IMP was purchased from the National Institutes for Food and Drug Control (Beijing, China) and its purity exceeds 99%. Epirubicin hydrochloride was obtained from Lunan Pharmaceutical (batch: 155160702). The Thermo Scientific RevertAid First cDNA Synthesis Kit was provided by Thermo Fisher Scientific. The Trizol RNA Extraction Kit was purchased from Ambion (Thermo Fisher Scientific, Beijing, China). The reverse transcription kit was purchased from Promega (Madison, WI, USA). Power SYBR Green PCR Mix was purchased from Life Technologies, Inc., (New York, NY, USA) PCR primers were synthesized by Shanghai Sangon Biotech Co. Ltd. (Shanghai, China). A mouse monoclonal anti-P-gp (MDR) antibody was obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA) A GAPDH mouse monoclonal antibody was purchased from Shanghai Bioleaf Biotech Co. Ltd. (Shanghai, China). Horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG), radio immunoprecipitation assay (RIPA) lysis buffer, protease inhibitors, phosphatase inhibitors, bicinchoninic acid (BCA) Protein Assay Reagent Kit, and enhanced chemiluminescence (ECL) Kit were purchased from Beijing Com Win Biotech Co. Ltd. (Beijing, China).

Human chronic myelogenous leukemia cell line K562 and its doxorubicin-resistant cell line K562/DOX, human ovary A2780 cell line and its Taxol-resistant cell line A2780/Taxol were all purchased from Shanghai Gefan Biotechnology. Co., Ltd. (Shanghai, China) Cells were grown in an atmosphere of 5% CO₂ at 37°C and cultured in RPMI 1640 medium containing 10% fetal bovine serum and 100 U/ml penicillin-streptomycin. Drug-resistant cell lines were maintained with suitable concentrations of drugs,, and the drugs were withdrawn 2 weeks before experiments.

Cell viability and proliferation was determined by Cell Counting Kit-8 (CCK-8) assay. Different cells were seeded into 96-well plates and incubated overnight. Drug at concentrations in the presence or absence of IMP (2.78, 5.56, 11.10 µM) were added to corresponding cells, respectively. After incubation for 48 h, 20 µl of the CCK-8 reagent was added to each well, and the absorbance was determined at 450 nm after 3 h. 50% inhibiting concentration (IC₅₀) was calculated from survival curves using the Bliss method. The resistance index (RI) was calculated by dividing the IC₅₀ value of K562/DOX cells by that of K562 cells. The reversal fold (RF) was calculated as the ratio between the IC₅₀ value alone to that of IMP combined with chemotherapeutic drugs.

K562 and K562/DOX cells were plated in 12-well plates and then incubated different concentration of drugs or Hank’s balanced salt solution (HBSS, 37°C, pH 7.4). Cells were incubated in a 5% CO₂ incubator at 37°C. The reaction was terminated after 60 min on ice. The solution was separated by centrifuging at 1,000 rpm for 5 min at 4°C, discarded the supernatant and washed for twice with cold HBSS. Rho 123 (5 µM) was added in different groups and incubated for 75 min. The accumulation was stopped by rinsing cells with ice-cold HBSS. Single-cell suspensions were prepared and centrifuged at 12,000 g for 10 min, and the protein concentrations were determined using BCA protein assay kit. The concentration of Rho 123 in each tube was divided by the total protein content of each tube to eliminate errors caused by the number of cells. In a parallel experiment, 200 µl of supernatant was mixed with 300 µl of acetonitrile followed by centrifugation at 14,000 g for 20 min. Rho 123 in the supernatant was detected by a previously established high-performance liquid chromatography (HPLC) method. Chromatographic separation was achieved on a Phenomenex-C18 (250 mm × 4.6 mm, 5 µm) column with acetonitrile/1% triethylamine (pH 3.0) under gradient conditions at a flow rate of 1 ml/min. Detection was performed using a fluorescence detector with an excitation wavelength of 485 nm and an emission wavelength of 546 nm [28].

Male SPF NOD/SCID mice (20 ± 2 g) were provided by the Nanjing Institute of Biomedicine of Nanjing University (Laboratory animal certificate number, 201901A014). Animals were housed under standard conditions of artificial light and dark cycles with free access to food and water, and the room had good ventilation with a temperature at 20°C–25°C. All animal studies were performed according to the approved protocols and guidelines of the Institutional Animal Ethical Care Committee.

K562/DOX cells (1 × 10⁷ cells/0.2 ml) were subcutaneously injected into the right flanks of 10 NOD/DSCID mice. When the tumor nodules were approximately 1 cm in diameter, they were subcultured to establish a transplanted tumor model. The model mice were randomly divided into the following 5 groups: model group (intraperitoneal injection of 0.9% saline once daily), DOX alone group (intraperitoneal injection of DOX at a dose of 2.5 mg/kg, twice a week), low-dose IMP combined with DOX group (oral administration of IMP at a dose of 5 mg/kg once daily, and intraperitoneal injection of DOX at a dose of 2.5 mg/kg, twice a week), medium-dose IMP combined with DOX group (oral administration of IMP at a dose of 10 mg/kg once daily, and intraperitoneal injection of DOX at a dose of 2.5 mg/kg, twice a week), and high-dose IMP combined with DOX group (oral administration of IMP at a dose of 20 mg/kg once daily, and intraperitoneal injection of DOX at a dose of 2.5 mg/kg, twice a week). Mice were continuously treated for 15 days, and tumor volumes were measured once a week. Animals were sacrificed, and the tumor tissues were removed and weighed after the last treatment.

Tumor tissues were immobilized in 4% formaldehyde solution, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E).

Tumor tissues were immobilized in 2.5% glutaraldehyde, dehydrated, embedded, cut into 80 nm sections, and stained with 2% uranium acetate and citrate. A JEM-1011 transmission electron microscope was used to observe the tumor ultrastructural changes.

Cell glycolysis, glutamine consumption, α-ketoglutaric acid (α-KG) production and ATP production were assessed by Glucose Assay Kit, Lactate Assay Kit, the Glutamine Assay Kit, α-KG Assay Kit and ATP Assay Kit according to the manufacturer’s instructions.

Cell extracellular acidification rate were performed to determine cellular aerobic glycolysis. In brief, 5 × 10⁴ Cells were seeded into the cell-culture dish overnight (96-well microplate), and in the 20 min added 10 mM of Glucose, 1 µM of Oligomycin add in 15 min, and 50 mM of 2-deoxy-D-glucose (2-DG) put in it another 15 min. Finally we used Glycolysis Rate Determination Kit to test, and then analyzed by Seahorse XFe Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA).

Tumor tissues were lysed in RIPA buffer containing phenylmethanesulfonyl fluoride and phosphatase inhibitor. The concentration of total protein was detected by an Enhanced BCA Protein Assay Kit. Equal quantities of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to PVDF membrane. Membranes were blocked in 5% bovine serum albumin) for 1 h and incubated with primary antibodies overnight. Membranes were then incubated with secondary antibodies at room temperature for 1 h. Proteins were detected by using an ECL Western blotting detection system.

The total RNA of tumor tissues was extracted by the RNA easy Total RNA mini kit and reverse transcribed to cDNA with reverse transcription kit according to the manufacturer’s protocol, respectively. PCR primer design and synthesis were performed by the primer design software provided by Applied Biosystems. The primer sequences were as follows: human MDR1, forward 5’-AAA GCG ACT GAA TGT TCA GTG G-3’, reverse 5’-TGC GTG TGG AGT ATT TGG ATG-3’; human hRpIP1v-F (internal control), forward 5’-CCC TCA TTC TGC ACG ACG AT-3’, reverse 5’-GGC TCA ACA TTT ACA CCG GC-3’. PCR amplification was performed using Power SYBR Green PCR Mix according to the manufacturer’s protocol.

All experiments were carried out at least three times. SPSS software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. All of the data were expressed as mean ± SD, and were subjected to statistical analysis by one-way ANOVA followed by a Student–Newman–Keuls post-hoc test. p < 0.05 was considered significant for all tests.

Both DOX and Taxol exhibited anti-proliferative activity on both parental and resistant cells dose-dependently (Fig. 2A, C), respectively. The IC₅₀ values of K562 was 1.63 ± 0.20 µM, whereas IC₅₀ values of K562/DOX was 38.20 ± 0.58 µM. The RI for K562/DOX cells was 23.43 (Fig. 2B, Table 1) (RI > 15). The IC₅₀ values of A2780 was 51.33 ± 1.76 µM, whereas IC₅₀ values of A2780/Taxol was 188.30 ± 4.63 µM.The RI for A2780/Taxol cells was 3.67 (Fig. 2D, Table 2). The results showed that the two kinds of resistant cell lines were all strongly resistant to corresponding chemotherapy drugs and can be used for follow-up research.

Treatments with IMP at concentrations of 2.78, 5.56, and 11.10 µM significantly increased the cytotoxicity of DOX in K562/DOX cells (p < 0.05), respectively, but had little effect in K562 cells (Fig. 3A, B and Table 1). IMP showed a dose-dependent reversal effect of drug resistance of K562/DOX cells. Treatments with IMP at concentrations of 7.40, 18.50, and 37.00 µM significantly increased the cytotoxicity of Taxol in A2780/Taxol cells (p < 0.05), respectively, while had little effect in A2780 cells (Fig. 3C, D and Table 2). IMP showed a dose-dependent reversal effect of drug resistance of A2780/Taxol cells. As shown in Table 1, as the concentration of IMP increased, the IC₅₀ value of DOX-IMP combinations reduced and resulting in the increased RF of IMP accordingly (RF from 3.33 to 9.00). We also can see in Table 2, with the concentration of IMP increased,the IC₅₀ value of Taxol-IMP combinations reduced and resulting in the increased RF of IMP accordingly (RF from 2.75 to 6.29). These results indicated that IMP could sensitiz the different tumor-resistant cell lines to chemotherapeutics.

Rho 123, a fat-soluble fluorescent substance and a substrate of P-gp, which can be transported from the cell membrane into the cytoplasm. While P-gp can discharge entered Rho 123 out of the cells. Therefore, the less amount of intracellular accumulation of Rho 123, the higher activity of P-gp. Verapamil is a P-gp inhibitor which could increase the amount of intracellular accumulation of Rho 123 [15]. We first examined the uptake of Rho 123 by K562/DOX cells. The results showed that the fluorescence intensity in K562/DOX cells increased with time, and gradually reached saturation state after a certain time (Fig. 4A). Therefore, 75 min was choosen as the coincubation time of Rho 123 and the impacts of IMP on the accumulation of Rho 123 in K562 cells and K562/DOX cells was observed. We further confirmed the effect of time on the absorption of IMP and verapamil (VER). The results showed that the fluorescence intensity of IMP increased with time just like VER, and gradually reached saturation state at 60 min (Fig. 4B). The HPLC detection showed that K562/DOX cell solution had no interference with the determination of Rho 123 (Fig. 4C). Next, we used HPLC detection method to observe the effects of IMP and VER on the accumulation of Rho 123 in K562 cells and K562/DOX cells. As shown in Fig. 4D, compared with K562 cells, there was a decreased accumulation and increased efflux of Rho 123 in K562/DOX cells, which is transported by P-gp (p < 0.05). IMP at concentrations of 2.78, 5.56, and 11.10 µmol/L significantly increased the intracellular Rho123 accumulation in K562/DOX cells (p < 0.01), implying that IMP may reduce the efflux activity of P-gp. The reference group VER at concentrations of 10.2, and 20.4 µM illustrated the same effect but more potently (p < 0.01).

Glycolysis and glutamine metabolism are important energy sources of cancer cells, and are also an important way for P-gp to inhibit drug efflux. We found that the expressin of P-gp was significantly increased in K562/DOX cells (Fig. 5A). We evaluated glycolysis and glutamine metabolism in K562/DOX cells by measuring glucose consumption and lactate production. The results revealed that IMP could significantly reduce the glucose consumption and lactate production of K562/DOX cells (Fig. 5B, C). Furthermore, IMP could also remarkably repress the glutamine consumption, α-KG production and ATP production of K562/DOX cells (Fig. 5D, E).

The tumor volume was measured every 7 days. Fig. 6A showed that both DOX alone and IMP combined with DOX treatments significantly reduced the average tumor volume at 14 days, but not obvious at 0 day and 7 days. IMP combined with DOX treatments tended to reduce the average tumor volume at 14 days, as compared with the DOX alone treatment. After 14 days, mice were sacrificed and the tumor tissues were weighed. Compared with the untreated model group, both DOX alone and IMP combined with DOX treatments significantly decreased the tumor weight. Moreover, the average tumor weight in groups of IMP at dosage of 10 mg/kg and 20 mg/kg combined with DOX was remarkably lower than that in DOX alone group, respectively (Fig. 6B, 6C).

H&E staining showed increased necrosis and apoptosis in tumor tissues collected from mice treated with DOX alone and IMP combined with DOX (Fig. 7A). IMP (10 mg/kg and 20 mg/kg) combined with DOX group showed increased necrosis compared to the other groups, and fibrous tissue hyperplasia was found between tumors in IMP (20 mg/kg) combined with DOX group. The blue arrow indicates that the cell structure was seriously damaged and the cell density was enhanced.

Dual staining with 2% uranium acetate and lead citrate were visualized with transmission electron microscopy. The red arrows indicated mitochondria, we could see the swelling or rupture of mitochondria. And the green arrows indicated normol cell structure, it showed that Mitochondria were uniform in size and clear in structure. As illustrated in Fig. 7B, obvious necrosis, cytoplasmic vacuole, and apoptotic characteristics appeared in some cells of both DOX alone and IMP (10 mg/kg and 20 mg/kg) combined with DOX groups, respectively.

We next examined whether IMP affected P-gp protein and mRNA expressions in K562/DOX xenograft tumor tissues in NOD/SCID mice through western blotting and quantitative RT-PCR experiments, respectively. As shown in Fig. 8, compared with the untreated model group, both DOX alone and IMP combined with DOX treatments significantly inhibited P-gp protein expression levels (Fig. 8A, B) and downregulated P-gp mRNA levels (p < 0.01, Fig. 8C). Moreover, IMP at dosage of 10 mg/kg and 20 mg/kg combined with DOX treatments further suppressed P-gp protein levels (Fig. 8A, B) and mRNA levels (Fig. 8C), as compared with the DOX alone treatment (p < 0.01), respectively. Our results indicated that IMP inhibited P-gp protein and mRNA expressions in K562/DOX xenograft tumor tissues in NOD/SCID mice.