Activation of peroxisome proliferator-activated receptor gamma is crucial for antitumoral effects of 6-iodolactone

Abstract

Background: Molecular iodine (I2) exhibits antiproliferative and apoptotic effects on in vivo and in vitro cancer models. These effects are thought to be mediated by an iodinated arachidonic acid derivative, 6-iodolactone (6IL), and one of the proposed mechanisms is that 6IL activates Peroxisome Proliferator-Activated Receptors type gamma (PPARG). These receptors have been implicated in the inhibition of carcinogenic processes, in addition to their classical role in maintaining lipid and glucose homeostasis. The aim of this study was to determine whether PPARG participates in the 6IL antiproliferative and apoptotic effects on the mammary cancer cell line MCF-7.

Methods: The 6IL/PPARG complex was inhibited by the PPARG antagonist GW9662, in both an endogenous and overexpressed (adenoviral vector infection) context, and stable PPARG-knockdown MCF-7 cells (RNA interference, confirmed with hydrolysis probes and Western blot), were used to corroborate the PPARG participation. 6IL effects on proliferation (measured by Trypan Blue exclusion) and apoptosis (phosphatidylserine identification by flow cytometer) were evaluated in conditions of chemical inhibition (GW9662) and silencing (RNA interference). A wound-healing assay was conducted on wild-type and stable PPARG-knockdown MCF-7 cells to evaluate the antimigrational effect of 6IL. Caspase-8 activity was evaluated to determine if the extrinsic pathway is involved in the effects of 6IL and I2 treatment.

Results: Antiproliferative and pro-apoptotic 6IL effects require the activation of PPARG. In addition, wound-healing assays show that 6IL is able to inhibit MCF-7 cell migration and that PPARG plays a role in this phenomenon. Finally, the data exclude the participation of the extrinsic apoptotic pathway in 6IL- and I2-induced apoptosis.

Conclusions: These results support the previously proposed mechanism, in which the I2 effects are mediated by 6IL, and they provide further support for the use of I2 as coadjuvant in breast cancer treatment.

Figures

Fig. 1

Antiproliferative and apoptotic effect of 6IL is blocked by GW9662 (GW). a MCF-7 cells were treated with increasing GW9662 concentrations to determine the optimal concentration that did not inhibit proliferation; 0.5 μM was selected for use in subsequent experiments. b Proliferation in the presence of 10 μM 6IL and after GW pretreatment was analyzed by the Trypan Blue exclusion assay. c Apoptosis was analyzed by flow cytometry (c, d). Results are expressed as percent change with respect to the control. Data are expressed as mean ± SD (n = 4 independent assays), and the asterisk indicates a significant difference with respect to the control (P < 0.05)

Fig. 2

Overexpression of PPARG enhances antiproliferative and apoptotic effects of 6IL, and these enhancements are partially prevented by GW9662 (GW). a Representative panels of the infection-efficiency assay. MCF-7 cells were infected (24 h) with AdGFP at increasing multiplicities of infection (MOIs). GFP expression is reported as a percentage of the number of events (%). bPPARG expression in AdPPARG-infected cells was analyzed by qPCR and normalized to ACTB expression. c A range of MOIs was analyzed to determine the optimal level with no effect on proliferation; an MOI of 50 was selected for subsequent experiments. d Fatty acid synthase (FASN) expression was analyzed by the qPCR assay, and the results were normalized to ACTB expression. e Proliferation in the presence of 10 μM 6IL and after GW pretreatment was analyzed by the Trypan Blue exclusion assay. f The apoptotic effect of 6IL was analyzed by flow cytometry, and the results are expressed as percent change with respect to the control. Data are expressed as mean ± SD (n = 4 independent assays); the asterisks indicate significant differences with respect to the control (P < 0.05), and different letters indicate significant differences between groups (P < 0.05)

Fig. 3

Knockdown of PPARG blocks the 6IL effects. MCF-7 cells were transfected with a retroviral silencing plasmid (HuSH™) containing a short, specific hairpin with RNA interference against PPARG (RNAi) or containing a scramble sequence (Scramble). a Representative microphotographs of GFP expression in stably transfected MCF-7 cells (20× objective, scale bar 50 μm). bPPARG expression was analyzed by qPCR and normalized to ACTB expression. c PPARG protein was quantified by Western blot and densitometry and is reported as percent change with respect to wild-type (WT) cells. d Proliferation in the presence of 10 μM 6IL was analyzed by Trypan Blue exclusion. e The apoptotic effect of 6IL was analyzed by flow cytometry, and the results are expressed as percent change with respect to the corresponding control. Data are expressed as mean ± SD (n = 4 independent assays), and different letters indicate significant differences between groups (P < 0.05)

Fig. 4

6IL inhibits MCF-7 migration and this effect is partially reverted by PPARG knockdown. Wild type, Scramble- and RNAi-PPARG transfected MCF-7 cells were treated with 10 μM 6IL for 24 h, and the number of cells that had migrated into the scratch during this time was determined (10× objective, scale bar 200 μm, average scratch width 377 μm). a Representative microphotographs of wound-healing assays. b Migrating cells (total number). Data are expressed as mean ± SD (n = 3 independent assays), two-way ANOVA and Tukey’s multiple comparison tests were performed; different letters indicate significant differences between groups (P < 0.05)

Fig. 5

Extrinsic apoptotic pathway is not involved in the induction of apoptosis by 6IL or I2. MCF-7 cells were treated with 10 μM 6IL or 100 μM I2 for 48 h. Caspase-8 activity was analyzed by a colorimetric assay and normalized for the amount of protein. The results are reported as percent change with respect to the control. Data are expressed as mean ± SD (n = 3 independent assays). There were no significant differences between groups