# Chondrosarcoma and Peroxisome  Proliferator-Activated Receptor

Authors: K. Nishida; T. Kunisada; Z. N. Shen; Y. Kadota; K. Hashizume; T. Ozaki
Journal: PPAR Research
 (2008)

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## Abstract

Induction of differentiation and apoptosis in cancer cells by ligands of PPARγ is a novel therapeutic approach to malignant tumors. Chondrosarcoma (malignant cartilage tumor) and OUMS-27 cells (cell line established from grade III human chondrosarcoma) express PPARγ. PPARγ ligands inhibited cell proliferation in a dose-dependent manner, and induced apoptosis of OUMS-27. The higher-grade chondrosarcoma expressed a higher amount of antiapoptotic Bcl-xL in vivo. The treatment of OUMS-27 by 15d-PGJ2, the most potent endogenous ligand for PPARγ, downregulated expression of Bcl-xL and induced transient upregulation of proapoptotic Bax, which could accelerate cytochrome c release from mitochondria to the cytosol, followed by induction of caspase-dependent apoptosis. 15d-PGJ2 induced the expression of CDK inhibitor p21 protein in human chondrosarcoma cells, which appears to be involved in the mechanism of inhibition of cell proliferation. These findings suggest that targeted therapy with PPARγ ligands could be a novel strategy against chondrosarcoma.


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## Body

## 1. Introduction

Cancers are associated with dysregulation of
differentiation and apoptotic cell death. Recent investigations have
demonstrated that induction of these cellular events by targeted therapy with
ligands of nuclear hormone receptors could be a novel strategy against cancers [1]. Peroxisome proliferator-activated receptor (PPAR)γ, a member of
the nuclear receptor superfamily, acts as a ligand-activated transcription
factor, and is involved in many processes important for homeostasis of cells
and tissues, including metabolism, immune and inflammatory controls, cell
proliferation and apoptotic cell death [2–6]. Because PPARγ is expressed
by many malignant tumors, activation of PPARγ by 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), the most potent endogenous ligand for PPARγ [7], and the synthetic PPARγ ligands (e.g., rosiglitazone, pioglitazone, troglitazone, and indomethacin)
have been regarded as a novel therapeutic approach for certain human
malignancies through growth inhibition, induction of apoptosis and terminal
differentiation, and inhibition of angiogenesis [8]. This
review will outline the inhibitory effects of synthetic and endogenous PPARγ ligands and discuss their potential therapeutic
effects on chondrosarcoma.


## 2. Clinical Features of Chondrosarcoma

Chondrosarcoma is a malignant tumor
of cartilage; the matrix formed by tumor cells is uniformly and entirely
chondroid in nature [9]. Human chondrosarcoma is a rare bone tumor, accounting for <10% of
primary malignant bone tumors. Chondrosarcoma also arises in pre-existing
benign lesions (e.g., osteochondromatosis, enchondromatosis) and is termed “secondary
chondrosarcoma.”Primary (conventional)
chondrosarcoma arises centrally in a previously normal bone, and mostly grows
slowly through the diaphyseal cortex. Most patients are aged >50 years.
Conventional chondrosarcoma is more common in men. The commonest sites are the
bones of the pelvis, followed by the femur and the humerus. Recognizable
histologic variants are clear cell, mesenchymal,
and dedifferentiated chondrosarcomas. On the basis of histologic features
(nuclear atypia and cellularity), conventional chondrosarcoma is further subdivided into three grades: I, II, and III [10]. The histologic grade of chondrosarcoma indicates the differentiation
status of tumor cells, and is one of the most important factors for prognosis [11]. Progression of a locally aggressive low-grade chondrosarcoma to a
metastasizing high-grade chondrosarcoma is associated with loss of
cartilaginous phenotype, genomic instability, and aneuploidy [12]. The grading of chondrosarcoma correlates well with clinical behavior,
although chondrosarcoma is one of the most difficult malignant tumors of bone
to diagnose [13].Most conventional chondrosarcomas
are grade I or II. For low-grade chondrosarcoma, surgical treatment with
adequate marginal resection is reported to be associated with better clinical
outcomes [14]. Only 5–10% of conventional
chondrosarcomas are grade-III lesions, which have definite metastatic
potential. The prognosis for high-grade chondrosarcoma is poor, despite
adequate surgery, because they are highly resistant to conventional
chemotherapy and radiotherapy [15]. These facts, that the differentiation status of chondrosarcoma is
predictive of clinical outcomes, suggest the favorable effects of the
modification of the differentiation status on clinical behavior. Recent
advances in understanding the progression or development of chondrosarcoma have
suggested several molecular targets for future development of new adjuvant
therapy [16], such as chondrocyte differentiation factors (PTHrP, CTGF) [17, 18], antiapoptotic gene (Bcl-2) [19, 20], tumor suppressor gene (p16, p53) [21, 22], and others (PDGF-α, VEGF, MDR-1)
[23–25].


## 3. Oums-27, A Chondrosarcoma Cell Line

A cell line derived from chondrosarcoma,
particularly from high-grade chondrosarcoma, can provide a useful model for the
investigation of cell development and treatment of chondrosarcoma [26–30]. The OUMS-27 cell line has been established from grade III human
chondrosarcoma [31]. The cells do not show contact inhibition after reaching confluence,
grow rapidly in multiple layers, and express proteoglycan, as well as collagen
type I, II, III, IX, and XI after 120 passages, showing stable maintenance of
the differentiated chondrocytic properties. The transplantation of OUMS-27
cells into athymic mice resulted in formation of grade II chondrosarcoma at the
injection site. There have been many studies on the etiology and treatment of
chondrosarcoma using this cell line [32–34].


## 4. Expression of PPARγ in Human  Chondrosarcoma and Oums-27

Subramanian et al. [35] investigated gene expression profiling of ten extraskeletal myxoid
chondrosarcomas (EMCs) using 42000 spot cDNA microarrays. Eighty-six genes that
distinguished EMC from the other sarcomas were identified by significance
analysis of microarrays with 0.25% likelihood of false significance. Of these,
PPARG and PPARGC1A, an interacting protein with PPARG and also a coactivator,
were highly expressed in EMCs.In vivo PPARγ protein
content was examined in conventional chondrosarcoma specimens from 28 patients
undergoing surgery [36]. Immunohistochemical study revealed that human chondrosarcoma cells
frequently express PPARγ protein. The
positivity (cutoff positivity of 10%) of chondrosarcoma cells were 65.0% in
grade I, 83.3% in grade II, and 50.0% in grade III; overall positivity was
67.9% (see Table 1). Expression of PPARγ in OUMS-27
cells at protein and mRNA levels was confirmed by immunocytochemistry and
reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, respectively
[36]. These data indicated that PPARγ is frequently
expressed in primary chondrosarcomas and chondrosarcoma cell line OUMS-27, and led
the authors to test the effect of PPARγ activators on
cell proliferation and survival of OUMS-27.Table 1
Summary of immunohistochemical study for PPARγ in human chondrosarcoma tissues.
Positive cellPathological grade of chondrosarcoma (%)ratios (%)I (n=20)II (n=6)III (n=2)<1035175010–40403350>4025500


## 5. Evidence of Apoptotic Cell Death  of Oums-27 Cells After Treatment by  PPARγ Ligands

In our previous report [36], OUMS-27 cells were treated with increasing concentrations of
pioglitazone (synthetic PPARγ ligand) and
15d-PGJ2 for up to 48 hours. The results of immunostain for Ki-67
(cell proliferation marker) and colorimetric MT T assay showed that treatment
with both pioglitazone and 15d-PGJ2 for 24
hours inhibited cell growth and reduced cell viability in a dose-dependent
manner, respectively. 15d-PGJ2 had more
noticeable effects on OUMS-27 cell growth than pioglitazone. It was unclear whether
the effects of ligands on OUMS-27 cells were strictly due to PPARγ activation. When
cells were treated with 15d-PGJ2 doses of
≥5 μg/mL, they showed relatively round shapes and some cells no longer adhered to the dish.Semithin sectioned, LR White-embedded cells stained by
toluidine blue revealed that many OUMS-27 cells treated with 15d-PGJ2 show apoptotic appearances with cell shrinkage and nuclear
condensation (see Figure 1). DNA fragmentations in OUMS-27 cells treated by
15d-PGJ2 (10 μg/mL) for 24 hours
were confirmed by DNA ladder formation and TUNEL staining. Transmission
electron microscopic study revealed sections of OUMS-27 cells treated with
15d-PGJ2 contained many cells consistent with morphological
apoptosis with condensed chromatin, many vacuoles in cytoplasm, and membrane
budding. Early apoptotic change and the translocation of phosphatidylserine
(PS) on the outer leaflet of the cell membrane were demonstrated by FACS
analysis. The population of apoptotic cells with PS at the outer membrane of
the cells (annexin-V-positive, PI-negative) was ~53.9% and 67.6% at 4 hours and
24 hours after coincubation with 15d-PGJ2, respectively.Cell morphology of chondrosarcoma cell line OUMS-27 after incubation with (b)
or without (a) 15d-PGJ2. Cells were treated
with 10 μg/mL of
15d-PGJ2 for 8 hours, and the cell pellet embedded in hydrophilic
resin. Semithin sections stained by toluidine blue show more apoptotic cells
with cell shrinkage and nuclear condensation (arrows) after treatment with
15d-PGJ2.
(a)(b)


## 6. Mechanism of Apoptotic Cell Death of  Oums-27 Cells by PPARγ Ligands

cDNA microarray analysis was carried out to
comprehensively explore the changes in gene expression pattern during OUMS-27
cell growth inhibition and possible cell cycle arrest caused by treatment with
15d-PGJ2 [37].
Among the 1081 genes analyzed, 52 genes were upregulated and 81 genes were
downregulated significantly in OUMS-27 cells after 8-hour treatment with 15d-PGJ2 (10 μg/mL). Microarray analysis is
shown in Table 2. Interestingly, the proapoptotic gene Bax was upregulated, and
the antiapoptotic gene Bcl-xL was downregulated. The other Bcl-2 members were
unchanged. These results were further confirmed by RT-PCR and real-time PCR
analysis.Table 2
Changes in apoptosis-related
gene expression in OUMS-27 cells after treatment with 15d-PGJ2.
Gene nameFoldClusterin+3.7Defender against cell death 1+3.8Tumor necrosis factor receptor 1+3.2State-induced state inhibitor 2+2.5Heat shock protein 60+2.4V-akt murine thymoma viral oncogene homolog 1+2.3Apoptosis regulator bax+2.3Interferon-induced RNA-dependent protein kinase+2.1Apoptosis regulator bcl-xl−2.2Calpain, small subunit 1−2.3Heat shock 70 KD protein 1−2.4Endothelin 2−2.4Insulin-like growth factor 1 receptor−2.5Death-associated protein 6−2.8Upregulation of Bax, concurrent with the
downregulation of Bcl-xL, can destabilize mitochondria, leading to the release
of several mitochondrial intermembrane space proteins such as cytochrome c,
AIF, Smac/DIABLO, Endo G, and Omi/HtrA2 into the cytosol, where they are
actively involved in apoptotic cell death [38]. This hypothesis is supported by
our observations of the release of cytochrome c from mitochondria into the
cytosol, and the activation of caspase-3 in 15d-PGJ2-treated OUMS-27 cells.
Coincubation of cells with the broad-spectrum caspase inhibitor Z-VAD-FMK
completely inhibited caspase activity, and prevented the cell death induced by
15d-PGJ2.
These results indicate that 15d-PGJ2 induced apoptosis in OUMS-27 cells through a
caspase-dependent signal transduction pathway which, at least in part, was
triggered by cytosolic release of cytochrome c [37].The decreased expression of antiapoptotic
Bcl-xL in OUMS-27 treated by 15d-PGJ2 led us to examine the expression in the
tissue of human chondrosarcoma samples to study the clinical application of
differentiation therapy by PPARγ activation. The result of
immunohistochemical study demonstrated that Bcl-xL was expressed in all three
grades of chondrosarcoma; the expression was strongest in grade III. These results indicated that higher-grade chondrosarcoma
cells may be resistant to apoptosis by overexpression of Bcl-xL, and 15d-PGJ2 might induce apoptotic cell
death by downregulation of Bcl-xL and transient upregulation of Bax [37].
Similar results were reported in renal cell carcinoma cells (786-O and A498
cells) showing the thiazolidinedione (TZD) induction of apoptosis
with increased Bax expression and decreased Bcl-2 expression [39].


### 6.1. Genetic and Epigenetic Alterations in Chondrosarcoma

Little is known about the role of genetic or epigenetic alterations in
tumor progression from low-malignant chondroblastic to highly malignant
anaplastic chondrosarcoma. The appearance of de novo aberrant DNA methylation is the commonest
molecular change in the cancer cell, which
inactivates many cellular pathways [40]. The most studied
change of DNA methylation in neoplasms is the silencing of tumor suppressor
genes by deoxy-cytidylatephosphate-deoxy-guanylate (CpG) island promoter
hypermethylation, which targets genes and molecules associated in cell
differentiation, such as p16(INK4a), BRCA1, and hMLH1 [41–43]. Röpke et al.
reported the p16 and E-cadherin promoter methylation in low-grade chondroid compartment of
dedifferentiated chondrosarcoma. Van Beerendonk et al. found p16 promoter methylation by
methylation-specific PCR in 5 of 30 tumors, but this did not correlate with
protein expression, or with loss of heterozygosity (LOH) at 9p21 region, one of
the few consistent genetic aberrations found in conventional chondrosarcoma [44]. In OUMS-27,
methylation was not detectable in the promoter of p16 gene (unpublished data).Some reports
suggested that p53 mutation and p53 loss of heterozygosity are involved [43, 45]. In
OUMS-27, we have previously shown that the p53 gene is mutated [31]. Asp et al. analyzed p16 and p53 in
cartilaginous tumor tissues and showed that the p16 gene was found to be partly
methylated in 5 high-grade chondrosarcomas and homozygously deleted in 1
chondrosarcoma, whereas the p53 gene revealed an unchanged structure in all 22
chondrosarcoma samples [46].


## 6.1. Genetic and Epigenetic Alterations in Chondrosarcoma

Little is known about the role of genetic or epigenetic alterations in
tumor progression from low-malignant chondroblastic to highly malignant
anaplastic chondrosarcoma. The appearance of de novo aberrant DNA methylation is the commonest
molecular change in the cancer cell, which
inactivates many cellular pathways [40]. The most studied
change of DNA methylation in neoplasms is the silencing of tumor suppressor
genes by deoxy-cytidylatephosphate-deoxy-guanylate (CpG) island promoter
hypermethylation, which targets genes and molecules associated in cell
differentiation, such as p16(INK4a), BRCA1, and hMLH1 [41–43]. Röpke et al.
reported the p16 and E-cadherin promoter methylation in low-grade chondroid compartment of
dedifferentiated chondrosarcoma. Van Beerendonk et al. found p16 promoter methylation by
methylation-specific PCR in 5 of 30 tumors, but this did not correlate with
protein expression, or with loss of heterozygosity (LOH) at 9p21 region, one of
the few consistent genetic aberrations found in conventional chondrosarcoma [44]. In OUMS-27,
methylation was not detectable in the promoter of p16 gene (unpublished data).Some reports
suggested that p53 mutation and p53 loss of heterozygosity are involved [43, 45]. In
OUMS-27, we have previously shown that the p53 gene is mutated [31]. Asp et al. analyzed p16 and p53 in
cartilaginous tumor tissues and showed that the p16 gene was found to be partly
methylated in 5 high-grade chondrosarcomas and homozygously deleted in 1
chondrosarcoma, whereas the p53 gene revealed an unchanged structure in all 22
chondrosarcoma samples [46].


## 7. Induction of Cell Cycle Arrest by  15d-pgJ2 in Oums-27

Ligands for PPARγ reportedly induce cell cycle
arrest in various cancer cells [39, 47–54]. 15d-PGJ2 induces G1 arrest and inhibits
cell growth of human anaplastic thyroid carcinoma through a p53-independent,
but p21- and p27-dependent, manner [55]. Activation
of PPARγ by troglitazone
inhibited cell growth and induced G1 arrest through the increase of
cycline-dependent kinase (CDK) inhibitor p27 in several cell lines, including
human pancreatic carcinoma cells, gastric cancer cells, and hepatocellular
carcinoma cells [56–58]. The effect of troglitazone on the proliferation of cancer cells was
inhibited by antisense for p27. Yang et al. also showed TZD decreased the
protein levels of proliferating cell nuclear antigen, pRb, cyclin D, and Cdk4,
but increased the levels of p21 and p27, in RCC cells [39].In OUMS-27, 15d-PGJ2 induced the expression of the CDK inhibitor p21 protein,
and it was increased within 24 hours. Expression of the other CDK inhibitors,
p16 and p27 proteins, were detected at time zero, and were not significantly
influenced by 15d-PGJ2 treatment [37].
15d-PGJ2-induced
p21 may exert cell cycle arrest in a p53-independent manner.Whether 15d-PGJ2 induces p21 expression in OUMS-27 cells through a PPARγ-dependent or -independent
pathway is unclear. It is possible that p21 expression is directly regulated by
PPARγ activation because p21 gene
contains a potentially conserved consensus PPARγ response element in the promoter
region [59]. Copland et al. reported [60] that RS5444, a novel high-affinity PPARγ agonist,
inhibits anaplastic thyroid cartinoma (ATC) tumor growth and angiogenesis in
mice. In DRO cells derived from ATC tumor, they demonstrated that upregulation
of p21 by RS5444 is PPARγ dependent,
and might be the major mechanism by which RS5444 inhibits DRO cell
proliferation. Han et al. demonstrated the link of PPARγ activation and p21 signaling to
cell growth inhibition in human lung cell cartinoma cells using p21 antisense oligonucleotides [61]. They also indicated the
induction of p21 expression by PPARγ ligands might be mediated
through increased Sp-1 and NF-interleukin 6 (IL6) CAAT/enhancer binding protein
(C/EBP)-dependent transcriptional activation.


## 8. Clinical Application of PPARγ Agonist for  Chondrosarcoma Suppression

Accumulating evidence suggests that PPARγ activators
might have clinical therapeutic benefit in the treatment of cancers. Although
initial clinical trials with troglitazone reported promising results in
liposarcomas [62] and prostate cancers [63], recent studies failed to show the expected therapeutic values of
rosiglitazone in liposarcomas [64] and early-stage breast cancers [65], and troglitazone in chemotherapy-resistant metastatic colorectal
cancers [66]. However, a single study of a phase-I clinical trial of
LY293111 in patients with advanced solid tumors reported a potential efficacy
of PPARγ agonist for
chondrosarcoma [67]. LY293111 is an orally stable leukotriene B4 (LTB4)
receptor antagonist, as well as a PPARγ agonist, as
demonstrated by activity in the rat ZDF diabetes model, and the induction of
adipocyte differentiation. One patient with progressive chondrosarcoma had
stable disease lasting ~336 days of LY293111 administration at the dose of 200 mg bd.


## 9. Future Direction

In chondrosarcoma, whether the cell
death and growth inhibitory effects induced by 15d-PGJ2 are PPARγ-dependent or -independent is
unknown. As 15d-PGJ2 at high doses is toxic for most of cell types independent
of PPARγ activation,
we examined the effects of the caspase inhibitor Z-VAD-FMK,
and the PPARγ antagonist GW9662, on caspase-3
activation and cell viability of OUMS-27 cells treated by 15d-PGJ2 [37].
15d-PGJ2 alone clearly increased cell
death; the addition of GW9662 partially inhibited cell death. Cell death was
inhibited almost to control level when Z-VAD-FMK was added to 15d-PGJ2. The activity of caspase-3 was
attenuated, though not completely, by stimulation of 15d-PGJ2 together with GW9662. These data indicate
that the greater proapoptotic effects of 15d-PGJ2 on chondrosarcoma cells may result from the
cumulative effects of PPARγ-dependent and -independent
pathways. Detailed analysis of the effects of ligands on cells
transfected with PPARγ siRNA should
provide important clues to understanding this phenomenon. Whether endogenous or
synthetic PPARγ ligands can
also induce tumor cell death in an experimentally transplanted chondrosarcoma
model remains to be examined before human trial.



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