Functional Characterization and Anti-Tumor Effect of a Novel Group II Secreted Phospholipase A2 from Snake Venom of Saudi Cerastes cerates gasperetti

Abstract

Secreted phospholipases A₂ are snake-venom proteins with many biological activities, notably anti-tumor activity. Phospholipases from the same snake type but different geographical locations have shown similar biochemical and biological activities with minor differences in protein sequences. Thus, the discovery of a new phospholipase A₂ with unique characteristics identified in a previously studied venom could suggest the origins of these differences. Here, a new Group II secreted phospholipase A₂ (Cc-PLA₂-II) from the snake venom of Saudi Cerastes cerastes gasperetti was isolated and characterized. The purified enzyme had a molecular weight of 13.945 kDa and showed high specific activity on emulsified phosphatidylcholine of 1560 U/mg at pH 9.5 and 50 °C with strict calcium dependence. Interestingly, stability in extreme pH and high temperatures was observed after enzyme incubation at several pH levels and temperatures. Moreover, a significant dose-dependent cytotoxic anti-tumor effect against six human cancer cell lines was observed with concentrations of Cc-PLA₂ ranging from 2.5 to 8 µM. No cytotoxic effect on normal human umbilical-vein endothelial cells was noted. These results suggest that Cc-PLA₂-II potentially has angiogenic activity of besides cytotoxicity as part of its anti-tumor mechanism. This study justifies the inclusion of this enzyme in many applications for anticancer drug development.

Keywords: anti-tumor effect; cytotoxicity; human cancer cell lines; secreted phospholipase A2; snake venom; stability.

Figure 1

(A) Chromatography of RP-HPLC using a C18 column and (B) SDS-PAGE profile (15%) of purified Cc-PLA₂-II. Lane 1: molecular mass markers, lanes 2 and 3: purified Cc-PLA₂-II, land 4: Wa-PLA₂-II. Blue line indicates linear acetonitrile gradient. (C) MALDI-TOF spectrum of Cc-PLA₂-II.

Figure 2

(A) pH profile and stability of purified Cc-PLA₂-II. Phospholipase activity was measured at various pH levels ranging from 6.0 to 12.0 at optimal temperature (55 °C). The pH stability was determined by incubating purified Cc-PLA₂-II in different buffers and at different pH values for 30 min at room temperature and residual enzyme activity was determined under standard assay conditions. (B) Effect of temperature on activity and stability of purified Cc-PLA₂-II. Activity was measured and stability was quantified after enzyme was incubated at different temperatures (20 °C to 90 °C) by measuring the residual activity under standard conditions. Results represent means of three independent experiments and are expressed as mean ± SD (n = 3).

Figure 3

(A) Effect of calcium concentration on purified Cc-PLA₂-II activity. Enzyme activity was measured at various concentrations of Ca²⁺ (0 to 10 mM) at pH 9 and 55 °C in the presence of 4 mM NaTDC. The absence of phospholipase activity in the presence of 10 mM EGTA/EDTA and the absence of Ca²⁺ is indicated by stars. (B) Effect of several metal ions on activity of Cc-PLA₂-II. Influence of 10 mM metal ions on activity in the absence or presence of 1 mM Ca²⁺ was investigated in the presence of 4 mM NaTDC using PC emulsion as a substrate at 55 °C and pH 9. Control represents 100% of PLA₂ activity at 8 mM Ca²⁺ under the same conditions. Results represent means of three independent experiments and are expressed as mean ± SD (n = 3).

Figure 4

Indirect hemolytic activity of Cc-PLA₂-II, Nm-PLA₂, and Wa-PLA₂ against human erythrocytes. Purified PLA₂ in different concentrations (0 to 2.5 nM) were incubated at 37 °C for 10 min with PC, erythrocytes, and PBS (1:1:8 v/v) to release Hb. Absorbance at 540 nm allowed the determination of free Hb released. Results represent means of three independent experiments and are expressed as mean ± SD (n = 3). Hb; hemoglobin.

Figure 5

Cytotoxic effect of Cc-PLA₂-II in normal and cancer cell lines. (A) MDA-MB231: human breast adenocarcinoma; (B) MCF-7: human breast-cancer; (C) HCT-116: human colorectal carcinoma; (D) RD: human rhabdomyosarcoma; (E) HT-29: adenocarcinoma; (F) NCI-292: mucoepidermoid carcinoma; (G) HUVECs: human umbilical-vein endothelial cells. All cells were treated with Cc-PLA₂-II concentrations ranging from 0.25 to 8 µM. Cytotoxicity assay was conducted using MTT reagent. Results represent means of three independent experiments. Viability was calculated as mean ± SD (n = 3).