Human TNBC cell line MDA-MB-231, the normal breast epithelial cell line MCF-10A or the ER-positive breast cancer cell line MCF-7 were maintained in DMEM culture medium(Gibco) that supplied with 10% fetal bovin serum (FBS, Biological Industries) and 1% penicillin-streptomycin liquid (Solarbio). Cells were placed in a 37°C humidified atmosphere with 5% CO₂. The corresponding reagents ferrostatin-1 (#HY-100579), Erastin (#HY-15763), and RSL3 (#HY-100218A) were purchased from MedChemExpress. VPA (#PHR1061) was purchased from Sigma.

Small interfering RNAs (siRNA) targeting FDFT1 (siFDFT1, target sequence: GCA​GAA​TCT​TCC​CAA​CTG​T#1, GTG​CCT​GAA​TGA​ACT​TAT​A#2, GGA​GCA​GGT​ATG​TTA​AGA​A#3) and negative control oligonucleotide was obtained from RiboBio (China). The oligonucleotides were transfected to cells by using riboFECT CP Transfection (RiboBio, China) following the manufacturer’s description. An adequate number of cells were seeded into the culture wells of 6-well plates, with transfection initiated when the cell density achieved a level of 30%–50%. Subsequently, 5 µL of 20 µM siRNA stock solution (RiboBio, China) was combined with 120 µL of 1X riboFECT™CP Buffer (RiboBio, China) per well, and the mixture was carefully agitated. Thereafter, 12 µL of riboFECT™CP Reagent (RiboBio, China) was added, and the resulting solution was gently mixed before being incubated at room temperature for a duration of 15 min to form the transfection complex. Thereafter, the transfection complex was added to each well containing 1863 µL of DMEM medium. Finally, the plates were placed in a 5% CO₂ incubator set at 37°C for cultivation.

Cell viability was evaluated using the CCK-8 purchased from Ape Bio (K1018). In brief, cells were planted in 96-well plates (5 × 10³ cells/well). After 24 h drug and transfection were added at the selected dose to the culture medium. The medium was replaced with 100 μL fresh medium containing 10 μL CCK-8 reagent and incubated in a humidified incubator (37°C, 5% CO₂) for 2 h. The absorbance at 450 nm was checked by using a microplate reader (PerkinElmer, USA).

The cells were seeded into a 24-well plate at a density of 1 × 10⁴ cells per well, and three replicate wells were prepared for each treatment. These cultures were maintained in an incubator overnight. On the subsequent day, the culture medium containing 0 or 4 mmol/L concentrations of VPA was supplemented externally. Subsequently, the existing culture medium in each well was replaced, and the cultures were allowed to continue. Following a 24-h incubation period post-cell administration, the cells subjected to 24 h of drug exposure were harvested and quantified utilizing the Automated Cell Counter (BIO-RAD). thereupon, the cell counts in the wells were conducted concurrently over four consecutive days.

Each six-well plate was inoculated with 5 × 10² cells, and three replicate wells were created within the same concentration cohort. Subsequently, the plate was situated within an incubator for the duration of the culturing process. Upon achieving adherence of the cells to the plate, a vehicle containing the therapeutic agents was introduced into the extracellular milieu, subsequently replacing the incumbent medium across all wells. The medium was exhausted approximately 14 days subsequent to the commencement of drug administration, at which point the medium within the six-well plate was discarded. Each well was delicately rinsed with 1 mL of phosphate-buffered saline (PBS) three times, followed by the addition of pre-chilled methanol, which was allowed to incubate at ambient temperature for a duration of 15 min. Post-methanol treatment, the solvent was discarded and 1 mL of 10% crystal violet (Solarbio) was added to each well, incubating for half an hour. Ultimately, the crystal violet was delicately washed with tap water, and the six-holed plate was subsequently dried on a sheet of white paper for photographic purposes.

The cells were seeded in Petri dish. After treatment with different concentrations of VPA (0, 2 or 4 mM) for 24 h, the supernatant was discarded, subsequent to double-washing the Petri dishes with 1 mL of PBS each time, cellular contents from three Petri dishes were aggregated into three separately designated centrifuge tubes, subsequent to which the malondialdehyde (MDA) content within the cell lysates was determined utilizing the Lipid Peroxidation MDA Assay Kit (S0131S, Beyotime, China), in strict accordance with the provider’s recommended protocols. Finally, the OD value was measured at 532 nm by a microplate reader (Molecular Devices SPECTRAMAX, United States), and MDA levels of cells were presented as a percentage of the control.

Each well was inoculated with 3 × 10⁵ cells and cultured overnight in a incubator. On the subsequent day, reagents were introduced to treat the cell within the pores for a duration of 24 h. Subsequently, each well was subjected to a single washing with 1 mL of PBS, and the cells were subsequently collected in a centrifuge tube. A concentration of 50 µM of C11-BODIPY dye (Invitrogen,D3861) was diluted to 5 µM in DMEM. Thereafter, the existing medium was exchanged with the diluted solution of 5 µM of C11-BODIPY dye, which was then incubated at a temperature of 37°C in the absence of light for a period of 30 min. Following three PBS washes, the sample was re-suspended in 500 µL of PBS for subsequent computer-based testing. Each set of samples was passed through a mesh screen, placed on ice, and then subjected to flow cytometric analysis utilizing a 488 nm laser at the FL1 detector via flow cytometer (Beckman).

The cells treated with VPA were harvested and subjected to centrifugation, subsequent to which the supernatant was discarded. Protein lysate was then added to the cell pellet, followed by mixing on ice for a duration of 40 min. Upon achieving complete lysis of the cells, the lysate was subjected to low-temperature centrifugation at 12,000 rpm for 20 min in a centrifuge (Thermo Fisher), with an appropriate volume of supernatant being aspirated and the precipitate discarded. Subsequently, the BCA kit (Solarbio) was employed for protein extraction from the cell lysates, and the protein concentration in each group was determined in accordance with the manufacturer’s instructions. Afterward, calculated amounts of loading buffer and PBS were added to the extracted proteins, and the proteins were denatured by boiling in a protein sample boiler for 5 min as per the instructions. Post-cooling, the proteins were stored at −20°C in the refrigerator. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed with buffer solutions tailored to the varying molecular weights of the proteins. Samples were loaded onto the SDS-PAGE gel, following which the power supply’s positive and negative terminals were connected. An electrophoretic voltage of 80V was chosen and maintained until the gel run was completed. The gel was cut to the desired protein molecular weight, and the polyvinylidenefluoride (PVDF) membrane was activated in methanol for 30 s before being immersed in the membrane transfer solution. The gel was positioned in the transfer apparatus with the cathode at the bottom, followed by two layers of sponge, two layers of filter paper, the PVDF membrane, two layers of filter paper, two layers of sponge, and finally the anode. Electrophoretic transfer was conducted at a voltage of 90V for 90 min (the duration of which may be adjusted based on the molecular weight of the target protein). Upon completion of the transfer, the PVDF membrane was washed with PBST three times for 5 min each. The membrane was then incubated in a solution containing 5% skim milk (Solarbio) at room temperature for 1 hour. The anti-stock solution was serially diluted with TBST in accordance with the manufacturer’s guidelines, and subsequent incubation was performed with the respective primary antibodies at 4°C overnight. The primary antibodies employed were GAPDH (Affinity, 1:10,000), β-actin (Affinity, 1:500), TUBULIN (Affinity, 1:1000), FDFT1 (Abcam, 1:1000), GPX4 (Abcam, 1:1000), and SLC7A11 (Abcam, 1:1000; Affinity, 1:1000). Following primary antibody incubation, the membrane was subjected to a series of washes with PBST, intermittently agitated at room temperature, and cleaned three times for 15 min each. Subsequently, the secondary antibody was allowed to incubate at room temperature for 1 hour. The secondary antibody used was an Anti-rabbit IgG (Affinity, 1:5000). Upon completion of the secondary antibody incubation, the membrane was re-introduced to a shaking platform at room temperature and subjected to additional PBST washes, triply for 15 min each. Prior to visualization, the ECL chemiluminescent solution (NCM Biotech) was prepared in a 1:1 ratio of solution A to solution B, with precautions taken to prevent light exposure. The prepared luminous solution was then evenly applied to the PVDF membrane, and visualization was achieved using a gel Imager (BIO-RAD).

The processed cells were subsequently transferred to an electron microscopy fixative (Solarbio) which was subsequently returned to room temperature. Thereafter, the cells were re-suspended, followed by fixation at 4°C for the purposes of preservation and transportation. And then wash the cells using 0.1 mL PBS for 3 times, 15 min each. Cells avoid light post fixed with 1% OsO₄ (Ted Pella Inc 18,456) in 0.1 mL PBS for 2 h at room temperature. After remove OsO₄, the cells are rinsed in 0.1 mL PBS for 3 times, 15 min each. Dehydrate at room temperature as followed: 30% ethanol (Sinaopharm Group Chemical Reagent Co. Ltd. 100,092) for 20 min; 50% ethanol for 20min; 70% ethanol for 20 min; 80% ethanol for 20 min; 95% ethanol for 20 min; Two changes of 100% ethanol for 20 min; Finally two changes of acetone (Sinaopharm Group Chemical Reagent Co. Ltd. 100,004) for 15 min. Resin penetration and embedding as followed: acetone:EMBed 812 (SPI 90529-77-4) = 1:1 for 2–4 h at 37°C; acetone:EMBed 812 = 1:2 overnight at 37°C; pure EMBed 812 for 5–8 h at 37°C; Pour the pure EMBed 812 into the embedding models and insert the tissues into the pure EMBed 812, and then keep in 37°C oven overnight. The embedding models with resin and samples were moved into 60°C oven to polymerize for more than 48 h. And then the resin blocks were taken out from the embedding models for standby application at room temperature. The resin blocks were sectioned into 1.5M slices using a semi-thin microtome, subsequently stained with toluidine blue, and positioned under a light microscope for positioning. The resin blocks were cut to 60–80 nm thin on the ultramicrotome (Leica, Leica UC7) and the tissues were fished out onto the 150 meshes cuprum grids with formvar film. 2% uranium acetate saturated alcohol solution avoid light staining for 8 min, rinsed in 70% ethanol for 3 times and then rinsed in ultrapure water for 3 times. 2.6% Lead citrate avoid CO₂ staining for 8 min, and then rinsed with ultrapure water for 3 times. After dried by the filer paper, the cuprum grids were put into the grids board and dried overnight at room temperature. The cuprum grids are observed under TEM (HITACHI HT7800/HT7700) and take images.

An analytical RNA-sequencing expression profile was retrieved from the publicly accessible database for breast cancer within the Cancer Genome Atlas (TCGA) repository via the university of Alabama at Birmingham cancer data analysis portal (UALCAN). This enabled the investigation of FDFT1 expression across normal human specimens as well as within breast cancer specimens. Subsequently, a continuous effort was made to derive additional RNA-sequencing expression profiles from the aforementioned TCGA breast cancer database within UALCAN, with the objective of examining the FDFT1 expression across diverse subtypes of breast cancer specimens.

Total RNA was extracted using 1 mL Triquick Reagent (Solarbio), and the RNA was reverse transcribed with a PrimeScript™ RT reagent Kit with gDNA Eraser tcDNA Synthesis Kit (Takara,RR047A). Quantitative real-time PCR was performed using TB Green^® Premix Ex Taq™ II Kit (Takara,RR820A). The threshold cycle (Ct) values for each gene were normalized to those of GAPDH, and the 2^−ΔΔCT method was used for quantitative analysis. Target genes were PCR amplified using the following primers: Q-FDFT1-F (GCA​ACG​CAG​TGT​GCA​TAT​TTT) and Q-FDFT1-R (CGC​CAG​TCT​GGT​TGG​TAA​AGG); Q-GAPDH-F (GGC​TCT​CCA​GAA​CAT​CAT​C) and Q-GAPDH-R (CTC​TTC​CTC​TTG​TGC​TCT​TG).

Eight SPF nude mice, aged 3–5 weeks and weighing between 16–20 g, were procured from Changsheng Bio-technology Co., Ltd. These mice were randomly allocated into two distinct groups and provided with SPF bedding and feed for a period of 1 week to ensure adaptive acclimation. To instigate subcutaneous tumor formation in the mice, a suspension of 2 × 10⁶ MDA-MB-231 cells was combined with 50 µL Matrigel (Corning) and 100 µL PBS. This cell mixture was then injected subcutaneously into the right posterior flank of the immunodeficient nude mice. Once the tumors were established, the mice were randomly assigned to two groups, each comprising five mice. One group received a daily intraperitoneal injection of valproic acid at a concentration of 150, 300 mg/kg, while the control group was administered with saline solution. Body weight and tumor dimensions were monitored daily for a duration of 10 days. Following 10 days of intraperitoneal injection, the mice were humanely euthanized. The tumors were then excised, their weights and volumes measured, and a section of each tumor was promptly fixed in 10% buffered formalin for subsequent hematoxylin-eosin (HE) staining and immunohistochemistry (IHC) analysis.

The tissue sections were dehydrated sequentially in xylene, followed by anhydrous ethanol and 75% ethanol. Subsequently, they were gently washed with tap water and subjected to a high-resolution constant dye pretreatment solution for 1 min. Thereafter, the sections were stained with hematoxylin dye solution (Servicebio) for three to 5 min and gently rinsed with tap water. Hematoxylin differentiation solution (Servicebio) was applied for 2–5 s followed by tap water rinsing. This was followed by treatment with hematoxylin return to blue solution (Servicebio) for 2–5 s. After rinsing with running water, the sections were dehydrated with 95% alcohol for 1 minute and then stained with eosin dye solution (Servicebio) for 15 s. Finally, the tissue sections were immersed in anhydrous ethanol, n-butanol, and xylene successively, and sealed with a neutral gum. Microscopic imaging and analysis were subsequently conducted.

Put the sections into environmentally friendly dewaxing solution I 10 min → Environmentally friendly dewaxing solution II 10 min → Environmentally friendly dewaxing solution III 10 min → anhydrous ethanol I 5 min → anhydrous ethanol II 5 min → anhydrous ethanol III 5 min → distilled water in turn. During this process, the buffer should be prevented from excessive evaporation and should not be dried. After natural cooling, the slide was placed in PBS and washed by shaking on the decolorizing shaker for 3 times, 5 min each time. The slices were placed in 3% hydrogen peroxide solution, incubated at room temperature away from light for 25 min, and the slides were placed in PBS and washed three times on a decolorizing shaking table for 5 min each time. The tissue was uniformly covered with 3% BSA in the tissue chemical circle and closed at room temperature for 30 min. Gently shake off the sealing solution, add PBS to the section in a certain proportion of primary antibody, and the section is placed flat in a wet box at 4°C for overnight incubation. The slide was placed in PBS and washed by shaking on the decolorizing shaker for 3 times, 5 min each time. After the slices were slightly dried, the tissue was covered with the secondary antibody (HRP label) of the corresponding species of the primary antibody, and incubated at room temperature for 50 min. The slide was placed in PBS and washed by shaking on the decolorizing table for 3 times, 5 min each time. After the sections were slightly dried, the freshly prepared DAB color developing solution was added into the circle. The color developing time was controlled under the microscope. The positive color was brown and yellow, and the sections were rinsed with tap water to terminate the color development. Hematoxylin re-staining for about 3 min, washing with tap water, hematoxylin differentiation solution for a few seconds, rinse with tap water, hematoxylin return to blue solution, and rinse with running water. Put the slices into 75% alcohol for 5 min → 85% alcohol for 5 min → anhydrous ethanol for 5 min → anhydrous ethanol for 5 min → n-butanol for 5 min → xylene for 5 min to dehydrate and transparent, take the slices out of xylene to dry slightly, and seal the slices with glue. The results are interpreted under a white light microscope.

The experimental data were analyzed employing Graphpad Prism version 6.01 software. The mean value of the recorded data is represented as the mean ± standard deviation. The independent two-sample t-test was conducted for the comparison between the two sets of data, while one-way analysis of variance (ANOVA) was utilized for the comparison among three or more groups of data. The p-value of less than 0.05 was considered indicative of statistically significant differences.

To detect the cytotoxicity of VPA toward TNBC cell line MDA-MB-231 and luminal A cell line MCF-7 in vitro, we treated breast cancer cells with various concentrations of VPA (from 2 to 8 mM) for 24 h and determined the cell viability using CCK-8 assays. VPA exerted dose-dependent toxicity and proliferation inhibitory effects on MDA-MB-231 and MCF-7 cells (Figures 1A, B) but not normal breast epithelial cell line MCF-10A (Figure 1C), indicating VPA possessed selective anti-breast cancer properties. We used cell counting analysis to find that VPA treatment inhibited 63.04% (MDA-MB-231) and 71.93% (MCF-7) cell numbers in Figures 1D, E. Colony formation analysis showed that VPA treatment significantly inhibited the proliferation of MDA-MB-231 (67.85%) and MCF-7 (72.26%) cells (Figures 1F, G). Therefore, these data suggest that VPA shows potent anti-breast cancer activity, suggesting its potential as a therapeutic drug.

To determine whether VPA exerted an anti-cancer effect by promoting ferroptosis in breast cancer cells, we first investigated the effects of VPA on the lipid peroxidation of ferroptosis. We measured the levels of MDA and oxidized glutathione disulfide (GSSG)/GSH ratio in MDA-MB-231 and MCF-7 cells upon VPA treatment given that the levels of MDA and GSSG/GSH ratio increased when lipid peroxidation occurs. As shown in Figures 2A, B, with increasing VPA concentration, MDA content increased by 19.7 and 4.1 μmol/mg in MDA-MB-231 and MCF-7 cells, respectively. And intracellular GSSG/GSH ratio increased by 57.34% (MDA-MB-231) and 26.01% (MCF-7) in Figures 2C, D. Additionally, we employed BODIPY 581/591 undecanoic acid to detect lipid ROS upon VPA treatment. As shown by flow cytometry, a more significant change in C11-BODIPY signaling was observed in MDA-MD-231 cells following VPA treatment compared to MCF-7 cells, indicating lipid ROS induction (Figures 2E, F). To elucidate the underlying mechanisms by which VPA promotes ferroptosis, we examined the expression levels of key ferroptosis pathway components, SLC7A11 and GPX4. Our findings indicated that VPA markedly diminished the protein levels of SLC7A11 and GPX4 in MDA-MB-231 cells, as compared to the MCF-7 cells. (Figures 2G, H). Collectively, these data demonstrate that VPA potently facilitates ferroptosis in MDA-MB-231 cells relative to MCF-7 cells, and that the induction of ferroptosis in breast cancer cells by VPA is mediated through the SLC7A11-GPX4 signaling pathway.

Based on the aforementioned discoveries, our research persists in examining MDA-MB-231 cells, which exhibit heightened sensitivity to VPA-induced ferroptosis. At first, we investigated the effects of VPA on the morphological characteristics of ferroptosis in MDA-MB-231 cells. One of the unique morphological characteristics of ferroptosis is a decrease in mitochondrial volume and an increase in membrane density (Stockwell et al., 2020). TEM showed that VPA induced a decrease in mitochondrial volume and an increase in membrane density in MDA-MB-231 cells (Figure 3A). To investigate the role of VPA-induced ferroptosis, MDA-MB-231 cells were pre-treated with Fer-1, a known inhibitor of ferroptosis. revealed a modest increase in cell viability (approximately 13.3% and 22.74%, respectively) as illustrated in Figures 3B, C. The above results prove that the anti-cancer activity of VPA via ferroptosis induction. Furthermore, when combined with Fer-1, VPA-induced the inhibition of the levels of SLC7A11 and GPX4 was abolished (Figure 3D). Collectively, these data demonstrate VPA affects the proliferation and viability of MDA-MB-231 cells through the ferroptosis pathway.

Ferroptosis inducers such as Erastin and RSL3 have been demonstrated to curtail SLC7A11 and GPX4 expressions, respectively, thereby facilitating ferroptosis (Dixon et al., 2012). To verify VPA treatment-induced ferroptosis, we asked whether VPA would increase MDA-MB-231 cells sensitivity to ferroptosis inducers, Erastin and RSL3. We performed cell viability assay and colony formation assay to assess the effect of treatment. In Figure 4A, the cell viability assay showed that when MDA-MB-231 cells treated with VPA were further exposed to Erastin, the cell proliferation rate was significantly higher than that of the group of cells treated with VPA only. Similarly, Figure 4B shows that cell viability was significantly increased after the addition of RSL3 to VPA-treated MDA-MB-231 cells, indicating enhanced cell proliferation. In the cell colony formation assay, Figure 4C shows that in MDA-MB-231 cells, the combination of VPA and Erastin resulted in a significant increase in the number and size of cell colonies, indicating a significant enhancement of cell proliferation. Correspondingly, Figure 4D shows that treatment of VPA-pretreated MDA-MB-231 cells with RSL3 also resulted in a significant increase in cell colony formation. Likewise, Erastin and RSL3 treatment synergized with VPA and significantly suppressed SLC7A11 and GPX4 expression, respectively (Figures 4E, F). Collectively, these data demonstrate the anti-TNBC activity of VPA via ferroptosis induction., and combine treatment with VPA and ferroptosis inducers result in synergistic response in MDA-MB-231 cells.

In order to acquire an initial insight into FDFT1 expression patterns in human breast cancer, we screened TCGA databases via UALCAN and discovered that the expression of FDFT1 is reduced in breast cancer when constrasted with that observed in normal breast cancer. Additionally, compared to non-TNBC, FDFT1 expression was potently downregulated in TNBC (Figures 5A, B). Furthermore, the levels of mRNA and protein for FDFT1 in the normal breast epithelial cell line MCF-10A, the ER-positive breast cancer cell line MCF-7, and the TNBC cell line MDA-MB-231 were determined via WB and qRT-PCR assays. The findings revealed that the expression levels of FDFT1 mRNA and protein were significantly decreased in MDA-MB-231 cells, whereas they were comparatively elevated in MCF-10A and MCF-7 cells (Figures 5C, D). To delve deeper into the functional significance of FDFT1 in modulating proliferation and ferroptosis in the TNBC cell line MDA-MB-231, we employed three distinct siRNAs, designated as 1, 2, and 3, to ablate FDFT1 expression in MDA-MB-231 cells. The experimental findings revealed that FDFT1 siRNA sequence No. 1 exerted the most pronounced impact on the suppression of FDFT1 protein expression compared to the negative control.in MDA-MB-231 cells (Figure 5E). As expected, the cell viabilities of MDA-MB-231 cells were induced by the treatment of the knockdown of FDFT1 (Figure 5F). Therefore, these findings indicate that FDFT1 may function as a tumor suppressor in TNBC cell line MDA-MB-231. Additionally, the levels of lipid ROS were decreased by the knockdown of FDFT1 in MDA-MB-231 cells (Figure 5G). The WB assay confirmed that the knockdown of FDFT1 markedly upregulated SLC7A11 levels in MDA-MB-231 cells (Figure 5H), this indicated that the knockdown of FDFT1 confers MDA-MB-231 cells resistant to ferroptosis, through the upregulation of SLC7A11 expression. Together these results indicate that FDFT1 represses proliferation and induces ferroptosis of MDA-MB-231 cells in vitro.

Our above findings suggest that VPA could induce TNBC cell ferroptosis and FDFT1 confers cells facilitative to ferroptosis. Consequently, it is speculated that a potential correlation may exist between VPA and FDFT1, our findings from qRT-PCR and WB assays indicated that VPA augmented the mRNA and protein levels of FDFT1 in MDA-MB-231 cells (Figures 6A, B). Next, we showed that the treatment of VPA stimulated lipid ROS levels and inhibited SLC7A11 levels of MDA-MB-231 cells while siRNA-mediated knockdown of FDFT1 was able to reverse the effect of VPA on MDA-MB-231 ferroptosis in vitro (Figures 6C, D). Taken together, these data suggest that inhibition of FDFT1 blocks VPA-induced ferroptosis of MDA-MB-231 cells, and VPA may facilitate ferroptosis in MDA-MB-231 cells by negatively modulating the expression of SLC7A11 via the upregulation of FDFT1 levels.

Our study examined whether VPA impedes the proliferation of the TNBC cell line MDA-MB-231 in vivo. MDA-MB-231 cells were implanted subcutaneously into the right flank of immunodeficient nude mice. One week later, tumor bearing mice were randomly divided into two groups and subsequently treated with physiological saline or VPA for 10 days. Throughout this interval, the body mass of nude mice and the histological features of the tumors were documented prior to each intraperitoneal injection. The results showed that there were significant differences in tumor characteristics compared to the control group. As shown in Figure 7A, visually, the tumor volume of the nude mice treated with VPA looked reduced, suggesting a potential inhibitory effect of VPA on tumor growth. Figure 7B plots the change in body weight of mice over time, further emphasizing the sustained weight loss of mice in the VPA-treated group compared to the control group. Meanwhile, Figure 7C shows that the tumor volume of mice in the VPA-treated group was subsequently reduced. Moreover, the body weight and tumor volume of mice showed a dose-dependent change with the concentration of VPA used (Figures 7A–C). Eventually, the tumor was excised and subsequent imaging, dimensional assessments, and weight determinations were conducted, unsurprisingly, VPA significantly suppressed tumor growth compared with the control group (Figures 7D, E). Furthermore, HE staining was conducted on mouse tumor sections, which revealed that the tumor cells in the control group were intact and densely packed with intense chromatin staining. Following VPA treatment, the tumor cells demonstrated a more disorganized arrangement, exhibited pale chromatin staining, and the majority of the tissues presented evident signs of degeneration and necrosis. Additionally, through IHC staining, we detected the expression of SLC7A11, FDFT1, and Ki67, a molecular marker of cellular proliferation. Our data demonstrated that mice treated with VPA exhibited reduced Ki67 expression in comparison to the control group. Moreover, in alignment with in vitro findings, mice subjected to VPA treatment exhibited reduced expression of SLC7A11 and elevated expression of FDFT1 (Figure 7F). The data indicate that VPA may facilitate ferroptosis in MDA-MB-231 cells by repressing the expression of SLC7A11, thereby contributing to the inhibition of tumor growth in vivo.