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Apoptosis

, Volume 16, Issue 1, pp 1–12 | Cite as

Survivin is released from cancer cells via exosomes

  • Salma Khan
  • Jessica M. S. Jutzy
  • Jonathan R. Aspe
  • Dalmor W. McGregor
  • Jonathan W. Neidigh
  • Nathan R. Wall
Original Paper

Abstract

Inhibitor of apoptosis (IAP) and Heat shock proteins (HSPs) provide assistance in protecting cells from stresses of hypoxia, imbalanced pH, and altered metabolic and redox states commonly found in the microenvironmental mixture of tumor and nontumor cells. HSPs are upregulated, cell-surface displayed and released extracellularly in some types of tumors, a finding that until now was not shared by members of the IAP family. The IAP Survivin has been implicated in apoptosis inhibition and the regulation of mitosis in cancer cells. Survivin exists in a number of subcellular locations such as the mitochondria, cytoplasm, nucleus, and most recently, the extracellular space. Our previous work showing that extracellular survivin was able to enhance cellular proliferation, survival and tumor cell invasion provides evidence that Survivin might be secreted via an unidentified exocytotic pathway. In the present study, we describe for the first time the exosome-release of Survivin to the extracellular space both basally and after proton irradiation-induced stress. To examine whether exosomes contributed to Survivin release from cancer cells, exosomes were purified from HeLa cervical carcinoma cells and exosome quantity and Survivin content were determined. We demonstrate that although proton irradiation does not influence the exosomal secretory rate, the Survivin content of exosomes isolated from HeLa cells treated with a sublethal dose of proton irradiation (3 Gy) is significantly higher than control. These data identify a novel secretory pathway by which Survivin can be actively released from cells in both the basal and stress-induced state.

Keywords

Survivin Exosomes Apoptosis Cancer Microenvironment 

Abbreviations

IAP

Inhibitor of apoptosis

HSPs

Heat shock proteins

AChE

Acetylcholinesterase

LAMP1

Lysosomal-associated membrane protein 1

cytoD

Cytochalasin D

XIAP

X-linked inhibitor of apoptosis

ATCC

American Type Culture Collection

IL2R

Interleukin-2 receptor

DTT

Dithiothreitol

CM

Conditioned medium

Notes

Acknowledgments

Grant Support: NCMHD Project EXPORT Program 5P20MD001632/Project 3 (N.R. Wall). Funding was also obtained as part of a start-up package from Loma Linda University’s Center for Molecular Biology and Gene Therapy, now the Center for Health Disparities Research and Molecular Medicine (NRW) and a National Merit Test Bed (NMTB) award sponsored by the Department of the Army under Cooperative Agreement Number DAMD17-97-2-7016 (NRW). Proton irradiation was accomplished at the Loma Linda University Radiobiology Proton Treatment Facility, now the James M. Slater, MD, Proton Treatment and Research Center. The authors would like to personally thank Dr. James Slater, Dr. Daila Gridley, Steven Rightnar and Celso Perez for all their help and we dedicate this work to our dear friend and colleague Dr. Lora Green who passed away unexpectedly.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10495_2010_534_MOESM1_ESM.pdf (125 kb)
Table S1. The Scaffold score for peptides and proteins represents the probability of a correct assignment as calculated by PeptideProphet (24) and ProteinProphet (25), respectively. The PeptideProphet probability represents a consensus of SEQUEST, Mascot, and X!Tandem scores when available for a given peptide. The Sequest Xcorr values of greater than 1.73 and 1.97 for single and double charged peptides, respectively, have been empirically shown to represent a 95% probability of a correct assignment (52). The expected number of hits for each peptide that will occur by random chance during a Mascot search of the database is shown in the last column (PDF 125 kb)
10495_2010_534_MOESM2_ESM.pdf (3 mb)
Figure S1. HeLaS/POZnSurvivin cells secrete Survivin and Hsp70-containing exosomes. (a) Isotype controls were used to show the specificity of the immunogold-conjugated antibodies. Exosomes were characterized from HeLaS/POZnSurvivin conditioned medium using electron microscopy. Immunoelectron microscopy on (b) anti-Hsp70- and (c) anti-Survivin- stained cryosections of HeLaS/POZnSurvivin cells shows that Survivin and Hsp70 localization in the exosome. Immunogold nanoparticles of 10 nm were conjugated to anti-Survivin pAb while 5 nm nanoparticles were conjugated to anti-Hsp70 mAb. Exosomes are cup-shaped and 50-150 nm in size. Bar, 100 nm (PDF 3,023 kb)
10495_2010_534_MOESM3_ESM.tif (95 kb)
Figure S2. Cancer cell lines disproportionately release Survivin in their exosomes compared with non cancer cells. (a) Western blotting shows that the cancer cell lines PozN-WT, Panc1 and PC3 have Survivin in their exosomes compared with cells and cell lines representing non-cancer cells: Peripheral Blood Mononuclear Cells (PBMC), Human Bone Marrow Stroma (HBMS), Prostate Stromal Cells (PrSC) and human embryonic kidney epithelial (A293) cells. Protein concentrations for Western blot loading were determined using the AChE activity. Exosome loading is controlled for by the Lysosome Associated Membrane Protein (LAMP1). Molecular-weight markers in kilodaltons (KDa) are shown on the left. Exosomes were collected after 24h of serum depletion. Protein quantitation and loading was accomplished by AChE for exosomes and BCA for whole cell lysates (WCL). (b) Exosomes were purified from control media after which AChE activity was determined as described under Methods and Materials. Values represent the means of three samples with measurements at 30 min recorded (TIFF 95 kb)
10495_2010_534_MOESM4_ESM.tif (260 kb)
Figure S3. Cytochalasin D disrupts actin microfilaments in HeLaS/POZnSurvivin cells. (a) HeLaS/POZnSurvivin cells and those treated with cytochalasin D were fixed and stained using alpha actin antibodies as described in the methods and materials and were visualized using fluorescence or phase-contrast light microscopy. Magnification under fluorescence and phase-contrast was x1000. (b) Propidium iodide (PI) staining was accomplished to define and quantitate the cytoD-induction of apoptosis in these cells. (c) Exosome presence was measured as AChE activity. Measured at 30 min, the addition of cytochalasin D (cytoD) or monensin (Mon.) to 3 Gy proton irradiation resulted in a marked reduction in exosome presence. (d) Western blots of Survivin and Hsp70 in the conditioned medium of exosome isolations (top) and whole cell lysates (bottom). Protein concentrations were determined using the BCA assay. Molecular-weight markers in kilodaltons (kDa) are shown on the left. AChE activity values represent the means of three samples. Exosomes were collected after 24 h of treatment. Abbreviations: CytoD = cytochalasin D, Mon. = Monensin (TIFF 260 kb)
10495_2010_534_MOESM5_ESM.tif (118 kb)
(TIFF 119 kb)

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Salma Khan
    • 1
    • 2
  • Jessica M. S. Jutzy
    • 1
    • 2
  • Jonathan R. Aspe
    • 1
    • 2
  • Dalmor W. McGregor
    • 1
    • 2
  • Jonathan W. Neidigh
    • 2
  • Nathan R. Wall
    • 1
    • 2
  1. 1.Center for Health Disparities Research and Molecular MedicineLoma Linda UniversityLoma LindaUSA
  2. 2.Department of Basic Sciences, Division of Biochemistry and MicrobiologyLoma Linda UniversityLoma LindaUSA

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