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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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Morphological changes caused by ZOL and small G proteins inhibitors. (a) Microphotographs of ZOL-treated cells as indicated (magnification, × 200). (b) Structure of actin fibers in EHMES-10 cells which were treated with ZOL, 1 μg/ml C3 transferase, 5 μM GGTI-298 or 1 mM NE10790 for 24 h and were stained with Alexa Fluor 488 phalloidin (magnification, × 630)
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fig5: Morphological changes caused by ZOL and small G proteins inhibitors. (a) Microphotographs of ZOL-treated cells as indicated (magnification, × 200). (b) Structure of actin fibers in EHMES-10 cells which were treated with ZOL, 1 μg/ml C3 transferase, 5 μM GGTI-298 or 1 mM NE10790 for 24 h and were stained with Alexa Fluor 488 phalloidin (magnification, × 630)

Mentions: Cells treated with ZOL showed morphological changes, altering fibroblastic into round-shaped configurations and being less adhesive to a culture plate (Figure 5a). ZOL treatments caused the changes in time- and dose-dependent manners and the alterations were inhibited with GGOH but not FOH supplement (Supplementary Figure S3). We then examined which small G proteins were responsible for ZOL-mediated changes using the inhibitors. We focused on the round-shaped configuration in EHMES-10 as an indicator of ZOL-induced morphological changes because MSTO-211H cells treated with ZOL became easily detached from a culture plate and thereby were difficult to judge the change. GGTI-298-treated EHMES-10 cells showed almost the same alterations as shown in ZOL-treated cells, whereas NE10790-treated cells did not exhibit the shape changes (Supplementary Figure S4). These data indicated that ungeranylgeranylated Rab family proteins were irrelevant to the ZOL-mediated morphological changes. EHMES-10 cells treated with C3 transferase or Cdc42 siRNA but not with NSC23766 showed the round-shaped morphology although degree of the spheroid shape was lesser than that of GGTI-298-treated cells (Supplementary Figure S5). We also examined actin stress fiber structures as an indicator of the ZOL-induced cytoskeletal deformation (Figure 5b). Phalloidin-stained cells showed that ZOL treatments suppressed the actin stress fiber formation, and that C3 transferase-treated and GGTI-298-treated cells exhibited similar fiber-disrupted patterns. In contrast, NE10790 treatments did not influence the actin stress fiber conformations. These data suggested that morphological changes induced by ZOL treatments were due to inhibition of RhoA and Cdc42.


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)

Morphological changes caused by ZOL and small G proteins inhibitors. (a) Microphotographs of ZOL-treated cells as indicated (magnification, × 200). (b) Structure of actin fibers in EHMES-10 cells which were treated with ZOL, 1 μg/ml C3 transferase, 5 μM GGTI-298 or 1 mM NE10790 for 24 h and were stained with Alexa Fluor 488 phalloidin (magnification, × 630)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: Morphological changes caused by ZOL and small G proteins inhibitors. (a) Microphotographs of ZOL-treated cells as indicated (magnification, × 200). (b) Structure of actin fibers in EHMES-10 cells which were treated with ZOL, 1 μg/ml C3 transferase, 5 μM GGTI-298 or 1 mM NE10790 for 24 h and were stained with Alexa Fluor 488 phalloidin (magnification, × 630)
Mentions: Cells treated with ZOL showed morphological changes, altering fibroblastic into round-shaped configurations and being less adhesive to a culture plate (Figure 5a). ZOL treatments caused the changes in time- and dose-dependent manners and the alterations were inhibited with GGOH but not FOH supplement (Supplementary Figure S3). We then examined which small G proteins were responsible for ZOL-mediated changes using the inhibitors. We focused on the round-shaped configuration in EHMES-10 as an indicator of ZOL-induced morphological changes because MSTO-211H cells treated with ZOL became easily detached from a culture plate and thereby were difficult to judge the change. GGTI-298-treated EHMES-10 cells showed almost the same alterations as shown in ZOL-treated cells, whereas NE10790-treated cells did not exhibit the shape changes (Supplementary Figure S4). These data indicated that ungeranylgeranylated Rab family proteins were irrelevant to the ZOL-mediated morphological changes. EHMES-10 cells treated with C3 transferase or Cdc42 siRNA but not with NSC23766 showed the round-shaped morphology although degree of the spheroid shape was lesser than that of GGTI-298-treated cells (Supplementary Figure S5). We also examined actin stress fiber structures as an indicator of the ZOL-induced cytoskeletal deformation (Figure 5b). Phalloidin-stained cells showed that ZOL treatments suppressed the actin stress fiber formation, and that C3 transferase-treated and GGTI-298-treated cells exhibited similar fiber-disrupted patterns. In contrast, NE10790 treatments did not influence the actin stress fiber conformations. These data suggested that morphological changes induced by ZOL treatments were due to inhibition of RhoA and Cdc42.

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