Highly sensitive method for assay of drug-induced apoptosis using fluorescence correlation spectroscopy.

Apoptosis plays a crucial role in many biological processes and pathogenesis of various malignancies and diseases of the immune system. In this paper, we described a novel method for sensitive detection of drug-induced apoptosis by using fluorescence correlation spectroscopy (FCS). The principle of this method is based on the assay of DNA fragmentation in the process of the drug-induced apoptosis. FCS is a single molecule method, and it can be used for sensitive and selective assay of DNA fragmentation without separation. We first developed a highly sensitive method for characterization of DNA fragments using a home-built FCS system and SYBR Green I as fluorescent DNA-intercalating dye, and then established a model of drug-induced apoptosis using human pancreatic cancer cells and a drug lidamycin. Furthermore, FCS method established was used to directly detect the fragmentation of DNA extracted from apoptotic cells or in the apoptotic cell lysate. In FCS assay, the single-component model and the multiple-components model were used to fit raw FCS data. The characteristic diffusion time of DNA fragments was used as an important parameter to distinguish the apoptotic status of cells. The obtained data documented that the characteristic diffusion time of DNA fragments from apoptotic cells significantly decreased with an increase of lidamycin concentration, which implied that DNA fragmentation occurred in lidamycin-induced apoptosis. The FCS results are well in line with the data obtained from flow cytometer and gel electrophoresis. Compared to current methods, the method described here is sensitive and simple, and more importantly, our detection volume is less than 1 fL, and the sample requirement can easily be reduced to nL level using a droplets array technology. Therefore, our method probably becomes a high throughput detection platform for early detection of cell apoptosis and screening of apoptosis-based anticancer drugs.

[1]  Yujin E. Kim,et al.  Monitoring apoptosis and neuronal degeneration by real-time detection of phosphatidylserine externalization using a polarity-sensitive indicator of viability and apoptosis , 2010, Nature Protocols.

[2]  Y. Ueno,et al.  The process of ultrastructural changes from nuclei to apoptotic body , 1998, Virchows Archiv.

[3]  Yafeng Wu,et al.  Signal amplification cytosensor for evaluation of drug-induced cancer cell apoptosis. , 2012, Analytical chemistry.

[4]  Y. Yamada,et al.  A new macromolecular antitumor antibiotic, C-1027. I. Discovery, taxonomy of producing organism, fermentation and biological activity. , 1988, The Journal of antibiotics.

[5]  H. Hug,et al.  Rhodamine 110-linked amino acids and peptides as substrates to measure caspase activity upon apoptosis induction in intact cells. , 1999, Biochemistry.

[6]  Lei Liu,et al.  Electrochemical approach to detect apoptosis. , 2008, Analytical chemistry.

[7]  D. Green,et al.  p53 Induces Apoptosis by Caspase Activation through Mitochondrial Cytochrome c Release* , 2000, The Journal of Biological Chemistry.

[8]  Regina Luttge,et al.  Apoptotic cell death dynamics of HL60 cells studied using a microfluidic cell trap device. , 2005, Lab on a chip.

[9]  Yuko Ito,et al.  Method of Specific Detection of Apoptosis Using Formamide-induced DNA Denaturation Assay , 2006, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[10]  A. Rosenzweig,et al.  Comparison of Comet Assay, Electron Microscopy, and Flow Cytometry for Detection of Apoptosis , 2003, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[11]  Ming Xu,et al.  Apoptotic DNA fragmentation and tissue homeostasis. , 2002, Trends in cell biology.

[12]  C. Reutelingsperger,et al.  Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. , 1998, Cytometry.

[13]  G Orlandini,et al.  Comparison of Annexin V and Calcein-AM as Early Vital Markers of Apoptosis in Adherent Cells by Confocal Laser Microscopy , 1998, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[14]  D. Xing,et al.  One-step homogeneous protein detection based on aptamer probe and fluorescence cross-correlation spectroscopy. , 2011, Analytical chemistry.

[15]  Joseph Irudayaraj,et al.  Quantitative investigation of compartmentalized dynamics of ErbB2 targeting gold nanorods in live cells by single molecule spectroscopy. , 2009, ACS nano.

[16]  S. Maiti,et al.  The Amyloid β Peptide (Aβ1-40) Is Thermodynamically Soluble at Physiological Concentrations† , 2003 .

[17]  P. Möller,et al.  Monoclonal antibody-mediated tumor regression by induction of apoptosis. , 1989, Science.

[18]  G. Salvesen,et al.  Caspase assays: identifying caspase activity and substrates in vitro and in vivo. , 2008, Methods in enzymology.

[19]  G. Häcker.  The morphology of apoptosis , 2000, Cell and Tissue Research.

[20]  A. Wyllie,et al.  Apoptosis: A Basic Biological Phenomenon with Wide-ranging Implications in Tissue Kinetics , 1972, British Journal of Cancer.

[21]  D. Pappas,et al.  Single molecule fluorescence correlation spectroscopy of single apoptotic cells using a red-fluorescent caspase probe. , 2012, In Analysis.

[22]  Chunlei Zhu,et al.  A potent fluorescent probe for the detection of cell apoptosis. , 2011, Chemical communications.

[23]  L. Attardi,et al.  The role of apoptosis in cancer development and treatment response , 2005, Nature Reviews Cancer.

[24]  A. Wyllie,et al.  Cell death: the significance of apoptosis. , 1980, International review of cytology.

[25]  R. Shao,et al.  Enediyne anticancer antibiotic lidamycin: chemistry, biology and pharmacology. , 2008, Anti-cancer agents in medicinal chemistry.

[26]  Jun Liu,et al.  Analysis of nonadherent apoptotic cells by a quantum dots probe in a microfluidic device for drug screening. , 2009, Analytical chemistry.

[27]  Y. Zhen,et al.  Antitumor efficacy of lidamycin on hepatoma and active moiety of its molecule. , 2005, World journal of gastroenterology.

[28]  R. V. Vega Thurber,et al.  Apoptosis in early development of the sea urchin, Strongylocentrotus purpuratus. , 2007, Developmental biology.

[29]  S. Muller,et al.  An ELISA for detection of apoptosis. , 1997, Nucleic acids research.

[30]  T. Tanaka,et al.  Solution structures of C-1027 apoprotein and its complex with the aromatized chromophore. , 2001, Journal of molecular biology.

[31]  O. Fackler,et al.  Cell motility through plasma membrane blebbing , 2008, The Journal of cell biology.

[32]  D. Galaris,et al.  DNA damage and apoptosis in hydrogen peroxide-exposed Jurkat cells: bolus addition versus continuous generation of H2O2 , 2002 .

[33]  Nibedita Pal,et al.  Fluorescence correlation spectroscopy: an efficient tool for measuring size, size-distribution and polydispersity of microemulsion droplets in solution. , 2011, Analytical chemistry.

[34]  P. Schwille,et al.  Fluorescence correlation spectroscopy in living cells , 2007, Nature Methods.

[35]  Jian-Rong Zhang,et al.  Toward the early evaluation of therapeutic effects: an electrochemical platform for ultrasensitive detection of apoptotic cells. , 2011, Analytical chemistry.

[36]  S. Van Cruchten,et al.  Morphological and biochemical aspects of apoptosis, oncosis and necrosis. , 2002 .

[37]  T. Kwok,et al.  Let-7a microRNA suppresses therapeutics-induced cancer cell death by targeting caspase-3 , 2008, Apoptosis.

[38]  E. Hogan,et al.  Combined TUNEL and double immunofluorescent labeling for detection of apoptotic mononuclear phagocytes in autoimmune demyelinating disease. , 2000, Brain research. Brain research protocols.

[39]  Randall D. Reif,et al.  Detection of apoptosis: A review of conventional and novel techniques , 2010 .

[40]  Y. Zhen,et al.  Down-regulation of the nuclear factor-kappaB by lidamycin in association with inducing apoptosis in human pancreatic cancer cells and inhibiting xenograft growth. , 2007, Oncology reports.

[41]  N. Periasamy,et al.  Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy. , 2003, Biophysical journal.

[42]  Randall D. Reif,et al.  Early detection of apoptosis in living cells by fluorescence correlation spectroscopy , 2010, Analytical and bioanalytical chemistry.

[43]  G. Kroemer,et al.  Chloromethyl-X-Rosamine is an aldehyde-fixable potential-sensitive fluorochrome for the detection of early apoptosis. , 1996, Cytometry.

[44]  S. Nagata.  Apoptotic DNA fragmentation. , 2000, Experimental cell research.