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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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Proliferation response of M14(5–16) with shRXRg infection and western blot of these cells. A – M14(5–16), the SCR infected and the shRXRγ infected subline was grown in 2% fetal bovine serum RPMI in the presence of 1 μmol/L of LGD1069, TTNPB or the combination for 6 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). Each treatment condition led to a significant decrease in proliferation compared to control (p < 0.001). The combination of LGD/TTNPB had a statistically significantly greater decrease in proliferation than each alone (*p = 0.02). Proliferation of the SCR infected M14(5–16) was compared to the native cell line to confirm a similar response and then the shRXRγ infected cell line was compared to the SCR condition for an assessment of attenuation of decreased proliferation. Proliferation was significantly attenuated compared to the M14(5–16) SCR subline monotherapy conditions (p ≤ 0.02). Columns, mean; bars, SEM. B – 60 μg of nuclear protein extract from M14(5–16), the SCR shRNA infected control cell and a clone of shRXRg infected cells 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 and RXRα primary antibodies and then secondary antibody with anti-rabbit IgG conjugated to horse-radish peroxidase as previously described. RXRγ is represented as a doublet because of cross-reaction of the RXRγ1 and RXRγ2 isoforms. PARP was measured as a loading control.
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Figure 6: Proliferation response of M14(5–16) with shRXRg infection and western blot of these cells. A – M14(5–16), the SCR infected and the shRXRγ infected subline was grown in 2% fetal bovine serum RPMI in the presence of 1 μmol/L of LGD1069, TTNPB or the combination for 6 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). Each treatment condition led to a significant decrease in proliferation compared to control (p < 0.001). The combination of LGD/TTNPB had a statistically significantly greater decrease in proliferation than each alone (*p = 0.02). Proliferation of the SCR infected M14(5–16) was compared to the native cell line to confirm a similar response and then the shRXRγ infected cell line was compared to the SCR condition for an assessment of attenuation of decreased proliferation. Proliferation was significantly attenuated compared to the M14(5–16) SCR subline monotherapy conditions (p ≤ 0.02). Columns, mean; bars, SEM. B – 60 μg of nuclear protein extract from M14(5–16), the SCR shRNA infected control cell and a clone of shRXRg infected cells 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 and RXRα primary antibodies and then secondary antibody with anti-rabbit IgG conjugated to horse-radish peroxidase as previously described. RXRγ is represented as a doublet because of cross-reaction of the RXRγ1 and RXRγ2 isoforms. PARP was measured as a loading control.

Mentions: We treated M14(5–16) with 1 μM LGD1069, TTNPB or the combination (500 nM of each) for 6 days. There was a modest but significant decrease in proliferation of 24%, 22% and 38% respectively compared to control conditions (p = 0.001). Additionally, the combination provided an additive response that was significant (16% reduction beyond either ligand alone compared to control, p = 0.02) (fig. 6a).


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)

Proliferation response of M14(5–16) with shRXRg infection and western blot of these cells. A – M14(5–16), the SCR infected and the shRXRγ infected subline was grown in 2% fetal bovine serum RPMI in the presence of 1 μmol/L of LGD1069, TTNPB or the combination for 6 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). Each treatment condition led to a significant decrease in proliferation compared to control (p < 0.001). The combination of LGD/TTNPB had a statistically significantly greater decrease in proliferation than each alone (*p = 0.02). Proliferation of the SCR infected M14(5–16) was compared to the native cell line to confirm a similar response and then the shRXRγ infected cell line was compared to the SCR condition for an assessment of attenuation of decreased proliferation. Proliferation was significantly attenuated compared to the M14(5–16) SCR subline monotherapy conditions (p ≤ 0.02). Columns, mean; bars, SEM. B – 60 μg of nuclear protein extract from M14(5–16), the SCR shRNA infected control cell and a clone of shRXRg infected cells 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 and RXRα primary antibodies and then secondary antibody with anti-rabbit IgG conjugated to horse-radish peroxidase as previously described. RXRγ is represented as a doublet because of cross-reaction of the RXRγ1 and RXRγ2 isoforms. PARP was measured as a loading control.
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Related In: Results  -  Collection

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Figure 6: Proliferation response of M14(5–16) with shRXRg infection and western blot of these cells. A – M14(5–16), the SCR infected and the shRXRγ infected subline was grown in 2% fetal bovine serum RPMI in the presence of 1 μmol/L of LGD1069, TTNPB or the combination for 6 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). Each treatment condition led to a significant decrease in proliferation compared to control (p < 0.001). The combination of LGD/TTNPB had a statistically significantly greater decrease in proliferation than each alone (*p = 0.02). Proliferation of the SCR infected M14(5–16) was compared to the native cell line to confirm a similar response and then the shRXRγ infected cell line was compared to the SCR condition for an assessment of attenuation of decreased proliferation. Proliferation was significantly attenuated compared to the M14(5–16) SCR subline monotherapy conditions (p ≤ 0.02). Columns, mean; bars, SEM. B – 60 μg of nuclear protein extract from M14(5–16), the SCR shRNA infected control cell and a clone of shRXRg infected cells 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 and RXRα primary antibodies and then secondary antibody with anti-rabbit IgG conjugated to horse-radish peroxidase as previously described. RXRγ is represented as a doublet because of cross-reaction of the RXRγ1 and RXRγ2 isoforms. PARP was measured as a loading control.
Mentions: We treated M14(5–16) with 1 μM LGD1069, TTNPB or the combination (500 nM of each) for 6 days. There was a modest but significant decrease in proliferation of 24%, 22% and 38% respectively compared to control conditions (p = 0.001). Additionally, the combination provided an additive response that was significant (16% reduction beyond either ligand alone compared to control, p = 0.02) (fig. 6a).

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