Studies On Lysionotin-mediated Apoptosis Of Hepatocellular Carcinoma Cells Via Caspase-3

As one of the most ordinary malignancies,liver cancer has been reported as the third cause of cancer-related deaths globally.The incidence and mortality of liver cancer have been remaining high for a long time.Due to its insidious onset and complex etiology,it is difficult for patients to be diagnosed at an early stage,and the prognosis is poor.Liver cancer,especially hepatocellular carcinoma,has become a global public health challenge that needs to be faced together.At present,chemoradiotherapy remains one of the most critical treatment strategies for liver cancer especially for patients with unresectable liver cancer.However,various adverse effects including liver dysfunction,liver toxicity,and immune system disorders would occur inevitably during chemotherapy.Therefore,it is of great significance to discover more efficient and less toxic treatment methods or drugs for liver cancer.Lysionotin,a flavonoid compound widely distributed in Lysionotus pauciflorus Maxim,has attracted considerable attention due to its multiple biological activities.Studies have proved that lysionotin has various effects,such as anti-tumor,anti-bacterial,anti-microorganism,anti-inflammatory,anti-tuberculosis,dissolves phlegm and arrests cough,anti-hypertensive,free radical scavenging and anti-allergic properties.However,the anti-liver cancer effect of lysionotin has not been systematically reported in cell and animal models.The purpose of this study was to investigate the anti-hepatocellular carcinoma activity and mechanism of lysionotin in Hep G2 and SMMC-7721 cells and their xenografted tumor mice model.First of all,the anti-hepatocarcinoma activity of lysionotin was investigated in Hep G2 and SMMC-7721 cells.The results indicated that lysionotin notably decreased the cell viability,increased cell apoptosis,inhibited cell migration and colony formation of liver cancer cells.In addition,lysionotin promoted the release of intracellular reactive oxygen species(ROS)levels and decreased mitochondrial membrane potential(MMP),resulting in the excessive accumulation of ROS content in cancer cells,which has an important impact on the proliferation and apoptosis of Hep G2 and SMMC-7721cells.The western blot results showed that the 24-h lysionotin incubation significantly enhanced the expression levels of Cleaved cysteinyl aspartate specific proteinase(caspase)-3,-8 and-9,and Cleaved poly ADP-ribose polymerase-1(PARP-1)in cancer cells.In addition,lysionotin could enhanced the expression levels of Fas ligand(Fas L),Bcl-2 antagonist of cell death(Bad)and Bcl-2-associated X protein(Bax),and reduced the expression levels of B-cell lymphoma-x L(Bcl-x L),NF-E2-related factor 2(Nrf2)and heme oxygenase-1(HO-1)in both Hep G2 and SMMC-7721 cells.In order to further study the anti-hepatocarcinoma effect and safety of lysionotin in vivo,the xenografted tumor mice model were investigated and administered lysionotin for 14 days.The body weight and tumor dimensions of the mice were monitored before every other day,and spleen,liver,kidney and tumor tissues were collected after the last administration.In this study,lysionotin administration had no significant effect on the body weight of nude mice,indicating that lysionotin had no significant toxicity as a therapeutic drug.With the increase of treatment days,the tumor volume of nude mice in the Hep G2 and SMMC-7721 blank control groups increased continuously,while the tumor volume in the lysionotin-administered group increased slowly.After two weeks of administration,the average volume of xenografted tumors in the Hep G2 blank control group was about 333.81 mm~3,that in the lysionotin-administration group was about 146.35 mm~3,and the average volume of xenografted tumors in the SMMC-7721 blank control group was about 334.37 mm~3,and about200.82 mm~3in the lysionotin-administration group,indicating that lysionotin can significantly inhibit the growth of liver cancer.Histopathological examination results showed that lysionotin administration failed to influence the structures of spleen,liver and kidney in nude mice,suggesting its efficacy and safety use in mice.Through western blot assay,we found that 14-day lysionotin treatment resulted in the enhancement in the expression levels of Cleaved PARP-1,Fas L,Bax,Bad,Cleaved caspase-3,-8 and-9,and the reduction in the expression levels of Nrf2,HO-1 and Bcl-x L in Hep G2-and SMMC-7721-xenografted tumor tissues.This result was consistent with the in vitro results.Ac-DEVD-CHO is a cell-permeable caspase-3 inhibitor,which can irreversibly inhibit the expression of caspase-3 and thereby inhibiting cell apoptosis.Our results indicated that the pre-treatment of Ac-DEVD-CHO strongly restored the low cell viability,the enhanced apoptosis rate,the dissipation of MMP caused by lysionotin exposure,as well as prevented the lysionotin-caused enhancement in expressions of apoptosis related proteins,especially Cleaved PARP-1,Fas L,Cleaved caspase-3 and Bax in both Hep G2 and SMMC-7721 cells,which further confirmed the leading role of caspase-3 during lysionotin-induced apoptosis in liver cancer cells.Altogether,the in vivo and in vitro results of this study indicate that,in Hep G2 and SMMC-7721 liver cancer cells and their-xenografted tumor bearing mice,lysionotin shows significant anti-liver cancer effects,which is,at least partially,related to caspase-3 mediated mitochondrial apoptosis.This provides a data basis for the potential clinical value of lysionotin in the treatment of liver cancer.