Effect Of PI3K/AKT Signaling Pathway And Related Regulatory Molecular Of HepG2Cell Intervened By Curcumin Combining With5-fluorouracil

Hepatocellular carcinoma (HCC) is one of the most common malignanttumors, with a high incidence in developing countries. Although in the pastfew decades, the treatment methods of liver cancer has increased, but becauseof insidious onset of liver cancer Intrahepatic metastasis earlier, recurrencerate is high,and the liver cancer cells are not sensitive to the commonly usedanticancer drugs and radiation therapy, the prognosis remains poor inhepatocellular carcinoma. Therefore, to find and develop new therapeuticstrategy is very necessary and urgent.5-fluorouracil (5-FU) is a pyrimidineanticancer drugs. Since its discovery,5-FU occupies the important status inthe field of cancer chemotherapy. It is very effective for malignant tumor andwidely used in a variety of cancers (such as liver, stomach, pancreatic, breastcancer). But the hepatocellular carcinoma cells is not particularly sensitive for5-FU treatment, So how to improve the sensitivity of5-FU is a very importantissue. Curcumin is a kind of pigment extraction from Zingiberaceae turmeric.Modern research has found that the main pharmacological action of curcuminhas antioxidant, inhibit inflammatory reaction and inhibit tumor growth. Inrecent years, many studies have shown that a wide variety of human tumors,such as gastric cancer, colorectal cancer, breast cancer, liver cancer, renal cellcarcinoma are closely related with PI3K/AKT signaling pathway. PI3K/AKTsignaling pathway can not only regulate tumor cell apoptosis, but also regulatemany kinds of cytokines and inflammatory factors expressions in tumormicroenvironment. Previous studies have found that PI3K/AKT signalingpathway can regulate the occurrence and development of immune cells orimmune molecules interfere with tumor. Akt is a serine/threonine proteinkinase, also called protein kinase B (PKB), is a key signaling molecule in PI3K/AKT signaling pathway and plays an important role in cell survival andapoptosis. PI3K belongs to the cytosolic phosphatidylinositol kinase familymembers, it can specifically the phosphatidyl inositol and phosphatidylinositolthird hydroxyl groups phosphorylation. PTEN (phosphatase and tensinhomolog deleted on chromosome ten), namely human chromosome tenthdeletion phosphatase and tensin homolog, is a newly discovered tumorsuppressor gene, located in chromosome tenth q23.3, transcripts for515KBmRNA. PTEN belongs to the PTP (protein tyrosine phosphatases) gene familymembers. The PTEN gene regulate the function of tumor suppressor cells, bypreventing excessive growth and division, and regulation of cell division cycle.Evidence suggested that PTEN also useful control cell adhesion，vascularformation and cell migration. In addition, PTEN also plays an important rolein maintaining the stability of cell genetic information. The PTEN gene is theevaluation index of numerous tumor prognoses, it is important to study themechanism of diagnosis and gene therapy of tumor. Toll like receptors(Toll-like, receptors, TLR) is a kind of important proteins involved innonspecific immunity, as well as a bridge to connect the non-specificimmunity and specific immunity. TLR can monitor and identify the variousdiseases associated molecular pattern (PAMP), is the first barrier againstinfectious diseases. The TLR is a single transmembrane non-catalytic protein,can the molecular recognition from microorganisms with conservativestructure. When a physical barrier to microbial breakthrough body, such asskin, mucous membrane, TLR can identify them and activate the immune cellresponse. TLR4can not only identify exogenous pathogens, but also toidentify endogenous substances and degradation products. Recent research hasshown that, TLR can combine some endogenous molecules produced by thebody. Immune adjuvant can enhance anti-tumor immunity, the molecular andcellular mechanism has been further clarified, TLR also plays an importantrole in this respect.Objective: To study the effect of5-FU alone or combined with curcuminon HepG2cells of PI3K/AKT signal pathway and related regulatory molecules AKT, PTEN and TLR4.Methods: the experiment was divided into two parts. The first part: theeffects of5-FU alone or combined with curcumin on HepG2cells ofPI3K/AKT signal pathway and related regulatory molecules AKT, PTEN andTLR4. Selection of HepG2hepatoma cells used in the study, HepG2cellswere cultured in vitro, the logarithmic growth phase were used in theexperiment, HepG2cells were inoculated in96holes plate, Drug interventionwas conducted after48hours of incubation with different concentrations ofcurcumin (5umol/L,10umol/L,20umol/L,40umol/L,50umol/L,60umol/L,80umol/L).HepG2has cultured for24,48hours after MTT examination,determined experimental concentration of curcumin. By using5fluorouracilalone or combined with curcumin as drug group of cultured HepG2cells,were cultured for24hours and48hours, MTT examination,5fluorouracilalone or combined effects of curcumin on HepG2cell survival rate, cellextraction of total RNA and total protein, the expression of RT-PCR methodwas used to detect mRNA levels of PTEN, AKT, TLR4, protein levels ofPTEN, AKT was detected by Western-blot method. The second part: theexpression of mRNA and protein of PTEN, AKT, TLR4in hepatocellularcarcinoma and cirrhosis tissues: hepatocellular carcinoma and liver fibrosistissues of the10cases, the protein levels of PTEN, AKT was detected byWestern-blot method. Total RNA was extracted from liver tissue and hepaticfibrosis, RT-PCR was used to detect the expression of mRNA levels of AKT,PTEN, TLR4.Results:1Different concentrations of curcumin effect on survival rate of HepG2cells:MTT results showed: in different time, at different concentrations(5umol/L,10umol/L,20umol/L,40umol/L,50umol/L,60umol/L,80umol/L)of the curcumin can inhibit cell, and in a time-and dose-dependent manner.25-FU alone and combined with curcumin effect on survival rate ofHepG2cells: MTT results showed: in the24,48hours and two hours, the use of5-FUalone (7.5umol/L) and5-FU combined with curcumin on HepG2cells inhibit.And in combination than medication alone inhibited, and the longerintervention time, the stronger inhibition.3Effects of5-FU alone and in combination with curcumin mRNA onHepG2cells in PTEN, AKT, TLR4: drug intervention in HepG2cells for24hours, the expression of5-FU group were significantly higher than those incontrol group, mRNA of PTEN and TLR4increased, mRNA of AKTdecreased obviously; curcumin combined with5-FU group compared with5-FU group and the expression of mRNA of PTEN and TLR4increased moresignificantly, expression of mRNA of AKT decreased also significantlymore. Compared with the results of24hours training, drug HepG2wereincubated for48hours in group5-FU mRNA of PTEN and TLR4wassignificantly increased in24hours, the expression of mRNA of AKT wassignificantly reduced in24hours; curcumin combined with5-FU groupcompared with the single5-FU group the expression trend more obvious.Each index of the two groups were statistically significant (P<0.05).45-FU alone or combined effect of curcumin on expression of AKTprotein in PTEN, HepG2cells and HepG2cells: drug intervention for24hours,5-FU group than in the control group increased the expression ofprotein of PTEN, decreased the expression of protein of AKT; curcumincombined with5-FU group compared with5-FU group the trend moreobvious. Drug intervention for48hours,5-FU alone or curcumin combinedwith5-FU group increased protein levels of PTEN and decreased proteinlevels of AKT protein more than the trend of24hours. Each index of the twogroups were statistically significant (P<0.05).5Expression of mRNA of PTEN, AKT, TLR4, and expression of proteinof PTEN, AKT, P-AKT in liver cancer and liver fibrosis tissue: the resultsshowed that the index of levels of mRNA of PTEN, AKT, TLR4and levelsof protein of PTEN, AKT,P-AKT were significantly higher in hepatocellularcarcinoma than in liver fibrosis tissue, the index has statistical significance between the two groups (P<0.05).Conclusions:1Curcumin can induce apoptosis of HepG2cells, with concentration andtime increasing apoptosis gradually clear. Apoptosis of5-FU is more obvious.2The effect of curcumin and5-FU through PI3K/AKT signaling reducedAKT levels and increased PTEN, TLR4level, thereby improving the antitumor effect.3Activity of PI3K/AKT signaling pathway increased significantly inliver cancer tissue than in liver fibrosis tissue.