Limits...
ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma.

Smit MA, Maddalo G, Greig K, Raaijmakers LM, Possik PA, van Breukelen B, Cappadona S, Heck AJ, Altelaar AF, Peeper DS - Mol. Syst. Biol. (2014)

Bottom Line: However, most patients eventually relapse due to drug resistance.We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3.Several proteins were down-regulated, including Rnd3, a negative regulator of ROCK1 kinase.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

No MeSH data available.


Related in: MedlinePlus

ROCK1 silencing sensitizes melanoma cells to BRAF or ERK inhibitionA Cells were transduced with sh-SCR(ambled) as a control or with one of the three different shRNAs against ROCK1 and analyzed for ROCK1 levels by Western blot analysis. β-actin served as a loading control.B, C Cells described in (A) were treated with a dilution series of PLX4720 (B) or SCH772984 (C) for 3 days. Cell viability was determined with a cell titer blue assay. The y-axis represents the percentage of living cells, normalized to cells expressing sh-SCR. Error bars represent standard error of the mean of one representative experiment done in triplicate. Dashed lines represent the change in IC50.D Cells described in (A) were plated and treated with 0.15 μM PLX4720 on the next day. After 3 days, cells were harvested (apoptotic cells in the supernatant were included in the analysis) and analyzed by Western blot. β-actin served as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4300494&req=5

fig05: ROCK1 silencing sensitizes melanoma cells to BRAF or ERK inhibitionA Cells were transduced with sh-SCR(ambled) as a control or with one of the three different shRNAs against ROCK1 and analyzed for ROCK1 levels by Western blot analysis. β-actin served as a loading control.B, C Cells described in (A) were treated with a dilution series of PLX4720 (B) or SCH772984 (C) for 3 days. Cell viability was determined with a cell titer blue assay. The y-axis represents the percentage of living cells, normalized to cells expressing sh-SCR. Error bars represent standard error of the mean of one representative experiment done in triplicate. Dashed lines represent the change in IC50.D Cells described in (A) were plated and treated with 0.15 μM PLX4720 on the next day. After 3 days, cells were harvested (apoptotic cells in the supernatant were included in the analysis) and analyzed by Western blot. β-actin served as a loading control.

Mentions: Four and two different hairpins were depleted for ROCK1 in the PLX4720 and SCH772984 sensitizer screens, respectively (Table1). We next validated this in an independent experiment. Silencing of ROCK1 with three different hairpins resulted in decreased levels of ROCK1 (Fig5A). Confirming our screen results, silencing of ROCK1 resulted in fewer viable cells upon treatment with either PLX4720 or SCH772984 as analyzed by dose response curves (Fig5B and C). To determine whether, indeed, ROCK1 silencing renders the cells more sensitive to death upon PLX4720 treatment, we analyzed the levels of the pro-apoptotic protein-cleaved caspase 3 in treated cells. Treatment of PLX4720 showed increased cleaved caspase 3 levels, consistent with dose response analysis. Although silencing ROCK1 had no effect on cleaved caspase 3 levels under normal conditions, upon treatment with PLX4720, cells with silenced ROCK1 had a further increase in cleaved caspase 3 levels (Fig5D).


ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma.

Smit MA, Maddalo G, Greig K, Raaijmakers LM, Possik PA, van Breukelen B, Cappadona S, Heck AJ, Altelaar AF, Peeper DS - Mol. Syst. Biol. (2014)

ROCK1 silencing sensitizes melanoma cells to BRAF or ERK inhibitionA Cells were transduced with sh-SCR(ambled) as a control or with one of the three different shRNAs against ROCK1 and analyzed for ROCK1 levels by Western blot analysis. β-actin served as a loading control.B, C Cells described in (A) were treated with a dilution series of PLX4720 (B) or SCH772984 (C) for 3 days. Cell viability was determined with a cell titer blue assay. The y-axis represents the percentage of living cells, normalized to cells expressing sh-SCR. Error bars represent standard error of the mean of one representative experiment done in triplicate. Dashed lines represent the change in IC50.D Cells described in (A) were plated and treated with 0.15 μM PLX4720 on the next day. After 3 days, cells were harvested (apoptotic cells in the supernatant were included in the analysis) and analyzed by Western blot. β-actin served as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300494&req=5

fig05: ROCK1 silencing sensitizes melanoma cells to BRAF or ERK inhibitionA Cells were transduced with sh-SCR(ambled) as a control or with one of the three different shRNAs against ROCK1 and analyzed for ROCK1 levels by Western blot analysis. β-actin served as a loading control.B, C Cells described in (A) were treated with a dilution series of PLX4720 (B) or SCH772984 (C) for 3 days. Cell viability was determined with a cell titer blue assay. The y-axis represents the percentage of living cells, normalized to cells expressing sh-SCR. Error bars represent standard error of the mean of one representative experiment done in triplicate. Dashed lines represent the change in IC50.D Cells described in (A) were plated and treated with 0.15 μM PLX4720 on the next day. After 3 days, cells were harvested (apoptotic cells in the supernatant were included in the analysis) and analyzed by Western blot. β-actin served as a loading control.
Mentions: Four and two different hairpins were depleted for ROCK1 in the PLX4720 and SCH772984 sensitizer screens, respectively (Table1). We next validated this in an independent experiment. Silencing of ROCK1 with three different hairpins resulted in decreased levels of ROCK1 (Fig5A). Confirming our screen results, silencing of ROCK1 resulted in fewer viable cells upon treatment with either PLX4720 or SCH772984 as analyzed by dose response curves (Fig5B and C). To determine whether, indeed, ROCK1 silencing renders the cells more sensitive to death upon PLX4720 treatment, we analyzed the levels of the pro-apoptotic protein-cleaved caspase 3 in treated cells. Treatment of PLX4720 showed increased cleaved caspase 3 levels, consistent with dose response analysis. Although silencing ROCK1 had no effect on cleaved caspase 3 levels under normal conditions, upon treatment with PLX4720, cells with silenced ROCK1 had a further increase in cleaved caspase 3 levels (Fig5D).

Bottom Line: However, most patients eventually relapse due to drug resistance.We found many proteins to be induced upon PLX4720 (BRAF inhibitor) treatment that are known to be involved in BRAF inhibitor resistance, including FOXD3 and ErbB3.Several proteins were down-regulated, including Rnd3, a negative regulator of ROCK1 kinase.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

No MeSH data available.


Related in: MedlinePlus