Abstract: Objective To investigate the mechanism of parthenolide for inducing necroptosis and ameliorating CD8+T cell exhaustion in colorectal cancer (CRC) and construct liposome nanoparticles for targeted delivery of parthenolide. Methods The inhibitory effect of parthenolide on proliferation of different CRC cell lines was examined using CCK8 assay, and ROS LDH detection and Western blotting were used to analyze the cell death pathways. In a mouse model bearing subcutaneous MC38 cell xenografts, the effects of 5 and 15 mg/kg parthenolide on tumor growth and CD8+ T cell depletion were observed. In a mouse model bearing orthotopic CRC cell xenograft in the ileocecal region, free parthenolide (100 μg/mL) or low (100 μg/mL) and high doses (200 μg/mL) of liposome nanoparticles loaded with parthenolide were injected via the tail vein, and the changes in CD8 expression in the xenografts were analyzed using immunohistochemistry. Results Treatment with parthenolide dose-dependently lowered the viability of the CRC cell lines SW480, DLD1, HCT116 and MC38 cells, and its effect was obviously antagonized by Nec-1. Immunoblotting analysis showed that parthenolide treatment resulted in increased RIP3 and MLKL phosphorylation in the CRC cells. In the mouse model bearing subcutaneous xenografts, parthenolide treatment at the high dose, but not at the low dose, significantly increased the number of infiltrating CD3+ CD8+ T cells and PD1hiTIM3+T cell percentage (P<0.01) and lowered the percentage of PD1loTIM3-T cells in the tumor tissue (P<0.01). In the mouse models bearing orthotopic CRC xenograft, intravenous injection of the liposomes loaded with parthenolide, especially at the high dose, significantly increased CD8 expression in the tumor tissue (P<0.01). Conclusion Parthenolide induces necroptosis in CRC and increases infiltrating CD8+ T cells to ameliorate CD8+T cell exhaustion in the tumor. Liposome nanoparticles for targeted delivery of parthenolide produce stronger, anti-tumor effect.

Key words: parthenolide; colorectal cancer; necroptosis; nanoparticles; T cell exhaustion