In HepG2 cells, although all retinoids examined significantly decreased sellectchem cell viability, only 9 cis retinoic acid induced weak caspase 3 cleavage. In Hep3B cells, six out of thirteen retinoids Inhibitors,Modulators,Libraries decreased cell viability, whereas another three retinoids increased cell number. 9 cis retinoic acid, fenretinide, TTNPB, and lutein induced strong caspase 3 cleavage in Hep3B cells. These findings indicate that fen retinide induces apoptosis in both Huh 7 and Hep3B cells, but Inhibitors,Modulators,Libraries not in HepG2 cells. To further confirm the differential responses of Huh 7 and HepG2 cells to fenretinide, both cell lines were treated with fenretinide and assessed for caspase 3 cleav age, DNA double strand breaks, and phosphatidylserine translocation in a time course study.
In Huh 7 cells, cas pase 3 cleavage was detected at as early as 24 hours after treatment, and the induction was sustained at Inhibitors,Modulators,Libraries 48 and 72 hours. In HepG2 cells, however, even after 72 hours treat ment, no obvious caspase 3 cleavage was detected. DNA double strand breaks, another hallmark of apoptosis, assessed by the TUNEL assay, were detected in Huh 7 cells after treatment. In contrast, no sig nificant increase in DNA double strand breaks was detected in HepG2 cells after treatment. Some background TUNEL staining was detected in HepG2 cells possibly due to the endogenous peroxidase activity. In addition, during apoptosis, membrane lipid phosphati dylserine translocates from the inner leaflet of the plasma membrane to the outer leaflet, resulting in loss of cell membrane asymmetry.
Fenretinide induced phosphati dylserine translocation in Huh 7 cells in a time depend ent manner, reaching 17 fold after 72 hours. However, fenretinide failed to induce such changes in HepG2 cells even after a 3 day treatment. Taken together, these findings convincingly demonstrate that Huh 7 cells are susceptible to fenretinide Inhibitors,Modulators,Libraries induced apoptosis, but HepG2 cells are resistant. High basal and inducibility of RAR gene expression in HCC cells is associated with their susceptibility to fenretinide induced apoptosis To determine whether nuclear receptors mediate the apoptotic effect of fenretinide in HCC cells, the basal mRNA levels of twelve nuclear receptors were assessed by real time PCR. Among the three cell lines, Huh 7 cells have the highest basal mRNA levels of RAR, RXR, and an orphan nuclear receptor Nurr1.
Hep3B cells have the second highest mRNA levels of RAR and Nurr1. In contrast, in HepG2 cells, Inhibitors,Modulators,Libraries the basal mRNA level of RAR is undetectable. On the other hand, in HepG2 cells, the basal mRNA levels of SXR and CAR, two xenobiotic sensors that mediate many xenobiotic responses, 17-AAG are the highest among the three cell clines. The regulation of these twelve nuclear receptor genes by fenretinide were then evaluated in Huh 7 and HepG2 cells by real time PCR.