Nomad Jellyfish Rhopilema nomadica Venom Induces Apoptotic Cell Death and Cell Cycle Arrest in Human Hepatocellular Carcinoma HepG2 Cells

Jellyfish venom is a rich source of bioactive proteins and peptides with various biological activities including antioxidant, antimicrobial and antitumor effects. However, the anti-proliferative activity of the crude extract of Rhopilema nomadica jellyfish venom has not been examined yet. The present study aimed at the investigation of the in vitro effect of R. nomadica venom on liver cancer cells (HepG2), breast cancer cells (MDA-MB231), human normal fibroblast (HFB4), and human normal lung cells (WI-38) proliferation by using MTT assay. The apoptotic cell death in HepG2 cells was investigated using Annexin V-FITC/PI double staining-based flow cytometry analysis, western blot analysis, and DNA fragmentation assays. R. nomadica venom displayed significant dose-dependent cytotoxicity on HepG2 cells after 48 h of treatment with IC50 value of 50 μg/mL and higher toxicity (3:5-fold change) against MDA-MB231, HFB4, and WI-38 cells. R. nomadica venom showed a prominent increase of apoptosis as revealed by cell cycle arrest at G2/M phase, upregulation of p53, BAX, and caspase-3 proteins, and the down-regulation of anti-apoptotic Bcl-2 protein and DNA fragmentation. These findings suggest that R. nomadica venom induces apoptosis in hepatocellular carcinoma cells. To the best of the authors’ knowledge, this is the first scientific evidence demonstrating the induction of apoptosis and cell cycle arrest of R. nomadica jellyfish venom.

Proteomic Changes during the Dermal Toxicity Induced by Nemopilema nomurai Jellyfish Venom in HaCaT Human Keratinocyte

Jellyfish venom is well known for its local skin toxicities and various lethal accidents. The main symptoms of local jellyfish envenomation include skin lesions, burning, prickling, stinging pain, red, brown, or purplish tracks on the skin, itching, and swelling, leading to dermonecrosis and scar formation. However, the molecular mechanism behind the action of jellyfish venom on human skin cells is rarely understood. In the present study, we have treated the human HaCaT keratinocyte with Nemopilema nomurai jellyfish venom (NnV) to study detailed mechanisms of actions behind the skin symptoms after jellyfish envenomation. Using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF/MS), cellular changes at proteome level were examined. The treatment of NnV resulted in the decrease of HaCaT cell viability in a concentration-dependent manner. Using NnV (at IC50), the proteome level alterations were determined at 12 h and 24 h after the venom treatment. Briefly, 70 protein spots with significant quantitative changes were picked from the gels for MALDI-TOF/MS. In total, 44 differentially abundant proteins were successfully identified, among which 19 proteins were increased, whereas 25 proteins were decreased in the abundance levels comparing with their respective control spots. DAPs involved in cell survival and development (e.g., Plasminogen, Vinculin, EMILIN-1, Basonuclin2, Focal adhesion kinase 1, FAM83B, Peroxisome proliferator-activated receptor-gamma co-activator 1-alpha) decreased their expression, whereas stress or immune response-related proteins (e.g., Toll-like receptor 4, Aminopeptidase N, MKL/Myocardin-like protein 1, hypoxia up-regulated protein 1, Heat shock protein 105 kDa, Ephrin type-A receptor 1, with some protease (or peptidase) enzymes) were up-regulated. In conclusion, the present findings may exhibit some possible key players during skin damage and suggest therapeutic strategies for preventing jellyfish envenomation.

Cloning and Expression of N-CFTX-1 Antigen from Chironex fleckeri in Escherichia coli and Determination of Immunogenicity in Mice

Background: Most jellyfish species are poisonous. Human victims of jellyfish sting each year are 120 million. Chironex fleckeri is a venomous box jellyfish that inflicts painful and potentially fatal stings to humans. The CfTX1 is one of the antigenic proteins of venom that is suggested to stimulate the immune system for treatment and vaccine. This study aimed to clone and express the CfTX-1 antigen in E. coli and then to determine the synthesis of related antibody in the mice. Methods: The study was performed in the Persian Gulf and Oman Sea Ecology Research Center, Bandar Abbas, Iran in autumn 2016. The synthetic CfTX-1 gene in PUC57 plasmid was purchased from Nedaye Fan Company. The 723 bp fragment of N-CfTX-1 was amplified by PCR, PUC57 plasmid containing CfTX-1 with BamHI SalI restriction enzyme sites were subcloned in pET28a [+] expression vector and transformed into E. coli BL21 (DE3). The CfTX-1 gene expression was induced by IPTG. Then antibody produced from the mice serum were isolated and confirmed by ELISA. After protein purification, resulted antigen was injected to mice in 4 repeats and then evaluated the rate of antibody in mice serum. Mice were challenged by the Carybdea alata. Results: The 726 bp of N-CfTX-1 were cloned in a vector of expression pET28a [+] and confirmed by PCR, sequencing and enzymatic analysis. Moreover, the recombinant protein was confirmed by SDS-PAGE and Western blotting. Then the antibody was isolated from mice serum and confirmed by ELISA test. The results showed that immunized mice tolerated 50x LD501 of jellyfish venom. Conclusion: The CfTX-1 recombinant protein was able to protect the BALB/c mice against jellyfish venom. The produced protein can be used as a candidate for vaccine against jellyfish venom.