The Mechanism Of Lithium-induced G2/M Phase Arrest In Hepatocellular Carcinoma Cells

Lithium has been used in the treatment of bipolar mood disorder for decades. Besides,lithium can exert a neuroprotective effect and has the capacity to inhibit cell proliferation.The possible preventive nature of lithium in human cancer development was reported recently.A study revealed that mental patients with lithium treatment have lower cancer prevalence than the general population.To date,several mammalian enzymes are known targets of lithium-including inositol monophosphatase(IMPase),glycogen synthase kinase-3(GSK-3),and several phosphomonoesterases;however,the mechanisms underlying its actions remain unclear.In the present study,we try to elucidate some mechanisms whereby lithium inhibits the growth of hepatocellular carcinoma cells.Firstly,we observed a negative effect of lithium on the growth of 7721 cells through MTT assay,not companied with the character of apoptosis staining with Hoechst 33258.Cell cycle profiles by flow cytometry showed that G2/M phase arrest was induced by lithium in a dose-dependent manner.The further staining of cells with Giemsa dye showed that G2 phase arrest was induced by lithium.The increase of p53, p21,and cyclin B1 by lithium was also observed through Western blot analysis,and the increase of phosphorylated cdc2 was observed with a dose dependent manner of lithium.SB216763,an inhibitor of GSK-3,failed to mimic the action of lithium,nor the inositol abrogated or weakened the effect of lithium.These results indicate that lithium inhibits the proliferation of 7721 cells by inducing G2 phase arrest.It is possible that the cell cycle arrest induced by lithium is associated with p53 pathway but not concerned with the activities of GSK-3 and IMPase.We next studied the mechanism of lithium-induced G2 phase arrest.To elucidate the role of p53,the effect of lithium on Huh7(mutant p53) and Hep3B(null p53) was investigated.Results showed that both of them were arrested in G2 phase after lithium treatment,implicating the dispensable role of p53.The further study revealed Chkl is associated with lithium-induced cell cycle arrest,because the effect of lithium on cell cycle was at least partially abrogated by SB218078,a potent Chkl inhibitor,as well as by Chk1 siRNA or the over-expression ofkinase dead Chk1.Furthermore, lithium-induced cdc25C phosphorylation and in vitro kinase assay showed that the activity of Chkl was enhanced after lithium treatment.Interestingly,the increase of Chk1 activity by lithium may be independent of ataxia telangiectasia mutated (ATM)/ATM and Rad3-related(ATR) kinase.This is because no elevated phosphorylation on Chkl(Ser-317 and Ser-345) was observed after lithium treatment. Moreover,caffeine,a known ATM/ATR kinase inhibitor,relieved the phosphorylation of cdc2(Tyr-15) by hydroxyurea,but not that by lithium.The more amazing is that lithium not only failed to induce phosphorylation of Chk1/Chk2,but also abrogated the phosphorylation of Chk1/Chk2 induced by hydroxyurea,etoposide, and UV.The underlying mechanism of this effect is not understood yet.In summary,the inhibitory effect of lithium on hepatocellular carcinoma cells is associated with G2 phase arrest.The G2 phase arrest induced by lithium is associated with two factors:involvement the role of Chkl and the downregulation of cdc25C. Given that Chk1 has been proposed to be a novel tumor suppressor,we suggest that the effect of lithium on Chk1 and cell cycle be useful in tumor prevention and therapy.