Reagents

BI-2865 (C-1443) was purchased from the Chemgood (Henrico, VA, USA). Paclitaxel (HY-B0015), Verapamil (HY-14,275), Vincristine (HY-N0488), Doxorubicin (HY-15,142 A) and Topotecan (HY-13,768) were acquired from MedChem Express. Mitoxantrone (S2485), Cisplatin (S1166), Ko143 (S7043) and Thiazolyl Blue (MTT agents, S6821) were bought from Selleck. PEG300, Tween-80 and DMSO were from Sigma-Aldrich. Antibodies for P-gp (sc-13,131), MRP1 (sc-18,835) and GAPDH (AMM22048N) were bought from the Santa Cruz. BCRP antibody (#AB40537) was got from AbSci. Antibodies for p-AKT (80455-1), AKT (60203-2), p-ERK1/2 (28733-1-AP) and ERK1/2 (28733-1-AP) were obtained from Proteintech. High-glucose DMEM, RPMI-1640, penicillin-streptomycin solution and trypsin-EDTA were bought from Gibco BRL. The Fetal Bovine Serum (FSP500) was got from ExCell Bio.

EZ-press RNA Purification Kit (B0004D), 4× Reverse Transcription Mix (EZB-RT2GQ), 2× SYBR qPCR Mix (A0001-R2) were purchased from the ZScience Biotechnology Corporation Limited on biotechnology (EZBioscience, USA).

Cell lines and culture

Both DMEM and RPMI-1640 were added with FBS (10%) and penicillin-streptomycin (1%) to mix for completed. Human oral cancer KB cell line and its P-gp overexpressed MDR KBv200 cell line, leukemia HL60 cell line and its MRP1-overexpressed MDR HL60/adr cell line were fostered with the completed RPMI-1640 medium. Human breast malignancy MCF-7 cell line and the corresponding P-gp overexpressed MCF-7/adr cell line, colon tumor S1 cell line and its BCRP-overexpressed S1-M1-80 cell line, and the human immortalized HEK293 cell lines stable-transfected with ABCB1, ABCG2-482-R2 or ABCG2-482-T7, and its corresponding HEK293/Vector cell line [30] were cultured with the completed DMEM medium. All mentioned cell lines were cultivated in an incubator of 37 ℃ temperature and 5% CO2.

Cytotoxicity and MDR reversal assessed by MTT assays

Planted 2000–3000 cells into the 96-well plates. When cells got attached, treated them with desired concentration of BI-2865 for 72 h, then incubated cells (200 µL medium) with 20 µL MTT (5 mg/mL) regents at 37 ℃ for another 4 h, followed by dissolving the crystal with 150 µL DMSO. Finally, detecting the OD absorption at 570/630 nm.

To evaluate the MDR reversal effect of BI-2865, cancer cells were treated with indicated BI-2865, Verapamil, Ko143 or MK571 at first, subsequently added gradient-diluted desired chemo-drugs. After incubation for 72 h, detecting the cell viability by MTT as described above.

Animal experiments

The ethical approval number (No. L102042023100C) of animal experiments in this study was authorized by Animal Ethics Committee of Sun Yat-sen University Cancer Center. All animal experiments were performed by strictly following the Declaration of Helsinki.

To assess the reversal action of BI-2865 in vivo, 3.5 × 10 6 KBv200 cells resuspended in PBS were subcutaneously seeded to the 3-week female BALB/C nude mice. As the tumors grew to nearly 100 mm3, randomly grouping mice into four and giving indicated administrations once every two days: (a) Saline (as Control); (b) paclitaxel intraperitoneal injection (15 mg/kg); (c) BI-2865 gavage (30 mg/kg, dissolved in solution of 10% DMSO, 40% PEG300, 5% Tween-80 and 45% Saline); (d) Co-administration of BI-2865 and paclitaxel. Simultaneously, recorded mice body weight and the tumor length and width. The tumor volume (V) was assessed by (length × width 2 /2). All mice were subject to euthanasia when tumor average volume in the saline group reached about 2000 mm3, and tumors were excised and weighed.

Doxorubicin accumulation and efflux assays

The excitation and emission wavelength of Doxorubicin (DOX) is correspondingly 475–485 nm and 575–585 nm. DOX amount in cells can be tested by Flow cytometry.

For analyzing the influence of BI-2865 on DOX accumulating in MDR cells, cells were pretreated by different doses of BI-2865 or vehicle for 3 h, followed by 10 µM Dox incubation for another 3 h. In the end, cells were collected using trypsin, washed and resuspended in PBS, followed by analyzation via Flow cytometry.

For analyzing the effect of BI-2865 on drug efflux in MDR cells, cells were pretreated by 10 µM Dox for 3 h, then discarded the medium and washed cells with PBS, added fresh medium with or without 8 µM BI-2865, incubated cells at 37 °C until cells were collected for detecting the residual DOX in cells through Flow cytometry at desired time points.

P-gp ATPase assay

The activity of P-gp ATPase was evaluated via performing a colorimetric assay as described previously [31]. Crude membranes were separated from P-gp overexpressing High Five insect cells. The crude membrane protein (100 µg protein/mL) was incubated with varying doses of BI-2865 (0–5 µM) with or without 0.3 mM of sodium orthovanadate (Na3VO4) in the pH 6.8 buffer (composed of 50 mM KCl, 2 mM EDTA, 10 mM MgCl2, 1 mM DTT and 5 mM sodium azide) at 37 °C for 5 min. Then added Mg-ATP solution (5 mM) to initiate the ATP hydrolysis, and the reaction was kept for 20 min at 37 °C. Afterwards, 30 µL of 10% SDS solution was added to end the reaction. Followed by an addition of detection reagent (containing 10% ascorbic acid, 15 mM zinc acetate and 35 mM ammonium molybdate), incubating another 20 min at 37 °C, then measuring the absorbance of the mixture at 750 nm via the 96-well Fisher Scientific Multiskan FC Microplate Reader (Pittsburgh, PA, USA). The release of inorganic phosphate was quantified at the standard curve. The final BI-2865-stimulated P-gp ATPase activation was defined as the variation between the released amounts of inorganic phosphate from ATP in the presence and absence of Na3VO4.

Photoaffinity labeling of P-gp with [125I]-iodoarylazidoprazosin (IAAP)

The photoaffinity labeling assay was conducted according to our previously established protocol [29]. Briefly, membrane protein was crudely extracted from the High Five insect cells expressing high P-gp, and taken 50 µg protein out for incubating with BI-2865 (0–5 µM) in the pH 7.5 Tris-HCl (50 mM) for 5 min at 25 °C. Under subdued light, added 3 nM [125I]-IAAP (2200 Ci/nmol) to the mixture for reacting another 5 min at 25 °C. Then radiolabeled samples with UV crosslinking on ice (365 nm). Afterwards, immunoprecipitated the radiolabeled P-gp with C219 antibody (Novus, Centennial, CO, USA). Finally, by using a Tris-acetate NuPAGE gel (7%), the samples were subjected to SDS-PAGE, dried and exposed overnight at -80 °C via Bio-Max MR film (Eastman Kodak Co., Rochester, NY). The IAAP labeling of P-gp captured radioactivity was assessed through the Storm 860 Phosphor Imager system (Molecular Dynamics, Sunnyvale, CA).

Western blotting

Cells were harvested, and the proteins were extracted in RIPA buffer. Then quantified protein samples by using Pierce™ BCA Protein Assay Kit. Same amounts of proteins were separated via SDS-PAGE electrophoresis, followed by transferring to the 0.2 μm PVDF membranes. After blocking in the 5% nonfat milk for 1–2 h, probed the membranes by using desired primary antibodies and subsequently applicable secondary antibodies, with a final visualization by the ECL chemiluminescent detection.

qRT-PCR

First, extracted the total RNA from cells by using the EZ-press RNA Purification Kit (B0004D). Reverse transcription assay was conducted by using the 4× Reverse Transcription Mix (EZB-RT2GQ). The obtained cDNA was used as templates in subsequent qRT-PCR assays by using the 2× SYBR Master Mix (A0001-R2). All steps were performed according to the corresponding instructions.

Primer sequences are presented here: P-gp (F: 5′-CAGGCTTGCTGTAATTACCCA-3′, R: 5′-TCAAAGAAACAACGGTTCGG-3′); GAPDH (F: 5′-GTCTCCTCTGACTTCAACAGCG-3′, R: 5′-ACCACCCTGTTGCTGTAGCCAA-3′).

Detection of P-gp on the cell surface

Cells were incubated with or without 8 µM BI-2865 for 48 h, followed by collection and washing with chilled PBS. Then resuspended cells in 30 µL PBS (containing 0.5% BSA), and probed it with the FITC-marked P-gp antibody (10 µL) at 4 °C (45 min). Finally, washed again and resuspended cells in 300 µL PBS for Flow cytometry analysis.

Immunofluorescence

KB, KBv200 cells (5 × 105) and MCF7, MCF7/adr cells (3 × 105) were planted to the glass bottom cell culture dishes. After attachment, cells were treated with 0 or 8 µM BI-2865 for 48 h, and then washed by PBS, fixed in 4% paraformaldehyde (PFA) for 20 min at room temperature (RT), additionally permeabilized in 0.1% Triton X-100 for 10 min. Cells were afterwards washed by using PBS again, followed by blocking with the 3% BSA at RT for 30 min and immunolabeling with the 1% P-gp antibody (22336-1-AP, Proteintech) overnight at 4 °C. Next, cells were in dark labeled with the 0.1% Alexa Fluor Plus 594-conjugated goat anti-rabbit IgG (A32740, Invitrogen) at RT for 1 h. Nuclei were stained by DAPI (1:1000) for 15 min. Finally, taken images by using Zeiss LSM 880 microscope with 63× oil lens.

Molecular docking with P-gp

Molecular docking is an important method widely used to predict the interaction of small molecules with proteins. In this study, we docked BI-2865, Paclitaxel and Vincristine to the drug-binding area of P-gp to further analyze their interaction. Firstly, obtained the P-gp crystal structure from web of Protein Data Bank (https://www.rcsb.org/). Then, docking was completed via Auto Dock Vina software, and the visualization of results was achieved via the PyMOL 1.8.

Accumulation of Paclitaxel in tumor analyzed by LC-MS

0.3 g tumor tissues were homogenized and extracted in 1500 µL methanol for 15 min at 70 Hz, then centrifuged at 12,000 rpm for 15 min to obtain the supernatants. Next, the supernatants were blown dry at room temperature, followed by the residues were dissolved again with 200 µL methanol, centrifuged again. Then, 2 µL of each sample’s final supernatant was injected into the high-resolution LC-MS (Synapt G2-Si, Waters) with PDA detector for further analysis.

A ACQUITY UPLC BEH C18 Column (1.7 μm, 2.1 × 100 mm) was used to separate the constituents in sample supernatant. The mobile phase (A) H2O with 0.1% formic acid, (B) acetonitrile were used in the following gradient elution profile: 0.00–1.00 min: 90% (A); 1.00–10.10 min: 10% (A); 10.10–12.00 min: 90% (A). Flow rate was maintained at 0.25 mL/min. Multiple reaction monitors (MRM) was used for quantification in both positive and negative modes.

Statistical analysis

Every experiment in this study was independently repeated more than three times and data here represent the Mean ± SD. The significance between two data was statistically analyzed by Two-tailed Student’s t-test. *p < 0.05, **p < 0.01 and ***p < 0.001 mean statistically significant.