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Romidepsin inhibits Ras-dependent growth transformation of NIH 3T3 fibroblasts and RIE-1 epithelial cells independently of Ras signaling inhibition.

Hanker AB, Healy KD, Nichols J, Der CJ - J Mol Signal (2009)

Bottom Line: Despite intensive effort, currently no effective anti-Ras therapies have successfully reached clinical application.Finally, we found that romidepsin did not inhibit Ras activation of the ERK and AKT effector pathways in NIH 3T3 and RIE-1 cells, suggesting that romidepsin does not directly antagonize Ras.Taken together, our results suggest that romidepsin is not selective for Ras-transformed cells and that the anti-tumor activity of romidepsin is not due to direct inhibition of Ras function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. cjder@med.unc.edu.

ABSTRACT

Background: Despite intensive effort, currently no effective anti-Ras therapies have successfully reached clinical application. Previous studies suggest that the histone deacetylatse (HDAC) inhibitor romidepsin, which is currently in clinical trials for the treatment of multiple malignancies, can block Ras-dependent signaling and growth transformation. These studies suggest that mutational activation of Ras may be a useful biomarker for sensitivity to romidepsin and that the anti-tumor activity of this HDAC inhibitor may involve inhibition of Ras effector-mediated signaling.

Results: To rigorously assess romidepsin as an antagonist of Ras, we utilized two well-characterized cell models for Ras transformation. We found that romidepsin blocked the anchorage-dependent and -independent growth of NIH 3T3 fibroblasts and RIE-1 epithelial cells transformed by all three Ras isoforms. However, romidepsin treatment also blocked growth transformation caused by other oncoproteins (B-Raf and ErbB2/Neu), suggesting that romidepsin is not selective for Ras. We also observed striking differences in romidepsin-mediated growth inhibition between transformed NIH 3T3 fibroblasts compared to RIE-1 epithelial cells, suggesting that the mechanism by which romidepsin blocks transformation is dependent on cellular context. Finally, we found that romidepsin did not inhibit Ras activation of the ERK and AKT effector pathways in NIH 3T3 and RIE-1 cells, suggesting that romidepsin does not directly antagonize Ras.

Conclusion: Taken together, our results suggest that romidepsin is not selective for Ras-transformed cells and that the anti-tumor activity of romidepsin is not due to direct inhibition of Ras function.

No MeSH data available.


Related in: MedlinePlus

Romidepsin more significantly alters the morphology of RIE-1 cells than that of NIH 3T3 cells. NIH 3T3 (A) or RIE-1 (B) cells stably expressing the indicated proteins were incubated in complete growth medium supplemented with DMSO (Vehicle), 5 nM romidepsin, or 10 μM U0126 for 48 h. Representative fields of cells were photographed under 10× magnification. Data shown are representative of at least two independent experiments.
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Figure 5: Romidepsin more significantly alters the morphology of RIE-1 cells than that of NIH 3T3 cells. NIH 3T3 (A) or RIE-1 (B) cells stably expressing the indicated proteins were incubated in complete growth medium supplemented with DMSO (Vehicle), 5 nM romidepsin, or 10 μM U0126 for 48 h. Representative fields of cells were photographed under 10× magnification. Data shown are representative of at least two independent experiments.

Mentions: A hallmark of Ras transformation of NIH 3T3 and RIE-1 cells is the appearance of dramatic changes in cell morphology, reflecting reduced cell attachment and spreading. This morphology can be reverted to a more normal morphology with pharmacologic inhibitors of Ras signaling, for example, inhibitors of MEK activation of ERK [33,34]. To determine whether romidepsin can cause morphological reversion of cells transformed by K- and N-Ras, we treated our NIH 3T3 stable cell lines with 1, 5 or 10 nM romidepsin for 48 h (Figure 5 and data not shown). While romidepsin caused slight cell flattening and changes in cell shape (as determined by phase contrast microscopy), it did not cause substantial morphological reversion in NIH 3T3 cells. In contrast, treatment with the MEK inhibitor U0126, which blocks ERK activation, caused more substantial morphological reversion in the Ras- and Raf-transformed NIH-3T3 cells (Figure 5A), suggesting that romidepsin functions by a mechanism distinct from inhibition of Ras- or Raf-mediated MEK-ERK activation.


Romidepsin inhibits Ras-dependent growth transformation of NIH 3T3 fibroblasts and RIE-1 epithelial cells independently of Ras signaling inhibition.

Hanker AB, Healy KD, Nichols J, Der CJ - J Mol Signal (2009)

Romidepsin more significantly alters the morphology of RIE-1 cells than that of NIH 3T3 cells. NIH 3T3 (A) or RIE-1 (B) cells stably expressing the indicated proteins were incubated in complete growth medium supplemented with DMSO (Vehicle), 5 nM romidepsin, or 10 μM U0126 for 48 h. Representative fields of cells were photographed under 10× magnification. Data shown are representative of at least two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2735739&req=5

Figure 5: Romidepsin more significantly alters the morphology of RIE-1 cells than that of NIH 3T3 cells. NIH 3T3 (A) or RIE-1 (B) cells stably expressing the indicated proteins were incubated in complete growth medium supplemented with DMSO (Vehicle), 5 nM romidepsin, or 10 μM U0126 for 48 h. Representative fields of cells were photographed under 10× magnification. Data shown are representative of at least two independent experiments.
Mentions: A hallmark of Ras transformation of NIH 3T3 and RIE-1 cells is the appearance of dramatic changes in cell morphology, reflecting reduced cell attachment and spreading. This morphology can be reverted to a more normal morphology with pharmacologic inhibitors of Ras signaling, for example, inhibitors of MEK activation of ERK [33,34]. To determine whether romidepsin can cause morphological reversion of cells transformed by K- and N-Ras, we treated our NIH 3T3 stable cell lines with 1, 5 or 10 nM romidepsin for 48 h (Figure 5 and data not shown). While romidepsin caused slight cell flattening and changes in cell shape (as determined by phase contrast microscopy), it did not cause substantial morphological reversion in NIH 3T3 cells. In contrast, treatment with the MEK inhibitor U0126, which blocks ERK activation, caused more substantial morphological reversion in the Ras- and Raf-transformed NIH-3T3 cells (Figure 5A), suggesting that romidepsin functions by a mechanism distinct from inhibition of Ras- or Raf-mediated MEK-ERK activation.

Bottom Line: Despite intensive effort, currently no effective anti-Ras therapies have successfully reached clinical application.Finally, we found that romidepsin did not inhibit Ras activation of the ERK and AKT effector pathways in NIH 3T3 and RIE-1 cells, suggesting that romidepsin does not directly antagonize Ras.Taken together, our results suggest that romidepsin is not selective for Ras-transformed cells and that the anti-tumor activity of romidepsin is not due to direct inhibition of Ras function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. cjder@med.unc.edu.

ABSTRACT

Background: Despite intensive effort, currently no effective anti-Ras therapies have successfully reached clinical application. Previous studies suggest that the histone deacetylatse (HDAC) inhibitor romidepsin, which is currently in clinical trials for the treatment of multiple malignancies, can block Ras-dependent signaling and growth transformation. These studies suggest that mutational activation of Ras may be a useful biomarker for sensitivity to romidepsin and that the anti-tumor activity of this HDAC inhibitor may involve inhibition of Ras effector-mediated signaling.

Results: To rigorously assess romidepsin as an antagonist of Ras, we utilized two well-characterized cell models for Ras transformation. We found that romidepsin blocked the anchorage-dependent and -independent growth of NIH 3T3 fibroblasts and RIE-1 epithelial cells transformed by all three Ras isoforms. However, romidepsin treatment also blocked growth transformation caused by other oncoproteins (B-Raf and ErbB2/Neu), suggesting that romidepsin is not selective for Ras. We also observed striking differences in romidepsin-mediated growth inhibition between transformed NIH 3T3 fibroblasts compared to RIE-1 epithelial cells, suggesting that the mechanism by which romidepsin blocks transformation is dependent on cellular context. Finally, we found that romidepsin did not inhibit Ras activation of the ERK and AKT effector pathways in NIH 3T3 and RIE-1 cells, suggesting that romidepsin does not directly antagonize Ras.

Conclusion: Taken together, our results suggest that romidepsin is not selective for Ras-transformed cells and that the anti-tumor activity of romidepsin is not due to direct inhibition of Ras function.

No MeSH data available.


Related in: MedlinePlus