Molecular Mechanism Of 10-hydroxy-2-decenoic Acid Induced Hepatocellular Carcinoma Cell Apoptosis,cycle Arrest And Migration Inhibition

Liver cancer,also known as hepatocellular carcinoma(HCC),is a common malignant tumor with strong malignant degree,high mortality rate and strong invasion and metastasis.Common chemotherapy drugs mainly include cisplatin,loplatin and 5-fluorouracil(5-FU),which have large side effects and high price.Therefore,it has become a research hotspot to find and develop a potential natural adjuvant drug or functional factor for anti-liver cancer with low toxicity and economic safety.10-Hydroxy-2-decenoic acid(10-HDA),a unique active ingredient in royal jelly,has a variety of biological activities such as anti-bacterial,anti-inflammatory,immunity,antitumor,anti-radiation and so on.To this end,the molecular mechanism of 10-HDA against liver cancer was explored at the cellular and molecular levels,and its effects on apoptosis induction,cycle arrest,regulation of reactive oxygen species(ROS)level,inhibition of migration and regulation of corresponding signal pathways of liver cancer cells were revealed.This study provides a new idea and theoretical basis for the research of functional factors for the prevention and treatment of liver cancer and the development of new adjuvant anti-tumor drugs.Taking 10-HDA as the research object and liver cancer cell line as the research model,the killing effect of 10-HDA on liver cancer cell line(Hep G2,Hep3 B,Huh7)and the toxicity effect of 10-HDA on normal liver,lung and stomach cells(L-02,IMR-90,GES-1)were detected by CCK-8 assay.The apoptotic effect of 10-HDA on Hep G2 cells was detected by Hoechst33342/PI double staining,Annexin V/PI staining and flow cytometry.Cell cycle arrest,the relationship between MAPK,STAT3,NF-?B signaling pathways and the expression of related proteins were detected by flow cytometry and Western blotting.The regulation of ROS-related signaling pathway and the expression of nuclear transcription factors STAT3 and NF-?B in Hep G2 cells were detected by DCFH-DA fluorescent probe and Western blotting.And the inhibitory effect of 10-HDA on the migration of Hep G2 cells and the expression of related proteins were detected by cell scratch assay and Western blotting.10-HDA had obvious killing effect on liver cancer cell lines.At the same time,5-FU significantly induced cytotoxicity in normal cell lines higher than that of 10-HDA.10-HDA inhibited the expression of Bcl-2,and promoted the expression of Bax,which activated the mitochondrial apoptosis pathway,decreased the mitochondrial membrane potential,and then released Cytochrome C(Cyt C),activated Caspase-3,and finally led to mitochondria-dependent apoptosis.In addition,10-HDA activated the mitogen-activated protein kinase(MAPK)and signal transduction and transcriptional activator(STAT)3 signaling pathways,which promoted the expression of P-JNK,P-P38,and I?B-?,and inhibited P-ERK,P-STAT3,and nuclear transcription factor(NF-?B),but they were blocked after MAPK inhibitors treatment.10-HDA also inhibited the expression of STAT3 and NF-?B nuclear transcription factors in the nucleus.10-HDA induced cycle arrest in G2/M phase by promoting the expression of P21 and inhibiting the expression of P-Akt,CDK1/2 and Cyclin A/B1.10-HDA induced ROS accumulation,and cell apoptosis and the expression of related-protein were significantly reversed after N-acetyl-L-cysteine(NAC)treatment.10-HDA inhibited cell migration by inhibiting the expression of TGF-?1,P-GSK-3?,Snail,Twist,N-cadherin,Vimentin and promoting the expression of E-cadherin.In conclusion,10-HDA regulated MAPK,STAT3 and NF-?B signaling pathways by upregulating intracellular ROS level,inducing mitochondria-dependent apoptosis of Hep G2 cells,and inhibited AKT signaling pathway,which regulated downstream Cyclin-related proteins,thus leading to G2/M phase cycle arrest of Hep G2 cells.In addition,10-HDA inhibited the TGF-?1signaling pathway,which inhibited the migration of Hep G2 cells.