Contributed equally
Recent developments in breast cancer therapy have significantly improved patient survival rate; however, recurrence remains a major problem. Systemic treatment of breast cancer with available therapies is not curative. Natural products can be potentially used for treating cancer. Recently, a wide range of pharmacological activities has been reported for Alismatis Rhizoma, a popular traditional Chinese medicine. However, the mechanisms via which its compounds act on breast cancer remain unclear. The present study aimed to investigate the potential of natural therapeutic agents from Alismatis Rhizoma for treating breast cancer. Human breast cancer MDA-MB-231 cells were treated with four main protostane triterpenes from Alismatis Rhizoma, including alisol A, alisol A 24-acetate, alisol B and alisol B 23-acetate. Among these, alisol A significantly inhibited cell viability. Alisol A induced cell apoptosis, G1 phase cell cycle arrest, autophagy, and intracellular reactive oxygen species (ROS) generation in MDA-MB-231 cells. The number of APE1-/γH2AX-/LC3-II positive cells was also significantly higher compared with that of negative control cells. All these results were dose-dependent. Cleaved caspase-3, cleaved caspase 9, Bcl-2, and p-p38 expression indicated cell apoptosis after alisol A treatment. The changes in cyclin A and cyclin D1 expression was associated with cell cycle arrest upon alisol A treatment. Furthermore, LC3-II expression upon alisol A treatment was indicative of autophagy. Alisol A treatment can induce autophagy-dependent apoptosis in human breast cancer cells via induction of ROS and DNA damage. Thus, Alisol A might serve as a new therapeutic agent against breast cancer.
Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-associated death among women in most countries (
Recent developments in breast cancer chemotherapy have significantly improved patient survival rates; however, the recurrence of breast cancer remains a major problem. Systemic treatment of breast cancer with available therapies is not curative (
In order to address the aforementioned challenges, new drugs for breast cancer therapy are urgently required. Natural products with a wide range of physiological activities can be used for treating specific diseases. Natural products have contributed significantly to the discovery and development of new drugs, especially those for cancer therapy (
In the present study, the effects of four main protostane triterpenes of Alismatis Rhizoma, including alisol A, alisol A 24-acetate, alisol B, and alisol B 23-acetate, were investigated in breast cancer cells. Alisol A showed significant anticancer effects in MDA-MB-231 breast cancer cells, which is a TNBC breast cancer cell line. The mechanisms of action of alisol A on this cell line were also investigated. The results will provide a comprehensive understanding regarding the anticancer effects of alisol A in human breast cancer cells.
The use of human specimens in the present study was approved by the Peking University Third Hospital Medical Science Research Ethic Committee (approval no. IRB00006761-M2019343). Informed consent was signed by all the patients.
Four protostane triterpenes, namely, alisol A, alisol A 24-acetate, alisol B, and alisol B 23-acetate, were purified from Alismatis Rhizoma by repeated chromatography on silica gel, reversed-phase C18, Sephadex LH-20, and semi-preparative RP-C18 high performance liquid chromatography (HPLC), and their structures were characterized based on comprehensive nuclear magnetic resonance, mass spectroscopy, and ultraviolet (UV) spectral analysis. The purity of the four compounds was greater than 98%, as assessed after normalization of the HPLC-UV peaks observed at 210 nm (
Human breast cancer MDA-MB-231 cells, from American Type Culture Collection, were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% (v/v) fetal bovine serum (FBS) (HyClone; Cytvia), 100 U/ml penicillin and streptomycin. MDA-MB-231 cells were cultured at 37°C in a humidified incubator in the presence of 5% (v/v) CO2. After the cells reached 80% confluence, cells were detached using trypsin (HyClone; Cytvia), counted, and plated at the necessary density for treatment. Primary human TNBC breast cancer cells were isolated from tumor specimens, lesion was 1×1×0.2 cm from a 56-year-old patient diagnosed with breast invasive ductal carcinoma in July 2019, and cultured in DMEM/F12 supplemented with 5% (v/v) FBS, 0.4 µg/ml hydrocortisone (Sigma-Aldrich; Merck KGaA), 1X Insulin-Transferrin-Selenium (Sigma-Aldrich; Merck KGaA), 10 ng/ml epidermal growth factor (EGF) (Invitrogen; Thermo Fisher Scientific, Inc.), 25 µg/ml adenine (Sigma-Aldrich; Merck KGaA), 10 ng/ml cholera toxin (Sigma-Aldrich; Merck KGaA), and 10 µmol/l Y-27632 (MedChemExpress). Alisol A, alisol A 24-acetate, alisol B, or alisol B 23-acetate were used to treat cells for 24 h.
A real-time cell proliferation assay was conducted using the ACEA RT-CES microelectronic cell sensor system (ACEA Bioscience, Inc.) to measure the numbers of living cells. This system works by measuring electrical impedance of sensor electrodes integrated on the bottom of microtiter E-plates as previously described (
Colony-formation assay was performed to determine the inhibitory effect of alisol A on MDA-MB-231 cells. Briefly, following treatment with 10 µM
MDA-MB-231 cells (1×105 cells) were seeded in 6-well plates. After treatment with 5, 10 or 20 µM alisol A for 24 h, an Annexin V-FITC/propidium iodide (PI) double-staining Apoptosis Detection kit (Becton, Dickinson and Company) was used to label the cells, according to the manufacturer's instructions. MDA-MB-231 cells treated with dimethyl sulfoxide (DMSO, 0.1% (v/v)) were used as negative control. Cells were washed twice with cold PBS, and then resuspended in 200 µl Annexin V binding buffer at room temperature. After the cells were stained with 10 µl FITC-labeled Annexin V and 5 µl PI at room temperature for 15 min in the dark, the samples were immediately analyzed using flow cytometry (Becton Dickinson FACS Calibur; Becton, Dickinson and Company).
To determine the effects of alisol A on DNA fragmentation in MDA-MB-231 cells, a DeadEnd™ fluorometric terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) system (Promega Corporation) was used. The TUNEL assay was processed according to the manufacturer's instructions. Cell nuclei was stained with 4′6-diamidino-2-phenylindole (DAPI) at room temperature for 10 min.
The MDA-MB-231 cells (1×105 cells) were seeded in 6-well plates. After treatment with 5, 10 or 20 µM alisol A for 24 h, the cells were harvested and washed twice with cold PBS. MDA-MB-231 cells treated with DMSO of 0.1% (v/v) were used as negative control. The cells were suspended in 0.5 ml 70% (v/v) ethanol and chilled at −20°C for 24 h. After extensive washing with PBS, the cells were resuspended in PBS containing 10 µg/ml 7-amino-actinomycin D (7AAD) (BD Biosciences) and 0.1 mg/ml RNase A and incubated at 37°C for 30 min. The cells were subsequently resuspended in PBS and analyzed using flow cytometry (Becton Dickinson FACS Calibur). The results were analyzed using ModFit LT 3.2 Software (Verity Software House, Inc.).
Intracellular ROS levels were measured using a cell-permeable fluorogenic probe as described previously (
Immunofluorescence was performed as previously described (
Proteins associated with cell cycle, apoptosis, and autophagy were detected by western blot analysis. Cells treated with alisol A or negative control cells were harvested individually. For protein extraction, cells were suspended in Cell lysis buffer for Western (cat. no. P0013; Beyotime Institute of Biotechnology) containing a protease inhibitor mixture and shaken on ice for 30 min. The cell lysate was centrifuged at 15,000 × g at 4°C for 10 min, and the supernatant was collected. The total protein concentration was measured using the Bradford method or the bicinchoninic acid (BCA) protein assay kit. Proteins (40 µg) were separated on 12% (w/v) SDS-PAGE gels and electrophoretically transferred onto polyvinylidene difluoride membranes (EMD Millipore). The membranes were blocked in 5% (w/v) fat-free milk in Tris-buffered saline-0.5% (v/v) Tween-20 at room temperature for 1 h and incubated overnight at 4°C with antibodies against caspase-3 (1:1,000; cat. no. 9662S; Cell Signaling Technology, Inc.), caspase-9 (1:1,000; cat. no. 9502S; Cell Signaling Technology, Inc.), Bcl-2 (1:1,000; cat. no. 15071S; Cell Signaling Technology, Inc.), p-p38 (1:100; cat. no. sc-166182; Santa Cruz Biotechnology, Inc.), cyclin A (1:100; cat. no. sc-271682; Santa Cruz Biotechnology, Inc.), cyclin D1 (1:500; cat. no. K0062-3; MBL International Co.), LC3-II (1:1,000; cat. no. 2775; Cell Signaling Technology, Inc.) or GAPDH (1:200; cat. no. sc-47724; Santa Cruz Biotechnology, Inc.). After three washes with PBS supplemented with 0.1% (v/v) Tween-20 (PBST) for 15 min, the membranes were incubated with goat anti-rabbit IRDye 680RD (1:5,000; cat. no. 926-68071; LI-COR Biosciences) or goat anti-mouse IRDye 800CW (1:5,000; cat. no. 926-32210; LI-COR Biosciences) for 1 h at room temperature. Proteins were identified by scanning the membranes using the Odyssey Imager (LI-COR Biosciences). ImageJ 1.8.0 (National Institutes of Health) was used to quantify the protein bands.
Data are presented as the mean ± standard deviation (SD) of three independent experiments. The significance of differences were analyzed using one-way analysis of variance (ANOVA) and post-hoc Tukey's test. The half maximal inhibitory concentration (IC50) of alisol A was calculated using Probit regression. All statistical analyses were performed using SPSS 23.0 software (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.
TNBC MDA-MB-231 cells were used to evaluate the cytotoxicity of Alismatis Rhizoma compounds on human breast cancer. Real-time cell proliferation assay and colony-formation assay were performed to evaluate the cytotoxicity of the four major protostane triterpenes of Alismatis Rhizoma. As shown in
To evaluate whether alisol A can induce cell apoptosis in MDA-MB-231 cells, annexin V/PI staining assay and TUNEL assay were used. After treatment with 5, 10 or 20 µM alisol A, the percentages of apoptosis-positive cells were 24.97±0.80, 31.81±0.36, or 33.87±0.65%, respectively, compared with 9.07±0.51% in the negative control group (
Cell cycle analysis showed that alisol A effectively induced G1 phase cell cycle arrest in human breast cancer MDA-MB-231 cells. After 24 h exposure to 5 µM of alisol A, the fraction of cells in the G1 phase increased from 26.67±1.45 to 38.67±0.88%. When treated with 10 or 20 µM alisol A, the fraction of cells in the G1 phase increased to 40.33±0.88 and 42.01±1.15%, respectively (
Recent reports have shown that autophagy can stimulate apoptosis (
DNA damage can promote cell autophagy and induce cell apoptosis via activation of the caspase pathway (
More than 2 million cases of breast cancer were diagnosed in 2018, and more than 626,000 people succumbed to breast cancer, establishing breast cancer as the second most common cancer and the third most common cause of cancer-associated deaths worldwide (
Alisols, triterpenes belonging to the protostane family, are known as the major bioactive ingredients of Alismatis Rhizoma (
In the present study, MDA-MB-231 cells were used to investigate the potential therapeutic effects of alisol A on TNBC. The results showed that alisol A can significantly inhibit the proliferation of MDA-MB-231 cells, using a real-time cell proliferation assay that measured the cell index in real time. While analyzing the cellular changes induced by alisol A treatment, it was observed that the percentage of apoptotic cells increased. Resistance to apoptosis is a major obstacle leading to chemotherapy failure during cancer treatment (
Cell cycle arrest at the G1 phase is one of the main triggers of apoptosis. It was found that the number of cells at the G1 phase was increased after alisol A treatment, indicating that the G1 cell cycle arrest was induced by alisol A. Cyclin A and cyclin D1, the two critical molecules involved in controlling cell cycle progression, were downregulated by alisol A treatment. Cyclins function as regulators of CDK to regulate mitotic events. Cyclin D1 acts to control the G1/S transition by regulating the activity of CDK4/CDK6. Defects in cyclin D1, which is under the complex regulation of upstream proteins, is sufficient to induce abnormal G1/S transition and G1 cell cycle arrest (
Basal levels of autophagy could ensure the physiological turnover of damaged organelles, while a large accumulation of autophagic vacuoles may induce cell death. Autophagy, which is induced in response to many types of stress, including chemotherapeutic intervention, could ultimately lead to apoptosis (
The induction of DNA damage is considered to be one of the important mechanisms of action of cancer therapeutics (
In conclusion, the present study suggests that alisol A from Alismatis Rhizoma may be used as a novel agent for breast cancer therapy. The results are consistent with the observations of a previous study on the effects of alisol A on breast cancer cells (
Not applicable.
This study was funded by the National Natural Science Foundation of China (grant nos. 81672610 and 81872978), and Major National Science and Technology Project of China (grant no. 2014ZX09304307-001-011).
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
YS, MW and PW performed the experiments and wrote the manuscript. TZ performed the western blot analysis. JY and LS performed the experiments for protostane triterpenes purification and assessment. ML, HW, QZ and HZ designed the study. All authors read and approved the final version of the manuscript.
The use of human specimens in the present study was approved by the Peking University Third Hospital Medical Science Research Ethic Committee (approval no. IRB00006761-M2019343). Informed consent was obtained from all the patients.
Not applicable.
The authors declare that they have no competing interests.
triple-negative breast cancer
terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
propidium iodide
apurinic/apyrimidinic endonuclease 1
phosphorylated H2AX
reactive oxygen species
deoxyribonucleic acid
high performance liquid chromatography
ultraviolet
fluorescein isothiocyanate isomer I
dimethyl sulfoxide
7-amino-actinomycin D
2′,7′-dichlorodihydrofluorescein diacetate
phosphate buffer saline
4′6-diamidino-2-phenylindole
phosphorylated p38
Alisol A decreases the cell viability of breast cancer MDA-MB-231 cells. (A and B) MDA-MB-231 cells were treated with 10 µM
Alisol A induces cell apoptosis in breast cancer MDA-MB-231 cells. (A) Annexin V/PI staining assay was conducted using flow cytometry, and the extent of apoptosis was calculated. (B) TUNEL assay was conducted to determine DNA fragmentation in MDA-MB-231 cells. (C and D) Effects of alisol A on the proteins associated with apoptosis in MDA-MB-231 cells. Cells were incubated with alisol A for 24 h, and cell lysates were subjected to western blot analysis for caspase-3, caspase-9 (C); Bcl-2, and p-p38 (D). GAPDH was used as the control. Data are shown as mean ± SD of three independent experiments. **P<0.01; ***P<0.001 vs. control.
Alisol A induces G1 phase cell cycle arrest in breast cancer MDA-MB-231 cells. (A) Cells at G1 phase were tested and analyzed using flow cytometry assay. (B) Proteins levels of cyclin A and cyclin D1 were measured using western blot analysis. Cells were incubated with alisol A for 24 h and the cell lysates were subjected to western blot analysis for cyclin A and cyclin D1. GAPDH was used as the control. Data are shown as mean ± SD of three independent experiments. **P<0.01; ***P<0.001 vs. control.
Alisol A induces autophagy in human breast cancer MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with alisol A for 24 h. Representative images showing LC3-II fluorescence. (B) LC3-II protein levels were measured using western blot analysis. GAPDH was used as the control. Data are show as mean ± SD of three independent experiments. **P<0.01; ***P<0.001 vs. control.
Alisol A induces intracellular ROS and DNA damage in human breast cancer MDA-MB-231 cells. (A) After cells were treated with alisol A for 24 h, the intracellular ROS levels in the breast cancer MDA-MB-231 cells were measured after DCF-DA molecular probe treatment, followed by fluorescence microscopy. Statistical analysis of ROS-positive cells per field are shown. (B) APE1 and (C) γH2AX expression was measured using immunofluorescence microscopy, after cells were treated with alisol A. Data are shown as the mean ± SD of three independent experiments. **P<0.01; ***P<0.001 vs. control. ROS, reactive oxygen species; APE1, apurinic/apyrimidinic endonuclease 1.