Metformin induces ferroptosis and suppresses malignant behaviors in diabetic breast cancer

Metformin induces ferroptosis and suppresses malignant behaviors in diabetic breast cancer | 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 Article Metformin induces ferroptosis and suppresses malignant behaviors in diabetic breast cancer Tao Chen, Xiaoxin Li, Yuanyuan Li, Chunyan Zhou, Chuangang Tang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4588932/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 This study investigates the potential role of metformin in breast cancer treatment, especially its impact on ferroptosis—an iron-dependent form of programmed cell death. Breast cancer is one of the most common malignancies globally, with limited treatment options, particularly for triple-negative breast cancer. The research involved analyzing tumor tissues from breast cancer patients. It was observed that the tumor tissues of diabetic patients treated with metformin had obvious iron accumulation, suggesting variations in the level of ferroptosis. Further analysis using gene transcription data from the TCGA database revealed correlations between diabetes-related genes and genes associated with ferroptosis. The experimental results indicated that metformin could evident inhibit the proliferation of breast cancer cells and induce ferroptosis in a diabetic model. Moreover, metformin was found to promote ferroptosis by affecting mitochondrial activity. In conclusion, the study suggests that metformin holds potential value in treating diabetic breast cancer, capable of suppressing tumor cell growth through the ferroptosis mechanism. These findings provide a new theoretical basis for using metformin as a treatment for breast cancer and lay the groundwork for future clinical applications. Biological sciences/Cancer/Breast cancer Biological sciences/Cell biology/Cell migration Metformin Breast Cancer Ferroptosis Diabetes Immunohistochemistry Bioinformatics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Breast cancer is the most prevalent malignancy worldwide and ranks second in cancer-related deaths, posing a seriously threat to public health 1 . Particularly in China, due to aging populations, dietary changes, and shifts in reproductive patterns, the incidence of breast cancer is rising above the global average 2 . The current treatment system for breast cancer is relatively mature, offering suitable treatments based on molecular subtyping. Due to the limited accessibility of genetic testing methods, IHC/FISH-based histopathological detection is commonly used in clinical practice to classify breast cancer into Luminal, Her2-overexpressing, and triple-negative subtypes 3 . Depending on the molecular subtype, patients will receive chemotherapy, endocrine therapy, and targeted therapy. The treatment options for triple-negative breast cancer are relatively limited, making the development of new treatment methods particularly important for breast cancer patients. Metformin, an insulin sensitizer, is the most widely used oral hypoglycemic drug in clinical practice. It reduces blood glucose levels by inhibiting hepatic gluconeogenesis. Further research into metformin has revealed its potential role in the onset and progression of tumors. Its anticancer effects can be achieved through various mechanisms. The induction of apoptosis in breast cancer cells by metformin has been confirmed, and this process can be inhibited by a high-glucose environment 4 , 5 . Additionally, studies have suggested that metformin induces apoptosis in breast cancer cells by regulating amino acid metabolism, the TCA cycle, the urea cycle, and the pentose phosphate pathway, as well as through a PRODH/POX-dependent mechanism 6 , 7 . The AMPK pathway is the most common target of metformin, independently affecting the cell cycle protein D1 and P53-induced apoptosis in breast cancer cells 8 . Moreover, metformin can enhance the sensitivity of breast cancer to chemotherapy and endocrine therapy by regulating angiogenesis, inducing apoptosis, and modulating the AKT/AMPK pathway 4 , 9 . Lipid peroxidation is a key process in ferroptosis, and under certain conditions, lipid oxidation can serve as a preliminary step. Metformin promotes the survival and immune evasion of breast cancer cells by regulating the AMPK signaling pathway and fatty acid oxidation 10 , 11 . As research into the mechanisms of metformin's action has progressed, it has shown unusual therapeutic potential in breast cancer ferroptosis. An increase in reactive oxygen species (ROS) leads to heightened oxidative stress within cells, resulting in lipid peroxidation and ferroptosis. Reports indicate that metformin can induce ROS production in breast cancer cells 12 , 13 . Further, metformin can target lncRNA H19 to induce ferroptosis in breast cancer cells, and inhibiting autophagy can regulate ferroptosis, further clarifying the relationship between autophagy and ferroptosis 14 . Other studies report that miR-324-3p, another non-coding RNA, is involved in the regulation of metformin-induced ferroptosis in breast cancer 15 . Additionally, metformin can regulate breast cancer cell ferroptosis by suppressing the ubiquitination level of SLC7A11 independently of the AMPK pathway 16 . However, as a hypoglycemic drug, these studies have not elucidated the role and mechanism of metformin in inducing ferroptosis in breast cancer under diabetic conditions. Regarding the impact of diabetes on the anticancer effects of metformin, scholars have shown that high glucose levels can promote the proliferation of breast cancer cells and inhibit the apoptosis induced by metformin 5 , 17 . Advanced glycation end-products (AGEs), pathological products formed from proteins, lipids, and nucleic acids in the Maillard reaction in the plasma of diabetic patients, not only play a crucial role in common macrovascular and microvascular complications of diabetes but can also promote the migration and invasion of breast cancer cells through the RAGE-mediated MEK-EMT and TLR4-MyD88 signaling pathways 18 , 19 . Thus, diabetes plays an important role in the development and progression of breast cancer. Recent clinical trials have indicated that metformin can improve the incidence and survival time of breast cancer in diabetic patients, but it has not had the same effect in non-diabetic patients, instead increasing the risk of new cancers in non-diabetic individuals 20 , 21 . These findings suggest that the anticancer effects of metformin on breast cancer need to be closely linked to diabetes. 2. Materials and Methods 2.1 Cell Lines and Culture The MCF-10A, MDA-MB-231, and MCF-7 cell lines were all acquired from Procell Life Science & Technology Co., Ltd. (Wuhan, China). The serum used for cell culture in this study was fetal bovine serum (Sigma, F0193). 2.2 Tissue Samples The experiment involved five tissue samples, all of which were obtained from breast cancer surgeries performed at the Xuzhou Central Hospital's Department of Breast Surgery between May and October 2023. The samples were preserved in the form of paraffin blocks within the hospital's Department of Pathology. The use of the specimens was approved by the ethical review board of Xuzhou Central Hospital. Furthermore, the use of the specimens conformed to the ethical committee's criteria for exemption from informed consent, thereby waiving the need for patient consent. 2.3 Establishment of Diabetic Breast Cancer Cell Model The triple-negative breast cancer cell line MDA-MB-231, non-triple-negative breast cancer cell line MCF-7, and normal mammary epithelial cell line MCF-10A were selected. Breast cancer cells were cultured in high-glucose DMEM or RPMI medium, supplemented with advanced glycation end products (AGEs) to a concentration of 200 mg/L, until cell density reached 80%-95% for passaging. This process was repeated three times. 2.4 Confocal Fluorescence Microscopy for Mitochondrial Activity To verify mitochondrial activity, mitochondrial fluorescent dye working solution was mixed with the base culture medium to prepare a 0.3 µM/L working staining solution. This solution was applied to the breast cancer cell slides after treatment. A 10 µL aliquot of anti-fade mounting medium was dropped onto a glass slide, and the cell slide was inverted onto the glass slide and placed on the stage of a confocal microscope. The intensity of red fluorescence and bright field light was adjusted. The average fluorescence intensity was quantitatively analyzed. 2.5 The glutathione (GSH) assay GSH assay utilized the Reduced Glutathione (GSH) Assay Kit from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. Cells were homogenized with PBS and lysed using ultrasonication. After centrifugation, the supernatant, GSH standards, and reagents were mixed and the optical density (OD) at 405 nm was measured in a 96-well plate. The GSH levels were calculated according to the formula provided in the manual. 2.6 The cellular malondialdehyde (MDA) Assay MDA content was determined using the Cell Malondialdehyde (MDA) Assay Kit produced by Nanjing Jiancheng Bioengineering Institute. Cells were mixed with reagents, lysed, and centrifuged to obtain the supernatant. The supernatant was then mixed with reagents and the OD value at 532 nm was measured colorimetrically against MDA standards. The concentration of MDA was calculated using the formula provided by the manufacturer. 2.7 Iron Ion Assay The intracellular iron ion content was measured using the Total Iron Content Colorimetric Assay Kit from Applygen Technologies, Beijing, China. Cells were thoroughly mixed with cell lysis buffer and reacted, followed by centrifugation to collect the supernatant. The supernatant was mixed with prepared 4.5% potassium permanganate and reagents, incubated at 60°C for 1 hour, and the iron detection reagent was added. The OD value at 550 nm was measured in a 96-well plate against standards, and the total iron ion concentration within the cells was calculated using the formula provided in the manual. 2.8 Cell Proliferation and Toxicity Detection by CCK8 Cultured cells were digested, centrifuged, and resuspended. A 10 µL aliquot of the cell suspension was added to a hemocytometer, and cells were counted under a microscope. The total cell count was multiplied by (5 \times 10^4/mL). A 96-well plate was prepared, with each well containing 200 µL of complete culture medium and 5,000 cells. After 24 hours, the medium was changed, and treatment drugs were added. After 48 hours, 180 µL of complete culture medium and 20 µL of CCK8 reagent were added to each well, mixed well, and incubated in a CO2 incubator for 1–4 hours. The absorbance at 450 nm was measured with a microplate reader and the results were analyzed. 2.9 Colony Formation Assay Cell suspension was counted, and a 12-well plate was prepared, with each well containing 1000 µL of complete culture medium and 3,000 cells, mixed well. After 3 days, the medium was changed, and treatment drugs were added. The cells were cultured in an incubator for 5–7 days. The culture medium was aspirated, and the wells were washed with PBS. A 1 mL aliquot of 4% paraformaldehyde was added for fixation for 20 minutes. The paraformaldehyde was aspirated, the wells were washed with PBS, and 1 mL of crystal violet staining solution was added for staining for 20 minutes. The wells were washed with PBS until the solution was no longer visibly purple, and images were taken under a transmitted light lamp to calculate the colony results. 2.10 Cell Scratch Assay Cells were treated with drugs for 48 hours and then switched to serum-free culture medium. A ruler and pipette were used to create a scratch. The bottom of the culture dish was marked with a marker pen to denote the location. The cells were then cultured in a carbon dioxide incubator, and photographs were taken now, and at 24 and 48 hours. The photos were appropriately processed to calculate the migration rate. 2.11 Transwell Migration Assay Cells were treated with serum-containing culture medium for 24 hours, after which the medium was discarded, and the cells were digested with trypsin to obtain a cell suspension. The cell suspension was centrifuged and resuspended for cell counting. A 12-well plate and Transwell chambers were prepared, with the upper chamber containing 400 µL of serum-free culture medium with 20,000 cells, and the lower chamber containing 600 µL of culture medium with 20% fetal bovine serum. The setup was placed in a CO2 incubator for 48 hours. The culture medium was discarded, and both chambers were fixed with 400 µL and 600 µL of 4% paraformaldehyde for 20 minutes, respectively, followed by a PBS wash. The lower chamber was stained with 800 µL of crystal violet staining solution for 10 minutes, the staining solution was discarded, and the chamber was washed with PBS until no obvious purple color remained. A cotton swab was used to gently remove the cells from the upper chamber, and the chamber was placed under a microscope for observation and analysis of the results. 2.12 HE Staining Dried paraffin sections were sequentially placed in xylene, xylene, 100% alcohol, 100% alcohol, 95% alcohol, 90% alcohol, 80% alcohol, and 70% alcohol. The sections were stained in hematoxylin staining solution for 5 minutes, rinsed with running water, differentiated with 1% hydrochloric acid in ethanol for 5 seconds, and rinsed with running water until the sections turned blue. The sections were then placed in eosin staining solution for 2 minutes, followed by 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, anhydrous ethanol, anhydrous ethanol, xylene, and xylene, dried thoroughly, and sealed with neutral resin. 2.13 Immunohistochemistry Paraffin sections were dried and sequentially placed in xylene, xylene, 100% alcohol, 100% alcohol, 95% alcohol, 90% alcohol, 80% alcohol, and 70% alcohol. After deparaffinization, the slides were rinsed under running water, soaked in 3% hydrogen peroxide solution for 10 minutes, washed twice with running water (1 minute each time), and then placed in citrate buffer. The slides were heated in a microwave oven for 3 minutes (medium heat) until boiling, cooled to room temperature, and then reheated in the microwave oven and cooled to room temperature again. The slides were blocked with 2.5% BSA solution for 1 hour. Primary antibodies were added, and the slides were stored overnight in a 4°C refrigerator. The slides were washed three times in PBS, each time for 3 minutes, followed by secondary antibodies, and incubated in a 37°C incubator for 30 minutes. The slides were washed three times in PBS, each time for 3 minutes, and DAB solution was added. The slides were rinsed with clean water for 1 minute, stained with hematoxylin staining solution until the desired color depth was achieved. The slides were then rinsed in clean water, placed in 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, 100% alcohol, 100% alcohol, xylene, and xylene, and placed in a fume hood. The slides were sealed with neutral resin. 2.13 Data and Bioinformatics Gene expression data and clinical data of breast cancer patients were downloaded from the TCGA database ( http://tcga-data.nci.nih.gov ). This included transcriptomic data, age, gender, estrogen receptor (ER) status, progesterone receptor (PR) status, human epidermal growth factor receptor 2 (Her2) status, and follow-up information. The R packages "rjson" and "tidyverse" were used to process gene expression data. "survival" and "survminer" were utilized to calculate Kaplan-Meier survival analysis results and to create visualizations. The log-rank test was employed to determine statistical significance. All other image creations and statistical analyses were performed using GraphPad Prism software ( https://www.graphpad-prism.cn ). Differences were assessed using analysis of variance (ANOVA) or t-tests, with P < 0.05 indicating statistical significance. 3. Results 3.1 Breast cancer patients using metformin experience ferroptosis Ferroptosis is an iron-dependent form of programmed cell death that regulates malignancies development and treatment 22 , 23 . Beyond apoptosis, ferroptosis provides a pathway for tumor principle research and offers effective guidance for the development of cancer treatment strategies. To understand the role of ferroptosis in breast cancer, we performed HE staining and immunohistochemical diagnosis on surgically removed tumor tissues from five breast cancer patients (Fig. 1 A). All of them had been diagnosed with diabetes and three of these patients had been treated with metformin. We examined the key enzymes in glycolysis and important antioxidants in ferroptosis, observing that metformin users had relatively lower expression of PKM2 (Pyruvate Kinase M2) (Fig. 1 B). Interestingly, GPX4 expression was evident higher in Luminal-type breast cancer tissues compared to other types, and in TNBC (triple-negative). samples, using of metformin was associated with reduced GPX4 expression. Affected by the small sample size, we can only speculate that GPX4 expression in breast cancer is obvious related to molecular subtyping, and metformin use may play a role (Fig. 1 C). To further understand if there is a difference in ferroptosis, we also measured the iron ion content in tumor tissue sections. The results were more pronounced this time, with the metformin group showing a obvious accumulation of iron ions compared to the non-metformin group, suggesting potential differences in the level of ferroptosis (Fig. 1 D). However, we have also observed in the images that the deposition of iron ions in the tumor stroma is more pronounced than in the breast cancer tissue itself. 3.2 Diabetes may inhibit ferroptosis from breast cancer To further investigate the expression of GPX4 in breast cancer and its direct connection with ferroptosis and diabetes, we analyzed gene transcription data of breast cancer tumor tissues from the TCGA database. We focused on the correlation between diabetes-related gene expression and ferroptosis-related gene expression. Our results indicate that IGF1 is negatively correlated with GPX4 and positively correlated with ACSL1, ACSL4, and LPCAT3, suggesting that IGF1 may promote the development of ferroptosis. Conversely, AGER showed the opposite trend, being positively correlated with GPX4 and negatively correlated with ACSL1, ACSL4, LPCAT3, and SLC7A11, which may be an important gene suppressing ferroptosis in diabetic patients (Fig. 2 A). Other genes related to glucose metabolism dysregulation, such as IGF1R, IGF2, IGF2R, IRS1, IRS2, and INSR, did not show a clear correlation with ferroptosis. In the previous test results, we found that GPX4 expression seemed to be overexpressed in luminal breast cancer tissues, so we verified the results in the database. The transcription level of GPX4 in Luminal-type breast cancer tissues was higher than in ERBB2+ (receptor tyrosine kinase 2) and TNBC subtypes, with significant differences (Fig. 2 B). The lowest expression of GPX4 was observed in TNBC, and based on previous immunohistochemical results and iron ion measurements, we believe that triple-negative breast cancer may be the most sensitive subtype to ferroptosis. Additionally, we analyzed the relationship between relevant gene expression and survival time. Through K-M analysis, we found that low expression of AGER was associated with extended survival time, while the expression of ferroptosis-related genes did not show a significant correlation with survival time (Fig. 2 C). 3.3 Metformin can induce the cell death of diabetic breast Advanced glycation end-products (AGEs), pathological products formed under the Maillard reaction from proteins, lipids, and nucleic acids in the plasma of diabetic patients, play a crucial role in common macrovascular and microvascular complications of diabetes and can also enhance the migration and invasion capabilities of breast cancer 18 , 19 , 24 . We investigated the impact of AGEs on the proliferative capacity of breast cancer cells to select an appropriate concentration for simulating a diabetic environment with high-glucose (25mM) culture medium. It was evident that even at a concentration of 800mg/L, AGEs did not exhibit obvious toxicity, even MCF-10A cells showed a trend of increased proliferative capacity (Fig. 3 A-C). Based on our laboratory's previous experience, we chose a concentration of 200mg/L AGEs as the suitable concentration for establishing a diabetic model. We evaluated the effect of metformin on cell proliferation using two breast cancer cell lines (triple-negative and luminal) and a normal mammary cell line. The results showed that metformin inhibited the proliferation of all cell lines in a concentration-dependent manner (Fig. 3 D). Notably, the IC50 values for metformin in the breast cancer cell lines (MCF-7, diabetic model MCF-7, MDA-MB-231, diabetic model MDA-MB-231, MCF-10A, diabetic model MCF-10A) were 36.29uM/L, 40.36uM/L, 19.31uM/L, 20.67uM/L, 50.52uM/L, and **( IC50 value cannot be calculated because the activity value is too high.), respectively(Fig. 3 E). Triple-negative cells were more sensitive, while normal mammary cell MCF-10A is the highest. Comparing cell types, under normal culture and diabetic model conditions, AGEs could resist the toxic effects of metformin to some extent, with this effect being most apparent in MCF-10A (Fig. 3 E). Thus, metformin exhibited toxic effects on both mammary and breast cancer cells. 3.4 Metformin's Inhibitory Effect May Be Related to Ferroptosis To further study the inhibitory effect of metformin on breast cancer cells without strong toxicity subsequent results, we selected concentrations of 5mM and 10mM metformin for the following experiments. Firstly, colony formation assays continued to support our previous view that metformin can inhibit cell proliferation to a certain extent. Additionally, under the diabetic model, not only could cells tolerate metformin better, but they also exhibited enhanced proliferative capacity. In MCF-10 cells, the trend was similar to that of MCF-7, but the increase of iron ions was slower (Fig. 4 A). On the other hand, the malignancy of tumors is also reflected in their migratory capacity. We verified the effects of metformin on MDA-MB-231 cells using scratch assays and Transwell migration experiments. Initially, advanced glycation end-products (AGEs) do not significantly alter the migratory ability of breast cancer cells; however, the effect of metformin is more evident. Concurrently, the inhibition of MDA-MB-231 cell migration by a 5mM concentration of metformin is gentler compared to that by a 10mM concentration of metformin (Figure S1A,S1B). Based on our previous immunohistochemical results and bioinformatics analysis, we hypothesized that ferroptosis occurred in breast cancer cells treated with metformin, whether in the control group or the diabetic model group. Therefore, we directly measured changes in the development process of ferroptosis. Starting with the iron ion levels in diabetic breast cancer cells, the results showed that the iron ion concentration in the MCF-7 diabetic model group was slightly higher than in the control group, and metformin significantly increased the iron ion levels in the diabetic model group, with the high-concentration metformin group showing higher iron ion levels than the low-concentration metformin group(Fig. 4 B). And the MCF-10A showed the same change (Fig. 4 C). During the development of ferroptosis, the level of oxidative stress within cells increases, lipid peroxidation begins, and the depletion of GSH is an important pathway. The GSH levels in MCF-7 cells were lower than in the control group, and metformin significantly inhibited GSH content(Fig. 4 D). In MDA-MB-231 cells, the GSH content in the diabetic model showed a trend opposite to MCF-7. Additionally, metformin's inhibitory effect on GSH was not pronounced (Fig. 4 E). Malondialdehyde (MDA) is one of the lipid peroxidation products formed during the ROS oxidation of biomembranes. Interestingly, in the lipid peroxidation (MDA) assay, we found a similar phenomenon to the GSH assay, with MDA levels in the MCF-7 diabetic model showing an increasing trend, and metformin's effect on increasing MDA levels was somewhat concentration gradient-dependent(Fig. 4 F). However, the MDA levels in the diabetic model MDA-MB-231 cells showed a decreasing trend, while metformin continued to significantly enhance lipid peroxidation capability (Fig. 4 G). Changes in mitochondrial morphology and activity are also characteristics of ferroptosis. Using the cationic fluorescent probe TMRM to detect mitochondrial membrane potential (MMP), which can specifically identify MMP and thus adhere to mitochondria, producing red fluorescence. Confocal microscopy analysis of fluorescence showed that in MDA-MB-231 cells, compared to the control group, the fluorescence intensity of the diabetic model group was relatively increased, while breast cancer cells treated with metformin had lower red fluorescence intensity, with the high-concentration metformin treatment group having the lowest (Fig. 4 H). These data are consistent with the above experimental results, indicating that metformin may induce breast cancer cell death through ferroptosis. 3.5 Metformin Inhibits Breast Cancer Cells Through Ferroptosis In the subsequent experiment, we introduced the ferroptosis inhibitor, ferrostatin-1 (Fer-1), to investigate its impact on cellular proliferation capabilities. Initially, in the assessment of clonogenic proliferation, the addition of metformin resulted in a clonal area that was smaller compared to the control group. However, with the application of ferrostatin-1, there was a noticeable increase in clonal area(Fig. 5 A). Similar outcomes were observed in the evaluation of cellular viability, where ferrostatin-1 exhibited a certain degree of resistance to the proliferation inhibition induced by metformin(Fig. 5 B-C). These results suggest that ferrostatin-1 may counteract the antitumor effects of metformin by inhibiting ferroptosis, thereby promoting cell proliferation. We also discovered that metformin significantly increases intracellular iron accumulation. Both the diabetic model and Fer-1 could reduce iron accumulation, and both had a synergistic effect (Fig. 5 D). In MDA-MB-231 cells, AGEs and GSH indeed showed the ability to upregulate GSH, consistent with previous results. Although Fer-1 also had this capability, it appeared to be less significant than AGEs (Fig. 5 E). Confocal microscopy analysis of red fluorescence indicated that metformin could reduce mitochondrial activity compared to the untreated group. The fluorescence intensity in the diabetes breast cancer model was relatively higher than in regular breast cancer cells, and Fer-1 could partially restore the red fluorescence reduction caused by metformin (Fig. 5 F). In summary, we believe that ferroptosis occurs in breast cancer cells and the diabetes breast cancer cell model under metformin treatment. Fer-1, as a ferroptosis inhibitor, can partially resist the ferroptosis induced by metformin. Similarly, the level of ferroptosis can also be resisted by our AGEs-established diabetic model. Neither can completely reverse this process. 4. Discussion More than a decade since the discovery of ferroptosis, a programmed cell death modality, its role in malignant tumors has gradually been recognized. Cancer cells, to promote growth, often have higher metabolic levels and require more iron compared to normal cells, making them more sensitive to ferroptosis 25 . In our study, we have demonstrated that metformin can selectively inhibit the growth of breast cancer through iron-dependent programmed cell death. Apart from breast cancer cells, normal mammary cell is less sensitive. Overall, our study indicates that metformin induces breast cancer death through ferroptosis. In our experiments with MCF-7 and MDA-MB-231 cells, both showed evident occurrence of ferroptosis with metformin treatment. However, With the addition of ages only, they diverged in the ferroptosis process. AGEs in MCF-7 cells showed a reduction in GSH, increase in MDA and total iron ions, a phenomenon completely opposite in the MDA-MB-231 cell line. We must consider the molecular typing of the two cells (MCF-7: ER+, PR+, HER2 0–1+; MDA-MB-231: ER-, PR-, HER2 0–1+) 26 . MFC-7 cells, being ER+, have lower sensitivity to ferroptosis due to the inhibitory effect of estrogen receptors on transferrin receptors 27 . Under the action of the estrogen receptor antagonist Fulvestrant, ER + breast cancer becomes more sensitive to breast cancer 28 . In GSH detection experiments, the GSH content of the MCF-7 control group was almost twice that of MDA-MB-231 cells, suggesting that ER + MCF-7 cells have stronger antioxidant capacity. Under the synergistic effect of AGEs and metformin, the downward trend of GSH was more pronounced. While in MDA-MB-231 cells, the downregulation of GSH under the antagonistic effect of AGEs and metformin on ferroptosis was more moderate. This phenomenon occurred in the opposite trend in the MDA detection results. The regulation of ferroptosis by AGEs does not fully align with our proliferation tests. We cannot entirely attribute the inhibitory effect of metformin on the proliferation of breast cancer cells to ferroptosis, as it is not solely ferroptosis at play, but also apoptosis, etc 4 – 7 . AGEs can not only promote the proliferation of breast cancer cells but also inhibit the toxic effects of metformin to a certain extent, consistent in both MCF-7 and MDA-MB-231. Research on AGEs and ferroptosis is scarce, but the receptor for advanced glycation end products (AGER) in type 2 diabetes is upregulated, and the polymorphism of AGER is manifested in diabetic complications 29 , 30 . Studies have reported that the absence of ager has shown to inhibit the sensitivity to ferroptosis in pancreatitis, while the insulin receptor (INSR) does not participate in the ferroptosis process 31 . However, TCGA data results are inconsistent, with AGER expression in breast cancer tissues positively correlated with GPX4, and negatively correlated with ACSL4, ACSL1, LPCAT3, etc. (Fig. 2 A), which is closer to our detection results in MDA-MB-231 cells. On the other hand, in both type 1 and type 2 diabetes patients, the insulin-like growth factor IGF-1 is often expressed as reduced, and supplementing IGF-1 can improve the symptoms of reduced glucose tolerance in type 2 diabetes patients 32 , 33 . According to our bioinformatics results, the reduction of IGF-1 may be accompanied by the decrease of ACSL1, ACSL4, LPCAT3, and the increase of GPX4. Therefore, under diabetic conditions, with increased serum Ager and decreased IGF-1, breast cancer tissues may be more likely to exhibit relative insensitivity to ferroptosis. Metformin, as the most common oral medication, primarily exerts its blood glucose-lowering function by regulating the mitochondrial pathway AMPK pathway 34 . In the field of tumors, metformin can inhibit tumor proliferation in vitro by activating the adenosine monophosphate-activated protein kinase AMPK or inhibiting the mammalian target of rapamycin mTOR. Furthermore, metformin can induce apoptosis and pyroptosis in breast cancer cells in vitro through AMPK, with mitochondrial dysfunction involved in these programmed cell death processes 4 , 35 . Although studies on metformin causing tumor cell ferroptosis are not numerous, some research indicates that metformin activation of AMPK phosphorylation has led to a decrease in AMPK expression, promoting the process of ferroptosis 16 . The role of AMPK in this aspect is somewhat different from what was imagined. Setting this aside, metformin-induced ferroptosis in breast cancer cells is related to epigenetics, including LncRNA, miRNA, and ubiquitination 14 – 16 . As time progresses, with the standardization of non-coding RNA technologies and functional research, these may have potential clinical applications. In conclusion, this study is the first to simulate a diabetic environment with high glucose levels using AGEs, exploring the impact of the diabetic environment on ferroptosis in breast cancer cells, and proving that metformin can induce ferroptosis in diabetic breast cancer. Additionally, AGEs exhibit different sensitivities to ferroptosis in various types of breast cancer cells. The results have been validated in human tissues, explaining the mechanistic reasons for metformin improving the prognosis of diabetic breast cancer patients, and indicating that metformin is a potential drug for breast cancer treatment. Declarations Acknowledgements Not applicable. Authors Contributions CL and XW conceived the study. XL was responsible for the experimental organization of tissue sections, TC completed the bioinformatics content, TC, YL, CZ, and CT were responsible for the cell experiments. TC processed the experimental data and created the statistical charts. TC and CL jointly analyzed the experimental results and wrote the manuscript. All authors participated in the revision of the manuscript to ensure the accuracy of its content. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding The work was supported by the Wu Jieping Medical Foundation clinical research fund (320.6750.2023-18-72) and Jiangsu Province university key laboratory open research project (XZSYSKF2022008). Ethics approval and consent to participate This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Xuzhou Central Hospital. Our research complies with the exemption from informed consent conditions set by the Ethics Committee of Xuzhou Central Hospital: 1. The medical records or biological specimens used in this study were obtained during past clinical diagnosis and treatment. 2. The risk to the subjects in this study does not exceed minimal risk * . 3. The exemption from informed consent will not adversely affect the rights and health of the subjects. 4. The privacy and personal identity information of the subjects are protected. 5. This study does not utilize medical records and specimens that patients/subjects have previously explicitly refused to use. *Minimal Risk: Refers to the anticipated risk in the trial, in terms of probability and magnitude, not exceeding that of everyday life, or the risk associated with routine physical examinations or psychological tests Consent to Participate Not applicable. References Sung, H. et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA A Cancer J Clinicians 71, 209–249 (2021). Lei, S. et al. Breast cancer incidence and mortality in women in China: temporal trends and projections to 2030. Cancer Biol Med 18, 900–909 (2021). Wolff, A. C. et al. Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Focused Update. Arch Pathol Lab Med 142, 1364–1382 (2018). Neamati, D. et al. Metformin synergistically increases the anticancer effects of lapatinib through induction of apoptosis and modulation of Akt/AMPK pathway in SK-BR3 breast cancer cell line. Iran J Basic Med Sci 24, 1529–1537 (2021). Varghese, S., Samuel, S. M., Varghese, E., Kubatka, P. & Büsselberg, D. High Glucose Represses the Anti-Proliferative and Pro-Apoptotic Effect of Metformin in Triple Negative Breast Cancer Cells. Biomolecules 9, 16 (2019). Huynh, T. Y. L. et al. Metformin Induces PRODH/POX-Dependent Apoptosis in Breast Cancer Cells. Front Mol Biosci 9, 869413 (2022). Huynh, T. Y. L. et al. Metformin Treatment or PRODH/POX-Knock out Similarly Induces Apoptosis by Reprograming of Amino Acid Metabolism, TCA, Urea Cycle and Pentose Phosphate Pathway in MCF-7 Breast Cancer Cells. Biomolecules 11, 1888 (2021). Yenmiş, G. et al. Metformin promotes apoptosis in primary breast cancer cells by downregulation of cyclin D1 and upregulation of P53 through an AMPK-alpha independent mechanism. Turk J Med Sci 51, 826–834 (2021). Wang, J.-C. et al. Metformin inhibits metastatic breast cancer progression and improves chemosensitivity by inducing vessel normalization via PDGF-B downregulation. J Exp Clin Cancer Res 38, 235 (2019). Hampsch, R. A. et al. AMPK Activation by Metformin Promotes Survival of Dormant ER + Breast Cancer Cells. Clin Cancer Res 26, 3707–3719 (2020). Cha, J.-H. et al. Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1. Mol Cell 71, 606–620.e7 (2018). Min, W. L. et al. A ROS/Akt/NF-κB Signaling Cascade Mediates Epidermal Growth Factor-Induced Epithelial-Mesenchymal Transition and Invasion in Human Breast Cancer Cells. World J Oncol 13, 289–298 (2022). Tan, M. et al. Inhibiting ROS-TFE3-dependent autophagy enhances the therapeutic response to metformin in breast cancer. Free Radic Res 52, 872–886 (2018). Chen, J. et al. Metformin may induce ferroptosis by inhibiting autophagy via lncRNA H19 in breast cancer. FEBS Open Bio 12, 146–153 (2022). Hou, Y., Cai, S., Yu, S. & Lin, H. Metformin induces ferroptosis by targeting miR-324-3p/GPX4 axis in breast cancer. Acta Biochim Biophys Sin (Shanghai) 53, 333–341 (2021). Yang, J. et al. Metformin induces Ferroptosis by inhibiting UFMylation of SLC7A11 in breast cancer. J Exp Clin Cancer Res 40, 206 (2021). Wahdan-Alaswad, R. et al. Glucose promotes breast cancer aggression and reduces metformin efficacy. Cell Cycle 12, 3759–3769 (2013). Pan, S. et al. Advanced glycation end products correlate with breast cancer metastasis by activating RAGE/TLR4 signaling. BMJ Open Diabetes Res Care 10, e002697 (2022). Kwak, T. et al. Targeting of RAGE-ligand signaling impairs breast cancer cell invasion and metastasis. Oncogene 36, 1559–1572 (2017). Mortimer, J. E. & Seewaldt, V. Who Will Benefit From Metformin? JAMA Oncol 8, 979–981 (2022). Goodwin, P. J. et al. Effect of Metformin Versus Placebo on New Primary Cancers in Canadian Cancer Trials Group MA.32: A Secondary Analysis of a Phase III Randomized Double-Blind Trial in Early Breast Cancer. J Clin Oncol 41, 5356–5362 (2023). Lee, J., You, J. H., Kim, M.-S. & Roh, J.-L. Epigenetic reprogramming of epithelial-mesenchymal transition promotes ferroptosis of head and neck cancer. Redox Biol 37, 101697 (2020). Ouyang, S. et al. Inhibition of STAT3-ferroptosis negative regulatory axis suppresses tumor growth and alleviates chemoresistance in gastric cancer. Redox Biology 52, 102317 (2022). Lee, J., Yun, J.-S. & Ko, S.-H. Advanced Glycation End Products and Their Effect on Vascular Complications in Type 2 Diabetes Mellitus. Nutrients 14, 3086 (2022). Dixon, S. J. & Stockwell, B. R. The role of iron and reactive oxygen species in cell death. Nat Chem Biol 10, 9–17 (2014). Subik, K. et al. The Expression Patterns of ER, PR, HER2, CK5/6, EGFR, Ki-67 and AR by Immunohistochemical Analysis in Breast Cancer Cell Lines. Breast Cancer (Auckl) 4, 35–41 (2010). Yu, H. et al. Sulfasalazine–induced ferroptosis in breast cancer cells is reduced by the inhibitory effect of estrogen receptor on the transferrin receptor. Oncol Rep 42, 826–838 (2019). Liang, D. et al. Ferroptosis surveillance independent of GPX4 and differentially regulated by sex hormones. Cell 186, 2748–2764.e22 (2023). Phimphilai, M., Pothacharoen, P. & Kongtawelert, P. Age-Influenced Receptors of Advanced Glycation End Product Overexpression Associated With Osteogenic Differentiation Impairment in Patients With Type 2 Diabetes. Front Endocrinol (Lausanne) 12, 726182 (2021). Lindholm, E. et al. Association between LTA, TNF and AGER Polymorphisms and Late Diabetic Complications. PLoS ONE 3, e2546 (2008). Yang, L. et al. Extracellular SQSTM1 exacerbates acute pancreatitis by activating autophagy-dependent ferroptosis. Autophagy 19, 1733–1744 (2023). Kolaczynski, J. W. & Caro, J. F. Insulin-like growth factor-1 therapy in diabetes: physiologic basis, clinical benefits, and risks. Ann Intern Med 120, 47–55 (1994). Palta, M., LeCaire, T. J., Sadek-Badawi, M., Herrera, V. M. & Danielson, K. K. The trajectory of IGF-1 across age and duration of type 1 diabetes. Diabetes Metab Res Rev 30, 777–783 (2014). Shaw, R. J. et al. The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science 310, 1642–1646 (2005). Zheng, Z., Bian, Y., Zhang, Y., Ren, G. & Li, G. Metformin activates AMPK/SIRT1/NF-κB pathway and induces mitochondrial dysfunction to drive caspase3/GSDME-mediated cancer cell pyroptosis. Cell Cycle 19, 1089–1104 (2020). Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.tif Supplementary Fig.1 A The triple negative breast cancer cell MDA-MB-231was treated with ages 200mg/L, ages 200mg/L+ metformin 5mM and ages 200mg/L+ metformin 10mM for 48h. Then href="https://www.baidu.com/s?wd=scratched&usm=2&ie=utf-8&rsv_pq=c52ec2f2003ecfb2&oq=scratch%E8%BF%87%E5%8E%BB%E5%BD%A2%E6%80%81&rsv_t=d1d5QoeuE5Vtfi%2B8FSfkv7j3xMVe1bRJb7j0%2BtNR0LNO5PZgXU9%2FvZxGDdA&sa=re_dqa_dda&icon=1" target="_self">scratched the cell-culture dishes and observe them. B The triple negative breast cancer cell MDA-MB-231was treated with ages 200mg/L, ages 200mg/L+ metformin 5mM and ages 200mg/L+ metformin 10mM for 48h. Then fixed, stained and observed Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-4588932","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":320644229,"identity":"839c4835-cf4b-44ac-9f7a-3cf10a87290d","order_by":0,"name":"Tao Chen","email":"","orcid":"","institution":"The Xuzhou Clinical College of Xuzhou Medical University, Xuzhou Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Chen","suffix":""},{"id":320644231,"identity":"9fdfb384-1d05-42d0-babb-89e7de646a39","order_by":1,"name":"Xiaoxin Li","email":"","orcid":"","institution":"Xuzhou Central Hospital, The Affiliated Xuzhou Hospital of Medical College of Southeast University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxin","middleName":"","lastName":"Li","suffix":""},{"id":320644233,"identity":"2227788f-bdc1-4aba-b346-9c687932b25a","order_by":2,"name":"Yuanyuan Li","email":"","orcid":"","institution":"The Xuzhou Clinical College of Xuzhou Medical University, Xuzhou Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Li","suffix":""},{"id":320644235,"identity":"c59b3e6a-c89d-45b6-8980-59ca85a56f50","order_by":3,"name":"Chunyan Zhou","email":"","orcid":"","institution":"The Xuzhou Clinical College of Xuzhou Medical University, Xuzhou Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chunyan","middleName":"","lastName":"Zhou","suffix":""},{"id":320644236,"identity":"dddca176-c77f-40f7-892e-c436364f6479","order_by":4,"name":"Chuangang Tang","email":"","orcid":"","institution":"Xuzhou Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chuangang","middleName":"","lastName":"Tang","suffix":""},{"id":320644237,"identity":"552ee118-800a-4ff6-89e7-763e7054a4d2","order_by":5,"name":"Xiang Wang","email":"","orcid":"","institution":"Xuzhou Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiang","middleName":"","lastName":"Wang","suffix":""},{"id":320644239,"identity":"50a9d9c6-5679-44f7-8cf9-8c34b9526da3","order_by":6,"name":"Changwen Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACPmYGNgaGCjY5BnbGBuK0sIG1nOEzZmAmWgsIMbbJJTYwE+swNnbmZ495zpil9zczt0nz7mCQ5xc7QMhhbObGPBVpuTMOMwK1nGEwnDk7gZAWHjbpnDPHchvAWtoYEgxuE6Mlt+1/ujypWtgSDEjQwmYm/ecMm+HGw4zNlnPbJAj7hZ//8DPJGRVs8nLH2x/eeNtmI88vTUALMmCRYGCQIF45CDB/IE39KBgFo2AUjBQAAI6LNckKTBFbAAAAAElFTkSuQmCC","orcid":"","institution":"Xuzhou Central Hospital","correspondingAuthor":true,"prefix":"","firstName":"Changwen","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2024-06-16 08:10:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4588932/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4588932/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60353579,"identity":"a4dd48ab-2866-4a17-8585-fb700d7ed4d8","added_by":"auto","created_at":"2024-07-15 23:41:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2497472,"visible":true,"origin":"","legend":"\u003cp\u003eBreast cancer patients using metformin experience ferroptosis. All experimental results in Figure 1 are from the same patient within the same column. From left to right, the molecular subtypes of breast cancer patients are as follows: Luminal A, HER2-enriched, Triple-negative, Triple-negative, Luminal B. \u003cstrong\u003eA \u003c/strong\u003eHematoxylin and Eosin (H\u0026amp;E) staining was used to examine the morphology of breast cancer cells, observed under a 4X microscope, with a scale bar of 500µm. \u003cstrong\u003eB-C \u003c/strong\u003eImmunohistochemistry was performed to detect the expression of PKM2 (Pyruvate kinase isozyme type M2) and GPX4 (Glutathione peroxidase 4) in breast cancer, observed under 4X and 20X microscopes, with scale bars of 500µm and 100µm, respectively. \u003cstrong\u003eD\u003c/strong\u003e Prussian blue staining was employed to observe iron ion deposition in breast cancer tissue, observed under 4X and 10X microscopes, with scale bars of 500µm and 200µm, respectively.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4588932/v1/aa060523c106dbacfe261df3.png"},{"id":60353946,"identity":"64e4faff-f305-4061-98d3-a7c64441b3af","added_by":"auto","created_at":"2024-07-15 23:49:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":209662,"visible":true,"origin":"","legend":"\u003cp\u003eDiabetes may inhibit ferroptosis from breast cancer. \u003cstrong\u003eA\u003c/strong\u003e Gene correlation analysis of breast cancer patient transcriptome data. The LinearRegression method was used, with P\u0026lt;0.05 indicating statistically significant. \u003cstrong\u003eB\u003c/strong\u003e Analysis of iron death-related gene expression in breast cancer molecular subtypes: Luminal, ERBB2+ (receptor tyrosine kinase 2, same as HER2-enriched), TNBC (triple-negative). The two-sample t-test method was used, with P\u0026lt;0.05 indicating statistically significant. P\u0026gt;=0.05 denoted as \"ns\", P \u0026lt; 0.05 as \"*\", P \u0026lt; 0.01 as \"**\", and P \u0026lt; 0.001 as \"***\". \u003cstrong\u003eC\u003c/strong\u003e In the transcriptomic data of breast cancer patients, gene expression levels in the top 50% are classified as High-expression, and the bottom 50% as low-expression. The Log-rank test method was used, with P \u0026lt; 0.05 indicating statistical significance.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4588932/v1/faac5f713e65b0c6d17d4d67.png"},{"id":60353577,"identity":"3d6b01a9-7b44-4db8-a47f-7dc31accfd6e","added_by":"auto","created_at":"2024-07-15 23:41:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":159936,"visible":true,"origin":"","legend":"\u003cp\u003eMetformin can induce the cell death of diabetic breast. \u003cstrong\u003eA -C\u003c/strong\u003e Breast cancer cell lines (MDA-MB-231 and MCF-7) and the mammary cell line (MCF-10A) treated with Ages (0,100,200,400,800mg/L) for 48h. \u003cstrong\u003eD\u003c/strong\u003e Dose effect of Metformin on the proliferation of the triple-negative breast cancer cell lines (MDA-MB- 231 and MCF-7) and the mammary cell line (MCF-10A) at 48 h. The diabetic model (DM) added Ages 200mg/L extra. \u003cstrong\u003eE\u003c/strong\u003e Mean IC\u003csub\u003e50\u003c/sub\u003e value of metformin in different cell lines (MDA-MB-231 , MCF-7 and MCF-10A).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4588932/v1/cc9f868b7abede157cd98397.png"},{"id":60353947,"identity":"105dc26e-4d70-4d1a-9fa2-bb8c643bdeff","added_by":"auto","created_at":"2024-07-15 23:49:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1662927,"visible":true,"origin":"","legend":"\u003cp\u003eMetformin's Inhibitory Effect May Be Related to Ferroptosis. \u003cstrong\u003eA\u003c/strong\u003e Colony formation of MDA-MB- 231 and MCF-7 cells treated with indicated concentration (ages 200mg/L, ages 200mg/L+ metformin 5mM and ages 200mg/L+ metformin 10mM) for 7 d. \u003cstrong\u003eB-G\u003c/strong\u003eThe triple negative breast cancer cell line MDA-MB-231 and the luminal cell line MCF-7 were treated with ages 200mg/L, ages 200mg/L+ metformin 5mM and ages 200mg/L+ metformin 10mM for 48h. The iron concentration, GSH concentration and MDA concentration were assayed. \u003cstrong\u003eH\u003c/strong\u003e The confocal fluorescence microscopy analysis of TMRM fluorescence of MDA-MB-231 cells line treated with indicated concentration (ages 200mg/L, ages 200mg/L+ metformin 5mM and ages 200mg/L+ metformin 10mM) for 48h.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4588932/v1/de1922f62c1f34f78ae352e3.png"},{"id":60353582,"identity":"4d258f3c-1abf-4188-b1e2-1715d66edeb6","added_by":"auto","created_at":"2024-07-15 23:41:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1030468,"visible":true,"origin":"","legend":"\u003cp\u003eMetformin Inhibits Breast Cancer Cells Through Ferroptosis. \u003cstrong\u003eA\u003c/strong\u003e Colony formation of MCF-7 cells treated with metformin (10mM) for 7d in the absence or presence of Ages (200mg/L) and Ferrostatin-1 (1 μM) for 7d. \u003cstrong\u003eB-E\u003c/strong\u003e MCF-7 cell line and MDA-MB-231 cell line treated with metformin (10mM) for 48h in the absence or presence of Ages (200mg/L) and Ferrostatin-1 (1 μM) for 48h. Then the cell viability , iron concentration and GSH concentration were assayed. \u003cstrong\u003eF\u003c/strong\u003eMDA-MB-231 cell line treated with metformin (10mM) for 48h in the absence or presence of Ages (200mg/L) and Ferrostatin-1 (1 μM) for 48h. The confocal fluorescence microscopy show the TMRM fluorescence.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4588932/v1/0f0d71b901edda8f97c8f4c8.png"},{"id":61997061,"identity":"30920632-1373-44fa-a0ce-57f7c919e962","added_by":"auto","created_at":"2024-08-08 04:47:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6669199,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4588932/v1/a8e5e0cd-0572-405b-bac7-4c53f762eda5.pdf"},{"id":60353581,"identity":"ded582ba-b8a5-43b8-9756-b774a0c31d80","added_by":"auto","created_at":"2024-07-15 23:41:57","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5028952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig.1\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e The triple negative breast cancer cell MDA-MB-231was treated with ages 200mg/L, ages 200mg/L+ metformin 5mM and ages 200mg/L+ metformin 10mM for 48h. Then \u003ca href=\"https://www.baidu.com/s?wd=scratched\u0026amp;usm=2\u0026amp;ie=utf-8\u0026amp;rsv_pq=c52ec2f2003ecfb2\u0026amp;oq=scratch%E8%BF%87%E5%8E%BB%E5%BD%A2%E6%80%81\u0026amp;rsv_t=d1d5QoeuE5Vtfi%2B8FSfkv7j3xMVe1bRJb7j0%2BtNR0LNO5PZgXU9%2FvZxGDdA\u0026amp;sa=re_dqa_dda\u0026amp;icon=1\" target=\"_self\"\u003escratched\u003c/a\u003e the cell-culture dishes and observe them. \u003cstrong\u003eB\u003c/strong\u003e The triple negative breast cancer cell MDA-MB-231was treated with ages 200mg/L, ages 200mg/L+ metformin 5mM and ages 200mg/L+ metformin 10mM for 48h. Then fixed, stained and observed\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4588932/v1/2ad83fe216ab479eb7c0988f.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metformin induces ferroptosis and suppresses malignant behaviors in diabetic breast cancer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBreast cancer is the most prevalent malignancy worldwide and ranks second in cancer-related deaths, posing a seriously threat to public health\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Particularly in China, due to aging populations, dietary changes, and shifts in reproductive patterns, the incidence of breast cancer is rising above the global average\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The current treatment system for breast cancer is relatively mature, offering suitable treatments based on molecular subtyping. Due to the limited accessibility of genetic testing methods, IHC/FISH-based histopathological detection is commonly used in clinical practice to classify breast cancer into Luminal, Her2-overexpressing, and triple-negative subtypes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Depending on the molecular subtype, patients will receive chemotherapy, endocrine therapy, and targeted therapy. The treatment options for triple-negative breast cancer are relatively limited, making the development of new treatment methods particularly important for breast cancer patients.\u003c/p\u003e \u003cp\u003eMetformin, an insulin sensitizer, is the most widely used oral hypoglycemic drug in clinical practice. It reduces blood glucose levels by inhibiting hepatic gluconeogenesis. Further research into metformin has revealed its potential role in the onset and progression of tumors. Its anticancer effects can be achieved through various mechanisms. The induction of apoptosis in breast cancer cells by metformin has been confirmed, and this process can be inhibited by a high-glucose environment\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Additionally, studies have suggested that metformin induces apoptosis in breast cancer cells by regulating amino acid metabolism, the TCA cycle, the urea cycle, and the pentose phosphate pathway, as well as through a PRODH/POX-dependent mechanism\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The AMPK pathway is the most common target of metformin, independently affecting the cell cycle protein D1 and P53-induced apoptosis in breast cancer cells\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Moreover, metformin can enhance the sensitivity of breast cancer to chemotherapy and endocrine therapy by regulating angiogenesis, inducing apoptosis, and modulating the AKT/AMPK pathway\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Lipid peroxidation is a key process in ferroptosis, and under certain conditions, lipid oxidation can serve as a preliminary step. Metformin promotes the survival and immune evasion of breast cancer cells by regulating the AMPK signaling pathway and fatty acid oxidation\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAs research into the mechanisms of metformin's action has progressed, it has shown unusual therapeutic potential in breast cancer ferroptosis. An increase in reactive oxygen species (ROS) leads to heightened oxidative stress within cells, resulting in lipid peroxidation and ferroptosis. Reports indicate that metformin can induce ROS production in breast cancer cells\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Further, metformin can target lncRNA H19 to induce ferroptosis in breast cancer cells, and inhibiting autophagy can regulate ferroptosis, further clarifying the relationship between autophagy and ferroptosis\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Other studies report that miR-324-3p, another non-coding RNA, is involved in the regulation of metformin-induced ferroptosis in breast cancer\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Additionally, metformin can regulate breast cancer cell ferroptosis by suppressing the ubiquitination level of SLC7A11 independently of the AMPK pathway\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. However, as a hypoglycemic drug, these studies have not elucidated the role and mechanism of metformin in inducing ferroptosis in breast cancer under diabetic conditions.\u003c/p\u003e \u003cp\u003eRegarding the impact of diabetes on the anticancer effects of metformin, scholars have shown that high glucose levels can promote the proliferation of breast cancer cells and inhibit the apoptosis induced by metformin\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Advanced glycation end-products (AGEs), pathological products formed from proteins, lipids, and nucleic acids in the Maillard reaction in the plasma of diabetic patients, not only play a crucial role in common macrovascular and microvascular complications of diabetes but can also promote the migration and invasion of breast cancer cells through the RAGE-mediated MEK-EMT and TLR4-MyD88 signaling pathways\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Thus, diabetes plays an important role in the development and progression of breast cancer. Recent clinical trials have indicated that metformin can improve the incidence and survival time of breast cancer in diabetic patients, but it has not had the same effect in non-diabetic patients, instead increasing the risk of new cancers in non-diabetic individuals\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. These findings suggest that the anticancer effects of metformin on breast cancer need to be closely linked to diabetes.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Cell Lines and Culture\u003c/h2\u003e \u003cp\u003eThe MCF-10A, MDA-MB-231, and MCF-7 cell lines were all acquired from Procell Life Science \u0026amp; Technology Co., Ltd. (Wuhan, China). The serum used for cell culture in this study was fetal bovine serum (Sigma, F0193).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Tissue Samples\u003c/h2\u003e \u003cp\u003eThe experiment involved five tissue samples, all of which were obtained from breast cancer surgeries performed at the Xuzhou Central Hospital's Department of Breast Surgery between May and October 2023. The samples were preserved in the form of paraffin blocks within the hospital's Department of Pathology. The use of the specimens was approved by the ethical review board of Xuzhou Central Hospital. Furthermore, the use of the specimens conformed to the ethical committee's criteria for exemption from informed consent, thereby waiving the need for patient consent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Establishment of Diabetic Breast Cancer Cell Model\u003c/h2\u003e \u003cp\u003eThe triple-negative breast cancer cell line MDA-MB-231, non-triple-negative breast cancer cell line MCF-7, and normal mammary epithelial cell line MCF-10A were selected. Breast cancer cells were cultured in high-glucose DMEM or RPMI medium, supplemented with advanced glycation end products (AGEs) to a concentration of 200 mg/L, until cell density reached 80%-95% for passaging. This process was repeated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Confocal Fluorescence Microscopy for Mitochondrial Activity\u003c/h2\u003e \u003cp\u003eTo verify mitochondrial activity, mitochondrial fluorescent dye working solution was mixed with the base culture medium to prepare a 0.3 \u0026micro;M/L working staining solution. This solution was applied to the breast cancer cell slides after treatment. A 10 \u0026micro;L aliquot of anti-fade mounting medium was dropped onto a glass slide, and the cell slide was inverted onto the glass slide and placed on the stage of a confocal microscope. The intensity of red fluorescence and bright field light was adjusted. The average fluorescence intensity was quantitatively analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 The glutathione (GSH) assay\u003c/h2\u003e \u003cp\u003eGSH assay utilized the Reduced Glutathione (GSH) Assay Kit from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. Cells were homogenized with PBS and lysed using ultrasonication. After centrifugation, the supernatant, GSH standards, and reagents were mixed and the optical density (OD) at 405 nm was measured in a 96-well plate. The GSH levels were calculated according to the formula provided in the manual.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 The cellular malondialdehyde (MDA) Assay\u003c/h2\u003e \u003cp\u003eMDA content was determined using the Cell Malondialdehyde (MDA) Assay Kit produced by Nanjing Jiancheng Bioengineering Institute. Cells were mixed with reagents, lysed, and centrifuged to obtain the supernatant. The supernatant was then mixed with reagents and the OD value at 532 nm was measured colorimetrically against MDA standards. The concentration of MDA was calculated using the formula provided by the manufacturer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Iron Ion Assay\u003c/h2\u003e \u003cp\u003eThe intracellular iron ion content was measured using the Total Iron Content Colorimetric Assay Kit from Applygen Technologies, Beijing, China. Cells were thoroughly mixed with cell lysis buffer and reacted, followed by centrifugation to collect the supernatant. The supernatant was mixed with prepared 4.5% potassium permanganate and reagents, incubated at 60\u0026deg;C for 1 hour, and the iron detection reagent was added. The OD value at 550 nm was measured in a 96-well plate against standards, and the total iron ion concentration within the cells was calculated using the formula provided in the manual.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Cell Proliferation and Toxicity Detection by CCK8\u003c/h2\u003e \u003cp\u003eCultured cells were digested, centrifuged, and resuspended. A 10 \u0026micro;L aliquot of the cell suspension was added to a hemocytometer, and cells were counted under a microscope. The total cell count was multiplied by (5 \\times 10^4/mL). A 96-well plate was prepared, with each well containing 200 \u0026micro;L of complete culture medium and 5,000 cells. After 24 hours, the medium was changed, and treatment drugs were added. After 48 hours, 180 \u0026micro;L of complete culture medium and 20 \u0026micro;L of CCK8 reagent were added to each well, mixed well, and incubated in a CO2 incubator for 1\u0026ndash;4 hours. The absorbance at 450 nm was measured with a microplate reader and the results were analyzed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Colony Formation Assay\u003c/h2\u003e \u003cp\u003eCell suspension was counted, and a 12-well plate was prepared, with each well containing 1000 \u0026micro;L of complete culture medium and 3,000 cells, mixed well. After 3 days, the medium was changed, and treatment drugs were added. The cells were cultured in an incubator for 5\u0026ndash;7 days. The culture medium was aspirated, and the wells were washed with PBS. A 1 mL aliquot of 4% paraformaldehyde was added for fixation for 20 minutes. The paraformaldehyde was aspirated, the wells were washed with PBS, and 1 mL of crystal violet staining solution was added for staining for 20 minutes. The wells were washed with PBS until the solution was no longer visibly purple, and images were taken under a transmitted light lamp to calculate the colony results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Cell Scratch Assay\u003c/h2\u003e \u003cp\u003eCells were treated with drugs for 48 hours and then switched to serum-free culture medium. A ruler and pipette were used to create a scratch. The bottom of the culture dish was marked with a marker pen to denote the location. The cells were then cultured in a carbon dioxide incubator, and photographs were taken now, and at 24 and 48 hours. The photos were appropriately processed to calculate the migration rate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Transwell Migration Assay\u003c/h2\u003e \u003cp\u003eCells were treated with serum-containing culture medium for 24 hours, after which the medium was discarded, and the cells were digested with trypsin to obtain a cell suspension. The cell suspension was centrifuged and resuspended for cell counting. A 12-well plate and Transwell chambers were prepared, with the upper chamber containing 400 \u0026micro;L of serum-free culture medium with 20,000 cells, and the lower chamber containing 600 \u0026micro;L of culture medium with 20% fetal bovine serum. The setup was placed in a CO2 incubator for 48 hours. The culture medium was discarded, and both chambers were fixed with 400 \u0026micro;L and 600 \u0026micro;L of 4% paraformaldehyde for 20 minutes, respectively, followed by a PBS wash. The lower chamber was stained with 800 \u0026micro;L of crystal violet staining solution for 10 minutes, the staining solution was discarded, and the chamber was washed with PBS until no obvious purple color remained. A cotton swab was used to gently remove the cells from the upper chamber, and the chamber was placed under a microscope for observation and analysis of the results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 HE Staining\u003c/h2\u003e \u003cp\u003eDried paraffin sections were sequentially placed in xylene, xylene, 100% alcohol, 100% alcohol, 95% alcohol, 90% alcohol, 80% alcohol, and 70% alcohol. The sections were stained in hematoxylin staining solution for 5 minutes, rinsed with running water, differentiated with 1% hydrochloric acid in ethanol for 5 seconds, and rinsed with running water until the sections turned blue. The sections were then placed in eosin staining solution for 2 minutes, followed by 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, anhydrous ethanol, anhydrous ethanol, xylene, and xylene, dried thoroughly, and sealed with neutral resin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Immunohistochemistry\u003c/h2\u003e \u003cp\u003eParaffin sections were dried and sequentially placed in xylene, xylene, 100% alcohol, 100% alcohol, 95% alcohol, 90% alcohol, 80% alcohol, and 70% alcohol. After deparaffinization, the slides were rinsed under running water, soaked in 3% hydrogen peroxide solution for 10 minutes, washed twice with running water (1 minute each time), and then placed in citrate buffer. The slides were heated in a microwave oven for 3 minutes (medium heat) until boiling, cooled to room temperature, and then reheated in the microwave oven and cooled to room temperature again. The slides were blocked with 2.5% BSA solution for 1 hour. Primary antibodies were added, and the slides were stored overnight in a 4\u0026deg;C refrigerator. The slides were washed three times in PBS, each time for 3 minutes, followed by secondary antibodies, and incubated in a 37\u0026deg;C incubator for 30 minutes. The slides were washed three times in PBS, each time for 3 minutes, and DAB solution was added. The slides were rinsed with clean water for 1 minute, stained with hematoxylin staining solution until the desired color depth was achieved. The slides were then rinsed in clean water, placed in 70% alcohol, 80% alcohol, 90% alcohol, 95% alcohol, 100% alcohol, 100% alcohol, xylene, and xylene, and placed in a fume hood. The slides were sealed with neutral resin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Data and Bioinformatics\u003c/h2\u003e \u003cp\u003eGene expression data and clinical data of breast cancer patients were downloaded from the TCGA database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://tcga-data.nci.nih.gov\u003c/span\u003e\u003cspan address=\"http://tcga-data.nci.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This included transcriptomic data, age, gender, estrogen receptor (ER) status, progesterone receptor (PR) status, human epidermal growth factor receptor 2 (Her2) status, and follow-up information. The R packages \"rjson\" and \"tidyverse\" were used to process gene expression data. \"survival\" and \"survminer\" were utilized to calculate Kaplan-Meier survival analysis results and to create visualizations. The log-rank test was employed to determine statistical significance. All other image creations and statistical analyses were performed using GraphPad Prism software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.graphpad-prism.cn\u003c/span\u003e\u003cspan address=\"https://www.graphpad-prism.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Differences were assessed using analysis of variance (ANOVA) or t-tests, with P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 indicating statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Breast cancer patients using metformin experience ferroptosis\u003c/h2\u003e \u003cp\u003eFerroptosis is an iron-dependent form of programmed cell death that regulates malignancies development and treatment\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Beyond apoptosis, ferroptosis provides a pathway for tumor principle research and offers effective guidance for the development of cancer treatment strategies. To understand the role of ferroptosis in breast cancer, we performed HE staining and immunohistochemical diagnosis on surgically removed tumor tissues from five breast cancer patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). All of them had been diagnosed with diabetes and three of these patients had been treated with metformin. We examined the key enzymes in glycolysis and important antioxidants in ferroptosis, observing that metformin users had relatively lower expression of PKM2 (Pyruvate Kinase M2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Interestingly, GPX4 expression was evident higher in Luminal-type breast cancer tissues compared to other types, and in TNBC (triple-negative). samples, using of metformin was associated with reduced GPX4 expression. Affected by the small sample size, we can only speculate that GPX4 expression in breast cancer is obvious related to molecular subtyping, and metformin use may play a role (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To further understand if there is a difference in ferroptosis, we also measured the iron ion content in tumor tissue sections. The results were more pronounced this time, with the metformin group showing a obvious accumulation of iron ions compared to the non-metformin group, suggesting potential differences in the level of ferroptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). However, we have also observed in the images that the deposition of iron ions in the tumor stroma is more pronounced than in the breast cancer tissue itself.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Diabetes may inhibit ferroptosis from breast cancer\u003c/h2\u003e \u003cp\u003eTo further investigate the expression of GPX4 in breast cancer and its direct connection with ferroptosis and diabetes, we analyzed gene transcription data of breast cancer tumor tissues from the TCGA database. We focused on the correlation between diabetes-related gene expression and ferroptosis-related gene expression. Our results indicate that IGF1 is negatively correlated with GPX4 and positively correlated with ACSL1, ACSL4, and LPCAT3, suggesting that IGF1 may promote the development of ferroptosis. Conversely, AGER showed the opposite trend, being positively correlated with GPX4 and negatively correlated with ACSL1, ACSL4, LPCAT3, and SLC7A11, which may be an important gene suppressing ferroptosis in diabetic patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Other genes related to glucose metabolism dysregulation, such as IGF1R, IGF2, IGF2R, IRS1, IRS2, and INSR, did not show a clear correlation with ferroptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the previous test results, we found that GPX4 expression seemed to be overexpressed in luminal breast cancer tissues, so we verified the results in the database. The transcription level of GPX4 in Luminal-type breast cancer tissues was higher than in ERBB2+ (receptor tyrosine kinase 2) and TNBC subtypes, with significant differences (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The lowest expression of GPX4 was observed in TNBC, and based on previous immunohistochemical results and iron ion measurements, we believe that triple-negative breast cancer may be the most sensitive subtype to ferroptosis. Additionally, we analyzed the relationship between relevant gene expression and survival time. Through K-M analysis, we found that low expression of AGER was associated with extended survival time, while the expression of ferroptosis-related genes did not show a significant correlation with survival time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Metformin can induce the cell death of diabetic breast\u003c/h2\u003e \u003cp\u003eAdvanced glycation end-products (AGEs), pathological products formed under the Maillard reaction from proteins, lipids, and nucleic acids in the plasma of diabetic patients, play a crucial role in common macrovascular and microvascular complications of diabetes and can also enhance the migration and invasion capabilities of breast cancer\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. We investigated the impact of AGEs on the proliferative capacity of breast cancer cells to select an appropriate concentration for simulating a diabetic environment with high-glucose (25mM) culture medium. It was evident that even at a concentration of 800mg/L, AGEs did not exhibit obvious toxicity, even MCF-10A cells showed a trend of increased proliferative capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Based on our laboratory's previous experience, we chose a concentration of 200mg/L AGEs as the suitable concentration for establishing a diabetic model.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe evaluated the effect of metformin on cell proliferation using two breast cancer cell lines (triple-negative and luminal) and a normal mammary cell line. The results showed that metformin inhibited the proliferation of all cell lines in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Notably, the IC50 values for metformin in the breast cancer cell lines (MCF-7, diabetic model MCF-7, MDA-MB-231, diabetic model MDA-MB-231, MCF-10A, diabetic model MCF-10A) were 36.29uM/L, 40.36uM/L, 19.31uM/L, 20.67uM/L, 50.52uM/L, and **( IC50 value cannot be calculated because the activity value is too high.), respectively(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Triple-negative cells were more sensitive, while normal mammary cell MCF-10A is the highest. Comparing cell types, under normal culture and diabetic model conditions, AGEs could resist the toxic effects of metformin to some extent, with this effect being most apparent in MCF-10A (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Thus, metformin exhibited toxic effects on both mammary and breast cancer cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Metformin's Inhibitory Effect May Be Related to Ferroptosis\u003c/h2\u003e \u003cp\u003eTo further study the inhibitory effect of metformin on breast cancer cells without strong toxicity subsequent results, we selected concentrations of 5mM and 10mM metformin for the following experiments. Firstly, colony formation assays continued to support our previous view that metformin can inhibit cell proliferation to a certain extent. Additionally, under the diabetic model, not only could cells tolerate metformin better, but they also exhibited enhanced proliferative capacity. In MCF-10 cells, the trend was similar to that of MCF-7, but the increase of iron ions was slower (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). On the other hand, the malignancy of tumors is also reflected in their migratory capacity. We verified the effects of metformin on MDA-MB-231 cells using scratch assays and Transwell migration experiments. Initially, advanced glycation end-products (AGEs) do not significantly alter the migratory ability of breast cancer cells; however, the effect of metformin is more evident. Concurrently, the inhibition of MDA-MB-231 cell migration by a 5mM concentration of metformin is gentler compared to that by a 10mM concentration of metformin (Figure S1A,S1B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBased on our previous immunohistochemical results and bioinformatics analysis, we hypothesized that ferroptosis occurred in breast cancer cells treated with metformin, whether in the control group or the diabetic model group. Therefore, we directly measured changes in the development process of ferroptosis. Starting with the iron ion levels in diabetic breast cancer cells, the results showed that the iron ion concentration in the MCF-7 diabetic model group was slightly higher than in the control group, and metformin significantly increased the iron ion levels in the diabetic model group, with the high-concentration metformin group showing higher iron ion levels than the low-concentration metformin group(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). And the MCF-10A showed the same change (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eDuring the development of ferroptosis, the level of oxidative stress within cells increases, lipid peroxidation begins, and the depletion of GSH is an important pathway. The GSH levels in MCF-7 cells were lower than in the control group, and metformin significantly inhibited GSH content(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In MDA-MB-231 cells, the GSH content in the diabetic model showed a trend opposite to MCF-7. Additionally, metformin's inhibitory effect on GSH was not pronounced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Malondialdehyde (MDA) is one of the lipid peroxidation products formed during the ROS oxidation of biomembranes. Interestingly, in the lipid peroxidation (MDA) assay, we found a similar phenomenon to the GSH assay, with MDA levels in the MCF-7 diabetic model showing an increasing trend, and metformin's effect on increasing MDA levels was somewhat concentration gradient-dependent(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). However, the MDA levels in the diabetic model MDA-MB-231 cells showed a decreasing trend, while metformin continued to significantly enhance lipid peroxidation capability (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eChanges in mitochondrial morphology and activity are also characteristics of ferroptosis. Using the cationic fluorescent probe TMRM to detect mitochondrial membrane potential (MMP), which can specifically identify MMP and thus adhere to mitochondria, producing red fluorescence. Confocal microscopy analysis of fluorescence showed that in MDA-MB-231 cells, compared to the control group, the fluorescence intensity of the diabetic model group was relatively increased, while breast cancer cells treated with metformin had lower red fluorescence intensity, with the high-concentration metformin treatment group having the lowest (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). These data are consistent with the above experimental results, indicating that metformin may induce breast cancer cell death through ferroptosis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Metformin Inhibits Breast Cancer Cells Through Ferroptosis\u003c/h2\u003e \u003cp\u003eIn the subsequent experiment, we introduced the ferroptosis inhibitor, ferrostatin-1 (Fer-1), to investigate its impact on cellular proliferation capabilities. Initially, in the assessment of clonogenic proliferation, the addition of metformin resulted in a clonal area that was smaller compared to the control group. However, with the application of ferrostatin-1, there was a noticeable increase in clonal area(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Similar outcomes were observed in the evaluation of cellular viability, where ferrostatin-1 exhibited a certain degree of resistance to the proliferation inhibition induced by metformin(Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). These results suggest that ferrostatin-1 may counteract the antitumor effects of metformin by inhibiting ferroptosis, thereby promoting cell proliferation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also discovered that metformin significantly increases intracellular iron accumulation. Both the diabetic model and Fer-1 could reduce iron accumulation, and both had a synergistic effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). In MDA-MB-231 cells, AGEs and GSH indeed showed the ability to upregulate GSH, consistent with previous results. Although Fer-1 also had this capability, it appeared to be less significant than AGEs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Confocal microscopy analysis of red fluorescence indicated that metformin could reduce mitochondrial activity compared to the untreated group. The fluorescence intensity in the diabetes breast cancer model was relatively higher than in regular breast cancer cells, and Fer-1 could partially restore the red fluorescence reduction caused by metformin (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eIn summary, we believe that ferroptosis occurs in breast cancer cells and the diabetes breast cancer cell model under metformin treatment. Fer-1, as a ferroptosis inhibitor, can partially resist the ferroptosis induced by metformin. Similarly, the level of ferroptosis can also be resisted by our AGEs-established diabetic model. Neither can completely reverse this process.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMore than a decade since the discovery of ferroptosis, a programmed cell death modality, its role in malignant tumors has gradually been recognized. Cancer cells, to promote growth, often have higher metabolic levels and require more iron compared to normal cells, making them more sensitive to ferroptosis\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. In our study, we have demonstrated that metformin can selectively inhibit the growth of breast cancer through iron-dependent programmed cell death. Apart from breast cancer cells, normal mammary cell is less sensitive. Overall, our study indicates that metformin induces breast cancer death through ferroptosis.\u003c/p\u003e \u003cp\u003eIn our experiments with MCF-7 and MDA-MB-231 cells, both showed evident occurrence of ferroptosis with metformin treatment. However, With the addition of ages only, they diverged in the ferroptosis process. AGEs in MCF-7 cells showed a reduction in GSH, increase in MDA and total iron ions, a phenomenon completely opposite in the MDA-MB-231 cell line. We must consider the molecular typing of the two cells (MCF-7: ER+, PR+, HER2 0\u0026ndash;1+; MDA-MB-231: ER-, PR-, HER2 0\u0026ndash;1+) \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. MFC-7 cells, being ER+, have lower sensitivity to ferroptosis due to the inhibitory effect of estrogen receptors on transferrin receptors\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Under the action of the estrogen receptor antagonist Fulvestrant, ER\u0026thinsp;+\u0026thinsp;breast cancer becomes more sensitive to breast cancer\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In GSH detection experiments, the GSH content of the MCF-7 control group was almost twice that of MDA-MB-231 cells, suggesting that ER\u0026thinsp;+\u0026thinsp;MCF-7 cells have stronger antioxidant capacity. Under the synergistic effect of AGEs and metformin, the downward trend of GSH was more pronounced. While in MDA-MB-231 cells, the downregulation of GSH under the antagonistic effect of AGEs and metformin on ferroptosis was more moderate. This phenomenon occurred in the opposite trend in the MDA detection results.\u003c/p\u003e \u003cp\u003eThe regulation of ferroptosis by AGEs does not fully align with our proliferation tests. We cannot entirely attribute the inhibitory effect of metformin on the proliferation of breast cancer cells to ferroptosis, as it is not solely ferroptosis at play, but also apoptosis, etc\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. AGEs can not only promote the proliferation of breast cancer cells but also inhibit the toxic effects of metformin to a certain extent, consistent in both MCF-7 and MDA-MB-231. Research on AGEs and ferroptosis is scarce, but the receptor for advanced glycation end products (AGER) in type 2 diabetes is upregulated, and the polymorphism of AGER is manifested in diabetic complications \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Studies have reported that the absence of ager has shown to inhibit the sensitivity to ferroptosis in pancreatitis, while the insulin receptor (INSR) does not participate in the ferroptosis process \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. However, TCGA data results are inconsistent, with AGER expression in breast cancer tissues positively correlated with GPX4, and negatively correlated with ACSL4, ACSL1, LPCAT3, etc. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), which is closer to our detection results in MDA-MB-231 cells. On the other hand, in both type 1 and type 2 diabetes patients, the insulin-like growth factor IGF-1 is often expressed as reduced, and supplementing IGF-1 can improve the symptoms of reduced glucose tolerance in type 2 diabetes patients\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. According to our bioinformatics results, the reduction of IGF-1 may be accompanied by the decrease of ACSL1, ACSL4, LPCAT3, and the increase of GPX4. Therefore, under diabetic conditions, with increased serum Ager and decreased IGF-1, breast cancer tissues may be more likely to exhibit relative insensitivity to ferroptosis.\u003c/p\u003e \u003cp\u003eMetformin, as the most common oral medication, primarily exerts its blood glucose-lowering function by regulating the mitochondrial pathway AMPK pathway\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In the field of tumors, metformin can inhibit tumor proliferation in vitro by activating the adenosine monophosphate-activated protein kinase AMPK or inhibiting the mammalian target of rapamycin mTOR. Furthermore, metformin can induce apoptosis and pyroptosis in breast cancer cells in vitro through AMPK, with mitochondrial dysfunction involved in these programmed cell death processes \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Although studies on metformin causing tumor cell ferroptosis are not numerous, some research indicates that metformin activation of AMPK phosphorylation has led to a decrease in AMPK expression, promoting the process of ferroptosis \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The role of AMPK in this aspect is somewhat different from what was imagined. Setting this aside, metformin-induced ferroptosis in breast cancer cells is related to epigenetics, including LncRNA, miRNA, and ubiquitination\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. As time progresses, with the standardization of non-coding RNA technologies and functional research, these may have potential clinical applications.\u003c/p\u003e \u003cp\u003eIn conclusion, this study is the first to simulate a diabetic environment with high glucose levels using AGEs, exploring the impact of the diabetic environment on ferroptosis in breast cancer cells, and proving that metformin can induce ferroptosis in diabetic breast cancer. Additionally, AGEs exhibit different sensitivities to ferroptosis in various types of breast cancer cells. The results have been validated in human tissues, explaining the mechanistic reasons for metformin improving the prognosis of diabetic breast cancer patients, and indicating that metformin is a potential drug for breast cancer treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCL and XW conceived the study. XL was responsible for the experimental organization of tissue sections, TC completed the bioinformatics content, TC, YL, CZ, and CT were responsible for the cell experiments. TC processed the experimental data and created the statistical charts. TC and CL jointly analyzed the experimental results and wrote the manuscript. All authors participated in the revision of the manuscript to ensure the accuracy of its content.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work was supported by the Wu Jieping Medical Foundation clinical research fund (320.6750.2023-18-72) and Jiangsu Province university key laboratory open research project (XZSYSKF2022008).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Xuzhou Central Hospital.\u003c/p\u003e\n\u003cp\u003eOur research complies with the exemption from informed consent conditions set by the Ethics Committee of Xuzhou Central Hospital:\u003c/p\u003e\n\u003cp\u003e1. The medical records or biological specimens used in this study were obtained during past clinical diagnosis and treatment. 2. The risk to the subjects in this study does not exceed minimal risk\u003csup\u003e*\u003c/sup\u003e. 3. The exemption from informed consent will not adversely affect the rights and health of the subjects. 4. The privacy and personal identity information of the subjects are protected. 5. This study does not utilize medical records and specimens that patients/subjects have previously explicitly refused to use.\u003c/p\u003e\n\u003cp\u003e*Minimal Risk: Refers to the anticipated risk in the trial, in terms of probability and magnitude, not exceeding that of everyday life, or the risk associated with routine physical examinations or psychological tests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung, H. \u003cem\u003eet al.\u003c/em\u003e Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. 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Science 310, 1642\u0026ndash;1646 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng, Z., Bian, Y., Zhang, Y., Ren, G. \u0026amp; Li, G. Metformin activates AMPK/SIRT1/NF-κB pathway and induces mitochondrial dysfunction to drive caspase3/GSDME-mediated cancer cell pyroptosis. Cell Cycle 19, 1089\u0026ndash;1104 (2020).\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":"Metformin, Breast Cancer, Ferroptosis, Diabetes, Immunohistochemistry, Bioinformatics","lastPublishedDoi":"10.21203/rs.3.rs-4588932/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4588932/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigates the potential role of metformin in breast cancer treatment, especially its impact on ferroptosis\u0026mdash;an iron-dependent form of programmed cell death. Breast cancer is one of the most common malignancies globally, with limited treatment options, particularly for triple-negative breast cancer.\u003c/p\u003e \u003cp\u003eThe research involved analyzing tumor tissues from breast cancer patients. It was observed that the tumor tissues of diabetic patients treated with metformin had obvious iron accumulation, suggesting variations in the level of ferroptosis. Further analysis using gene transcription data from the TCGA database revealed correlations between diabetes-related genes and genes associated with ferroptosis. The experimental results indicated that metformin could evident inhibit the proliferation of breast cancer cells and induce ferroptosis in a diabetic model. Moreover, metformin was found to promote ferroptosis by affecting mitochondrial activity.\u003c/p\u003e \u003cp\u003eIn conclusion, the study suggests that metformin holds potential value in treating diabetic breast cancer, capable of suppressing tumor cell growth through the ferroptosis mechanism. These findings provide a new theoretical basis for using metformin as a treatment for breast cancer and lay the groundwork for future clinical applications.\u003c/p\u003e","manuscriptTitle":"Metformin induces ferroptosis and suppresses malignant behaviors in diabetic breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-15 23:41:52","doi":"10.21203/rs.3.rs-4588932/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":"b0ef62ad-2df5-4c20-835e-1c5b0702a7d5","owner":[],"postedDate":"July 15th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33901537,"name":"Biological sciences/Cancer/Breast cancer"},{"id":33901538,"name":"Biological sciences/Cell biology/Cell migration"}],"tags":[],"updatedAt":"2024-08-08T04:38:55+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-15 23:41:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4588932","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4588932","identity":"rs-4588932","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}