The Therapeutic Effect Of Tyroservatide On Hepatocarcinoma And Its Mechanism By Inhibiting Histones Of Tumor Cells

Objective:To investigate of the inhibitory effects of tripeptide tyroservatide (YSV) onhuman hepatocarcinoma and explore its possible mechanisms by which inhibitsexpression of histones to inhibit the proliferation of tumor cells and induce apoptosisand necrosis of tumor cells.Methods:1. The MTS Assay and Cellmax Implant Membrane Hollow Fiber implantationwere applied for in vitro and in vivo proliferation assessment of YSV on fivehepatocarcinoma cells, BEL-7402, SMMC-7721, Hep3B, Hep G2, SK-HEP-1,and two normal liver cells, Chang liver, HL-7702 and human normal lymphocyte.The human hepatocarcinoma BEL-7402 xenografts in nude mice were used toestimate the antitumor effects of YSV in vivo and the effects on the ultrastructureof the tumor cells using electron microscopy.2. The effects of YSV on the cell cycle of BEL-7402 cells were determined by flowcytometry. Annexin V/PI double staining, DNA fragmentation and NucleosomeELISA were used to investigate apoptosis in BEL-7402 cells in vitro.3. RT-PCR and Western blot were used to observe the effects of YSV on expressionof five histones (H1, H2A, H2B, H3 and H4). Samples were extracted fromhuman hepatocarcinoma BEL-7402 xenografts in nude mice, the livers of nudemice bearing xenografts in vivo and human hepatocarcinoma cells BEL-7402,Hep G2 and normal liver cells HL-7702, human normal lymphocytes.4. Nuclear Run-on Assay was used to address the contribution of transcriptioninitiation to the regulation of five histones by YSV. Actinomycin D inhibitor studies were used to detect the mRNA half-life of five histones. SLBP weremeasured by Western blot assay to identify regulation of YSV on Pre-mRNAsplicing.5. H2A.X phosphorylation flow cytometry assay were used to estimate thedouble-stranded DNA breaks (DSBs) of BEL-7402 cells induced by YSV.Giemsa staining was used to observe the effects of YSV on structure ofchromosome in BEE-7402. H3 phosphorylation flow cytometry assay wereused to observe the effects of YSV on mitosis index of BEL-7402 cells.Results:1. YSV (0.2～1.6mg/ml in vitro and 160, 320μg/kg/d in vivo) could significantlyinhibit proliferation of five hepatocarcinoma cells (P＜0.05), and had no obviouseffects on normal liver cells Chang liver, HL-7702 and human normallymphocytes (P＞0.05) .Amino acid mix of YSV had no effects on growth of fivehepatocarcinoma cells, which indicated the distinct structure of YSV play a vitalrole in sustaining antitumor effects of YSV. YSV (320, 640μg/kg/d) significantlyinhibited the growth of BEL-7402 transplanted in nude mice. Observations underelectron microscopy showed that YSV destroyed the structure of chromatin,damaged cell organelles of tumor cells, and induced apoptosis and necrosis intumor cells.2. YSV (0.8mg/ml) could repress the cell cycle of BEL-7402 cells by inducing Sphase arrest and decreasing the percentage of G2/M phase cells. YSV (0.8mg/ml)could significantly induce the apoptosis in BEL-7402 cells: apoptotic cellsamount in sample treated with YSV are higher than that of control group, and theDNA ladder and smear were detected in BEL-7402 cells treated with YSV. Andthe Nucleosome ELISA assay showed that the mono- and oligo-nucleosomes in apoptotic cells increased significantly in BEL-7402 cells by YSV (P＜0.05).3. YSV could decrease mRNA and protein expression of five histones (H1, H2A,H2B, H3 and H4) in human hepatocarcinoma BEL-7402 in nude mice andBEL-7402 and Hep G2 cells in vivo (P＜0.05). YSV show no obvious effects onmRNA and protein expression of five histones in liver tissue from nude micebearing human hepatocarcinoma BEL-7402 and human normal liver cellHL-7702 and human normat lymphocyte.4. YSV (0.8mg/ml) significantly inhibit transcription initiation of histone H3;decrease half-life of histone H2B, H3 and H4. And YSV inhibited the proteinexpression of SLBP, which play an important role in Pre-mRNA splicing ofhistones.5. YSV (0.8mg/ml) could induce DSBs in BEL-7402 cells and damage thechromosome. And YSV significantly reduce percentage of pho-Histone H3positive cells in BEL-7402 cells.Conclusions:YSV could significantly inhibit the growth of hepatocarcinoma cells in vivo andin vitro, stop the cell cycle of tumor cells, and induce those cells apoptosis andnecrosis. At the same dosage, YSV showed no effect on normal cells. The mechanismof its antitumor effects may be as follows: YSV could inhibit the mRNA synthesis ofhistones to reduce histones, which induced DSBs and damaged chromosome to killtumor cells. Also the reducing of histones could activate S phase checkpoint, whichinduced S phase arrest to stop cell cycle of tumor cells.