Dissecting the dilemma betweenin vitroandin vivodrug screening: treating HepG2 cells with Artesunate as a model

AbstractThe dilemma betweenin vitroandin vivodrug screening results persisted to hinder preclinical anti-cancer drug development. In this study, drug screening was initially performed on monolayer cultures of HepG2 cells, whereasin vivodrug testing was performed on the subcutaneous xenograft mouse model of HepG2 cells in athymic nude mice. Results showed that Artesunate inhibited HepG2 cell growthin vitro, but was ineffectivein vivo. Hence, we investigated the difference betweenin vitroandin vivosettings using this cell-drug combination as a model. Immuno-staining of hepatocellular carcinoma (HCC) biomarkers showed that α-fetoprotein (AFP) was unaffected by Artesunate treatmentin vitroorin vivo, suggesting that AFP was neither a biomarker nor response indicator. Alternatively, high albumin was detected in both monolayer and organoid cultures; contrariwise, the xenograft tumors prevailed low albumin, in consistency to human HCC with poor prognosis. Artesunate treatment reduced intracellular albumin expressionin vitro; Artesunate did not alter tissue and serum albumin in xenograft mice, in coincidence with its irresponsivenessin vivo. However, Artesunate binding to albumin was undetectable. Instead, we observed a transient stimulation of Erk1/2 phosphorylation followed by DAPK1 dephosphorylation and apoptosis. Combined treatment of Artesunate with U0126 revoked Erk1/2-DAPK1 phosphorylation and exerted modest proliferative advantage at early time points, but eventually did not rescue HepG2 cells from death. U0126 induced multiploid formation independent of Artesunate, resulting in cell cycle arrest within 24 h post-treatment.SignificanceSystematic investigation of HCC biomarkers AFP and albumin among thein vitromodels of monolayer and organoid cultures, and thein vivomodels of subcutaneous and liver implantation xenograft mice was conducted.