The online version of this article (doi:10.1186/s12943-015-0336-y) contains supplementary material, which is available to authorized users.
Patents are pending for the intellectual property disclosed in this manuscript. X. Qi is listed as an inventor on the patent for SapC-DOPS technology that is the subject of this research. Consistent with current Cincinnati Children’s Hospital Medical Center policies, the development and commercialization of this technology has been licensed to Bexion Pharmaceuticals, LLC, in which X. Qi, holds a minor (<5%) equity interest. The other authors declared no conflict of interest.
Conceived and designed the experiments: SMK, ZC, RSF, XQ. Performed the experiments: SMK, ZC, XQ. Analyzed the data: SMK, ZC, VMB, SDV, RSF, XQ. Contributed reagents/materials/analysis tools: SMK, ZC, RSF, XQ. Wrote the paper: SMK, XQ. Edited and approved the manuscript: SMK, ZC, VMB, SDV, RSF, XQ.
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.
Additional file 1: Figure S1. Evaluation of necrosis and mitochondrial superoxide formation. A) MTT assay of SK-N-SH cells treated with DOPS. B) Necrosis measured by G6PD release in SK-N-SH cells following treatment with SapC, DOPS and SapC-DOPS for 24 h. C) Mean MitoSox-Red fluorescence following 50 μM SapC-DOPS treatment of neuroblastoma cells for 24 h. Pos CTL stands for pre-treatment with 20 μM Antimycin A. D) Quantification of fold changes in MitoSox intensity after treatment with 50 μM SapC, 350 μM DOPS or SapC-DOPS (25, 50 μM) for 24 h.12943_2015_336_MOESM1_ESM.tiff
Additional file 2: Figure S2. Protein expression analysis in SK-N-SH cells. A) Expression of apoptotic proteins following treatment with 350 μM DOPS. Fractions indicate fold-change estimated by densitometric analysis of proteins normalized to β-Actin corresponding to the lane. B) ShRNA-mediated knockdown of Smac in SK-N-SH cells. Percentages represent reduction in Smac normalized to β-Actin.12943_2015_336_MOESM2_ESM.tiff
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- SapC-DOPS nanovesicles induce Smac- and Bax-dependent apoptosis through mitochondrial activation in neuroblastomas
Mahaboob K Sulaiman
Victor M Blanco
Subrahmanya D Vallabhapurapu
Robert S Franco
- BioMed Central
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