ITZ (HY-17514) and rapamycin (HY-10219) were purchased from MedChemExpress (United States).

The human triple-negative breast cancer cell lines MDA-MB-231 and BT-549 were from the cell bank of the Chinese Academy of Sciences (Shanghai). MDA-MB-231 cells were cultured in Dulbecco’s modified Eagle’s medium plus 10% fetal bovine serum (FBS, GIBCO, Brazil) and 1% penicillin-streptomycin antibiotics (Beyotime, China). BT-549 cells were cultured in RPMI 1640 medium (GIBCO, Brazil) plus 10% FBS and 1% penicillin-streptomycin. All cells were cultured in a 37°C incubator containing 5% carbon dioxide.

Determination of IC50 concentration: 5 × 10³ cells were inoculated into each well of a 96-well plate. After 24 h, culture medium containing a series of drug dilutions was changed and cells were further cultured for 48 h. Then, 10 µL CCK-8 reagent (Invigentech, United States) and 100 µL culture medium were added to each well and cells were cultured an additional 2 h before absorbance was measured at 450 nm. Growth inhibition was calculated as Inhibition rate% = (OD value of control group-OD value of experimental group)/(OD value of control group-OD value of the blank) × 100%. The SPSS 19.0 software was used for statistical analysis of data to obtain half maximal growth inhibitory concentration (IC50) value of drugs, each experiment was repeated for three times.

Quantitation of cell proliferation: cells were inoculated at 1 × 10³ cells per well into 96-well plates and cultured at 37C, 5% CO₂ and allowed to attach for 24 h. Drugs at their half maximal growth inhibitory concentration (IC50) concentrations were added. After 24, 48, 72, 96, and 120 h, 10 μL CCK-8 reagent and 100 μL medium were added for 2 h at 37°C. Then absorbance of each well was measured at 450 nm using a spectrophotometer.

Cells were inoculated into a 60 mm dish with drug at the IC50 concentration for 48 h. Then, cells were resuspended and seeded at 1 × 10³ cells per well in 6-well plates and cultured for 14 days. Cells were fixed with methanol and stained with 0.1% crystal violet. Count the size and number of cell colonies in each group.

Chambers with 8-µM pore membranes were used for migration assays. Cells were initially cultured for 48 h in media without and with different concentrations of drugs. Then, cells were digested with pancreatin and diluted in serum-free medium and inoculate into the upper transwell chambers at a density of 2 × 10⁴ cells per chamber. Complete medium was added to the bottom chamber. After 24 or 48 h, migrated cells were stained with 0.1% crystal violet.

For quantitation of apoptosis, Annexin V-FITC Detection Kit (C1063, Beyotime, China) was used and cells were inoculated into a 60 mm dish with drugs at IC50 concentrations for 48 h. After washing the cells twice with PBS, the cells were digested and resuspended in binding buffer containing 5 µL of an Annexin V-FITCAfter incubation at room temperature for 20 min, 10 µL PI was added to the suspension and incubated for another 10 min. Then the degree of apoptosis was detected by flow cytometry (Accuri C6, Becton-Dickinson, United States).

For quantitation of cell cycle phases, Cell cycle Detection Kit (BestBio, China) was applied and cells were incubated with drugs for 24 h, then digested and collected into 70% ethanol at -20°C and allowed to fix for 2 h. The fixed cells were centrifuged at 1000 rpm and then washed with PBS, resuspended in staining buffer (0.5 ml) containing PI (25 µL) and RNase A (10 µL), and incubated at 37°C for 30 min in the dark. Finally, the DNA level was measured by flow cytometry.

Cell cultures were incubated in 60 mm dishes with different concentrations of drug for 48 h and then washed twice with PBS for digestion and cell collection. The cells were lysed for 30 min in ice-cold RIPA buffer with phenylmethylsulfonyl fluoride and Protein phosphatase inhibitor (Solarbio, China). Then, lysates were ultrasonicated three times (4 s each) and centrifuged at 12,000 rpm for 15 min to remove the precipitate, and the protein concentration was determined using a bicinchoninic acid assay (BCA) kit (GenStar, China). After denaturation, 30 µg of protein was subjected to SDS-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and blocked with 5% milk. The PVDF membrane was incubated with primary antibodies overnight to detect the expression of mTOR pathway-related proteins and cycle-related proteins. Primary antibodies and diluted concentration were followed: Akt (1:1000, #4691, CST, United States), p-Akt (1:1000, #4060, CST, United States), mTOR (1:1000, #2983, CST, United States), p-mTOR (1:1000, #5536, CST, United States), p70S6K (1:1000, #9202, CST, United States), p-p70S6K (1:1000, #9205, CST, United States), cyclin D1 (1:1000, #2978, CST, United States), GAPDH (1:1000, #TA309157, ZSGB-BIO, China), p21 (1:1000, #2947, CST, United States), β-Actin (1:1000, #TA-09, ZSGB-BIO, China). After incubation with primary antibody, membranes were washed, then incubated with HRP-conjugated secondary antibody (1:2000) at room temperature for 120 min. Enhanced chemiluminescence chemicals were used to visualize target proteins and chemiluminescence western blotting detection system (Bio-Rad ChemiDoc XRS+, United States) was used for protein detection.

To investigate the potential function and mechanism of ITZ in TNBC, the concentration of ITZ required for treating TNBC cells was determined by CCK-8 assay to identify IC50 for MDA-MB-231 and BT-549 cells. Results showed that with increasing concentration of ITZ, the survival rate of TNBC cells decreased in a dose-dependent manner (Figure 1A), with IC50s of 4.917 μM for MDA-MB-231 and 4.367 μM for BT-549 obtained.

The anti-cancer activities of ITZ were examined by measuring the effects of ITZ on the proliferation and motility of TNBC cells. In the CCK-8 assay, TNBC cells were exposed with or without ITZ at the IC50 concentration for 24, 48, 72, 96, and 120 h. ITZ treatment inhibited the proliferation of both MDA-MB-231 and BT-549 cells (Figure 1B). Colony formation was also decreased by ITZ, compared with the control group, in both MDA-MB-231 and BT-549 cells (Figure 1C). Furthermore, transwell assays suggested that treatment with ITZ suppressed the migratory ability of MDA-MB-231 and BT-549 cells (Figure 1D).

To explore the underlying mechanism of ITZ in TNBC cells, protein levels of AKT/mTOR signaling pathway components were examined by western blotting. Interestingly, the activated forms of AKT and mTOR were decreased with ITZ treatment, while the expression levels of AKT and mTOR were not suppressed in the ITZ-treated groups. To confirm the involvement of the AKT/mTOR signaling pathway, phosphorylation of the mTOR downstream target p70S6K was also examined. Phosphorylation of p70S6K was inhibited in the ITZ-treated groups, consistent with reduced p-AKT and p-mTOR levels (Figures 1E,F).

The ability of rapamycin to inhibit growth of TNBC cells was also determined by CCK-8 assay, which showed IC50s of 12.2 μM for MDA-MB-231 and 15.9 μM for BT-549 cells (Figure 2A). Not surprisingly, the proliferation and colony formation of both MDA-MB-231 and BT-549 cells were suppressed by rapamycin treatment at the IC50 concentration (Figures 2B,C). The number of migratory cells in the transwell assay was lower in the rapamycin-treated group than that in the control (Figure 2D). As an mTOR inhibitor, rapamycin treatment of TNBC cells resulted in low activity of the AKT/mTOR signaling pathway, as demonstrated by decreased expression of p-AKT, p-mTOR, and p-p70S6K (Figures 2E,F).

To investigate the potential function of combined ITZ with rapamycin in TNBC cells, CCK-8, colony formation and transwell assays were used to evaluate the malignant behavior of TNBC cells. In Figure 3A, the proliferation of both MDA-MB-231 and BT-549 cells was suppressed in both the ITZ- and rapamycin-treated groups, compared with the control groups, while the combined treatment inhibited the proliferation of TNBC cells dramatically compared to treatment with either drug alone. Consistently, colony formation was decreased by ITZ or rapamycin treatment alone, but the decline was even greater in the combined treatment groups than that in the groups treated with either drug alone (Figures 3B,C). Regarding the migratory ability of TNBC cells, transwell assays showed that the inhibition of TNBC cell motility was the most obvious in the combined treatment of ITZ and rapamycin rather than treatment with ITZ or rapamycin alone (Figure 3D).

The activity of the AKT/mTOR signaling pathway was also evaluated by the expression of biomarkers. In Figures 3E,F, although the expression level of AKT, mTOR, and p70S6K was not decreased following treatment with ITZ and rapamycin alone or in combination, their activated forms, i.e. p-mTOR, and p-p70S6K, were decreased in the single drug-treated groups, and were even lower in the combined treatment group.

In order to investigate the combined effect of ITZ and rapamycin in TNBC, the percentage of apoptotic cells was evaluated by flow cytometry after ITZ and rapamycin treatment. Apoptosis was induced by ITZ treatment and rapamycin treatment alone in MDA-MB-231 cells (Figure 4A), whereas for BT-549 cells, ITZ but not rapamycin induced apoptosis (Figure 4A).

For the combined treatment of ITZ and rapamycin, the cells were collected after a 48 h treatment and analyzed by flow cytometry. Although in the ITZ-treated group, apoptosis was induced accordingly, the percentage of apoptotic cells in the combined treatment group was not increased compared with the single drug groups (Figure 4B), indicating that the synergistic inhibition of ITZ and rapamycin on TNBC cells was not through inducing apoptosis.

As apoptosis was not synergistically induced by ITZ and rapamycin in combination, the question arose as to how the proliferation and motility of TNBC cells was inhibited. Cell cycle analysis of TNBC cells was conducted to investigate the underlying mechanism. Interestingly, the proportion of TNBC cells in G0/G1 phase increased with ITZ and rapamycin treatment alone, but was especially high in the combined treatment group (Figure 5A).

Concomitant with cell cycle arrest in G0/G1 phase, the number of TNBC cells entering S and G2 phases was decreased accordingly. To confirm the synergistic effect of ITZ and rapamycin on blocking cell cycling, cell cycle-related proteins were detected by western blotting, which showed that treatment with ITZ (5 μM) or rapamycin (15 μM) alone suppressed the expression of cyclin D1, an indicator of G1/S phase transition (Figures 5B,C). Importantly, upon combined treatment with ITZ (2 μM) and rapamycin (10 μM), the expression of cyclin D1 was the lowest compared with control and single drug-treated groups (Figures 5D,E).