Activation of the JNK-c-Jun pathway in response to irradiation facilitates Fas ligand secretion in hepatoma cells and increases hepatocyte injury

It is well established that some irradiated liver non-parenchymal cells secrete pro-inflammatory cytokines to facilitate the development of radiation-induced liver disease. However, little is known on whether the irradiated hepatoma cells-mediated non-irradiated hepatocyte injury occurs in HCC patients. Here, we elucidated the roles of the irradiated hepatoma cells in driving non-irradiated hepatocyte injury and its underlying mechanism.

In HCC cells, the JNK-c-Jun pathway plays an important role in mediating irradiation- induced FasL expression, which may be critical in determining non-irradiated hepatocyte injury.

SMMC7721 cells were cultured and divided into irradiated (4-Gy X-ray, R) and non-irradiated (NR) groups. At 24th hour after irradiation, conditioned medium (CM) from these cultures was mixed with normal culture medium in specific proportions, and termed as 7721-R-CM and 7721-NR-CM. Following incubation with these CM compound, the biological characteristics of L02 cells related to liver cell injury including viability, apoptosis and liver dysfunction indices were comparatively analyzed. Simultaneously, the levels of proliferation- and apoptosis-related cytokines in irradiated and non-irradiated SMMC7721 cells were also measured. FasL as a cytokine with significantly differential expression, was selected to clarify its effects on L02 apoptosis. Subsequently, FasL expression following irradiation was examined in SMMC7721 and other HCC cells with varying malignant potentials, as well as in HCC tissues, the related mechanism of higher expression of FasL in irradiated HCC cells was further investigated.

Apoptosis and liver dysfunction indices were all significantly enhanced in L02 cells treated with 7721-R-CM, whereas viability was suppressed, compared to those with 7721-NR-CM stimulation. FasL was identified as a leading differential cytokine in the irradiated SMMC7721 cells. Higher proportion of apoptosis was also found in L02 cells following FasL incubation. A recombinant Fas-Fc protein, which blocks Fas-FasL interaction, ameliorated 7721-R-CM-induced apoptosis in L02 cells. FasL was highly expressed in a dose-dependent manner, and peaked at the 24th hour post-irradiation in different HCC cells and their culture supernatant. Meanwhile, phosphorylation levels of JNK, ERK, Akt, and p38 were all upregulated significantly in irradiated HCC cells. But, only JNK inhibition was validated to block radiation-induced FasL expression in HCC cells. c-Jun, the target transcription factor of JNK, was also activated.