The Effects Of Inositol Hexaphosphate-induced Apoptosis In HT-29 Human Colon Carcinoma Cells, Concentration Of Calcium And Expression Of Apoptosis Associated Gene And Protein

Objective: To investigate the effects of inositol hexaphosphate(IP₆)in HT-29 human colon carcinoma cells and its mechanisms of anti-cancer.Methods:1 HT-29 cells were treated with IP₆ in different concentrations(0mM,1.8mM,3.3mM,5.0mM,8.0mM,13.0mM)during different time(0-6days).The effect of IP₆on cells proliferation was evaluated by MTT assay.2 HT-29 cells were treated with IP₆ in different concentrations(0mM,1.8mM,3.3mM)during different time(3days,3months,6 months).The effect of apoptosis was detected by transmission electron microscopy (TEM); the rate of cells apoptosis was assayed by flow cytornetrycytometric; the concentration of intracellular-free calcium was determined by Fura-2/AM; Caspase-3 expression state in cells were determined by reverse transcription polymerase chain reaction(RT-PCR); the expression of Bc1-2 and NF-кB was detected by Immunocytochemical stain.Results:1 MTT assay indicated a significant dose-as well as time-dependent growth inhibition was observed in IP₆-treated HT- 29 cells.2 Morphological examination by transmission electron microscopy(TEM) showed cells shrinkage, and the presense of "apoptopic bodies".3 Flow cytometric analysised that different concentration of IP₆ increased the rate of cells apoptosis(P<0.05),and indicate time-dependent effect.4 Fura-2/AM indicated concentzation of intracellular- free calcium was strongly increased (P<0.05).5 Reverse transcription polymerase chain reaction(RT-PCR) showed that TP₆ increased the expression of Caspase-3mRNA (P< 0.05),and indicate time-dependance.6 The immunocytochemical stain showed that different concentration of IP₆ decreased the expression of Bcl-2 and NF-кB (P<0.05) ,and the effect on Bcl-2 issignificant time-dependance. Conclusion:1 The results showed that IP₆ had a significant dose-dependent (0-13.0mM) as well as time-dependent(0-6days) inhibition effect on proliferation of HT-29 cells.2 IP₆ could induce apoptosis and that might involve regulating cells signal transduction and expression of apoptosis associated gene. IP₆ and secondary phosphorylation IP₆ can increase the concentration of intracellular-free calcium and induce apoptosis mediated calcium overload,involving caspaseses non-activating proenzyme activation, cascade amplification reaction of caspases-3. IP₆ decreased the expression of Bcl-2 and NF-кB,induced apoptosis and exert its anticancer effects.3 IP₆ had a time-dependent effect on inducing apoptosis of HT-29 cells,and showed IP₆-treated for long time can exert its more effective anticancer effects.