The Therapeutic Effect Of Tyroserleutide On Hepatocarcinoma And Its Mechanism By Affecting Signal Transduction In Tumor Cell

Objective:To explore the anticancer effect of Tyroserleutide (YSL) on humanhepatocellular carcinoma and to approach its preliminary mechanisms by inhibitingproliferation and inducing death.Methods:1. The hepatocellular carcinoma cell lines BEL-7402, SMMC-7721, Hep3B,HepG2, SK-HEP-1 and normal hepatocyte cell lines Chang's Liver and HL7702were used to explore the effect of YSL on cell proliferation. In vitro effect wasassayed by MTS method, and in vivo effect was assay by hollow fiber tumormodel. Nude mouse model of implanted BEL-7402 tumor was set up to observedthe inhibitory effect of YSL on growth of tumor. The ultra structure change oftumor cells was observed by electron microscopy.2. The effect of YSL on cell cycle of human hepatocellular carcinoma BEL-7402cells was assayed by FCM method. The apoptosis-inducing function of YSL onhuman hepatocellular carcinoma BEL-7402 cells was assayed by FCM, DNALadder and Nucleosome ELISA methods.3. Human gene-chip analysis was used to explore the effect of YSL on geneexpression of BEL-7402 tumor implanted to nude mice.4. The effects of YSL on mitochondrial permeability transition pore opening;mitochondrial transmembrane potential and change of cytoplasmic free calciumin BEL-7402 cells were explored by FCM and microplate fluorometer.5. Western Blot were used to observed the effects of YSL on protein expression ofapoptosis inhibitor Bcl-2, Bcl-X_L, calcium binding protein--Calreticulin, andCaspase-12. Cytochrome C releasing and activation of Caspase-3 were assayed by Western Blot, too.6. By Real-time PCR and Western Blot methods, effect of YSL on mRNA andprotein expression, and also the phosphorylation of regulators and effectors inPI3K-Akt pathway were observed, including PTEN, Akt, PDK1, P21, P27,MDM2, P53 and Bad.Results:1. YSL (0.4～3.2mg/ml in vitro and 320～640μg/kg/d in vivo) can remarkably inhibitproliferation of human hepatocellular carcinoma cells BEL-7402, SMMC-7721,Hep3B, HepG2, SK-HEP-1(P＜0.05), and had no obvious effect on normalhepatocyte Chang's liver and HL7702 (P＞0.05). YSL (320, 640μg/kg/d)significantly inhibited the growth of HCC BEL-7402 transplanted in nude mice,with inhibition rate 40.04ï¼…and 52.85ï¼…, respectively. The constituting aminoacids mixture of YSL could not inhibit the growth of the human hepatocarcinomacell lines in vitro. Observations under electron microscopy showed that YSLdestroyed the structure of chromatin, damaged cell organelles in tumor cells, andinduced apoptosis and necrosis in tumor cells.2. Human gene-chip analysis suggested that YSL might regulate gene expression oftumor cell through many pathways, including inhibited the genes responsible formitochondria respiratory chain and tumor cell proliferation, and signaltransduction, as well as the oncogene/anti-oncogene expression.3. YSL (0.8, 1.6mg/ml) could stop the cell cycle of tumor cells by remarkablyincreasing the percentage of G₀/G₁ phase cells, decreasing the percentage of Sphase cells (P＜0.05); YSL could induce the apoptosis in BEL-7402 cells:apoptotic cells amount in sample treated with YSL were more than that of controlgroup; the DNA Ladders were detected in BEL-7402 cells treated with YSL; andthe Nucleosome ELISA assay showed that the mono- and oligo-nucleosomes of apoptotic cells increased significantly in groups treated with YSL.4. YSL could activate the opening of mitochondrial permeability transition pore,decrease mitochondrial transmembrane potential, and increase the concentrationof cytoplasmic free calcium in BEL-7402 cells.5. YSL could decrease protein expression of apoptosis inhibitor Bcl-2 and Bcl-XL,promote Cytochrome C releasing and increase amount of activated Caspase-3fragment in BEL-7402 cells. Calreticulin is a type of calcium binding protein inendoplasmic reticulum, and plays an important role in calcium homeostasisregulation. YSL could increase protein expression of Calreticulin in BEL-7402cells. YSL also could increase protein expression of Caspase-12 which is themember of ER-associated death pathway.6. YSL could increase the mRNA and protein expression of anti-oncogene PTEN,up-regulate its function, and inhibit expression of oncogene Akt, down-regulatethe function of Akt and its associated kinase PDK1. YSL could increase themRNA and protein of cell cycle regulator P21and P27. YSL could inhibit thephosphorylation of MDM2, and then increase the protein level of P53. YSL alsocould decrease the phosphorylation of Bad, which may cause mitochondriadamage and apoptosis.Conclusion:YSL can inhibit the proliferation of hepatocellular carcinoma cells in vivo and invitro, stop the cell cycle of tumor cells, and induce those cells apoptosis. At the samedosage, YSL showed no effect on normal heaptocyte. The mechanism of itsanticancer effect may be as follows: YSL can injury tumor cell mitochondria, activatemitochondrial permeability transition pore opening, decrease mitochondrialtransmembrane potential, and increase the concentration of cytoplasmic free calciumin tumor cells. These effects could induces the Cytochrome C, Ca²⁺ and other apoptosis promoter releasing from mitochondria, activate mitochondria deathpathway and ER-associated death pathway. YSL also can regulate the expressionbalance between oncogene and anti-oncogene, mediates inhibition of tumor growththrough inhibition of the PI3K pathway.