Total RNA was extracted from the fresh leaves of V. fordii (collected from the test field at Hefei Institutes of Physical Science, Chinese Academy of Science, Hefei, China) using an E. Z. N. A. Plant RNA kit (OMEGA Biotek, Inc., Norcross, GA, USA). Subsequently, RNA samples with an optical density (OD) 260/280 wavelength ratio between 2.0 and 2.2 were used for primary cDNA synthesis with a GoScript Reverse Transcription kit (Promega Corporation, Madison, WI, USA). Specific primers were designed based on the V. fordii RNA-Seq database (accession no. GSE98631) to obtain V. fordii (vf)-RIP and its mature peptide (fordin), as follows: vf-RIP, forward, 5′-ATTAGAAACCATCTCCTCCCTCT-3′ and reverse, 5′-CTTAGTTTGAAGCCCTTCTACTT-3′; fordin, forward, 5′-AATGGATCCAGAGTTAGCCCTTTACTA-3′ and reverse, 5′-GGGAAGCTTCTACTTCTTTACATACAGC-3′. The underlined bases in the primers were designated as BamHI and HindIII restriction sites, respectively. The polymerase chain reaction (PCR) was performed in 50-µl reaction mixtures (Takara Biotechnology Co., Ltd., Dalian, China), each mixture included: 0.5 µl PrimeSTAR HS DNA Polymerase, 10 µl 5X PCR Buffer, 4 µl cDNA, 4 µl dNTPs, 2 µl primers, and 29.5 µl ddH₂O, according to the following procedure: 94°C for 2 min; 30 cycles at 98°C for 10 sec, 58°C for 90 sec, and 68°C for 45 sec; and final extension at 68°C for 10 min. The PCR products were analyzed by 1.2% agarose electrophoresis. The fragments of expected size were purified with an Agarose Gel Extraction kit (OMEGA Biotek, Inc.), and then cloned into the pEASY-T1 vector (Beijing TransGen Biotech Co., Ltd., Beijing, China) for sequencing. Multiple sequence alignment was performed using DNAMAN software 8.0 (LynnonBiosoft Bioinformatics solutions, San Ramon, CA, USA).

The coding sequence of fordin was cloned into the pET28b-SUMO vector, constructed by Professor Fan (University of Anhui Agriculture, Anhui, China) (27,28). The recombinant plasmid (pSUMO-Fordin) was transformed into Escherichia coli Rosetta host strain (DE3) competent cells (Novagen/Merck, Darmstadt, Germany). E. coli harboring the recombinant plasmid was incubated with shaking at 200 rpm at 37°C until the OD 600 of the culture reached 0.8. The expression of SUMO-Fordin was induced by the addition of 0.5 mM IPTG. Following further incubation at 16°C for 24 h, the cells were harvested and sonicated in binding buffer containing 50 mM NaH₂PO₄ (pH 8.0) and 300 mM NaCl. The cell-free supernatant was applied to a Ni²⁺ resin column (2.0×15 cm). The resin was washed with the binding and washing buffers, containing 50 mM NaH₂PO₄, (pH 8.0), 300 mM NaCl, and 20 mM imidazole. The fusion protein was eluted with elution buffer containing 50 mM NaH₂PO₄ (pH 8.0), 300 mM NaCl and 250 mM imidazole. To cleave the His-SUMO tag by protease ULP, the fusion protein was then processed with buffer through ultrafiltration columns (EMD Millipore, Billerica, MA, USA) with membrane cartridges having molecular weight cut-offs of 3,000 Da. The cleaved protein (fordin) was purified by Ni²⁺ resin again. Finally, fordin was pooled and measured using a BCA protein assay kit (Sangon Biotech Co., Ltd., Shanghai, China). The purity of fordin was analyzed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blot analysis using rabbit polyclonal antibodies specific for 6X His-tag (1:1000, cat. no. D110002; Sangon Biotech Co., Ltd.) was performed to confirm the presence of the fusion protein. The identification of fordin was assessed by liquid chromatography-mass spectrometry with a MALDI TOF/TOF analyzer (ABI; Thermo Fisher Scientific, Inc., Waltham, MA, USA).

The assay was performed according to the method described by Wu et al (29) with several revisions. Total RNA was extracted from Sapium sebiferum leaves (collected from the test field at Hefei Institutes of Physical Science) by the method mentioned above. Subsequently, 3 µg total RNA was treated with 0.4 µg of fordin. The mixture was incubated at 30°C for 30 min, and then treated with 2 M fresh aniline/acetate (pH 4.5). RNA was recovered by ethanol precipitation and examined by 2.0% agarose electrophoresis.

Human osteosarcoma (U-2 OS), hepatoblastoma (HepG2) (30), cervical carcinoma (HeLa), lung carcinoma (A549) and fetal lung fibroblast (MRC-5) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in DME/F-12 medium (GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; BioWest, Miami, FL, USA). The cells were incubated in a humidified atmosphere with 5% CO₂ at 37°C.

The cell viability was assessed using a Cell Counting Kit-8 (CCK-8) assay (Sangon Biotech, Co., Ltd.). The cell suspension (100 µl) was dispensed (7×10³ cells/well) into 96-well plates and exposed to various concentrations of fordin (0.2–10 µM) for 24, 48 and 72 h. CCK-8 solution was added (10 µl/well) and incubated for 30 min. The absorbance was measured at 450 nm using a microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA). Cell viability was calculated as the percentage of untreated controls. Inhibitory concentrations (ICs) of fordin were calculated using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). In a subsequent experiment, to avoid antiproliferative effects, relatively low concentrations of fordin (1 µM and IC₂₀) were used for investigating the scratch-healing and invasive behavior of U-2 OS and HepG2 cells. Low (IC₂₀), medium (IC₅₀), and high (IC₇₀) fordin concentrations were used to evaluate concentration-dependent effects on U-2 OS and HepG2 cells via nuclear staining, flow cytometry, and reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses.

The U-2 OS and HepG2 cells were seeded into 6-well plates. The confluent monolayers of cells were scratched using 200-µl sterile pipette tips. Following removal of the floating cells, the cells were cultured in medium without serum. The effect of fordin (1 µM and IC₂₀) on the scratch-healing response was determined under an inverted microscope (Olympus, Tokyo, Japan). A total of six randomly selected images were selected per sample at 0 and 24 h.

The effect of fordin on cell invasion ability was investigated using a Transwell assay. Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) diluted 1:8 in cold serum-free culture media was added to each 24-well Transwell chamber with an 8-µm pore polycarbonate membrane (Corning Incorporated, Corning, NY, USA) and incubated under standard conditions for at least 12 h to gel. The cells treated with fordin (1 µM and IC₂₀) for 24 and 48 h were seeded onto the Matrigel. Each lower chamber contained 600 µl of culture media containing 10% FBS. Following incubation for 24 h, transmembrane cells were stained by 0.1% crystal violet. Six fields were randomly selected under the inverted fluorescence microscope, and the number of transmembrane cells was counted.

The selected cells were seeded into 12-well plates containing sterilized coverslips. Following treatment with fordin (IC₂₀, IC₅₀, or IC₇₀) for 24 h, the cells were fixed with 4% paraformaldehyde, and stained with 4 µg/ml of Hoechst 33342 solution (Invitrogen; Thermo Fisher Scientific, Inc.). The coverslips with cells were inverted onto slides. Six random images were captured with the inverted fluorescent microscope.

Following treatment with fordin (IC₂₀, IC₅₀, or IC₇₀) for 24 h, the U-2 OS and HepG2 cells were harvested, and the extent of apoptosis was determined through flow cytometry using an Annexin V-FITC/Dead Cell Apoptosis kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cell apoptosis was analyzed in triplicate for each treatment using a FACSCalibur platform (BD Biosciences) and FlowJo software 7.6 (Tree Star, Inc., Ashland, OR, USA).

NF-κB/p65 siRNA was purchased from GenePharma (Shanghai, China). Non-silencing siRNA was used as the negative control. U-2 OS and HepG2 cells were transfected with p65 siRNA or control siRNA using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h according to the manufacturer's protocol. Following transfection, the cells were treated with or without fordin (IC₅₀) for 24 h. Subsequently, certain proteins involved in the NF-κB signaling pathway were analyzed by western blot analysis.

Total RNA was extracted using the E. Z. N. A RNA kit from cells treated with fordin (IC₂₀, IC₅₀, or IC₇₀) for 24 h. cDNA corresponding to 1 µg of total RNA was used to examine the mRNA levels of the selected genes by a Light Cycler 96 Real-Time PCR system (Roche Diagnostics GmbH, Mannheim, Germany). FastStart Essential DNA Green Master mix (Roche Diagnostics GmbH) was applied for the RT-qPCR analysis. Each reaction mixture included 10 µl 2x Master Mix, 1 µl cDNA, 0.8 µl primers, and 8.2 µl ddH₂O. The reactions were performed according to the following conditions: 94°C for 30 sec, 40 cycles at 94°C for 5 sec, the optimal temperature for primers of the selected genes (Table I) for 15 sec and 72°C for 10 sec. After 40 cycles, the dissociation curve was determined. All samples were performed as triplicate. The 2^−ΔΔCq method (31) was applied to calculate the relative mRNA expression of the selected genes using GAPDH to normalize the data.

Following the various treatments, the cells were maintained in lysis buffer for 10 min on ice for preparing the whole cell lysates. Nuclear and cytoplasmic extracts were prepared using NE-PER™ nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The protein concentrations were measured using a BCA Protein Assay kit. Equal quantities (50 µg) of protein were separated on a 12% SDS-PAGE gel and then transferred onto a PVDF membrane (EMD Millipore), which was then blocked in 5% skim milk for 1 h at room temperature. The membrane was then incubated overnight with primary antibodies at 4°C. The primary antibodies were as follows: Anti-Bcl-2-associated X protein (Bax; 1:2,000, cat. no. sc-70408) and anti-Bcl-2 (1:1,000, cat. no. sc-130308) from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The other primary antibodies were anti-Bcl-2-related protein A1 (Bfl-1; 1:200, cat. no. ab45413), anti-MMP-2 (1:500, cat. no. ab92536), anti-MMP-9 (1:500, cat. no. ab76003), anti-p65 [(phosphorylated S536; 1:1,000, cat. no. ab76302) and total (1:1,000, cat. no. ab76311)], anti-IKKα (1:1,000, cat. no. ab32518), anti-IKKβ (1:1,000, cat. no. ab124957), and anti-IκB [(phosphorylated S36; 1:1,000, cat. no. ab133462) and total (1:1,000, cat. no. ab32518)], all of which were purchased from Abcam (Cambridge, MA, USA). The membrane was incubated with the secondary antibody (goat anti-mouse IgG-HRP, 1:1,000, cat. no. sc-2005; goat anti-rabbit IgG-HRP, 1:1,000, cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature and visual-ized by chemiluminescence. β-actin and Histone H3 were used as internal controls for the whole cell lysates, cytosolic extracts and nuclear extracts, respectively.

The statistical analysis was performed using GraphPad Prism 6 software (GraphPad Software, Inc., La Jola CA, USA). All data are expressed as the mean ± standard deviation. Statistical analysis between groups was performed by one-way analysis of variance followed by the LSD post hoc test. P<0.05 was considered to indicate a statistically significant difference.

The coding sequence of the vf-RIP gene (GenBank accession no. MF521485) is 840 bp in length with translation initiation (ATG) and stop (TAG) codons. The open reading frame encodes a 279-amino acid polypeptide, of which the putative molecular mass is 30.8 kDa. An endoplasmic reticulum signal peptide (residues 1–21) was identified, followed by the mature peptide (fordin; residues 22–279) with a molecular mass of 28.3 kDa (Fig. 1). The results of sequence alignment showed that fordin showed similarity with other type I RIPs, including TCS (from Trichosanthes kirilowi), curcin (from Jatropha curcas), momorcharin (from Momordica charantia) and ricin A-chain (from Ricinus communis) with respect to the conserved region. The fordin amino acid residues, Tyr-109 (Y), Tyr-148 (Y), Glu-197 (E), Arg-200 (R), and Trp-229 (W), aligned perfectly with the consensus active site sequences of RIPs (Fig. 1) (32).

E. coli strains harboring the recombinant plasmid (pSUMO-Fordin) and non-recombinant plasmid (pSUMO) were incubated for expression analysis. The recombinant protein with an expected band of ~45 kDa was detected in the soluble fraction (Fig. 2A), which consisted of the SUMO protein with a molecular weight of 20 kDa fused to fordin. The His-SUMO tag allowed the fused protein to be purified by Ni-NTA affinity chromatography and be confirmed by western blot analysis using the rabbit polyclonal anti-His antibody (Fig. 2B). The His-SUMO tag was cleaved with protease ULP, and fordin was measured. The purity of the fused protein and fordin was identified via the presence of a single band at ~45 and 28 kDa on the gel, respectively (Fig. 2A). The yields of the fused protein and fordin were ~25 and 13 mg of protein/l of bacterial culture, respectively. The expressed protein was characterized by mass spectrometry (MS) analysis, and the theoretical molecular mass obtained by MS methods was 28.3 kDa. The sequence from MS analysis had 54% sequence identity with the putative polypeptide based on the vf-RIP gene, with 21 unique matching peptides (partially in Table II).

The N-glycosidase activity of fordin was examined against Sapium sebiferum rRNA. A specific endo-fragment was generated following fordin treatment due to the N-glycosidase activity of fordin (Fig. 2C). The negative control (no fordin treatment), did not yield the same fragment.

The selected tumor and normal cell lines were treated with increasing concentrations of Fordin (0.2–10 µM) for 24, 48 and 72 h. Following fordin exposure, effective cytotoxic activity was observed as a decline in survival rates in a dose- and time-dependent manner in the four cancer cell lines (Fig. 3A–D). The ICs of fordin were calculated based on the viability assay (Table III). Considering the IC₅₀ values, fordin was more toxic against the four tumor cell lines, particularly the U-2 OS and HepG2 cells, than the normal cell line (Fig. 3E). Specifically, a sharp decline in survival was observed in the U-2 OS and HepG2 cells at a fordin concentration >1 µM at the 24- and 48-h time intervals, as shown in Fig. 3A and B.

To confirm fordin induces apoptosis, the nuclear morphology of U-2 OS, Hep G2 and MRC-5 cells was determined by Hoechst 33342 staining using fluorescence microscopy. Exposure of the cells to fordin (IC₂₀, IC₅₀, or IC₇₀) for 24 h resulted in clear destructive changes (chromatin condensation and nuclear fragmentation) in the cancer cell nuclei. At the fordin IC₂₀, nuclear shrinkage and chromatin condensation were noted, whereas at IC₅₀ or IC₇₀, nuclear fragmentation was also observed in the cells (Fig. 4A). The apoptotic effects in response to fordin exposure were more profound in U-2 OS and HepG2 cells compared with MRC-5 cells (Fig. 4A and B).

To further verify fordin as an inducer of apoptosis, an Annexin V-based assay was performed. The cells treated with fordin (IC₂₀, IC₅₀, or IC₇₀) for 24 h were examined by FACS analysis. Significant increases, from 2.03 to 35.65% and 1.30 to 13.95%, occurred in Annexin V-FITC-positive cells with increasing concentrations of fordin in U-2 OS and HepG2 cells, respectively (Fig. 4C). However, this increase was lower (from 0.84 to 2.90%) in the MRC-5 cells.

Following fordin treatment for 24 h, apoptosis-related gene expression of the U-2 OS and HepG2 cells was examined by RT-qPCR and western blot analyses. The results showed the potential of fordin to influence the expression of Bcl-2 family genes (Fig. 5). Fordin exposure significantly increased the mRNA level of Bax (maximum, 2.6-fold), and inhibited the mRNA level of Bfl-1 (maximum, 46.0-fold) in U-2 OS cells. The mRNA expression levels of Bcl-2 and Bcl-xL in the treated cells were essentially unchanged compared with those in the untreated cells. Similar effects were observed in HepG2 cells, in which maximum induction was 2.7-fold for the Bax gene and maximum inhibition was 47.9-fold for the Bfl-1 gene. These results are consistent with the western blot data. The expression of Bax was increased in a concentration-dependent manner in the U-2 OS (Fig. 5C and D) and HepG2 cells (Fig. 5E and F), with maximum increases of 2.3- and 3.1-fold, respectively (P<0.001). The expression of Bfl-1 was decreased in the U-2 OS (maximum, 1.5-fold, P<0.05) and HepG2 (maximum, 7.6-fold, P<0.05) cells. The expression of Bcl-2 did not differ between the treated cells and untreated control cells.

The Transwell assay was used to evaluate the effects of fordin on the invasive activity of U-2 OS and HepG2 cells. The invasive behavior of the cells was examined following 24 and 48 h treatment with fordin (1 µM and IC₂₀). The two cell lines exhibited a reduction in the number of trans-membrane cells (Fig. 6). The number of U-2 OS cells was reduced by 23% (P<0.05) and 74% (P<0.001) following 24 h treatment with fordin (1 µM and IC₂₀), respectively. The reduction was more pronounced for the cells treated with fordin (1 µM and IC₂₀) for 48 h (74 and 92%, respectively; P<0.001). The number of HepG2 cells was decreased only by 16% (P=0.0548) and 35% (P<0.001) following 24 and 48 h treatment with fordin (1 µM), respectively. Following 24 and 48 h treatment with fordin (IC₂₀), the reduction in invasive cell number increased to 28% (P<0.001) and 73% (P<0.001), respectively.

A scratch assay was used to monitor the ability of U-2 OS and HepG2 cells to cover the vacant scuffed area introduced by scratching for 24 h. As shown in Fig. 7, the ability of cells treated with fordin to cover the scratched area was reduced. No apparent morphologic signs of treatment-related toxicity were shown at a low concentration of fordin (1 µM and IC₂₀). U-2 OS and the HepG2 control cells covered 1,427±201 and 629±36 µm distances between the scratched edges following a 24-h incubation period, respectively. Fordin (1 µM) exposure inhibited the migration potential by 20% (1,143±77 µm) and 51% (310±51 µm) in the U-2 OS and HepG2 cells, respectively. The effects were more marked at a higher concentration of fordin (IC₂₀), with a 49% (730±79 µm) and 77% (143±43 µm) decrease of the migration potential of U-2 OS and HepG2 cells, respectively.

RT-qPCR and western blot analysis were used to examine the effects of fordin on several invasion-relevant genes (Fig. 8). Following fordin exposure of the U-2 OS cells (Fig. 8A, C and D), the maximum inhibition of the mRNA and protein levels of the MMP-9 gene with migratory potential was 35- and 4.8-fold (P<0.001), respectively. A maximum induction of 3.5-fold for the mRNA level of tissue inhibitor of metalloproteinase (Timp)-3, an inhibitor of the MMP family, was also observed. In the HepG2 cells (Fig. 8B, E and F), the mRNA and protein levels of MMP-9 were decreased in a dose-dependent manner with a maximum reduction of 19- and 6.2-fold (P<0.001), respectively. The mRNA level of Timp-3 was not affected by fordin treatment. The mRNA and protein levels of MMP-2 gene did not differ significantly between control and treated groups in either cell line. In addition, in the two cell lines, fordin had inhibitory effects on the mRNA levels of cathepsin B (maximum, 1.0- and 2.0-fold), urokinase plasminogen activator (uPA; maximum, 5.1- and 11.7-fold), and uPA receptor (maximum, 3.8- and 1.6-fold) (Fig. 8A and B), which have significant roles in tumor invasion. Downregulation of the aforementioned metastasis-related genes may be important in the suppression of U-2 OS and HepG2 cell invasion and migration caused by fordin.

To investigate the effects of fordin on NF-κB activation, the protein levels of p65 (total and phosphorylated) were determined in U-2 OS and HepG2 cells. As shown in Fig. 9A and B, the expression of phosphorylated p65 was significantly decreased in the treated cells; however, the total p65 expression was not changed. To further verify the effects of fordin on the inhibition of p65 activation, lipopolysac-charide (10 µg/ml) was used to induce NF-κB activation. The protein levels of intra-nuclear p65 (phosphorylated and total) were markedly suppressed (maximum, 10.3- and 9.6-fold, respectively; P<0.001) in U-2 OS cells treated with LPS and fordin (IC₂₀, IC₅₀, or IC₇₀) compared with the cells treated with LPS alone (Fig. 9C). Similar findings were observed in the HepG2 cells, in which the maximum reductions in intra-nuclear p65 (phosphorylated and total) expression were 8.5- and 2.6-fold (P<0.001) (Fig. 9D), respectively. In the two cell lines, the protein levels of total p65 in the cytoplasm showed negligible change; however, the expression of phosphorylated p65 in the cytoplasm was downregulated in Hep G2 cells (maximum, 3.2-fold, P<0.05). These results demonstrated that fordin inhibited the nuclear accumulation and phosphorylation of NF-κB in cancer cells.

In addition, understanding whether NF-κB is involved in the regulation of the expression of Bfl-1 and MMP-9 mediated by fordin, LPS and p65 siRNA were used to regulate the activation of NF-κB in U-2 OS and HepG2 cells. As shown in Fig. 9C and D, upregulation of the intra-nuclear expression of p65, Bfl-1 and MMP-9 due to LPS was reduced by fordin treatment in the U-2 OS (maximum, 8.6-, 1.2- and 8.2-fold; P<0.05) and HepG2 (maximum, 2.2-, 2.4- and 4.0-fold, respectively; P<0.001) cells. In Fig. 9E and F, the knockdown of p65 reduced the protein levels of MMP-9 and Bfl-1 in U-2 OS (6.5- and 4.3-fold, respectively; P<0.001 vs. control siRNA transfected cells) and HepG2 cells (2.4- and 1.7-fold, respectively; P<0.05 vs. control siRNA transfected cells). The effects of fordin on the expression of MMP-9 and Bfl-1 were weaker in the p65 siRNA-transfected cells (U-2 OS: 1.7- and 0.5-fold; HepG2: 0.3- and 0.4-fold, respectively; P>0.05) than in control siRNA-transfected cells (U-2 OS: 4.5- and 1.0-fold; Hep G2: 1.9- and 1.1-fold, respectively; P<0.05). These findings revealed that NF-κB regulated the expression of Bfl-1 and MMP-9 in U-2 OS and HepG2 cells, and fordin downregulated the expression of Bfl-1 and MMP-9, at least in part, associated with the inactivation of NF-κB.

The finding that fordin inhibited the nuclear accumulation and phosphorylation of NF-κB in cancer cells was verified by a corollary study (Fig. 10A–F). Fordin treatment decreased the mRNA levels of IKKα (maximum, 2.2- and 5.0-fold) and IKKβ (maximum, 2.6- and 4.7-fold) in the U-2 OS and HepG2 cells, respectively. The protein levels of IKKα and IKKβ were decreased by a maximum of 4.5- and 4.3-fold (P<0.001) in U-2 OS cells, respectively. The maximum reduction in IKKα and IKKβ was 0.9- and 2.1-fold (P<0.001) in the HepG2 cells, respectively. The protein levels of phosphorylated IκB were also reduced in U-2 OS and HepG2 cells (maximum, 6.6- and 11.6-fold, respectively; P<0.001).