Study On The Anti-hepatocarcinoma Activity Of Erianin And Its Transferrin Targeted Liposomes

Cancer has now been recognized as a global problem,however there is no effective and feasible solution for it.Cancer is already the second leading cause of death after cardiovascular diseases,and the morbidity and mortality rates are increasing year by year.Liver cancer(LC)is the most common cancer and has a high incidence in China.At present there are several methods for LC,such as surgery,radiotherapy,chemotherapy,immunotherapy and traditional Chinese medicine treatment,but each treatment has its limitations.And the immune system disorder and organ damage which caused by the treatments still plague the majority of patients.Erianin,a natural compound derived from Dendrobium candidum,shows various pharmacological activities Studies in the last two decades have shown that erianin is a potential anti-cancer drug.However,the poor water solubility and low bioavailability of maolansu greatly affect the development of maolansu as a therapeutic drug.Therefore,it is particularly important to select the appropriate drug delivery carrier.Therefore,this paper mainly studied the anti-tumor activity of erianin on Hep G2 and SMMC-7721 cells,designed and prepared transferrin-liposomes-erianin(Tf-LP-Erianin)and evaluated its anti-hepatocellular cancer activity.In vitro,studies have found that erianin could induce apoptosis of Hep G2 and SMMC-7721 cells through ROS-mediated mitochondrial pathway.Specifically,erianin inhibited cell viability,colony formation and cell migration of Hep G2 and SMMC-7721 cells.Erianin enhanced the activity of Caspase-3,-8 and-9,induced apoptosis of liver cancer cells and caused cell arrest in G2/M phase.At the same time,erianin also increased the levels of reactive oxygen species(ROS)in the cell and reduced the mitochondrial membrane potential(MMP).Western blot experiments showed that erianin caused the activation of caspase in Hep G2 and SMMC-7721 cells,the caspase cascade reaction and the cleavage of its substrate poly ADP-ribose polymerase-1(PARP-1).At the same time,erianin reduced the expression of B-cell lymphoma-2(Bcl-2),and enhanced the expression of Bcl-2 associated X protein(Bax),Bcl-2 associated death promoter(Bad),Bcl-2 interacting mediator of cell death(Bim)and p53 upregulated modulator of apoptosis(PUMA).In the in vivo study of Hep G2-and SMMC-7721-xenografted nude mouse models,erianin inhibited tumor growth of mice,without significant effect on its body weight.The results of pathological section showed that the liver cells and spleen cells in the administration group were uniform compared with the blank group,and there was no obvious inflammatory infiltration,indicating that the toxic effect of erianin on normal tissues was not obvious.After 14 days of treatment with erianin,Cleaved caspase-3,-8,and-9 were significantly increased in tumor tissues of nude mice,and Cleaved PARP-1 was also significantly increased.Erianin also increased the expression levels of Bax,Bad,Bim,and PUMA in tumor tissues and decreased the expression levels of Bcl-2,which was consistent with in vitro results.In Hep G2-and SMMC-7721-xenografted BALB/c mouse models,erianin could reduce the levels of oxidative stress in spleen cells and inhibit the phosphorylation of nuclear factor kappa-B(NF-?B),thereby regulating the secretion of inflammatory factors and enhancing the body's anti-tumor immune response.Specifically,erianin inhibited the growth of tumors in BALB/c mice,and had no obvious effect on the body weight and tissue structure of the mice.The cytokine antibody array analysis showed that erianin caused changes in some cytokines in tumor tissues,namely there were 38 and 15 kinds of cytokines that changed more than 50% in the tumor tissues of Hep G2 and SMMC-7721 tumor-bearing mice.Results of enzyme-linked immunosorbent assay(ELISA),showed that erianin enhanced the serum levels of tumor necrosis factor(TNF)-? and granulocyte-macrophage colony stimulating factor and reduce the expression levels of matrix metalloproteinase-2 and matrix metalloproteinase-9,as well as,interleukin(IL)-6,IL-10,chemokine C-C motif ligand(CCL)2,CCL11,CCL21,chemokine(C-X-C motif)ligand(CXCL)11,CXCL13 and CXCL16 in tumor-bearing mice.Erianin contains multiple methoxy groups and can be dissolved in organic solvents such as methanol and ethanol,but its solubility in water is low,which greatly affects its development as a therapeutic drug.Therefore,we designed erianin liposome as a drug delivery system,and modified it with transferrin.LP erianin liposomes were prepared by ethanol injection method.Firstly,(2,3-dioleoyl-propyl)-trimethylamine,egg yolk phosphatidylcholine,cholesterol,phosphatidylethanolamine polyethylene glycol 2000 maleimide were dissolved in ethanol,and then the above solutions were mixed according to a certain amount of material ratio and injected into phosphate buffer saline.Afterwards,LP-Erianin was modified with transferrin(Tf)to obtain Tf-LP-Erianin.LP-Erianin and Tf-LP-Erianin showed smooth surface and uniform particle size under field emission scanning electron microscopy,and their particle sizes were 62.60 nm and 88.63 nm,respectively,and their dispersion coefficients index were less than 0.3,which met the pharmacopoeia standard.And within 48 hours,the particle size stability is good.The ? potential of LP-Erianin and Tf-LP-Erianin were 14.5 ± 1.21 MV and 2.36 ± 0.36 MV,respectively.The envelopment efficiency of erianin in LP-Erianin and Tf-LP-Erianin is 69.5% and68.5%,respectively.The results show that LP-Erianin and Tf-LP-Erianin have reasonable physicochemical properties,which are suitable for in vivo and in vitro activity study.The study evaluated the antitumor activity of Tf-LP-Erianin in vivo and in vitro.In vitro,Tf-LP-Erianin inhibited the cell viability of Hep G2 and SMMC-7721 cells,reduced the MMP,induced cells apoptosis and enhanced the uptake of erianin by tumor cells.In vivo,studies have demonstrated that the Tf-LP-Erianin as a drug delivery system successfully delivered erianin and enhanced the anti-tumor effect of erianin in Hep G2-and SMMC-7721-xenografted tumor model in BALB/c nude mice and BALB/c mice.Specifically,both LP-Erianin and Tf-LP-Erianin inhibited the growth of ectopic tumors and improved the tissue distribution of erianin in BALB/c nude mice.In vivo imaging results showed that with the prolonged administration time,Tf-LP-Erianin were more distributed in tumor tissues,reducing damage to other tissues.In Hep G2-and SMMC-7721-xenografted BALB/c mouse models,LP-Erianin and Tf-LP-Erianin could also reduce the levels of oxidative stress in the spleen,enhance its immunoregulatory function and regulate the expression levels of matrix metalloproteinases(MMPs),interleukins and chemokines,which enhanced the anti-tumor immune response of erianin.In summary,the in vivo and in vitro studies in this article have proved that erianin could induce Hep G2 and SMMC-7721 cells apoptosis through ROS-mediated mitochondrial pathway.At the same time,erianin also reduced the oxidative stress level in spleen cells,inhibited the phosphorylation of NF-?B,thereby regulated the secretion of inflammatory factors and enhanced the anti-tumor immune response.In addition,this study successfully established Tf-LP-Erianin as a drug delivery system,and proved the anti-liver cancer activity of Tf-LP-Erianin in vivo and in vitro,which enhanced the anti-tumor effect of erianin.All in all,drug delivery system of Tf-LP-Erianin is a potential strategy for the treatment of liver cancer.