Cellular Mechanism of Newly Synthesized Indoledione Derivative-induced Immunological Death of Tumor Cell

Abstract

Background: EY-6 is one of the newly synthesized indoledione derivatives to induce tumor cell-specific cell death. In this study, we investigated the mechanism of immunological death induced by EY-6 at mouse colon cancer cell as well as at the normal immune cell represented by dendritic cell.

Methods: C57BL/6 mouse syngeneic colon cancer cell MC38 was treated with EY-6, and analyzed by MTT for viability test, flow cytometry for confirming surface expressing molecules and ELISA for detection of cytokine secretion. Normal myeloid-dendritic cell (DC) was ex vivo cultured from bone marrow hematopoietic stem cells of C57BL/6 mice with GM-CSF and IL-4 to analyze the DC uptake of dead tumor cells and to observe the effect of EY-6 on the normal DC.

Results: EY-6 killed the MC38 tumor cells in a dose dependent manner (25, 50 and 100 µM) with carleticulin induction. And EY-6 induced the secretion of IFN-γ but not of TNF-α from the MC38 tumor cells. EY-6 did not kill the ex-vivo cultured DCs at the dose killing tumor cells and did slightly but not significantly induced the DC maturation. The OVA-specific cross-presentation ability of DC was not induced by chemical treatment (both MHC II and MHC I-restricted antigen presentation).

Conclusion: Data indicate that the EY-6 induced tumor cell specific and immunological cell death by modulation of tumor cell phenotype and cytokine secretion favoring induction of specific immunity eliminating tumor cells.

Keywords: Carleticulin; Dendritic cell; Immunological death of tumor cell; Indoledione derivatives.

Figure 1

EY-6 induced tumor-specific killing. MC38 cell (A) or cultured BM-DC (B) was treated with different doses of EY-6 (25, 50 and 100 µM) for 48 hr at 37℃. At the end of the incubation, supernatants were removed and MTT solution was added to analyze the cell death. Asterisks indicate the statistical significance at p<0.05.

Figure 2

Surface expression of immunogenicity-inducing molecules on the tumor cells killed by EY-6. MC38 cells were overnight incubated with or without EY-6 25 µM at 37℃. Then cells were stained with FITC or PE-tagged antibodies against surface molecules as was described in the "Materials and Methods" section.

Figure 3

Increased DC uptake of EY-6 treated MC38 cells. MC38 cells were overnight incubated with or without EY-6 25 µM at 37℃, then stained with CRT-FITC. Cultured-DC was stained with CD11c-PE. Two cells were co-incubated for 6 hr at 37℃ before observe the proportion of tumor cell uptake DCs by measuring CD11c⁺CRT⁺ double positive cells.

Figure 4

Cytokine secretion from the EY-6 treated MC 38 cells. MC38 cells were treated with EY-6 25 µM at 37℃. At several incubation time points, supernatants were obtained to measure the secreted cytokines (IFN-γ or TNF-α) by ELISA following manufacturer's protocol. Asterisks indicate the statistical significance at p<0.05.

Figure 5

Effect of EY-6 on the DC maturation. Cultured BM-DC by the methods described in the "Materials and Methods" section, was treated with 25 µM EY-6 for 18~24 hr at 37℃. At the end of the incubation, harvested and stained the cells with FITC or PE-tagged antibodies for the surface molecules to identify DCs.

Figure 6

Effect of EY-6 on the DC for the OVA-specific cross presentation to CD4⁺(MHC II) and CD8⁺(MHC I) T cells. Cultured BM-DC was treated with EY-6 25 µM for overnight, then introduced with OVA before start the co-culture with MHC-restricted and OVA-specific T cell hybridomas. Details of methods are described in the "Materials and Methods" section. (A) IL-2 (pg/ml) secretion from the MHCII-restricted DOBW cells were measured by ELISA. (B) Color change induced by lacZ activity in the MHC I-restricted B3Z cells was measured. Numbers in Y-axes represent the absorption at OD580 nM. None of the responses were statistically significant.