Effects Of Rocaglamide On Apoptosis Of Hepatocellular Carcinoma Cells And Its Mechanism

BackgroundHepatocellular carcinoma（HCC）is the most common and aggressive gastr ointestinal tumor in the world.Due to the characteristics of occulting onset,ra pid development and easy metastasis of HCC,the overall therapeutic effect is still unsatisfactory^[1].Therefore,exploring the potential mechanisms of tumor g enesis,proliferation and metastasis has become a hot topic in anti-tumor resear ch.Hepatocellular carcinoma cells proliferate rapidly and form a local hypoxic microenvironment,in which the metabolic pattern of tumor glycolysis can impr ove the tolerance of cells to hypoxic ischemia and promote the invasion and metastasis of tumor cells^[2].HexokinaseⅡ（HK2）is a key enzyme that catalyze s the first step of glycolysis,helping to combine ATP formation and glucose p hosphorylation in mitochondria,and is involved in cance0r cell growth,survival and metastasis.HK2 expression in hepatocellular carcinoma has been found to be related to tumor grade,stage and increased mortality of liver cancer and d rug resistance^[3].Rocaglamide（Roc-A for short）is an antitumor substance deriv ed from the plant Aglaia elliptifolia,which acts on eukaryotic translation initiat ion factor 4A（e IF4A）by acting on A bimolecular lumina between specific RN A sequences^[4].Roc-A can inhibits glycolysis by inhibiting the expression of he at shock factor（HSF1）,increasing the expression of thioredoxin interacting prot ein（TXNIP）and reducing glucose uptake.Apoptosis was also induced by activ ating MAPK P38 and JNK signaling pathways and inhibiting RAS-CRAf-MEK-ERK signaling pathways^[5].Roc-A can inhibits the occurrence and development of tumor through A variety of pathways,but does it promote the apoptosis of HCC cells by inhibiting HK2 expression?At present,there are few studies on relevant mechanisms.ObjectiveTo investigate the apoptosis-inducing effect of Roc-A on human hepatoma cell line Hep G2（Hep G2 cells）and its possible mechanism.Methods1.The apoptosis of Hep G2 cells cultured with different concentrations of Roc-A was observed:Hep G2 cells in the logarithmic phase were divided into f ive groups and were added with Roc-A solution of 0（blank control）,25,50,100 and 200 nmol/L,respectively.Cells in each group were cultured for 24 h,and apoptosis was observed by YF488-Annexin V/PI fluorescence double staini ng.2.The hexokinaseⅡ（HK2）and anti-apoptotic factor analogue（BCL2L1）m RNA in Hep G2 cells cultured with different concentrations of Roc A were detected:Hep G2 cells in the logarithmic growth phase were divided into two groups and were added with 0 and 100 nmol/L Roc-A solution,respectively.HK2 and BCL2L1 m RNA were detected by RT-q PCR after 24 hours of cultur e.3.Hep G2 cells treated with Roc-A at different concentrations were collecte d for detection of HK2 and BCL2L1 proteins expression:Hep G2 cells were di vided into 3 groups which were then added with 0,100 and 200 nmol/L Roc-A,respectively.HK2 and BCL2L1 proteins of Hep G2 cells were detected by proteomics mass spectrometry after 24-hour culture.Hep G2 cells in the logarith mic growth phase were divided into five groups and were added with Roc-A s olution of 0,25,50,100 and 200 nmol/L,respectively.HK2 and BCL2L1 pro teins were detected by Western blotting at 24 hours of culture.4.Analysis of the relationship between HK2 expression in cancer tissues a nd survival time of patients with liver cancer:Kaplan Meier Plotter cancer dat abase was used to analyze the relationship between HK2 expression and surviv al time of liver cancer patientsResults1.Hep G2 cell apoptosis rates were 0.83%±0.21%,9.46%±2.34%,24.68%±4.22%,39.60%±2.16%and 58.63%±1.62%in Roc-A solution of 0,25,50,100and 200 nmol/L,respectively.F=8807,Р﹤0.0.05.In this experiment,cell apo ptosis rate increased with the increase of Roc-A solution concentration.2.Compared with blank control,HK2 and BCL2L1 m RNA relative expres sion levels of Hep G2 cells cultured for 24 h with 100 nmol/L Roc-A solution were decreased（allР﹤0.05）.3.Compared with blank control,HK2 and BCL2L1 protein relative expres sion levels of Hep G2 cultured with different concentrations of Roc-A were dec reased（allР﹤0.05）.4.Compared with liver cancer patients with high expression of HK2,liver cancer patients with low expression of HK2 had a longer survival time（Р﹤0.05）.Conclusion1.Roc-A promotes apoptosis of Hep G2 cells,and apoptosis is positively c orrelated with Roc-A concentration.2.The mechanism of Roc-A promoting apoptosis of Hep G2 cells may be that Roc-A inhibits the expression of HK2 and BCL2L1 m RNA and protein in Hep G2 cells.