Structural and functional characterisation of a novel peptide from the Australian sea anemone Actinia tenebrosa.

Sea anemone venoms have long been recognised as a rich source of peptides with interesting pharmacological and structural properties. Our recent transcriptomic studies of the Australian sea anemone Actinia tenebrosa have identified a novel 13-residue peptide, U-AITx-Ate1. U-AITx-Ate1 contains a single disulfide bridge and bears no significant homology to previously reported amino acid sequences of peptides from sea anemones or other species. We have produced U-AITx-Ate1 using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The solution structure of U-AITx-Ate1 was determined based on two-dimensional nuclear magnetic resonance spectroscopic data. Diffusion-ordered NMR spectroscopy revealed that U-AITx-Ate1 was monomeric in solution. Perturbations in the 1D 1H NMR spectrum of U-AITx-Ate1 in the presence of dodecylphosphocholine micelles together with molecular dynamics simulations indicated an interaction of U-AITx-Ate1 with lipid membranes, although no binding was detected to 100% POPC and 80% POPC: 20% POPG lipid nanodiscs by isothermal titration calorimetry. Functional assays were performed to explore the biological activity profile of U-AITx-Ate1. U-AITx-Ate1 showed no activity in voltage-clamp electrophysiology assays and no change in behaviour and mortality rates in crustacea. Moderate cytotoxic activity was observed against two breast cancer cell lines.

Novel combination of sorafenib and biochanin-A synergistically enhances the anti-proliferative and pro-apoptotic effects on hepatocellular carcinoma cells.

Sorafenib (SOR) is the first-line treatment for hepatocellular carcinoma (HCC). However, its use is hindered by the recently expressed safety concerns. One approach for reducing SOR toxicity is to use lower doses in combination with other less toxic agents. Biochanin-A (Bio-A), a promising isoflavone, showed selective toxicity to liver cancer cells. We postulated that combining SOR and Bio-A could be synergistically toxic towards HCC cells. We further evaluated the underlying mechanism. Cytotoxicity assay was performed to determine the IC50 of Bio-A and SOR in HepG2, SNU-449 and Huh-7 cells. Then, combination index in HepG2 was evaluated using Calcusyn showing that the concurrent treatment with lower concentrations of SOR and Bio-A synergistically inhibited cell growth. Our combination induced significant arrest in pre-G and G0/G1 cell cycle phases and decrease in cyclin D1 protein level. Concomitantly, SOR/Bio-A reduced Bcl-2/Bax ratio. Furthermore, this co-treatment significantly increased caspase-3 &-9 apoptotic markers, while decreased anti-apoptotic and proliferative markers; survivin and Ki-67, respectively. Active caspase-3 in HepG2, SNU-449 and Huh-7 confirmed our synergism hypothesis. This study introduces a novel combination, where Bio-A synergistically enhanced the anti-proliferative and apoptotic effects of SOR in HCC cells, which could serve as a potential effective regimen for treatment.