Chemicals

Falnidamol, doxorubicin, paclitaxel, mitoxantrone, cisplatin, verapamil, Ko143, and other reagents were purchased from MedChemExpress (Shanghai, China). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Thermo Fisher Inc (Shanghai, China). MTT kit, anti-ABCB1, anti-AKT, anti-p-AKT, anti-ERK, anti-p-ERK, anti-GAPDH and the secondary antibodies were bought from Beyotime Biotechnology (Shanghai, China).

Cell lines and cell culture

The following cell lines were used in this study: The human cervical cancer cell line HELA and its ABCB1-overexpressed resistant cell line HELA-Col selected by colchicine. The human colon cancer cell line SW620 and its ABCB1-overexpressed resistant cell line SW620-Adr selected by doxorubicin. The human embryonic kidney cell line HEK293 and their stably transfected cell lines HEK293-Vector (transfected with empty plasmid), HEK293-ABCB1 (transfected with ABCB1 plasmid) and HEK293-ABCG2 (transfected with ABCG2 plasmid). The cell lines, including HELA, SW620 and HEK293, were obtained from Shanghai Cell Bank. The cell lines, including HELA-Col, SW620-Adr, HEK293-Vector and HEK293-ABCB1 were constructed by our laboratory. All cell lines were cultured in DMEM supplemented with 10% FBS.

Cytotoxicity and reversal experiments

The cytotoxicity and reversal experiments were conducted using MTT assay as previously reported [15]. Briefly, cells were seeded into 96-well plates (5 × 103 cells per well) and maintained overnight. For the cytotoxicity assay, different concentrations of falnidamol were added into the wells. For the reversal assay, after pre-incubating with falnidamol or verapamil for 2 h, different concentrations of conventional chemotherapeutic medicines, including doxorubicin, paclitaxel, or cisplatin, were added into the wells. The cells were cultured for 72 h, then MTT was added, and the absorbance was detected using an enzyme reader at 570 nm. Verapamil was utilized as a positive control inhibitor. Cisplatin, a non-substrate agent of ABCB1, was utilized as a negative control anti-cancer agent.

Colony formation and 3D spheroid assays

Colony formation and 3D spheroid assays were performed as described in the previous study [16]. In brief, for colony formation assay, HELA-Col cells were seeded in 6 well plates (1000 cells per well) and treated with falnidamol (5 µM, 6 h), paclitaxel (1 µM, 4 h) or the combination (falnidamol for 2 h followed by paclitaxel for 4 h). After removal of the agents, the cells were cultured for further 10 days. Finally, colonies were stained using 0.1% crystal violet for 30 min after being fixed using methanol for 30 min, and then colonies were counted under a microscope. For 3D spheroid assay, HELA-Col cells were seeded in 6-well plates (1000 cells per well) and treated with falnidamol (5 µM, 6 h), paclitaxel (1 µM, 4 h) or the combination (falnidamol for 2 h followed by paclitaxel for 4 h). After removal of the agents, the cells were seeded into 96-well plates with minimal surface adhesion, and cultured for further 7 days. Finally, the diameter of the 3D spheroid was measured.

Western blot and immunofluorescence assays

Western blot was performed as our previous report [17]. Briefly, HELA-Col cells were treated with different concentrations of falnidamol for different times, and lysed. Subsequently, proteins were transferred to PVDF membrane after SDS-PAGE. After sequential incubation of the membrane with primary antibody (1:1000) and secondary antibody (1:1000), the protein bands were measured using an ECL kit. Immunofluorescence was carried out as previously described [18]. In short, HELA-Col cells were seeded in a confocal dish, and treated with 5 µM falnidamol for 0–72 h. Cells were fixed in 4% formaldehyde for 20 min and incubated overnight with anti-ABCB1 antibody (1:250). Subsequently, cells were incubated with a secondary antibody (1:500) and DAPI and photographed with a confocal microscopy.

Doxorubicin accumulation and efflux assays

Doxorubicin accumulation assay was assessed as in previous study [19]. In brief, HELA and HELA-Col cells were seeded in 6-well plates (1 × 106 cells per well) for 24 h, and then treated with 5 µM falnidamol or verapamil for 2 h. Subsequently, 10 µM doxorubicin was added and incubated for another 2 h. Finally, cells were collected for measurement by flow cytometry. Doxorubicin efflux assay was evaluated as reported previously [20]. In short, HELA and HELA-Col cells were seeded in 6-well plates (1 × 106 cells per well) for 24 h, and then treated with 10 µM doxorubicin for 30 min. Afterwards, cells were incubated with 5 µM falnidamol or verapamil, and harvested at various time points for flow cytometry examination.

ATPase assay

The ATPase activity of ABCB1 was tested as described in previous study [8]. Briefly, cell membranes overexpressed ABCB1 were incubated with various concentrations of falnidamol for 5 min. Subsequently, the ATPase reaction was initiated by the addition of 5 mM Mg2+ ATP. At last, luminescence signals of Pi were detected after 30 min incubation. The variations in relative light units were calculated by comparing the samples treated with Na3VO4 with those treated with falnidamol.

Docking analysis

Docking analysis was performed using Maestro v11.1 (Schrödinger LLC, MA, USA) as previously described [16]. In short, human ABCB1 protein model (7A69) was downloaded from RCSB Protein Data Bank, and the molecular structure of falnidamol was downloaded from PubChem. After receptor/ligand preparation, glide XP docking was performed, followed by induced fit docking using the default protocol.

Cellular thermal shift assay

The cellular thermal shift assay was carried out as mentioned previously [21]. HELA-Col cells (2 × 107 cells) were washed, resuspended in PBS, and then the cell suspension was frozen-thawed five times with liquid nitrogen. Protein samples were harvested by centrifugation at 10,000 × g for 10 min at 4 ℃, and then treated with 50 µM of falnidamol or DMSO for 30 min at room temperature, respectively. Next, the same quantities of protein were taken and treated at various temperatures for 3 min. Finally, protein samples were analyzed by western blot.

In vivo assay

In short, HELA-Col cells (1 × 107 cells per mouse) were inoculated subcutaneously in nude mice. Subsequently, the mice were divided into four groups and given one of the following treatments every 3 days: (1) normal saline control group (30 mL/kg, p.o); (2) falnidamol group (30 mg/kg, p.o.); (3) paclitaxel group (15 mg/kg, i.p.); (4) combination of falnidamol (30 mg/kg, p.o.) and paclitaxel group (15 mg/kg, i.p.). The tumor’s growth was monitored every 3 days, and the tumor’s volume was determined by the following formula: volume (mm3) = length × width2 × 0.5. After the mice were executed by deep anesthesia, and the tumors were harvested and weighed, while the liver and kidney were collected for histopathological examination.

Histopathological examination

Histopathological examination was performed as mentioned previously [22]. In short, tissue samples were fixed with 10% neutral buffered formalin. After paraffin embedding, the samples were sectioned at approximately 4 μm thick and stained with hematoxylin-eosin (HE).

Statistical analysis

Data were analyzed by ANOVA using SPSS 18.0 software. Data were expressed as mean ± SD, with P < 0.05 representing statistical significance. The experiments were repeated three times.