Figure 1.
pNK and NK-92 cells sensitized tumor cells.
1×105 of various tumor cells were seeded in 96-well tissue-culture plates, co-cultured with 2.5×105 pNK cells for 4 h, washed and then exposed to 800 cGy of irradiation and evaluated 48 h late for cell proliferation by the MTS (A). C, cancer cell alone; C/NK, cancer cell and NK coculture; C+RT, cancer cell treated with 800 cGy radiation; C/NK+RT, cancer cell and pNK coculture followed by radiation. The apoptosis of CNE-1 cells after irradiation 48 h under various co-culture conditions that described previously was analyzed by (B) Annexin-V assay and (C) cell cycle analysis. (D) CNE-1 cells were incubated with NK-92 cells in 1∶2.5 ratios for 2, 4, and 8 h and irradiated at indicated doses. (E) 1×105 CNE-1 cells were cultured in the lower chambers of transwells, and 2.5×105 NK-92 cells were cultured in the upper chambers for 4 and 8 h. Both of (D) and (E) were assayed using Annexin-V to detect apoptotic cells (AnnexinV+). (*, p<0.05).
Figure 2.
Effect of NK-92-treated CNE-1 cells on Fas blockage.
CNE-1 cells were co-cultured with 2.5 fold NK-92 cells for 4 h in presence of anti-FasL blocking antibody (10 µg/ml). The percentage of apoptotic cells after irradiation 48 h under various co-culture conditions was analyzed by Annexin-V assay (AnnexinV+, D). Results from 3 independent experiments are shown; bars indicate mean ± SD.
Figure 3.
The caspase signaling pathway was induced after co-culture.
CNE-1 cells were co-cultured with 2.5 fold of NK-92 cells for 4 h, then NK-92 cells were washed away, and CNE-1 cells were exposed to 800 cGy of radiation. Control cells of CNE-1 alone or CNE-1 cells that had been co-cultured were not irradiated. After 24 h of incubation, cells were harvested for western blot analysis of procaspase/caspase-3, procaspase-8, and procaspase-9 protein in lysates of CNE-1 alone (lane C), CNE-1 cells cultures with NK-92 cells (lane C/N). The arrows indicate cleaved (activated) caspase 3 at about 17 kDa and its precursor, pro-caspase 3, at about 43 kDa; precaspase 8, at about 55kDa; procaspase 9, at about 45kDa. β-actin was used as the internal control.
Figure 4.
Granzyme B was secreted by NK-92 cells and penetrated into CNE-1 cells during co-culture.
(A) Levels of Granzyme B and perforin mRNA expression in NK-92 cells co-cultured with or without CNE-1 cells for 4 h were measured by real-time RT-PCR. The amounts of mRNA are expressed relative to the amount of MBD-4 in each sample and are shown as the mean ± SD of 3 separate experiments. Significant differences in the expression in the presence or absence of the stimulators are indicated as * (p<0.05). (B) Quantitative analysis of Granzyme B protein in lysates of CNE-1 alone by western blotting (lane C); CNE-1 treated with NK-92 cells for 4 h then NK-92 cells removed (lane C/N); NK-92 cells that had been washed from CNE-1 cell co-culture (lane NK92). CD56 was used to demonstrate exclusion of contamination with NK-92 cells, and β-actin was used as the internal control. (C) DCIC pretreated with CNE-1 cells for 1 h, then co-cultured with 2.5 fold of NK92 cells for 4 h. After washing NK92 cells away, and CNE-1 cells were exposed to 800 cGy of radiation and analyzed for the ratio procaspase/caspase-3 by ImageJ (a decreased ratio is indicative of apoptosis). The bar chart was average of three independent experiments. (D) Annexin-V assay. (*, p<0.05).
Figure 5.
Caspase-3 was inhibited by XIAP and XIAP was downregulated by binding of Smac after radiation.
CNE-1 cells (lane C) were treated with NK-92 cells for 4 h (lane C/N) before combined treatment with 800 cGy of radiation. (A) Cell lysates were immunoprecipitated with anti-XIAP antibody and immunoblotted with anti-Smac, anti-caspase-3 or anti-XIAP antibody. (B) CNE-1 cells were transfected with 80 nM of XIAP siRNA for 16 h and co-cultured with NK-92 cells for 4 h before NK-92 cells were washed away. The cells were assayed using Annexin-V to determine the percentage of apoptotic cells (AnnexinV+). (C) Cell lysates were immunoprecipitated by anti-Smac antibody and detected with anti-XIAP antibody by Western blot. (D) CNE-1 cells were treated with NK-92 cells for 4 h (C/N) before combined treatment with 800 cGy of radiation (C/N+RT) or CNE-1 treated with 800 cGy of radiation alone (C+RT). After treatment, cells were further incubated for 0 min, 15 min, 2 h, or 24 h, then harvested and fractionated into cytosolic (Cyto) and mitochondrial (Mito) fractions for assay by western blot. β-actin was used as the loading control for each fraction. Density of the XIAP normalized with β-actin was assayed by Image J. The bar chart was average of three independent experiments.
Figure 6.
Primary NK cells sensitized tumor cells with same pathway.
CNE-1 cells were transfected with 80 nM of XIAP siRNA for 16 h and co-cultured with pNK cells for 4 h before pNK cells were washed away. The cells were assayed using Annexin-V to determine the percentage of apoptotic cells.
Figure 7.
Mechanism of reciprocal interaction between NK cells and radiation in target cells.
NK cells damage target cell through perforin/granzyme B and death receptor/caspase mediated pathway. The radiosensitisation effect through NK cell depends more on the perforin/granzyme B pathway. Without radiation, the suboptimal activation of NK cells cause up-regulation of XIAP. With radiation, the mitochondria releases Smac to neutralize XIAP and enhances NK cell-mediated cytotoxicity.