Methotrexate was obtained from Pude Pharmaceutical Company Ltd. (Shanxi, China). The novel synthetic histone deacetylase inhibitor (FA17) was kindly provided by Professor Jie Zhang from Health Science Center of Xi'an Jiaotong University. Verapamil was purchased from China Pharmaceutical Biological Products Analysis Institute. The Annexin V FLUOS staining kit was purchased from Invitrogen (USA). The primary antibodies against NPM, breast cancer resistance protein (BCRP), dihydrofolate reductase (DHFR), Bcl-2 and Bax were from Epitomics (USA), while phosphatase and tensin homolog deleted on chromosome 10 (PTEN), total Akt (t-Akt), phospho-Akt (p-Akt), caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP) antibodies were purchased from Cell Signaling Technology (USA). P-glycoprotein (P-gp) and reduced folate carrier (RFC) antibodies were obtained from Abcam (UK), and multidrug resistance associated protein 1 (MRP1) was from GeneTax (USA). The β-actin antibody was purchased from Biosynthesis Biotechnology and the horseradish peroxidase-conjugated goat anti-rabbit IgG was from Cwbiotech (Beijing, China).

The MCF-7/S sensitive human breast cancer cell line was obtained from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences (Shanghai, China). The methotrexate-resistant breast cancer cell line (MCF-7/MTX) was successfully established as previously described (5) with the concentration of 220 nM MTX. Both cell types were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (PAA, Austria) and 1% penicillin/streptomycin in a humidified atmosphere of 5% CO₂ at 37°C.

Protein samples were extracted from whole-cell, the protein concentration was detected by BCA reagent (Beyotime, China) and then separated by 10 or 12% sodium dodecyl sulfate-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, USA). Then the membranes were blocked with 5% fat-free milk at room temperature for 2 h, and incubated with diluted antibodies NPM (1:1,000, ab109546), PTEN (1:1,000, #9188), p-Akt (1:800, #4090), t-Akt (1:1000, #4691), Bcl-2 (1:2,500, #1017-1), Bax (1:2,500, #1063-1), caspase-9 (1:800, #9502), caspase-3 (1:800, #9662), PARP (1:800, #9542), P-gp (1:500, ab98322), MRP1 (1:600, GTX116046), BCRP (1:800, #3765-1), RFC (1:1,500, ab62302), DHFR (1:20,000, ab124814) and β-actin (1:800, bs-0061R) overnight at 4°C. The second antibody conjugated to horseradish peroxidase incubation was developed at room temperature for 2 h. Protein bands were tested using the SuperSignal West Pico kit (Pierce, Thermo Scientific). All samples were performed in triplicates and the protein expression was analyzed using a quantitative analysis system (Image-Pro Plus).

For in vitro knockdown experiments, the double-stranded small interfering RNA (siRNA) against human NPM1 and non-specific siRNA (control siRNA) were purchased from Shanghai GenePharma Company Ltd. (China). They were used for transient transfection as previously described (5). MCF-7/MTX cells were seeded at a density of 5×10⁵ per well into a 6-well plate and cultured in the medium until cell confluence reached ~40%. The following day, cells were transfected with 50 nM NPM1 siRNA and Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions.

Total cellular mRNA was extracted using RNAfast2000 kit (Fastagen) and reverse transcription to cDNA was performed using PrimeScript RT Master Mix Perfect Real-Time kit (Takara). Quantitative real-time PCR (qRT-PCR) was conducted using SYBR Premix Ex Taq II (Takara) and the primer sequences and product lengths are listed in Table I. The assay was performed on the Bio-Rad CFX96™ Real-time system with the following procedure: 95°C for 30 sec; 95°C for 5 sec and 60°C for 30 sec (40 cycles); and 95°C for 15 sec, 60°C for 30 sec, 95°C for 15 sec. Each sample was normalized on the basis of β-actin. Three independent experiments were run to analyze the gene expressions and each sample was repeated in triplicate.

The cell viability under drug treatment was measured using MTT assay. Cells were seeded at a density of 5×10⁴ per well into 96-well plates and treated with cytotoxic agents at increasing concentrations for 72-h incubation, then MTT (5 mg/ml) was added to each well for an additional 4 h. After discarding the medium, the blue formazan was dissolved in 150 μl of DMSO. The absorbance was tested at 490 nm on a microplate reader (BioTek, USA). The resistance factor (RF) was calculated using the equation: RF = (IC₅₀ value of MCF-7/MTX)/(IC₅₀ value of MCF-7/S). The data represent three independent experiments.

The reversal effect of FA17 on methotrexate resistance was evaluated in MCF-7/MTX cells using MTT assay. MCF-7/MTX cells were seeded at a density of 5×10⁴ per well into 96-well plates for 24 h, then methotrexate at increasing concentrations in combination with FA17 were added and cultured for 72 h. The reversal index (RI) was calculated using this equation: RI = IC₅₀ of methotrexate/IC₅₀ of methotrexate plus FA17. The data represent three independent experiments.

For the cell cycle assay, cells were harvested after treatment and fixed in 70% cold ethanol at 4°C overnight. After suspension with PBS, cells were stained with RNase at 37°C for 15 min and with propidium iodide on ice for 30 min away from light. Then cell cycle distribution was detected immediately using flow cytometry (BD Bioscience, USA).

Cell apoptosis was measured by Annexin V FLUOS staining kit according to the manufacturer's instructions. Cells from different samples were analyzed for live, necrotic, early and late apoptotic cells by flow cytometry. All experiments were run in triplicate.

Female BALB/c nude mice (18±4 g, 4–6 weeks) were purchased from Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. All animal experiments were carried out in accordance with guidelines and approval of the Institutional Animal Care and Use Committee of Xi'an Jiaotong University. MCF-7/MTX cells (200 μl, 1×10⁷ cells) were injected subcutaneously into mice, and when they all developed tumors with a size ~100 mm³, mice were randomly assigned into four groups (n=6) and treated intraperitoneally with vehicle, MTX (9 mg/kg), FA17 (10 mg/kg) or in combination every three days for a total of three weeks. Tumor volume was measured on alternate day and calculated with the formula a × b²/2 (a, the largest diameter; b, the smallest diameter), additionally, mouse weight was monitored three times per week. Upon termination, mice were sacrificed and tumors were harvested. Protein were exacted from tumors for western blot analysis. The other tumor tissues were fixed with formalin and embedded with paraffin, which were analyzed for regular hematoxylin and eosin (H&E) stain and immunohistochemistry. These tissue sections were incubated with primary antibody (NPM, 1:200 dilution), and control group with PBS. The immunostaining intensities or percentages of positive cells were evaluated as previously described under bright-field microscopy (16). Images from 6 random fields of each section were used for quantitative analysis (Image-Pro Plus).

All data were expressed as mean ± standard deviation (SD). Statistical analyses were performed using one-way ANOVA, and P-value <0.05 was considered as statistical significance.

Our previous study identified that NPM was significantly overexpressed in the MCF-7/MTX cells, and served as a novel drug-resistant target in breast cancer using proteomics technology (5). Similarly, some reports found upregulation of NPM accounted for MDR phenotype in various cancers (10,11,17). In addition, PI3K/Akt signaling pathway played a significant role in carcinogenesis and development of malignant tumors, so activation of this pathway emerged in a majority of tumors and even facilitated the formation of drug-resistance (18–21). We detected NPM and related factors of PI3K/Akt signaling pathway both in MCF-7/S and MCF-7/MTX cells by western blotting. As shown in Fig. 2A and B, NPM was obviously overexpressed in MCF-7/MTX cells compared with MCF-7/S cells. The decreased PTEN level and increased p-Akt expression were both observed in MCF-7/MTX cells, which indicated the PI3K/Akt pathway was activated in MCF-7/MTX cells. Furthermore, the downstream pro-survival factor Bcl-2 was upregulated and pro-apoptotic factor Bax was downregulated, which significantly suppressed cells apoptosis in MCF-7/MTX cells.

To better understand the effect of drug resistance on cell apoptosis, the interrelated factors of mitochondrial apoptosis pathway were also investigated. As Akt augments phosphorylation, the downstream apoptotic molecules including cleaved-caspase-9, cleaved-caspase-3 and cleaved-PARP were all downregulated in MCF-7/MTX cells (Fig. 2C and D). The above demonstrated that NPM-induced resistance not only activated PI3K/Akt pathway, but also inhibited the apoptotic function of resistant cells.

To further explore the interaction of NPM and PI3K/Akt pathway, we determined the changes in expression of related factors after knockdown of NPM. We predicted that the reduction of NPM could restrain the PI3K/Akt pathway and accelerate its downstream pro-apoptotic effect. Decreasing NPM expression by siRNA in MCF-7/MTX cells, mRNA level of NPM was reduced by >70% compared with the siRNA controls (Fig. 3A). Additionally, decreasing NPM expression could accelerate upregulation of PTEN and attenuate Akt phosphorylation while t-Akt was not apparently changed in siNPM-resistant cells. Then the expression of downstream pro-apoptotic factor Bax was elevated, and the pro-survival factor Bcl-2 was remarkably decreased (Fig. 3B and C). Moreover, mitochondrial apoptosis molecules cleaved-caspase-9, cleaved-caspase-3 and cleaved-PARP were all increased (Fig. 3D and E). These results declared that function of pro-apoptosis was elevated by devitalizing PI3K/Akt pathway with NPM knockdown in MCF-7/MTX cells.

A novel synthetic histone deacetylase inhibitor FA17 was reported to display a promising profile as an anti-tumor candidate (15), but whether or not FA17 could reverse breast cancer MDR was not clear. We detected intrinsic cytotoxicity of FA17 in MCF-7/S and MCF-7/MTX cells by MTT analysis. As shown in Fig. 4A, FA17 obviously inhibited growth of sensitive and resistant cells in a dose-dependent manner, and IC₅₀ was 16.6±2.43 and 23.4±2.23 μM, respectively, these data indicated that MCF-7/MTX cells did not produce resistance to FA17 which may serve as a candidate chemicals to reverse MDR.

The reversal effect of FA17 on the MDR of MCF-7/MTX cells was analyzed by MTT assay. Three concentrations including 0.85, 1.7 and 3.4 μM under the lower-toxic dose (inhibition rate <15%) were chosen to evaluate efficacies of FA17 on NPM-mediated methotrexate resistance in breast cancer cells, and verapamil was used as a positive control. The results are presented in Table II, RIs of these three doses to MCF-7/MTX cells was 2.60, 3.80 and 4.38, respectively, thus, the effect of FA17 higher concentration was close to verapamil and its dose was markedly less than control. This phenomenon demonstrated strong reversal activity of FA17 in MCF-7/MTX cells.

To further investigate effects of FA17 on cell cycle, MCF-7/MTX cells were treated with methotrexate (2 μM), FA17 (3.4 μM), and two drug combinations using flow cytometry for cell-cycle detection. Compared with control, methotrexate and combination group caused a significant decrease of cells in the G₀/G₁ phase and a corresponding increased proportion of S-phase cells, while FA17 alone treatment apparently added G₀/G₁-phase cells and reduced S-phase cells (Fig. 4B and C). The experimental results revealed that the role of methotrexate and combination treatment blocked S-phase cells, but G₀/G₁-phase arrest appeared in the FA17 group.

In addition, to assess the effect of FA17 on methotrexate induced resistance, an apoptosis assay was performed in MCF-7/MTX cells treated with methotrexate or FA17 alone and combination group, verapamil was used as a positive control again. Treatment of cells with FA17 and combination groups significantly increased cells apoptosis rates compared with methotrexate group, and FA17 strengthened methotrexate-induced MCF-7/MTX cell apoptosis, its intensity was apparently greater than the effect of positive drug verapamil (Fig. 4D). Thus it was proven that FA17 had the ability to induce resistant cell apoptosis, while increasing the sensitivity of MCF-7/MTX cells to methotrexate.

As previous studies have demonstrated that NPM, membrane transport proteins including P-gp, MRP1, BCRP and methotrexate resistance-associated RFC and DHFR all had abnormal expression in resistant cells (5), we determined if resistant factors were modulated by FA17 in MCF-7/MTX cells using qRT-PCR and western blot assays. After 48-h treatment, both mRNA and protein expressions of NPM, P-gp, MRP1, BCRP, DHFR were downregulated while RFC level was upregulated in MCF-7/MTX cells (Fig. 4E and F). The results validated that FA17 levels partially reversed MDR characteristics of MCF-7/MTX cells at gene and protein levels.

We have discovered that the occurrence of drug resistance was correlated with the activation of PI3K/Akt pathway, however, to clarify whether FA17 reversed methotrexate-induced breast cancer resistance by PI3K/Akt pathway, we determined expressions of PTEN, Akt and downstream mitochondrial apoptosis related factors with western blotting. As shown in Fig. 5A and B, Akt phosphorylation was suppressed along with the intensifying of PTEN level, and it emerged evidently in a dose-dependent manner, then enervated relevant factor Bcl-2 level and enhanced Bax expression, which eventually reduced MDR phenotype of MCF-7/MTX and induced cell apoptosis.

Next, we investigated the inhibitory effect of FA17 on apoptotic process of resistant cells, the variation of mitochondrial apoptosis signaling pathway molecule expression was detected in MCF-7/MTX cells. As expected, caspase-9, caspase-3, and PARP proteins obvious cleaving belts depended on the dose under FA17 treatment (Fig. 5C and D), which predicted appearance of cells apoptosis. Taken together, these results illuminated that FA17 may reverse drug resistance of MCF-7/MTX cells through inhibiting the activation of PI3K/Akt and accelerating mitochondrial apoptosis.

Our research has suggested that FA17 reversed MDR of MCF-7/MTX cells by PI3K/Akt pathway in vitro. To further confirm this effect in vivo, we evaluated FA17 alone or combined with methotrexate treatment in nude mouse xenograft model when tumors were palpable and continued for 14 days. Animals bearing MCF-7/MTX cells displayed a tumor growth profile (Fig. 6A and B), comparing with controls, tumor volumes were significantly decreased after methotrexate and FA17 combined treatment, and Table III shows the most effective in attenuating tumor burden (51% tumor growth inhibition), whereas methotrexate or FA17 alone group also possessed a certain degree of suppression of tumor growth, and inhibition rates were 19 and 29%, respectively. In addition, it did not cause conspicuous reduction in body weight and any other abnormities under drug therapy.

To further explore influence of FA17 and methotrexate treatment on tumor tissues of nude mice, H&E staining was performed to observe morphological variations and immunohistochemical analysis was applied to detect NPM expression. As shown in Fig. 6C, tumor necrosis and histomorphology of tumor tissues were not obviously observed after drug treatment. Whereas, NPM expression was strongly positive in the control group, and its level was downregulated in methotrexate or FA17 alone group, but NPM showed almost negative expression in the combined treatment (Fig. 6D). These results manifested that methotrexate and FA17 combined therapy significantly inhibited drug-resistant target NPM and lowered resistance of nude mice.

To clarify the underlying resistance mechanism of combination where methotrexate and FA17 are more effective than either drug alone, we next detected expressions of PI3K/Akt pathway related factors. The immunoblotting revealed that intensity of PTEN was augmented under drug treatment accompanied by downregulation of NPM level, especially in the combined group. Furthermore, drug action inhibited Akt phosphorylation and downstream Bcl-2 expression, and reinforced the role of Bax, which led to resistant tumor cells apoptosis, eventually blocking the growth of methotrexate-resistant tumors in nude mice (Fig. 6E and F). These experiments clarified the molecular mechanism by which FA17 reversed MDR of breast cancer in vivo.