All experimental reagents and antibodies were purchased from Sigma-Aldrich (Merck KGaA) unless otherwise stated. Bafilomycin A1 (Baf A1) was purchased from Sangon Biotech Co., Ltd. The antibodies used in this study included anti-FOXO1 antibody (cat. no. 2880; Cell Signaling Technology, Inc.), anti-p-FOXO1 antibody (cat. no. WL03634; Wanleibio Co., Ltd.) and anti-Lamin AC antibody (cat. no. 10298-1-AP; ProteinTech Group, Inc.).

MDA-MB-231 TNBC cell lines were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences and HCC-1937 cell lines were provided by the Kunming Institute of Zoology, Chinese Academy of Sciences. The cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM; cat. no. C11995500BT; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; cat. no. 10099-141; Gibco; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 µg/ml) (Invitrogen; Thermo Fisher Scientific, Inc.) in a humidified atmosphere of 5% CO₂ at 37°C.

Exponentially growing MDA-MB-231 and HCC-1937 cells were transferred to 12-well plates at a density of 2×10⁴ cells/well and cultured at 37°C in a 5% CO₂ atmosphere. The cells were treated with 0, 10, 20 and 30 nM PTX (cat. no. 48248; MedChemExpress) at 37°C. When the cells reached 60-70% confluency, they were rinsed twice with PBS and supernatant was discarded and cells were used for morphological observation. Images were acquired using an Olympus IX 71 microscope (Olympus Corporation). Next, MDA-MB-231 cells were treated with different concentrations of PTX (0, 0.03, 0.1, 0.3, 1, 3, 10 and 30 nM) at 37°C, 5% CO₂ for 24 h to perform cell proliferation assay using cell counting kit (CCK-8) (cat. no. P10330; TransGen Biotech) according to manufacturer's instruction.

For detecting PTX-induced cell apoptosis, MDA-MB-231 cells were treated with 0, 10, 20 and 30 nM PTX at 37°C, 5% CO₂ for 24 h. For detecting PTX-induced cell apoptosis under FOXO1 inhibition, MDA-MB-231 cells were transfected with small interfering RNA at 37°C for 7 h, and then were treated with or without 20 nM PTX at 37°C, 5% CO₂ for 24 h. The apoptosis assay was performed using an Annexin V-FITC/PI test kit (cat. no. FXP018-100; Beijing 4A Biotech Co., Ltd.), according to the manufacturer's instructions. Flow cytometry analysis was performed using a Beckman CytoFLEX flow cytometer (Beckman Coulter, Inc.). The cells undergoing apoptosis were defined as reported previously (16) early (Annexin V-FITC-positive/PI-negative, Q3) + late apoptotic cells (Annexin V-FITC-positive/PI-positive; Q2).

For detecting LC3, MDA-MB-231 cells were pretreated with Baf A1 (0 and 10 nM) at 37°C, 5% CO₂ for 24 h and then treated with or without 20 nM PTX at 37°C, 5% CO₂ for 24 h. Cells were treated with 0, 10 and 30 nM PTX combined with or without 5 nM 3-methyladenine (3-MA; 5 nM; a PI3K inhibitor that is a widely used inhibitor of autophagy via its inhibitory effect on class III PI3K) at 37°C, 5% CO₂ for 24 h. For detecting FOXO1, MDA-MB-231 cells were treated with 0, 10 and 20 nM PTX at 37°C, 5% CO₂ for 24 h. Immunofluorescence analysis of light chain 3 (LC3) and FOXO1 proteins was performed as described in our previous study (17). In brief, the treated cells were fixed in 95% methanol at room temperature for 10 min and blocked in a buffer containing 1% BSA (cat. no. A8020; Beijing Solarbio Science & Technology Co., Ltd.) and 0.1% Triton X-100 at RT for 1 h. Then, the fixed cells were incubated with primary antibodies against LC3B (1:200) and FOXO1 (1:100) at 4°C overnight. Next, the cells were incubated with secondary fluorescence-conjugated antibodies (Green for LC3, 1:200, cat. no. 111-545-003; Red for FOXO1, 1:200, cat. no. 711-605-152, Jackson ImmunoResearch Inc.) at room temperature for 1 h for visualization via laser confocal microscopy (Olympus FV 1000; Olympus Corporation).

This assay was performed according to our previous study (18). Briefly, adherent MDA-MB-231 cells (250 cells/plate) were treated with or without 3-MA and/or PTX (0, 10, 30 nM) at 37°C for 72 h and then cultured for 15 days. Thereafter, the cells were fixed with 4% paraformaldehyde (cat. no. P1110; Solarbio) at RT for 30 min and stained with 10% Giemsa (cat. no. G1015; Beijing Solarbio Science & Technology Co., Ltd.) at RT for 15 min. The colonies were washed, air dried, imaged and counted (>100 cells as one colony). Finally, the colony formation ratio was calculated according to the following formula: Colony formation ratio=number of colonies/number of seeded cells ×100%.

MDA-MB-231 cells (2×10⁶ cells/well) were seeded into 96- or 6-well plates. Then, control random siRNA (cat. no. sc-37007; proprietary sequence) or FOXO1-targeted siRNA (cat. no. sc-35382) that included a pool of three sequences are in Table SI (Santa Cruz Biotechnology, Inc.; 100 pmol/well) were transfected into MDA-MB-231 cells using an siRNA transfection reagent (Santa Cruz Biotechnology, Inc.) according to the manufacturer's protocol. The cells were transfected at 37°C for 7 h, washed with PBS and then were immediately treated with 0 and 20 nM PTX at 37°C for 24 h. Then, the cells were immediately collected, and cell lysates were prepared for reverse transcription-quantitative PCR (RT-qPCR) and western blotting. Cells were also used for apoptosis analyses.

The MDA-MB-231 cells treated with 0, 10 and 20 nM PTX at 37°C for 24 h were used for the analysis of mRNA expression of the autophagy-regulated genes. The MDA-MB-231 cells transfected with siRNA at 37°C for 7 h were treated with 0 and 20 nM PTX at 37°C, 5% CO₂ for 24 h and used for the analysis of mRNA expression of the autophagy-related genes (ATG). The mRNA expression levels of genes were assessed via qPCR, including the autophagy-regulated genes, sestrin 1 (SESN1), phosphatase and tensin homolog (PTEN), mTOR, ectopic P-granules autophagy protein 5 homolog (EPG5), TSC complex subunit 1 (TSC1), serine/threonine kinase 1 and 11 (AKT and LKB1), FOXO1, lamin A/C (LMNA), autophagy and beclin 1 regulator 1 (AMBRA1), DNA damage regulated autophagy modulator 1 and 2 (DRAM1 and DRAM2), as well as, the ATGs, ATG5, class III phosphoinositide 3-kinase vacuolar protein sorting 34 (VPS34), autophagy related 4B cysteine peptidase (ATG4B), BECN1 and microtubule associated protein 1 light chain 3β (MAP1LC3B). Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA was synthesized from total RNA using a Prime-Script RT reagent kit (Takara Bio, Inc.) according to the manufacturer's protocol. The obtained cDNA was used as a template for TB Green-based qPCR kit (cat. no. RR820B; Takara Bio, Inc.) on a CFX-96 system (Bio-Rad Laboratories, Inc.). GAPDH was used for normalization. The primer sequences are shown in Table SII. Thermocycling conditions were as follows: Initial denaturation, 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec, 60°C for 30 sec, and 72°C for 30 sec. Finally, the data was analyzed using 2^−∆∆Cq method (19).

MDA-MB-231 and HCC-1937 cells were treated with 0, 10, 20 and 30 nM PTX at 37°C, 5% CO₂ unless otherwise stated for 24 h. At the end of the designed treatment time, the cells were washed twice with PBS and collected. The cells were lysed in RIPA lysis buffer (cat. no. BB-3201-2; Bestbio) with protease inhibitors (cat. no. D1201-2; TransGen Biotech) at 4°C. Then, the total protein concentrations of the cell lysates were determined using a BCA Protein Assay kit (cat. no. P0009-1 & 2; Beyotime Institute of Biotechnology). The cytosolic and nuclear proteins were extracted using Nuclear and Cytoplasmic Protein Extraction kit (cat. no. P0028-1; Beyotime Institute of Biotechnology) according to manufacturer's instruction. Protein samples (50 µg/lane) were separated via 12% SDS-PAGE and subsequently transferred onto PVDF membranes. The membranes were incubated at RT for 30 min in 5% BSA buffer (Beijing Solarbio Science & Technology Co., Ltd.) with gentle shaking to block non-specific binding before incubation with the diluted primary antibody (Bcl-2, 1:1,000, cat. no. AF6139, Affinity; Bax, 1:1,000, cat. no. WL01637, Wanlei, China; LC3B, 1:2,000, cat. no. L7543; Sigma-Aldrich; Merck KGaA; P62; 1:2,000, cat. no. P0067; Sigma-Aldrich; Merck KGaA; FOXO1, 1:1,000; p-FOXO1, 1:1,000; AKT, 1:1,000, cat. no. 4691S; Cell Signaling Technology, Inc.; p-AKT, 1:1,000, cat. no. 9271S; Cell Signaling Technology, Inc.; Lamin AC, 1:2,000; β-actin, 1:5,000, cat. no. A5441; Sigma-Aldrich; Merck KGaA) overnight at 4°C. Subsequently, membranes were incubated with horseradish peroxidase-conjugated secondary antibody (rabbit IgG, 1:5,000, cat. no. HAF008; mouse IgG, 1:10,000, cat. no. HAF007; R&D Systems) for 90 min at RT. Membranes were washed three times in PBS for 10 min each time. Then, the membranes were treated for 3 min in the dark with reagent from an Easysee Western Blot kit (TransGen Biotech) and visualized using an imaging system (Bio-Rad Laboratories, Inc.). The densitometry of protein bands was performed using Image Lab Software (version 6.1; Bio-Rad Laboratories, Inc.).

All numerical data are representative of at least three independent experiments and are presented as the mean ± standard deviation (SD) on the basis of three independent experiments. The data from the different concentrations of PTX were analyzed by one-way ANOVA, while the data from the combined treatment with PTX + Baf A1 or PTX + si-FOXO1 were analyzed by two-way ANOVA followed by a Tukey's post hoc test. The data from PTX and PTX + 3-MA treatment were analyzed using unpaired Student's t-test. Analysis of all numerical data was performed using SPSS version 22 software (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

PTX is a chemotherapeutic agent that promotes the polymerization of tubulin, which disrupts normal microtubule dynamics, leading to cell death (20). To determine the effect of PTX on MDA-MB-231 cell viability, morphological observations, and CCK-8 assays were performed after PTX treatment. Morphological changes revealed that MDA-MB-231 cell death increased with increasing doses of PTX (Fig. 1A). Furthermore, the CCK-8 assay showed that the cell survival rate also decreased with increasing PTX doses, and the half maximal inhibitory concentration (IC₅₀) of PTX fell between 10 and 30 nM (Fig. 1B). These results indicated that PTX induced MDA-MB-231 cell death in a dose-dependent manner.

To further determine whether the observed cell death was caused by PTX inducing the apoptotic pathway, the expression of apoptotic markers was evaluated and the results indicated that the expression levels of the apoptosis markers Bcl-2 and Bax, especially expression levels of Bcl-2, were decreased in a dose-dependent manner (Fig. 1C). Further, PI and Annexin V staining coupled with flow cytometry were also performed and it was observed that PTX exposure resulted in a dose-dependent increase in the apoptosis rate (Fig. 1D and E). Collectively, these results indicated that PTX induced the apoptosis of MDA-MB-231 cells.

In response to PTX-induced MDA-MB-231 cell apoptosis, the effect of PTX on autophagy was investigated. A significant increase in LC3-II/LC3-I and a decrease in P62 in response to 30 nM PTX was observed (Fig. 2A-C), indicating an increased level of autophagy. Considering that the IC₅₀ value of PTX was close to 20 nM, it was speculated whether 20 nM PTX could induce autophagy. Therefore, autophagic flux levels in MDA-MB-231 cells treated with 20 nM PTX were assessed after blocking autophagosome-lysosome fusion and degradation with Baf A1. Immunoblotting results revealed a significant increase in LC3 puncta in the presence of PTX + Baf A1 (Fig. 2D and E), suggesting that 20 nM PTX induced increased autophagic flux. Taken together, 20 and 30 nM PTX induced autophagy, while simultaneously inducing apoptosis in MDA-MB-231 cells.

In addition, another TNBC cell line (HCC-1937) were also treated with 0, 10, 20 and 30 nM PTX and it was observed that HCC-1937 cells were more sensitive to PTX treatment than MDA-MB-231 cells because more HCC-1937 cells died compared with MDA-MB-231 cells at the same dose of PTX (Fig. S1A). A significant decrease in the Bcl-2/Bax ratio (Fig. S1B and C) and a significant increase in the LC3-II/LC3-I ratio (Fig. S1F and G) was found, although no change in P62 protein expression (Fig. S1D and E) was found at 30 nM PTX, suggesting that PTX-induced apoptosis and autophagy in TNBC cells is a common phenomenon.

Given that PTX enabled the simultaneous induction of apoptosis and autophagy in MDA-MB-231 cells, it was next investigated whether inhibition of autophagy contributed to improving PTX-induced apoptotic cell death. After treatment with PTX + 3-MA, inhibition of autophagy was clearly observed, including a significant decrease in the LC3-II/LC3-I ratio at 10 and 30 nM PTX (Fig. 3A and B) and an increase in P62 expression at 30 nM (Fig. 3A and C). In addition, LC3 puncta also markedly decreased at 10 and 30 nM PTX after treatment with 3-MA (Fig. 3D). Furthermore, the formation of cell colonies was effectively inhibited at 10 and 30 nM PTX + 3-MA (Fig. 3E and F). These results indicated that inhibition of autophagy with 3-MA promoted PTX-induced apoptosis in MDA-MB-231 cells.

Autophagy is regulated by autophagy-regulated genes, such as the FOXO1, PTEN, LKB1, mTOR, SESN1, EPG5, TSC1, AKT, LMNA, AMBRA1 and DRAM1 genes, which are essential for autophagy signaling pathways (21–23). The mRNA levels of these genes were measured via RT-qPCR and it was found that 20 nM PTX induced a 2.7-fold increase in FOXO1 mRNA in MDA-MB-231 cells (Fig. 4A). Therefore, the role of FOXO1 in 20 nM PTX-treated MDA-MB-231 cells was the focus of subsequent assays. First, FOXO1 and p-FOXO1 protein expression in response to PTX treatment was assessed. The results showed that PTX induced a significant decline in the p-FOXO1/FOXO1 ratio in a dose-dependent manner, suggesting that FOXO1 rather than p-FOXO1 played an important role in PTX-induced autophagy in MDA-MB-231 cells (Fig. 4B and C). In addition, the upstream inhibitor of FOXO1, p-ATK, exhibited a significantly downregulated pattern (Fig. 4D and E). Thus, it was speculated that the decreased p-FOXO1/FOXO1 ratio was related to decreased p-AKT levels. Since FOXO1 is a transcription factor, it was assessed whether FOXO1 exerted a transcriptional function in PTX-mediated autophagy of MDA-MB-231 cells. Therefore, the localization of FOXO1 was further detected via western blotting and immunofluorescence. The results showed that FOXO1 was primarily distributed in the nucleus, further confirming that FOXO1 exerted its autophagy-regulated function in a transcriptional activation pattern (Fig. 5A and B).

To investigate the function of FOXO1 in PTX-induced autophagy in MDA-MB-231 cells, FOXO1 expression was knocked down using siRNA. At the protein level, the expression of FOXO1 was successfully inhibited after treatment with siRNA (Fig. 6A and B). Knocking down the FOXO1 gene led to significantly decreased LC3II and significantly increased P62 protein expression levels in PTX-treated MDA-MB-231 cells, suggesting reduced autophagy levels (Fig. 6A, C and D). A number of autophagy-related genes, such as ATG5, VPS34 (PI3KC3), ATG4B, BECN1 and MAP1LC3B, are transcriptionally regulated by FOXO1 (24). In this study, it was also found that these genes were suppressed in response to FOXO1 knockdown in PTX-treated MDA-MB-231 cells (Fig. 6E). Importantly, knockdown of FOXO1 enhanced PTX-induced apoptotic cell death (Fig. 6F and G). These results illustrated that FOXO1 played a role in PTX-induced autophagy in MDA-MB-231 cells and that targeting FOXO1 may improve the efficacy of PTX in TNBC therapy.