Curcumol Induces Apoptosis Through Raf/MEK/ERK Pathway In Hepatocelluar Carcinoma Cells

Hepatocellular carcinoma(HCC)as a primary cancer originating from the epithelium or mesenchyme of the liver,is one of the most common malignant tumors with the highest fatality rate worldwide.In China,about 383 thousand people die from HCC annually,50%of the world’s total.HCC usually concealed onset and the progression is rapid,this severe situation has brought a heavy burden on society and medical care.The clinical treatment of early liver cancer is mainly by surgical resection,but because of the majority of patients with liver cancer complicated by cirrhosis,recurrence after surgical resection is still easy.In addition,as new therapeutic techniques,local ablation,radiological intervention,and biologic gene therapy provide patients a variety of options,however,each method is limited to their respective indications,and can not be used in all patients,so it is particularly important to explore a new anti-hepatoma drugs.Chinese medicine is the material basis for the development of traditional medicine science.Thousands of years of continuous absorption and integration of advanced technologies and ideologies of different periods have gradually become an important means of curing illnesses and safeguarding health.Under the guidance of the fundamental theory of traditional Chinese medicine,Chinese medicine has made enormous progress in the treatment of cancer.There are a large number of reports on the anti-tumor effect of traditional Chinese medicine both home and abroad.It has been widely used to improve the quality of life of patients due to its characteristics of multi-target,multi-link,long-lasting effect,low toxic and low side effects.Curcumol with the structure of a guaiane-type sesquiterpenoid hemiketal,is the bioactive component of Rhizoma Curcumae,has antibacterial,antiviral,anticancer and other physiological properties.Taking into account its anti-tumor mechanism is not yet completely clear,this study made a further research on the mechanism of curcumol anti-hepatoma.Experimental thesis consists of three parts.The first part:the anti-hepatoma effect of curcumol in vitroMTT-based proliferation assay was used to investigate the viability of cells,which including hepatocellular carcinoma cell line Bel-7402,SMMC-7721,HepG2 and Human normal liver L02 cells.The result showed that curcumol exhibited a significant growth inhibition on hepatocellular carcinoma cells.In contrast,L02 cells showed only minor death even in high concentration of curcumol,suggesting that the antineoplastic activity of curcumol appeared to be more specific to hepatoma cells.The second part:the anti-hepatoma effect of curcumol in vivoWe determined the LD50 of curcumol by acute toxicity test,and based on this set the dose to carried out tumor-bearing nude mice experiment.Considering good pharmacodynamic results of Bel-7402 in vitro,subsequent experiments were performed by use it.Nude mice were inoculated subcutaneously with Bel-7402 cells to establish the subcutaneous tumor model of hepatocellular carcinoma.Nude mice were treated with curcumol and elemene,respectively.The length and the breadth of tumor were measured with vernier slide caliper every other day,all the data were used to portray the growth curve ultimately.By calculating the tumor volume and mass inhibition rate,we found that curcumol was effective in vivo and better than elemene.The third part:the anti-hepatoma mechanism of curcumolIn this part,formation of apoptotic bodies were observed using Hoechst 33258 staining.The decrease of mitochondrial membrane potential was detected by JC-1 probe.Furthermore,to confirm the inhibition effect of curcumol on Bel-7402 cells is associated with induced apoptosis of tumor cells,the cellular apoptotic response was investigated via Annexin V-FITC/PI staining assay.The result indicated that curcumol induces apoptosis of Bel-7402 cells in a does-dependent manner.Western blot analysis showed that increasing concentrations of curcumol distinctly decreased the expressions of p-Raf,p-MEK1/2,p-ERK1/2,especially p-ERK1/2.The apoptosis of Bel-7402 could be greatly reduced by adding agonist of p-ERK.Moreover,the experiment also found that curcumol decreased the expression of Bcl-2 by inhibiting the activation of Raf,thereby restraining the anti-apoptotic function of Bcl-2.IHC staining of tumor tissue showed that the mechanism of anti-tumor activity of curcumol in vivo is also related to the suppression of Raf/MEK/ERK signaling pathway.Meanwhile,propidium iodide(PI)staining was used to detect the effect of curcumol on the cell cycle.The results showed that curcumol could block the cell cycle of Bel-7402 in G0/G1 phase and thus inhibit cell proliferation.In summary,curcumol has significant anti-hepatocellular carcinoma effect in vitro and in vivo.Curcumol inhibits the growth of Bel-7402 cells via triggering apoptosis.The underlying mechanisms may be associated with the suppression of Raf/MEK/ERK signaling pathway.