Effects And Mechanisms Of Differentiation Induced By Tachyplesin On The Human Hepatocarcinoma SMMC-7721 Cells

In this study, we used tachyplesin, a low molecular weight peptide which isolated from the hemocytes of horseshoe crab (Tachypleus tridentatus), together with a polar compound HMBA as control group of differentiation induce and their combination, to treat the human hepatocarcinoma SMMC-7721 cells for investigating the biological effects and the antitumor mechanisms of tachyplesin by the methods of cell biology, immune biology, biochemistry and molecular biology.The results revealed that the proliferation of SMMC-7721 cells were inhibited apparently after being treated with 3.0ug/mL tachyplesin or 5.0mmol/L HMBA or 1.5ug/mL tachyplesin combinated with 2.0mmol/L HMBA respectively, the growth of cells were inhibited, the cell mitotic index and the abilities of cell colony forming were declined obviously. Biochemistry, cytochemistriy and immunocytochemistry assays showed that the expression of a -fetoprotein and proliferating cell nuclear antigen and the enzymes activities of Y -glutamyl-transpeptidase and alkaline phosphatase were declined significantly while the enzyme activity of tyrosine- a -ketoglutarate transaminase were increased significantly in the cells treated with tachyplesin , HMBA or their combination respectively. Light microscopy, scanning and transmission electron microscopy showed that SMMC-7721 cells, which induced with tachyplesin or HMBA or their combination, had undergone the restorational alteration in morphology and ultrastructure which were different from those of nontreated cells but were similar to those of normal cells, and the changes were as follows: the cells turned to be flat and spread, the nucleo-cytoplasmic ratio lessened and nuclear shape became rather regular, the number of nucleous reduced and its volume lessened, euchromatin increased while heterchromatin decreased in nucleus. In the cytoplasm, mitochondria grew in number with relatively consistent structure and well-developed mitochondria cristae, Golgi complex turned to be well-developed and typical, rough endoplasmic reticulum increased, polyribosome decreased while free ribosome increased. The microvilli at cellular surface were rare and the filopodia reduced while lamellipodia increased at the cell edge. Light microscopy, laser confocal scanning microscopy and immunofluorometric assays revealed that the cytoskeleton and the nuclear matrix had undergone obvious changes in the cells treated with tachyplesin or HMBA or theircombination respectively. The cytoskeleton filaments were well-distributed and arranged regularly, microfilaments were decreased while microtubule and intermediate filaments were increased, the well-distributed nuclear matrix filaments and the intermediate filaments were connected closely by the ununiformly thick nuclear lamina and interlaced into a regular network throughout the whole cell region. Flow cytometry analysis showed tachyplesin and HMBA and their combination could arrest SMMC-7721 cells in Go/Gi phase, with an accumulation of the cells in Go/d phase while a decrease of the cells in S phase. Immunocytochemistry and in situ hybridization with digoxigenin-labelled cRNA probes detection revealed that the expression of p21WAF1/CIP1, p!6 proteins and p2iWAF1/CIPI mRNA were upregulated significantly while the products of mutant p53, Cyclin Dl, CDK4, Rb, c-myc proteins and c-myc mRNA were downregulated significantly in the cells treated with tachyplesin or HMBA or their combination. In the meantime, tachyplesin and HMBA and their combination also could downregulate the proteins expression of NF- K B, PKC and pi 85 effectively and declined the expression of bcl-2 protein slightly, but couldn't alter the protein expression of TGF- 3 RII.Taken together, the results of the current study showed that tachyplesin could effectively inhibit the proliferation of the human hepatocarcinoma SMMC-7721 cells, alter the activities of metabolism enzymes and the expression of antigens associated with hepatocellular carcinoma, reverse the malignant morphological and ultrastructural characteristics, alter the co...