Volume 44, Issue 1, Pages 142-150 (January 2006) Pamidronate induced anti-proliferative, apoptotic, and anti-migratory effects in hepatocellular carcinoma  Akira Wada, Koji Fukui, Yoshiyuki Sawai, Kazuho Imanaka, Shinichi Kiso, Shinji Tamura, Iichiro Shimomura, Norio Hayashi  Journal of Hepatology  Volume 44, Issue 1, Pages 142-150 (January 2006) DOI: 10.1016/j.jhep.2005.09.022 Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 1 Effects of pamidronate on human HCC cell proliferation. PLC/PRF/5, HepG2, Hep3B, Huh7, and Mahlavu cells (5×104/well) were seeded in 96-well plates and after 24h treated with the indicated concentrations of pamidronate or vehicle control for 72h. Proliferation was measured by means of (A) MTT assay and (B) BrdU assay. The growth of control cells was set as 100% and the proliferation of treated cells was calculated as a percentage of the control. The values represent the mean of three experiments±SD. Journal of Hepatology 2006 44, 142-150DOI: (10.1016/j.jhep.2005.09.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 2 Effects of pamidronate on the Ras/ERK 2 pathway in PLC/PRF/5 cells by immunoblotting. Pamidronate prevents the Ras from being located on the cell membrane. The membrane was also probed with β-actin to confirm equal loading. Pamidronate inhibited the phosphorylation of ERK 2 in total cell lysates while no changes in the expression of ERK 2 were found. Journal of Hepatology 2006 44, 142-150DOI: (10.1016/j.jhep.2005.09.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 3 Effects of pamidronate on apoptosis in HCC cells. (A) Representation morphological changes in apoptosis in PLC/PRF/5 cells for apoptotic nuclei were obtained by DAPI staining. Chromatin condensation, one of the key features of apoptosis, was induced after pamidronate treatment. (B) Time course of pamidronate induced apoptosis valued for DAPI staining (n=4). The means±SD are shown. P<0.0001 vs. control. (C) (D) DNA-fragmentation in HCC cells evaluated for 24–72h was quantified using histone ELISA assay detecting cytoplasmic nucleosomes (n=4). (C) PLC/PRF/5, (D) Huh7. The means±SD are shown. P<0.0001 vs. control. (E) The effect of farnesol on apoptosis induced by pamidronate. PLC/PRF/5 cells were pretreated with 10μM pamidronate. Farnesol, a mevalonate pathway intermediate was added at a level of 100μM to the MTT assay, and the inserts were incubated for 72h. The apoptosis of control cells was set as 100% and the apoptosis of treated cells was calculated as a percentage of the control. The values represent the mean of three experiments±SD. Journal of Hepatology 2006 44, 142-150DOI: (10.1016/j.jhep.2005.09.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 4 Caspase dependencies of pamidronate-induced apoptosis in PLC/PRF/5 cells. Caspase-3 and caspase-9 in PLC/PRF/5 cells after pamidronate treatment for 48h were detected by immunoblotting analysis. Bcl-2 was not involved in the pamidronate-induced apoptosis of HCC. Journal of Hepatology 2006 44, 142-150DOI: (10.1016/j.jhep.2005.09.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 5 (A) Inhibition of the RhoA/ROCK-1 migration pathways by pamidronate in PLC/PRF/5 cells. Pamidronate prevents the RhoA from being located on the cell membrane, and inhibits the expression of ROCK-1 in total cell lysates. The membranes were also probed with β-actin to confirm equal loading. (B) (C) Inhibition of migration in HCC cells by pamidronate. HCC cells were incubated in DMEM supplemented with 0–1–10–20μM pamidronate with b-FGF (100ng/ml). After incubation, migration was measured as described in Section 2 (n=4). The ratio of migrated cells to viable cells at control cells were set as 100% and the ratio of migrated cells to viable cells at treated cells were calculated as a percentage of the control. (B) PLC/PRF/5, (C) Huh7. The values represent the mean of three experiments±SD. *P<0.0001 and **P<0.003 vs. control. Journal of Hepatology 2006 44, 142-150DOI: (10.1016/j.jhep.2005.09.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions

Fig. 6 (A) Tumor growth inhibition by pamidronate in vivo. PLC/PRF/5 cells (1×107) were inoculated s.c. into the dorsal region of nude mice (15 mice in each group). After visualizing the tumor, 1–4mg/kg/day of pamidronate was injected i.p. at daily intervals into the mice. Tumor volumes (mm3) were expressed as width2×length×0.52. Tumor growth rate was the ratio at the time-beginning of tumor volumes. The means±SE are shown. Each pamidronate injection group P<0.01 vs. control vehicle at three weeks. Open circles denote controls; open squares pamidronate at 1mg/kg/day, open triangles pamidronate at 2mg/kg/day, open diamonds pamidronate at 4mg/kg/day. *P<0.05 and **P<0.01 vs. control. (B) Microscopic findings of the tumor and liver stained by the TUNEL method following treatment with pamidronate. Original magnification: ×200. Journal of Hepatology 2006 44, 142-150DOI: (10.1016/j.jhep.2005.09.022) Copyright © 2005 European Association for the Study of the Liver Terms and Conditions