Antiviral and antitumor activities of the lectin extracted from Aspidistra elatior

2.1 Plant material, cell lines, virus strains, chemicals and reagents

The rhizomes of A. elatior Blume were collected on the campus of Sichuan University (Chengdu, China) in August. The Vero cell line (African green monkey kidney) and human tumor cell lines Pro-01 (prostate), Lu-04 (lung), Bre-04 (breast), HepG2 (liver), Hep3B (p53-null; liver), and HeLa (cervix) were purchased from Di’ao Group (Chengdu, China).

Vesicular Stomatitis Virus, Coxsacke Virus B4, Respiratory Syncytial Virus, Sindbis Virus, and Reovirus-1 were provided by the Medical Sciences Center of West China, Sichuan University, China. Meanwhile, (S)-9-(2,3-Dihydroxypropyl)adenine [(S)-DHPA] and ribavirin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Carboxymethyl Sepharose^TM and Sephacryl^TM S-100 were purchased from Pharmacia (Uppsala, Sweden).

2.2 Purification of agglutinin from rhizomes of A. elatior

The procedure of Xu et al. [18] was followed. In brief, the 80% ammonium sulfate saturated crude protein extract from A. elatior Blume rhizomes, after dialysis against 20 mM Tris-HCl (pH 8.0), was applied to a diethylaminoethyl-Sepharose column equilibrated with the same buffer. Then, the proteins were eluted with a linear 0–0.5 M NaCl gradient. The fractions showing hemagglutinating activity were pooled, concentrated to approximately 4 mg/mL and then loaded on a Sephacryl S-100 column (2 cm×100 cm) equilibrated with 10 mM phosphate-buffered saline (PBS; pH 7.2).

2.3 Homogeneity of AEL and determination of molecular mass

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in 15% (w/v) acrylamide gels. Protein bands were visualized by Coomassie Brilliant Blue R-250 staining. The molecular mass of the native lectin was determined to be 56 kDa by gel filtration chromatography on the same Sephacryl S-100 column that had been used for purification (see above). Two adjacent bands of 13.5 kDa and 14.5 kDa were seen in SDS-PAGE, representing the two subunits of AEL [18].

2.4 Anti-viral assay

Vero cells were grown in RPMI medium supplemented with 10% fetal calf serum (FCS), 100 μg/mL penicillin (Chongqing Yaoyou Pharmaceutical Co., China), and 100 μg/mL streptomycin (Shanghai 4th Pharmaceutical Co.). Vero cells were seeded onto a 96-well plate with a concentration of 5×10⁴ cells per mL and a volume of 20 μL per well. After incubation at 37 °C with 5% CO₂ for 1 h, cells were then washed three times with phosphate buffered saline (PBS). Different concentrations of virus samples were then added to the cultures well in 1–10⁶ dilutions. AEL samples and other drugs were serially diluted with PBS, after which aliquots of each dilution were adsorbed on a Vero cell monolayer. At the end of the adsorption, the virus inoculum was removed and cells were replenished with fresh medium. After incubation at 37 °C with 5% CO₂ for 72 h, cells were observed and examined by a light microscopy. Virus replication in infected cells was assessed by the 50% tissue culture infection dose (TCID₅₀) [19].

2.5 In vitro anti-proliferative potential of AEL against human tumor cell lines

The inhibitory potential of AEL against various human tumor cell lines (Pro-01, Lu-04, Bre-04, HepG2, Hep3B, and HeLa) was trested using the method of Kaur et al. [20]. Cells were seeded at 10⁴ cells/well in 100 μL of RPMI 1640 medium containing 10% FCS in a 96-well tissue culture plate. Next, cells were suspended as single cells in the medium and incubated for 24 h in a CO₂ incubator. Subsequently, 100 μL of lectin solution (50 μg/mL), prepared in RPMI 1640 medium, was added to the cells, and the cultures were incubated for 48 h. After the incubation period, adherent cells were fixed in situ by adding 50 μL of 50% (v/v) trichloroacetic acid (final concentration 10%) and incubated for 1 h at 4 °C. Next, the supernatant was discarded and the plates were washed five times with deionized water before allowing them to dry. About 100 μL of sulforhodamine B (0.4% w/v in acetic acid) were added to each well, and the cultures were incubated for 30 min at room temperature. The unbound sulforhodamine was removed by washing with 1% acetic acid and the plates were air-dried. The dye bound to basic amino acids of the cell membrane was solubilized with Tris buffer (10 mM, pH 10.5), and the absorption was measured at 540 nm using an enzyme-linked immunosorbent assay reader. This was done to determine the relative viability in the treated and untreated cells.

2.6 Cell cycle measurement

Tumor cells were treated with or without 10 μg/mL AEL at 37 °C for 24 h and then harvested. FACScan flow cytometry was performed as previously described [21]. The percentages of cells at different cell cycle phases, or those that were undergoing apoptosis, were evaluated using Calibur FACScan flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).

2.7 Western blot analysis

After treatments, tumor cells were collected and the cell pellets were resuspended in lysis buffer and lysed at 4 °C for 1 h. The lysis buffer consisted of 50 mM Hepes, pH 7.4, 1% Triton X-100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM acetic acid, 1 mM phenylmethanesulfonyl fluoride (PMSF), 10 mg/L aprotinin, and 10 mg/L leupeptin (Sigma-Aldrich, St. Louis, MO, USA). After centrifugation at 12,000 g for 15 min, the supernatant protein content was determined using the Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of total protein were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes, after which the membranes were soaked in blocking buffer (5% skim milk). Antibodies against p21, p53, and actin B were purchased from Santa Cruz Biotech (Santa Cruz, CA, USA). Proteins were visualized using horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG and 3,3-diaminobenzidine tetrahydrochloride (DAB) as the HRP substrate [22].

2.8 Statistical analysis

All results presented here were confirmed in at least three independent experiments. Data were expressed as mean±SD. Statistical comparisons were made by Student’s t-test, and the least-significant difference (LSD) test was used to estimate significant differences between the mean values of the treatments. A value of p<0.05 was considered statistically significant.

3.1 Anti-virus activity of AEL

On the one hand, treatments with 20 μg/mL AEL caused microscopically detectable alterations of the normal cell morphology, thus indicating cytotoxicity. The minimum inhibitory concentrations (to reduce virus-induced cytopathogenicity by 50%) of AEL against Vesicular Stomatitis Virus, Coxsackie Virus B4, and Respiratory Syncytial Virus were all the same at about 4 μg/mL. On the other hand, AEL was rather ineffective against Sindbis Virus and Reovirus-1 (Table 1). The minimum inhibitory concentrations for these two viruses were higher than 100 μg/mL (the minimum cytotoxic concentration; therefore, higher concentrations of AEL were not used).

Minimum inhibitory concentrations of two anti-viral drugs, (S)-DHPA [23] and ribavirin [24], were higher than those of AEL. The concentrations varied for the different viruses, but the variation patterns differed from that of AEL (Table 1). (S)-DHPA and ribavirin are ribonucleoside analogs that directly inhibit viral RNA polymerase activity [24], whereas AEL may inhibit virus replication through different mechanisms (see Discussion for details).

3.2 Observation of cellular morphology

Marked apoptotic morphological changes, such as membrane blebbing, cell volume reduction and rounding, were obvious in AEL-treated Bre-04, Lu-04, HepG2, and Pro-01 cells (Figure 1). However, AEL did not cause apoptotic morphology in HeLa or Hep3B cells (Figure 1). These results indicate that AEL selectively induced apoptosis in Bre-04, Lu-04, HepG2 and Pro-01 cells, but not in HeLa or p53-deficient Hep3B cells, respectively.

Figure 1:

Effects of AEL on the morphology of human tumor cell lines.

The indicated human tumor cells were treated with or without 50 μg/mL AEL for 24 h, and their morphologies were examined under a phase contrast microscopy. We used size bar in one of the panels.

3.3 In vitro anti-proliferative potential of AEL on human tumor cell lines

The anti-proliferative effect of the lectin on human tumor cells was determined over a range of 10–50 μg/mL. At 50 μg/mL, AEL had the highest anti-proliferative effect against Bre-04 (76%), followed by Lu-04, HepG2 and Pro-01, in which 72%, 68% and 61% growth inhibitions were observed, respectively, at the 5th day (Figure 2). At a concentration of 25 μg/mL, growth inhibition rates were 58%, 53%, 50%, and 20% for Bre-04, Lu-04, HepG2 and Pro-01, respectively. At 10 μg/mL, the growth inhibitions were 42%, 40%, 28%, and 12% in Bre-04, Lu-04, HepG2 and Pro-01 cells, respectively. At 1 μg/mL, AEL was almost ineffective against all tumor cell lines. The lectin was found to be inactive against the HeLa or p53-deficient Hep3B cell lines at all concentrations and for the entire duration studies (Figure 2). This finding is consistent with the earlier reported variation in the anti-proliferative potential of a variety of lectins with tumor cell lines as a function of their doses [19, 21, 22].

Figure 2:

Anti-proliferative effect of 50 μg/mL AEL on human tumor cell lines as a function of time.

Bars indicate SD.

3.4 AEL arrested tumor cell cycles

As demonstrated in Table 2, AEL markedly increased the proportion of the sub-G₁ phase in Bre-04, Lu-04, HepG2 and Pro-01 cells, indicating that AEL-induced apoptosis occurred in these tumor cells. Moreover, as evident from Table 2, AEL also triggered G₂/M phase cell-cycle arrest. However, AEL neither enhanced the sub-G₁ proportion nor triggered G₂/M phase cell-cycle arrest in HeLa or p53- deficient Hep3B cells (Table 2).

3.5 Analyses of cell cycle related proteins

Due to the AEL-induced arrest of the sub-G₁ and G2/M phase, respectively, the levels of cell cycle-related proteins were investigated. Western blot data demonstrated that treatment of Bre-04, Lu-04, HepG2, and Pro-01 cells with AEL resulted in upregulation of p53 (Figure 3). Considering that p53 is an important regulator of p21 [22], p21 levels were also assessed. As seen in Figure 2, p21 was increased in parallel to p53. No p53 and only a trace of p21 could be detected in the p53- deficient Hep3B cell line. Likewise, almost no changes in the levels of p53 and p21 were observed in HeLa or Hep3B cells, indicating their insensitivity to the lectin (Figure 3).

Figure 3:

p53 and p21 protein levels of human tumor cells treated with or without 50 μg/mL AEL. Western blots of actin B (ACTB) were used as the loading control.