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. Author manuscript; available in PMC: 2021 Sep 1.
Published in final edited form as: Head Neck. 2020 Jun 9;42(9):2542–2554. doi: 10.1002/hed.26286

Effects of PPAR-γ agonists on oral cancer cell lines: potential horizons for chemopreventives and adjunctive therapies

Jeffrey Adam Hall 1,^, Mark Rusten 2,^, Raed D Abughazaleh 3, Beverly Wuertz 3, Vannesa Souksavong 4, Paul Escher 4, Frank Ondrey 3
PMCID: PMC7657659  NIHMSID: NIHMS1621744  PMID: 32519370

Abstract

Background:

Peroxisome proliferator-activated receptor-gamma (PPAR-γ) activators have anti-cancer effects. Our objective was to determine the effect of PPAR-γ ligands 15-deoxy-D12,14-Prostaglandin J2 (15-PGJ2) and ciglitazone on proliferation, apoptosis, and NF-κB in human oral squamous cell carcinoma cell lines.

Methods:

NA and CA9-22 cells were treated in vitro with 15-PGJ2 and ciglitazone. Proliferation was measured by MTT colorimetric assay and cell cycle analysis performed via flow cytometry, apoptosis by caspase-3 colorimetric assay and poly-(ADP-ribose) polymerase cleavage on Western blot, and NF-κB activation by luciferase assays.

Results:

MTT assays demonstrated dose-dependent decreases after 15-PGJ2 treatment in both cell lines, and S-phase cell cycle arrest was also demonstrated. NF-κB luciferase reporter gene activity decreased 7-fold and 8-fold in NA and CA9-22 cells, respectively. Caspase-3 activity increased 2-fold and 8-fold in NA and CA9-22 cells, respectively.

Conclusions:

Our results suggest these agents, in addition to activating PPAR-γ, can downregulate NF-κB and potentiate apoptosis in oral cancer cells.

Keywords: PPAR-γ; 15-deoxy-D12,14-Prostaglandin J2 (15-PGJ2); Ciglitazone; NF-κB; Squamous cell carcinoma

1. Introduction

Head and neck squamous cell carcinoma (HNSCC) is a devastating disease affecting 65,000 patients and resulting in 14,000 deaths each year in the United States, with the majority of both new cases and deaths attributable to oral carcinoma [1]. Despite advances in surgical and medical treatment with multi-modality protocols and improved understanding of HNSCC molecular biology, the combined five-year survival rate has improved little over several decades [2,3], with improvements in the most recent decade likely being attributable to improvements in screening [4]. Given the extreme morbidity and mortality caused by oral carcinoma and its surgical treatment, as well as the relative stagnancy of survival rates, development of new pharmacologic therapies is of pressing importance.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors belonging to the nuclear hormone receptor family [5]. PPARs heterodimerize with the retinoid X receptors (RXR) and bind to a direct repeat sequence (DR-1) on the six nucleotide direct repeat PPAR response element (PPRE) [6,7], AGGTCA, which is separated by a single base pair (AGGTCA-n-AGGTCA) [8].

In vitro PPAR-γ activation via agonists such as 15-deoxy-D12,14-Prostaglandin J2 (15-PGJ2), troglitazone and pioglitazone has been associated with growth inhibition, cellular differentiation, and cell cycle changes in a variety of solid tumors. 15-PGJ2 has shown favorable effects such as decreased proliferation, increased cell death and cell cycle arrest in studies of prostatic, gastric, and lung adenocarcinoma, as well as esophageal, renal and breast cancer [9-15]. Various thiazolidinediones have shown similar cell-level effects in esophageal adenocarcinoma, renal cell carcinoma, and breast cancer [13-15].

NF-κB is a cytoplasmic transcription factor, which upon activation has numerous procarcinogenic effects via increased transcription of angiogenic factors, growth factors, cellular adhesion molecules, and proteases [16]. Importantly, it promotes apoptosis resistance when constitutively expressed in cancer cells [17]. Further, we have seen upregulation of NF-kB and its downstream targets by cigarette smoke products in aerodigestive cancer cell lines [16,18]. Therefore, abrogation of both constitutive and stimulated NF-kB elevations in aerodigestive cancers could be of therapeutic importance.

In the present study, we expand upon previous work with a variety of PPAR-γ activators to focus on the effects of 15-PGJ2 and ciglitazone, a prototypical thiazolidinedione, on cell growth, cell cycle arrest and NF-κB activity in NA and CA9-22 oral cavity cancer cell lines. These cell lines are common oral cancer models, and have been shown in our prior work to express PPAR-γ [19,20]. Given their PPAR-γ expression and the effects of PPAR-γ activation in other solid tumors, study of the cellular responses of these lines to the agents in question will help to determine whether PPAR-γ agonists may be a useful therapeutic class for the treatment of HNSCC.

2. Materials and methods

2.1. Reagents

10xTBE (ThermoFisher, Waltham, MA), electrophoresis grade acrylamide and bis-acrylamide, PMSF, DTT, NaCl (Sigma, St. Louis, MO). MgCl2, KCl (Quality Biological Corp., Gaithersburg, MD), EDTA (ThermoFisher), 1 M HEPES, Glycerol (ThermoFisher), Ammonium Persulfate, TEMED, 10x TBE (Bio-Rad Laboratories, Richmond, CA). 15-deoxy-delta-12-14-PGJ2 (Cayman Chemical Company, Ann Arbor, MI), Ciglitazone, ETYA (Enzo, Farmingdale NY).

2.2. Cell culture

Mycoplasma free CA9-22 and NA cell lines derived from human oral cavity squamous cell carcinoma tumor specimens [19] were cultured at 37 °C and 5% CO2 as adherent monolayer cultures in RPMI 1640 media supplemented with 2 mM glutamine, 10% heat-inactivated FBS (ThermoFisher), 50 U/mL penicillin, and 50 μg/mL streptomycin.

2.3. MTT assay

Cell proliferation rates of CA9-22 and NA cells in vitro were determined using the MTT reaction assay (Roche, Indianapolis, IN). Ninety-six-well plates, containing 5000 cells/well, were maintained in complete media overnight. The following morning, complete media was replaced with 0%-5% FBS containing media and ciglitazone or 15-PGJ2 in DMSO, or DMSO alone. Cells were incubated for 24 to 96 hours prior to the lysis and assay according to manufacturer instructions using a Tecan plate spectrophotometer at 560 nm using Tecan software. The values were corrected for background level absorbance. All experiments were performed with six replicate data points yielding similar results with at least three repeated experiments.

2.4. Cell cycle distribution analysis

Cell cycle analysis was performed on cells treated with 1, 2.5 and 5 μM 15-PGJ2, and 5 and 10 μM ciglitazone or the vehicle (DMSO) for 24 hours. Experiments evaluating reversibility of cell cycle changes were conducted in media containing 2.5% FBS. Cells were collected by centrifugation, washed with PBS, and fixed in 70% ethanol for 2 hours on ice. Cells were pelleted, washed with PBS, and resuspended in 20 μg/mL propidium iodide (PI) in PBS-containing 200 μg/mL DNAse. Samples were then incubated for 15 minutes at 37°C and analyzed using a FACSCalibur flow cytometer equipped with CELLQuest Pro software (BD Biosciences, San Jose, CA). Assays were performed in triplicate.

2.5. Western blotting

Technique was exactly as previously published with the following modifications [20]. Blots were incubated in primary antibody at 1:1000 (goat anti-involucrin, mouse anti-involucrin, mouse anti-fillagrin, mouse anti-leptin, rabbit anti-aP2) in blocking solution at 4°C overnight. Blots were briefly rinsed, then incubated in secondary antibody 1:2000 (Santa Cruz goat anti-mouse HRP, etc.) for 1 hour at RT. After a brief rinse, the blots were developed using Santa Cruz chemiluminescence reagents according to manufacturer’s recommendations and exposed to film.

2.6. Caspase-3 colorimetric assays

Caspase-3 activity was quantified with a colorimetric assay kit (R&D Systems, Minneapolis, MN) to assess for apoptosis after treatment with 15-PGJ2. NA and CA9-22 cells were grown to 80% confluence and treated with 2.5, 5 and 10 μM 15-PGJ2 in serum-free media for 6 hours along with DMSO controls. Cells were trypsinized and lysed and cytoplasmic extracts were aliquoted in triplicate to 96-well plates. Caspase-3 kit reagent was added (DEVD substrate peptide conjugated to p-nitroaniline) and incubated for 90 minutes. Plates were read at 405 nm on a Tecan model 530 plate spectrophotometer.

2.7. Western Blot for PARP cleavage

Cells were grown in 75 cm2 flasks to 80% confluency in standard media. Cells were treated with ETYA (10 and 20 μM), ciglitazone (9 and 27 μM) and 15-PGJ2 (2 and 5 μM) serum-free media for 12 hours. Nuclear and cytosolic extracts were prepared as previously described [21]. 20 μg nuclear extract were prepared and run exactly as above except on 1.5 mm 10% polyacrylamide gel and run at 20 mA for 2 hours at 4°C. After transfer and blocking, blots were incubated in primary Santa Cruz mouse anti-human PARP antibody, a standard antibody for cleaved analysis, at 1:1000 in blocking solution overnight [21]. Blots were briefly rinsed, then incubated with a secondary antibody at 1:2000 (Santa Cruz goat anti-mouse HRP). Following rinse, the blots were developed using chemiluminescence technique per Santa Cruz reagent’s manufacturer protocol and exposed to film.

2.8. Luciferase reporter gene assays

Assays were performed exactly as described [20] with the NF-κB-luciferase reporter gene structures previously published [16] with the following modifications. Cells were incubated for 48 hours after transfection in standard media and then treated with 2.5, 5, and 10 μM 15-PGJ2 for 6 hours in serum-free media with DMSO controls and then assayed

2.9. Electromobility shift assays

NA and CA9-22 SCCA cell lines were grown to 80% confluence in 75 cm2 cell culture flasks and treated with 15-PGJ2 (2.5, 5 or 10 μM) for 6 hours with DMSO control. The remainder of techniques for extract preparation, binding reaction, and EMSA were precisely as previously published [17]. Anti-NF-κB p65 (RelA) subunit (rabbit) (Rockland, Gilbertsville, PA) supershift antibodies were incubated with selected samples for 1 hour prior to binding with oligonucleotide probes. The binding complexes from the reactions were then resolved on 5% polyacrylamide gels in 0.5x TBE buffer at 20°C run for 90 minutes at 200V. The gels were dried and imaged by phosphorimaging with a Packard Instant Imager™ (Packard Technologies, Downers Grove, IL). Experimental groups were analyzed by densitometry to quantify oligonucleotide probe binding for each concentration of agent and percent decrease from control were calculated. Assays were performed in triplicate.

2.10. Statistical analysis

Levels of statistical significance were evaluated via student’s t-test. Analysis was done using GraphPad software (GraphPad Prism, Carlsbad, CA). P-value <0.05 was considered statistically significant. Bars on graphs are ± SEM.

3. Results

3.1. Serum dependence of reduced proliferation with ligands

First, we confirmed prior findings with both cell lines and demonstrated significant dose-dependent decreased cellular proliferation with either ciglitazone or 15-PGJ2 for 24 to 96 hours. Figure 1A shows by day 4, CA9-22 cells treated with ciglitazone (5, 10, and 20 μM) showed growth inhibition of 7%, 32%, and 74%, respectively, compared to control cells (DMSO), with a five day IC50 of 9.0 μM. Similar data is seen in Figure 1C when NA cells were treated, showing growth inhibition of 20%, 39%, and 45%, respectively, with a five day IC50 of 14.5 μM. Figure 1B and 1D show growth inhibition upon treatment with 15-PGJ2 (1.25, 2.5, 5, and 10 μM). In Figure 1B, growth of CA9-22 cells were inhibited by 13% and 85% at doses of 1.25 and 2.5 μM, respectively, with a five day IC 50 of 2.8 μM. When treated with 5 and 10 μM, the treatment was cytotoxic. In Figure 1D, growth of NA cells was inhibited by 22%, 67%, and 100% at doses of 1.25, 2.5, and 5 μM, respectively, with a five day IC50 of 6.4 μM. At 10 μM, the dose was cytotoxic. These experiments confirm prior analyses we have performed in these cell lines and established a basis for subsequent experiments focusing on NF-κB effects of the PPAR activators.

Figure 1. Effect of ciglitazone or 15-PGJ2 on growth of CA9-22 cells and NA cells.

Figure 1

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Cells were treated with 4 days of control (DMSO) and either ciglitazone (C; 5, 10, and 20 μM) or 15-PGJ2 (P; 1.25, 2.5, 5, and 10 μM). Significantly decreased proliferation was seen with increasing drug concentration when compared to the control in both cell lines *, ( p<0.05). **, (p<0.001). A) CA9-22 cells treated with ciglitazone B) CA9-22 cells treated with 15-PGJ2 C) NA cells treated with ciglitazone D) NA cells treated with 15-PGJ2

Next, we tested the serum dependence of the effects or cytoprotective effects from serum. In experiments using 0%, 2.5% and 5% serum-containing media, the growth inhibiting effects of each treatment were decreased in the presence of increasing concentrations of serum-containing media (Fig. 2 A-D). These findings confirm oral cancer cells benefit from the presence of serum in the culture media, either from serum binding of drug or other cytoprotection afforded by serum, consistent with published data utilizing varying concentrations of serum-containing media, as well as serum-free media [22, 23]. Further, this indicates increased serum concentrations of either agent might be necessary in vivo.

Figure 2. Effects of serum concentration on CA9-22 and NA cells treated with ciglitazone and 15-PGJ2.

Figure 2

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Cells were treated for 5 days in varying doses of ciglitazone (0-50 μM) or 15-PGJ2 (0-20 μM) in culture medium containing varying concentrations of serum (0%, 2.5% and 5%) and were analyzed by MTT cellular proliferation assay. Most concentrations and doses showed significantly different proliferation, with more proliferation observed with increased serum concentration. *, values significantly different from the other concentration of serum at the given drug concentration (p<0.05). **, value significantly different compared to 0% serum condition (p<0.05). A) CA9-22 cells treated with ciglitazone B) CA9-22 cells treated with 15-PGJ2 C) NA cells treated with ciglitazone D) NA cells treated with 15-PGJ2

3.2. Decreased proliferation with PPAR-γ activation is not reversible

We next examined whether the antiproliferative effects of the agents were reversible. Both cell lines were independently treated in culture medium with 2.5% FBS with 15-PGJ2 (2.5 μM), ciglitazone (10 μM), or DMSO control for 48 hours. At 48 hours, culture medium was changed to either control condition or continued treatment condition for an additional 48 hours. In both cell lines at day 4, treatment was withdrawn and fresh media was replenished and at 48 hours showed similar decreases in proliferation. In cell lines treated with 2.5 μM 15-PGJ2, there was a small, statistically significant, recovery. This demonstrates that these agents have irreversible effects on oral cancer cells.

3.3. Ligands of PPAR-γ induce changes in cell cycle profile

We treated both cell lines for 24 hours with vehicle alone (DMSO), 15-PGJ2 (1, 2.5, and 5 μM), or ciglitazone (5 and 10 μM) in serum-free media. Cell cycle analysis demonstrated up to a threefold increase in the proportion of cells in S-phase, correlating with proliferation decreases. The greatest increase in S-phase proportion was seen in CA9-22 cells treated with 5 μM 15-PGJ2 (Fig. 3B). Figure 3C shows results with NA cells treated with 5 μM and 10 μM ciglitazone. Cell cycle analysis was done in at least duplicate and yielded similar results. Data from representative plots are shown in Table 1 A, B, C, and D. These results indicate an S-phase arrest-type phenomenon is achieved by these agents.

Figure 3. Effects on cell cycle profile of 15-PGJ2 and ciglitazone in CA9-22 and NA cells within 24 hours.

Figure 3

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Cells were treated for 24 hours with DMSO (control) and varying concentrations of 15-PGJ2 (1, 2.5 and 5 μM) and ciglitazone (5 and 10 μM), independently. Experiments were done in duplicate, at minimum. Representative data is shown and reveals a two to nearly three-fold increase in the percentage of cells in S-phase. A) CA9-22 cells treated with ciglitazone B) CA9-22 cells treated with 15-PGJ2 C) NA cells treated with ciglitazone D) NA cells treated with 15-PGJ2

Table 1A).

Ca9-22 cells treated with ciglitazone, percentage of cells in each cell cycle phase in duplicate assays

Condition
(μM)		 G1 S G2
Control 76.9 87.4 8.2 6.8 14.9 5.8
C5 57.3 59 24.6 28.9 18.1 12.1
C10 58.1 76.4 22.3 5.6 19.6 18
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Table 1B).

Ca9-22 cells treated with 15-PGJ2, percentage of cells in each cell cycle phase in duplicate assays

Condition
(μM)		 G1 S G2
Control 76.9 87.4 8.2 6.8 14.9 5.8
P1 59.4 77.8 19.2 13.5 21.4 8.7
P2.5 58.9 69.6 22.8 18.1 18.3 12.3
P5 64.7 63.5 23.2 22.9 12.1 13.6
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Table 1C).

NA cells treated with ciglitazone, percentage of cells in each cell cycle phase in duplicate assays

Condition
(μM)		 G1 S G2
Control 89.2 89.2 9.4 9.4 1.5 1.5
C5 79.4 86.7 18 12.5 2.7 0.81
C10 80.6 84.9 15.1 10 4.4 5.1
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Table 1D).

NA cells treated with 15-PGJ2, percentage of cells in each cell cycle phase in duplicate assays

Condition
(μM)		 G1 S G2
Control 92.2 89.2 7.3 9.4 0.5 1.5
P1 91.4 83.6 8 10.3 0.6 6
P2.5 90 85 10 12 0 3
P5 85.49 87.1 8.55 8 5.96 4.9
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To determine if cell cycle changes were reversible, both cell lines were treated with the two ligands for 24 hours, and changed to control conditions for an additional 24 hours. These cells were compared to cells in control conditions and cells which continued under treatment conditions for 48 hours. The cell cycle profile 24 hours after return to control conditions was similar to cells continuing treatment. Figure 4D shows NA cells treated with 15-PGJ2 demonstrate irreversible cell cycle profile change at 48 hours. Figure 4A shows similar results for CA9-22 cells treated with ciglitazone. In all experiments, cells treated and returned to control conditions more closely resembled cells continuing treatment compared with cells only exposed to control conditions.. This indicates the prolonged S-phase arrest we observed after ciglitazone/15-PGJ2 treatment may represent irreversible alteration of the cell cycle.

Figure 4. Reversibility of cell cycle changes with ciglitazone and 15-PGJ2 treatment of CA9-22 and NA cells.

Figure 4

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Cells were treated for 48 hours with DMSO (control) or ciglitazone 10 μM (C10 and C10-removed) or 15-PGJ2 2.5 μM (P2.5 and P2.5-removed). At 48 hours, control cells (DMSO) and C10 cells or P2.5 continued treatment under the same conditions. CA C10-removed or P2.5-removed cells were changed back to control conditions (DMSO). Mean values of MTT data are shown. A) CA9-22 cells treated with ciglitazone. There was no significant difference between the cells continuing treatment for the entire 4 days compared to cells changed back to control conditions. B) CA9-22 cells treated with 15-PGJ2 C) NA cells treated with ciglitazone D) NA cells treated with 15-PGJ2. NA P2.5-removed cells showed increased proliferation at day 4 compared to NA P2.5 cells, *p<0.05, however they continued to more closely resemble cells which continued treatment.

3.4. 15-PGJ2 causes an increase in caspase-3 activity in NA and CA9-22 cells

We next examined whether 15-PGJ2 could initiate apoptosis as a mechanism for the cell death we noted in prior experiments. The effect of 15-PGJ2 on caspase-3 activity was examined with various concentrations of 15-PGJ2 in both cell lines for 6 hours (Fig. 5). Increased caspase-3 activity was observed at 2.5 μM compared to DMSO controls in the NA cells to a maximum inhibition of 2-fold at 10 μM in a dose-dependent manner. A similar dose-dependent increase in caspase-3 activity was observed in CA9-22 cells starting at 2.5 μM with a maximum 8-fold increase at 10 μM. Results with each concentration were analyzed independently by t-test. P-values of ≤0.0127 were calculated for all groups in both cell lines. This indicates that beyond S-phase arrest, apoptosis, as judged by caspase activation, is significantly augmented after treatment with ciglitazone and 15-PGJ2.

Figure 5. Caspase-3 colorimetric assay of CA9-22 (A) and NA (B) cells after treatment with 15-PGJ2.

Figure 5

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Cells were treated for 6 hours with 2.5, 5, and 10 μM 15-PGJ2. Cytoplasmic extracts were then assayed for caspase-3 activity. An 8-fold and 2-fold dose-dependent decrease in caspase-3 activity was seen in CA9-22 and NA cells, respectively.

3.5. 15-PGJ2 causes PARP cleavage by Western blot analysis in CA9-22 cells

To confirm the effects of 15-PGJ2 on caspase 3, we investigated whether PARP cleavage could be augmented. In CA9-22 cells, western blots of nuclear extracts after treatment with 2.5 μM and 5 μM 15-PGJ2 for 12 hours demonstrated PARP cleavage (Fig. 6). Additionally, a dose-dependent response was seen when comparing concentrations of 2.5 μM and 5 μM 15-PGJ2.

Figure 6. Effect of ciglitazone and 15-PGJ2 on PARP cleavage on western blot analysis in CA9-22 cells.

Figure 6

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Western blot analysis of nuclear extracts after treatment with various concentrations of PPAR-ligands: ETYA (10, 20 μM), ciglitazone (9, 27 μM), and 15-PGJ2 (2, 5 μM) for 12 hours with DMSO control. PARP cleavage is seen as two distinct bands in Lanes 7 and 8, confirming the induction of apoptosis in this group. PARP remains uncleaved in Lanes 2-6 (control, ETYA and ciglitazone). There is a dose-dependent response when comparing 2 μM with 5 μM of 15-PGJ2.

3.6. 15-PGJ2 causes decreased activity of NF-κB in NA and CA9-22 cells

Attacking active NF-κB is one putative mechanism for augmenting apoptosis with cancer treatment drugs. Therefore, we examined whether 15-PGJ2 downregulates NF-κB. We performed functional tests of NF-κB activity during the 24-hour period where we observed increased caspase activity (i.e. < 24 hours). Figure 7 shows the results of treatment of both cell lines with varying 15-PGJ2 concentrations for 6 hours. NA cells showed decreased NF-κB activity starting at 2.5 μM and continuing in a dose-dependent manner to a maximum 7-fold inhibition (p ≤ 0.001). CA9-22 cells treated with 15-PGJ2 showed a similar dose-dependent decrease in NF-κB activity to a maximum inhibition of 8-fold at 10 μM (p ≤ 0.001). These data suggest decreasing NF-κB constitutive activation with 15-PGJ2 treatment in oral cancer cells is one possible mechanism to create a permissive environment for apoptosis via caspase activation/PARP cleavage.

Figure 7. NF-κB luciferase reporter gene assays of CA9-22 (A) and NA (B) cells after treatment with 15-PGJ2.

Figure 7

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Cells were transfected with a luciferase reporter plasmid containing an NF-κB recognition sequence. Transfected cells were then treated for 6 hours with 15-PGJ2 (5, 10, and 20 μM). Luciferase activity was assayed using a Dual-Light reporter gene assay system. A dose-dependent 8-fold and 7-fold increase in NF-κB luciferase reporter activity was seen in CA9-22 and NA cells, respectively. **, (p<0.001)

3.7. 15-PGJ2 causes decreased binding of NF-κB oligonucleotide in a dose-dependent manner

In addition to functional reporter gene assay, we examined 15-PGJ2 treatment of NA and CA9-22 cells via EMSA. As shown in Figure 8, treatment of NA cells with various concentrations of 15-PGJ2 for 6 hours followed by nuclear extract analysis shows dose-dependent decreased binding of labeled NF-κB oligonucleotide in cells. Specificity of the oligonucleotide was demonstrated by inhibition of binding by unlabeled (cold) oligonucleotide (lanes 3,7,10,13), but not mutant oligonucleotide (lanes 4,8,11,14). Specificity of binding was further demonstrated by supershift assay utilizing an antibody to the p65 subunit of NF-κB in the control and 2.5 μM treatment group (lanes 5, 15). Inhibition is observed at all concentrations of 15-PGJ2. Inhibition of NF-κB activity was not consistently observed in CA9-22 cell line with EMSA analysis. Bands in lanes 2,6,9, and 12 were analyzed via densitometry revealing a 27%, 40% and 81% decrease in oligonucleotide binding compared to control in the 2.5, 5, and 10 μM groups, respectively.

Figure 8. Electrophoretic mobility shift assay (EMSA) of NA cells treated with 15-PGJ2.

Figure 8

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NA cells were treated with 5, 10, and 20 μM 15-PGJ2 for 6 hours. Nuclear extracts were run on 5% acrylamide gel with P32 labeled NF-κB oligonucleotide and unlabeled (cold) competition, mutant oligonucleotide competition, and NF-κB P65 subunit antibody for supershift. NA cells showed decreased NF-κB expression with 15-PGJ2 treatment. Assays were performed in triplicate.

4. Discussion

Both 15-PGJ2 and ciglitazone administration were associated with decreased proliferation and cell cycle changes. This contrasts with work by Nikitakis et al. showing thiazolidinediones do not have similar effects to 15-PGJ2 in SCCA cells [24]. 15-PGJ2 was more potent than ciglitazone in decreasing proliferation in both cell lines on a molar basis and this data is in agreement with results reported for other solid tumors [10,13,14,25]. This suggests drug class differences come into play when PPAR-γ activators are investigated as cancer therapies, and J-series prostaglandins may act differently than thiazolidinediones.

Dose-dependent proliferation decreases were observed with both drugs. Further, serum-dependence was observed, as judged by IC50 differences when serum concentrations were altered. This varying bioavailability with serum concentration has been studied in more detail in other models [26]. Serum-containing media was used in experiments in which cells were treated for greater than 24 hours, as this was necessary to maintain a sufficient number of viable cells for evaluation. The drug dosages used for the cell cycle analysis included the doses for which significant decreased proliferation was observed via MTT assay. We have reproduced similar effects on proliferation in other cell lines in prior work with ciglitazone, specifically PC-3 (IC50=9-18 μM) and HeLa (IC50=5-10 μM). We have also reproduced similar effects in prior work with the second-generation thiazolidinediones, rosiglitazone and pioglitazone, in HeLa cells, with both showing an IC50=10 μM [27].

15-PGJ2 also caused apoptosis, as determined by colorimetric assay for caspase-3 activity and PARP cleavage. Our cell lines had dose-dependent increases in caspase-3 activity with increasing 15-PGJ2 concentrations. Apoptosis resistance is a significant barrier to improved outcomes of advanced and recurrent HNSCC, and therefore any agent which can facilitate apoptosis would be a promising treatment for this disease.

15-PGJ2 also caused decreased functional activation of NF-κB in both cell lines by reporter gene activation and EMSA. These findings concur with other reports of 15-PGJ2 inhibiting NF-κB in multiple cancer cell lines [28-31]. Since cancer cells are more radiosensitive and chemosensitive during certain phases of the cell cycle, we wanted to investigate whether the cell cycle was altered by ligand treatment. This strategy has been employed, for example, in prior HNSCC clinical trials with paclitaxel and radiation [32]. PPAR-γ agonist administration was associated with an increased number of cells in S-phase. These data, combined with the decreased proliferation observed via MTT, suggest an S-phase arrest in two cultured oral cavity-derived SCCA cell lines as an additional mechanism for decreased cell proliferation. Studies in gastric carcinoma, esophageal adenocarcinoma and breast cancer cell lines have described G1-phase arrest cells treated with 15-PGJ2 and various thiazolidinediones [10,13,14]. We did not observe this in over 10 cytometry experiments with this agent. The accumulation of cells in the S-phase we observed, however, is similar to the cell cycle changes observed by Butler et al. in prostate carcinoma cell lines treated with 15-PGJ2 [9]. S-phase arrest is much more unusual than G1-phase arrest. The cell cycle control is heavily influenced by cyclins and cyclin-dependent kinases, which allow cells to progress through the phases of the cell cycle by phosphorylating substrates. A possible future area of study could be examining cells for evidence of cyclin D1, cyclin B1 or cyclin E changes after treatment with PPAR-γ agonists to clarify the causes of the cell cycle profile alterations.

The cell cycle-altering effects on cultured SCCA cells observed with administration of PPAR-γ agonists could show a possible novel mechanism for treatment improvements for HNSCC in clinical settings. Increased percentages of cells in S-phase has been reported as a possible means of radiosensitization in several different models [33-35], however, in contrast it is also purported cells going through S-phase can be radioresistant [36]. In our previous work, we have also found effect with ciglitazone,15-PGJ2, and other PPAR-γ ligands on decreased cell proliferation, clonogenic potential, and NF-kB activity, providing additional evidence for the utility of these agents as adjunct therapies [20,37,38]. Clearly, further study would be necessary to characterize and optimize these effects; however, PPAR-γ mediator treatment of HNSCC may offer an additional complementary treatment modality with other forms of chemotherapy or radiotherapy.

Previous studies and clinical trials have also examined the possibility of using PPAR-γ agonists as chemopreventives. Reductions in oral cancer formation have been observed in rats treated with pioglitazone and troglitazone [39,40], and others have demonstrated decreased incidence of head and neck, as well as lung cancers, in diabetics taking thiazolidinediones [41,42]. Our group demonstrated in a mouse model that dietary pioglitazone decreased lung adenoma formation when given early after carcinogen exposure [43]. These results were borne out when pioglitazone was given as an aerosol, this time with efficacy in administration both early and late following carcinogen exposure [44]. Lyon, et al. showed reversal of premalignant lung lesions by rosiglitazone in mice [45], however, this reversal effect was not clearly shown in a recent phase II trial of pioglitazone for lung cancer chemoprevention by Keith, et al. [46]. Again, further research is warranted to fully examine these effects and understand their applicability to oral cancer in determining the role of PPAR-γ agonists as chemopreventives. However, these prior studies give reason to hypothesize our in vitro results could indeed be borne out in vivo, which is the next important horizon for investigation. One challenge using 15-PGJ2 in vivo is the relatively short half-life of prostaglandins [47]. This difficulty has been addressed by prior researchers via chemical modification of prostaglandins, an approach which may prove critical for future study of these compounds in head and neck cancer [48,49]. Additionally, the efficacy seen in mouse models of aerosolized pioglitazone to reduce lung adenoma formation suggests topical administration of PPAR-γ agonists should be further investigated. While existing data do not suggest PPAR-γ agonists would effectively reverse existing dysplastic changes, it is worth investigating whether topical agents in the form of a mouthwash or another topical formulation in high-risk groups might be an efficacious means of chemoprevention.

Concerns regarding PPAR-γ agonists, particularly thiazolidinediones, have been raised due to several high-profile adverse effects in which patients suffered hepatic failure, in some cases resulting in death or necessitating transplantation [50,51]. These cases were almost entirely in patients receiving troglitazone, and such severe hepatotoxicity has only been noted in two patients receiving rosiglitazone, and none in pioglitazone [51]. There is also concern over the cardiovascular risks of thiazolidinediones, particularly myocardial infarction (MI), congestive heart failure exacerbation, and peripheral edema. Peripheral edema is documented in 15.3% of patients who take rosiglitazone [50]. However, recent evidence has called into question the risks of MI and death from cardiovascular causes [52]. As with any cancer pharmacotherapy, the side effect profile of the agents must be considered. In the case of PPAR-γ agonists, the risk-benefit analysis certainly does not rule out further investigation of these agents as therapy for oral cancer, particularly when considering the morbidity of the disease and paucity of effective medical adjuncts to surgical and radiotherapies.

5. Conclusions

Treatment of cultured SCCA cell lines with ciglitazone and 15-PGJ2, two PPAR-γ agonists, causes decreased cellular proliferation associated with an accumulation of cells in S-phase of the cell cycle. Cells which had treatment withdrawn showed similar decreases in proliferation with no statistical difference, excepting NA cells treated with 15-PGJ2, indicating these changes are not reversible after cessation of treatment. NA and CA9-22 cells treated with 15-PGJ2 showed increased caspase-3 activity, and western blot analysis showed PARP cleavage in CA cells treated with 15-PGJ2. Additionally, EMSA of 15-PGJ2-treated NA cells showed decreased NF-κB expression. These findings indicate anticancer effects in head and neck cancer cell lines demonstrated via decreased cellular proliferation, increased apoptosis, and decreased NF-κB expression. This mechanism of 15-PGJ2 action may be through direct covalent modification of the NF-κB binding domain or IKK resulting in decreased cell growth and apoptosis. Ciglitazone and 15-PGJ2 represent two agents worth further investigation as potential pharmacologic chemopreventive agents and/or adjuncts to current oral cancer treatment regimens.

Acknowledgments

Funding Sources:

Lion’s 5M Hearing Center Grant (Minnesota) and P30 CA77598-07 NCI/NIH

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