Synergy between PPARgamma ligands and platinum-based drugs in cancer - PubMed (original) (raw)

Synergy between PPARgamma ligands and platinum-based drugs in cancer

Geoffrey D Girnun et al. Cancer Cell. 2007 May.

Abstract

PPARgamma is a member of the nuclear receptor family for which agonist ligands have antigrowth effects. However, clinical studies using PPARgamma ligands as a monotherapy failed to show a beneficial effect. Here we have studied the effects of PPARgamma activation with chemotherapeutic agents in current use for specific cancers. We observed a striking synergy between rosiglitazone and platinum-based drugs in several different cancers both in vitro and using transplantable and chemically induced "spontaneous" tumor models. The effect appears to be due in part to PPARgamma-mediated downregulation of metallothioneins, proteins that have been shown to be involved in resistance to platinum-based therapy. These data strongly suggest combining PPARgamma agonists and platinum-based drugs for the treatment of certain human cancers.

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Figures

Figure 1

Figure 1. PPARγ Activation Enhances Carboplatin Growth Inhibition in A549 Lung Adenocarcinoma Cells

(A) A549 cells treated with 0.5 μM of rosiglitazone and indicated doses of carboplatin alone or in combination. (B) A549 cells were treated with 1 μm rosiglitazone, 250 nM GW1929, or carboplatin, alone or in combination. (C) A549 cells were treated with 0.5 μM rosiglitazone alone or in combination with 250 nM GW9662, with and without carboplatin. (D) Interaction of rosiglitazone with different platinum-based drugs. A549 cells treated with 0.5 μM rosiglitazone, 10 μM carboplatin, 1 μM cisplatin, or 1 μM oxaliplatin alone or in combination. Cell number was determined after 7–10 days and expressed as a percent of control cells. Representative experiments, n = 3, mean ± SD. *p < 0.05, **p < 0.01.

Figure 2

Figure 2. Rosiglitazone and Carboplatin Synergize to Suppress Growth in Multiple Cell Types

Cells were treated with the indicated concentrations of carboplatin and 0.5 μM rosiglitazone or (B) 0.2 μM rosiglitazone. (A and B) NSCLC adenocarcinoma, (C) squamous cell carcinoma, (D) adenosquamous cell carcinoma, (E) OVCA420, or (F) OVCA429 ovarian epithelial cancer cell lines were treated with 1 μM rosiglitazone and 10 μM carboplatin alone or in combination. Cell number was determined after 7–10 days and expressed as a percent of control cells. Representative experiments, n = 3 mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Figure 3. Combination Treatment Increases G2-M Arrest and Apoptosis

A549 cells were treated with 0.5 μM rosiglitazone or 10 μM carboplatin alone or in combination. (A) Cell-cycle analysis was determined by PI staining using FACS. n = 3 ± SD. (B) Immunoblotting for cleaved PARP-1. 115 kDa, uncleaved PARP1; 85 kDa, cleaved PARP1. (C) Percentage of apoptotic cells as determined by annexin V-positive cells using FACS analysis. Representative experiments, n = 3 mean ± SD. *p < 0.05, **p < 0.01.

Figure 4

Figure 4. Rosiglitazone Suppresses Several Members of the Metallothionein Gene Family

(A) Heat diagram of cluster analysis for heavy metal binding proteins from microarray data of RNA following treatment with rosiglitazone, carboplatin, or a combination of the two. (B) Real-time PCR for the expression of metallothioneins 1G, 1H, 1X, and IIA following treatment of A549 cells with 1 μM rosiglitazone for 24 hr. (C) Real-time PCR for the expression of metallothioneins 1G, 1H, 1X, and IIA following treatment of A549 cells with 250 nM GW1929 for 24 hr. (D) Metallothionein protein expression in A549 cells following treatment with PPARγ agonists GW1929 (250 nM) or rosiglitazone (1 μM) for 24 hr. (E) A549 cells were treated with 1 μM rosiglitazone alone or in combination with the PPARγ antagonist GW9662 for 24 hr, and real-time PCR was carried out for MT1G, MT1H, MT1X, and MTIIA. *p < 0.05 rosi + GW9662 versus rosiglitazone alone. (F) Ectopic expression of MT1H blunts the synergistic effect of rosiglitazone and carboplatin. A549 cells stably transduced with a control or retrovirus expressing MT1H were treated with 0.5 μM rosiglitazone or 10 μM carboplatin alone or in combination, and the cell number was determined using a hemocytometer after 7 days. Representative experiment, n = 3 mean ± SD. *p < 0.05.

Figure 5

Figure 5. Rosiglitazone and Carboplatin Synergize In Vivo to Reduce Tumor Growth

1 × 107 A549 cells were injected subcutaneously into the flank of nude mice. Once the tumor reached 50–75 mm3, treatments were initiated. (A) Control chow, chow containing 5 mg/kg/day rosiglitazone, 10 mg/kg carboplatin IP two times per week or in combination. (B) Control chow, chow containing 20 mg/kg rosiglitazone/day, 50 mg/kg carboplatin IP two times per week or in combination. Tumor growth was measured two times per week. n = 10 mice per group, mean ± SD. *p < 0.05. (C) Average TUNEL-positive cells per field from paraffin-embedded sections of animals treated with either drug alone or in combination. n = 5–8 tumors per group, 4 fields per tumor section, mean ± SD. (D) PPARγ suppresses MT gene expression in vivo. RNA was isolated from tumors of mice treated with rosiglitazone and carboplatin and expression of the indicated metallothioneins determined. n = 6–9 tumors per group, mean ± SD. *p < 0.05, **p < 0.0005.

Figure 6

Figure 6. Rosiglitazone and Carboplatin Synergize In Vivo in a Spontaneous Tumor Model

Mice were treated with 10 mg/kg AOM once a week for 6 weeks. Following an additional 12 weeks, mice were treated as described in the Experimental Procedures for 6 weeks. Mice were then euthanized and examined for tumor incidence (A) or number of polyps per mouse (B). n = 8–9 mice per group, mean ± SD. *p < 0.05 versus control, rosiglitazone alone, or carboplatin alone.

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