The Effects And Mechanisms Of Inositol Hexaphosphate (IP₆) On Cell Cycle Of HT-29 Human Colon Carcinoma Cell Line

Objective:To investigate the effects and mechanisms of inositol hexaphosphate on cell cycle of HT-29human colon carcinoma cell line.Methods: cells were exposed to various concentrations (0.0mM , 1.8mM,3.3mM,5.0mM ,8.0mM,13.0mM) of IP₆ for certain time. Its effects on the cells were evaluated by detecting followindexes:1. Cells were treated continuously and uncontinuously respectively with IP₆. The effect of IP₆ on cells proliferation was evaluated by MTT assay.2. The effect of IP₆ on cells cycle was evaluated by Flow Cytometry.3. Immunocytochemical stain was used to detect the expression of mutant type p53 protein , p21 protein and proliferating cell nuclear antigen(PCNA).4. CyclinD1 and CDK4 mRNA expression state in cells were determined by reverse transcription polymerase chain reaction (RT-PCR).Results: 1. Continous treatment with IP₆ had an inhibition effect on the proliferation of HT-29 cells.The OD Value of each IP₆ group was lower than that of control group ( P<0.05 ) . The results showed that the inhitition effects of IP₆ was both concentration-dependent and time-dependent. The experiment of uncontinuous treatment with IP₆ also showed a time-dependent inhitition. 2. The Flow cytometry showed that defferent concentration of IP₆ had a significantly increase of G0-G1 phase cells ( P<0.01 ) .After 48 and 72 hours' exposure to IP₆, the effects was showed by concentration-dependent and time-dependent style. 3. The RT-PCR showed that the expression of cyclinD1 and CDK4 mRNA was decreased by 1.8mM-3.3mM(.P<0.01). 4. The immunocytochemical stain showed that defferent concentration of IP₆ decreased the expression of mutant type p53 gene , induced the expression of p21 ( P<0.05) . The expression of PCNA was decreased by 3.3-5.0 mM (P<0.05)Conclusion: These results show that IP₆ controls the progression of HT-29 cells through the cycle by decreasing S- phase and arresting cells in the G0/G1-phase of the cell cycle. The mechanisms of the effect might be related to many links, such as inhibiting the expression of oncogene mutant type p53 gene while increasing the expression of wild type p53 gene. Wild p53 can induce the expression of cell cycle inhibitor p21 thus blocking the cell cycle by combinding cyclinDl/CDK4 and PCNA. The rising of P21-cyclinD1/CDK4 may decrease activity of CDK-cyclin kinase, decrease DNA synthesis and inhibit cell proliferation.