Combination of 7-O-geranylquercetin and microRNA-451 enhances antitumor effect of Adriamycin by reserving P-gp-mediated drug resistance in breast cancer

MiRNAs reversed ADR-resistance in MCF-7/ADR cells

It was shown that miR-326, miR-328 and miR-451 expressed at low levels in ADR-resistant cells while miR-155 expressed at a high level. On the contrary, miR-326, miR-328 and miR-451 showed high levels in MCF-7 cells, while miR-155 showed a low level (Figure 1A). This demonstrated that these four miRNAs might be related to ADR-resistance in breast cancer cells.

Figure 1. MiRNAs reversed ADR-resistance in MCF-7/ADR cells. (A) Expression of miR-326, miR-328, miR-451 and miR-155 in MCF-7 and MCF-7/ADR cells were detected by Real-time RT-PCR. ^***P < 0.001, comparing with MCF-7 cells. (B) MCF-7/ADR cells and MCF-7/ADR cells transfected with miRNAs were treated with ADR (43 μM) for 48 h. Cell viability was measured by CCK-8 assay. All data represent the means ± SD of three independent experiments. ^**P < 0.01, ^***P < 0.001, comparing with non-transfection group.

When ADR-resistant cells were subjected to ADR for 48 h, the proliferation rate of miR-NC-transfected cells was similar to that of untransfected cells, but the proliferation rates of the cells transfected with miR-326, miR-328, miR-451 and the inhibitor of miR-155 dropped from 60-80% to 19%, 18%, 17% and 27%, respectively (Figure 1B). The results showed that these miRNAs reversed drug resistance in ADR-resistant cells.

We also detected the reversal folds of miRNAs to ADR-resistance. IC₅₀ values of ADR to MCF-7 and ADR-resistant cells were respectively 5.73 μM and 61.51 μM, and the resistance fold was 10.73. Drug resistance reversal folds of miR-326, miR-328, miR-451 and the inhibitor of miR-155 to ADR-resistant cells were 3.15, 3.0, 3.76, 2.02, respectively (Table 1). These results indicated that miR-451, miR-326, miR-328 and the inhibitor of miR-155 could reverse ADR-resistance, and miR-451 showed the highest reversal effect.

MiRNAs regulated the expression of MRP1, BCRP, MDR1 genes and MRP1, BCRP, P-gp proteins

To disclose the mechanism of miRNAs reversing drug resistance, the expression levels of MRP1, BCRP, MDR1 genes and MRP1, BCRP, P-gp proteins in ADR-resistant cells were tested after transfection of miRNAs. Real-time RT-PCR assay showed that miR-326 and miR-328 down-regulated the expression levels of MRP1, BCRP respectively, miR-451 and the inhibitor of miR-155 down-regulated the expression level of MDR1 (Figure 2). Western blot showed that miR-326 and miR-328 suppressed the expression of MRP1 and BCRP, while miR-451 and the inhibitor of miR-155 suppressed the expression of P-gp. MiR-451 showed the strongest inhibition effect on gene and protein expression (Figure 3). Above results revealed that miRNAs attenuated the expression of transporters MRP1, BCRP and P-gp by suppressing the expression of MRP1, BCRP and MDR1 genes in ADR-resistant cells.

Figure 2. MiRNAs regulated the expression of MRP1, BCRP, MDR1 genes. The expression levels of MRP1, BCRP and MDR1 in MCF-7/ADR cells and MCF-7/ADR cells transfected with miRNAs were detected by Real-time RT-PCR. All data represent the means ± SD of three independent experiments. ^**P < 0.01, ^***P < 0.001, comparing with control group.

Figure 3. MiRNAs regulated the expression of MRP1, BCRP and P-gp proteins. (A) Western blot was conducted to detect the expression of MRP1, BCRP, P-gp and β-actin in MCF-7/ADR cells and MCF-7/ADR cells transfected with miRNAs. (B) Quantitative analysis of the results in (A). All data represent the means ± SD of three independent experiments. ^*P < 0.05, ^***P < 0.001, comparing with control group.

Combination of GQ and miR-451 enhanced the inhibitory action of ADR on proliferation and migration of MCF-7/ADR cells

With transfection of miR-451 into ADR-resistant cells, the IC₅₀ of ADR decreased from 61.51 μM to 16.34 μM, and then to 2.93 μM with additional treatment of GQ. Drug resistance reversal fold of miR-451 to ADR was 3.76, while that of GQ combining miR-451 was 21.04 (Table 1), indicating that combination of GQ and miRNA-451 more effectively reversed ADR-resistance in MCF-7/ADR cells.

In scratch experiment, ADR, GQ, miR-451 alone or GQ combining miR-451 showed slight inhibition effect on the migration of ADR-resistant cells. The inhibitory action of ADR was enhanced via being combined with miR-451 or GQ, and ADR combined with GQ and miR-451 showed the strongest inhibition effect on cell migration (Figure 4). These data demonstrated that GQ combining with miR-451 exerted synergistic effect on reversing ADR-resistance in MCF-7/ADR cells.

Figure 4. Effect of GQ and miR-451 on the migration inhibition of ADR to MCF-7/ADR cells. MCF-7/ADR cells or miR-451-transfected MCF-7/ADR cells were seeded on 6-well plates with monolayer and divided on average with a UV-sterilized ruler and pipette tips. Then the cells were treated with GQ (10 μM), ADR (43 μM) or their combination for 48 h and photographed using an inverted microscope.

Combination of GQ and miR-451 inhibited the expression of MDR1 and P-gp in MCF-7/ADR cells

Real-time RT-PCR showed that both miR-451 and GQ significantly inhibited MDR1 expression in ADR-treated MCF-7/ADR cells, and the inhibitory action of GQ combining miR-451 was more potent than that of GQ or miR-451 (Figure 5A). Western blot showed that miR-451 and GQ markedly inhibited P-gp expression in ADR-treated MCF-7/ADR cells, the inhibitory action of GQ combining miR-451 was stronger than that of GQ or miR-451 alone (Figure 5B). These indicated that GQ combining miR-451 inhibited MDR1 and P-gp expression, and had a stronger capability of reversing drug resistance as compared to either of them.

Figure 5. Combination of GQ and miR-451 inhibited the expression of MDR1 and P-gp. MCF-7/ADR cells and miR-451-transfected MCF-7/ADR cells were subjected to ADR (43 μM) or ADR (43 μM) combining GQ (10 μM) for 48 h. (A) Expression levels of MDR1 were detected by Real-time RT-PCR. The MCF-7/ADR cells only treated with ADR were regarded as control. (B) Expression levels of P-gp were detected by western blot. All data represent the means ± SD of three independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, comparing with ADR group; ^#P < 0.05, ^###P < 0.001, comparing with miR-451, GQ and ADR combination group.

Combination of GQ and miR-451 enhanced the inhibition effect of ADR on tumor growth

After proving the drug resistance reversing effect of GQ combining miR-451 in ADR-resistant cells, we then evaluated their synergetic drug resistance reversing effect on xenograft in mice. The tumor in mice treated only with GQ, ADR or miR-451 continuously grew during the treatment, whereas the tumor growth rate in mice treated with GQ or ADR was slower than that in miR-451-transfected mice or control mice. The tumor volume of mice treated with ADR combined with GQ or/and miR-451 reduced obviously. The tumor growth inhibition effect of ADR combined with GQ and miR-451 was stronger than that of ADR combined with GQ or miR-451 (Figure 6A, 6B). The body weight of mice treated with ADR or ADR combined with miR-451 showed a slight downward trend while that of mice in other groups had no significant change during the treatment, revealing that GQ, miR-451 and liposome CDO14 did not induce apparent toxicity in mice (Figure 6C). All of these suggested that GQ and miR-451 augmented tumor suppressive action of ADR in vivo.

Figure 6. GQ and miR-451 enhanced the inhibitory effect of ADR on tumor growth in mice. Tumor-bearing mice were treated with ADR (3 mg/kg) or the complex of miR-451 (0.55 mg/kg) and liposome CDO14 (1.5 mg/kg) via tail vein injection, or treated with GQ (18 mg/kg) by gavage for 6 times at an interval of 3 days. (A) Tumors removed from the mice after treatment. (B) Volume of tumor in mice during the treatment. (C) Body weight of mice during the treatment. All data represent the means ± SD, n=3. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, comparing with control group.

Combination of GQ and miR-451 depressed P-gp expression in tumor tissues

Immunohistochemistry (IHC) assay showed that P-gp-positive area in tumor tissues of mice subjected to GQ, ADR combined with GQ or/and miR-451 was obviously less than that in control group, and P-gp-positive area was the least in the group treated with ADR combined with GQ and miR-451 (Figure 7A, 7B). Western blot assay meanwhile demonstrated that the expression level of P-gp was the lowest in the tumors of ADR combined with GQ and miR-451 treatment group (Figure 7C). So, it was confirmed that GQ and miR-451 reversed ADR-resistance in MCF-7/ADR cells by depressing P-gp expression, thereby increasing tumor suppression effect.

Figure 7. Effect of GQ and miRNA on the expression of P-gp in tumor tissues. (A) P-gp expression levels in tumor tissues were detected by IHC assay, the bar indicated 100 μm; (B) Quantitative analysis of the results in (A); (C) P-gp expression levels in tumor tissues were detected by western blot. All data represent the means ± SD of three independent experiments. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, comparing with control group. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, comparing with miR-451, GQ and ADR combination group.

Cell lines and cell culture

Human breast cancer cell line MCF-7 was purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). MCF-7/ADR (ADR-resistant) cell line was obtained from Pharmacy College of Dalian Medical University (Dalian, China). The cells were cultured in RPMI-1640 medium supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (Seven Biotech, Beijing, China) at 37° C under a humidified atmosphere with 5% CO₂ [12].

Animals

Four to six weeks old female BALB/c-nude mice were purchased from Liaoning Changsheng Biotechnology Co., LTD, China (SCXK: 2020-0001). Mice feeding and experiment were under a specific pathogen-free condition and in accordance with the Experimental Animal Management Law of China. Experiment protocol was approved by the Animal Ethics Committee of Dalian Medical University.

Transfection of miRNA in MCF-7/ADR cells

ADR-resistant cells were seeded in 6-well plates (2×10⁵ cells per well) and incubated for 24 h until cell density reached 80~90%. MiRNAs and their inhibitors (GenePharma, Shanghai, China) displayed in Table 2 were mixed with cationic liposome CDO14 (synthesized in our lab) [34] at a weight ratio of 1:3 at 4° C for 20 min. The complexes (500 μL per well) were transfected into the cells and incubated for 6 h in serum-free RPMI-1640 medium. Then the cells were incubated in normal medium overnight and subjected to subsequent treatments.

Cell viability assay

Cell counting kit-8 (CCK-8) assay was performed to measure cell viability for evaluating the role of miRNAs on ADR-resistance. ADR-resistant cells were cultured in 96-well plates at the initial density of 2×10⁴ cells per well for 18 h. MiR-NC, miR-326, 328, 451 and 155 and their inhibitors (1.5 μg, 500 μL) were respectively transfected into the cells for 6 h. MiRNAs-transfected or untransfected ADR-resistant cells were subsequently subjected to 43 μM ADR (Haizheng Pharmaceutical Co, Ltd, Zhejiang, China) for 48 h. Ten microliter of CCK-8 (Biotool, Shanghai, China) was added into each well and the cells were incubated for 2 h. A microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect absorbance at 450 nm.

Determination of drug-resistant folds

CCK-8 assay was also used to determine half inhibition concentration (IC₅₀) values of ADR to MCF-7 and ADR-resistant cells. The cells were seed in 96-well plates (2×10⁴ cells per well) and cultured for 24 h. MiR-326, 328, 451 and miR-155 inhibitor (1.5 μg, 500 μL) were respectively transfected into ADR-resistant cells for 6 h. The transfected and untransfected ADR-resistant cells, MCF-7 cells were administrated with ADR at various concentrations (decreasing proportionally from 172 μM) for 48 h. CCK-8 assay was performed as described above. IC₅₀ values of ADR to MCF-7 cells, ADR-resistant cells, miRNAs-transfected ADR-resistant cells, and drug-resistant folds were calculated. Drug-resistant fold was the IC₅₀ value of ADR against ADR-resistant cells (or miRNAs-transfected ADR-resistant cells) divided by that against MCF-7 cells. Reversal fold was the drug-resistant fold of ADR-resistant cells divided by that of miRNAs-transfected ADR-resistant cells.

Effect of GQ and miR-451 on ADR-resistance in MCF-7/ADR cells was detected. The cells were transfected with miR-451 (1.5 μg, 500 μL) for 6 h and then administrated with 10 μM of GQ (synthesized in our lab) [37] and ADR at various concentrations for 48 h. CCK-8 assay was used to detect cell viability and IC₅₀ value was calculated.

Detection of gene expression

Real-time RT-PCR assay was performed to detect the expression levels of miR-328, 326, 451, 155 in MCF-7 and ADR-resistant cells. The cells were incubated in 6-well plates at an initial density of 2×10⁵ cells per well for 24 h. Then cell lysis, RNA extraction and reverse transcription reaction were conducted as reported methods [12]. Real-time qPCR was performed according to the manufacturer’s recommendations and our previous study [12]. Endogenous U6 snRNA was used to normalize gene expression levels, the relative expression of miRNAs was quantified using 2^-ΔΔCt method [38].

Effect of miR-328, 326, 451, 155 and their inhibitors on the expression of MRP1, BCRP and MDR1 in ADR-resistant cells was detected by Real-time RT-PCR according to above methods [12]. β-actin mRNA was used to normalize the mRNA levels of MRP1, BCRP and P-gp.

Real-time RT-PCR was also performed to investigate the influence of GQ and miR-451 on the expression of MDR1 gene in ADR-resistant cells. The cells were seed in 6-well plates with a density of 2×10⁵ cells each well and incubated for 18 h. MiR-451 (1.5 μg, 500 μL) was transfected into the cells for 6 h. Both miR-451-transfected and untransfected ADR-resistant cells were administrated with ADR (43 μM) or ADR (43 μM) combining GQ (10 μM) for 48 h. The expression level of MDR1 gene in the cells was detected by Real-time RT-PCR as described above. The sequences of the above gene primers (GenePharma, Shanghai, China) were shown in Table 3.

Detection of protein expression

Western blot was performed to detect the effect of miRNAs on the expression of P-gp, BCRP and MDR1 in ADR-resistant cells. The cells (2×10⁵ cells per well) were cultured in 6-well plates overnight and subsequently transfected with miR-326, 328, 451, 155 and their inhibitors (1.5 μg, 500 μL) for 48 h. After that, the cells were collected and lysed as the described methods [36]. The protein samples were quantified using BCA assay kit (Beyotime, Shanghai, China) and stored at -80° C.

The influence of GQ and miR-451 on the expression of P-gp was also investigated by western blot. ADR-resistant cells were cultured in 6-well plates at the initial density of 2×10⁵ cells per well overnight following by transfection of miR-451 (1.5 μg, 500 μL) for 6 h. MiR-451-transfected and untransfected ADR-resistant cells were administrated with ADR (43 μM) or ADR (43 μM) combining GQ (10 μM) for 48 h. Then the cells were lysed and kept as described above. The protein samples were subjected to SDS-PAGE at 30 μg per lane, and then to western blot as reported methods with β-actin as an internal control [39].

Scratch test

ADR-resistant cells were incubated in 6-well plates with the initial density of 2×10⁵ cells per well for 12 h. The cells in each well were divided on average with a UV-sterilized ruler and pipette tips, and washed twice to remove detached cells and debris. Some cells were transfected with miR-451 (1.5 μg, 500 μL) for 6 h before treatments of GQ (10 μM), ADR (43 μM) or their combination for 48 h while others subjected to the same treatments directly. Then the cells were photographed under an inverted microscope (GE, USA) to observe the width of scratches.

Tumor inhibition in mice

The mixture of ADR-resistant cells (5×10⁷ cells/mL) and matrigel at equal volume was subcutaneously inoculated in the right armpit of female BALB/c nude mice (200 μL per mouse). Volume of the xenograft tumor was measured and calculated as follows: Volume = L×W²/2 (L and W respectively represented the length and width of the tumor) [12]. Twenty-four mice with tumor about 150 mm³ were divided into eight groups in random, and respectively treated with normal saline (Control), GQ, ADR, miR-451, ADR combining GQ, ADR combining miR-451, ADR combining GQ and miR-451, ADR combining GQ and miR-NC. The complex of liposome CDO14 and miR-451 (0.55 mg/kg) with the ratio of 3:1, ADR (3 mg/kg) and normal saline were injected via the tail vein, and GQ (18 mg/kg) was administered by gavage [39]. Tumor-bearing mice were administered once every three days for 6 times. Body weight, tumor volume and physical state were recorded daily until the mice were killed at 18th day. Tumors stripped from the mice were photographed and then subjected to western blot and IHC assay for detecting the expression of P-gp in tumor tissues.

A part of tissues was cut into small pieces and lysed in RIPA buffer to obtain protein for western blot assay according to reported methods [36]. Another part of tumor tissues were fixed in formalin fixative and penetrated in paraffin. Paraffin sections were made and IHC assay was performed as reported methods [12].

Statistical analysis

Statistical analysis was performed using SPSS 21.0 (SPSS, Inc, Chicago, IL, USA). All data are presented as the mean ± standard deviation (SD) of at least three independent replicates. Student’s t-test was applied to assess the difference among groups. P < 0.05 was considered statistically significant.