TMC was prepared as previously described (referred as compound 3 h) (Peng et al., 2016a), and its purity was >98%. Xylene, eosin Y, hematoxylin, and other commonly used chemicals were purchased from Sigma (St. Louis, MO). Primary and secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA). ECL Advance reagent was purchased from Merck-Millipore (St. Louis, MO). RNAiso Plus reagent, SYBR Green master mix, and the PrimeScript RT Reagent Kit with gDNA Eraser were obtained from TaKaRa (Bio Inc., Shiga, JP). The MiRNeasy Mini Kit was obtained from Qiagen (Hilden, DE) and the TaqMan MicroRNA Reverse Transcription Kit was obtained from Ambion (Life Technologies, MA, USA).

MDA-MB-231, BT549, MCF-10A, 4T1, 293T, and MCF-7 cells were incubated at 37°C in a 5% CO₂ incubator purchased from American Type Culture Collection (ATCC, USA). MDA-MB-231, BT549 and 4T1 cells are TNBC cell lines. MCF-7 cells are hormone-receptor positive breast cancer cells. MCF-10A cells are normal human mammary epithelial cells. All growth mediums were supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin from Gibico (Life Technologies, MA, USA), except for MCF-10A cells. MCF-10A cells were cultured in special keratinocyte serum-free medium. MDA-MB-231, BT549, and 293T cells were cultured in DMEM, while 4T1 and MCF-7 cells were cultured in RPMI 1640 medium.

MDA-MB-231 and BT549 cells were seeded in 96-well plates at a density of 5 × 10³ cells/well. Two breast cancer cell lines were exposed to TMC for 24 h and 48 h. Cell viability was assessed using the CCK-8 kit obtained from MedChemExpress (NJ, USA) according to the manufacturer’s instructions. After TMC treatment, the cells were treated with 10 μl of the reagent for 2 h in a 5% CO₂ incubator, and the absorbance at 450 nm was determined by enzyme-linked immunosorbent assay (ELISA) on a plate reader. These experiments were performed in triplicate.

Foci formation assays were performed as previously described (Tang et al., 2018). Briefly, MDA-MB-231 and BT549 cells were seeded at a density of 1 × 10³ cells in six-well plates. Clones were stained with 0.5% crystal violet, and foci numbers were counted. The experiments were performed in triplicate.

MDA-MB-231 and BT-549 cells were seeded at a density of 3 × 10⁵ cells/ml into Culture-Inserts (Ibidi GmbH, Martinsried, DE) according to the manufacturer’s instructions. Images were recorded at the beginning and end of the experiment using a Zeiss Axio Lab A1 microscope (Oberkochen, DE). These experiments were performed in triplicate.

Chamber migration and invasion assays were performed for according to the instructions from Corning Inc (NY, USA). MDA-MB-231, and BT549 cells were seeded on the upper surface at a density of 1.25 × 10⁵. After TMC treatment, migrating and invading cells were fixed with 4% PFA before 0.5% crystal violet staining. The remaining cells were imaged using a Zeiss Axio Lab A1 microscope and counted in triplicate in different fields of view.

The apoptosis analysis kit, equipment, and software were purchased from BD Company (CA, USA). MDA-MB-231 and BT549 cells were seeded in 6-well plates at a density of 5 × 10⁵ cells/well. The percentage of apoptotic cells after 24-h TMC treatment was detected using the Annexin V/PI staining apoptosis analysis kit according to the manufacturer’s instructions. Stained samples were analyzed by FACSAria SORP within 1 h. The experiments were performed in triplicate.

Protein extraction was performed using the Cell lysis buffer from Sigma. Protein (20 μg) was resolved on 10% SDS-PAGE gels and transferred onto PVDF membranes from Merck-Millipore (St. Louis, MO). After blocking with 5% bovine serum albumin (BSA), the membranes were incubated with primary antibodies against Bax, Bcl-2, and β-actin (loading control) at 4°C overnight. After probing with secondary antibody, Western blots were developed using ECL Advance reagent and evaluated using Image Lab Software from Bio-Rad (Kidlington, UK).

Total RNA was extracted using the RNAiso Plus reagent. Reverse-transcription of mRNA was performed using the PrimeScript RT Reagent Kit with gDNA Eraser, and real-time PCR experiments were performed using the SYBR Green master mix. GAPDH was selected as the loading control for mRNA expression analyses. miRNAs were extracted using the miRNeasy Mini Kit. miRNA reverse-transcription was conducted using the TaqMan MicroRNA Reverse Transcription Kit, and real-time PCR experiments were performed using the SYBR Green master mix. U6 was selected as the loading control for miRNA expression analyses. The corresponding primers for detecting BAX, GAPDH and U6 are listed in Table S2.

MDA-MB-231 cells (2 × 10⁶) were inoculated into mammary gland fatpads of 4-week-old nude mice. After successful modeling, nude mice were randomly divided (n = 5) into a control group, low dose group (TMC, 20 mg/kg/d) and high dose group (TMC, 40 mg/kg/d). Twenty-one days after the intraperitoneal injection of TMC, the mice were euthanized and the tumor weights and volumes were measured. All animal experiments were performed according to the standard institutional guidelines of Sichuan University and Chengdu University of Traditional Chinese Medicine.

MiRNA extraction, reverse transcription, and real-time PCR reactions were performed as previously described. PCR arrays were performed using the miRNA miRNome PCR Panel from Qiagen according to the manufacturer’s instructions.

The pMIR-REPOR miRNA Expression Reporter Vector System was used. We inserted the miR-374a-binding sequence of BAX (with or without mutation) to mimic the regulation of BAX by miR-374a through its 3′UTR. The primers used in these experiments are listed in Table S3. Dual-Glo Luciferase assays were performed according to the instructions from Promega (Madison, WI). The Renilla luciferase activity and firefly luciferase activity were recorded using a SpectraMax M5 (MD, USA).

MirVana miRNA-374a mimic and mirVana miRNA Mimic Negative Control were obtained from Ambion. Lipofectamine 2000 Transfection Reagent was purchased from Invitrogen (Life Technologies, MA, USA). Transfections were performed according to the manufacturer’s instructions. MiR-374a mimic or negative control were mixed with diluted Lipofectamine 2000 Transfection Reagent as 1:1 ratio and incubated for 15 min at room temperature. MiRNA-lipid complexes were added to MDA-MB-231 and BT549 cells (60%–80% coverage) on the plate. The cells were incubated at 37°C in a 5% CO₂ incubator for 24 h.

Statistical analysis was performed with GraphPad Prism 7.0 (USA). Two-tailed Student’s t-tests and one-way ANOVAs were used to evaluate statistical significance (*p < 0.05, **p < 0.01).

We used CCK-8 assays to determine the effects of varying TMC concentrations (1, 5, 10, 20, 40, 80, and 100 μM) on MDA-MB-231 and BT549 cells (Figure 2A). The results suggested that TMC (> 5 μM) significantly inhibited TNBC cell proliferation after treatment for 24 and 48 h (p < 0.01). The IC₅₀ values of 24-h TMC treatment of MDA-MB-231 and BT549 cells were 8.696 and 14.28 μM, respectively. The cytotoxic effect of TMC was further investigated through foci formation assays. As shown in Figures 2B, C, TMC also suppressed the foci forming ability of MDA-MB-231 and BT549 cells after 24-h treatment. These results suggest that TMC has an inhibitory effect on TNBC proliferation.

The effect of TMC on TNBC cell movement was determined by wound healing and chamber invasion assays. Results demonstrated that 24-h TMC treatment (> 1 μM) critically suppressed wound closure in MDA-MB-231 and BT549 cells, and the inhibitory effect increased with increasing drug concentration (p < 0.01) (Figures 3A, B). Chamber invasion assays demonstrated that MDA-MB-231 and BT549 cells had obvious invasive capacities, and 24-h TMC treatment reduced the number of invading cells (Figures 3C, D). TMC (2.5 μM) significantly inhibited the invasive ability of TNBC cells, however its suppressive effect was more mild in MDA-MB-231 cells than BT549 cells (p < 0.01). Taken together, these results indicate that TMC significantly inhibits the migratory and invasive capacities of MDA-MB-231 and BT549 cells.

We utilized the Annexin V/PI staining apoptosis analysis kit TMC to quantify apoptotic cells. The results demonstrated that 24-h TMC treatment (> 5 μM) increased the percentage of apoptotic MDA-MB-231 and BT549 cells (p < 0.01) (Figures 4A, B). The proapoptotic effect of TMC was enhanced with increased drug dosage. Further, Western blot analyses demonstrated that 24-h TMC treatment promoted Bax expression and repressed Bcl-2 expression in a dose-dependent manner in TNBC cells (Figure 4C). Bax is a proapoptotic factor that promotes mitochondrial apoptosis. BCL-2 is an anti-apoptotic factor that inhibits proapoptotic proteins and promotes cellular survival. Real-time PCR confirmed that TMC increased Bax mRNA levels in a dose-dependent manner (p < 0.01) (Figure 4D). Collectively, these findings suggest that TMC triggers apoptosis in MDA-MB-231 and BT549 cells by increasing Bax and decreasing Bcl-2 expression.

The in vivo anti-cancer effect of TMC was investigated using MDA-MB-231 xenografts. TMC was administered to nude mice through intraperitoneal injection at 20 mg/kg/d (low dose group) and 40 mg/kg/d (high dose group) for 21 days. The mice weights and tumor volumes were recorded every three days. Tumor volumes were calculated according to a standard formula: (mm³) = L × W²/2. The tumors and other corresponding tissues were collected and measured after sacrificing the mice. Results exerted that TMC drastically suppressed TNBC tumor volumes in both the low dose and high dose groups (p < 0.01) (Figures 5A, C). In addition, TMC administration significantly reduced tumor weights after 21-day treatment (p < 0.01) (Figure 5D). Mouse weight did not decrease dramatically after TMC administration, even in the high dose group (Figure 5B). TMA could increase Bax protein and mRNA levels, and decrease Bcl-2 protein and mRNA levels in vivo (Figures 5E, F). Altogether, these findings suggest that TMC significantly inhibits TNBC tumor growth without obvious cytotoxic effects on normal tissues.

We used PCR arrays to determine the changes in miRNAs expression levels (at least 1.5-fold) in TNBC cells after exposure to 10 μM TMC for 24 h. MiR-374a decreased most significantly in all the varied miRNAs after TMC interference. Results displayed that TMC decreased the high expression of miR-374a in both MDA-MB-231 and BT549 cells (Figure 6A). Real-time PCR analysis confirmed that TMC significantly inhibited miR-374a in a dose-dependent manner in TNBC cells (p < 0.01) (Figure 6C). Additionally, miR-374a was expressed at low levels in normal breast cells and high levels in breast cancer cells, especially in TNBC cells (Figure 6B). Data from Array Express (https://www.ebi.ac.uk/arrayexpress/) showed that miR-374a was upregulated in TNBC tumor tissues compared to their peritumor breast tissues (Figure S2). Taken together, these results indicate that miR-374a is a potential oncogenic miRNA downregulated by TMC.

Rnahybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybrid) predicted the direct interaction of miR-374a and the BAX 3′UTR, suggesting miR-374a as a negative regulator of BAX expression. Luciferase assays confirmed that miR-374a directly binded to the 3′UTR of BAX (Figure 7A). Real-time PCR analysis confirmed successful transfection of MDA-MB-231 and BT549 cells with the miR-374a mimic and miRNA negative control (Figure 7B). Data from Oncomine (https://www.oncomine.org/) demonstrated that BAX was expressed at low levels, while BCL2 was expressed at high levels in breast cancer tumors compared to normal tissues (Figures 7C, S3). Western blot results demonstrated that TMC increased Bax protein expression, and that upregulation of miR-374a partly reversed the effect of TMC (Figure 7D). Real-time PCR analysis exerted that TMC promoted Bax mRNA expression, and miR-374a upregulation partially blocked the TMC-induced increase in BAX (Figure 7E). Taken together, these data suggest that TMC increases Bax protein and mRNA levels through downregulation of miR-374a in TNBC cells.