Cytotoxicity of Southeast Asian snake venoms

Cytotoxicity of venoms from eleven medically important snakes found in Southeast Asia (Naja kaouthia, Naja siamensis, Naja sumatrana, Ophiophagus hannah, Bungarus candidus, Bungarus fasciatus, Enhydrina schistosa, Calloselasma rhodostoma, Trimeresurus purpureomaculatus and Tropidolaemus sumatranus) was determined, based on the MTS cytotoxicity assay, which determines the survival of viable cells in monolayer MDCK and Vero cell cultures upon exposure to the snake venoms. Snake venom toxicity was expressed as the venom dose that killed 50% of the cells (CTC50) under the assay conditions. Venoms of C. rhodostoma (2.6 µg/mL, 1.4 µg/mL) and O. hannah were the most cytotoxic (3.8 µg/mL, 1.7 µg/mL) whereas N. siamensis venom showed the least cytotoxicity (51.9 µg/mL, 45.7 µg/mL) against Vero and MDCK cells, respectively. All the viper venoms showed higher cytotoxic potency towards both Vero and MDCK cell lines, in comparison to krait and cobra venoms. E. schistosa did not cause cytotoxicity towards MDCK or Vero cells at the tested concentrations. The cytotoxicity correlates well with the known differences in the composition of venoms from cobras, kraits, vipers and sea snakes

A Preliminary Study in Search of Potential Peptide Candidates for a Combinational Therapy with Cancer Chemotherapy Drug

Cancer which caused by the growth and spreading of abnormal cells in an uncontrolled manner remains a major cause of death affecting millions of people. The current cancer chemotherapy treatment modalities have several disadvantages, mostly related to their undesirable side effects. In this study, we explore the potencies of selected cell penetrating antimicrobial peptides in combination with the widely used chemotherapy drug, Doxorubicin (DOX), to increase the specificity of anticancer chemotherapy. Screening the potential peptide candidates to be developed into chemotherapy drug combination led to identification of two most potent peptides, Tachyplesin 1 (TCH) and Latarcin 1 (LTC). Cell viability of normal liver cells was reduced to 50% by 20 µM of TCH or LTC, while liver cancer cell lines lost 50% of their viability at approximately 4 µM of these peptides. The combination of DOX with TCH peptide showed the higher levels of lactate dehydrogenase (LDH) leakage from cancer cells (80%) compared to normal cells (30%). The combinational treatment DOX-peptide showed significant (P < 0.01) increase in caspase 3/7 activity compared to DOX alone. Pre-treatment of the cells with TCH peptides prior to DOX treatment considerably increased the Caspase 3/7 activities in both cell types with significant increase (P < 0.05) in cancer cells compared to normal cells. Our study demonstrate that TCH peptide is a potential anticancer peptide that could be used in combinational therapy with cancer chemotherapy drugs. © 2017, Springer Science+Business Media, LLC, part of Springer Nature

Cellular uptake of the peptide-fusion protein (TACH-MAP30-LATA) and MAP30 by cancer and normal cells.

<p><b>A)</b> Fluorescence ELISA-like cell-based assay at 0 to 2.5 μM showed the peptide-fusion protein was internalized more efficiently than MAP30 into cancer cells (HepG2 and MCF-7) compared with normal cells (WRL68 and ARPE19) in a dose-dependent manner<b>. B)</b> Confocal laser microscopy analysis showed TACH-MAP30-LATA was preferentially uptaken by HepG2 and MCF-7, compared with normal cells (WRL68 and ARPE19), whereas the cellular uptake of MAP30 was less in all cell lines.</p

The anti-proliferative activity of the peptides.

<p>The individual components of the fusion protein (TACH, LATA and MAP30) showed higher inhibition against cancer cells compared with normal cells in an overall dose range of 0–200 μM based on the maximal toxic concentration of each peptide. The 50% cytotoxic concentration (CC₅₀) of the peptide-fusion protein (TACH- MAP30-LATA) towards HepG2 (0.35±0.1 μM) and MCF-7 (0.58±0.1 μM) was significantly lower than WRL68 (1.83±0.2 μM) and ARPE19 (2.5±0.1 μM). Two Way-ANOVA, P<0.001.</p

Treatment of liver normal cells (WRL68) and liver cancer cells (HepG2) with combinations of doxorubicin and increasing concentrations of the peptide-fusion protein.

<p>The dose of doxorubicin (2.5 μM) that showed 100% of normal cell viability and approximately 80% of cancer cells viability was used with increasing concentrations (0–1.4 μM) of TACH-MAP30-LATA. The results showed significant reduction in cancer cell viability compared with normal cells (Two Way ANOVA, P<0.001).</p

Purification of inclusion bodies by solubilization and refolding in alkaline-based buffer containing redox agents.

<p><b>A)</b> Semi-solubilization of inclusion bodies in dH₂O (pH 12) after 10 min of incubation. L1, the isolated inclusion bodies; L2, purified protein (final product); L3, removing of host cell proteins; L4, remaining insoluble aggregates after final solubilization (each line duplicated). <b>B)</b> Refolding of soluble protein in buffer containing oxidized and reduced glutathione to reform the disulphide bridges of TACH. The refolded protein showed different levels under reducing and non-reducing conditions of the SDS-PAGE, indicating the formation of disulphide bridges. <b>C)</b> SDS-PAGE analysis of the fusion protein. L1, a single band at the expected size for the monomer. L2, double bands of the fusion protein. The upper faint band at the dimer size and the lower thick band at the monomer size (arrows).</p

Design and production of the recombinant peptide-fusion protein.

<p><b>A)</b> The short peptides TACH and LATA were fused to MAP30 as a central protein using a six amino-acid linker. The TACH-MAP30-LATA expression cassette including 6XHis tag at the N-terminal of fusion protein was cloned into the appropriate <i>E</i>. <i>coli</i> expression vector and transformed into <i>E</i>. <i>coli</i> BL21. <b>B)</b> SDS-PAGE was done to determine the molecular weight of the purified peptide-fusion protein (~37 kDa). <b>C)</b> Immunoblotting using anti-6XHis tag antibody.</p

Production of the recombinant peptide-fusion protein (TACH- MAP30-LATA) and MAP30 by a recombinant <i>E</i>. <i>coli</i>.

<p><b>A)</b> Total dried biomass of <i>E</i>. <i>coli</i> after the chemical induction at all of the time points of the fermentation process. <b>B)</b> Relative total recombinant protein to the biomass of TACH-MAP30-LATA and MAP30. <b>C)</b> Recombinant protein yield from inclusion bodies. <b>D)</b> Western blot analysis of the purified recombinant peptide-fusion protein at each time point of the fermentation process (Two Way-ANOVA, p>0.05).</p

Effect of Ottoman Viper (Montivipera xanthina (Gray, 1849)) Venom on Various Cancer Cells and on Microorganisms

WOS: 000334174300009PubMed ID: 23381026Cytotoxic and antimicrobial effects of Montivipera xanthina venom against LNCaP, MCF-7, HT-29, Saos-2, Hep3B, Vero cells and antimicrobial activity against selected bacterial and fungal species: Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, E. coli O157H7, Enterococcus faecalis 29212, Enterococcus faecium DSM 13590, Staphylococcus epidermidis ATCC 12228, S. typhimirium CCM 5445, Proteus vulgaris ATCC 6957 and Candida albicans ATCC 10239 were studied for evaluating the potential medical benefit of this snake venom. Cytotoxicity of venom was determined using MTT assay. Snake venom cytotoxicity was expressed as the venom dose that killed 50 % of the cells (IC50). The antimicrobial activity of venom was studied by minimal inhibitory concentration (MIC) and disc diffusion assay. MIC was determined using broth dilution method. The estimated IC50 values of venom varied from 3.8 to 12.7 or from 1.9 to 7.2 mu g/ml after treatment with crude venom for 24 or 48 h for LNCaP, MCF-7, HT-29 and Saos-2 cells. There was no observable cytotoxic effect on Hep3B and Vero cells. Venom exhibited the most potent activity against C. albicans (MIC, 7.8 mu g/ml and minimal fungicidal concentration, 62.5 mu g/ml) and S. aureus (MIC, 31.25 mu g/ml). This study is the first report showing the potential of M. xanthina venom as an alternative therapeutic approach due to its cytotoxic and antimicrobial effects