Functional Enhancement of Exosomes Derived from NK Cells by IL-15 and IL-21 Synergy Against Hepatocellular Carcinoma Cells: The Cytotoxicity and Apoptosis in Vitro Study

Exosomes are released by various cells, including natural killer (NK) cells and transport signaling molecules for the intercellular communication. Hepatocellular carcinoma (HCC), also known as primary liver cancer, is often inoperable and difficult to accurate diagnosis. Notably, the prognosis and underlying mechanisms of HCC are not fully understood. Exosomes-derived NK cells (NK-exos) express unique cytotoxic proteins with a killing ability in tumors and can easily penetrate tumor tissues to improve their targeting ability. NK cell functions, inducing cellular cytotoxicity are modulated by cytokines such as interleukin (IL)-15 and IL-21. However, the mechanisms and effects of cytokines-stimulated NK-exos for the treatment of liver cancer, including HCC, are not well known. In this study, we aimed to investigate the synergistic anti-tumor effects of NK-exos stimulated with IL-15 and IL-21 (NK-exos) in Hep3B cells. Our findings revealed that NK-exos expressed cytotoxic proteins (perforin and granzyme B) and contained typical exosome markers (CD9 and CD63) within the size range of 100-150 nm. Moreover, we demonstrated that NK-exos induced the enhancement of cytotoxicity and apoptotic activity in Hep3B cells by activating the specific pro-apoptotic proteins (Bax, cleaved caspase 3, cleaved PARP, perforin, and granzyme B) and inhibiting the anti-apoptotic protein (Bcl-2). In summary, our results suggest that NK-exos regulate strong anti-tumor effects of HCC cells, by increasing the cytotoxicity and apoptosis through the activation of specific cytotoxic molecules.