Inhibiting Autophagy Can Potentiate The Inhibitory Effect Of Sodium Aescinate In Hepatocellular Carcinoma

Objective Hepatocellular carcinoma（HCC）always keeps the high morbidity and mortality,and its therapeutic effect is not satisfying as well,including mainly high recurrence after the operation,low response rate to the drugs,and strong drug resistance.Therefore,the more effective treatment is urgently needed to relieve the patients’pain and save their lives.Our previous studies have found that sodium aescinate can inhibit the HCC cells growth and induce HCC cells early apoptosis.This article aims to demonstrate that sodium aescinate can induce the autophagic flux in HCC cells,and inhibiting autophagy can enhance the antitumor effect of sodium aescinate.Methods（1）The culture of three HCC cell lines（Huh7,Hep G2和Hep3B）.（2）In order to form the xenografts,Huh7 cells were injected into the subcutaneous tissue of 24 nude mice.10 days later,12 nude mice forming the xenografts（similar size）were assigned randomly to 4 groups:control,sodium aescinate,chloroquine,and sodium aescinate+chloroquine,with three mice in every group.These mice were injected with saline or drugs intraperitoneally every 2 days from the 14^th day.4 weeks later,the xenografts were grafted for taking a photo and analysis.（3）Transfections of LC3B plasmid and ATG5small interfering RNA（si RNA）in Huh7 cells.（4）Western blot was used to detect the proteins related to autophagy and its signaling pathway:LC3B,Beclin-1,P62,PI3K,AKT,P-AKT（Ser473）,m TOR,P-m TOR（Ser2448）,4E-BP1,Phospho-4E-BP1（Thr37/46）,ATG5.（5）Observe the autophagosomes and autolysosomes after sodium aescinate treatment,using the confocal laser scanning microscope and transmission electron microscope.（6）MTT assay was used to examine the cell viability after drug treatment.（7）Annexin V-FITC/PI staining was used to examine the cell early apoptosis after drug treatment.（8）Colony formation assay was used to examine the colony formation ability of single cell after drug treatmentResults（1）Sodium aescinate upregulated the expressions of LC3B protein（I and II）in three HCC cell lines and xenografts of nude mice.（2）Sodium acscinate increased the numbers of autophagosomes and autolysosomes by the observation of confocal laser scanning microscope and transmission electron microscope.（3）Compared with single drug treatment,the combination of sodium aescinate and chloroquine could induce more expressions of LC3B protein（I and II）in three HCC cell lines.（4）Sodium aescinate inhibited the expressions of proteins related to the PI3K/AKT/m TOR signaling pathway.（5）Using the insulin-like growth factors 1（IGF-1）to activate the PI3K/AKT/m TOR signaling pathway could attenuate the inhibitory effect of sodium aescinate to the HCC cells.（6）Compared to the alone drug treatment,the co-administration of sodium aescinate and chloroquine could decrease the cell viability,increase the cell early apoptosis,and prohibit the colony formation ability of HCC cells to a greater extent.（7）Downregulating the expression of ATG5 protein using si RNA could enhance further the effect of sodium aescinate in reducing the cell viability and inducing the cell early apoptosis.（8）The combined administration of sodium aescinate and chloroquine had a stronger inhibition effect against the xenografts than the sodium aescinate alone in the subcutaneous xenograft model of nude mice.Conclusion In the experiments of HCC cell lines and nude mice,the autophagic flux induced by sodium aescinate is protective for HCC cells,which counteracts the partial inhibitory effect of sodium aesciante in HCC,and inhibiting autophagy by chloroquine or ATG5 si RNA potentiates the effect of sodium aescinate in HCC.