SPARC overexpression decreases aggressiveness of human hepatocellular carcinoma

CATALINA ATORRASAGASTI; MARIANA MALVICINI; LAURA ALANIZ; MIGUEL RIZZO; JORGE AQUINO; OSVALDO PODHAJCER; MARCELO SILVA; GUILLERMO MAZZOLINI

Boston. MA, USA

Congreso; The Liver Meeting 2008; 2008

American Association for the Study of Liver Diseases: AASLD

Background and aim: Hepatocellular carcinoma (HCC) is now the sixth most common cancer and the third leading cause of cancer-related death worldwide. The incidence and mortality associated to HCC is increasing in the lasts decades. Current therapeutic options are extremely disappointing and only a minority of patients can receive a curative therapy. Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular glycoprotein involved in many biological processes including cell adhesion, wound healing, differentiation and proliferation. Overexpression of SPARC in malignant cells is frequently associated with increased aggressiveness, although its role in the development of HCC remains unknown. Hereby we have analyzed the effects that changes in SPARC expression levels have in HCC aggressiveness.Methods: Adenoviral vectors carrying hSPARC sense (AdsSPARC) and -galactosidase codingsequence (Ad gal) were used. HepG2 cell line was used for in vitro and in vivo studies. Transduced and non-transduced HepG2 cells viability was measured using a colorimetric MTT assay. Quantitative determination of apoptotic and necrotic cells was obtained after double staining with orange acridine and ethidium bromide by using epifluorescence microscopy. Three-dimensional spheroids were prepared in 96-well plates coated with 1% agarose in PBS using HepG2 alone or infected with the different adenoviruses. Cell migration was analyzed using Boyden chamber. Cell colony formation assayswere performed plating 10e3 cells in 6-well plates and counting colonies at day 15. Five-eight week-old athymic nude-mice were subcutaneously injected, in the right hind flank, with 1.5x10e6 ex vivo AdsSPARC or Ad gal-transduced HepG2 cells. Results: SPARC protein was identified in HepG2 cell line. Positive modulation of SPARC levels did not affect HepG2 cells proliferation until day 3 in vitro. Morphological changes indicative of apoptosis in HepG2 cells after AdsSPARC infection were observed at day 5. SPARC overexpression had a profound inhibitory effect on spheroid growth whichwas more pronounced at day 6. AdsSPARC decreased the rate of HepG2 cells migration in the Boyden chamber assay compared with Ad gal or untreated cells. In addition, SPARC overexpression reduced HCC cell colony formation. In vivo experiments revealed that SPARC overexpression in HCC cells inhibits tumor growth and increases animal survival. Conclusions: Our results suggest that SPARC overexpression has profound effects on HCC aggressiveness by inducing apoptosis and decreasing tumor cell migration, colony formation and tumorigenesis. These evidences suggest SPARC as a novel target for HCC treatment.