Biochemical and Biophysical Research Communications
Anti-chondrosarcoma effects of PEDF mediated via molecules important to apoptosis, cell cycling, adhesion and invasion
Introduction
Chondrosarcoma is the third most common primary malignancy of bone after myeloma and osteosarcoma. The overproduction of chondrocytes and subsequently cartilage matrix by a heterogeneous group of neoplasms results in chondrosarcomas (WHO). Majority of these tumours are mild and slow-growing, however it still constitutes a significant morbidity risk, with a 5-year survival rate of 64–77% [1], [2], [3]. Most chondrosarcoma cases are diagnosed around the age of 40 years with the preferential location deeper in regions such as the pelvis, proximal femur, proximal humerus, distal femur and the ribs. At present, the most favourable treatment for intermediate to high-grade tumours is wide surgical excision as traditional chemotherapy and radiotherapy are not as effective [4]. This is mostly attributed to their extracellular matrix, low percentage of dividing cells and poor vascularity. Novel methods that do not rely on chemo- or radiotherapy for chondrosarcoma treatment are thus required [5].
PEDF is a 50-kDa secreted glycoprotein first discovered as a factor secreted by the pigment epithelium of the human foetal eye. In addition to its neurotrophic properties [6], it has been identified to be the most potent of any known endogenous inhibitors of angiogenesis, boasting potency twice that of angiostatin and seven times of endostatin [7]. PEDF induces its anti-angiogenic signals in two ways, by either activating endothelial cell apoptosis through the Fas/FasL death pathway or through disruption of the balance of pro- and anti-angiogenic factors, thereby decreasing the expression of pro-angiogenic factors such as vascular endothelial growth factor (VEGF), FGF-1, FGF-2 and interleukin-8 [7], [8], [9]. Despite the widespread expression of PEDF in most tissues, it is not critical for viability as PEDF-deficient mice were born alive and healthy, although further examination did reveal an increased stromal microvessel density in the pancreas, kidney and prostate [10]. Clinical studies examining tumours have also shown that decreased expression of PEDF correlated with a higher intra-tumoural microvessel density (MVD) and subsequently acquiring a more metastatic phenotype [11], [12]. PEDF anti-angiogenic activity is also uniquely selective as it only targets the neovasculature without affecting the pre-existing vessels, poising it as an ideal candidate for targeted cancer therapy [13].
PEDF also influences cell proliferation through regulation of the cell cycle, induction of apoptosis and inducing tumour cell differentiation towards a more mature phenotype [14], [15], [16]. Based on findings by Filleur et al. [16], PEDF was able to induce tumour cell apoptosis directly, due to a distinct functional epitope on the PEDF protein. Filleur showed that the 34-mer PEDF peptide sequence reduced prostate proliferation in vitro unlike the 44-mer peptide, which was found to induce differentiation in neuroblastomas and prostate-carcinoma cells [15], [16]. Collectively, full-length PEDF protein significantly reduced tumour cell proliferation in both human melanoma and osteosarcoma cells [14], [17]. Additionally, Crawford et al. 2001 speculated that the low expression of PEDF in several tumours seemed to correlate clinically with a poorer prognosis and the development of metastasis, in part related to the degree of differentiation of the neoplastic cells [15]. Moreover, as the degree of differentiation is often inversely related to the rate of proliferation, the ability of PEDF to reduce cell proliferation and trigger cell differentiation might be intimately related.
To date, PEDF has been examined in prostate, ovarian and pancreatic cancers, melanoma, glioma and osteosarcoma (reviewed in [18]). In this paper, the effects of PEDF as a potential anti-cancer agent on the human chondrosarcoma JJ012 cell line are examined.
Section snippets
Cell culture
Human chondrosarcoma cell line JJ012 (Dr. J. A. Block, Rush University Medical Centre, Chicago) was cultured in α-MEM/GlutaMAX (Invitrogen, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (Invitrogen, Carlsbad, CA, USA) and 1% antibiotic/antimycotic (Invitrogen, Carlsbad, CA, USA), at 37 °C in a humidified 5% CO2 atmosphere. PEDF was obtained as lyophilised powder (BioProducts MD, Middletown, MD, USA) and re-constituted in 1× phosphate buffered saline (PBS) pH 7.2 (Invitrogen,
PEDF results in anti-proliferative activity and induces apoptosis
The anti-proliferative activity of PEDF was first evaluated on human chondrosarcoma cells JJ012 (Fig. 1A). JJ012 cells were not affected when treated with low concentrations of PEDF (5 and 25 nM), having no change or causing a slight increase in cell viability. However, an increased treatment dose of 100 and 200 nM PEDF significantly decreased JJ012 cell viability by as much as 38%. At the lowest most effective dose of 100 nM PEDF, there was a 33% decrease in cell viability and thus was chosen as
Discussion
Belonging to the broad family of sarcomas – cancers of connective tissues, research into new treatments for osteosarcoma (cancers originating from osteoblasts) has benefited from the use of PEDF. PEDF suppressed primary osteosarcoma growth and prevented the development of macroscopic pulmonary metastases in an orthotopic mouse model of human osteosarcoma [19], [20]. Thus, the use of PEDF for chondrosarcoma (cancers originating from chondrocytes) treatment was examined.
PEDF is a 50-kDa secreted
Conclusion
Human JJ012 chondrosarcoma cells treated with PEDF demonstrated a significant decrease in cell viability that was accompanied by increased expression of apoptotic cell death signals and electron microphotographs displaying apoptotic cell death characteristics. Ki-67 staining of PEDF-treated cells showed a significant decrease in high Ki-67 expressing cells with results from immunoblotting showing evidence of decreased cell proliferation. The adhesion and migration assays both showed evidence of
Acknowledgments
We thank Dr. J. A. Block for JJ012 cells and the Australian Orthopaedics Association for funding.
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