HEPATOCELLULAR-CARCINOMA CELLS RESIST NECROSIS DURING ANOXIA BY PREVENTING PHOSPHOLIPASE-MEDIATED CALPAIN ACTIVATION

Although hepatocellular carcinoma (HCC) cells are more resistant to an oxic injury than normal hepatocytes, the mechanisms responsible for th is differential sensitivity remain obscure. Because enhanced calpain p rotease activity contributes to hepatocyte necrosis, we tested the hyp othesis that HCC cells resist anoxia by preventing calpain activation. Cell viability in two rat HCC cell lines (N1S1 and McA-RH7777 cells) was fourfold greater compared to rat hepatocytes after 4 h of anoxia. Although calpain activity increased twofold in rat hepatocytes during anoxia, no increase in calpain activity occurred in HCC cells. Western and Northern blot analysis revealed greater or equivalent expression of calpains and calpastatin in HCC cells compared to hepatocytes. Beca use increases in cytosolic free Ca++ (Ca-i(++)) and phospholipid degra dation products regulate calpains in vitro, we measured Ca-i(++) and p hospholipid degradation. Ca++, did not change in any cell types during 60 min of anoxia. In contrast, phospholipid degradation was fourfold greater in hepatocytes compared to HCC cells. Melittin, a phospholipas e A(2) activator, increased calpain activity and cell necrosis in all cell types; melittin-induced cell necrosis was ameliorated by a calpai n protease inhibitor. In summary, these data demonstrate for the first time 1) calpain activation without a measureable increase in Ca-i(++) , 2) phospholipase-mediated calpain activation in hepatocytes and HCC cells, and 3) the adaptive mechanism responsible for the resistance of HCC cells to anoxia-an inhibition of phospholipid-mediated calpain ac tivation. Interruption of phospholipase-mediated calpain activation ma y be a therapeutic strategy for preventing anoxic cell injury. (C) 199 6 Wiley-Liss, Inc.