SapC-DOPS nanovesicles induce Smac- and Bax-dependent apoptosis through mitochondrial activation in neuroblastomas

Abstract

BackgroundHigh 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.MethodsIn 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.ResultsSapC-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.ConclusionsTaken 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0336-y) contains supplementary material, which is available to authorized users.

SapC-DOPS – a Phosphatidylserine-targeted Nanovesicle for selective Cancer therapy

“…Importantly, the higher the PS expression on the surface of a cell, the more effectively SapC-DOPS binds to the cell and triggers the ceramide cascade, ultimately resulting in apoptosis ( Fig. 1) [15,26]. Binding of SapC to PS is favored at acidic pH.…”

“…Studies in neuroblastoma reveal that SapC-DOPS-induced apoptosis involves cytosolic release of second mitochondria-derived activator of caspases (Smac) and cytochrome c, as well as mitochondrial translocation and polymerization of Bax (Fig. 1) [26]. Studies of saposin C membrane fusion revealed that although saposin C-induced fusion occurred with a mixture of anionic saturated and unsaturated acyl chains, the fusion process was much slower than that with synthetic unsaturated DOPS, thus DOPS enhances saposin C fusion, especially at acidic pH [30].…”

“…The effect of SapC-DOPS has also been assessed in the pediatric cancers using neuroblastoma and peripheral neuroblastic tumors [17,26]. SapC-DOPS effectively targeted and inhibited the growth of neuroblastoma and pNTS in vitro.…”

“…Furthermore, xenograft mice were used to demonstrate in vivo therapeutic efficacy. In these mice, SapC-DOPS exhibited specific targeting of neuroblastoma tumors and induced apoptotic cell death [17,26].…”

“…The unique appeal of SapC-DOPS as a cancer therapy include its consistent selective targeting and killing of cancer cells while being tolerated in healthy cellsthis phenomenon has been reflected in results of phase I clinical trials where SapC-DOPS showed a strong safety profile [24,25]. In addition, SapC-DOPS [1] utilizes multiple mechanisms to induce cancer cell death including caspase 9 cleavage and lysosomal membrane permeability [2] is capable of crossing the blood-brain tumor barrier and [3] enhances the effects of existing therapies [9,10,23,26]. Therapeutic assessment of SapC-DOPS and other PStargeting nanovesicles by other researchers indicate that, as a class, they are a promising therapeutic option for the treatment of several types of cancers.…”