrophotometer, and calculate the enzyme activity. For LDH enzyme activity, 100 µL of standard solution was taken for concentration dilution, a standard curve was made, the test solution was prepared according to the steps in the instruction manual, and the absorbance value was detected at 570 nm by an enzyme meter to calculate the LDH enzyme activity. After that, PGK1 enzyme activity was detected using ELISA kit. Briefly, 3×10 4 cells were centrifuged at 1000 rpm for 10 min and the supernatant was taken for experimental detection. Dilute the standard solution by 50 µL, add 50 µL each of different concentrations of the samples to be tested and detection dilutions, add 100 µL of horseradish peroxidase (HRP)-labeled detection antibody to each well, and incubate for 60 min at 37℃. After that, add 350 µL of washing solution to wash the plate 5 times, add 50 µL of washing reagent, protect from light, and incubate for 15 min at 37℃. Finally, add the stop buffer 50 µL, the OD values of each experimental group were detected at 450 nm with enzyme-labelling measuring. ATP content assay When the BT-549 cell fusion reached 80%~90% in a sterile 12-well plate, 150 µL ATP lysis buffer was added to each well, and centrifuged at 4ºC 12000rpm for 5 min, and the supernatant was taken for experiments. Dilute the ATP standard solution in multiplicity, prepare the standard curve, and set a blank control. Dilute the test reagent according to the ratio of 1:4, add 100 µL to each well, and let it stand at room temperature for 5 min, after that, add 20 µL each of the sample and the standard, and select the luminometer program of the enzyme marker to determine the RLU value of the sample. Cell migration and invasion test The inhibitory effect of resveratrol on the migration and invasion ability of breast cancer BT-549 cells was detected by wound healing, transmembrane migration and invasion assays. In the wound healing experiment, BT-549 cells (1×10 5 /well) were cultured in a 6-well plate for 24 h and allowed to reach 70% and 80% cell fusion, then the tip of a sterile 200 µL gun was used to scratch the cells perpendicular to the plane of the 6-well plate, and then the cells were washed with PBS for 3 times and the cell migration was monitored under an inverted microscope for more than 24 h. Finally, the ImageJ software was used for analysis, and the scratch width was measured to calculate the healing rate. Scratch healing rate (%) = [(0 h-24 h / 48 h scratch area) / 0 h scratch area] × 100%. For transwell invasion, a transwell chamber (Corning, USA) with an 8 µm hole polycarbonate filter was used. Briefly, dilute 50 mg/L of Materiel (BD, Biosciences, USA) with sterile PBS in a 1:8 ratio, and 50 µL was added to the upper chamber, which was allowed to solidify at 37℃ for 2 h. After that, 100 µL of medium containing 1% FBS was added to the upper chamber, and the incubation was performed at 37℃ for about 30 min. Then, the cells were incubated with resveratrol (20, 40, and 60 µg/mL) for 24 h and digested with RPMI-1640 serum-free medium. A cell suspension of 100 µL (medium containing 1% FBS) containing 2.5×10 4 /mL invasive cells was inoculated into the upper chamber coated with 50 µL of matrigel, and 600 µL of serum-containing medium was added to the lower chamber for 24 h of incubation, and non-migratory cells at the top of the upper chamber were removed with a cotton swab. The cells were fixed with 4% paraformaldehyde (Beyotime, China) and stained with 0.1% crystal violet for 1h, and then imaged by microscope. For migration experiments, BT-549 cells treated with different concentrations of resveratrol were inoculated into top chambers without matrigel coating, and other steps were the same as for invasion experiments. Apoptosis assay Apoptosis was detected using the Muse Caspase-3/7 kit. BT-549 cells (1×10 5 ~ 5×10 6 cells/mL) were suspended in 1×BA assay buffer and 50 µL was added to each experimental tube. Positive and negative control staining was performed, followed by the addition of 5 µL of Muse Caspase-3/7 reagent working solution to each tube. The tubes were then incubated at 37℃ in a 5% CO 2 incubator for 30 min. Subsequently, 150 µL of Muse Caspase7-AAD working solution was added to each tube and mixed. The tubes were incubated at room temperature for 5 min, avoiding light. Apoptosis was detected using a Muse flow cytometer (Merck-Millipore, Germany). Western blotting analysis BT-549 cells were treated with resveratrol (20, 40, 60 µg/mL) for 24 h, the cells were collected and lysed, and then quantified by BCA protein assay kit (Beyotime, China). The protein was separated by SDS-PAGE, transferred to PVDF membrane and sealed in 5% skimmed milk powder solution for 1 h. After incubating the blotting membrane with the corresponding primary antibody, and then incubating with the second antibody bound to horseradish peroxidase, the target protein band was detected by enhanced chemiluminescence under the scanning ™MP imaging system (Bio-Rad, USA). Real-time qPCR Total RNA was extracted from BT-549 cells by applying TRIzol reagent (Invitgen, USA). cDNA was synthesized using Prime SCRIPT RT kit (Takara, Japan) in order to detect the gene expression level. qRT-PCR reaction was performed using SYBR Green (Takara, Japan). β-actin gene as an internal reference, qRT-PCR was performed to detect the expression of PGK1 and c-Myc genes, the relative expression was calculated by 2 −∆∆ct . PGK1 (F, 5'-GTGAAGATTACCTTGCCTGTT-3'; R, 5'-GCTTCCCATTCAAATACCC-3'), c-Myc(F, 5'-CCGCCTGCGATGATTTATAC-3'; R, 5'-CAGCCGAGCACTCTAGCTCT-3'),β-actin (F, 5'-GAGCGGGAAATCGTGCGTGACAT-3'; R, 5'-CAGGAAGGAAGGCTGGAAGAGTG-3'). Tumor growth and metastasis in mice with breast cancer 4T1 murine mammary carcinoma cells were purchased from the Institute of Basic Medical Science, Chinese Academy of Medical Sciences (Beijing, China). Mouse breast cancer 4T1 cells (2×10 6 cells/0.2 mL) were subcutaneously injected into the right axilla of female BALB/c mice to establish a 4T1 tumor mouse (age: 4 ~ 6 weeks; body weight: 18 ~ 22g, n = 6, SPF grade) model. The mice were randomly divided into 5 groups, and when the tumor volume was about 300–400 mm 3 , Each group was intraperitoneally injected with 0.2 mL of normal saline 0.2 mL/kg, CTX solution 2.5 mg/kg, resveratrol solution (20, 50, 100 mg/kg). After 21 days of continuous injection, the mice were killed by spinal dislocation method, histological examination was performed by hematoxylin-eosin staining, and the expression of PGK1, PCNA and MMP2/9 protein in tumor tissue was detected by immunohistochemistry. Statistical analysis The experimental data were completed in 3 sessions and statistical differences were expressed as mean ± standard deviation. Comparisons between groups were made using analysis of variance and t-tests, with differences considered statistically significant at P < 0.05 and significant at P < 0.01. Results PGK1 could be a better glycolytic target for breast cancer Abnormal expression of metabolic enzymes in glycolysis is one of the main causes of Warburg effect, which is one of the hallmarks of cancer. Thus, we firstly analyzed the expression levels of different glycolytic enzymes between different breast cancer tissues and normal tissues through GEPIA database (Fig. 1 A). The results indicated that Phosphofructokinase (PFKP) and Lactate dehydrogenase (LDHA) showed no significance between breast cancer and normal epithelial tissues, and Hexokinase 2 (HK2) only showed higher expression levels in HER2 breast cancers. However, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and PGK1 were highly expressed in Basal-like, HER2, and Luminal-B breast cancers and M-type Pyruvate kinase (PKM) was PKM could be more effective targets for most breast cancer. In addition, the immunohistochemistry results of GAPDH, PGK1 and PKM from Human Protein Atlas database indicated that the ratio of breast cancer tissues performing “Medium/High” staining was higher than that of normal tissues, which mainly performed “Not detected/Low” staining results (Fig. 1 B-D), further proved our prediction. To further elucidate the research significance of above targets, we analyzed their correlation with the prognosis of breast cancer patients through GEPIA and The Human Protein Atlas database (Fig. 1 E-G). The analysis results of these two databases showed that there is a significant correlation between the poor prognosis and higher PGK1 expression level, but not that of GAPDH. About PKM, only The Human Protein Atlas database suggested a negative correlation between PKM expression and patients’ prognosis. The results suggested that PGK1 and PKM may serve as a promising target for breast cancer, especially for PGK1. Resveratrol inhibits PGK1 and glycolysis in breast cancer As a classical antineoplastic drug, resveratrol has been reported to inhibit some of glycolysis enzymes, including PFKP, HK2 and PKM. However, to the best of our knowledge, effects of resveratrol on PGK1 remains unknown. Thus, we detected effects of resveratrol on PGK1, both from the expression and activity point of view. qRT-PCR and Western-blot results suggested that resveratrol can inhibit mRNA and protein expression in a dose dependent manner (Fig. 2 A-B). In addition, the molecular docking results indicated that resveratrol can also combine with multiple amino acids of PGK1 automatically with a binding energy less than − 5.0 kcal·mol − 1 , among the combined amino acids, 291 and 292 located in the same active pocket with amino acids from 273 to 276 (the substrate binding sites of PGK1), with a minimum distance of 8.3Å (Fig. 2 C). This indicated that in addition to PGK1 expression, resveratrol may also inhibit PGK1 activity through their direct combination. Consistent with above results, resveratrol inhibited the catalytic activity of PGK1 in a dose dependent manner (Fig. 2 D). As PGK1 is the key rate limited enzymes of glycolysis, we further detected the effects of PGK1 on glycolysis. According to Fig. 2 E and Fig. 2 F, resveratrol can obviously inhibit catalytic activities of PK and LDH, which located downstream of PGK1. What’s more, resveratrol can inhibit ATP contents in glycolysis (Fig. 2 G). All the above results proved the inhibitory effects of resveratrol on PGK1 and glycolysis. Resveratrol inhibits PGK1-related c-Myc/PI3K/AKT aixs In order to gain a thorough understanding of the potential mechanism by which resveratrol inhibits PGK1 expression, we assessed the effects of resveratrol on c-Myc, which is a well-known transcription factor that can regulate PGK1 expression [ 11 ]. Our results revealed that resveratrol reduced c-Myc mRNA and protein expression levels (Fig. 3 A-B). Moreover, Resveratrol showed synergistic inhibitory effects on the expression level of c-Myc and PGK1, indicating that Resveratrol inhibits PGK1 partially through the inhibition of c-Myc (Fig. 3 C-D). As a well-known oncogene, PGK1 can active PI3K/AKT signaling pathway [ 12 ], which leads to the phosphorylation and inactivation of GSK-3β and prevents GSK-3β from binding to β-catenin. Consequently, β-catenin accumulates and translocates into the nucleus, inducing transcription of target genes [ 13 ]. In addition, PGK1 can also contribute the stabilization of GSK-3β, and then promotes β-catenin expression and maintains the stemness of breast cancer cells [ 14 ]. Our results demonstrated that resveratrol significantly inhibited the phosphorylation of PI3K and AKT, and the expression of GSK-3β, and β-catenin (Fig. 3 E-F). These findings suggest that resveratrol could exert anti-breast cancer effects by regulating PGK1 related c-Myc/PI3K/AKT aixs. Resveratrol inhibits cell proliferation and induce apoptosis in breast cancer The glycolytic pathway is one of the main hallmarks of cancer, which provides rapid ATP supplementation for cell proliferation. In addition, PGK1 is also a kind of oncogene which involves the initiation and development of several cancers. Thus, based on the inhibition effects on PGK1, we discussed anti-tumor effects of resveratrol in BT-549 cells, which has not been reported before. MTT methods revealed that resveratrol can inhibit BT-549 cell viability in a dose and time dependent manner, with an IC 50 of 40.55 ± 3.39 µg/mL at 24h (Fig. 4 A). In addition, resveratrol also reduced the expression level of proliferation marker PCNA (Fig. 4 B), which confirmed the inhibitory effect of resveratrol on cell proliferation together with MTT results. Furthermore, our flow cytometry experiments demonstrated that resveratrol increased the proportion of apoptotic cells in a dose dependent manner (Fig. 4 C-D). Moreover, apoptosis related proteins such as the Bax/Bcl-2 ratio, cleaved Caspase-3 and cleaved Caspase-7 were also promoted by resveratrol (Fig. 4 E-F). All the results above revealed that resveratrol can inhibit cell proliferation and induce apoptosis in breast cancer. Resveratrol inhibits cell migration and invasion in breast cancer It has been reported that targeting PGK1-mediated Warburg effect could suppress breast tumor metastasis [ 15 ]. Though resveratrol has been reported to inhibit migration and metastasis of some breast cancer cells including MDA-MB-231 [ 16 ], related effects on BT-549 cells remains unknown. Thus, we detected the effects of resveratrol on BT-549 cell migration and invasion. The scratch-wound experiment demonstrated that after scratched for 24 hours, the wound healing rate of BT-549 cells was 59.19 ± 3.585%, indicating its highly metastatic potential. However, treatment with resveratrol significantly inhibited the wound healing rate of BT-549 cells (Fig. 5 A-B). In addition, the Trans-Well experiment without the matrix also confirmed the inhibitory effects of resveratrol on BT-549 cells (Fig. 5 C-D). Number of invasion cell decreased from 198 ± 6.07 (control group) to 98.33 ± 5.074 (20 µg/mL), 73 ± 6.245 (40 µg/mL), and 44.62 ± 4.163 (60 µg/mL) respectively (Fig. 5 E-F). What’s more, Western-blot experiments revealed that resveratrol dose-dependently inhibited the expression levels of migratory invasion markers MMP2 and MMP9 (Fig. 5 G-H). Collectively, these findings provided evidence for the anti-breast cancer effects of resveratrol. In vivo anti-tumor effects of resveratrol on breast cancer Finally, we investigated the in vivo anti-mammary effects of resveratrol. We conducted experiments using breast cancer cells to induce mouse tumors (model group), which were then treated with different doses of resveratrol (treatment group). We used the well-known anti-tumor drug CTX as a positive control. The results from HE staining revealed that the tumor tissue in the model group exhibited vigorous growth, with a large number of tightly packed cancer cells showing irregular morphology and darker nuclear staining color. However, after treatment with resveratrol at concentrations of 20, 50, and 100 mg/kg, the dense structure of the tumor was damaged, leading to a gradual restriction of tumor tissue growth. The tumor cells also exhibited varying degrees of shrinkage and rounding, and the arrangement of the tumor cells became progressively sparse. The CTX group showed the most significant inhibition of tumor growth in the breast cancer hormone mice, with a large area of necrosis and dissolution observed in the tissue (Fig. 6 A). In addition, IHC staining results demonstrated that resveratrol can inhibit the positive staining of proteins including PGK1, PCNA, and MMP9 (Fig. 6 B-C). Notably, the above effects of 100 mg/kg resveratrol was similar to that of the positive control drug CTX. Finally, we observed that resveratrol can significantly inhibit the tumor volume (Fig. 6 D-E) and tumor weight (Fig. 6 F) while did not affect the body weight (Fig. 6 G) of the tumor bearing mice. Discussion Breast cancer is a malignant tumor that originates in the epithelial tissue of the breast gland. Recent epidemiological investigations have shown a steady increase in the incidence of breast cancer. In fact, as of 2020, breast cancer has surpassed lung cancer to become the most common cancer globally, particularly among female patients [ 17 ]. The current dominant approach in Western medicine for treating breast cancer is cytotoxic drug chemotherapy, which includes anthracyclines and albumin-binding paclitaxel [ 18 , 19 ]. However, these conventional therapies have significant drawbacks, such as severe adverse effects and invasiveness, leading to a low 5-year survival rate and poor prognosis [ 20 , 21 ]. Consequently, it is imperative to develop highly effective and low-toxicity treatments for breast cancer. These treatments include induced cell cycle blockade, signaling pathway and mRNA-targeted therapies, immunotherapy, endocrine therapies, and the integration of botanical medicines as natural anticancer agents. These advancements offer a promising outlook for long-term disease control of breast cancer. In recent years, resveratrol has gained significant attention from scholars both domestically and internationally due to its effective anti-tumor activity. Resveratrol has been found to inhibit breast cancer both in vivo and in vitro, and its mechanism of action is closely related to various molecular targets including cell proliferation, epithelial-mesenchymal transition, chemosensitization, invasion and metastasis, apoptosis, and epigenetics [ 22 , 23 ]. Numerous preclinical experiments have demonstrated that resveratrol inhibits the expression of histone transferase EZH2 by regulating the dephosphorylation of protein kinase ERK1/2, resulting in the inhibition of growth and proliferation of breast cancer cells [ 24 ]. Resveratrol also blocks the G0/G1 phase of breast cancer MCF-7 cells and reduces the expression of invasive and metastatic markers MMP2 and MMP9, thereby inhibiting the invasion and metastasis of breast cancer cells [ 25 ]. Furthermore, resveratrol has been shown to inhibit the invasion and migration of human breast cancer MCF-7 cells through the PI3K/Akt and Wnt/β-catenin signaling pathways [ 26 , 27 ]. Resveratrol has been found to reduce the expression of POLD1, inhibiting PCNA and Bcl-2 expression, while increasing the expression of the apoptotic index caspase-3, these effects activate the apoptotic pathway, promoting apoptosis in breast cancer cells [ 28 ]. Resveratrol exhibits a range of pharmacological activities and shows promising potential in the field of medicine and health care. However, its clinical application is limited due to issues such as low bioavailability, poor water solubility, and poor stability. Researchers face the challenging task of modifying its structure to develop resveratrol derivatives with enhanced biological activity and higher bioavailability. PGK1, the first key enzyme in the glycolytic pathway, plays a crucial role in ATP production, breast cancer cell growth, and lactate production. It is considered a significant target gene for potential breast cancer treatment [ 29 , 30 ]. Notably, PGK1 acts not only as a metabolic enzyme but also as a protein kinase, facilitating tumor growth, migration, and invasion by phosphorylating essential substrates [ 31 , 32 ]. Previous research has demonstrated that MiR-16-1-3P suppresses tumor cell growth, invasion, and metastasis by directly targeting the 3'-UTR region of PGK1, this inhibition leads to reduced glucose uptake, lactate and ATP production, and extracellular acidification rates, effectively inhibiting aerobic glycolysis [ 33 ]. Additionally, LINC00926 negatively regulates PGK1 expression through STUB1-mediated ubiquitination, offering promising clinical outcomes in breast cancer, this regulation of breast cancer glycolysis, tumor growth, and lung metastasis has been observed both in vivo and in vitro [ 15 ]. In conclusion, targeting PGK1 expression presents a novel strategy to inhibit tumor growth, making it a potential biomarker for targeted tumor therapy. Conclusion In conclusion, resveratrol demonstrates inhibitory effects on breast cancer cell proliferation, invasive and migration, as well as induction of apoptosis in vitro. It also inhibits the growth of mouse breast cancer transplantation tumors in vivo and exhibits oncostatic effects both in vitro and in vivo. Moreover, resveratrol reduces the expression of PGK1 in breast cancer BT-549 cells by regulating the transcription factors c-Myc. This regulation leads to the blockage of cellular glycolysis pathways, ultimately inhibiting the malignant biological behavior of breast cancer cells. Declarations Ethics approval and consent to participate This study was approved by the Ethics Committee of Qiqihar Medical University. (Ethical code QMU-AECC-2022-143), Informed consent to participate in this study was provided by all patients. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding This work was supported by grants from the National Natural Science Foundation of China (No.81972491), Natural Science Foundation of Heilongjiang Province (ZD2023H007), the Qiqihar Science and Technology Plan Joint guidance Project (No.LSFGG-2022033). Author Contribution Xiuli Gao,Yu Gao and Liling Yue contributed to the conception and design; Yaoyao Wang contributed to the experiments sumpletment after major revision; Baodi Wang, Wenbin Zhu and Yu Gao were response for experiment execution; Qunying Hu and Jirui Jiang contributed to bioinformatic analysis; Bo Feng contributed to collection of tissue samples; Likun Liu contributed to preparation of materials. The first draft of the manuscript was written by Yu Gao and was revised by Xiuli Gao and Liling Yue. All authors read and approved the final manuscript. Acknowledgements Not applicable. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5210318","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":363924873,"identity":"a814ecd8-0bcb-4daf-8bb0-8c311d4cb415","order_by":0,"name":"Yu Gao","email":"","orcid":"","institution":"The Third Affiliated Hospital of Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Gao","suffix":""},{"id":363924874,"identity":"4ed5fe20-d73b-40e9-9a95-ae28cf676983","order_by":1,"name":"Yaoyao Wang","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yaoyao","middleName":"","lastName":"Wang","suffix":""},{"id":363924876,"identity":"830d4c98-f809-4170-b4a5-fa2279d0795b","order_by":2,"name":"Baodi Wang","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Baodi","middleName":"","lastName":"Wang","suffix":""},{"id":363924878,"identity":"04ee75bb-c475-4329-8898-3504b589ff00","order_by":3,"name":"Qunying Hu","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qunying","middleName":"","lastName":"Hu","suffix":""},{"id":363924879,"identity":"66cb7eef-43a9-4099-bddb-7504eda92d13","order_by":4,"name":"Jirui Jiang","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jirui","middleName":"","lastName":"Jiang","suffix":""},{"id":363924880,"identity":"3288631e-b7ee-4394-82d5-20641aa15735","order_by":5,"name":"Bo Feng","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Feng","suffix":""},{"id":363924882,"identity":"0dc9eddd-e5a6-4e1c-8408-e5f5fa796ee7","order_by":6,"name":"Xiuli Gao","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiuli","middleName":"","lastName":"Gao","suffix":""},{"id":363924883,"identity":"2b5cf166-ab0e-4fef-a49a-1565f63f16fa","order_by":7,"name":"Likun Liu","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Likun","middleName":"","lastName":"Liu","suffix":""},{"id":363924885,"identity":"3ca900a4-e1c0-4649-8f09-d9ac7721a6a8","order_by":8,"name":"Wenbin Zhu","email":"","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenbin","middleName":"","lastName":"Zhu","suffix":""},{"id":363924886,"identity":"68960068-cb2a-4bfd-a697-81d0876c374b","order_by":9,"name":"Liling Yue","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyUlEQVRIiWNgGAWjYHACxgMPDCTk7NsbGx98IFbPgYQCG2MDnsPNhjOI1/IhLXGDRHqbNAcxyg1uJB84kGBwmHG75MMGaQYGOzndBoJa0hJAWpgtZyc2GBcwJBubHSCoJccApIWN4XZiQ/IMhgOJ2whryf8A0sLDcPNgA5AkSksO0PsGaRIGNxgbm4nSInnmGchhNgaSPYnNjDMMiPAL3/Hkhw8+/JGo72c//vzHhwo7OYJaFFAVGBBQDgLyDUQoGgWjYBSMghEOAMXHTa4gb2TPAAAAAElFTkSuQmCC","orcid":"","institution":"Qiqihar Medical University","correspondingAuthor":true,"prefix":"","firstName":"Liling","middleName":"","lastName":"Yue","suffix":""}],"badges":[],"createdAt":"2024-10-05 19:23:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5210318/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5210318/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":68884489,"identity":"e28e4e28-e8af-4017-a256-96214a7b83c7","added_by":"auto","created_at":"2024-11-13 06:26:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3874978,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of survival and prognosis of glycolytic enzymes in different breast cancers by database. \u003cstrong\u003eA\u003c/strong\u003e Relationship between expression levels of glycolytic metabolic enzyme（HK2, PFKP, GAPDH, LDHA, PKM and PGK1）of breast cancer. \u003cstrong\u003eB\u003c/strong\u003eHuman Protein Atlas analyzes the immunohistochemical expression of PGK1 enzymes in breast cancer. \u003cstrong\u003eC\u003c/strong\u003e Immunohistochemical expression of GAPDH enzymes in breast cancer. \u003cstrong\u003eD\u003c/strong\u003e Immunohistochemical expression of PKM2 enzymes in breast cancer. \u003cstrong\u003eE\u003c/strong\u003e GEPIA and Human Protein Atlas analysis of relationship between expression levels of glycolytic metabolic enzyme PGK1 and overall survival of breast cancer patients. \u003cstrong\u003eF\u003c/strong\u003e Relationship between expression levels of glycolytic metabolic enzyme GAPDH and overall survival of breast cancer patients. \u003cstrong\u003eG\u003c/strong\u003e Relationship between expression levels of glycolytic metabolic enzyme PKM2 and overall survival of breast cancer patients.\u003c/p\u003e","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5210318/v1/83eb2b368d0819167c112f00.png"},{"id":68884488,"identity":"0e4ac249-2cd7-4ae5-8ccd-52db10ad6566","added_by":"auto","created_at":"2024-11-13 06:26:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":7953147,"visible":true,"origin":"","legend":"\u003cp\u003eInhibitory effect of resveratrol on PGK1 and glycolytic enzymes. \u003cstrong\u003eA\u003c/strong\u003e Inhibition of PGK1 mRNA expression by resveratrol. \u003cstrong\u003eB\u003c/strong\u003e Inhibition of PGK1 protein expression by resveratrol, α-tubulin was used as loading control. \u003cstrong\u003eC\u003c/strong\u003e Molecular docking of resveratrol with PGK1. \u003cstrong\u003eD\u003c/strong\u003eInhibitory effect of resveratrol on PGK1 enzyme activity. \u003cstrong\u003eE\u003c/strong\u003e Inhibitory effect of resveratrol on PK enzyme activity. \u003cstrong\u003eF\u003c/strong\u003e Inhibitory effect of resveratrol on LDH enzyme activity. \u003cstrong\u003eG\u003c/strong\u003e Inhibitory effect of resveratrol on ATP content.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5210318/v1/c6a44b95b3d3392b35eb8510.png"},{"id":68885193,"identity":"1c00c086-6eef-41f8-98fa-0b9c90f5c8ab","added_by":"auto","created_at":"2024-11-13 06:34:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":14305734,"visible":true,"origin":"","legend":"\u003cp\u003eResveratrol inhibits PGK1-related c-Myc/PI3K/AKT aixs. \u003cstrong\u003eA\u003c/strong\u003e Resveratrol (20, 40 and 60 μg/ml) on the expression of c-Myc mRNA in BT-549 cells. \u003cstrong\u003eB\u003c/strong\u003e Resveratrol (20, 40 and 60 μg/ml) on the expression of c-Myc protein in BT-549 cells. \u003cstrong\u003eC\u003c/strong\u003ec-Myc protein expression induced by resveratrol (40 μg / mL) and 10058-F4 (90 μM) alone and in combination.\u003cstrong\u003eD\u003c/strong\u003e PGK1 protein expression induced by resveratrol (40 μg / mL) and 10058-F4 (90 μM) alone and in combination. \u003cstrong\u003eE\u003c/strong\u003eWestern blot detection for phosophrylation related proteins in the PI3K/AKT/GSK-3β/β-catenin pathway. \u003cstrong\u003eF\u003c/strong\u003e Quantification of Fig. 3E. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, n=3\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-5210318/v1/0e6ee154f2c56bd2bf94a784.png"},{"id":68884492,"identity":"43525900-8551-40a4-9b91-3f4efd0a9571","added_by":"auto","created_at":"2024-11-13 06:26:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18200546,"visible":true,"origin":"","legend":"\u003cp\u003eResveratrol inhibits cell proliferation and induce apoptosis in breast cancer. \u003cstrong\u003eA\u003c/strong\u003e Inhibitory effects of resveratrol on BT-549 cells detected by MTT methods, IC\u003csub\u003e50\u003c/sub\u003e values were calculated through Graphpad Prism. \u003cstrong\u003eB\u003c/strong\u003e Resveratrol (20, 40 and 60 μg/ml) on PCNA protein expression in BT-549 cells. \u003cstrong\u003eC\u003c/strong\u003e Resveratrol (20, 40 and 60 μg/ml) on apoptosis rate of BT-549 cells. \u003cstrong\u003eD\u003c/strong\u003e Quantification of Fig. 4C. \u003cstrong\u003eE\u003c/strong\u003e Resveratrol (20, 40 and 60 μg/ml) on the protein expression of BAX, Bcl-2, Caspase-3 and Caspase-7 in BT-549 cells, α-tubulin was used as loading control. \u003cstrong\u003eF\u003c/strong\u003e Quantification of Fig. 4E. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, n=3\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-5210318/v1/33abe75acbb11ec74a5240e4.png"},{"id":68884496,"identity":"329a9b01-ca16-4316-85a7-98ae473b7747","added_by":"auto","created_at":"2024-11-13 06:26:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":137497398,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of resveratrol on migration and invasion of BT-549 cells. \u003cstrong\u003eA \u003c/strong\u003eScratch assay to detect the effect of resveratrol (20, 40 and 60 μg/ ml) on the wound scratching ability of BT-549 cells. \u003cstrong\u003eB\u003c/strong\u003e Quantification of Fig. 5A. \u003cstrong\u003eC\u003c/strong\u003e TransWell assay to detect the effect of resveratrol (20, 40 and 60 μg/ml) on the migration ability of BT-549 cells. \u003cstrong\u003eD\u003c/strong\u003eQuantitative results of Fig. 5C. \u003cstrong\u003eE\u003c/strong\u003e TransWell assay to detect the effect of resveratrol (20, 40 and 60 μg/ml) on the invasive metastatic ability of BT-549 cells. \u003cstrong\u003eF\u003c/strong\u003e Quantitative results of Fig. 5E. \u003cstrong\u003eG\u003c/strong\u003e Resveratrol (20, 40 and 60 μg/ml) on the expression of MMP2 and MMP9 proteins in BT-549 cells, β-actin was used as loading control. \u003cstrong\u003eH\u003c/strong\u003eQuantitative results of Fig. 5G. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001, n=3\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-5210318/v1/a2010137d3dde3aef5ff0845.png"},{"id":68884497,"identity":"79ddd164-6027-451f-920b-be7338e3391a","added_by":"auto","created_at":"2024-11-13 06:26:05","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":155873242,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-tumor effect of resveratrol in vivo. \u003cstrong\u003eA\u003c/strong\u003ePathological examination of tumor tissue in vivo detected by HE staining. \u003cstrong\u003eB\u003c/strong\u003eIHC staining to detect the differences in the expression of relevant proteins in tumor tissues. \u003cstrong\u003eC\u003c/strong\u003e Quantitative results of Fig. 6B. \u003cstrong\u003eD\u003c/strong\u003e Effect of resveratrol (20, 50 and 100 mg/kg) and CTX on the tumor volumes of loaded mise. \u003cstrong\u003eE\u003c/strong\u003e Quantitative results of Fig. 6D. \u003cstrong\u003eF\u003c/strong\u003e Effect of resveratrol (20, 50 and 100 mg/kg) and CTX on tumor weight in hormonal mice. \u003cstrong\u003eG\u003c/strong\u003e Effects of resveratrol (20, 50 and 100 mg/kg) and CTX on the body weights of the mise. *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, n=3\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-5210318/v1/eed38dc0ca9bba8d1c6d0650.png"},{"id":68884487,"identity":"28c2a2c8-18ea-4a53-97cb-7388ddce3678","added_by":"auto","created_at":"2024-11-13 06:26:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":568811,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5210318/v1/1e2e7db0-7511-4c82-b0f7-c538792fa292.pdf"},{"id":68884491,"identity":"be0c6d8d-d60d-4b0c-a5a5-7302af2bd259","added_by":"auto","created_at":"2024-11-13 06:26:03","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":264475,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-5210318/v1/d04857983c310c5f14e8e177.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mechanism of resveratrol affecting biological functions of breast cancer through glycolysis pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBreast cancer is a prevalent malignant disease among women, surpassing lung cancer in 2020 with a high incidence of 2.26\u0026nbsp;million cases and becoming the most common cancer worldwide. Surprisingly, the incidence rate of breast cancer among Chinese women of childbearing age accounts for 25% and represents 10% of all malignant tumors, This rate shows an increasing trend every year [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The etiology and pathogenesis of breast cancer have not yet been fully understood, and current treatment options primarily include chemotherapy, radiotherapy, and surgery. However, once tumor metastasis occurs, the chances of survival are significantly reduced. Despite advancements in treatment that have reduced patient mortality, breast cancer remains the leading cause of death among women.\u003c/p\u003e \u003cp\u003eResveratrol is a natural polyphenolic compound that can be found in various sources including blueberries, mulberries, Polygonum multiflorum, and cassia seeds [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It possesses numerous pharmacological activities such as anti-inflammatory, antioxidant, anti-tumor, metabolism regulation, and immune system modulation. Resveratrol has been shown to inhibit the growth of various types of tumors including colon cancer, pancreatic cancer, ovarian cancer, and breast cancer [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The anti-tumor effect of resveratrol has gained significant attention in research. However, the complete understanding of its action mechanism is still lacking and requires further investigation. Studies have shown that resveratrol can inhibit the formation and metastasis of abnormal crypt lesions by influencing cancer cell growth, apoptosis, and angiogenesis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. It exerts its anti-tumor activity by directly targeting relevant factors. These findings serve as a valuable reference for the development of new anti-tumor drugs and demonstrate significant inhibitory effects on various types of tumors [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Additionally, resveratrol block the cell cycle by activating the oncogenes p53 and suppressor gene PTEN, while also down-regulating PI3K. Ultimately, this leads to the induction of apoptosis in cancer cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, the potential of resveratrol as a cancer preventive agent in humans has been widely recognized.\u003c/p\u003e \u003cp\u003ePGK1 is an essential enzyme in glycolysis, serving as both a metabolic enzyme and a protein kinase. It plays a crucial role in tumor growth, invasion, and metastasis by phosphorylating key substrates. As a metabolic enzyme, the primary function of PGK1 is to participate in the glycolysis reaction. This involves transferring the high-energy phosphate group from its substrate to ADP, generating ATP. This process is vital for sustaining cellular energy production, particularly in hypoxic conditions. PGK1 is a crucial molecular target in tumor therapy and has gained significant attention in recent years [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Recent studies have demonstrated that PGK1 plays a role in promoting breast cancer cell growth and lactate production, making it a potential target gene for breast cancer treatment [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Moreover, downregulation of PGK1 has been shown to significantly inhibit the invasive ability of breast cancer cells, reverse the process of epithelial-mesenchymal transition, and enhance the Warburg effect [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. PGK1 is highly expressed in metastatic and invasive ductal breast cancer cells. It is significantly correlated with advanced tumor stage, suggesting its close association with breast cancer onset, progression, and metastasis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Moreover, PGK1 overexpression is linked to poorer prognosis in breast cancer patients. Detecting PGK1 expression before chemotherapy can help predict the sensitivity of chemotherapeutic drugs, benefiting patients who are less responsive to drugs by avoiding unnecessary drug toxicity. In conclusion, reducing PGK1 expression offers a promising strategy to inhibit tumor growth in the future. Therefore, PGK1 can serve as an important biomarker for targeted tumor therapy.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eResveratrol (\u0026ge;\u0026thinsp;99%, HPLC) was obtained from the National Institutes for Food and Drug Contro (Beijing, China). RPMI 1640 medium was purchased from Biosharp (Anhui, China). HK, PK and LDH activity assay reagents were purchased from Solarbio Company (Beijing, China). PGK1 ELISA reagents were purchased from Jianglai Biological (Shanghai, China). Muse Caspase-3/7 Kit was purchased from Luminex Company (Shanghai, China). Enhanced ATP detection reagent was purchased from Beyotime Biotechnology (Shanghai, China). PGK1, c-Myc, BAX and MMP2/9 antibodies were purchased from Cell Signaling Technology (Boston, USA). Antibodies to Caspase-3 and Caspase-7 were purchased from Wanlei Biotechnology (Shenyang, China).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCorrelation between PGK1 expression in breast cancer and patient prognosis analyzed by GEPIA and The Human Protein Atlas\u003c/b\u003e \u003c/p\u003e \u003cp\u003eGEPIA (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gepia.cancer-pku.cn\u003c/span\u003e\u003cspan address=\"http://gepia.cancer-pku.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the Gene Expression Profiling Interactive Analysis, is able to predict PGK1 expression in breast cancer samples, in order to explore the expression level of PGK1 in breast cancer and its relationship with patients' prognostic survival. The Human Protein Atlas (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.proteinatlas.org\u003c/span\u003e\u003cspan address=\"http://www.proteinatlas.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) by analyzing the correlation between the expression level of human gene mRNA in tumor tissue and the prognostic data of cancer patients, the immunohistochemical (IHC) map of cancer tissue and the relationship between the expression of specific genes and the occurrence and development of specific tumors were obtained.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMolecular docking predicts the binding energy of resveratrol and PGK1\u003c/h3\u003e\n\u003cp\u003eMolecular docking of resveratrol with PGK1 was performed by AutoDock software to detect the docking binding energy. The chemical structure of the ligand was downloaded from the ZINC database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://zinc.docking.org/\u003c/span\u003e\u003cspan address=\"http://zinc.docking.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the Auto Dock Tools tool was used to set up a Grid Box centered on the ligand, and good docking active sites were obtained in the Autogrid module.\u003c/p\u003e\n\u003ch3\u003eCell culture\u003c/h3\u003e\n\u003cp\u003eHuman breast cancer cell BT-549 is from the Shanghai Cell Bank of the Chinese Academy of Medical Sciences. It is regularly cultured in RPMI-1640 medium containing 10% fetal bovine serum, in which 57.5 \u0026micro;L/100 mL insulin was added, and cultured at 37℃ with 5% CO\u003csub\u003e2\u003c/sub\u003e for 1\u0026thinsp;~\u0026thinsp;3 days for passage treatment.\u003c/p\u003e\n\u003ch3\u003eCell proliferation assay\u003c/h3\u003e\n\u003cp\u003eThe MTT assay was employed to investigate the impact of varying concentrations of resveratrol on the proliferation of BT-549 breast cancer cells. Briefly, resveratrol final concentrations of 2, 4, 8, 16, 32, 64 and 128 \u0026micro;g/mL were used in sterile 96-well plates inoculated with breast cancer BT-549 cells (6 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well) for 24 h. For the experiment, we utilized 180 \u0026micro;L of serum-free cell culture medium and added 20 \u0026micro;L of a 5 mg/mL MTT solution (Beyotime, China) to continue incubation for 4 h. Afterwards, 150 \u0026micro;L of DMSO (Sigma-Aldrich, USA) was added to each well, and the crystals were dissolved by shaking on a shaker for 10 min. The absorbance value was measured at 570 nm using a enzyme-labelling measuring, and the cell half inhibition rate (IC\u003csub\u003e50\u003c/sub\u003e) was calculated.\u003c/p\u003e\n\u003ch3\u003eDetermination of activity of glycolytic enzymes and ATP content\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetects HK, PK, LDH and PGK1 enzyme activities\u003c/h2\u003e \u003cp\u003eThe activities of HK and PK enzymes were measured using a UV spectrophotometer assay, while the LDH enzyme activity was measured using the enzyme-labelling measuring instrument. Firstly, breast cancer BT-549 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells were taken for the experiment, and the cells were lysed by adding appropriate amount of cell extract, centrifuged at 4℃, 10000 rpm for 15 min, and the supernatant was taken for the assay. Secondly, for HK and PK enzyme activity assay, mix the cell supernatant and the assay reagent according to the instructions, detect the absorbance value at 340 nm by UV spectrophotometer, and calculate the enzyme activity. For LDH enzyme activity, 100 \u0026micro;L of standard solution was taken for concentration dilution, a standard curve was made, the test solution was prepared according to the steps in the instruction manual, and the absorbance value was detected at 570 nm by an enzyme meter to calculate the LDH enzyme activity. After that, PGK1 enzyme activity was detected using ELISA kit. Briefly, 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells were centrifuged at 1000 rpm for 10 min and the supernatant was taken for experimental detection. Dilute the standard solution by 50 \u0026micro;L, add 50 \u0026micro;L each of different concentrations of the samples to be tested and detection dilutions, add 100 \u0026micro;L of horseradish peroxidase (HRP)-labeled detection antibody to each well, and incubate for 60 min at 37℃. After that, add 350 \u0026micro;L of washing solution to wash the plate 5 times, add 50 \u0026micro;L of washing reagent, protect from light, and incubate for 15 min at 37℃. Finally, add the stop buffer 50 \u0026micro;L, the OD values of each experimental group were detected at 450 nm with enzyme-labelling measuring.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eATP content assay\u003c/h3\u003e\n\u003cp\u003eWhen the BT-549 cell fusion reached 80%~90% in a sterile 12-well plate, 150 \u0026micro;L ATP lysis buffer was added to each well, and centrifuged at 4\u0026ordm;C 12000rpm for 5 min, and the supernatant was taken for experiments. Dilute the ATP standard solution in multiplicity, prepare the standard curve, and set a blank control. Dilute the test reagent according to the ratio of 1:4, add 100 \u0026micro;L to each well, and let it stand at room temperature for 5 min, after that, add 20 \u0026micro;L each of the sample and the standard, and select the luminometer program of the enzyme marker to determine the RLU value of the sample.\u003c/p\u003e\n\u003ch3\u003eCell migration and invasion test\u003c/h3\u003e\n\u003cp\u003eThe inhibitory effect of resveratrol on the migration and invasion ability of breast cancer BT-549 cells was detected by wound healing, transmembrane migration and invasion assays. In the wound healing experiment, BT-549 cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e/well) were cultured in a 6-well plate for 24 h and allowed to reach 70% and 80% cell fusion, then the tip of a sterile 200 \u0026micro;L gun was used to scratch the cells perpendicular to the plane of the 6-well plate, and then the cells were washed with PBS for 3 times and the cell migration was monitored under an inverted microscope for more than 24 h. Finally, the ImageJ software was used for analysis, and the scratch width was measured to calculate the healing rate. Scratch healing rate (%) = [(0 h-24 h / 48 h scratch area) / 0 h scratch area] \u0026times; 100%.\u003c/p\u003e \u003cp\u003eFor transwell invasion, a transwell chamber (Corning, USA) with an 8 \u0026micro;m hole polycarbonate filter was used. Briefly, dilute 50 mg/L of Materiel (BD, Biosciences, USA) with sterile PBS in a 1:8 ratio, and 50 \u0026micro;L was added to the upper chamber, which was allowed to solidify at 37℃ for 2 h. After that, 100 \u0026micro;L of medium containing 1% FBS was added to the upper chamber, and the incubation was performed at 37℃ for about 30 min. Then, the cells were incubated with resveratrol (20, 40, and 60 \u0026micro;g/mL) for 24 h and digested with RPMI-1640 serum-free medium. A cell suspension of 100 \u0026micro;L (medium containing 1% FBS) containing 2.5\u0026times;10\u003csup\u003e4\u003c/sup\u003e/mL invasive cells was inoculated into the upper chamber coated with 50 \u0026micro;L of matrigel, and 600 \u0026micro;L of serum-containing medium was added to the lower chamber for 24 h of incubation, and non-migratory cells at the top of the upper chamber were removed with a cotton swab. The cells were fixed with 4% paraformaldehyde (Beyotime, China) and stained with 0.1% crystal violet for 1h, and then imaged by microscope.\u003c/p\u003e \u003cp\u003eFor migration experiments, BT-549 cells treated with different concentrations of resveratrol were inoculated into top chambers without matrigel coating, and other steps were the same as for invasion experiments.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis assay\u003c/h2\u003e \u003cp\u003eApoptosis was detected using the Muse Caspase-3/7 kit. BT-549 cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e ~ 5\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL) were suspended in 1\u0026times;BA assay buffer and 50 \u0026micro;L was added to each experimental tube. Positive and negative control staining was performed, followed by the addition of 5 \u0026micro;L of Muse Caspase-3/7 reagent working solution to each tube. The tubes were then incubated at 37℃ in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 30 min. Subsequently, 150 \u0026micro;L of Muse Caspase7-AAD working solution was added to each tube and mixed. The tubes were incubated at room temperature for 5 min, avoiding light. Apoptosis was detected using a Muse flow cytometer (Merck-Millipore, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting analysis\u003c/h2\u003e \u003cp\u003eBT-549 cells were treated with resveratrol (20, 40, 60 \u0026micro;g/mL) for 24 h, the cells were collected and lysed, and then quantified by BCA protein assay kit (Beyotime, China). The protein was separated by SDS-PAGE, transferred to PVDF membrane and sealed in 5% skimmed milk powder solution for 1 h. After incubating the blotting membrane with the corresponding primary antibody, and then incubating with the second antibody bound to horseradish peroxidase, the target protein band was detected by enhanced chemiluminescence under the scanning \u0026trade;MP imaging system (Bio-Rad, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eReal-time qPCR\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from BT-549 cells by applying TRIzol reagent (Invitgen, USA). cDNA was synthesized using Prime SCRIPT RT kit (Takara, Japan) in order to detect the gene expression level. qRT-PCR reaction was performed using SYBR Green (Takara, Japan). β-actin gene as an internal reference, qRT-PCR was performed to detect the expression of PGK1 and c-Myc genes, the relative expression was calculated by 2\u003csup\u003e\u0026minus;∆∆ct\u003c/sup\u003e. PGK1 (F, 5'-GTGAAGATTACCTTGCCTGTT-3'; R, 5'-GCTTCCCATTCAAATACCC-3'), c-Myc(F, 5'-CCGCCTGCGATGATTTATAC-3'; R, 5'-CAGCCGAGCACTCTAGCTCT-3'),β-actin (F, 5'-GAGCGGGAAATCGTGCGTGACAT-3'; R, 5'-CAGGAAGGAAGGCTGGAAGAGTG-3').\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTumor growth and metastasis in mice with breast cancer\u003c/h2\u003e \u003cp\u003e4T1 murine mammary carcinoma cells were purchased from the Institute of Basic Medical Science, Chinese Academy of Medical Sciences (Beijing, China). Mouse breast cancer 4T1 cells (2\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/0.2 mL) were subcutaneously injected into the right axilla of female BALB/c mice to establish a 4T1 tumor mouse (age: 4\u0026thinsp;~\u0026thinsp;6 weeks; body weight: 18\u0026thinsp;~\u0026thinsp;22g, n\u0026thinsp;=\u0026thinsp;6, SPF grade) model. The mice were randomly divided into 5 groups, and when the tumor volume was about 300\u0026ndash;400 mm\u003csup\u003e3\u003c/sup\u003e, Each group was intraperitoneally injected with 0.2 mL of normal saline 0.2 mL/kg, CTX solution 2.5 mg/kg, resveratrol solution (20, 50, 100 mg/kg). After 21 days of continuous injection, the mice were killed by spinal dislocation method, histological examination was performed by hematoxylin-eosin staining, and the expression of PGK1, PCNA and MMP2/9 protein in tumor tissue was detected by immunohistochemistry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe experimental data were completed in 3 sessions and statistical differences were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Comparisons between groups were made using analysis of variance and t-tests, with differences considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePGK1 could be a better glycolytic target for breast cancer\u003c/h2\u003e \u003cp\u003eAbnormal expression of metabolic enzymes in glycolysis is one of the main causes of Warburg effect, which is one of the hallmarks of cancer. Thus, we firstly analyzed the expression levels of different glycolytic enzymes between different breast cancer tissues and normal tissues through GEPIA database (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The results indicated that Phosphofructokinase (PFKP) and Lactate dehydrogenase (LDHA) showed no significance between breast cancer and normal epithelial tissues, and Hexokinase 2 (HK2) only showed higher expression levels in HER2 breast cancers. However, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and PGK1 were highly expressed in Basal-like, HER2, and Luminal-B breast cancers and M-type Pyruvate kinase (PKM) was PKM could be more effective targets for most breast cancer. In addition, the immunohistochemistry results of GAPDH, PGK1 and PKM from Human Protein Atlas database indicated that the ratio of breast cancer tissues performing \u0026ldquo;Medium/High\u0026rdquo; staining was higher than that of normal tissues, which mainly performed \u0026ldquo;Not detected/Low\u0026rdquo; staining results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D), further proved our prediction. To further elucidate the research significance of above targets, we analyzed their correlation with the prognosis of breast cancer patients through GEPIA and The Human Protein Atlas database (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE-G). The analysis results of these two databases showed that there is a significant correlation between the poor prognosis and higher PGK1 expression level, but not that of GAPDH. About PKM, only The Human Protein Atlas database suggested a negative correlation between PKM expression and patients\u0026rsquo; prognosis. The results suggested that PGK1 and PKM may serve as a promising target for breast cancer, especially for PGK1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eResveratrol inhibits PGK1 and glycolysis in breast cancer\u003c/h2\u003e \u003cp\u003eAs a classical antineoplastic drug, resveratrol has been reported to inhibit some of glycolysis enzymes, including PFKP, HK2 and PKM. However, to the best of our knowledge, effects of resveratrol on PGK1 remains unknown. Thus, we detected effects of resveratrol on PGK1, both from the expression and activity point of view. qRT-PCR and Western-blot results suggested that resveratrol can inhibit mRNA and protein expression in a dose dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). In addition, the molecular docking results indicated that resveratrol can also combine with multiple amino acids of PGK1 automatically with a binding energy less than \u0026minus;\u0026thinsp;5.0 kcal\u0026middot;mol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, among the combined amino acids, 291 and 292 located in the same active pocket with amino acids from 273 to 276 (the substrate binding sites of PGK1), with a minimum distance of 8.3\u0026Aring; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This indicated that in addition to PGK1 expression, resveratrol may also inhibit PGK1 activity through their direct combination. Consistent with above results, resveratrol inhibited the catalytic activity of PGK1 in a dose dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). As PGK1 is the key rate limited enzymes of glycolysis, we further detected the effects of PGK1 on glycolysis. According to Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, resveratrol can obviously inhibit catalytic activities of PK and LDH, which located downstream of PGK1. What\u0026rsquo;s more, resveratrol can inhibit ATP contents in glycolysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). All the above results proved the inhibitory effects of resveratrol on PGK1 and glycolysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eResveratrol inhibits PGK1-related c-Myc/PI3K/AKT aixs\u003c/h2\u003e \u003cp\u003eIn order to gain a thorough understanding of the potential mechanism by which resveratrol inhibits PGK1 expression, we assessed the effects of resveratrol on c-Myc, which is a well-known transcription factor that can regulate PGK1 expression [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Our results revealed that resveratrol reduced c-Myc mRNA and protein expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). Moreover, Resveratrol showed synergistic inhibitory effects on the expression level of c-Myc and PGK1, indicating that Resveratrol inhibits PGK1 partially through the inhibition of c-Myc (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). As a well-known oncogene, PGK1 can active PI3K/AKT signaling pathway [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], which leads to the phosphorylation and inactivation of GSK-3β and prevents GSK-3β from binding to β-catenin. Consequently, β-catenin accumulates and translocates into the nucleus, inducing transcription of target genes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, PGK1 can also contribute the stabilization of GSK-3β, and then promotes β-catenin expression and maintains the stemness of breast cancer cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our results demonstrated that resveratrol significantly inhibited the phosphorylation of PI3K and AKT, and the expression of GSK-3β, and β-catenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F). These findings suggest that resveratrol could exert anti-breast cancer effects by regulating PGK1 related c-Myc/PI3K/AKT aixs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eResveratrol inhibits cell proliferation and induce apoptosis in breast cancer\u003c/h2\u003e \u003cp\u003eThe glycolytic pathway is one of the main hallmarks of cancer, which provides rapid ATP supplementation for cell proliferation. In addition, PGK1 is also a kind of oncogene which involves the initiation and development of several cancers. Thus, based on the inhibition effects on PGK1, we discussed anti-tumor effects of resveratrol in BT-549 cells, which has not been reported before. MTT methods revealed that resveratrol can inhibit BT-549 cell viability in a dose and time dependent manner, with an IC\u003csub\u003e50\u003c/sub\u003e of 40.55\u0026thinsp;\u0026plusmn;\u0026thinsp;3.39 \u0026micro;g/mL at 24h (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In addition, resveratrol also reduced the expression level of proliferation marker PCNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), which confirmed the inhibitory effect of resveratrol on cell proliferation together with MTT results. Furthermore, our flow cytometry experiments demonstrated that resveratrol increased the proportion of apoptotic cells in a dose dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). Moreover, apoptosis related proteins such as the Bax/Bcl-2 ratio, cleaved Caspase-3 and cleaved Caspase-7 were also promoted by resveratrol (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-F). All the results above revealed that resveratrol can inhibit cell proliferation and induce apoptosis in breast cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eResveratrol inhibits cell migration and invasion in breast cancer\u003c/h2\u003e \u003cp\u003eIt has been reported that targeting PGK1-mediated Warburg effect could suppress breast tumor metastasis [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Though resveratrol has been reported to inhibit migration and metastasis of some breast cancer cells including MDA-MB-231 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], related effects on BT-549 cells remains unknown. Thus, we detected the effects of resveratrol on BT-549 cell migration and invasion. The scratch-wound experiment demonstrated that after scratched for 24 hours, the wound healing rate of BT-549 cells was 59.19\u0026thinsp;\u0026plusmn;\u0026thinsp;3.585%, indicating its highly metastatic potential. However, treatment with resveratrol significantly inhibited the wound healing rate of BT-549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-B). In addition, the Trans-Well experiment without the matrix also confirmed the inhibitory effects of resveratrol on BT-549 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC-D). Number of invasion cell decreased from 198\u0026thinsp;\u0026plusmn;\u0026thinsp;6.07 (control group) to 98.33\u0026thinsp;\u0026plusmn;\u0026thinsp;5.074 (20 \u0026micro;g/mL), 73\u0026thinsp;\u0026plusmn;\u0026thinsp;6.245 (40 \u0026micro;g/mL), and 44.62\u0026thinsp;\u0026plusmn;\u0026thinsp;4.163 (60 \u0026micro;g/mL) respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F). What\u0026rsquo;s more, Western-blot experiments revealed that resveratrol dose-dependently inhibited the expression levels of migratory invasion markers MMP2 and MMP9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG-H). Collectively, these findings provided evidence for the anti-breast cancer effects of resveratrol.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eIn vivo anti-tumor effects of resveratrol on breast cancer\u003c/h2\u003e \u003cp\u003eFinally, we investigated the in vivo anti-mammary effects of resveratrol. We conducted experiments using breast cancer cells to induce mouse tumors (model group), which were then treated with different doses of resveratrol (treatment group). We used the well-known anti-tumor drug CTX as a positive control. The results from HE staining revealed that the tumor tissue in the model group exhibited vigorous growth, with a large number of tightly packed cancer cells showing irregular morphology and darker nuclear staining color. However, after treatment with resveratrol at concentrations of 20, 50, and 100 mg/kg, the dense structure of the tumor was damaged, leading to a gradual restriction of tumor tissue growth. The tumor cells also exhibited varying degrees of shrinkage and rounding, and the arrangement of the tumor cells became progressively sparse. The CTX group showed the most significant inhibition of tumor growth in the breast cancer hormone mice, with a large area of necrosis and dissolution observed in the tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). In addition, IHC staining results demonstrated that resveratrol can inhibit the positive staining of proteins including PGK1, PCNA, and MMP9 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C). Notably, the above effects of 100 mg/kg resveratrol was similar to that of the positive control drug CTX. Finally, we observed that resveratrol can significantly inhibit the tumor volume (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-E) and tumor weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF) while did not affect the body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG) of the tumor bearing mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eBreast cancer is a malignant tumor that originates in the epithelial tissue of the breast gland. Recent epidemiological investigations have shown a steady increase in the incidence of breast cancer. In fact, as of 2020, breast cancer has surpassed lung cancer to become the most common cancer globally, particularly among female patients [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The current dominant approach in Western medicine for treating breast cancer is cytotoxic drug chemotherapy, which includes anthracyclines and albumin-binding paclitaxel [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, these conventional therapies have significant drawbacks, such as severe adverse effects and invasiveness, leading to a low 5-year survival rate and poor prognosis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Consequently, it is imperative to develop highly effective and low-toxicity treatments for breast cancer. These treatments include induced cell cycle blockade, signaling pathway and mRNA-targeted therapies, immunotherapy, endocrine therapies, and the integration of botanical medicines as natural anticancer agents. These advancements offer a promising outlook for long-term disease control of breast cancer.\u003c/p\u003e \u003cp\u003eIn recent years, resveratrol has gained significant attention from scholars both domestically and internationally due to its effective anti-tumor activity. Resveratrol has been found to inhibit breast cancer both in vivo and in vitro, and its mechanism of action is closely related to various molecular targets including cell proliferation, epithelial-mesenchymal transition, chemosensitization, invasion and metastasis, apoptosis, and epigenetics [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Numerous preclinical experiments have demonstrated that resveratrol inhibits the expression of histone transferase EZH2 by regulating the dephosphorylation of protein kinase ERK1/2, resulting in the inhibition of growth and proliferation of breast cancer cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Resveratrol also blocks the G0/G1 phase of breast cancer MCF-7 cells and reduces the expression of invasive and metastatic markers MMP2 and MMP9, thereby inhibiting the invasion and metastasis of breast cancer cells [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Furthermore, resveratrol has been shown to inhibit the invasion and migration of human breast cancer MCF-7 cells through the PI3K/Akt and Wnt/β-catenin signaling pathways [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Resveratrol has been found to reduce the expression of POLD1, inhibiting PCNA and Bcl-2 expression, while increasing the expression of the apoptotic index caspase-3, these effects activate the apoptotic pathway, promoting apoptosis in breast cancer cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Resveratrol exhibits a range of pharmacological activities and shows promising potential in the field of medicine and health care. However, its clinical application is limited due to issues such as low bioavailability, poor water solubility, and poor stability. Researchers face the challenging task of modifying its structure to develop resveratrol derivatives with enhanced biological activity and higher bioavailability.\u003c/p\u003e \u003cp\u003ePGK1, the first key enzyme in the glycolytic pathway, plays a crucial role in ATP production, breast cancer cell growth, and lactate production. It is considered a significant target gene for potential breast cancer treatment [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Notably, PGK1 acts not only as a metabolic enzyme but also as a protein kinase, facilitating tumor growth, migration, and invasion by phosphorylating essential substrates [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Previous research has demonstrated that MiR-16-1-3P suppresses tumor cell growth, invasion, and metastasis by directly targeting the 3'-UTR region of PGK1, this inhibition leads to reduced glucose uptake, lactate and ATP production, and extracellular acidification rates, effectively inhibiting aerobic glycolysis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Additionally, LINC00926 negatively regulates PGK1 expression through STUB1-mediated ubiquitination, offering promising clinical outcomes in breast cancer, this regulation of breast cancer glycolysis, tumor growth, and lung metastasis has been observed both in vivo and in vitro [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In conclusion, targeting PGK1 expression presents a novel strategy to inhibit tumor growth, making it a potential biomarker for targeted tumor therapy.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, resveratrol demonstrates inhibitory effects on breast cancer cell proliferation, invasive and migration, as well as induction of apoptosis in vitro. It also inhibits the growth of mouse breast cancer transplantation tumors in vivo and exhibits oncostatic effects both in vitro and in vivo. Moreover, resveratrol reduces the expression of PGK1 in breast cancer BT-549 cells by regulating the transcription factors c-Myc. This regulation leads to the blockage of cellular glycolysis pathways, ultimately inhibiting the malignant biological behavior of breast cancer cells.\u003c/p\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e This study was approved by the Ethics Committee of Qiqihar Medical University. (Ethical code QMU-AECC-2022-143), Informed consent to participate in this study was provided by all patients.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (No.81972491), Natural Science Foundation of Heilongjiang Province (ZD2023H007), the Qiqihar Science and Technology Plan Joint guidance Project (No.LSFGG-2022033).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eXiuli Gao,Yu Gao and Liling Yue contributed to the conception and design; Yaoyao Wang contributed to the experiments sumpletment after major revision; Baodi Wang, Wenbin Zhu and Yu Gao were response for experiment execution; Qunying Hu and Jirui Jiang contributed to bioinformatic analysis; Bo Feng contributed to collection of tissue samples; Likun Liu contributed to preparation of materials. The first draft of the manuscript was written by Yu Gao and was revised by Xiuli Gao and Liling Yue. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eZhang X, Ge X, Jiang T, Yang R, Li S. Research progress on immunotherapy in triple-negative breast cancer (review). Int J Oncol. 2022;61(2):95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChhabra G, Singh CK, Amiri D, Akula N, Ahmad N. Recent advancements on immunomodulatory mechanisms of resveratrol in tumor microenvironment. 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Front Cell Dev Biol. 2021;9:649787.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"resveratrol, breast cancer, glycolysis, invasion, apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-5210318/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5210318/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cb\u003eBackground\u003c/b\u003e Phosphoglycerate kinase 1 (PGK1) plays a crucial role in the glycolytic pathway and its overexpression has a negative impact on tumor development and prognosis. Resveratrol, a natural polyphenolic compound, has gained significant attention in recent years due to its anti-inflammatory, antioxidant, and anti-tumor properties. However, the mechanism by which resveratrol inhibits breast cancer growth, invasion, and metastasis through the PGK1 glycolytic pathway is still not fully understood. Therefore, this study aimed to investigate the inhibitory effects of resveratrol on breast cancer cell proliferation and invasive migration, as well as its ability to promote apoptosis in vitro. Additionally, the study examined the inhibitory effects of resveratrol on the growth of mouse breast cancer graft tumors in vivo.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMethods\u003c/b\u003e We analyzed the expression levels of glycolytic enzymes in different breast tissues and their correlation with the prognosis of breast cancer patients through GEPIA and The Human Protein Atlas database. Wound healing assay, Transwell migration and invasion assay were used to study the effect of resveratrol on the biological functions of breast cancer. RT-qPCR and Western blot\u003c/p\u003e \u003cp\u003emethods were used to explore the potential molecular mechanism of resveratrol inhibiting the development of breast cancer through the PGK1 glycolysis pathway. In vivo experiments explore the biological mechanisms behind PGK1-mediated breast cancer proliferation and invasion.\u003c/p\u003e \u003cp\u003e \u003cb\u003eResults\u003c/b\u003e High expression of PGK1 is significantly associated with poor prognosis in breast cancer patients, and resveratrol induces breast cancer cell apoptosis in a dose-dependent manner, inhibiting PK, PGK1, LDH enzyme activity and ATP content. Resveratrol inhibits PGK1 expression and its related c-Myc/PI3K/AKT signaling pathway. Western blot experiments show that resveratrol affects the biological functions of breast cancer by inhibiting PCNA, MMP2 and MMP9 proteins and activating BAX/Bcl-2 and Caspase3/7 proteins. In addition, in vivo experiments show that resveratrol significantly inhibits the growth of transplanted tumors in breast cancer mice and inhibits breast cancer tumor proliferation, invasion and metastasis by downregulating PGK1 expression.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusion\u003c/b\u003e Resveratrol demonstrates inhibitory effects on breast cancer cell proliferation, invasive and migration, as well as induction of apoptosis in vitro. It also inhibits the growth of mouse breast cancer transplantation tumors in vivo and exhibits oncostatic effects both in vitro and in vivo. Moreover, resveratrol reduces the expression of PGK1 in breast cancer BT-549 cells by regulating the transcription factors c-Myc. This regulation leads to the blockage of cellular glycolysis pathways, ultimately inhibiting the malignant biological behavior of breast cancer cells.\u003c/p\u003e","manuscriptTitle":"Mechanism of resveratrol affecting biological functions of breast cancer through glycolysis pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-13 06:25:58","doi":"10.21203/rs.3.rs-5210318/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":"3be16775-1451-4079-adb5-7bc2981b1f42","owner":[],"postedDate":"November 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-11-13T06:26:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-13 06:25:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5210318","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5210318","identity":"rs-5210318","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}