Reciprocal complementation of the tumoricidal effects of radiation and natural killer cells

Abstract

The tumor microenvironment is a key determinant for radio-responsiveness. Immune cells play an important role in shaping tumor microenvironments; however, there is limited understanding of how natural killer (NK) cells can enhance radiation effects. This study aimed to assess the mechanism of reciprocal complementation of radiation and NK cells on tumor killing. Various tumor cell lines were co-cultured with human primary NK cells or NK cell line (NK-92) for short periods and then exposed to irradiation. Cell proliferation, apoptosis and transwell assays were performed to assess apoptotic efficacy and cell viability. Western blot analysis and immunoprecipitation methods were used to determine XIAP (X-linked inhibitor of apoptosis protein) and Smac (second mitochondria-derived activator of caspase) expression and interaction in tumor cells. Co-culture did not induce apoptosis in tumor cells, but a time- and dose-dependent enhancing effect was found when co-cultured cells were irradiated. A key role for caspase activation via perforin/granzyme B (Grz B) after cell-cell contact was determined, as the primary radiation enhancing effect. The efficacy of NK cell killing was attenuated by upregulation of XIAP to bind caspase-3 in tumor cells to escape apoptosis. Knockdown of XIAP effectively potentiated NK cell-mediated apoptosis. Radiation induced Smac released from mitochondria and neutralized XIAP and therefore increased the NK killing. Our findings suggest NK cells in tumor microenvironment have direct radiosensitization effect through Grz B injection while radiation enhances NK cytotoxicity through triggering Smac release.

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.