Dihydroartemisinin induced caspase-dependent apoptosis through inhibiting the specificity protein 1 pathway in hepatocellular carcinoma SK-Hep-1 cells

Aims: Dihydroartemisinin (DHA) is a semi-synthetic derivative of artemisinin, well known for a safe and effective first-line antimalarial agent. This study investigated whether and how DHA induces apoptosis focusing on the specificity protein 1 (Sp1) pathway in hepatocellular carcinoma (HCC) SK-Hep-1 cells.

Main methods: The cell viability was evaluated by MTT assay. Cell cycle analysis was performed after PI staining by flow cytometry system. Apoptosis was confirmed by DAPI staining and by detecting cytoplasmic histone-associated-DNA-fragments using a cell death detection ELISA^PLUS kit. The expression of proteins involved in apoptosis was evaluated by Western blot. The nuclear localization of Sp1 was evaluated by immunofluorescence assay.

Key findings: DHA exerted potent cytotoxicity against HCC SK-Hep-1 cells compared with normal hepatocyte AML12 cells. The sub-G₁ DNA content and apoptosis index were increased by DHA, which was accompanied by nuclei condensation and fragmentation. DHA activated caspase 3, caspase 8, and caspase 9 and cleaved poly (ADP-ribose) polymerase (PARP). DHA-induced apoptotic cell death, activation of caspases and cleavage of PARP were dramatically inhibited by pan caspase inhibitor Z-VAD-FMK. DHA down-regulated protein expression and nuclear localization of Sp1, which in turn decreased Sp1 downstream target protein, X-linked inhibitor of apoptosis. Decreased Sp1 protein expression by DHA was restored by proteasome inhibitor MG132. DHA led to a down-regulation of phospho-ERK, -p38 and -JNK without affecting their total forms.

Significance: These results demonstrate that DHA induces caspase-dependent apoptosis in HCC SK-Hep-1 cells by proteasome-dependent degradation of Sp1, which is involved in mitogen-activate protein kinase pathway.