OR Oncology Reports 1021-335X 1791-2431 D.A. Spandidos 10.3892/or.2023.8630 OR-50-5-08630 Articles Pseudolaric acid B induces apoptosis associated with the mitochondrial and PI3K/AKT/mTOR pathways in triple‑negative breast cancer YangKe 1 * WangJun-Qi 2 * LiKai 3 ChenSu-Ning 1 YuFei 1 Department of Traditional Chinese Medicine, Shengjing Hospital of China Medical University, Shenyang, Liaoning 110004, P.R. China Department of General Surgery, Shengjing Hospital of China Medical University, Shenyang, Liaoning 110004, P.R. China Department of Oncology, Shengjing Hospital of China Medical University, Shenyang, Liaoning 110004, P.R. China Correspondence to: Professor Fei Yu, Department of Traditional Chinese Medicine, Shengjing Hospital of China Medical University, 36 Sanhao Street, Heping, Shenyang, Liaoning 110004, P.R. China, E-mail: vivian.nisu@gmail.com yuf1@sj-hospital.org

Contributed equally

 11 2023 14 09 2023 50 5 193 18052023 18082023 Copyright: © Yang et al. 2023 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Pseudolaric acid B (PAB), a diterpene acid isolated from the root bark of Pseudolarix kaempferi, has been shown to exert strong antitumor properties. The aim of the present study was to investigate the mechanisms underlying the proposed antitumor properties of PAB in the triple-negative breast cancer cells, MDA-MB-231. The cell processes evaluated included cell proliferation by Cell Counting Kit-8 assay, colony formation and EdU assay, apoptosis by Annexin V-FITC/PI apoptosis assay, cell migration by Transwell migration assay and invasion by Transwell invasion assay. PAB significantly inhibited the proliferation of MDA-MB-231 cells through a mechanism that was considered to be associated with cell cycle arrest at the G2/M phase. There was decreased protein expression levels of CDK1 and cyclin B1 and increased protein expression levels of p53 and p21. However, there were no well-defined inhibitory effects on the normal breast cell line MCF10A. PAB also triggered apoptosis in a concentration-dependent manner through the mitochondrial apoptosis pathway. It caused collapse of mitochondrial membrane potential, accumulation of reactive oxygen species and release of cytochrome c, as well as upregulation of cleaved caspase-3, cleaved caspase-9, cleaved PARP and Bax, and downregulation of Bcl-2 and Bcl-xl. The migration and invasion ability of MDA-MB-231 cells were inhibited by decreasing the expression levels of the epithelial-mesenchymal transition-related markers N-cadherin and vimentin and increasing the expression of E-cadherin. Moreover, the expression levels of PI3K (p110β), phosphorylated (p)-AKT (ser473) and p-mTOR (ser2448) were downregulated and LY294002, a PI3K inhibitor, could interact additively with PAB to induce apoptosis of MDA-MB-231 cells. Overall, the present results demonstrated that PAB induced apoptosis via mitochondrial apoptosis and the PI3K/AKT/mTOR pathway in triple-negative breast cancer. It also inhibited cellular proliferation, migration and invasion, suggesting that PAB may be a useful phytomedicine for the treatment of triple-negative breast cancer.

 pseudolaric acid B apoptosis migration invasion mitochondrial PI3K/AKT/mTOR Science and Technology Project of Liaoning Province 2014226033 This work was supported by a grant from the Science and Technology Project of Liaoning Province (grant no. 2014226033). Introduction

According to global cancer statistics published by the International Agency for Research on Cancer of the World Health Organization, breast cancer surpassed lung cancer in 2020 to become the most diagnosed cancer in the world (1). Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by negative expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 (2). Although this subtype accounts for 15–20% of all types of breast cancer, it lacks effective therapeutic targets and treatment options are limited (3). Therefore, it is critical to find novel therapeutic targets for the treatment of TNBC.

Pseudolaric acid B (PAB) is a diterpene acid derived from the cortex of Pseudolarix kaempferia (golden larch) that exhibits diverse properties, including anti-inflammatory (4), antifungal (5), antiangiogenic (6), pro-apoptotic (7) and microtubule-destabilizing (8) effects. In previous years, researchers have shown that PAB has antitumor effects in liver cancer (9), gastric cancer (10), medulloblastoma (7), lung cancer (11) and leukemia (12). However, the mechanisms underlying these antitumor effects remain unclear.

Apoptosis, also known as programmed cell death, is a form of self-cleaning in which the body removes damaged cells in an orderly and efficient manner. The dysregulation of apoptosis is a major reason for the unlimited proliferation exhibited by tumor cells (13). Thus, dysregulation of apoptosis is considered one of the hallmarks of cancer. The mitochondrial apoptotic pathway comprises the core apoptotic signaling pathway in vertebrates and is triggered by a change in mitochondrial outer membrane permeabilization (14). Subsequently, cytochrome c is released from the mitochondria into the cytoplasm, stimulating caspase-3 and other mechanisms of apoptosis (15). Previous studies demonstrated that PAB induces apoptosis through the mitochondrial and death receptor pathway in numerous types of cancer, including hepatocellular (9), cervical (16), head and neck (17) and colorectal cancer (18). However, to the best of our knowledge, the apoptotic mechanism of PAB in TNBC has not been studied.

The PI3K/AKT/mTOR signaling pathway plays a key role in several processes of tumor development, such as apoptosis, proliferation, metabolism and metastasis (19). It can also enhance the malignancy of various types of tumor cells (20). Previous studies have demonstrated that PI3K/AKT/mTOR signaling affects the mitochondrial apoptosis pathway by regulating proteins in the Bcl-2 family (21–23).

The present study aimed to explore the anticancer activity and related mechanisms of PAB in the TNBC MDA-MB-231 cell line. Effects of PAB on migration and invasion were also investigated.

 Materials and methods <sec> <title>Reagents and antibodies

PAB was purchased from Beijing Solarbio Science & Technology Co., Ltd. Primary antibodies for caspase-3 (1:1,000; cat. no. 19677-1-AP), caspase-9 (1:1,000; cat. no. 10380-1-AP), Bax (1:2,000; cat. no. 50599-2-Ig), Bcl-2 (1:2,000; cat. no. 60178-1-Ig), p53 (1:5,000; cat. no. 80077-1-RR), p21 (1:2,000; cat. no. 10355-1-AP) and GAPDH (1:8,000; cat. no. 10494-1-AP), and HRP-conjugated Affinipure goat anti-rabbit (cat. no. SA00001-2; 1:8,000) and anti-mouse (cat. no. SA00001-1; 1:8,000) IgG secondary antibodies were purchased from ProteinTech Group, Inc. In addition, the primary antibodies for PARP (cat. no. T40050); Bcl-xl (cat. no. T40057), Cytochrome c (cat. no. T55734), PI3K (cat. no. T55224), AKT (cat. no. T55561), p-AKT (cat. no. T40067), mTOR (cat. no. T55306), p-mTOR (cat. no. T56571) (all 1:1,000) were purchased from Abmart Pharmaceutical Technology Co., Ltd. Antibodies for CDK1 (1:1,000; cat. no. PTM-6521), cyclin B1 (1:1,000; cat. no. PTM6659), Vimentin (1:1,000; cat. no. PTM5376), E-cadherin (1:2,000; cat. no. PTM6222) and N-cadherin (1:1,000; cat. no. PTM5221) were purchased from PTM Biolabs, Inc. Annexin V-FITC/PI Apoptosis Detection kit was purchased from Vazyme Biotech Co., Ltd. Cell Counting Kit-8 (CCK-8) was purchased from MedChemExpress. Mitochondrial membrane potential (MMP) assay kit with JC-1 (cat. no. C2006) and western stripping buffer (cat. no. P0025N) were purchased from Beyotime Institute of Biotechnology.

 Cell culture

The TNBC cell line MDA-MB-231 and human breast cell line MCF-10A were donated by the Department of Oncology, Shengjing Hospital of China Medical University (Shenyang, China). MDA-MB-231 cells were cultured in Leibovitz's L-15 medium (Procell Life Science & Technology Co., Ltd.) containing 5% fetal bovine serum (FBS; Procell Life Science & Technology Co., Ltd.) and 1% antibiotics (100 U/ml penicillin and 100 U/ml streptomycin). MCF10A cells were cultured in DMEM/F12 (Procell Life Science & Technology Co., Ltd.) containing 5% HS, 20 ng/ml epidermal growth factor, 0.5 µg/ml Hydrocortisone, 10 µg/ml insulin, 1% non-essential amino acid and 1% antibiotics (100 U/ml penicillin and 100 U/ml streptomycin). MDA-MB-231 cells were grown free of CO2 in a cell culture incubator at 37°C. MCF-10A cells were cultured at 37°C with 5% CO2.

 Cell viability assay

The effect of PAB on cellular viability was measured using a CCK-8 assay. Cells (5×103/well) were cultured in a 96-well plate overnight, then treated with different concentrations of PAB (0, 2.5, 5, 7.5, 10, 12.5 and 15 µM) at 37°C for 24, 48 and 72 h. After treatment, the medium was removed and a mixture of 90 µl medium (Leibovitz's L-15 or DMEM/F12) and 10 µl CCK-8 reagent was added to each well (24). After 2 h of incubation at 37°C, the absorbance at 450 nm was measured using a microplate reader.

 Colony formation assay

To explore the effect of PAB on cellular proliferation, the colony formation assay was conducted. MDA-MB-231 and MCF-10A cells (1×103/well) were seeded in six-well plates overnight, then treated with different concentrations of PAB (5, 7.5 and 10 µM) at 37°C for 48 h before changing back to a drug-free medium (Leibovitz's L-15 or DMEM/F12). Cells were cultured for an additional 14 days, then fixed with 4% polyoxymethylene at room temperature for 15 min and stained with 0.1% crystal violet at room temperature for 15 min. After removing the crystal violet, plates were washed twice with phosphate-buffered saline (PBS) and the number of colonies was counted and analyzed by Image J 1.8.0 software (National Institutes of Health). A colony with more than 50 cells is defined as a colony.

 EdU staining assay

The BeyoClick™ EdU-488 cell proliferation kit (Beyotime Institute of Biotechnology) was used to evaluate the effect of PAB on cellular proliferation. MDA-MB-231 cells (2×104/well) were seeded in 12-well plates and cultured at 37°C overnight. The kit was used according to manufacturer's instructions. Briefly, cells were incubated with medium containing EdU (10 µM) at 37°C for 2 h, then fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were then stained with the Click reaction solution at room temperature for 30 min in darkness. Next, cell nuclei were stained using Hoechst 33342 and images were captured using a fluorescence microscope (Nikon Corporation) at a magnification of 100.

 Cell cycle analysis

Cells were treated with 5, 7.5 and 10 µM PAB at 37°C for 48 h. After harvesting and washing with pre-cold PBS, cells (1×106/ml) were resuspended with cold 70% ethanol and fixed at 4°C overnight. Cells were washed twice with PBS and centrifuged at 1,000 × g at 4°C for 5 min to remove residual ethanol, then stained with 50 µg/ml propidium iodide (PI) and 100 µg/ml RNase solution (Beijing Solarbio Science & Technology Co., Ltd.) at 37°C for 20 min (25). The cell cycle arrest in G2-M phase was measured using flow cytometry (FACS Calibur; BD Biosciences) and analyzed with FlowJo 7.6 software (FlowJo LLC).

 Apoptosis assay

The rate of apoptosis was detected using the annexin V-FITC/PI apoptosis kit (Vazyme Biotech Co., Ltd.) according to the manufacturer's instructions. Briefly, MDA-MB-231 cells (2×105) were seeded in six-well plates overnight, then cultured with PAB (5, 7.5 and 10 µM), LY294002 (40 µM) or both (7.5 µM PAB + 40 µM LY294002) at 37°C for 48 h. Next, cells were harvested with EDTA-free trypsin and washed twice with chilled PBS. Cell precipitates were resuspended with 1X binding buffer and then stained with 5 µl annexin V-FITC and 5 µl PI staining solution in the dark for 10 min at room temperature (24,26). Finally, the rate of apoptosis was analyzed by flow cytometry (FACS Calibur; BD Biosciences).

 Assessment of MMP and (reactive oxygen species) ROS

Considering that a decline in MMP is a key trigger to activate the mitochondrial apoptosis pathway, MMP assay kit with JC-1 was used to detect the MMP level of MDA-MB-231 cells. JC-1 forms aggregates in the mitochondrial matrix when the MMP is high, resulting in red fluorescence. When the MMP is low, JC-1 cannot aggregate in the mitochondrial matrix and cells fluoresce is green. Briefly, after treatment with PAB (5, 7.5 and 10 µM) at 37°C for 48 h, cells were harvested and resuspended in 500 µl JC-1 working solution, in the darkness at 37°C for 20 min. Cells were then washed twice with chilled JC-1 staining buffer (1X) and immediately examined using flow cytometry (FACS Calibur; BD Biosciences).

To assess ROS levels, cells were cultured with PAB (5, 7.5 and 10 µM) at 37°C for 48 h, then collected and incubated with 10 µM DCFH-DA fluorescent probe for 20 min at 37°C in the darkness. The cells were washed twice with FBS-free Leibovitz's L-15 medium (Procell Life Science & Technology Co., Ltd.) and the cellular ROS level was detected using flow cytometry (FACS Calibur; BD Biosciences).

 DAPI staining

Cells (1×104/well) were seeded on glass coverslips in 24-well plates overnight, then treated with PAB (5, 7.5 and 10 µM) at 37°C for 48 h. The cells were fixed with 4% paraformaldehyde at room temperature for 15 min and permeabilized with 0.5% Triton X-100 at room temperature for 20 min. After washing three times with PBS, the cells were stained with fluorescent dye DAPI in a darkroom at room temperature for 15 min. The nuclear morphology was observed using a fluorescence microscope at a magnification of 200.

 Transwell migration and invasion assays

Cell migration and invasion assays were performed using Transwell chambers with a pore size of 8 µm (27). Matrigel (Corning Biocoat; Corning Life Sciences) was used for the cell invasion assay but not for the cell migration assay. A mixture of Martrigel and FBS-free Leibovitz's L-15 medium was added to the upper chamber and placed at 37°C for 1 h. Cells were cultured with serum-free Leibovitz's L-15 medium overnight, then suspended and diluted to a density of 1×105 with various concentrations of PAB (5, 7.5 and 10 µM). A 200 µl cell suspension containing 2×104 cells was added to the upper chamber on the 24-well plate. The lower chamber was filled with 500 µl Leibovitz's L-15 medium supplemented with 10% FBS. After incubation at 37°C for 48 h, the cells that passed through the chambers were fixed with 4% paraformaldehyde at room temperature for 20 min, stained with 0.1% crystal violet at room temperature for 30 min and then non-penetrating cells were wiped off with a cotton swab. Cells were observed under light microscope at a magnification of 100.

 Wound healing assays

MDA-MB-231 cells were grown in six-well plates until the cell confluence reached 100%. A wound was made by scratching the adherent cell layer with a 200-µl pipette tip (25). Shed cells were washed off with PBS and the remaining cells were treated with serum-free Leibovitz's L-15 medium containing different concentrations of PAB (5, 7.5 and 10 µM) for 48 h. Cells were observed under light microscope at a magnification of 200. Data analysis was performed using ImageJ 1.8.0 software (National Institutes of Health). The rate of wound healing=[(the wound width of 0–48 h)/0 h wound width] ×100%.

 Western blotting

Protein expression levels were evaluated using western blotting as previously described (26,28). Cells were treated with 5, 7.5 and 10 µM PAB at 37°C for 48 h, then lysed with RIPA buffer (Beyotime Institute of Biotechnology) for protein extraction. Cells were centrifuged at 10,000 × g for 15 min at 4°C, and protein concentrations were measured using a BCA Protein Assay kit (Beyotime Institute of Biotechnology). Protein samples (30 µg/lane) were separated using 10–12.5% SDS-PAGE gels before being transferred to the PVDF membrane (MilliporeSigma). PVDF membranes were horizontally cut to probe proteins with different molecular weights. After blocking with 5% skim milk diluted with Tris-buffered saline with Tween (TBST) containing 0.1% Tween at room temperature for 2 h, the PVDF membranes were incubated with primary antibodies at 4°C overnight. Blots were washed three times with TBST and incubated with secondary antibodies for 2 h at room temperature. Stripping buffer was used to remove the antigen-antibody complex from the PVDF membrane in order to re-probe other antibodies on the same membrane. The blots were measured using SuperFemto ECL Chemiluminescence kit (Vazyme Biotech Co., Ltd) through the chemiluminescence detection system of the Amersham Imager 600 (GE Healthcare Production).

 Statistical analysis

All data were obtained from three independently replicated experiments and presented as mean ± standard deviation. One-way analysis of variance was used to analyze statistical significance for multiple comparisons, followed by Tukey's multiple comparisons test. P<0.05 was considered to indicate a statistically significant difference.

 Results <sec> <title>PAB inhibits the proliferation of TNBC cells

To investigate the effect of PAB on the viability of TNBC cells, the CCK-8 assay was performed to evaluate the influence of various concentrations of PAB for 24, 48 and 72 h. PAB inhibited the proliferation of MDA-MB-231 cells in dose- and time-dependent manners, with IC50 values of 19.3, 8.3 and 5.76 µM at 24, 48 and 72 h respectively (Fig. 1A). However, the result of CCK-8 showed that PAB had no obvious side-effects on normal cells line MCF10A (Fig. 1B). Colony formation assays further confirmed that PAB suppressed the proliferation of MDA-MB-231 cells in a dose-dependent manner, but not MCF10A cells (Fig. 1C-F). Furthermore, according to the EdU assay, compared with the control, all three different concentrations (5, 7.5 and 10 µM) of PAB can significantly reduce the number of EDU positive MDA-MB-231 cells (Fig. 2A), indicating that PAB could suppress the proliferative capacity of these cells (Fig. 2B).

 G<sub>2</sub>/M cell cycle arrest of MDA-MB-231 cells is induced by PAB

The cell cycle distribution was analyzed by flow cytometry after propidium iodide staining. MDA-MB-231 cells treated with various concentration of PAB for 48 h were harvested for cell cycle analysis. As shown in Fig. 2C and D, it induced a significant increase in the number of cells in G2/M phase. Meanwhile, western blotting showed that the protein expression levels of CDK1 and cyclin B1 were significantly reduced compared with the control, while those of p53 and p21 were increased after PAB treatment (Fig. 2E and F). These results indicate that PAB may induce cell cycle arrest by altering the expression of cell recycle regulators.

 PAB induces apoptosis via the mitochondrial pathway

After treatment with various concentration of PAB for 48 h, flow cytometry showed that PAB induced apoptosis in MDA-MB-231 cells in a dose-dependent manner (Fig. 3A and B). The effect of PAB on the nuclear status of MDA-MB-231 cells was tested using DAPI staining. Apoptotic cells presented with nuclear condensation and DNA fragmentation (Fig. 3C).

A collapse of MMP is an important factor leading to apoptosis mediated by the mitochondrial apoptosis pathway. JC-1 staining showed that PAB induced a dose-dependent loss of MMP in MDA-MB-231 cells (Fig. 3D and E). To further explore the mechanisms of PAB-induced apoptosis, the related protein expression was measured and it was revealed that PAB significantly induced Cytochrome c release from the mitochondria into the cytosol (Fig. 4A and B), upregulated the expression of cleaved-caspase3, cleaved-caspase9, cleaved-PARP and Bax, and downregulated the expression of Bcl-2 and Bcl-xl (Fig. 4C and D). All these changes were statistically significant (P<0.05). Therefore, the results demonstrated that PAB induced apoptosis mediated by mitochondrial apoptosis pathway in TNBC.

 PAB increases ROS levels

It is well known that ROS production is related to mitochondrial pathway-associated apoptosis. Therefore, ROS production was detected by the fluorescent probe DCFH-DA. As shown in Fig. 3F and G, flow cytometry demonstrated that the ROS accumulation was directly related to PAB concentration.

 PAB inhibits migration and invasion by regulating the epithelial-mesenchymal transition (EMT) pathway

Migration of MDA-MB-231 cells was measured by the wound healing and Transwell migration assays. PAB significantly inhibited wound healing ability (Fig. 5A and B) and Transwell migration ability (Fig. 5C and D) in a dose-dependent manner. As shown in Fig. 5E and F, PAB significantly inhibited cell invasion. Protein levels of N-cadherin and vimentin were reduced by PAB, while the protein level of E-cadherin was increased (Fig. 5G and H). Overall, these data suggested that PAB inhibited migration, invasion and EMT in MDA-MB-231 cells.

 PAB inhibits the PI3K/AKT/mTOR signaling pathway

To investigate the role of the PI3K/AKT/mTOR signaling pathway in the anticancer effect of PAB on MDA-MB-231 cells, the activation of PI3K, AKT and mTOR were evaluated by western blotting. PAB inhibited PI3K (p110β), the phosphorylation of AKT and the phosphorylation of mTOR in a dose-dependent manner and PAB did not influence total levels of AKT and mTOR (Fig. 6A and B). Furthermore, the apoptotic rate of PAB and LY294002 co-treated cells exceeded that of PAB or LY294002 alone (Fig. 6C and D). These results suggested that the PI3K/AKT/mTOR signaling pathway was involved in PAB-induced apoptosis in MDA-MB-231 cells.

 Discussion

PAB, the main medicinal component of Cortex pseudolaricis, is a natural plant product with potential antifungal, immunosuppressive and anticancer properties. Previous studies have shown that PAB has multi-target anticancer effects in different types of tumors (9,29–31). To investigate the anticancer properties and mechanisms of PAB on TNBC, the present study investigated the effects of PAB on the proliferation, apoptosis, invasion and migration of MDA-MB-231 cells. CCK-8, EdU and colony formation assays demonstrated that PAB inhibited the proliferation of MDA-MB-231 cells in a dose- and time-dependent manner.

Excessive cell division is an important reason for the continuous proliferation of tumor cells (32). Recently, researchers have indicated that numerous natural drugs exert their anticancer effects by regulating the cycle checkpoints, which can lead to arrest of the tumor cell cycle (33–36). Among these checkpoints, cyclin B1 and CDK1 are necessary for the cell cycle to switch from the S to the G2/M phase (37). The tumor suppressor p53 induces G2/M phase arrest by activating the downstream transcriptional target p21, which is a CDK inhibitor (38,39). The present results indicated that PAB induced G2/M phase arrest of MDA-MB-231 cells by upregulating the expression of p53 and p21, resulting in the downregulation of cyclin B1 and CDK1 proteins.

Flow cytometry showed that PAB induced apoptosis in MDA-MB-231 cells in a concentration-dependent manner. Mitochondria are the hubs for cellular energy metabolism, and mitochondrial dysfunction is the main cause for activation of the mitochondrial apoptosis pathway (40). ROS are mainly produced in mitochondria, and its excessive accumulation can damage mitochondria and activate the mitochondria-mediated intrinsic apoptotic pathway (41). To further investigate the mechanism of apoptosis, nuclear morphology, MMP level, ROS levels and protein expression were evaluated. DAPI nuclear staining indicated that PAB led to chromatin condensation, cellular shrinkage and DNA fragmentation. It also caused a decrease of MMP and an increase of ROS. Furthermore, western blotting showed that it upregulated the proapoptotic protein Bax and downregulated the antiapoptotic proteins Bcl-2 and Bcl-xl. Cytochrome c is released from mitochondria into the cytosol and subsequently activates caspase-9 and caspase-3, which help to initiate the mitochondrial apoptosis pathway (42). The present experiments showed that PAB could promote the release of cytochrome c from mitochondria into the cytoplasm. In addition, levels of cleaved caspase-3 and caspase-9, as well as PARP, were increased. Western blotting further demonstrated that it could downregulate the anti-apoptosis proteins Bcl-2 and Bcl-xl and increased the pro-apoptosis protein Bax. These results suggested that PAB induced apoptosis of MDA-MB-231 cells via the mitochondrial apoptosis pathway (Fig. S1).

The PI3K/AKT/mTOR signaling pathway, an important regulator of tumor proliferation, apoptosis, invasion and migration, is closely related to the expression of Bcl-2 family proteins in the mitochondrial apoptosis pathway (22). Western blotting showed that treatment of TNBC cells with PAB significantly decreased levels of PI3K (p110β), p-AKT and p-mTOR. Its apoptosis effect was also significantly elevated when combined with LY294002, a PI3K inhibitor. These data demonstrated that PI3K/AKT/mTOR signaling may be the target of PAB-induced apoptosis in MDA-MB-231 cells.

TNBC is the most aggressive and malignant type of breast cancer, and is more likely to develop lung and brain metastases (43–45). The present study revealed that EMT is critical for regulating the proliferation, invasion and metastasis of carcinoma cells (46). Activation of the EMT mechanism depends on loss of the epithelial marker E-cadherin and the upregulation of mesenchymal markers N-cadherin and vimentin, thereby prompting tumor cells through multiple steps in the process of invasion and metastasis (47,48). Results of the present wound-healing and Transwell assays suggested that PAB inhibited the ability of cells to migrate and invade. Further experiments demonstrated that it decreased the levels of N-cadherin and vimentin and increased the level of E-cadherin. These results suggested that EMT in MDA-MB-231 cells could be inhibited by PAB treatment.

There are several limitations and challenges to the present research. First, it was only verified that the mechanism of PAB-induced apoptosis in MDA-MB-231 cells was related to the mitochondrial apoptosis pathway, but it was not verified whether it was dependent on the mitochondrial apoptosis pathway. Second, the present study demonstrated that the PI3K/AKT/mTOR signaling pathway had a superpositioned effect on PAB-induced apoptosis, but the specific mechanism of action has not been clearly explored. Future studies will hopefully explore the specific role of PI3K/AKT/mTOR signaling pathway in PAB-induced apoptosis. Third, although the aim of the present study was to demonstrate how PAB plays an anticancer role in TNBC, the specific mechanism of PAB in vivo studies remains to be determined. In addition, a previous study has revealed that PAB-induced autophagy of breast cancer cells line MCF-7 inhibits apoptosis and promotes cell survival, which indicates that the combination of autophagy inhibitors may improve the anticancer effect of PAB (49).

In conclusion, the present results demonstrated that PAB exhibited anticancer effects against TNBC and that the mechanism was related to multiple pathways (Fig. 7). The present study revealed that PAB significantly inhibited the proliferative ability of MDA-MB-231 cells by arresting the cell at the G2/M phase. The pro-apoptotic activity of PAB in TNBC was demonstrated through activation of the mitochondrial apoptosis pathway and inhibition of the PI3K/AKT/mTOR signaling pathway. PAB also demonstrated an anticancer effect on TNBC by inhibiting cell migration and invasion, through a mechanism related to the suppression of EMT. Overall, these results provided evidence that PAB exerted multiple anticancer activities through multiple targets in TNBC.

 Supplementary Material Supporting Data Acknowledgements

The group would like to thank Professor Caigang Liu from Shengjing Hospital Cancer Research Center (Shenyang, China) for the cell support. The group would like to thank Professor Yuxin Tong, Medical Research Center, Shengjing Hospital of China Medical University (Shenyang, China) for technical guidance.

 Availability of data and materials

The data used and/or analyzed during the present study are available from the corresponding author on reasonable request.

 Authors' contributions

FY, SNC and KL designed the study and revised the manuscript. KY and JQW performed the experiments and drafted the manuscript together. All authors read and approved the final manuscript. KY and JQW confirm the authenticity of all the raw data.

 Ethical approval and consent to participate

Not applicable.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

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PAB inhibits the growth of MDA-MB-231 cell. (A) MDA-MB-231 and (B) MCF10A cells were incubated with various concentration of PAB for 24, 48 and 72 h, and cell viability was determined using CCK-8 assay. Results of colony formation assays of (C) MDA-MB-231 and (D) MCF10A cells. Quantification of (E) MDA-MB-231 and (F) MCF10A cell colony formation assays. ****P<0.0001 vs. control. CCK-8, Cell Counting Kit-8; PAB, Pseudolaric acid B; ns, not significant.

PAB induces cell arrest at the G2/M phase. (A) MDA-MB-231 cell proliferation was assessed using an EdU assay (magnification, ×100). (B) Quantification of the proportion of EdU-positive cells induced by PAB (C) Percentage of cells in G0-G1, G2-M and S phase in MDA-MB-231 cells with or without the treatment of PAB for 48 h. (D) Quantitative data of PAB-induced cell cycle distribution. (E) Western blotting showed Cyclin B1 and CDK1 were inhibited by PAB. p53 and p21 expression are both enhanced as PAB concentration increased. (F) Quantitative data of the relative protein expression. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 vs. control. PAB, Pseudolaric acid B.

PAB induces apoptosis in MDA-MB-231 cells. (A) Cells were pretreated with PAB (5, 7.5, 10 µM) for 48 h. Annexin V-FITC and PI staining were used to identify the apoptosis, and the data were analyzed by flow cytometry. (B) Quantitative data of PAB-induced apoptosis. (C) Cell nuclei were observed by confocal microscope (magnification, 400×) after 48 h of PAB treatment by DAPI staining. Typical apoptosis morphological changes were shown in treated cells including chromatin condensation and DNA fragmentation. (D) Cell mitochondrial membrane potential was determined using flow cytometry with JC-1 staining after 48 h of treatment with 0, 5, 7.5 and 10 µM PAB, respectively. (E) Red/green fluorescence ratio. The ratio of red to green fluorescence represents the percentage of decreased MMP. (F) Fluorescence in the cell is represented as the percentage of ROS production (G) analyzed using flow cytometry. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. control. PAB, Pseudolaric acid B; MMP, mitochondrial membrane potential; ROS, reactive oxygen species.

Effect of PAB on mitochondria-mediated apoptotic pathway. (A) PAB significantly induced Cytochrome c released from mitochondria into the cytosol. (B) Quantitative data of the protein expression of Cytochrome c. (C) Western blotting analysis result of the expression levels of Bcl-2, Bcl-xl, Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP. (D) Quantitative data of the relative protein expression. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. control. PAB, Pseudolaric acid B.

PAB suppresses MDA-MB-231 cells migration, invasion and epithelial-mesenchymal transition. (A) Wound healing assay (magnification, ×200) and (B) quantitative analysis data. (C) Transwell migration assay (magnification, ×100) was performed and (D) quantified to detect the migration ability of cells treated by PAB. (E) Transwell invasion assay (magnification, ×100) was performed and (F) quantified to detect the invasion ability of cells treated by PAB. (G) Expression of markers for epithelial-mesenchymal transition in MDA-MB-231 cells with PAB treatment by western blotting. (H) Quantitative data of the relative protein expression. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. control. PAB, Pseudolaric acid B.

PAB inhibits the PI3K/mTOR/AKT signaling pathway. (A) MDA-MB-231 cells were exposed to the indicated doses of PAB for 48 h. The expression levels of PI3K, p-AKT (ser473), AKT, p-mTOR (ser3448) and mTOR were assessed by western blotting. (B) Quantitative data of the relative protein expression. (C) Cells were pretreated with PAB (5 µM), LY294002 (40 µM) or both for 48 h. Annexin V-FITC and PI staining were used to identify apoptosis rate, and (D) the data were analyzed by flow cytometry. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. control. PAB, Pseudolaric acid B; p-, phosphorylated.

Plausible delivery mechanisms through which pseudolaric acid B exerts multiple anticancer activities through multiple targets in triple negative breast cancer. ROS, reactive oxygen species; EMT, epithelial-mesenchymal transition.