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

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1

Preparation and tumor-targeting potential of SapC-DOPS in mice xenografts. A) Treatment of athymic nude mice bearing neuroblastoma (CHLA-20) xenografts with CVM-labeled SapC-DOPS (1), CVM-DOPS (2) or SapC-CVM (3). Animals were imaged 24 h after injection with exposure time of 1 s. Saposin C, 4.2 mg/kg; dioleoylphosphatidylserine, 2 mg/kg; CellVue Maroon, 6 μmol. B) Evaluation of tumor burden in SapC-DOPS treated mice xenografts. Mice xenografts (n = 15) were treated with five intratumoral injections of SapC-DOPS (SapC 4 mg/kg, DOPS 2 mg/kg) or PBS every 3 days and followed for tumor growth. C) Histological examination of the neuroblastoma tumor tissue in mice after intratumoral injection of DOPS and SapC-DOPS. Original magnification: 1000x. D) Evaluation of apoptosis by TUNEL staining (arrows) in DOPS- and SapC-DOPS treated tumors. Original magnification: 40x.

Figure 2

SapC-DOPS induces apoptosis in neuroblastoma cell lines. A) Viability of SK-N-SH, IMR-32 and human Schwann cells measured by the MTT assay. **represents the lowest concentration from which P value is significant i.e., < 0.001. B) Microscopic images of SK-N-SH cells after treatment with 350 μM DOPS or 50 μM SapC-DOPS. Arrows indicate morphological changes such as cell shrinkage and chromatin condensation. Original magnification: 20x). C) Changes in cell cycle following treatment of SK-N-SH cells with 50 μM SapC-DOPS for 24 h. The value represents percentage of cells in Sub-G1 region. *P < 0.05, **P < 0.001 when compared to control. Points, mean of three to five independent experiments; bars, SE.

Figure 3

SapC-DOPS induces loss of mitochondrial potential (ΔΨM). A) Dose- and time-dependent changes in ΔΨM evaluated by JC-1 ratio of red and green fluorescence following SapC-DOPS treatment in SK-N-SH and IMR-32 cells. B) ΔΨM measured by JC-1 ratio in bongkrekic acid-pretreated SK-N-SH cells following SapC-DOPS treatment for 24 h. C) Viability measured by MTT assay in SK-N-SH cells following pre-treatment with bongkrekic acid (BA) and subsequent treatment with 50 μM SapC-DOPS for 72 h. *P < 0.05, **P < 0.001 when compared to control. Points, mean of three to five independent experiments; bars, SE.

Figure 4

SapC-DOPS treatment causes redistribution of apoptogenic proteins and Bax oligomerization in mitochondria of neuroblastoma cells. A) Immunoblots from whole cell extracts showing apoptotic protein expression changes following treatment with 50 μM SapC-DOPS. Fractions indicate fold-change estimated by densitometric analysis of proteins normalized to β-Actin corresponding to the lane. Fold change indicated for caspase-3 corresponds to the cleaved 19 kDa fragment. Right lanes: (−) refers to negative control (Schwann cells treated with SapC-DOPS) and (+) refers to positive control (SK-N-SH cells treated with 10 μM staurosporine) for 24 h. B) Relocation of Smac and Cyto c in SK-N-SH cells. Cox4 and Tubulin served as loading control for the mitochondrial and cytoplasmic fractions, respectively. C) Bax redistribution in neuroblastoma cells following SapC-DOPS treatment. D) Bax oligomerization in neuroblastoma cells. **P < 0.001. Points, mean of three to five experiments; bars, SE. Western blots are representative of three independent experiments.

Figure 5

Apoptotic redistribution of Smac and Cyto c is independent of ROS formation, Cyclophilin D activity and Ca ²⁺ . A) Effect of N-acetyl cysteine (NAC)-mediated ROS inhibition on viability of SK-N-SH cells following SapC-DOPS treatment. B) Effect of pretreatment with 2 mM N-acetyl cysteine (NAC) following SapC-DOPS (50 μM) treatment on Smac and Cyto c relocation in SK-N-SH cells. C) Effect of 1 μM cyclosporine A (CsA)-pretreatment on Smac and Cyto c relocation following SapC-DOPS (50 μM) treatment in SK-N-SH cells. D) Flow cytometric measurement of Ca²⁺ using Fluo-3 AM in SK-N-SH cells treated with 50 μM SapC-DOPS. Values represent geometric mean of fluorescence. Points, mean of three to five experiments; bars, SE. Western blots are representative of three independent experiments.

Figure 6

Smac plays an important role in SapC-DOPS-induced apoptosis. A) SK-N-SH cell viability assessed by MTT assay after treatment with 50 nM control scrambled shRNA (empty bars) or 50 nM Smac shRNA (filled bars) and subsequent SapC-DOPS treatment B) Evaluation of ΔΨM by JC-1 assay in Smac-knockdown SK-N-SH cells following 50 μM SapC-DOPS treatment for 24 h. Pos CTL refers to treatment with 50 μM 2-[2-(3-Chlorophenyl)hydrazinylyidene]propanedinitrile (CCCP). C) Redistribution of apoptogenic proteins following 50 μM SapC-DOPS treatment for 24 h in Smac-knockdown SK-N-SH cells. (D) Caspase-3 activation in SapC-DOPS (50 μM) treated Smac-knockdown SK-N-SH cells. Densitometry graph shows relative changes in cleaved caspase-3 fragment expression normalized to β-Actin. *P < 0.05, **P < 0.001. Points, mean of three to five experiments; bars, SE. Western blots are representative of three independent experiments.

Figure 7

Bax inhibition reduces SapC-DOPS-induced apoptosis. A) MTT assay in control peptide-treated (empty bar) and V5 peptide-treated (Bax-inhibited; filled bars) SK-N-SH cells following SapC-DOPS treatment. B) ΔΨM measured by JC-1 assay in SK-N-SH cells following SapC-DOPS treatment for 24 h. C) Apoptotic protein expression in control-peptide and Bax-V5-peptide pre-treated SK-N-SH cells following 50 μM SapC-DOPS treatment for 24 h. D) Changes in Smac expression in cytosolic and mitochondrial extracts of Bax-inhibited SK-N-SH cells treated with 50 μM SapC-DOPS. *P < 0.05, **P < 0.001. Points, mean of three to five experiments; bars, SE. Western blots are representative of three independent experiments.