1. Zoledronic Acid Produces Antitumor Effects on Mesothelioma Through Apoptosis and S- Phase Arrest in p53-Independent and Ras prenylation-Independent Manners 
Shinya Okamoto, MS, Kiyoko Kawamura, MS, Quanhai Li, PhD, Makako Yamanaka, MD, PhD, Shan Yang, MS, Toshihiko Fukamachi, PhD, Yuji Tada, MD, PhD, Koichiro Tatsumi, MD, PhD, Hideaki Shimada, MD, PhD, Kenzo Hiroshima, MD, PhD, Hiroshi Kobayashi, PhD, Masatoshi Tagawa, MD, PhD 
Journal of Thoracic Oncology 
Volume 7, Issue 5, Pages (May 2012)
DOI: /JTO.0b013e31824c7d43
Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

2. FIGURE 1
Zoledronic acid (ZOL)-induced cytotoxicities by inducing apoptosis or S-phase arrest. (A) Cells were treated with ZOL for 5 days and the cell viabilities were measured with the WST assay. The relative viabilities were calculated based on the absorbance without any treatments. Means of triplicated samples and SE bars are shown. (B) Flow cytometrical analyses of cell cycles of mesothelioma cells treated with ZOL for 48 hours. (C) Western blot analyses to detect caspase cleavages and cyclin expressions in ZOL-treated cells. Cleaved caspases were undetectable in EHMES-10 cells. (D) Electrophoresis of genomic DNA from MSTO-211H cells treated with ZOL (50 µM) or CDDP (20 µM). A positive control was DNA of apoptotic U937 cells provided by the manufacturer.
Journal of Thoracic Oncology 2012 7, DOI: ( /JTO.0b013e31824c7d43)
Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

3. FIGURE 2
Zoledronic acid (ZOL)-mediated p53 activation and small interfering RNA (siRNA)-mediated p53 down-regulation. (A, B) Cells were treated with (A) CDDP (20 µM) or (B) ZOL and then subjected to Western blot analyses. (C) Cell-cycle analyses of p53-mutated mesothelioma cells 48 hours after ZOL treatments. (D, E) Cells were transfected with 5 nM p53-siRNA or nonspecific control-siRNA for 24 hours and then treated with 50 µM ZOL for 48 hours. They were subjected to (D) Western blot analyses or (E) cell-cycle analyses.
Journal of Thoracic Oncology 2012 7, DOI: ( /JTO.0b013e31824c7d43)
Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

4. FIGURE 3
Geranylgeranylated small G proteins were involved in zoledronic acid (ZOL)-induced effects. (A, B) Cells were treated with (A) ZOL or (B) first treated with farnesol (FOH) or geranylgeraniol (GGOH) for 3 hours and then with ZOL for 48 hours in MSTO-211H cells or for 72 hours in EHMES-10 cells. The cell lysate was subjected to Western blot analyses. Two bands with high (arrow) and low (dotted arrow) molecular weights correspond to unprenylated and prenylated form of Ras and Rap1A. #MSTO-211H cells treated with 50 µM ZOL for 24 hours as a control. (C) Cells were treated with various concentrations of ZOL alone or together with 10 µM GGOH or FOH for 5 days and the relative viabilities were measured with the WST assay. Means of triplicated samples with SE bars are shown. (D) Cell-cycle analyses of cells treated with 50 µM ZOL or 10 µM GGOH alone, or both agents together for 48 hours. (E) Flow cytometrical analyses for δψm. Cells were first treated with GGOH for 3 hours and then with ZOL for 48 hours. Cells were also treated with 0.1% dimethyl sulfoxide as a solvent control or with 50 µM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 1 hour as a control for mitochondrial depolarization. Means of triplicated samples with SE bars are shown. *p < 0.01.
Journal of Thoracic Oncology 2012 7, DOI: ( /JTO.0b013e31824c7d43)
Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

5. FIGURE 4
PD98059 effects on zoledronic acid (ZOL)-induced apoptosis and S-phase arrest. (A, B) Cells were treated with 20 µM PD98059 for 2 hours and then further treated with 50 µM ZOL for 48 or 72 hours. They were subjected to (A) Western blot analyses or (B) cell-cycle analyses (data at 48 hours are shown).
Journal of Thoracic Oncology 2012 7, DOI: ( /JTO.0b013e31824c7d43)
Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions

6. FIGURE 5
Zoledronic acid (ZOL)-mediated antitumor effects in an orthotopic animal model. (A, B) Cells (1 × 106) were inoculated into the pleural cavity in BALB/c nu/nu mice and then ZOL or phosphate buffered saline (PBS) as a control was intrapleurally administered on (A) day 3 or (B) 10. (A) Tumor weights were measured on day 25 (MSTO-211H cells) or 35 (EHMES-10 cells) (n = 6 or 7). (B) Some of the mice intrapleurally injected with MSTO-211H cells (n = 6) were confirmed to bear the tumor development on day 10. The rest of mice were treated with ZOL (n = 8) or PBS (n = 7) on day 10 and were examined for the tumor weight on day 25. SE bars are also shown. *p < 0.05, **p < 0.01.
Journal of Thoracic Oncology 2012 7, DOI: ( /JTO.0b013e31824c7d43)
Copyright © 2012 International Association for the Study of Lung Cancer Terms and Conditions