Study of the biological behavior of caspases in hepatocellular carcinoma cells

Background: Hepatocellular carcinoma (HCC) is the fifth most common malignancy in the world and is estimated to cause approximately half a million deaths annually. Octreotide, a somatostatin analogue, has been used to treat inoperable hepatocellular carcinoma. Clinical trials have demonstrated a survival benefit of patients with inoperable HCC treated with octreotide, but also negative studies have been published and recently criticized. Several reports indicate, also, that octreotide inhibits the proliferation and induces apoptosis of HCC cells in vitro The mechanisms of apoptosis induction however are not well understood.. Moreover, clinical observations suggest that ursodeoxycholate (UDCA) may protect from hepatocellular carcinoma in cirrhotic patients. Increased apoptosis of malignant cells is a candidate mechanism. Decreased apoptosis of cholangiocytes has been proposed as a mechanism for the favourable effect of UDCA in primary biliary cirrhosis. Aims: 1) To investigate the role of octreotide on cellular proliferation, apoptosis and caspases activities of HepG2 carcinoma cells. 2) To investigate the effects of different concentrations of UDCA on HepG2 cell proliferation, apoptosis and caspases activities. Methods: We studied the occurrence of cellular proliferation, apoptosis and the possible internal caspase-mediated apoptosis pathway involved, after treatment of HepG2 carcinoma cells with octreotide, in comparison with the apoptosis caused by TNF-alpha. We, also, studied the apoptotic features and the caspases activities, after treatment of HepG2 cells with different concentrations of UDCA alone or in combination with TNF-alpha. Activities of caspase-3, caspase-9, caspase-8 and caspase-2 were studied, while apoptosis was investigated through detection of DNA fragmentation and through identification of apoptotic cells with the Annexin-V/PI flow cytometric method. Results: Cellular proliferation was decreased, after treatment of HepG2 cells with octreotide or TNF-alpha alone but, in contrast to TNF-alpha, octreotide decreased proliferation only at concentration of 10-8M, while lower concentrations increased proliferation. Early and late apoptosis were significantly increased with both substances. Octreotide significantly increased caspase-3, caspase-8 and caspase-2 activity. TNF-alpha significantly increased only caspase-2. UDCA significantly inhibits cell proliferation only at high concentrations, but increases apoptosis at low concentrations and protects from apoptosis at higher concentrations. TNF-alpha induced DNA fragmentation is potentiated by UDCA, but flow cytometry indicates protection from early apoptosis and increase in cell survival by low and intermediate UDCA concentrations. UDCA differentially activates initiator and effector caspases in different concentrations. Conclusions: Our results support the induction of a caspase-mediated apoptotic pathway by octreotide in hepatocellular carcinoma cells, implicating both the receptor-mediated and the mitochondrial-apoptotic pathway. The correlation of specific apoptotic, caspase-mediated pathways, with the expression of sst receptors in HCC cells, need more investigation, to better define and clarify the intracellular mechanisms of the antiproliferative effects of octreotide. Also, our findings indicate that measurements of serum octreotide levels may be important, at least in clinical trials, to verify optimal therapeutic drug concentrations. Moreover, our data demonstrate that the effect of UDCA on caspase activation and apoptosis of HepG2 cells is concentration-dependent and activation of the caspase cascade is not always translated into increased apoptosis. Serum levels of UDCA should be possibly monitored and dosage of the drug adjusted according to the required effect