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Phospholipase A 2 From Krait Bungarus fasciatus Venom Induces Human Cancer Cell Death in Vitro

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Phospholipase A 2 From Krait Bungarus fasciatus Venom Induces Human Cancer Cell Death in Vitro

Thien V Tran et al. PeerJ.

Abstract

Background: Snake venoms are the complex mixtures of different compounds manifesting a wide array of biological activities. The venoms of kraits (genus Bungarus, family Elapidae) induce mainly neurological symptoms; however, these venoms show a cytotoxicity against cancer cells as well. This study was conducted to identify in Bungarus fasciatus venom an active compound(s) exerting cytotoxic effects toward MCF7 human breast cancer cells and A549 human lung cancer cells.

Methods: The crude venom of B. fasciatus was separated by gel-filtration on Superdex HR 75 column and reversed phase HPLC on C18 column. The fractions obtained were screened for cytotoxic effect against MCF7, A549, and HK2 cell lines using colorimetric assay with the tetrazolium dye MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The primary structure of active protein was established by ultra high resolution LC-MS/MS. The molecular mechanism of the isolated protein action on MCF7 cells was elucidated by flow cytometry.

Results: MTT cell viability assays of cancer cells incubated with fractions isolated from B. fasciatus venom revealed a protein with molecular mass of about 13 kDa possessing significant cytotoxicity. This protein manifested the dose and time dependent cytotoxicity for MCF7 and A549 cell lines while showed no toxic effect on human normal kidney HK2 cells. In MCF7, flow cytometry analysis revealed a decrease in the proportion of Ki-67 positive cells. As Ki-67 protein is a cellular marker for proliferation, its decline indicates the reduction in the proliferation of MCF7 cells treated with the protein. Flow cytometry analysis of MCF7 cells stained with propidium iodide and Annexin V conjugated with allophycocyanin showed that a probable mechanism of cell death is apoptosis. Mass spectrometric studies showed that the cytotoxic protein was phospholipase A2. The amino acid sequence of this enzyme earlier was deduced from cloned cDNA, and in this work it was isolated from the venom as a protein for the first time. It is also the first krait phospholipase A2 manifesting the cytotoxicity for cancer cells.

Keywords: Breast cancer; Cytotoxicity; Human cancer cells; Krait; Lung cancer; Mass spectrometry; Phospholipase A2; Venom.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1. Fractionation of B. fasciatus venom.
(A) Gel filtration of crude venom on the Superdex® 75 10/300 GL column (1 × 30 cm) equilibrated with the 0.1 M ammonium acetate buffer (pH 6.2). Flow rate 0.5 ml/min. The eluate was monitored by spectrophotometry (OD = 226 nm). Horizontal bars indicate the collected fractions. (B) Reversed phase chromatography of Fraction 3 (From A) on the Jupiter C18 column (10 × 250 mm) in a gradient of 25–40% acetonitrile in 75 min in the presence of 0.1% trifluoroacetic acid, at a flow rate of 2.0 ml/min. The eluate was monitored by spectrophotometry (OD = 275 nm). Horizontal bars indicate the collected fractions.
Figure 2
Figure 2. Cytotoxicity of fractions obtained from B. fasciatus venom by gel-filtration and reversed phase chromatography against human MCF7 and A549 cell lines.
Cell viability was examined using the colorimetric MTT assay. The percentage of viable cells was determined from a comparison with untreated control. All results are presented as the mean ± SEM (standard error of the mean). n = 3. The significance of differences between experimental and control groups was analyzed by t-test: Two-Sample Assuming Equal Variances using the Microsoft Excel 2016 MSO program. The complete statistical data are reported in Supplemental Materials 1 and 2. Here, р < 0.05, p < 0.01 and p < 0.001 are indicated by *, ** and ***, respectively. (A–F) Gel-filtration fractions. BF1–BF5 correspond to fractions 1–5 from Fig. 1A. BF—B. fasciatus venom. (G–L) Fractions obtained after reversed phase chromatography (Fig. 1B). BF3.1–BF3.4 correspond to fractions 3.1–3.4 from Fig. 1B. BF3 fraction 3 from Fig. 1A.
Figure 3
Figure 3. MALDI mass spectroscopy analysis of B. fasciatus venom fractions.
MALDI-TOF mass spectrometry measurements were performed using Ultraflex TOF/TOF mass spectrometer. The mass spectrometry data were processed using Bruker Daltonics Flex Analysis 2.4 software. BF3—fraction 3 obtained after gel-filtration of crude venom on Superdex® 75 10/300 GL column (Fig. 1A). BF3.1, BF3.2 and BF3.3—fractions 3.1, 3.2 and 3.3, respectively obtained by reversed phase chromatography of gel-filtration fraction 3 on Jupiter C18 column (Fig. 1B).
Figure 4
Figure 4. Cytotoxicity test of fractions obtained from B. fasciatus venom against normal human kidney HK2 cells.
Cell viability was examined using the colorimetric MTT assay. The percentage of viable cells was determined from a comparison with untreated control. All results are presented as the mean ± SEM (standard error of the mean). (A–C) Gel-filtration fractions. BF1–BF5 correspond to fractions 1–5 from Fig. 1A. BF—B. fasciatus venom. (D–F) Fractions obtained after reversed phase chromatography (Fig. 1B). BF3.1–BF3.4 correspond to fractions 3.1–3.4 from Fig. 1B. BF3 fraction 3 from Fig. 1A. The significance of differences between experimental and control groups was analyzed by t-test: Two-Sample Assuming Equal Variances. No differences were found.
Figure 5
Figure 5. Cytotoxicity of cisplatin to human A549 (A), MCF7 (B) and HK2 (C) cell lines.
Cell viability was examined using the colorimetric MTT assay. The percentage of viable cells was determined from a comparison with untreated control. All results are presented as the mean ± SEM (standard error of the mean). The significance of differences between experimental and control groups was analyzed by t-test: Two-Sample Assuming Equal Variances. Here p < 0.01 and p < 0.001 are indicated by ** and ***, respectively.
Figure 6
Figure 6. Morphological changes of the A549 (A–B) and MCF7 (D–F) cells after treatment with fractions BF3 (B and E) or BF3.3 (C and F) for 72 h detected by phase contrast microscopy.
Changes in the cell morphology were observed using phase contrast microscopy with Zeiss Axio Vert 25C. Here are the representative images of cells untreated (A and D) or treated with fractions BF3 (B and E) or BF3.3 (C and F) at 100 μg/ml. All experiments were performed in triplicate and gave similar results.
Figure 7
Figure 7. The fragment of high-resolution mass spectrum of the protein from the fraction BF3.3.
(A) High-resolution mass spectrum at Z = 10. The horizontal bar indicates the isotopomers corresponding to the main component. (B) Enlarged fragment of the spectrum corresponding the main izotopomers (Z = 10).
Figure 8
Figure 8. Flow cytometry analysis of Ki-67 expression in MCF7 cells.
Cells were treated with of PLA2 for 24 h then stained with Ki-67 antibody and analyzed by flow cytometry. Histograms show differences in the expression level of Ki-67 in phospholipase A2 (PLA2) treated and control cells. (A) Gray dotted histogram—isotype control; black histogram—control cells; gray histogram—cells treated with 7.63 μM of phospholipase A2. The numbers in the histograms represent the mean fluorescence intensities in a population of Ki-67 positive cells. (B) Changes in the level of expression of Ki-67 in control cells and cells treated with different doses of phospholipase A2. A dose-dependent decrease in the level of cell fluorescence with increasing concentration of phospholipase A2 is shown.
Figure 9
Figure 9. Flow cytometry analysis of MCF7 cells treated with various doses of phospholipase A2 for 24 h.
(A) Untreated cells. (B), (C), (D) Cells treated with 7.63, 0.76 and 0.076 μM of phospholipase A2, respectively. Representative figures show population of viable (Q4, Annexin V/PI), early apoptotic (Q3, Annexin V+/PI), late apoptotic (Q2, Annexin V+/PI+) and necrotic (Q1, Annexin V/PI+) cells. Exposure to PLA2 induces apoptosis in the MCF7 breast carcinoma cell line.

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Grant support

The work was supported by the Vietnam Academy of Science and Technology (research project QTRU01.03/18-19) and Russian Foundation for Basic Research (Project No: 18-54-54006). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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