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Introduction

In recent years, cytokine research has been at the forefront of cancer research and cytokine approaches are involved in the treatment of various carcinomas. Cytokine approaches for cancer therapy have three potential mechanisms of action. They can i) directly induce cell death programs in tumor cells, ii) increase the number or activity of immune effector cells, or iii) increase the recognition of tumor cells by the immune system (1).

Interferons (IFNs) are one of the most important cytokines. They are naturally secreted glycoproteins produced by almost every cell type as a mechanism of host defense in response to microbial attack (2). The IFN family includes three different groups. IFNα belongs to the type I IFN group, and was discovered 50 years ago. It was the first cytokine to be produced by recombinant DNA technology, and it is used as an important regulator of cell growth and differentiation, affecting cellular communication and signal transduction pathways as well as immunological control (3). More recently, IFNα has been applied in the treatment of multiple carcinomas including leukemia, hepatocellular carcinoma, bladder cancer, and osteosarcoma (4–7).

Breast cancer is a leading cause of cancer-related death in women worldwide. According to GLOBOCAN 2012, there are approximately 1.67 million newly diagnosed breast cancer patients every year (8). In China, breast cancer is the most commonly diagnosed cancer, and the number of breast cancer patients has been increasing annually (9). Surgery, chemotherapy, radiotherapy, endocrinotherapy and molecular targeting therapy are the major treatment modalities for breast cancer. However, challenges remain in the treatment of breast cancer, and novel therapeutic approaches are urgently needed.

Although the use of IFN in clinical practice is widely recommended, its antineoplastic activity and clinical efficacy for breast cancer is still unclear and controversial (10,11). In this study, an NEB Ph.D.-12 peptide library was employed to select a short peptide that specifically binds to the cell membrane of MCF-7 cells, and a high-affinity IFNα-MCF-7 fusion molecule IMBP was constructed. Our aim was to investigate whether this reconstructed cytokine had enhanced cell growth inhibition activity compared to the wild-type IFNα.

Materials and methods

Cell culture

Human breast cancer cell line MCF-7, lung cancer cell line A549 and prostate cancer cell line PC-3 were kept in our lab. Human embryonic kidney 293T cell line was used for lentiviral packaging. All cell lines were routinely cultivated in Dulbecco's modified eagle's medium (DMEM, Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (Invitrogen), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen) at 37°C in a humidified atmosphere containing 5% CO2.

Phage display library screening of breast cancer binding peptides

Phage display library (Ph.D.-12 library, #E8110S, New england Biolabs, Ipswich, MA, USA) was employed to screen a short 12-peptide that specifically binds to MCF-7 breast cancer cells (12,13). Briefly, 1×1011 phage in 1 ml DMEM medium was incubated with 1×106 MCF-7 cells at room temperature (RT) for 1 h to develop phage-cell complexes. After binding, cells were washed 5 times with 5 ml TBST [TBS (10 mmol/l Tris pH 7.5, 150 mmol/l NaCl) containing 0.1% Tween-20] to remove the unbound phages. Cells were collected by centrifuging at 3200 × g. The surface-bound phages were eluted in 1 ml elution buffer [0.2 N Glycine-HCl (pH 2.2), 1 mg/ml BSA] for 20 min and was neutralized by 150 μl 1 N Tris-HCl, pH 9.0. The eluted phages were reproduced by infecting E. coli ER2738 and purified using polyethylene glycol (PEG)-8000/NaCl solution (20% PEG-8000 and 2.5 N NaCl).

After 4 rounds of surface panning, the DNA sequences of MCF-7-binding peptide (MBP) were amplified by PCR (Fig. 1A). Primers used for PCR amplification were as follows: 5′-CTTTAGTGGTACCTTTCTATTCTCGAGTCT-3′ (forward primer with Xho I) and 5′-CTTTCAACAGTTTCGTCTAGAACCTCCACC-3′ (reverse primer with XbaI). The PCR production was purified from 3% agrose gel and cloned into pJet vector using CloneJET PCR Cloning kit (Thermo Fisher Scientific, Waltham, MA, USA) for sequencing and constructing the engineered IFNα molecules.

Figure 1 Construction of the IFNα/MCF-7-binding peptide fusion protein. (A) Ph.D.-12 peptide library screening of the MCF-7 breast cancer cell binding peptides. MCF-7 cells were incubated with phage display library and the cell-specific binding phages were screened following the manufacturer's protocol. After 4 rounds of surface panning, the DNA sequences encoding the MBPs were cloned and sequenced. (B) The amino acid sequence of MBP that was selected for the cell study. (C) Cell binding affinity assay of the screened MBP-carrying phage particles. MBP-phage particles were incubated with A549, PC-3 and MCF-7 cells. The binding affinity of the MBP-carrying phage was quantitated by phage-ELISA method. *P<0.05 as compared with A549 and PC-3 cells. (D) Construction of IMBP fusion protein expression vector. pCMV, CMV promoter; IFNα, wild-type interferon α; MBP, the control construct that contains only MCF-7 binding phage-display peptide sequence; IMBP, IFNα and MBP fusion protein expression vector; Vector, empty vector control. (e) The predicted protein structure of the synthetic IMBP. The putative structure of IMBP was predicted using software on the website: http://zhanglab.ccmb.med.umich.edu. Arrow indicates MBP fused into wild-type IFNα.

Phage-ELISA assay

Phage-ELISA was carried out to determine the binding affinity of the isolated MBP. Approximately 2×104 MCF-7 cells were collected into a 96-well V-bottom tissue culture plate and incubated with blocking buffer (5% BSA/PBS, Sigma, Shanghai, China) at 37°C for 1 h. For comparison, A549 lung cancer and PC-3 prostate cancer cells were used as negative controls. Cells were pelleted by centrifuging at 3,200 × g and resuspended in 100 μl PBS buffer (Thermo Fisher Scientific, Beijing, China) containing 1×109 phage particles. After incubated at RT for 1 h, cells were pelleted and washed with PBST for 5×5 min. For quantitation, cells were incubated with 100 μl PBS buffer containing HRP conjugated anti-M13 monoclonal antibody (1:5,000, GE Healthcare, Piscataway, NJ, USA) at 37°C for 45 min. Cells were centrifuged and washed by PBST for 5×5 min. Then 100 μl of TMB solution (BD Biosciences, San Jose, CA, USA) was added to each well and the plate was kept in the dark for 15 min until blue color was developed. Reaction was stopped by adding 50 μl 1 N sulfuric acid. The absorbance of 450 nm and 630 nm were measured by microplate reader (Bioteck, Beijing, China). Final optical density (OD) was designated as OD450-OD630-ODblank.

Recombinant plasmid construction

After the phage library screening and sequencing, one of the MBP sequence was selected for the following cell studies. We ligated MBP to the C-terminus of IFNα to construct an engineered synthetic IFNα fusion protein. The fusion protein was generated by ligating the MBP sequence into IFNα vector at the XhoI/XbaI restriction sites and was designated as IMBP. The wild-type IFNα expression plasmid was prepared as previous described (14,15) and kept in our laboratory. The vector containing the isolated MBP sequence and the empty lentivirus vector were constructed in parallel and used as control groups. All plasmid constructs were confirmed by DNA sequencing.

The putative structure of the IMBP

The putative structure of the synthetic IMBP was predicted using online I-TASSER server (http://zhanglab.ccmb.med.umich.edu).

Lentivirus production

293T cells were seeded into a 6-well plate at a proper density 24 h pre-transfection. Lentiviruses were packaged by the co-transfection of the plasmids constructed above and pSPAX2/pMD2.G packing vectors (System Biosciences, Mountain View, CA, USA) using Lipofectamine 3000 reagent (Thermo Fisher Scientific). The viral supernatants were collected at 24 and 48 h post-transfection and used for cell transfection as previously described (16,17).

Infection of recombinant lentivirus

Approximately 2×105 MCF-7 cells were plated into a 6-well plate 24 h pre-transfection. Cells were infected with lentiviruses carrying MBP, IFNα, IMBP and empty vector DNA in the presence of 8 μg/ml polybrene (Sigma). After two rounds of infection with lentiviruses at 37°C for 24 h, the tissue culture medium was replaced with fresh complete medium. Three days after viral infection, the stable MCF-7 cells were selected by puromycin and used for the following cell experiments.

Detection of the secreted IMBP fusion protein

On day 5 post-transduction, 100 μl supernatant of MCF-7 cells was collected and the secreted IFNα and IMBP fusion protein were quantitated with IFNα assay kit according to the manufacture's instruction (Cusabio, Hubei, China).

Western blotting

Protein were extracted with RIPA buffer (KeyGEN Biotech, Jiangsu, China) supplemented with cocktail protease inhibitor (Roche, Shanghai, China) and quantified with BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China). Whole cell lysates were resolved on 5–10% or 5–12% SDS polyacrylamide gel electrophoresis (SDS-PAGE). To detect the secreted IFNα and IMBP proteins in cell supernatants, on day 5 post-transduction, 100 μl MCF-7 cell supernatant was collected and condensed to 20 μl in a vacuum-freeze dryer (Boyikang, Beijing, China). Solutions were resolved on Mini-PROTEIN TGX gradient gel (Bio-Rad, Beijing, China). Proteins were transferred to 0.45 μm PVDF membranes (Roche) and immunoblotted at 4°C overnight or at RT for 1 h with the following antibodies: IFNα (1:1,000, Abcam, Shanghai, China), STAT1 (phospho Y701, 1:1,000, Abcam), Cleaved CASP-9 (1:1,000, Cell Signaling Technology, Beijing, China), p53 (1:1,500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p21 (1:500, Santa Cruz Biotechnology), CDK2 (1:1500, Cell Signaling Technology), Cyclin A (1:1,500, Abcam) and β-actin (1:3,000, Santa Cruz Biotechnology). Membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:3,000, ZSGB-BIO, Beijing, China) at 37°C for 1 h before chemiluminescence reading. Protein expression levels were determined semi-quantitatively by densitometric analysis with the Quantity One software (Bio-Rad). Western blotting was performed in triplicate, and data showed a representative finding of these triplicate analyses.

Cell binding assay of IMBP

The binding affinity of the engineered hybrid molecule to the MCF-7 cell membrane was measured by FACS. MCF-7 cells were collected and stained with Trypan blue to make sure that viable cells were more than ninety percent. Approximately 1×106 normal MCF-7 cells were incubated with equal amount of the secreted IFNα or IMBP at 37°C for 1 h. After washed with PBS, cells were incubated with FITC-conjugated IFNα antibody (PBL, Piscataway, NJ, USA). The FITC-conjugated mouse IgG (Abcam) was used as the isotype control. Cells were analyzed using BD LSRII Fortesa flow cytometer (BD Biosciences) and FlowJo software (FlowJo, OR, Ashland, USA) to calculate the fluorescence intensity (14,15).

Cell viability assay

On day 7 post-transfection, approximately 5×103 viable stable MCF-7 cells transduced by lentivirus carrying IMBP, IFNα, MBP and empty vector DNA were seeded into a 96-well plate 24 h before cell viability assay. Then, cell growth was analyzed by WST-1 Cell Proliferation Reagent (Roche). According to the manufacturer's instructions, 20 μl WST-1 reagent was added to 200 μl cell culture medium and incubated at 37°C in the dark for 2 h. The absorbances of 450 and 630 nm were measured with microplate reader (Bioteck). Final OD was designated as OD450-OD630-ODblank.

Cell cycle assay

On day 7 post-transfection, 1×106 stable MCF-7 cells were collected and washed by cold PBS. Cells were resuspended in 1 ml fixation solution (300 μl PBS and 700 μl ethanol). After incubation at 4°C for 4 h, cells were centrifuged and the fixation solution was removed. After washed twice with PBS, cells were pelleted, stained with 0.5 ml propidium iodide (PI, Sigma) staining solution [50 μg/ml PI, 20 μg/ml RNase A (Takara, Liaoning, China) and 0.2% Triton X-100 (Sigma)], and incubated in the dark at 37°C for 10 min. Cell suspensions were filtered with a 400-mesh sieve. Cell cycle was analyzed by BD LSRII Fortesa flow cytometer.

Flow cytometry for cell apoptosis

Annexin V-FITC and PI double staining flow cytometry analyses were used for cell apoptosis assay. Approximately 1×106 stable MCF-7 cells were collected and washed three times with cold PBS and binding buffer. Then cells were stained with Annexin V-FITC and PI (Annexin V-FITC Apoptosis Detection kit, BD Bioscience) for apoptosis detection. Briefly, MCF-7 cells were first resuspended in 1 ml binding buffer. Then, 5 μl of Annexin V-FITC was added to the tubes, and cells were incubated for 10 min at RT followed by the addition of 5 μl PI. After 15 min incubation in PI buffer at RT, cells were immediately analyzed with a flow cytometer (BD Biosciences). The cells in the different portions represented the different cell states as follows: the late-apoptotic cells were present in the upper right portion, the viable cells were present in the lower left portion, and the early apoptotic were cells present in the lower right portion.

Treatment with chemotherapeutic drug doxorubicin

The anticancer drug doxorubicin (DOX, Sigma) was dissolved in water. We harvested 12 ml of cell supernatant containing the secreted interferons from the stable MCF-7 cells transduced by lentivirus carrying IMBP, IFNα, MBP and empty vector. The cell supernatant was centrifuged at 3,500 × g for 5 min to remove cell debris. Then the supernatant was successively passed through two centrifugal filter units with MW cut-off 30 and 10 kDa (Millipore, Temecula, CA, USA) to remove large and small molecules. The interceptions were dissolved in 6 ml DMEM complete medium and kept in 4°C. Normal MCF-7 cells were seeded into 6-well plates at a density of 3×105 cells/well. After 24 h, cell medium was changed to the interception-DMEM supplemented with 0.1 μg/ml DOX. MCF-7 cells were treated with this therapeutic medium for 3 days, with the medium changed every day. After that, cells were harvested and applied for the next step of cell viability assay or molecule detection.

Real-time PCR

Total cellular RNA was isolated using an Eastep Super total RNA isolation kit (Promega, Beijing, China). First-strand cDNA was synthesized from 800 ng of total RNA using Transcriptor First Strand cDNA Synthesis kit (Roche). Real-time PCR was performed using an aliquot of first-strand cDNA as a template in a 20 μl reaction system containing 10 μl 2X SYBR premixed buffer (Roche), 2 μl forward and reverse primers. The primers were as follows: IFIT1 (interferon induced protein with tetratricopeptide repeats) sense 5′-TCTCAGAGGAGCCTGGCTAA-3′, IFIT1 antisense 5′-CCAGACTATCCTTGACCTGATGA-3′; MX1 (MX dynamin-like GTPase 1) sense: 5′-CTTTCCAGTCCAGCTCGGCA-3′, MX1 antisense: 5′-AGCTGCTGGCCGTACGTCTG-3′); STAT1 sense: 5′-GGCACCAGAACGAATGAGGG-3′, STAT1 antisense: 5′-CCATCGTGCACATGGTGGAG-3′ (18); IFITM1 (interferon-induced transmembrane protein 1) sense: 5′-ACAGGAAGATGGTTGGCGAC-3′, IFITM1 antisense: 5′-ATGGTAGACTGTCACAGAGC-3′; β-actin sense: 5′-TCACCCACACTGTGCCCATCTACGA-3′, β-actin antisense: 5′-CAGCGGAACCGCTCATTGCCAA TGG-3′ (19). The PCR amplification process was one cycle at 95°C 10 min, 40 cycles at 95°C for 10 sec and 60°C for 30 sec (ABI StepOnePlus, Beijing, China).

Statistical analysis

All data and results were calculated from at least three replicate measurements and presented as means ± SD. The significance was determined by SPSS 20.0 (IBM). Student's t-test was used to compare statistical differences for variables among treatment groups. P<0.05 was considered to indicate a statistically significant difference.

Results

Construction of the IFNα/MCF-7-binding peptide fusion protein

In order to enhance the antitumor activity of IFNα in breast cancer therapy, we used a NEB Ph.D.-12 peptide library to select a short peptide with 12 amino acids that specifically binds to the cell membrane of MCF-7 cells. Our aim is to add a short, high-affinity peptide to the C-terminus of IFNα and construct an IFNα-MCF-7 fusion molecule (IMBP), which may facilitate the binding of IFNα to the MCF-7 cell membranes and enhance the cell growth inhibition activity of IFNα.

According to the manual of NEB Ph.D.-12 peptide library, MCF-7 cells were incubated with 1×1011 phage particles in DMEM tissue culture medium. The unbounded phage particles were stripped off by stripping buffer. The bound phages on the cell membrane were eluted and recovered for the second round of screening. After four rounds of screening, the phages specifically binding to MCF-7 cells were enriched and identified (Fig. 1A). After cloning and sequencing, one sort of phage encodes short peptide with the sequence of 'C E H I K D E L V C Q N' was selected for further cell studies (Fig. 1B). Using phage-ELISA, we showed that this phage displayed short peptide was able to bind preferentially to MCF-7 cells compared to A549 lung cancer and PC-3 prostate cancer cells (Fig. 1C).

In order to examine the role of this short peptide and compare the antitumor activity of wild-type IFNα and IMBP, we linked the short peptide to the C-terminus of IFNα and the IMBP fusion molecule was synthesized (Fig. 1D). Then we used software on the website (http://zhanglab.ccmb.med.umich.edu) to predict the protein structure of the synthetic IMBP. The structure of wild-type IFNα and the putative structure of IMBP is illustrated in Fig. 1E. Arrow indicates the short peptide fused into wild-type IFNα.

Detection of the secreted IFNα and IMBP fusion protein

We used 293T cells to produce lentivirus carring the DNA sequences of IMBP, wild-type IFNα, MBP and empty vector. Then, MCF-7 cells were transducted by lentiviruses and the stable cells were selected with puromycin. On day 5 post-transduction, 100 μl supernatant of MCF-7 cells was collected and the secreted IFNα and IMBP fusion protein were quantitated with IFNα assay kit. Based on the result of IFNα ELISA, the concentration of secreted wild-type IFNα in MCF-7 supernatant was 885 pg/ml and that of the IMPB fusion molecule was 763 pg/ml (Fig. 2A). We further used western blotting to confirm the secretion of wild-type IFNα and IMPB fusion molecule. The blots are shown in Fig. 2B.

Figure 2 Detection of the secreted IFNα and IMBP fusion protein. (A) Detection of the secreted IFNα and IMBP fusion protein with IFNα ELISA kit. 293T cells were transfected with IMBP, wild-type IFNα, MBP DNA sequence and empty vector. At 24 and 48 h post-transfection, cell supernatant containing lentiviruses was harvested. MCF-7 cells were transducted by lentiviruses and stable cells were selected with puromycin. At day 5 post-transduction, 100 μl MCF-7 cell supernatant was collected and the secreted IFNα and IMBP fusion protein were quantitated with IFNα assay kit. (B) Detection of the secreted IFNα and IMBP fusion protein with western blotting. At day 5 post-transduction of MCF-7 cells, 100 μl cell supernatant was collected and condensed to 20 μl in a vacuum-freeze dryer. Then the cell supernatant was resolved by Mini-PROTEIN TGX gradient gel for western blot analysis. IMBP: 23 kDa, IFNα: 21 kDa as estimated by ExPASy (http://web.expasy.org/cgi-bin/compute_pi/pi_tool). (C) Analyses of the cell membrane binding of IFNα and IMBP with FACS. Approximately 1×106 MCF-7 cells were inchubated with stable cell supernatant containing equal amount of secreted IFNα and IMBP. The FITC conjugated-IFNα antibody was used for FACS quantitation of the cell binding ability assay. The FITC conjugated mouse IgG was used as the isotype control. The fluorescence intensity was measured to evaluate the cell binding ability.

Since the phage particles bind to MCF-7 cell membrane specifically, we used FACS to further examine whether the IMBP fusion protein binds to MCF-7 cell surface easier than the wild-type IFNα. Therefore, FITC conjugated-IFNα antibody was used in FACS assay and the binding affinity of the two proteins was quantitatively evaluated according to the fluorescence intensity in flow cytometry (Fig. 2C). Quantitative analysis result showed that the binding affinity of the IMBP fusion protein was 1.5-fold increase compared to the wild-type IFNα (P<0.05), and the binding ability of both the wild-type IFNα and the IMBP fusion protein was significantly higher than the isotype control (Fig. 2C, P<0.05).

Synthetic IMBP fusion protein inhibits cell growth of MCF-7 cells

We compared the cell growth inhibition effect between the IMBP and the wild-type IFNα. MCF-7 breast cancer cells were transfected with lentiviruses carrying IMBP, IFNα, MBP and empty vector, respectively. Cell proliferation was determined by WST-1 assay. We found that although both the secreted IFNα and IMBP inhibited the growth of MCF-7 cells, IMBP was superior to IFNα (Fig. 3A, P<0.05).

Figure 3 The synthetic IMBP fusion protein inhibits cell growth of MCF-7 breast cancer cells. (A) Cell viability of MCF-7 breast cancer cells. MCF-7 cells were transduced by lentiviruses carrying IMBP and IFNα, MBP and empty vector sequences, and the stable cells were selected by puromycin. On day 7 post-transduction, approximately 5×103 viable stable MCF-7 cells were seeded in 96-well and cell viability was determined by WST-1 method. (B) Cell apoptosis of the stable MCF-7 breast cancer cells were analyzed on day 7 after viral transduction by flow cytometry. Cell morphology were recorded on day 7 after viral transduction. *P<0.05 as compared with the IFNα group.

Then we examined cell apoptosis of the stable transfected MCF-7 cells with flow cytometry. As was shown in Fig. 3B, we found that the transfection of both IFNα and IMBP induced an increased apoptosis ratio, but the IMBP group showed a significantly higher apoptosis ratio than the IFNα group (26 vs. 18%, P<0.05). The cell morphology also showed a similar result (Fig. 3C).

IMBP potentiates the therapeutic efficacy of doxorubicin-based chemotherapy

Since doxorubicin is one of widely used anthracyclines to treat breast cancer, we examined the synergistic effect on cell killing by the combined use of DOX and IMBP fusion molecule. First, using WST-1 cell proliferation assay, we tested a serial working concentration of DOX including 0.02, 0.05, 0.1, 0.2, 0.5 and 1.0 μg/ml for the cell growth inhibition of MCF-7 cells. We selected a relative low concentration of 0.1 μg/ml of DOX with a cell growth inhibition ratio approximately 25% for the chemotherapy (data not shown). Then, we investigated the cell killing ability of the co-administration of DOX/IFNα or DOX/IMBP. The WST-1 assay result showed that cell proliferation in both the DOX/IFNα and DOX/IMBP treatment groups was apparently inhibited compared to the PBS control group and the DOX group. Of note, cell killing ability of the co-administration of DOX and IMBP was superior to the co-administration of DOX and IFNα (Fig. 4A, 48 vs. 32%, P<0.05).

Figure 4 IMBP potentiates the therapeutic effect of doxorubicin-based chemotherapy. (A) MCF-7 breast cancer cells were treated with chemotherapeutic medium for 3 days and cell growth was measured by WST-1 assay. (B) Cell cycle distrubution of the treated MCF-7 breast cancer cells was analyzed by flow cytometry. (C) Western blot analyses of cell cycle and apoptosis related genes. (D) Relative band density of western blotting. 1: PBS control group; 2: DOX group; 3: IFNα plus DOX group; 4: IMBP plus DOX group. *P<0.05 as compared with the IFNα plus DOX group.

Then we examined the cell cycle distribution in the treated MCF-7 cells with flow cytometry. We found that the co-administration apparently induced an S phase cell cycle arrest, and the DOX/IMBP treatment group showed a more obvious arrest effect than the DOX/IFNα group (Fig. 4B, 43 vs. 37%, P<0.05).

To unveil the underlying molecular mechanism that explains the enhanced therapeutic efficacy of the combined use of DOX and IMBP in chemotherapy, we first examined the expression of the cell cycle pathway related genes by western blotting. We found that the expression of p53 and p21 in the DOX/IMBP chemotherapy group was dramatically up-regulated compared to the DOX/IFNα group; the expression of CDK2 and cyclin A was dramatically down regulated (Fig. 4C left panel and D, P<0.05). We then examined the expression of the cell apoptosis pathway-related genes and found that the caspase 9 and Bcl-2/Bax apoptosis pathways were activated (Fig. 4C right panel and D, P<0.05).

IMBP fusion molecule activates the STAT1 pathway

To further delineate the mechanism of the enhanced activity of the combination chemotherapy, we used real-time PCR to examine the expression of the interferon pathway genes in the treated MCF-7 cells. The quantitation result showed that both the DOX/IMBP and DOX/IFNα co-administration activate several inducible genes of the STAT1 pathway, including STAT1, IFIT1, IFITM1, and MX1. However, DOX/IMBP group showed a stronger activation effect than the DOX/IFNα group (Fig. 5A). The western blot analysis result also showed an activation of the phosphorylated STAT1 (Y701, Fig. 5B).

Figure 5 IMBP activates the STAT1 signal pathway in MCF-7 chemotherapy. (A) Real-time PCR analyses of the activation of the STAT1 signal pathway. (B) Western blot analyses of the STAT1 expression and the relative band density of western blots. 1: PBS control group; 2: DOX group; 3: IFNα plus DOX group; 4: IMBP plus DOX group. *P<0.05 as compared with the IFNα plus DOX group.

Discussion

Anthracyclines, one type of anticancer therapy, are commonly used to treat both early and metastatic breast cancer; however, their toxicity, especially cardiac toxicity, is high (20,21). Additionally, frequent use of single therapeutic agent may result in chemoresistance, which is a major obstacle to the successful treatment of breast cancer. To minimize the toxicity of DOX and maximize the therapeutic efficiency of DOX, a combination therapeutic strategy is important.

The functions of IFNs are represented by three major biological activities, antiviral activity, antitumor activity and immunoregulatory activity (22). Although IFNα is widely used in the treatment of various types of cancer, its antineoplastic potency for breast cancer is low. Therefore, improvements in this cytokine are valuable and further investigation is still needed.

In our previous study, using a cDNA in-frame library screening approach, we identified a short peptide derived from placental growth factor-2 (PLGF-2). We demonstrated that fusing this short peptide to IFNα and IFNγ induced greater activity than the wild-type counterparts in inhibiting of tumor cell growth, invasion and colony formation (14,15). In this study, using a Ph.D.-12 peptide library, we selected a short peptide that specifically binds to the cell membrane of MCF-7 cells, and we synthesized a high-affinity IFNα-MCF-7 fusion molecule (IMBP). Using lentiviral DNA delivery system, we obtained a stable MCF-7 breast cancer cell line that secretes IMBP. Compared with wild-type IFNα, the IMBP fusion molecule demonstrated higher antitumor activity potency in the inhibition of cell growth, promotion of cell cycle arrest and induction of cell apoptosis. Recently, using the same approach, we also identified a short peptide that specifically binds to Jurkat T lymphocyte leukemia cells (JBP). We demonstrated that a JBP and IFNα fusion protein (IFNP) also had significantly better antitumor activity than wild-type IFNα (unpublished data). Through this series study, we evaluated the value of engineering an IFNα fusion molecule, which may serve as a novel antitumor agent in cancer treatment.

IFNs bind to receptors on the cell surface, activate specific signaling pathways and exert antitumor actions (23). Unfortunately, we still do not know the function of how this Ph.D.-12 peptide binds to the cell membrane. According to the result of 'Blastp' on the NCBI website, this 12-peptide is not homologous to any existing protein sequences. We speculate that it may bind to some unknown receptor(s) or biomacromolecule(s) on the surface of the cell membrane. Using a cell binding assay, we demonstrated that MBP promoted the adherence affinity of IFNα to its receptor (Fig. 2C), which is probably why we detected a lower level of free IMBP in the cell supernatant (Fig. 2A). We speculate that because more IMBP molecules adhere to the cell surface of MCF-7 cells than IFNα, the IMBP possesses a higher tumor cell killing ability than wild-type IFNα.

The STAT pathway plays a critical role in the anti-infection role of IFNs (24–27). When binding to its receptor on the cell surface, IFNα initiates the phosphorylation of TYK2 and JAK1 kinases, which is followed by the activation of STAT family transcription factors (28,29). Several IFN inducible genes can be co-activated in the STAT pathway, including IFIT1, IFIT3, MX1, IFITM1, OAS1 (2′–5′-oligoadenylate synthetase 1), PARP9 and PARP12 [poly(ADP-ribose) polymerase family] (30). It was reported that IFIT3 promotes IFNα adjuvant therapeutic effects by strengthening IFNα effector signaling in hepatocellular carcinoma patients (31); IFNγ inhibits the growth of human liver cancer cells through activating IFITM1, which enhances the transcriptional activity of p53 and stabilizes the p53 protein by inhibiting p53 phosphorylation on Thr55 (19).

The JAK-STAT signaling pathway also plays an important role in doxorubicin-based breast cancer chemotherapy (32). It was demonstrated that the combined use of doxorubicin and IFNs may have a significant synergistic effect. Thomas et al showed that doxorubicin potentiates STAT1 activation in response to IFNγ, and the combination of doxorubicin and IFNγ enhances apoptosis in breast cancer cells (33). Hannesdóttir et al reported that STAT1 is crucial to the susceptibility of breast cancer cells to chemotherapeutic drugs (e.g., doxorubicin) by contributing to the induction of a productive antitumor immune response, which is based on IFNγ-producing T cells (34). In this study, we found that a combination of low dose DOX antitumor drug (0.1 μg/ml) and endogenous secretory IFNα had an advantage over the single use of either DOX or IFNα in inhibiting breast cancer cell growth. Of note, we found that low dose DOX combined with synthetic IMBP fusion protein had a significantly better therapeutic effect than the DOX/IFNα combination. When we examined the molecular mechanism of this enhanced antitumor activity, we observed that the DOX/IMBP combination activated cell cycle arrest and cell apoptosis pathway genes more efficiently than the DOX/IFNα combination (Fig. 4), which was also correlated with the activation of STAT1 pathway target genes (Fig. 5).

In summary, by using a phage library screening, we have identified a short peptide that specifically binds to MCF-7 breast cancer cells. Fusion of this short peptide to IFNα significantly enhanced the antitumor activity. The combined use of DOX and IMBP fusion protein potentiated the effectiveness of chemotherapy. Since using lentivirus to deliver molecules into cells is hard to apply in clinical gene therapy, construction of an in vitro expression and purification system to produce purified IMBP recombinant protein is needed in our further study. We consider this type of purified IMBP molecule may have good clinical application prospect and the co-administration of DOX and IMBP provides new insight into breast cancer treatment.

Acknowledgments

The present study was supported by grants from the National Science Foundation of China (81302380, to D.-H.Y.) and Grant from the Health and Family Planning Commission in Jilin Province of China (2016Q035, to L.Z.).

References