Total RNA of 5×10⁶ Plasmodium yoelii 265BY sporozoite (gifted from the Academy of Military Medical Sciences of China, Beijing, China) was extracted using TRIzol™ Reagent (Thermo Fisher Scientific, MA, USA) and reverse transcribed to cDNA using PrimeScript™ RT reagent kit with gDNA Eraser (Takara Bio, Inc., Otsu, Japan). The full-length CSP coding sequence was amplified using polymerase chain reaction (PCR) from the cDNA with the primers PCSP3 (5′-CCCaagcttGGGAAGAAGTGTACCATTTTAG-3; the Hind III restriction site is underlined) and PCSP1284 (5′-CGggatccCGTTAATTAAAGAATACTAATAC-3′, the BamH I restriction site is underlined). The amplified fragment, which was 1281 bp in length, was cloned into the pFLAG-CMV8 plasmid, resulting in the recombinant plasmid pFLAG-CMV8-CSP. The PCR conditions consisted of an initial predenature at 94°C for 2 min, denaturation at 98°C for 10 sec, annealing at 50°C for 30 sec followed by amplification for 40 cycles of 45 sec at 68°C. A second plasmid, pFLAGCMV8-CSP NLS, was constructed through ligation of the annealing nuclear location signal (NLS) oligonucleotide to the Hind III and BamH I restriction sites of pFLAG-CMV8. To construct the recombinant plasmid pFLAG-CMV8-CSPΔNLS, the CSP lacking the NLS was obtained by overlapping PCR using a KOD FX kit (Toyobo Life Science, Osaka, Japan) and then cloned into the Hind III and BamH I restriction sites of pFLAG-CMV8. Two fragments of CSP without NLS were amplified from the full CSP coding sequence (1284 bp). The primer sequences used are as follows: first fragment (1–1119 bp), forward, 3′-AAGCTTGGGAAGAAGTGTACCATTTTAGTTG-5′, and reverse, 3′-TTCTGGTTGCTTGTTACCAGAACCACAGGTTAC-5′, and second fragment (1147–1284 bp), forward, 3′-AACAAGCAACCAGAAAATTTGACCTTAGAGG-5′, and reverse, 3′-GGATCCTTAATTAAAGAATACTAATACTAATAATATTAC-5′.

The PCR conditions for the two fragments consisted of pre-denaturation at 94°C for 3 min; 35 cycles of denaturation at 94°C for 30 sec, annealing at 50° for 30 sec and extension at 68°C for 90 sec, followed by a final extension at 68°C for 5 min. The two fragments were purified using a Gel Extraction Kit (Omega Bio-Tek, Inc., Norcross, GA, USA). The splicing CSPΔNLS (1257 bp) fragment was amplified using the following primers: forward, 5′-CTGTGGTTCTGGTAACAAGCAACCAG-3′ and reverse, 5′-CTGGTTGCTTGTTACCAGAACCACAG-3′. The overlapping PCR conditions consisted of an initial pre-denaturation at 94°C for 3 min; 35 cycles of denaturation at 94°C for 30 sec, annealing at 42° for 30 sec and extension at 68°C for 90 sec, followed by a final extension at 68°C for 5 min. The correct orientation of all the recombinant plasmids was confirmed using DNA sequencing.

A549 cell line was purchased from the Japanese Cancer Research Bank (JCRB, Tokyo, Japan). A549 cells were maintained in 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) supplemented Dulbecco's modified Eagle's medium (Nacalai Tesque, Kyoto, Japan), 100 U/ml penicillin and 100 µg/ml streptomycin. The culture conditions were at 37°C with a 5% CO₂ atmosphere and 85% humidity were achieved by adjusting the incubator (ASTEC, Fukuoka, Japan).

The B cell epitope QGP GAQ GPG AQG PGA P of the CSP was synthesized and purified via high-performance liquid chromatography (HPLC) by using Shimadzu Inertsil ODS-SP column (4.6×250 mm ×5 µm; Shimadzu Corporation) on a Shimadzu Corporation Prominence HPLC system (Kyoto, Japan). The column was operated at 30°C, and proteins were detected at 214 nm. Samples were analyzed at a volume of 60 µl. The composition of mobile phase (two solvents) was as follows: buffer A: 0.1% TFA (A501480, Sangon Biotech, China) in 100% water and buffer B: 0.1% TFA in 100% acetonitrile (A506811, Sangon Biotech, China). The flow rate was 1.0 ml/min, and the gradient elution condition was as follows: the running time was 27.01 min, the initial mobile phase was consist of 92% buffer A and 8% buffer B, and the final mobile phase was consist of 5% buffer A and 95% buffer B. The consist of mobile phase was changed by the system with time in the gradient elution.

Then the peptide was conjugated with keyhole limpet hemocyanin (KLH). The peptide was produced by Beijing Biosynthesis Biotechnology Co., Ltd., (Beijing, China; cat. no., 080825). The resulting conjugated peptide was emulsified with Freund's adjuvant (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and immunized in a specific-pathogen-free grade rabbit at 0, 2 and 4 weeks. After the final immunization, rabbit serum was collected, and the titer of antibody against the epitope was detected using ELISA (cat. no., 44-2404-21, Nunc MaxiSorp™ flat-bottom, Nalge Nunc International, Penfield, NY, USA). Ethical approval for animal experiments was obtained from the Animal Ethical and Welfare Committee of The Third Military Medical University (Chongqing, China).

Following transfection with 0.8 µg pFLAG-CMV8-CSP or a control plasmid using Lipofectamine 2000TM for 24 h, A549 cells, which were grown on a coverslip in 24-well plate, were incubated with rabbit anti-CSP serum (1:10) for 1 h. After two washes with phosphate-buffered saline (PBS), the cells were labeled with goat anti-rabbit FITC-immunoglobulin G (H+L) for 20 min and observed under fluorescence microscopy.

Alamar blue is a sensitive oxidation reduction indicator that fluoresces and undergoes a color change upon reduction in living cells. A549 cells were transfected with or without pFLAG-CMV8-CSP (0.2, 0.4, 0.8, 1.2 or 1.6 µg), and after 24 h 120 µl SunBioTMAm-Blue was added to each well and incubated for 2 h at 37°C. The fluorescence was read at 570 and 600 nm. Cell proliferation was expressed as optical density (OD) ratio of OD570/OD600.

A549 cells were transfected using Lipofectamine™ 2000 in 24-well plates. Each well received 200 ng pBIIx-luc report vector containing two κB sites upstream of the c-fos promoter (gift from Dr Sankar Ghosh, Yale University, Connecticut, USA) and 2 ng TK-RL (Promega Corporation, Madison, WI, USA), together with 600 ng Pflag-CMV8-CSP, pFLAG-CMV8-CSP NLS or pFLAG-CMV8-CSPΔNSL. After 24 h, the cells were stimulated in the presence or absence of 100 ng/ml human recombinant TNF-α (hTNF-α; PeproTech, New Jersey, USA) and/or lipopolysaccharide (Invitrogen; Thermo Fisher Scientific, Inc.) for 6 h. Next, the cells were lysed, and the activity of firefly and Renilla luciferase was determined using the Dual Luciferase Assay kit (Promega Corporation). The data were expressed as the ratio of firefly luciferase to Renilla luciferase.

After A549 cells were transfected with or without 0.8 µg pFLAG-CMV8-CSP for 24 h, the cells were stimulated with 100 ng/ml hTNF-α for 6 h. Next, the cells were collected and incubated with Annexin V-FITC (Beyotime Institute of Biotechnology, Haimen, China) and propidium iodide (PI) for 10 min on ice and analyzed using flow cytometry. The data were analyzed by FlowJo software 10.4 (FlowJo LLC, Ashland, OR, USA).

Following transfection with or without 4 µg pFLAG-CMV8-CSP NLS for 24 h, A549 cells were re-stimulated with 100 ng/ml hTNF-α for 30 min. The cells (2×10⁶) were washed with cold PBS and suspended in 0.4 ml hypotonic lysis buffer containing protease inhibitors for 30 min. The cells were lysed with 12.5 µl 10% Nonidet P-40 (Beyotime Institute of Biotechnology). The homogenate was centrifuged, and the supernatant containing the cytoplasmic extracts was stored at −80°C. The nuclear pellet was resuspended in 25 µl ice-cold nuclear extraction buffer. After 30 min of intermittent mixing, the extract was centrifuged at 15,000 × g for 30 min, and the supernatants containing nuclear extracts were collected. Protein concentration in cytoplasmic extracts and nuclear extracts were then determined with the BCA protein assay kit (Beyotime Institute of Biotechnology). Equal amounts 20 µg of cytoplasmic and nuclear extracts were separated using SDS-PAGE (10% gel), transferred to a PVDF membrane, then washed by TBST buffer (TBS buffer with 0.1% tween-20) and blocked by 5% non-fat-dried milk in TBST buffer at room temperature for 1 h. After blocking, the membrane was probed with primary antibodies against NF-κB p105/p50 (1:2,000; cat. no., 12540; Cell Signaling Technology, Inc., Danvers, MA, USA) and GAPDH (1:4,000; cat. no., 5174; Cell Signaling Technology, Inc.) for 16 h at 4°C, and secondary goat anti-rabbit HRP-conjugated antibody (1:20,000; cat. no., sc-2004; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature, then the bands were detected by using ECL reagent (Pierce; Thermo Fisher Scientific, Inc.).

All data were analyzed using Student's t-test or one-way analysis of variance, and P<0.05 was considered statistically significant. SPSS 19.0 software (IBM Corp., Armonk, NY) was used for the statistical analysis.

For CSP to be able to promote the development of sporozoite in hepatocytes, it has to be transferred from the parasitophorous vacuole into the cytoplasm (18). To mimic this process, the full length of the CSP coding sequence was amplified from the Plasmodium yoelii 265BY sporozoite, and cloned into the pFLAG-CMV8 plasmid. The resulting recombinant plasmid, pFLAG-CMV8-CSP, was then transfected into the human lung cancer cell line A549, and its intracellular distribution was determined using indirect immunofluorescence assay using anti-CSP serum. CSP was expressed primarily in the cytoplasm. CSP was not present in A549 cells transfected with pFLAG-CMV8 (Fig. 1A), but was present in the membrane of A549 cells following transfection of recombinant plasmid pFLAG-CMV8-CSP (Fig. 1B).

CSP in the cytoplasm is important for the development of the malaria sporozoite (18). However, the effects of CSP on cancer cell proliferation are unclear. Thus, the Alamar Blue assay was used to investigate the effect of the CSP on proliferation of A549 cells. As shown in Fig. 2, transfection with 0.4 µg pFLAGCMV8-CSP was able to significantly suppress the proliferation of A549, compared with proliferation following transfection with the control pFLAG-CMV8. When the concentration of the transfected pFLAG-CMV8-CSP was increased from 0.4 to 1.2 µg, cell viability and proliferation decreased in a dose-dependent manner. Following transfection with 1.2 µg pFLAG-CMV8-CSP, cell proliferation was <30% (OD value, 0.105 vs. 0.375). Thus, the data demonstrated the inhibitory role of CSP on the proliferation of A549 cells.

To investigate whether CSP is also able to induce the apoptosis of A549 cells, an Annexin V-FITC apoptosis assay was performed following the transfection of cells with 0.8 µg pFLAG-CMV8-CSP and then stimulated with hTNF-α. As indicated in Fig. 3, the apoptotic rate of A549 cells transfected with the control plasmid pFLAG-CMV8 was 3.5%, comparable with the rate of cells that were transfected with pFLAG-CMV8 or pFLAG-CMV8-CSP, or stimulated with hTNF-α alone. However, the apoptotic rate of hTNF-α-stimulated A549 cells transfected with pFLAG-CMV8-CSP was significantly higher compared with the rate of cells that were transfected with pFLAG-CMV8 (17.4 vs. 3.5%, P<0.01). Therefore, the data indicated that CSP was able to induce the apoptosis of A549 cells.

As NF-κB is important for survival and proliferation of tumor cells (8), the effect of the CSP on NF-κB activation in A549 cells was investigated. A NF-κB reporter gene assay indicated that transfection with pFLAG-CMV8-CSP was able to suppress the activation of NF-κB in hTNF-α-induced A549 cells (P=0.008; Fig. 4A). CSP is a critical protein for the invasion of sporozoites into hepatocytes and includes important motifs, including region I, region II+ and region III (Fig. 3B) (19). Previously, a NLS motif with a sequence of VRVRKNVN was reported in CSP (18) (Fig. 4B). To determine the region responsible for NF-κB inactivation by CSP, the recombinant plasmids pFLAGCMV8-CSP NLS (containing only the NLS motif) and pFLAG-CMV8-CSPΔNLS (CSP lacking the NLS motif) were constructed (Fig. 3B), and their inhibitory role on the activation of NF-κB in hTNF-α-induced A549 cells was observed. As shown in Fig. 4C, transfection of pFLAGCMV8-CSPΔNLS was not able to inhibit NF-κB activation in hTNF-α-stimulated A549 cells, compared with that transfected with pFLAG-CMV8 or pFLAG-CMV8-CSP. However, transfection with pFLAG-CMV8-CSP or pFLAG-CMV8-CSP NLS was able to reduce NF-κB activity of A549 cell by >3.5 fold, when compared with pFLAG-CMV8 plasmid. Therefore, the data supported the essential role of NLS for the inactivation of NF-κB by CSP.

Proteins with NLS are imported into the cell nucleus through the nuclear pore complex (20). It was hypothesized that CSP may inhibit the nuclear translocation of NF-κB through its NLS motif. The cytoplasmic and nuclear extracts were probed with NF-κB p105/p50 antibody following separation on SDS-PAGE. As indicated in Fig. 5, although the cytoplasmic NF-κB p50 was at a comparable level in A549 cells transfected with either pFLAG-CMV8 or pFLAG-CMV-CSPNLS, the level of NF-κB p50 in the nuclear extract of A549 cells transfected with pFLAG-CMV-CSP NLS was markedly lower compared with cells transfected with pFLAG-CMV8. Therefore, the data suggested that NLS did not affect the phosphorylation of IκB, and the subsequent release of NF-κB from the inhibitory complex, but it may inhibit the nuclear translocation of the activated NF-κB.