Research On NDGA's Inducing Apoptosis Of Colon Cancer Cell Line HT-29, Mechanis Of This Apoptosis And Influence On Its Metastasis

Background: Colon cancer is a common kind of malignant tumor. Due to the factors like the improvement of people's living standard , the increasing of fat content in people's everyday diet, the incidence rate of this diease is on the rise in recent years. The episode age is gradually declining and metastasis is easily seen in early stage. Apart from surgical operation, treatments of it include chemotherapy and radiotherapy. However, the effects of traditional chemotherapy drugs such as 5-FU are unsatisfying, with too many side-effects and poor tolerance of patients. Therefore, scientists are now vigorously engaged in seeking an new effective drug to treat colon cancer.The focus of research, domestic or abroad, is on the metabolism pathway of arachidonic acid in tumor cell. Arachidonic acid can be classified into pathways of cyclooxygenase (COX) and lipoxygenase(LOX). COX is the key factor in the COX pathway and consists of COX-1 and COX-2. Many scholars agree that inhibiting the pathway of COX can induce tumor cell apoptosis and affect its metastasis. But there is little agreement and few reports on whetherinhibiting the pathway of LOX can inhibit colon cancer's growth, induce its apotosis and suppress its metastasis.Objective: To find out the effect of NDGA, the lipoxygenase inhibitor, on colon cancer cell line HT-29 in vitro from the aspects of cell apoptosis, apoptosis mechanism, and metastasis.Materials and Methods: The colon cancer cell line HT-29 was cultivated in RPMI1640 medium, which contained 10% calf blood serum and was placed in a constant temperature oven of 37°C and 5% CO₂. Then NDGA of different concentrations was used to dispose the cancer cell. We applied respectively. a.) MTT to draw the growth curve. b.) inverted phase contrast microscope to observe morphologic change of cells as well as scanning electron microscope to observe changes of cell's ultra-microstructure and apoptotic body. c.) flow cytometry to examine the apoptosis and periodic changes of cells. d.) Study of cell's apoptosis mechanisms: RT-PCR to detect the expression of 5-LOX messenger ribonucleic acid (5-LOXmRNA) and that of messenger ribonucleic acid about human telomerase reverse transcriptase (hTERTmRNA) respectively; flow cytometry to detect the expression of Bcl-2; confocal laser scanning electron microscope to examine freeing calcium of cell. e.) influence of cell metastasis: RT-PCR to detect VEGF, scanning electron microscope to observe cell's microvilli, flow cytometry to detect expression of CD44v6, confocal laser scanning electron microscope detect cytoskeleton change.The data was statistically processed with the software SPSS12.0,with the value of P smaller than 0.05 being considered statistically significant.Results: a.)Cancer cell's growth apoptosis examination: We used different concentration of NDGA to dispose cancer cell independently. Accompanying the rise in drug's concentration,apoptosis rate of cells rose gradually with difference of notable significance, b.) Morphologic change of cells: In the control group, cancer cells showed pararound shape, some of them showing fusiform due to extension. After the drug's disposing, morphologic of cells became round, the volume became small and they abscised from the inner surface of the bottle. With the passage of time, the trend became increasingly apparent. Apoptotic body could be found through scanning electron microscope after NDGA (100M-mol/L) had been used for 48 hours, c.) Using flow cytometry to examine cell's apoptosis, we found that the rate of cancer cell apoptosis rise following the drug's concentration change and the growth of cells was blocked on the stage of Go/Gi. In contrast to the control section, the difference was obvious, d.) Mechanism of cell apoptosis: colon cancer cell lines HT-29 showed positive expression of5-LOXmRNA.This expression became weaker following the rising of the drug's concentration and so were hTERTmRNA and bcl-2. Freeing calcium of cell increased following the rise of cell's apoptosis. e.) Cell's metastasis affected by NDGA: The expression of CD44v6 went down following the rising of drug concentration, and so was VEGF detected with RT-PCR. Through scanning electron microscope, it was found that cell's microvilli curled to microball after the drug ( lOP-mol/L) had been used for 48 hours. Confocal laser scanning electron microscope was used to detect cytoskeleton change. It was also observed that after the cells had been disposed with NDGA for 36 hours, the actin showed depolymerization, the intensity of fluorescence became weaker and the annular structure around cell nuclei disappeared.Conclusions: NDGA can inhibit colon cancer cell line HT-29 growth and induce its apoptosis, the rate of which rises following the...