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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 in A375(DRO) resistant cell lines. 3a: 60 μg of nuclear protein extract from the resistant A375(DRO) sublines was size-separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with 10% nonfat milk and incubated with RXRγ (MS-1343-P NeoMarkers) and RXRα (sc D-20) antibodies at a concentration of 1:500 and PPARγ (H-100) rabbit polyclonal ab (sc-7196, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:500. Secondary antibodies were anti-rabbit IgG conjugated to horse-radish peroxidase at a 1:5000 dilution for RXRs and 1:1000 for PPARγ (GE Healthcare UK). β-Actin was measured as a loading control. 3b: PPARγ receptor to β-Actin ratio was calculated using an Alpha Innotech alpha imager.
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Figure 3: Western blot of nuclear hormone receptors in A375(DRO) resistant cell lines. 3a: 60 μg of nuclear protein extract from the resistant A375(DRO) sublines was size-separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with 10% nonfat milk and incubated with RXRγ (MS-1343-P NeoMarkers) and RXRα (sc D-20) antibodies at a concentration of 1:500 and PPARγ (H-100) rabbit polyclonal ab (sc-7196, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:500. Secondary antibodies were anti-rabbit IgG conjugated to horse-radish peroxidase at a 1:5000 dilution for RXRs and 1:1000 for PPARγ (GE Healthcare UK). β-Actin was measured as a loading control. 3b: PPARγ receptor to β-Actin ratio was calculated using an Alpha Innotech alpha imager.

Mentions: We next examined nuclear protein levels of RXRα, RXRγ and PPARγ in each of the drug resistant sub-lines of A375(DRO) (fig 3a). Interestingly, RXRα and RXRγ protein levels were unaffected, but PPARγ was lower in all 3 sublines, with the LGD/TZD R cells having 71% less PPARγ receptor relative to DMSO R (fig 3b).


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 in A375(DRO) resistant cell lines. 3a: 60 μg of nuclear protein extract from the resistant A375(DRO) sublines was size-separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with 10% nonfat milk and incubated with RXRγ (MS-1343-P NeoMarkers) and RXRα (sc D-20) antibodies at a concentration of 1:500 and PPARγ (H-100) rabbit polyclonal ab (sc-7196, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:500. Secondary antibodies were anti-rabbit IgG conjugated to horse-radish peroxidase at a 1:5000 dilution for RXRs and 1:1000 for PPARγ (GE Healthcare UK). β-Actin was measured as a loading control. 3b: PPARγ receptor to β-Actin ratio was calculated using an Alpha Innotech alpha imager.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Western blot of nuclear hormone receptors in A375(DRO) resistant cell lines. 3a: 60 μg of nuclear protein extract from the resistant A375(DRO) sublines was size-separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with 10% nonfat milk and incubated with RXRγ (MS-1343-P NeoMarkers) and RXRα (sc D-20) antibodies at a concentration of 1:500 and PPARγ (H-100) rabbit polyclonal ab (sc-7196, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:500. Secondary antibodies were anti-rabbit IgG conjugated to horse-radish peroxidase at a 1:5000 dilution for RXRs and 1:1000 for PPARγ (GE Healthcare UK). β-Actin was measured as a loading control. 3b: PPARγ receptor to β-Actin ratio was calculated using an Alpha Innotech alpha imager.
Mentions: We next examined nuclear protein levels of RXRα, RXRγ and PPARγ in each of the drug resistant sub-lines of A375(DRO) (fig 3a). Interestingly, RXRα and RXRγ protein levels were unaffected, but PPARγ was lower in all 3 sublines, with the LGD/TZD R cells having 71% less PPARγ receptor relative to DMSO R (fig 3b).

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