Journal: The Journal of Biological Chemistry

Article Title: Anticancer Drugs Cause Release of Exosomes with Heat Shock Proteins from Human Hepatocellular Carcinoma Cells That Elicit Effective Natural Killer Cell Antitumor Responses in Vitro *

doi: 10.1074/jbc.M112.340588

Figure Lengend Snippet: Secretion of HSP60, HSP70, and HSP90 by HepG2 and PLC/PRF/5 cells is increased under stress conditions. HSP60 (A), HSP70 (B), and HSP90 (C) secretion before and after heat shock and anticancer drugs was detected by ELISA. The findings showed no cell line-specific differences and enhancements of HSP60, HSP70, and HSP90 secretion into the extracellular medium under stress conditions. Resistant anticancer drugs (irinotecan hydrochloride and carboplatin) markedly increased HSP60, HSP70, and HSP90 production by HepG2 and PLC/PRF/5 cells compared with under basal (control) and other stress conditions (p < 0.05). The results are of one representative experiment of three. Mean values ± S.D. are calculated from triplicate experiments with similar results.

Article Snippet: Granzyme B ELISA Granzyme B released by NK cells during the stimulation period of 4 days, either with low dose IL-2 alone (100 IU/ml) or with IL-2 in combination with exosomal proteins (5, 10, or 20 μg/ml), was measured using a human granzyme B ELISA kit (CSB-E08718h, Cusabio Biotech) according to manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Control

Journal: The Journal of Biological Chemistry

Article Title: Anticancer Drugs Cause Release of Exosomes with Heat Shock Proteins from Human Hepatocellular Carcinoma Cells That Elicit Effective Natural Killer Cell Antitumor Responses in Vitro *

doi: 10.1074/jbc.M112.340588

Figure Lengend Snippet: Anticancer drugs enhance the positive effect of HSP-bearing exosomes on NK cell-mediated cytotoxic response through granzyme B release concomitant with an altered cell surface density of several NK cell receptors. NK cells were incubated either with low dose IL-2 alone or with IL-2 plus exosomes derived from HepG2 cells, treated with paclitaxel or carboplatin, for 4 days. A, NK cell-mediated cytotoxic activity was measured in a standard lactate dehydrogenase release assay. A comparable, strong lysis was detected against K562 target cells if NK cells were stimulated with IL-2 plus exosomes. After stimulation with IL-2 alone, cytotoxic activity was weaker (p < 0.05). Data are from one experiment representative of seven separate experiments with similar results (means ± S.D.). B, after 4 days, culture supernatants were harvested, and the amount of released granzyme B was estimated by ELISA. Columns, mean values of three independent experiments; error bars, S.D. *, statistical significance (p < 0.05). C, after 4 days, NK cells were stained with anti-NKG2D, anti-CD69, anti-CD94, or anti-NKp44 antibodies and analyzed by flow cytometry. The geometric MFI of NK cells stimulated with IL-2 plus exosomes was significantly different from IL-2-stimulated controls (p < 0.05). Data represent mean values ± S.D. (error bars) from three independent experiments with similar results.

Article Snippet: Granzyme B ELISA Granzyme B released by NK cells during the stimulation period of 4 days, either with low dose IL-2 alone (100 IU/ml) or with IL-2 in combination with exosomal proteins (5, 10, or 20 μg/ml), was measured using a human granzyme B ELISA kit (CSB-E08718h, Cusabio Biotech) according to manufacturer's instructions.

Techniques: Incubation, Derivative Assay, Activity Assay, Release Assay, Lysis, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry