Apigenin promotes apoptosis of 4T1 cells through PI3K/AKT/Nrf2 pathway and improves tumor immune microenvironment in vivo

Abstract

The 2022 US Cancer Statistics show that breast cancer is one of the most common cancers in women. Epidemiology has shown that adding flavonoids to the diet inhibits cancers that arise in particular women, such as cervical cancer, ovarian cancer, and breast cancer. Although there have been research reports on apigenin (API) and breast cancer, its anti-tumor effect and potential mechanism on breast cancer have not yet been clarified. Therefore, in this study, we used 4T1 cells and a 4T1 xenograft tumor mouse model to investigate the antitumor effect of API on breast cancer and its underlying mechanism. In vitro, we used MTT, transwell, staining, and western blotting to investigate the inhibitory effect of apigenin on 4T1 and the underlying molecular mechanism. In vivo by establishing a xenograft tumor model, using immunohistochemistry, and flow cytometry to study the inhibitory effect of apigenin on solid breast tumors and its effect on the tumor immune microenvironment. The results showed that API can induce breast cancer cell apoptosis through the PI3K/AKT/Nrf2 pathway and can improve the tumor immune microenvironment in mice with breast tumors, thereby inhibiting the growth of breast cancer. Thus, API may be a promising agent for breast cancer treatment.

Introduction

In 2022, one of the most vulnerable cancers in women is breast cancer, which has the highest prevalence and incidence.¹ Current treatments for breast cancer include surgical resection, chemotherapy, or radiation therapy. Although effective in reducing the mortality rate of breast cancer patients, the negative effects of these treatments are also numerous, and even breast cancer survivors are accompanied by many sequelae, such as persistent pain, cognitive fatigue, and handicap.^2–4 Therefore, it is very important to elucidate the mechanisms leading to the malignant progression of breast cancer and to develop new therapeutic drugs.

Apoptosis, a form of programmed cell death, has been identified as a highly regulated and controlled process that promotes tumor death.^5,6 Bcl-2 family members are major players in the intrinsic mitochondrial apoptotic program.^7,8 In addition, the caspase protein family, especially, caspase-3 is a key factor in the process of apoptosis. The mechanism of apoptosis mediated by the intrinsic mitochondrial pathway is associated with changes in mitochondrial membrane potential. Mitochondria are the most important source of reactive oxygen species in cells. Studies have shown that ROS is an important factor leading to the reduction of mitochondrial membrane potential.⁹

PI3K/AKT signaling pathway plays an important role in breast cancer, and is often abnormally activated in breast cancer, thereby promoting tumorigenesis and progression, leading to reduced survival.^10–12 The Nuclear factor erythroid 2-like 2 (Nfe2l2/NRF2) is a transcription factor. In healthy cells, Nrf2 expression is low and maintains resistance to oxidative stress, however, cancer cells overexpress Nrf2, which has been implicated in various phenomena such as the development of drug resistance, angiogenesis, cancer stem cell development, and metastasis. Aberrant Nrf2 expression reduces the toxicity and efficacy of therapeutic anticancer drugs and provides cytoprotecting to cancer cells. Nrf2 is a key downstream factor of PI3K/AKT and is involved in the regulation of oxidative stress. Therefore, the PI3K/AKT pathway may play an important role in the activation of Nrf2.^13,14

Regulatory T cells, a subset of tumor-infiltrating lymphocytes, play a key role in immune tolerance and are one of the suppressive immune cells that can inhibit the proliferation of effector T lymphocytes, thereby suppressing anti-tumor immune responses. Studies have demonstrated that high numbers of tumor-infiltrating Tregs are associated with poor prognosis, suggesting that Tregs abundance may be a prognostic biomarker in breast cancer.^15–19

Apigenin(API), chemical name 4′, 5, 7, -trihydroxy flavonoid, is a common dietary brass, abundant in many fruits, vegetables, and Chinese herbal medicines.²⁰ Currently, API is widely studied for its anti-cancer effects and low toxicity. More and more studies have proved that API has a wide range of anti-cancer effects, such as cervical cancer, colorectal cancer, and lung cancer.^21–23 There is also evidence that API can produce anti-tumor immune effects and have a certain impact on the tumor immune microenvironment. In a mouse pancreatic cancer model, API treatment can enhance CD4+CD8+ T cells and reduce the percentage of Tregs, thereby improving mouse survival time, reduce tumor weight, and prevent splenomegaly.²⁴ These fully demonstrate that API can inhibit various cancers in vivo and in vitro, and is a promising cancer therapeutic agent. Therefore, in this study, we investigated the effect of API on breast cancer using mouse breast cancer cells and a xenograft mouse model and surveyed the potential mechanism of action.

Materials and methods

Chemicals

API was purchased from MedChem Express (HY-N1201, MCE, USA). Doxorubicin (DOX) was purchased from Aladdin (D107159, Aladdin). Roswell Park Memorial Institute 1,640 medium (RPMI-1640), fetal bovine serum (FBS), penicillin, streptomycin, and Hank’s balanced salt solution (HBSS) were all procured from Gibco (Invitrogen, USA).

Cell culture

The 4T1 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). 4T1 cells were maintained in RPMI 1640 supplemented with 10% FBS and 1% penicillin–streptomycin and incubated at 37 °C in a humidified 5% CO₂ atmosphere.

Cell viability assay

4T1 cells (5 × 10³) were seeded onto 96-well plates in 100 μL of culture medium. After 24 h of incubation to allow cells to adhere, cells were treated with various concentrations of API (0 ~ 20 μM) for 24 h and then subjected to a cell-viability assay (MTT assay, Promega, Madison WI). The absorbance was measured at 570 nm using a microplate reader.

Plate clonogenic assay

4T1 cells were diluted and seeded at 500 cells/well in six-well plates and incubated for 24 h. Subsequently, various concentrations (0 ~ 20 μM) of API were added for 24 h, and then continuously incubated in a new fresh medium at 37 °C. After incubation for 14 days, cells were stained with 0.1% crystal violet, and a colony was defined as consisting of more than 50 cells.

Wound healing assays

4T1 cells were grown to full confluence in 6-well plates and a small area was then disrupted by scratching the monolayer with a 200-μL plastic pipette tip. Cells were washed twice with phosphate-buffered saline (PBS) and replaced with a complete medium containing various concentrations of API, and the wound was observed after 24 h. Images were immediately captured under a microscope at 40× magnification.

Transwell migration and invasion assays

Cell motility was analyzed with the aid of a transwell. To analyze cell migration, 4 × 10⁴ cells in 0.2 ml of serum-free medium were seeded in an uncoated top chamber (12-well insert, pore size, 8 μm, Corning), adding medium supplemented with API and 10% FBS to the lower chamber. An invasion assay was conducted following the same procedure, with the exception that 5 × 10⁴ cells were plated in a Matrigel (40,183, Yeason)-coated top chamber. After 24 h of incubation, cells were fixed in 100% methanol for 5 min, stained in 0.1% crystal violet for 30 min, and rinsed in PBS. The number of cells migrating and invasion were visualized and counted under a light microscope (40×, three random fields per well).

Hoechst 33342—propidium iodide co-staining assay

Cell apoptosis was detected by Hoechst 33342 and propidium iodide (PI) staining. After treatment with API for 4T1 cells on the confocal dish, Hoechst 33342 dye solution (B8040, Beyotime, China) was added, then keep in an incubator for 15 min. After washing the cells with PBS, the cells were incubated with PI dye solution (P8080, Beyotime, China) on ice for 15 min in dark. After washing the cells with PBS, the cells were sealed. The confocal dish was photographed at 20× magnification using a confocal microscope (NanoZoomer S60, Hamamatsu, Japan).

Annexin V—propidium iodide co-staining assay

Apoptosis of 4T1 cells induced by API was analyzed using an Annexin-V/FITC apoptosis detection kit I (C1062S, Beyotime, China). The cells (2 × 10⁵ cells) were seeded on six-well plates and treated with API (0, 5, 10, and 20 μM) for 24 h in an incubator. Supernatants were removed and cells were detached with trypsin-EDTA. The cells were collected by centrifugation, washed with PBS, and resuspended using 1× binding buffer. Then, incubated with 5 μL FITC Annexin V and 5 μL PI for 15 min at room temperature in the dark. Finally, 400 μL of 1× binding buffer was added to a 5 mL culture tube. Fluorescence intensity was analyzed using a FACS Calibur (BD Biosciences).

Mitochondrial membrane potential (ΔΨm) assessment

Transmembrane ΔΨm was determined using the JC-1 assay. The 4T1 cells were plated in a confocal dish at a density of 1.5 × 10⁵/mL, and pretreated with API at the concentration of 0 ~ 20 μM for 24 h. The medium was removed and 5 μg/mL JC-1 dye (C2006, Beyotime, China) was added for 20 min at room temperature. The cells were washed gently with PBS. Then the cells were photographed at 20× magnification using a fluorescence microscope (Nikon E100, Tokyo, Japan). In addition, ΔΨm was measured using a fluorescence plate reader.

Determination of ROS

The 4T1 cells were plated in a confocal dish at a density of 1.5 × 10⁵/mL, and pretreated with API at the concentration of 0 ~ 20 μM for 24 h. After removing the medium, 2′, 7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) solution (Beyotime, China) was added in the wells for 30 min-induction at 37 °C in dark. The cells were washed gently with PBS, then the cells were photographed at 20× magnification using a fluorescence microscope (Nikon E100, Tokyo, Japan). In addition, the ROS level was detected by a multifunction microplate reader.

Western blotting

Proteins were collected from cells in a lysis buffer prepared with 98% RIPA Lysis Buffer (Beyotime, China), 1% PMSF (Beyotime, China), and 1% phosphatase inhibitor (HY-K0021, MCE, USA), and kept on ice for 30 min. Cell sediment was removed via centrifugation (4 °C, 12,000 rpm) for 30 min, and the supernatant was obtained. Its concentration was detected using a BCA assay kit according to the manufacturer’s instructions. An equal number of denatured proteins (20 μg) were separated on a 10%–15% SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) separation gel and transferred to 0.22 μm PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% nonfat milk for 1 h at room temperature. Then, these membranes were incubated with specific primary antibodies overnight at 4 °C the specific antibodies include BAX (2772, 1:1000, CST), Bcl-2 (3498, 1:1000, CST), Caspase-3 (9662, 1:1000, CST), Cleaved-caspase-3 (9661, 1:1000, CST), PI3K (4292, 1:1000, CST), P-PI3K (4228, 1:1000, CST), AKT (9272, 1:1000, CST), P-AKT(4060, 1:1000, CST) and Nrf2 (16396-1-AP, 1:1000, Proteintech). The target protein was incubated with a secondary antibody (Beyotime, China) for 2 h. The related proteins were visualized by Enhanced Chemiluminescence (ECL) kit (Fdbio Science, China). Finally, the density of the target proteins was circled and quantified using the Carestream Molecular Imaging system (Carestream Health, Inc., USA).

Animal experiments

Femail BALB/c mice (five-week-old, 16–20 g) were purchased from the GemPharmatech (Nanjing, China). The mice were housed in a clean and sterile plastic cage at 24–26 °C, 50%–60% humidity, and maintained a 12 h light-12 h dark cycle. The animals have free access to water and food for a week to adapt to the environment. All animal ethical welfare and experimental procedures were complied with and approved by the Research Ethics Committee of Jinan University (Permit No.2019883). Animal experiments were performed in compliance with the Guiding Principles for the Care and Use of Laboratory Animals, approved by the Japanese Pharmacological Society.

4T1 cells were injected subcutaneously into female nude mice. Xenograft mice were randomly assigned to 5 groups, with six mice in each group: the model group (40% PEG400, and 60% double-distilled water respectively), the experimental groups (treated with 25 mg/kg, 50 mg/kg, and 100 mg/kg of API, and API was formulated in 40% PEG400, and 60% double-distilled water respectively via intragastric administration), and the positive group (treated with 2 mg/kg/week of DOX, and DOX was formulated in saline). All the treatments were injected once daily. All surgery procedures were performed under anesthesia using isoflurane. At the end of the experiments, animals were humanely killed by CO₂ asphyxiation. In animal studies, both the carer of the animals and the assessor of the results are blinded.

Flow cytometry analysis

Immune profiling of TDLN with flow cytometry. In brief, the CD4+ and Treg cells in TDLN were determined via flow cytometry. Single-cell suspensions were generated from isolated TDLN of mice from each group. Single-cell suspensions of TDLNs were generated by mechanically dispersing the lymph nodes through a 70-μM nylon mesh cell strainer (BD Biosciences). The following mAbs were used to characterize mouse lymphocyte subpopulations: Fluorescein isothiocyanate (FITC)-labeled anti-CD4 (11-0041-82, eBioscience), Phycoerythrin (PE)-labeled anti-FoxP3 (12-5773-82, eBioscience), Allophycocyanin (APC)-labeled anti-CD25 (17-0251-82, eBioscience). Before surface staining with predetermined optimal concentrations of each mAb, cell samples were blocked by incubation within the flow buffer and anti-mouse CD16/CD32 (14-0161-82, eBioscience) for 30 min at 4 °C. For intracellular FoxP3 staining, the PE-labeled anti-mouse/rat Treg Staining Kit (eBioscience) was used according to the manufacturer’s instructions. Three-color flow cytometry was performed on a BD FACSCanto II flow cytometer (Becton Dickinson, US) and data was analyzed using FlowJo software (Tree Star, USA). Each analysis shown represents ≥10,000 events within the live lymphocyte gate.

Immunohistochemistry

Paraffin-embedded tissues sections (4-μm-thick) were deparaffinized and dehydrated, subjected to antigen retrieval, blocked endogenous nonspecific antigen and peroxidase activity with goat serum and 3% H₂O₂, and incubated for 2 h at room temperature with primary antibodies for PCNA (10205-2-AP, 1:200, Proteintech). After three times washes with PBS, the immune chromogenic reagent kit (GK500705, Gene Tech, China) was used for secondary antibody incubation and DAB immune chromogenic reaction under the manufacturer’s protocol. The color reaction of the slice was observed under the microscope at ×200 magnification, and five random areas were photographed for quantitative analysis. The IHC results for tissues were scored by two independent observers according to both the percentage of positively stained cells (scored from 0 to 4) and the staining intensity (scored from 0 to 3). The final staining scores were obtained by multiplying the intensity and the extent scores. For statistical analysis, tumor tissues with final staining scores of <8 indicated low expression, and ≥8 indicated high expression.

Statistical analysis

All results were shown as mean ± standard error of the mean (SEM). Statistical analysis of experimental data was determined by GraphPad Prism 8 software (GraphPad Software Inc., Avenida, CA, USA). The experimental data were analyzed by one-way ANOVA, followed by a least significant difference (LSD) test. Animal survival time was determined utilizing a Kaplan-Meier survival analysis and log-rank test. A P-value of <0.05 was considered a statistically significant difference.

Results

API suppresses 4T1 cells proliferation

In this study, the effect of API on the proliferation ability of 4T1 cells was analyzed by MTT (Fig. 1A). The experimental results showed that apigenin could inhibit the proliferation of 4T1 cells. Furthermore, the effect of API on colony formation of 4T1 cells was analyzed by colony formation (Fig. 1D and F). The experimental results showed that the number of cell clones in the API group decreased significantly. Taken together, these results indicate that API inhibits 4T1 cell proliferation and colony formation.

Fig. 1.

API suppresses 4T1 cells proliferation. A) 4T1 cells were treated with API (0–20 μM) for 24 h, and cell viability was measured via the MTT assay. DOX was used as a positive control. (n = 8) (B) IC50 values of API after 24 h treatment in 4T1 cells, as determined by the MTT assay. C) IC50 values of DOX after 24 h treatment in 4T1 cells, as determined by the MTT assay. D) Representative images of colony formation assay of the 4T1 cells. E) The change in the morphology of 4T1 cells after 24 h of API treatment. F) Summary of the number of colonies in 4T1 cells. (n = 3) (G) Chemical structure of API. The results shown are representative of at least three independent experiments. Data are shown as the means ± SEM. (^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 compared with the control group) API: Apigenin; DOX: Doxorubicin.

API restraints 4T1 cells migration and invasion

To determine whether API treatment affects cancer cell migration and invasion, the effect of API on inhibiting 4T1 cells migration and invasion was evaluated by wound scratch assay and transwell assay. As shown in Fig. 2, our results demonstrated that cells in the control group had a higher cell migration ability (Fig. 2A and D). Similarly, transwell migration test results are consistent with wound scratch results (Fig. 2B and E). The transwell invasion assay revealed the apparent decline in the invasion ability of 4T1 cells treated with API (Fig. 2C and F). These results suggested API showed a potent effect on inhibiting the invasion, and migration of 4T1 cells.

Fig. 2.

API restraints 4T1 cells migration and invasion. A) Representative images from the wound-healing assay of 4T1 cells are displayed for 24  h. (bar = 100 μm) (B) Representative images of the transwell migration assay are shown for 24 h after cell seeding. (200×, bar = 100 μm) (C) Representative images of the transwell invasion assay are shown for 24 h after cell seeding. (200×, bar = 100 μm) (D). Quantitative form of data shown in (A). (n = 3) (E) Quantitative form of data shown in (B) about cell number. (n = 5) (F) Quantitative form of data shown in (C) about cell number. (n = 5) The results shown are representative of at least three independent experiments. Data are shown as the means ± SEM. (^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 compared with the control group) API: Apigenin; DOX: Doxorubicin.

API triggers 4T1 apoptosis and modulates the expression levels of apoptotic-associated proteins in 4T1 cells

To assess the effect of API on 4T1 cell apoptosis, Annexin V-FITC/PI double staining was used to measure 4T1 cell apoptosis. The present results showed that API significantly promoted both early and late apoptosis in 4T1 cells (Fig. 3A and B). To further verify apoptosis induction, nuclear morphological changes of treated and untreated cells were analyzed using Hoechst 33342/PI double staining (Fig. 3C). Western blotting was used to evaluate the expression of apoptosis-related proteins in 4T1 cells treated with different concentrations of API for 24 h. The results showed that the ratios of Bcl-2/Bax and caspase-3/cleaved-caspase-3 were decreased in API-treated 4T1 cells (Fig. 3D–F).

Fig. 3.

API triggers 4T1 apoptosis and modulates the expression levels of apoptotic-associated proteins in 4T1 cells. A) Annexin V-PI co-staining assay to evaluate apoptosis in 4T1 cells. B) Percent of apoptotic cells. (n = 5) (C) Hoechst 33342-PI co-staining assay to evaluate apoptosis in 4T1 cells. (800×, bar = 20 μm) (D) The protein levels of Bcl-2, Bax, caspase-3, and cleaved-caspase-3 were detected by Western blotting in 4T1 treated with API (0–20 μM) and DOX for 24 h. E) The expression of Bcl-2/Bax in 4T1 cells. (n = 3) (F) The expression of caspase-3/cleaved-caspase-3 in 4T1 cells. (n = 3) The results shown are representative of at least three independent experiments. Data are shown as the means ± SEM. (^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 compared with the control group) API: Apigenin; DOX: Doxorubicin.

API triggers the depolarization of ΔΨm in 4T1 cells

Depolarization of MMP is another representative feature of apoptosis. To determine whether API-induced cell apoptosis was associated with mitochondrial dysfunction, we investigated MMP dysfunction in 4T1 cells by staining with JC-1 and analyzing the cells with a fluorescent microscope and a microplate reader. The results show that API triggered ΔΨm depolarization, demonstrated by the presence of green signals (Fig. 4A). The results of OD values detected by the microplate reader showed that API (20 μM) treatment for 24 h significantly decreased the MMP of 4T1 cells (Fig. 4B). The results presented in Fig. 3. 3 demonstrate a significant decrease in the ratios of Bcl-2/Bax and caspase-3/cleaved-caspase-3 in 4T1 cells upon treatment with API. The Bcl-2 family and caspases are well-known participants in the mitochondrial apoptotic pathway. These results suggest that API enhances mitochondrial dysfunction within 4T1 cells, thereby increasing cell apoptosis.

Fig. 4.

API triggers the depolarization of ΔΨm in 4T1 cells. A) JC-1 staining detected changes in mitochondrial membrane potential through API treatment. (800×, bar = 20 μm) (B) Quantitative analysis data. The results shown are representative of at least three independent experiments. Data are shown as the means ± SEM. (n = 8) (^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 compared with the control group) API: Apigenin; DOX: Doxorubicin.

API induces ROS generation

We hypothesized that the API-induced apoptosis occurred via intracellular ROS generation. 4T1 cells were treated with various concentrations of API for 24 h and stained with the cell-permeable dye H₂DCFH-DA, we studied ROS production in 4T1 cells by analyzing the cells with a fluorescence microscope and a microplate reader. Fluorescence results showed that API stimulated 4T1 cells to produce ROS. The detection results of the microplate reader are consistent with the results of the fluorescence microscope (Fig. 5). These results demonstrate that API markedly elevated ROS levels in a dose-dependent manner.

Fig. 5.

API induces ROS generation. A) Changes in ROS levels after treatment with different concentrations of API. (800×, bar = 20 μm) (B) Quantitative analysis data. The results shown are representative of at least three independent experiments. Data are shown as the means ± SEM. (n = 8) (^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 compared with the control group) API: Apigenin; DOX: Doxorubicin.

API regulates PI3K/AKT/Nrf2 signal transduction pathways in 4T1 cells

The study focused on assessing the expressions of proteins associated with the PI3K/AKT/Nrf2 pathway, recognizing its crucial role in breast cancer development. The examined proteins included p-PI3K, p-AKT, Nrf2. The results revealed a significant downregulation of p-PI3K/PI3K, p-AKT/AKT, and Nrf2 in 4T1 cells following API treatment (Fig. 6A–D). This suggests that API specifically targets and suppresses the phosphorylation of PI3K and AKT, key components of the PI3K/AKT pathway, as well as downregulates Nrf2 expression. These findings imply that API may exert its anti-tumor effects in breast cancer cells by modulating the PI3K/AKT/Nrf2 pathway. The downregulation of p-PI3K and p-AKT suggests inhibition of the pathway’s activation, and the decrease in Nrf2 expression further supports the potential regulatory role of API in this signaling cascade. The specific targeting of these pathway components by API could represent a promising therapeutic strategy for breast cancer.

Fig. 6.

API regulates PI3K/AKT/Nrf2 signal pathways in 4T1 cells. A) The protein levels of PI3K, P-PI3K, Akt, P-AKT, and Nrf2 in 4T1 cells were determined by Western blotting. B) The expression of P-PI3K/PI3K in 4T1 cells. C) The expression of P-AKT/AKT in 4T1 cells. D) The expression of Nrf2 in 4T1 cells. The results shown are representative of at least three independent experiments. Data are shown as the means ± SEM. (n = 8) (^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 compared with the control group) API: Apigenin; DOX: Doxorubicin.

API exhibits anti-tumor activity in mice

To further confirm the in vitro findings, we investigated the effects of API in a 4T1 xenograft tumor model using BALB/c mice. Our data demonstrated that API significantly inhibited the tumor growth of 4T1 xenografts (Fig. 7B–D). After treatment for 21 days, the xenograft tumors decreased in the API-treated group (Fig. 7E). Furthermore, the immunohistochemistry study demonstrated significantly decreased expressions of PCNA in API-treated tumors (Fig. 7F and G). The results of animal survival experiments show that API can appropriately prolong the survival of mice with mammary gland tumors (Fig. 7H). These results further indicated that API acted as an anti-proliferative in vivo.

Fig. 7.

API exhibits anti-tumor activity in mice. A) Schematic representation of the experiment. B) Representative image of a tumor. (n = 6) (C) Quantitative analysis data of body weight. (n = 6) (D) Quantitative analysis data of average tumor volume. (n = 6) (E) Tumor weights of model mice, API-treated mice (25, 50, 100 mg/kg/day), DOX (2 mg/kg/week) on the last day of the study. F) Tumor tissue samples were analyzed by hematoxylin, eosin staining, and immunohistochemistry to examine the histopathology and expression levels of PCNA (H&E, 400×, bar = 50 μm; IHC, 400×, bar = 50 μm). G) Quantitative analysis data of PCNA. (H) Survival curve of mice with mammary tumor. Kaplan-Meier survival curve shows model mice, API-treated (50 mg/kg) mice, and DOX-treated mice per group. The survival curve graph represents at least three independent experiments. (n = 7) The results shown are representative of at least three independent experiments. Data are shown as the means ± SEM. (^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 compared with the model group) API: Apigenin; DOX: Doxorubicin.

API improves anti-tumor responses in BALB/c mice

To determine whether API had any effect on the shift in T cell numbers in mice, we measured effector T cell (CD4+ and Treg) percentages in mice. Flow cytometry results show that TDLN from model mice had a significant reduction in CD4+ (Fig. 8B) but an increase in Treg percentages (Fig. 8C). We also found the proportion of Treg cells (CD4 + CD25 + FoxP3 + T cell) in TDLN was decreased in the API-treated group.

Fig. 8.

API improves anti-tumor responses in BALB/c mice. A) Flow cytometry analysis of CD4+ T cells and CD4 + CD25 + Foxp3+ Treg percentages in TDLN from control, model, API-treated mice, and DOX-treated mice. B) Percentage of CD4 + T cells in TDLN. C) Percentage of Treg in TDLN. The results shown are representative of at least three independent experiments. Data are shown as the means ± SEM. (n = 6) (^***P < 0.001 compared with the control group) (^#P < 0.05, ^##P < 0.01 and ^###P < 0.001 compared with the model group) API: Apigenin; DOX: Doxorubicin.

Discussion

Long-term survival of breast cancer patients after surgery or chemotherapy remains low due to the high risk of breast cancer recurrence and metastasis.²⁵

As a flavonoid in many fruits and plants, API has various biological activities, among which the anticancer activity has attracted much attention. In 4T1 cells, the effect of API on cell viability was detected by MTT assay. In addition, the effect of API on the proliferation ability of 4T1 cells was detected by plate clone formation assay, and the results showed that API could significantly inhibit the proliferation ability of 4T1 cells.

Metastasis is an important aspect of cancer and is the leading cause of death in approximately 90% of cancer patients.²⁶ The migratory and invasive functions of cancer cells can lead to significant effects such as tumor metastasis and poor prognosis.²⁷ Therefore, in addition to investigating the effect of API on cell proliferation, this study also investigated the effect of API on the migration and invasion of 4T1 cells. Through wound healing experiments and transwell experiments, we found that the migration and invasion of 4T1 cells were significantly inhibited. As expected, API could limit 4T1 cells migration and invasion to some extent.

This study demonstrated by flow cytometry that several concentrations of API treatment could induce apoptosis of 4T1 cells. This may be related to the massive generation of ROS. A large amount of ROS may be related to oxidative damage. As expected, this study by demonstrating that when 4T1 cells were treated with API, ROS significantly increased, resulting in oxidative damage. Another side, ROS may alter mitochondrial membrane function, leading to apoptosis.²⁸ Loss of MMPs is considered a prominent feature in the mitochondrial apoptotic pathway. Our results demonstrate that API induces a decrease in MMPs in 4T1 cells. Furthermore, ROS regulates the translocation, phosphorylation, and cleavage of pro-apoptotic Bcl-2 members, thereby triggering apoptosis.²⁹ Bax and Bcl-2 can form homologous protein dimers as molecular switches in cell death signaling pathways. When the ratio of Bcl-2/Bax dimer was decreased, it induced cell apoptosis. The present results show that after treatment with API, Bcl-2 was significantly decreased, and the Bax/Bcl-2 ratio was increased. An elevated Bax/Bcl-2 ratio stimulates changes in mitochondrial permeability, driving caspase initiation Apoptosis.^30,31 Our results showed that cleaved-caspase 3 was significantly upregulated by API treatment, demonstrating that API increased caspase-3 function in 4T1 cells, thus indicating cell death is regulated by mitochondria. Unfortunately, we did not assess the expression of cleaved-PARP protein, which also holds significant relevance in elucidating the mechanisms of apoptosis. Further investigations are required to provide additional evidence regarding the changes in cleaved-PARP protein.

Phosphatidylinositol 3-kinases (PI3Ks) signaling is involved in processes such as cell proliferation, differentiation, and glucose transport. In recent years, it has been found that the signaling pathway composed of type IA PI3K and its downstream molecular protein kinase B (PKB or AKT) is related to human tumorigenesis, cancer cell migration, adhesion, and tumor angiogenesis.³² In this study, API significantly reduced the expression of PI3K and AKT, thereby downregulating Nrf2 expression in 4T1 cells and inhibiting the growth of these cells.

In addition, this study evaluated the efficacy of API in the treatment of breast cancer at the animal level by establishing xenografts in BALB/c mice. The experimental results showed that API can inhibit breast tumor growth in vivo. Immunosuppressive regulatory T cells (Tregs) are a subset of CD4+ T cells.³³ Their primary function is to maintain peripheral immune tolerance to self- and foreign antigens by suppressing CD4+ and CD8+ T cell responses.³⁴ The percentage of Tregs is elevated in BC from human patients as well as in mouse models of BC.^35–37 In this study, we evaluated the effect of API on Treg cell immune responses. API may help deplete Treg cells in tumor-draining lymph nodes and shape the tumor microenvironment into a favorable antitumor immune state. Our findings suggest that API may be a possible therapeutic agent for maintaining T cell homeostasis in BC mice.

The mutagenicity and toxicity of API, compared to other flavonoids, are relatively low. Although there has been research on the anti-breast cancer effects of API, most studies have been confined to the cellular level. In this study, we not only elucidate the impact and mechanisms of API on breast cancer using an in vitro 4T1 cell model but also establish a xenograft mouse model to illustrate the inhibitory effects of API on breast cancer from both in vitro and in vivo perspectives. Importantly, we conducted the first assessment of API’s influence on the tumor microenvironment in 4T1 xenograft mice. API demonstrated the ability to reduce the expression of Treg cells in the tumor microenvironment, thereby improving the immune milieu of the tumor. This dual impact on cancer cells and the surrounding immune response sets API apart from compounds that may solely target cancer cells directly, suggesting its potential to enhance therapeutic efficacy in a more comprehensive manner.

Overall, our findings support the application of API in anticancer therapy. Apigenin inhibits the proliferation, migration, and invasion of 4T1 cells. It also triggers the mitochondrial apoptosis pathway by promoting the production of ROS in 4T1 cells and reducing the mitochondrial membrane potential to reduce the ratio of caspase-3/cleaved-caspase-3 and Bcl-2/Bax. And it plays an anti-breast cancer effect by inhibiting the PI3K/AKT/Nrf2 signaling pathway. API can affect the tumor immune microenvironment by depleting Treg cells in tumor-draining lymph nodes, shaping the tumor microenvironment into a favorable environment. Our study may provide a new research direction for harnessing novel natural compounds in breast cancer treatment.

Conclusion

Apigenin can induce the apoptosis of breast cancer cells through the PI3K/AKT/Nrf2 pathway, improve the tumor immune microenvironment in mice with breast tumors, and thus inhibit the growth of breast tumors. Therefore, API may be a promising drug for breast cancer treatment.

Author contributions

Chu Zhang (Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing—original draft, Writing—review & editing, Supervision). Yupei Liao (Resources, Formal analysis, Visualization, Validation, Writing—review & editing), Tangjia Li (Formal analysis, Investigation, Writing—review & editing, Supervision). Haijing Zhong (Formal analysis, Writing—review & editing, Supervision). Luchen Shan (Formal analysis, Writing—review & editing, Supervision). Pei Yu (Resources, Formal analysis, Writing—review & editing, Supervision). Chenglai Xia (Writing—review & editing, Supervision), Lipeng Xu (Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Writing—original draft, Writing—review & editing, Supervision).

Funding

This work is supported by grants from the National Natural Science Foundation of China (No. 81673496).

 

Conflict of interest statement. The authors declare no conflict of interest, financial or other-wise.