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Retinoid and thiazolidinedione therapies in melanoma: an analysis of differential response based on nuclear hormone receptor expression.

Klopper JP, Sharma V, Berenz A, Hays WR, Loi M, Pugazhenthi U, Said S, Haugen BR - Mol. Cancer (2009)

Bottom Line: A375(DRO) tumor growth was significantly inhibited by either ligand alone and the combination had an additive effect.A375(DRO) sublines resistant to rexinoid, TZD and combination were generated and all three sublines had reduced PPARgamma expression but preserved RXR expression. shRNA knockdown of PPARgamma or RXRgamma attenuated the rexinoid, TZD and combination ligand-mediated decreased proliferation in A375(DRO) cells.Rexinoid (LGD1069) and retinoid (TTNPB) treatment of M14(5-16) cells resulted in decreased proliferation that was additive with combination of both rexinoid and retinoid. shRNA knockdown of RXRgamma resulted in a decreased response to either ligand.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Division of Endocrinology, Metabolism and Diabetes, University of Colorado Denver, Aurora, CO, USA. joshua.klopper@ucdenver.edu

ABSTRACT

Background: Metastatic melanoma has a high mortality rate and suboptimal therapeutic options. Molecular targeting may be beneficial using the rexinoid LGD1069, a retinoid x receptor selective agonist, and thiazolidinediones (TZD), PPARgamma selective ligands, as novel treatments.

Results: Mouse xenograft models with human melanoma cell lines [A375(DRO) or M14(5-16)] were treated for 4 weeks with daily vehicle, RXR agonist (rexinoid, LGD1069, 30 mg/kg/d), PPARgamma agonist (TZD, rosiglitazone, 10 mg/kg/d) or combination. A375(DRO) tumor growth was significantly inhibited by either ligand alone and the combination had an additive effect. M14(5-16) tumors only responded to LGD1069 100 mg/kg/day. A375(DRO) sublines resistant to rexinoid, TZD and combination were generated and all three sublines had reduced PPARgamma expression but preserved RXR expression. shRNA knockdown of PPARgamma or RXRgamma attenuated the rexinoid, TZD and combination ligand-mediated decreased proliferation in A375(DRO) cells. Rexinoid (LGD1069) and retinoid (TTNPB) treatment of M14(5-16) cells resulted in decreased proliferation that was additive with combination of both rexinoid and retinoid. shRNA knockdown of RXRgamma resulted in a decreased response to either ligand.

Conclusion: A375 (DRO) melanoma cell growth is inhibited by rexinoid and TZD treatment, and this response is dependent on RXR and PPARgamma receptor expression. M14 (5-16) melanoma cell growth is inhibited by rexinoid and retinoid treatment, and this response is dependent on RXR expression. These findings may help guide molecular-based treatment strategies in melanoma and provide insight for mechanisms of resistance to nuclear receptor targeted therapies in certain cancers.

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Western blot of nuclear hormone receptors and proliferation in A375(DRO) cells with shPPARγ infection. A. – 60 μg of nuclear protein extract from A375(DRO), the SCR shRNA infected control cell and two clones of shPPARγ infections. Proteins were size-separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with 10% nonfat milk and incubated with RXRγ, RXRα and RARβ primary antibodies and then secondary antibody with anti-rabbit IgG conjugated to horse-radish peroxidase as previously described. β-actin was measured as a loading control. B. – A375(DRO), the SCR infected and two shPPARγ infected sublines were grown in 2% fetal bovine serum RPMI in the presence of 1 umol/L of LGD1069, TZD or the combination for 9 days. Cell growth was analyzed using a nonradioactive cell proliferation assay. Proliferation was compared to that of cells grown in volume equivalent vehicle (DMSO – represented by the line). Proliferation of the SCR infected A375(DRO) was compared to the native cell line to confirm a similar response and then the shPPARγ cell lines were compared to the SCR condition for an assessment of decreased proliferation. Proliferation was statistically significantly attenuated compared to the A375(DRO) SCR subline in all treatment conditions (p < 0.001). Columns, mean; bars, SEM.
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Figure 4: Western blot of nuclear hormone receptors and proliferation in A375(DRO) cells with shPPARγ infection. A. – 60 μg of nuclear protein extract from A375(DRO), the SCR shRNA infected control cell and two clones of shPPARγ infections. Proteins were size-separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with 10% nonfat milk and incubated with RXRγ, RXRα and RARβ primary antibodies and then secondary antibody with anti-rabbit IgG conjugated to horse-radish peroxidase as previously described. β-actin was measured as a loading control. B. – A375(DRO), the SCR infected and two shPPARγ infected sublines were grown in 2% fetal bovine serum RPMI in the presence of 1 umol/L of LGD1069, TZD or the combination for 9 days. Cell growth was analyzed using a nonradioactive cell proliferation assay. Proliferation was compared to that of cells grown in volume equivalent vehicle (DMSO – represented by the line). Proliferation of the SCR infected A375(DRO) was compared to the native cell line to confirm a similar response and then the shPPARγ cell lines were compared to the SCR condition for an assessment of decreased proliferation. Proliferation was statistically significantly attenuated compared to the A375(DRO) SCR subline in all treatment conditions (p < 0.001). Columns, mean; bars, SEM.

Mentions: Western blot analysis demonstrated loss of PPARγ protein in two distinct shPPARγ clones compared with scrambled shRNA control (SCR) or non-infected cells (fig 4a). We confirmed decreased mRNA by qRT-PCR to verify the loss of protein expression was occurring at the mRNA level (data not show). shPPARγ had no effect on RXRα, RXRγ or RARβ protein levels.


Retinoid and thiazolidinedione therapies in melanoma: an analysis of differential response based on nuclear hormone receptor expression.

Klopper JP, Sharma V, Berenz A, Hays WR, Loi M, Pugazhenthi U, Said S, Haugen BR - Mol. Cancer (2009)

Western blot of nuclear hormone receptors and proliferation in A375(DRO) cells with shPPARγ infection. A. – 60 μg of nuclear protein extract from A375(DRO), the SCR shRNA infected control cell and two clones of shPPARγ infections. Proteins were size-separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with 10% nonfat milk and incubated with RXRγ, RXRα and RARβ primary antibodies and then secondary antibody with anti-rabbit IgG conjugated to horse-radish peroxidase as previously described. β-actin was measured as a loading control. B. – A375(DRO), the SCR infected and two shPPARγ infected sublines were grown in 2% fetal bovine serum RPMI in the presence of 1 umol/L of LGD1069, TZD or the combination for 9 days. Cell growth was analyzed using a nonradioactive cell proliferation assay. Proliferation was compared to that of cells grown in volume equivalent vehicle (DMSO – represented by the line). Proliferation of the SCR infected A375(DRO) was compared to the native cell line to confirm a similar response and then the shPPARγ cell lines were compared to the SCR condition for an assessment of decreased proliferation. Proliferation was statistically significantly attenuated compared to the A375(DRO) SCR subline in all treatment conditions (p < 0.001). Columns, mean; bars, SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654861&req=5

Figure 4: Western blot of nuclear hormone receptors and proliferation in A375(DRO) cells with shPPARγ infection. A. – 60 μg of nuclear protein extract from A375(DRO), the SCR shRNA infected control cell and two clones of shPPARγ infections. Proteins were size-separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with 10% nonfat milk and incubated with RXRγ, RXRα and RARβ primary antibodies and then secondary antibody with anti-rabbit IgG conjugated to horse-radish peroxidase as previously described. β-actin was measured as a loading control. B. – A375(DRO), the SCR infected and two shPPARγ infected sublines were grown in 2% fetal bovine serum RPMI in the presence of 1 umol/L of LGD1069, TZD or the combination for 9 days. Cell growth was analyzed using a nonradioactive cell proliferation assay. Proliferation was compared to that of cells grown in volume equivalent vehicle (DMSO – represented by the line). Proliferation of the SCR infected A375(DRO) was compared to the native cell line to confirm a similar response and then the shPPARγ cell lines were compared to the SCR condition for an assessment of decreased proliferation. Proliferation was statistically significantly attenuated compared to the A375(DRO) SCR subline in all treatment conditions (p < 0.001). Columns, mean; bars, SEM.
Mentions: Western blot analysis demonstrated loss of PPARγ protein in two distinct shPPARγ clones compared with scrambled shRNA control (SCR) or non-infected cells (fig 4a). We confirmed decreased mRNA by qRT-PCR to verify the loss of protein expression was occurring at the mRNA level (data not show). shPPARγ had no effect on RXRα, RXRγ or RARβ protein levels.

Bottom Line: A375(DRO) tumor growth was significantly inhibited by either ligand alone and the combination had an additive effect.A375(DRO) sublines resistant to rexinoid, TZD and combination were generated and all three sublines had reduced PPARgamma expression but preserved RXR expression. shRNA knockdown of PPARgamma or RXRgamma attenuated the rexinoid, TZD and combination ligand-mediated decreased proliferation in A375(DRO) cells.Rexinoid (LGD1069) and retinoid (TTNPB) treatment of M14(5-16) cells resulted in decreased proliferation that was additive with combination of both rexinoid and retinoid. shRNA knockdown of RXRgamma resulted in a decreased response to either ligand.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, Division of Endocrinology, Metabolism and Diabetes, University of Colorado Denver, Aurora, CO, USA. joshua.klopper@ucdenver.edu

ABSTRACT

Background: Metastatic melanoma has a high mortality rate and suboptimal therapeutic options. Molecular targeting may be beneficial using the rexinoid LGD1069, a retinoid x receptor selective agonist, and thiazolidinediones (TZD), PPARgamma selective ligands, as novel treatments.

Results: Mouse xenograft models with human melanoma cell lines [A375(DRO) or M14(5-16)] were treated for 4 weeks with daily vehicle, RXR agonist (rexinoid, LGD1069, 30 mg/kg/d), PPARgamma agonist (TZD, rosiglitazone, 10 mg/kg/d) or combination. A375(DRO) tumor growth was significantly inhibited by either ligand alone and the combination had an additive effect. M14(5-16) tumors only responded to LGD1069 100 mg/kg/day. A375(DRO) sublines resistant to rexinoid, TZD and combination were generated and all three sublines had reduced PPARgamma expression but preserved RXR expression. shRNA knockdown of PPARgamma or RXRgamma attenuated the rexinoid, TZD and combination ligand-mediated decreased proliferation in A375(DRO) cells. Rexinoid (LGD1069) and retinoid (TTNPB) treatment of M14(5-16) cells resulted in decreased proliferation that was additive with combination of both rexinoid and retinoid. shRNA knockdown of RXRgamma resulted in a decreased response to either ligand.

Conclusion: A375 (DRO) melanoma cell growth is inhibited by rexinoid and TZD treatment, and this response is dependent on RXR and PPARgamma receptor expression. M14 (5-16) melanoma cell growth is inhibited by rexinoid and retinoid treatment, and this response is dependent on RXR expression. These findings may help guide molecular-based treatment strategies in melanoma and provide insight for mechanisms of resistance to nuclear receptor targeted therapies in certain cancers.

Show MeSH
Related in: MedlinePlus