Growth arrest and apoptosis of human hepatocellular carcinoma cells induced by hexamethylene bisacetamide.

AIM: To investigate the cellular effects of hybrid polar compound hexamethylene bisacetamide (HMBA) on the growth and apoptosis of human hepatocellular carcinoma cells and to provide the molecular mechanism for potential application of HMBA in the treatment of liver cancer. METHODS: Effects of HMBA on the growth of human hepatocellular carcinoma SMMC-7721 cells were assayed by MTT chronometry. Apoptosis induced by HMBA was detected by phase-contrast microscopy, flow cytometry, propidium iodide staining and immunocytochemical analysis. RESULTS: The growth of SMMC-7721 cells was significantly inhibited by HMBA, and the growth inhibitory rate was 51.1%, 62.6%, 68.7% and 73.9% respectively after treatment with 5.0, 7.5, 10.0 and 12.5 mmol/L of HMBA. In the cells treated with 10 mmol/L of HMBA for 72 h, the population of cells at sub-G(1) phase significantly increased, and the apoptotic bodies and condensed nuclei were detected. Moreover, treatment of SMMC-7721 cells with 10 mmol/L of HMBA down-regulated the expression of Bcl-2 anti-apoptotic protein, while slightly up-regulated the level of pro-apoptotic protein Bax. CONCLUSION: Treatment with 10.0 mmol/L of HMBA can significantly inhibit the growth and induce apoptosis of human hepatocellular carcinoma SMMC-7721 cells by decreasing the ratio of Bcl-2 to Bax.

Effects of tachyplesin on the regulation of cell cycle in human hepatocarcinoma SMMC-7721 cells.

AIM: To investigate the effects of tachyplesin on the cell cycle regulation in human hepatcarcinoma cells. METHODS: Effects of tachyplesin on the cell cycle in human hepatocarcinoma SMMC-7721 cells were assayed with flow cytometry. The protein levels of p53, p16, cyclin D1 and CDK4 were assayed by immunocytochemistry. The mRNA levels of p21(WAF1/CIP1) and c-myc genes were examined with in situ hybridization assay. RESULTS: After tachyplesin treatment, the cell cycle arrested at G0/G1 phase, the protein levels of mutant p53, cyclin D1 and CDK4 and the mRNA level of c-myc gene were decreased, whereas the levels of p16 protein and p21(WAF1/CIP1) mRNA increased. CONCLUSION: Tachyplesin might arrest the cell at G0/G1 phase by upregulating the levels of p16 protein and p21(WAF1/CIP1) mRNA and downregulating the levels of mutant p53, cyclin D1 and CDK4 proteins and c-myc mRNA, and induce the differentiation of human hepatocacinoma cells.

Effects of tachyplesin on proliferation and differentiation of human hepatocellular carcinoma SMMC-7721 cells.

AIM: To investigate the antitumor activities of tachyplesin on human hepatocellular carcinoma (HCC) cells. METHODS: Tachyplesin, isolated from acid extracts of Chinese horseshoe crab (Tachypleus tridentatus) hemocytes, was used to treat the human HCC cell line SMMC-7721. Effects of tachyplesin on the proliferation of SMMC-7721 cells were measured with trypan blue dye exclusion test and HE staining. The morphology and ultrastructure of the cells were examined by light microscopy and transmission electron microscopy, respectively. The activities of gamma-glutamyltransferase (gamma-GT) and tyrosine aminotransferase (TAT) were assayed with biochemical methods. The levels of alpha fetoprotein (alpha-FP), proliferating cell nuclear antigen (PCNA), p21( WAF1/CIP1) and c-myc were examined by immunocytochemistry. RESULTS: After treatment with tachyplesin 3.0 mg/L, the proliferation of SMMC-7721 cells was inhibited significantly, with the cell growth inhibitory rate amounted to 55.57 % and the maximum cell mitotic index declined by 43.68 %. The morphology and ultrastructure underwent restorational alteration. The activity of gamma-GT declined while TAT activity increased obviously, and the levels of alpha-FP and PCNA decreased. Moreover, the expression of p21(WAF1/CIP1) protein was up-regulated and that of c-myc protein was down-regulated. CONCLUSION: Tachyplesin could effectively inhibit the proliferation of hepatocarcinoma cells, reverse the malignant morphological and ultrastructural characteristics, alter the levels of enzymes and antigens, regulate the expression of differentiation-associated oncogene and tumor suppressor gene, and induce hepatocarcinama cell differentiation.

[Tachyplesin-induced differentiation of human hepatocarcinoma cell line SMMC-7721].

BACKGROUND & OBJECTIVE: The study on antitumor activities of marine bioactive substances is an important field in exploiting marine bioactive substances and antitumor drugs. And the induction of tumor cell differentiation is a new strategy for drug therapy of tumors. So the authors used tachyplesin, a marine bioactive substance, to investigate its effects on the differentiation of human hepatocarcinoma SMMC-7721 cells for further studying its antitumor activities and mechanisms. METHODS: Tachyplesin, which was isolated from hemocytes of horseshoe crab (Tachypleus tridentatus), was used to treat human hepatocarcinoma SMMC-7721 cells. Light microscopy and transmission electron microscopy were applied to examine the changes in morphology and ultrastructure of SMMC-7721 cells, respectively. The activities of alkaline phosphatase or the expression of AFP and PCNA were assessed by cytochemistry or immunocytochemistry. RESULTS: In the cells treated with 3.0 micrograms/ml tachyplesin, the morphology and ultrastructure returned to be normal, the activity of alkaline phosphatase was decreased and the levels of AFP and PCNA were downregulated. CONCLUSION: Tachyplesin might effectively reverse the malignant morphology and ultrastructure, change the activity of enzyme and the levels of antigens associated with hepatocarcinoma cell, and induce the differentiation of the human hepatocarcinoma SMMC-7721 cells.

[Effects of HMBA on the expression of cell-cycle-associated genes in human hepatocarcinoma SMMC-7721 cells].

In this study, the effects of HMBA on the expression of G0/G1 phase arrest-associated genes in human hepatocarcinoma SMMC-7721 cells were investigated. Immunocytochemistry and in situ hybridization assay revealed that the levels of p21WAF1/CIP1 and p16 proteins and the level of p21WAF1/CIP1 mRNA were increased while the levels of CDK4 and Cyclin D1 proteins and c-myc mRNA were decreased in the cells treated with HMBA. These results showed that HMBA could up-regulate the expression of p21WAF1/CIP1 and p16 genes, down-regulate the activity of Cyclin D1-CDK4 and the transcription of c-myc gene which were necessary for cells entering into S phase, and so arrest the cells in G0/G1 phase, and induce the differentiation of human hepatocarcinoma SMMC-7721 cells.

Effects of tachyplesin on the morphology and ultrastructure of human gastric carcinoma cell line BGC-823.

AIM:To investigate the morphological and ultrastructural changes in the human gastric carcinoma cell line BGC-823 after being treated with tachyplesin.METHODS:Tachyplesin was isolated from acid extracts of Chinese horseshoe crab (Tachypleus tridentatus) hemocytes. BGC-823 cells and the cells treated with 2.0mg/L tachyplesin were examined respectively under light microscope, scanning and transmission electron microscope.RESULTS: BGC-823 cells had undergone the restorational alteration in morphology and ultrastructure after tachyplesin treatment. The changes were as follows: the shape of cells was unanimous, the volume enlarged and cells turned to be flat and spread, the nucleo-cytoplasmic ratio lessened and nuclear shape became rather regular, the number of nucleolus reduced and its volume lessened,heter-chromatin decreased while euchromatin increased in nucleus. In the cytoplasm, mitochondria grew in number with consistent structure relatively, Golgi complex turned to be typical and well-developed,rough endoplasmic reticulum increased and polyribosome decreased. The microvilli at cellular surface were rare and the filopodia reduced while lamellipodia increased at the cell edge.CONCLUSION:Tachyplesin could alter the malignant morphological and ultrastructural charact-eristics of human gastric carcinoma cells effectively and have a certain inducing differen-tiation effect on human gastric carcinoma cells.