Biological Activities of Cyclic and Acyclic B-Type Laxaphycins in SH-SY5Y Human Neuroblastoma Cells

Abstract

Laxaphycins are a family of non-ribosomal lipopeptides that have been isolated from several cyanobacteria. Some of these compounds have presented cytotoxic activities, but their mechanism of action is poorly understood. In this work, the already described laxaphycins B and B3, and acyclolaxaphycins B and B3 were isolated from the marine cyanobacteria Anabaena torulosa. Moreover, two new acyclic compounds, [des-(Ala4-Hle5)] acyclolaxaphycins B and B3, were purified from the herviborous gastropod Stylocheilus striatus, with this being the first description of biotransformed laxaphycins. The structure of these new compounds was elucidated, together with the absolute configuration of acyclolaxaphycins B and B3. The bioactivities of the six peptides were determined in SH-SY5Y human neuroblastoma cells. Laxaphycins B and B3 were cytotoxic (IC50: 1.8 and 0.8 µM, respectively) through the induction of apoptosis. In comparison, acyclic laxaphycins did not show cytotoxicity but affected mitochondrial functioning, so their effect on autophagy-related protein expression was analyzed, finding that acyclic peptides affected this process by increasing AMPK phosphorylation and inhibiting mTOR. This work confirms the pro-apoptotic properties of cyclic laxaphycins B and is the first report indicating the effects on autophagy of their acyclic analogs. Moreover, gastropod-derived compounds presented ring opening and amino-acids deletion, a biotransformation that had not been previously described.

Combined Effect of Caspase-Dependent and Caspase-Independent Apoptosis in the Anticancer Activity of Gold Complexes with Phosphine and Benzimidazole Derivatives

“…SH-SY5Y cells were seeded into 96-well plates at a density of 5 × 10 4 cells per well and allowed to grow for 24 h. Cells were treated with compounds 1 and 2 at different concentrations (0.01, 0.1, 0.5, 1, 2.5, 5, 7.5, and 10 μM) for 6 and 24 h. The cytotoxic effect of compounds was evaluated with MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay [ 54 , 55 , 56 ]. SH-SY5Y cells were rinsed with saline solution and 200 μL of 500 μg/mL MTT (Merck, Darmstadt, Germany) dissolved in saline buffer were added to each well.…”

“…The cell death type induced by compounds was determined with an Annexin V-FITC Apoptosis detection kit (Immunostep, Salamanca, Spain) following the manufacturer’s instructions [ 56 ]. Cells were seeded in 12-well plates at 1 × 10 6 cells per well and treated with compounds at IC 50 concentrations for 6 and 24 h. Then, cells were washed, resuspended in PBS (Phophate-Buffered Saline), and 5 μL of Annexin V-FITC and Propidium Iodide (PI) were added to each tube.…”

“…To further confirm if apoptotic cell death was occurring, SH-SY5Y cells were preincubated with the pan-caspase inhibitor Z-VAD-FMK (Merck) for 24 h. Then, the assay was carried out as described above. Staurosporine (STS) (Merck) was used as a positive control in all the experiments [ 56 ].…”

“…Analysis of caspase-3 activity in SH-SY5Y cells after exposure to gold complexes was carried out using the EnzChek Caspase-3 Assay Kit (Thermo Fisher Scientific), following the manufacturer’s instructions [ 56 ]. Cells were seeded in 12-well plates at 1 × 10 6 cells per well and treated with compounds at IC50 concentrations for 6 and 24 h. Then, cells were lysed with 50 μL lysis buffer (10 mM TRIS, pH 7.5, 0.1 M NaCl, 0.01% CHAPS (3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate), 1 mM EDTA and 0.01% TRITON™ X-100), resuspended and centrifuged.…”

Blue-Print Autophagy in 2020: A Critical Review

“…Alvariño et al have reported the isolation of four acyclic peptides— acyclolaxaphycin B and acyclolaxaphycin B3 (originally found in the marine cyanobacteria Anabaena torulosa [ 125 ]), as well as [des-(Ala 4 -Hle 5 )]acyclolaxaphycin B and [des-(Ala 4 -Hle 5 )]acyclolaxaphycin B3 —from herbivorous gastropod Stylocheilus striatus [ 126 ]. The authors examined their activity in human neuroblastoma SH-SY5Y cells.…”