cytochrome-c-t and 1-2-dioleoylphosphatidylserine

High toxicity, morbidity and secondary malignancy render chemotherapy of neuroblastoma inefficient, prompting the search for novel compounds. Nanovesicles offer great promise in imaging and treatment of cancer. SapC-DOPS, a stable nanovesicle formed from the lysosomal protein saposin C and dioleoylphosphatidylserine possess strong affinity for abundantly exposed surface phosphatidylserine on cancer cells. Here, we show that SapC-DOPS effectively targets and suppresses neuroblastoma growth and elucidate the molecular mechanism of SapC-DOPS action in neuroblastoma in vitro.. In vivo targeting of neuroblastoma was assessed in xenograft mice injected intravenously with fluorescently-labeled SapC-DOPS. Xenografted tumors were also used to demonstrate its therapeutic efficacy. Apoptosis induction in vivo was evaluated in tumor sections using the TUNEL assay. The mechanisms underlying the induction of apoptosis by SapC-DOPS were addressed through measurements of cell viability, mitochondrial membrane potential (ΔΨM), flow cytometric DNA fragmentation assays and by immunoblot analysis of second mitochondria-derived activator of caspases (Smac), Bax, Cytochrome c (Cyto c) and Caspase-3 in the cytosol or in mitochondrial fractions of cultured neuroblastoma cells.. SapC-DOPS showed specific targeting and prevented the growth of human neuroblastoma xenografts in mice. In neuroblastoma cells in vitro, apoptosis occurred via a series of steps that included: (1) loss of ΔΨM and increased mitochondrial superoxide formation; (2) cytosolic release of Smac, Cyto c, AIF; and (3) mitochondrial translocation and polymerization of Bax. ShRNA-mediated Smac knockdown and V5 peptide-mediated Bax inhibition decreased cytosolic Smac and Cyto c release along with caspase activation and abrogated apoptosis, indicating that Smac and Bax are critical mediators of SapC-DOPS action. Similarly, pretreatment with the mitochondria-stabilizing agent bongkrekic acid decreased apoptosis indicating that loss of ΔΨM is critical for SapC-DOPS activity. Apoptosis induction was not critically dependent on reactive oxygen species (ROS) production and Cyclophilin D, since pretreatment with N-acetyl cysteine and cyclosporine A, respectively, did not prevent Smac or Cyto c release.. Taken together, our results indicate that SapC-DOPS acts through a mitochondria-mediated pathway accompanied by an early release of Smac and Bax. Specific tumor-targeting capacity and anticancer efficacy of SapC-DOPS supports its potential as a dual imaging and therapeutic agent in neuroblastoma therapy.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-2-Associated X Protein; Caspase 3; Cell Line, Tumor; Cell Survival; Cyclophilins; Cyclosporine; Cytochromes c; Humans; Intracellular Signaling Peptides and Proteins; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitochondria; Mitochondrial Proteins; Nanoparticles; Neuroblastoma; Peptidyl-Prolyl Isomerase F; Phosphatidylserines; Reactive Oxygen Species; Saposins

Activation of peroxidase catalytic function of cytochrome c (cyt c) by anionic lipids is associated with destabilization of its tertiary structure. We studied effects of several anionic phospholipids on the protein structure by monitoring (1) Trp59 fluorescence, (2) Fe-S(Met80) absorbance at 695 nm, and (3) EPR of heme nitrosylation. Peroxidase activity was probed using several substrates and protein-derived radicals. Peroxidase activation of cyt c did not require complete protein unfolding or breakage of the Fe-S(Met80) bond. The activation energy of cyt c peroxidase changed in parallel with stability energies of structural regions of the protein probed spectroscopically. Cardiolipin (CL) and phosphatidic acid (PA) were most effective in inducing cyt c peroxidase activity. Phosphatidylserine (PS) and phosphatidylinositol bisphosphate (PIP2) displayed a significant but much weaker capacity to destabilize the protein and induce peroxidase activity. Phosphatidylinositol trisphosphate (PIP3) appeared to be a stronger inducer of cyt c structural changes than PIP2, indicating a role for the negatively charged extra phosphate group. Comparison of cyt c-deficient HeLa cells and mouse embryonic cells with those expressing a full complement of cyt c demonstrated the involvement of cyt c peroxidase activity in selective catalysis of peroxidation of CL, PS, and PI, which corresponded to the potency of these lipids in inducing cyt c's structural destabilization.

Topics: Animals; Apoptosis; Cardiolipins; Cytochromes c; Electron Spin Resonance Spectroscopy; Enzyme Activation; Etoposide; Fluorescence; Heme; Humans; Mice; Peroxidase; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Phosphates; Phosphatidylserines; Phospholipids; Protein Structure, Tertiary; Tryptophan

The interaction of phenethyl alcohol with model membranes and its effect on translocation of the chemically prepared mitochondrial precursor protein apocytochrome c across a lipid bilayer was studied. Phenethyl alcohol efficiently penetrates into monolayers and causes acyl chain disordering judged from deuterium nuclear magnetic resonance measurements with specific acyl chain-deuterated phospholipids. Translocation of apocytochrome c across a phospholipid bilayer was stimulated on addition of phenethyl alcohol indicating that the efficiency of translocation of this precursor protein is enhanced due to a disorder of the acyl chain region of the bilayer.

Topics: Apoproteins; Biological Transport; Cytochrome c Group; Cytochromes c; Ethanol; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Lipids; Mitochondria; Phenylethyl Alcohol; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Protein Precursors; Trypsin

Deuterium and phosphorus nuclear magnetic resonance techniques were used to study the interaction of the mitochondrial precursor protein apocytochrome c with headgroup-deuterated (dioleoylphosphatidyl-L-[2-2H1]serine) and acyl chain deuterated (1,2-[11,11-2H2]dioleoylphosphatidylserine) dispersions. Binding of the protein to dioleoylphosphatidylserine liposomes results in phosphorus nuclear magnetic resonance spectra typical of phospholipids undergoing fast axial rotation in extended liquid-crystalline bilayers with a reduced residual chemical shift anisotropy and an increased line width. 2H NMR spectra on headgroup-deuterated dioleoylphosphatidylserine dispersions showed a decrease in quadrupolar splitting and a broadening of the signal on interaction with apocytochrome c. Addition of increasing amounts of apocytochrome c to the acyl chain deuterated dioleoylphosphatidylserine dispersions results in the gradual appearance of a second component in the spectra with a 44% reduced quadrupolar splitting. Such large reduction of the quadrupolar splitting has never been observed for any protein studied yet. The lipid structures corresponding to these two components could be separated by sucrose gradient centrifugation, demonstrating the existence of two macroscopic phases. In mixtures of phosphatidylserine and phosphatidylcholine similar effects are observed. The induction of a new spectral component with a well-defined reduced quadrupolar splitting seems to be confined to the N-terminus since addition of a small hydrophilic amino-terminal peptide (residues 1-38) also induces a second component with a strongly reduced quadrupolar splitting. A chemically synthesized peptide corresponding to amino acid residues 2-17 of the presequence of the mitochondrial protein cytochrome oxidase subunit IV also has a large perturbing effect on the order of the acyl chains, indicating that the observed effects may be a property shared by many mitochondrial precursor proteins. In contrast, binding of the mature protein, cytochrome c, to acyl chain deuterated phosphatidylserine dispersions has no effect on the deuterium and phosphorus nuclear magnetic resonance spectra, thereby demonstrating precursor-specific perturbation of the phospholipid order. The inability of holocytochrome c to perturb the phospholipid order is due to folding of this protein, since unfolding of cytochrome c by heat or urea treatment results in similar effects on dioleoylphosphatidylserine bilayers, as obs

Topics: Animals; Apoproteins; Centrifugation, Density Gradient; Chemical Phenomena; Chemistry, Physical; Cytochrome c Group; Cytochromes c; Horses; Magnetic Resonance Spectroscopy; Mitochondria; Phosphatidylserines; Protein Conformation; X-Ray Diffraction

(1) The interaction of apocytochrome c with different molecular species of phosphatidylserine was studied using monolayers at constant surface area or constant surface pressure. The protein inserted readily into dioleoylphosphatidylserine monolayers up to a limiting pressure of 50 mN/m, whereas the interaction decreased with increasing molecular packing of the phosphatidylserine species, indicating the importance of the hydrophobic core of the lipid layer for the interaction. (2) The high affinity of apocytochrome c for dioleoylphosphatidylserine is indicated by the low Kd of 0.017 microM. There is little or no interaction with phosphatidylcholines. The importance of charge interactions is underlined by its ionic strength and pH dependency. (3) Experiments using 14C-labelled apocytochrome c indicate that cholesterol can enhance the protein binding. (4) It was demonstrated that apocytochrome c monomers penetrate the monolayer whereas oligomers can be formed in an adsorbed layer and washed off without changing the surface pressure. Preincubation of apocytochrome c in 3 M guanidine, to obtain the monomeric form, was essential to measure the full effect of interfacial interaction. (5) The molecular area of apocytochrome c changed from 1200-1300 A2/molecule in the absence of lipid to 700-900 A2/molecule after penetration of dioleoylphosphatidylserine monolayers. (6) Apocytochrome c-dioleoylphosphatidylserine interactions are only possible when the monolayer is approached from the subphase. It is concluded that the charge interactions are required for binding and penetration of the protein.

Topics: Apoproteins; Cholesterol; Cytochrome c Group; Cytochromes c; Hydrogen-Ion Concentration; Membrane Lipids; Mitochondria; Phosphatidylcholines; Phosphatidylserines; Temperature