Volume 42, Issue 2, Pages 230-237 (February 2005) Potent induction of apoptosis in human hepatoma cell lines by targeted cytotoxic somatostatin analogue AN-238  Malika Lasfer, Nathalie Vadrot, Andrew V. Schally, Attila Nagy, Gabor Halmos, Dominique Pessayre, Gérard Feldmann, Florence J. Reyl-Desmars  Journal of Hepatology  Volume 42, Issue 2, Pages 230-237 (February 2005) DOI: 10.1016/j.jhep.2004.10.014 Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 1 (A) Representative Scatchard plot derived from specific radioiodinated RC-160 binding to the membrane fraction isolated from Hep3B human hepatoma cells. Specific binding was determined as described [24]. Each point represents the mean of triplicate determinations. (B) Representative displacement of radioiodinated RC-160 binding to membrane fractions of Hep3B human hepatoma cells by increasing concentrations of cytotoxic somatostatin analogue AN-238. One hundred percent specific binding is defined as difference between binding in the absence and that in the presence of 10μM of RC-160. Each data point represents mean of duplicate or triplicate determinations. Journal of Hepatology 2005 42, 230-237DOI: (10.1016/j.jhep.2004.10.014) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Analysis by RT-PCR of the expression of hsst2A,5 in HepG2 and Hep3B cell lines (A) and rsst5,3,2A in rat tumoral H4IIE cell line and normal isolated hepatocytes (B). PCR products obtained from HepG2, Hep3B, H4IIE cells or hepatocytes were separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. Sizes of expected PCR products were: 1128bp (hsst2A), 1119bp (hsst5), 1140bp (rsst2A), 1170bp (rsst5). M: 1kb Plus DNA ladder markers (Invitrogen, Cergy Pontoise, France). Journal of Hepatology 2005 42, 230-237DOI: (10.1016/j.jhep.2004.10.014) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 3 AN-238 is more powerful than AN-201 in inducing the sub-G1 phase in HepG2 and Hep3B cell-lines. Cells were exposed or not (control) for 15min to 10nM AN-201 or AN-238. After 24h of growth, DNA was stained with propidium iodide and the proportion of cells in different cycles was analyzed by flow cytometry. The sub-G1 phase was indicated by an arrow. One representative of three experiments is shown (right panel). The proportion of cells in the sub-G1 phase relative to total cells was estimated (left panel). Mean values±SEM of three experiments are shown. Significantly different from AN-201 (***P<0.001). Journal of Hepatology 2005 42, 230-237DOI: (10.1016/j.jhep.2004.10.014) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 4 AN-238 induces DNA fragmentation that can be blocked by the peptide carrier RC-121 in HepG2 (left panel) and Hep3B (right panel) cell lines. Exposure of cells to 10nM AN-201 and AN-238 for 15min was performed in the absence or presence of 10μM RC-121.Control cells received no treatment. After 24h of growth, low molecular weight DNA fragments were isolated and subjected (20μg) to electrophoresis on 1% agarose gel. M: 1kb Plus DNA ladder markers (Invitrogen, Cergy Pontoise, France). Journal of Hepatology 2005 42, 230-237DOI: (10.1016/j.jhep.2004.10.014) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 5 AN-238 induces the formation of nuclear bodies and PARP cleavage. HepG2 (upper panel) and Hep3B (lower panel) cell lines were exposed or not (control) for 15min to 10nM AN-238. After 24h of growth, cells were stained with DAPI and the presence of nuclear bodies (indicated by white arrows) was shown by UV microscopy. The PARP cleavage into a fragment of 85kDa (see black arrows) was also examined from nuclear extracts by western blotting with anti-PARP antibody. Journal of Hepatology 2005 42, 230-237DOI: (10.1016/j.jhep.2004.10.014) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 6 AN-201 and AN-238 induce ROS production. HepG2 and Hep3B cell lines were exposed or not (control) for 15min to 10nM AN-201 or AN-238. ROS production was measured as the percentage relative to that obtained with 5mM tert-butylhydroperoxide used to determine the 100%. Mean values±SEM of three experiments with three replicates are presented. Significantly different from control (***P<0.001 and **P<0.01). Significantly different from AN-201 (*P<0.01 and **P<0.001). Journal of Hepatology 2005 42, 230-237DOI: (10.1016/j.jhep.2004.10.014) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions

Fig. 7 Cell survival (A) and DNA fragmentation (B) of rat normal isolated hepatocytes and H4IIE hepatocellular carcinoma cell line. Cells were exposed or not (control) for 15min to 10nM RC-121, DOX, AN-201 and AN-238. After 24h, cell survival was measured by MTT assay. Mean values±SEM of three experiments with four replicates are represented. Significantly different from control (***P<0.001 and **P<0.01, respectively). DNA fragmentation from hepatocytes and H4IIE cells was examined by agarose gel electrophoresis. M: 1kb Plus DNA ladder markers (Invitrogen, Cergy Pontoise, France). One of three representative experiments is shown. Journal of Hepatology 2005 42, 230-237DOI: (10.1016/j.jhep.2004.10.014) Copyright © 2004 European Association for the Study of the Liver Terms and Conditions