Carbohydrate-Binding Properties and Antimicrobial and Anticancer Potential of a New Lectin from the Phloem Sap of Cucurbita pepo

Abstract

A Cucurbita phloem exudate lectin (CPL) from summer squash (Cucurbita pepo) fruits was isolated and its sugar-binding properties and biological activities were studied. The lectin was purified by affinity chromatography and the hemagglutination assay method was used to determine its pH, heat stability, metal-dependency and sugar specificity. Antimicrobial and anticancer activities were also studied by disc diffusion assays and in vivo and in vitro methods. The molecular weight of CPL was 30 ± 1 KDa and it was stable at different pH (5.0 to 9.0) and temperatures (30 to 60 °C). CPL recovered its hemagglutination activity in the presence of Ca²⁺. 4-nitrophenyl-α-D-glucopyranoside, lactose, rhamnose and N-acetyl-D-glucosamine strongly inhibited the activity. With an LC₅₀ value of 265 µg/mL, CPL was moderately toxic and exhibited bacteriostatic, bactericidal and antibiofilm activities against different pathogenic bacteria. It also exhibited marked antifungal activity against Aspergillus niger and agglutinated A. flavus spores. In vivo antiproliferative activity against Ehrlich ascites carcinoma (EAC) cells in Swiss albino mice was observed when CPL exerted 36.44% and 66.66% growth inhibition at doses of 3.0 mg/kg/day and 6.0 mg/kg/day, respectively. A 12-day treatment by CPL could reverse their RBC and WBC counts as well as restore the hemoglobin percentage to normal levels. The MTT assay of CPL performed against human breast (MCF-7) and lung (A-549) cancer cell lines showed 29.53% and 18.30% of inhibitory activity at concentrations of 128 and 256 µg/mL, respectively.

Keywords: Cucurbita lectins; antibiofilm; antifungal; antitumor; bacteriostatic; chitin binding.

Figure 1

Purification, hemagglutination activity and toxicity of CPL. (A) Purification of CPL by affinity chromatography. The minimum concentration of CPL required to agglutinate mice erythrocytes was 31.25 μg/mL. The green line indicates the lectin fraction with hemagglutinating activity. The red frame shows the hemagglutination titer of CPL. (B) SDS-PAGE of purified CPL. Lane 1: purified CPL. Lane 2 (M): standard markers of phosphorylase b (94 kDa), serum albumin (66 kDa), ovalbumin (42 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (21 kDa) and lysozyme (14 kDa). Lane 3 and 4: undialyzed CPL. (C) Toxicity of CPL against brine shrimp nauplii. Results are presented as means ± SD (n = 3). Statistical significance was determined with p < 0.01, with the data showing significance denoted as * p < 0.05 and ** p < 0.01.

Figure 2

Effect of temperature and pH on CPL hemagglutination activity. (A) CPL thermostability. (B) Influence of pH on CPL hemagglutination activity. For both experiments, the results are presented as the mean ± standard deviation (n = 3), with error bars indicating the standard error from the triplicate trials.

Figure 3

Bactericidal and antibiofilm properties of CPL. Antibacterial effectiveness of CPL against (A) Escherichia coli (ATCC 27853) and (B) Shigella dysenteriae (ATCC 238135). In this context, T and C indicate the sample disc with a high concentration of CPL (100 µg/disc) and the control disc (without CPL), respectively. A standard antibiotic disc (ciprofloxacin 5 µg) is used in both tests. (C) Antibiofilm activity of CPL against E. coli. Error bars: data represent the mean ± SD (n = 3) from triplicate experiments, with standard errors (SEs) shown. Statistical significance is noted as p < 0.01, with data represented as * p < 0.05, ** p < 0.01 and *** p < 0.001 to indicate the levels of significance.

Figure 4

(A) Bacteriostatic effects of CPL on Bacillus cereus (ATCC 14579), Shigella sonnei (ATCC 29930), E. coli (ATCC 27853), Shigella dysenteriae (ATCC 238135), Shigella boydii (ATCC 231903) and Staphylococcus aureus (ATCC 25923) at concentrations ranging from 6.25 to 200 µg/mL. (A) Results after 24 h of treatment. (B) Results after 48 h of treatment. The graph uses blue, orange, black, yellow, dark blue and green to represent different CPL concentrations (200, 100, 50, 25, 12.5 and 6.25 µg/mL, respectively). For each bacterium, there were one negative control and six positive controls. Results are presented as means ± SD (n = 3). Statistical significance was determined at p < 0.01, with data indicated as * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 5

Antifungal activity of CPL. (A) Lectin-untreated spores of Aspergillus flavus. (B) Spores of Aspergillus flavus agglutinated by 100 µg/mL of CPL. (C) Disc diffusion assay demonstrating the fungistatic effect of CPL on Aspergillus niger. C: negative control disc (soaked with TBS only), T: test disc (200 µg/disc of CPL), S: positive control disc with 1% clotrimazole. (D) Magnified view of the test disc (T). Scale bar: 50 µm.

Figure 6

Anticancer activity (in vivo) of CPL against EAC cells. (A) Lectin-untreated (control) and CPL-treated Swiss albino mice. (B) In vivo inhibitory effects of CPL on EAC cell growth at low (3 mg/kg/day) and high (6 mg/kg/day) doses compared to control mice. Data are presented as means ± SD (n = 3). Number and appearance of (C) lectin-untreated and (D) CPL-treated EAC cells. (E) EAC cells from control mice. (F) Agglutination of lectin-treated EAC cells. Scale bar: 50 µm. Here, p < 0.01 was considered to be statistically significant and the data are represented as * p < 0.05 and ** p < 0.01 to indicate the levels of significance.

Figure 7

Morphology of CPL-treated EAC cells. (A) EAC cells from control mice exhibit a regular round shape. Treated EAC cells display irregular size and shape, with apoptotic morphological changes evident at (B) low dose (3 mg/kg/day) and (C) high dose (6 mg/kg/day) of CPL. Scale bar: 50 µm.

Figure 8

Impact of CPL on blood parameters in EAC-bearing (control) and normal mice. (A) Hemoglobin percentage in CPL-treated mice compared to EAC-bearing control and normal mice. (B) RBC count increased in CPL-treated mice with rising lectin concentrations (3 to 6 mg/kg/day) relative to control and normal mice. (C) WBC count significantly decreased in lectin-treated mice compared to control mice, approaching levels observed in normal mice. Results are shown as means ± SD (n = 3). Here, p < 0.01 was considered to be statistically significant and the data are represented as * p < 0.05, ** p < 0.01 and *** p < 0.001.

Figure 9

In vitro antiproliferative activity of CPL against human cancer cell lines. (A) CPL exhibited significant antiproliferative effects on human breast cancer cells (MCF-7) within the concentration range of 32–256 µg/mL. (B) CPL demonstrated milder antiproliferative effects on the human lung cancer cell line (A-549). Results are presented as means ± SD (n = 3). Here, p < 0.01 was considered to be statistically significant and the data are represented as * p < 0.05, ** p < 0.01 and *** p < 0.001.