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Zoledronic acid induces apoptosis and S-phase arrest in mesothelioma through inhibiting Rab family proteins and topoisomerase II actions.

Okamoto S, Jiang Y, Kawamura K, Shingyoji M, Tada Y, Sekine I, Takiguchi Y, Tatsumi K, Kobayashi H, Shimada H, Hiroshima K, Tagawa M - Cell Death Dis (2014)

Bottom Line: ZOL-treated cells decreased a ratio of membrane to cytoplasmic fractions in RhoA, Cdc42 and Rab6 but less significantly Rac1 proteins, indicating that these proteins were possible targets for ZOL-induced actions.ZOL suppressed an endogenous topoisomerase II activity, which was associated with apoptosis and S-phase arrest in respective cells because we detected the same cell cycle changes in etoposide-treated cells.These data demonstrated that anti-tumor effects by ZOL were attributable to inhibited functions of respective small G proteins and topoisomerase II activity, and suggested that cellular factors were involved in the differential cell cycle changes.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chiba, Japan [2] Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan.

ABSTRACT
Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, produced anti-tumor effects through apoptosis induction or S-phase arrest depending on human mesothelioma cells tested. An addition of isoprenoid, geranylgeraniol but not farnesol, negated these ZOL-induced effects, indicating that the ZOL-mediated effects were attributable to depletion of geranylgeranyl pyrophosphates which were substrates for prenylation processes of small guanine-nucleotide-binding regulatory proteins (small G proteins). ZOL-treated cells decreased a ratio of membrane to cytoplasmic fractions in RhoA, Cdc42 and Rab6 but less significantly Rac1 proteins, indicating that these proteins were possible targets for ZOL-induced actions. We further analyzed which small G proteins were responsible for the three ZOL-induced effects, caspase-mediated apoptosis, S-phase arrest and morphological changes, using inhibitors for respective small G proteins and siRNA for Cdc42. ZOL-induced apoptosis is due to insufficient prenylation of Rab proteins because an inhibitor of geranlygeranyl transferase II that was specific for Rab family proteins prenylation, but not others inhibitors, activated the same apoptotic pathways that ZOL did. ZOL suppressed an endogenous topoisomerase II activity, which was associated with apoptosis and S-phase arrest in respective cells because we detected the same cell cycle changes in etoposide-treated cells. Inhibitors for geranlygeranyl transferase I and for RhoA produced morphological changes and disrupted actin fiber structures, both of which were similar to those by ZOL treatments. These data demonstrated that anti-tumor effects by ZOL were attributable to inhibited functions of respective small G proteins and topoisomerase II activity, and suggested that cellular factors were involved in the differential cell cycle changes.

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Influence of geranylgeranyl transferase I and II inhibition on cell cycle. (a) Cells treated with agents as indicated were subjected to western blot analyses. (b) Lysates of cells treated with agents for 48 h were separated into cytoplasm (c) or membrane (m) fraction and probed with respective antibodies. (c) Differential expression ratios between cytoplasm and membrane fractions in (b) were determined with an imaging analyzer. (d and e) Cells treated with agents as indicated were examined for cell cycle with flow cytometry
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fig3: Influence of geranylgeranyl transferase I and II inhibition on cell cycle. (a) Cells treated with agents as indicated were subjected to western blot analyses. (b) Lysates of cells treated with agents for 48 h were separated into cytoplasm (c) or membrane (m) fraction and probed with respective antibodies. (c) Differential expression ratios between cytoplasm and membrane fractions in (b) were determined with an imaging analyzer. (d and e) Cells treated with agents as indicated were examined for cell cycle with flow cytometry

Mentions: We further investigated the effects of inhibitors for geranylgeranylation, GGTI-298 and NE10790, which inhibit geranylgeranyl transferase I and II, respectively (Supplementary Figure S1). GGTI-298 treatments increased unprenylated Rap1A and whole RhoA levels as shown in ZOL-treated cells, but did not influence Rab6 expression (Figure 3a). The majority of RhoA in GGTI-298-treated cells was distributed in cytoplasmic fractions but that of Rab6 remained in membrane fractions (Figures 3b and c). In addition, GGTI-298 treatments upregulated Rac1 expression with enhanced GTP-binding Rac1 in MSTO-211H and less significantly in EHMES-10 cells (Figure 2c). NE10790 treatments did not influence the levels of unprenylated Rap1A or total RhoA expression, or fractionated RhoA distributions (Figures 3a–c). In contrast, NE10790-treated cells showed downregulation of Rab6 translocation to membrane in MSTO-211H and EHMES-10 cells although the expression levels were variable depending on the cells tested. We also examined cell cycle progression with these inhibitors. GGTI-298 increased sub-G1 populations in time- and dose-dependent manners in MSTO-211H cells but scarcely influenced the cell cycle in EHMES-10 cells (Figure 3d). NE10790 also augmented sub-G1 fractions in MSTO-211H cells but had little effects on cell cycle in EHMES-10 cells (Figure 3e). These data indicated that ungeranylgeranylation of Rho and/or Rab family proteins was responsible for ZOL-induced apoptosis but not for S-phase arrest.


Zoledronic acid induces apoptosis and S-phase arrest in mesothelioma through inhibiting Rab family proteins and topoisomerase II actions.

Okamoto S, Jiang Y, Kawamura K, Shingyoji M, Tada Y, Sekine I, Takiguchi Y, Tatsumi K, Kobayashi H, Shimada H, Hiroshima K, Tagawa M - Cell Death Dis (2014)

Influence of geranylgeranyl transferase I and II inhibition on cell cycle. (a) Cells treated with agents as indicated were subjected to western blot analyses. (b) Lysates of cells treated with agents for 48 h were separated into cytoplasm (c) or membrane (m) fraction and probed with respective antibodies. (c) Differential expression ratios between cytoplasm and membrane fractions in (b) were determined with an imaging analyzer. (d and e) Cells treated with agents as indicated were examined for cell cycle with flow cytometry
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260733&req=5

fig3: Influence of geranylgeranyl transferase I and II inhibition on cell cycle. (a) Cells treated with agents as indicated were subjected to western blot analyses. (b) Lysates of cells treated with agents for 48 h were separated into cytoplasm (c) or membrane (m) fraction and probed with respective antibodies. (c) Differential expression ratios between cytoplasm and membrane fractions in (b) were determined with an imaging analyzer. (d and e) Cells treated with agents as indicated were examined for cell cycle with flow cytometry
Mentions: We further investigated the effects of inhibitors for geranylgeranylation, GGTI-298 and NE10790, which inhibit geranylgeranyl transferase I and II, respectively (Supplementary Figure S1). GGTI-298 treatments increased unprenylated Rap1A and whole RhoA levels as shown in ZOL-treated cells, but did not influence Rab6 expression (Figure 3a). The majority of RhoA in GGTI-298-treated cells was distributed in cytoplasmic fractions but that of Rab6 remained in membrane fractions (Figures 3b and c). In addition, GGTI-298 treatments upregulated Rac1 expression with enhanced GTP-binding Rac1 in MSTO-211H and less significantly in EHMES-10 cells (Figure 2c). NE10790 treatments did not influence the levels of unprenylated Rap1A or total RhoA expression, or fractionated RhoA distributions (Figures 3a–c). In contrast, NE10790-treated cells showed downregulation of Rab6 translocation to membrane in MSTO-211H and EHMES-10 cells although the expression levels were variable depending on the cells tested. We also examined cell cycle progression with these inhibitors. GGTI-298 increased sub-G1 populations in time- and dose-dependent manners in MSTO-211H cells but scarcely influenced the cell cycle in EHMES-10 cells (Figure 3d). NE10790 also augmented sub-G1 fractions in MSTO-211H cells but had little effects on cell cycle in EHMES-10 cells (Figure 3e). These data indicated that ungeranylgeranylation of Rho and/or Rab family proteins was responsible for ZOL-induced apoptosis but not for S-phase arrest.

Bottom Line: ZOL-treated cells decreased a ratio of membrane to cytoplasmic fractions in RhoA, Cdc42 and Rab6 but less significantly Rac1 proteins, indicating that these proteins were possible targets for ZOL-induced actions.ZOL suppressed an endogenous topoisomerase II activity, which was associated with apoptosis and S-phase arrest in respective cells because we detected the same cell cycle changes in etoposide-treated cells.These data demonstrated that anti-tumor effects by ZOL were attributable to inhibited functions of respective small G proteins and topoisomerase II activity, and suggested that cellular factors were involved in the differential cell cycle changes.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Pathology and Cell Therapy, Chiba Cancer Center Research Institute, Chiba, Japan [2] Department of Respirology, Graduate School of Medicine, Chiba University, Chiba, Japan.

ABSTRACT
Zoledronic acid (ZOL), a nitrogen-containing bisphosphonate, produced anti-tumor effects through apoptosis induction or S-phase arrest depending on human mesothelioma cells tested. An addition of isoprenoid, geranylgeraniol but not farnesol, negated these ZOL-induced effects, indicating that the ZOL-mediated effects were attributable to depletion of geranylgeranyl pyrophosphates which were substrates for prenylation processes of small guanine-nucleotide-binding regulatory proteins (small G proteins). ZOL-treated cells decreased a ratio of membrane to cytoplasmic fractions in RhoA, Cdc42 and Rab6 but less significantly Rac1 proteins, indicating that these proteins were possible targets for ZOL-induced actions. We further analyzed which small G proteins were responsible for the three ZOL-induced effects, caspase-mediated apoptosis, S-phase arrest and morphological changes, using inhibitors for respective small G proteins and siRNA for Cdc42. ZOL-induced apoptosis is due to insufficient prenylation of Rab proteins because an inhibitor of geranlygeranyl transferase II that was specific for Rab family proteins prenylation, but not others inhibitors, activated the same apoptotic pathways that ZOL did. ZOL suppressed an endogenous topoisomerase II activity, which was associated with apoptosis and S-phase arrest in respective cells because we detected the same cell cycle changes in etoposide-treated cells. Inhibitors for geranlygeranyl transferase I and for RhoA produced morphological changes and disrupted actin fiber structures, both of which were similar to those by ZOL treatments. These data demonstrated that anti-tumor effects by ZOL were attributable to inhibited functions of respective small G proteins and topoisomerase II activity, and suggested that cellular factors were involved in the differential cell cycle changes.

Show MeSH
Related in: MedlinePlus