Ginsenoside panaxatriol reverses TNBC paclitaxel resistance by inhibiting the IRAK1/NF-κB and ERK pathways

Chemicals and reagents

Ginsenoside panaxatriol was obtained from Must Bio-Technology (Chengdu, China). PTX was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM) (11995-040), F-12 nutrient mixture (Ham) and fetal bovine serum (FBS) were bought from Life Technologies (Grand Island, NY, USA). MammoCul medium (human) and supplements were purchased from STEMCELL Technologies (Vancouver, BC, Canada). CellTiter-Glo luminescent cell viability assay kits were purchased from Promega Corporation (Madison, WI, USA). iScript gDNA Clear cDNA Synthesis Kits and iTaq Universal SYBR Green Supermix Kits were purchased from Bio-Rad Laboratories (Hercules, CA, USA). p-IRAK1 S376, IRAK1, p-P65 S536, P65, p-ERK1/2, ERK1/2, BAX, BCL-2 and MCL-1 antibodies were supplied by Cell Signaling Technology (Danvers, MA, USA). Beta-actin antibody was purchased from Sigma–Aldrich (St. Louis, MO, USA).

Cell culture and viability assay

MDA-MB-231 cells and SUM159 cells were obtained from ATCC. MDA-MB-231 PTX resistant (MB231-PR) cells and SUM159 PTX resistant (SUM159-PR) were established as previously described (Wee et al., 2015). Briefly, cells were treated with increasing concentrations of PTX for over a period of 3 months. Then, MB231-PR cells were cultured in DMEM supplemented with 75 nM PTX, 1% penicillin/streptomycin, and 10% FBS at 37 °C with 5% CO₂. SUM159-PR cells were maintained in F-12 supplemented with 300 nM PTX, 5% FBS, 10 mM HEPES, 10 μg/ml hydrocortisone, 5 μg/ml insulin and 1% penicillin/streptomycin. For cell viability assay, 1,000 cells/well in 90 μl medium were seeded into Costar 96-well white plates. The next day, different concentrations of drugs in 10 μl medium were added and incubated for the indicated times. Then, cells were lysed with 50 μl CellTiter-Glo reagent and the chemiluminescent signals were detected with a PerkinElmer VICTOR X4 plate reader.

Cell cycle and apoptosis assay

Cell cycle and apoptosis analysis was performed by DNA content quantification to quantify the subG1 population, which is a reflective of the extent of cell death. Briefly, floating and adherent cells were harvested together after 48 h and 72 h treatment respectively. Then, cells were fixed by 70% ethanol at 4 °C overnight. After washing with phosphate buffered saline (PBS), cells were resuspended in 100 μl of 100 μg/ml RNase A. 5 min later, 400 μl of 50 μg/ml propidium iodide was added, and cells were incubated for 30 min in dark area. Finally, the stained cells were analyzed by FACScalibur and quantified using CellQuest software.

Colony formation assay

A total of 1,000 cells/well were seeded into 12-well plates. The next day, drugs were added and incubated for 12 days. Medium was changed every 3 days. Then, cells were washed with PBS and fixed with methanol for 10 min. Finally, cells were stained with 0.1% crystal violet at room temperature for 10 min and photographed.

Tumor sphere formation assay

A total of 3,000 cells/well in 180 μl medium were seeded into Corning 96-well spheroid microplates in complete MammoCul medium. The next day, drugs in 20 μl medium were added and incubated for 12 days. Pictures were taken on day 6 and day 12. Finally, on day 12, cells were lysed with 100 μl CellTiter-Glo reagent and the chemiluminescent signal was detected with a PerkinElmer VICTOR X4 plate reader.

Transwell invasion assay

A total of 10,000 cells in 100 μl serum-free DMEM containing DMSO, PTX, GPT, or combination were added into Corning Transwell polycarbonate membrane inserts coated with Matrigel (300 μg/mL). And medium containing 10% FBS was added to the bottom chamber. After 24 h incubation, the cells that remained on the above surface of the insert membrane were scraped off with a cotton swab. The cells that passed through Matrigel to the bottom of the insert were fixed with paraformaldehyde and stained with 0.1% crystal violet in methanol. The inserts were photographed, and the cells were counted.

Quantitative-PCR (qPCR) assay

RNA extraction and purification were performed according to the instructions from Zymo Research (R2052). A total of 750 ng RNA was used to synthesize cDNA. And qPCR was performed using the Applied Biosystems 7500 Fast Real-Time PCR system. All primers are listed in Table 1. For quantification of mRNA levels, 18S was used as the internal control, and the expression of target genes were analyzed using the 2^−ΔΔCt method.

Western blot assay

Western blot was performed using whole-cell extracts in protein lysis buffer with freshly added protease inhibitor cocktail. Proteins were separated on 8–10% SDS polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% non-fat dry milk in tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST). The membrane was then incubated with primary antibody (1:1,000 dilution) in 5% bovine serum albumin overnight. After washed three times with TBST, the membrane was incubated with secondary antibody (1:2,000 dilution) in 5% non-fat dry milk at room temperature for 1 h. Then, SuperSignal West Femto Maximum Sensitivity Substrate was added, and images were taken using the ChemiDoc MP System.

Statistical analysis

Data are shown as mean ± SD. The t test was used to determine whether there are any statistically significant differences between two groups. P < 0.05 was considered statistically significant.

GPT promotes cytotoxicity of PTX in MB231-PR cells

To explore whether GPT can promote cytotoxicity of PTX in TNBC resistant cells, MB231-PR was constructed and used as cell model. Firstly, we conducted CellTiter-Glo assay to observe different concentration of GPT on cell viability. As shown in Fig. 1A, GPT treatment significantly decreased cell viability of MB231-PR cells in a dose dependent manner, with the half maximal inhibitory concentration (IC50) 21.39 μM. Secondly, we combined GPT with PTX to check whether they have synergistic effects. Results showed that the combination caused dramatic cell death in a dose and time dependent manner, comparing to either single use group (Fig. 1B). Interestingly, the synergistic effects didn’t apply to MB231 parental (MB231-PT) cells, although MB231-PT cells were sensitive to PTX (Fig. S1) and showed more sensitive to GPT when treated with the same concentration (Fig. 1C). Notable, the clinical using drug GRg3 didn’t cause significant cell death in single or combination treatment group (Fig. S2). In addition, colony formation assay confirmed the synergistic cytotoxicity effects of the combination on MB231-PR cells (Fig. 1D; Fig. S3).

Since chemotherapy resistance appears partly due to aberrant changes of signaling pathways that endowed cells with the abilities to escape apoptosis, restoring apoptosis is a very important therapeutic strategy for antitumor therapy (Baig et al., 2016; Plati, Bucur & Khosravi-Far, 2008). Therefore, next, we used flow cytometry to measure subG1 changes after the combination treatment, which is marker of apoptosis. Not surprisingly, GPT combined with PTX significantly increased subG1 cell accumulation both after 48 h and 72 h (Fig. 1E; Fig. S4). Taken together, these results suggested GPT as a very effective molecular to reverse PTX resistance in TNBC cells.

The combination treatment activates mitochondria mediated apoptosis

The alteration of pro-apoptotic proteins and anti-apoptotic proteins play important roles in the determination of cancer cells apoptosis, and are associated with chemoresistance (Campbell & Tait, 2018; Warren, Wong-Brown & Bowden, 2019). Thus, we observed the protein expression of BAX and BCL-2 after treatment, two key mediators of apoptotic response to chemotherapy. As shown in Figs. 2A and 2B, GPT combined with PTX significantly increased BAX and decreased BCL-2 expression in a dose and time dependent manner.

Besides BAX and BCL-2, MCL-1 was recently reported to be associated with poor prognosis in TNBC patients and can be used as a therapeutic target (Campbell et al., 2018). Notably, we have shown that IRAK1 inhibitor can decrease MCL-1 expression in MB321-PR cells to induce cell apoptosis (Wee et al., 2015). Therefore, we also evaluated the protein expression of MCL-1 after treatment. As shown in Figs. 2A and 2B, the combination treatment also resulted in down-regulation of MCL-1 expression. These results together suggested that the combination treatment activated mitochondria mediated apoptosis to reverse PTX resistance.

The combination treatment inhibits IRAK1/NF-κB and ERK pathways

To further clarify the signaling pathways that involved in GPT effects, gene expression profiling was conducted in MB231-PR cells treated with DMSO, PTX, GPT and combination, respectively. Results showed that NOD-like receptor signaling pathways played an important part in GPT activity in MB231-PR cells (data not shown). Interestingly, through loss and gain of function study, we have previously reported that activation of IRAK1, a key kinase of NOD-like receptor signaling pathway, is associated with PTX resistance in TNBC cells (Wee et al., 2015) Moreover, target IRAK1 using pharmacologic inhibitor can induce MB231-PR cells apoptosis, when combined with PTX (Wee et al., 2015). Thus, consideration was given to IRAK1 and its downstream signaling pathways. Results showed that the combination treatment can significantly inhibit the phosphorylation of IRAK1, P65, ERK1/2, and increase the expression of IκB-alpha in a dose and time dependent manner (Figs. 2A and 2B).

To additionally characterize the functional effects of IRAK1 mediated pathways, we investigated the mRNA expression of NF-κB target genes by qPCR, including interleukin 6 (IL6), IL8, chemokine (C-X-C motif) ligand 1 (CXCL1), and chemokine (C-C motif) ligand 2 (CCL2). The above cytokines were shown to be distinctly expressed among different group in our gene expression profiling experiment, and were reported to be critical for the anchorage independent growth of TNBC cells (Hartman et al., 2013). As shown in Figs. 2C–2F, (Tables S1 and S3), compared to DMSO, PTX significantly promoted the expression of IL6, IL8, CXCL1 and CCL2. However, this induction can be significantly attenuated when combined with GPT.

Except these target cytokines, we previously published that IRAK1 and S100A7/8/9 formed a feedback loop to drive the malignancy of TNBC cells (Goh et al., 2017). Here, we also showed that the combination treatment significantly decreased S100A7 and S100A9 mRNA expression (Figs. 2G and 2H; Tables S1 and S3), although S100A8 mRNA expression level was too low to be detected. These results together suggested that the combination treatment overcome PTX resistance by inhibiting IRAK1 mediated NF-κB and ERK pathways.

The combination treatment inhibits cancer stem cell-related genes expression and impairs tumor sphere growth and invasion ability

Accompanied with killing cancer cell, PTX treatment has been reported to induce cancer stem cell (CSC) enrichment, another key mechanism suggested to be responsible for chemoresistance and cancer metastasis (Bousquet et al., 2017; Zhang et al., 2019). And drug that can target cancer stemness are proposed as new strategies for clinical cancer treatment (Saygin et al., 2019; Sun et al., 2019). In order to testify the effect of combination therapy on characteristics of CSC, firstly, qPCR was used to check the expression of a group of CSC-related genes. As shown in Figs. 3A– 3E, (Tables S2, S4 and S5), compared to PTX, the combination treatment significantly lead to down-regulation of octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), NANOG, aldehyde dehydrogenase 1 (ALDH1), and CD44 gene expression. Secondly, transwell invasion and tumor sphere assay were conducted to assess CSC properties. As shown in Figs. 3F and 3G–3J, the combination treatment significantly suppressed MB231-PR cell invasion ability, and impaired tumor sphere growth both in MB231-PR and SUM159-PR cells.