Effects Of Ketone Bodies On Growth And Apoptosis Of HeLa Cells

Normal cells can utilize glucose,fatty acids and ketone body as their main source of energy supply.Normal cells rely mainly on mitochondrial oxidative phosphorylation for their required energy supply,however,cancer cells utilize aerobic glycolysis pathway for their energetic need.Recent studies have shown that cancer cells are largely dependent upon glucose for energy and are not able to use ketone body as a primary energy source.ketones inhibit the proliferation and viability of cultured tumor cells.The ketogenic diet is a high-fat,adequate-protein,low-carbohydrate diet.If there is very little carbohydrate in the diet,the liver converts fat into fatty acids and ketone body.In human beings and most other mammals,acetyl-CoA formed in the liver during oxidation of fatty acids may enter the citric acid cycle or it may be converted to the "ketone body" acetoacetate,D-?-hydroxybutyrate,and acetone for export to other tissues.The studies on cells and molecular levels on the ketogenic diet are too few.Therefor,we analysed the effect of ketone body on growth and apoptosis of hela cells.Preliminary research on the apoptosis of Hela cell induced by ketone body.The results are as follows:Methods:1.To screen the optimal treating concentration by MTT.Select the 25 mmol/L as the concentration of ketone body..2.The HeLa cells were co cultured with human umbilical cord cells.The cell morphology was observed by inverted microscope.AO/EB Double Staining for detection of apoptosis.3.The cell viability was measured by MTT.The cell morphology of was observed by inverted microscope.4.AO/EB Double Staining for detection of apoptosis.Single cell gel electrophoresis:Detection of DNA damage at different levels of sensitivity.Mitochondria were observed by mitochondrial staining.Flow cytometry in analysis of cell cycle.5.The cell cycle and apoptotic proteins were detected by Western Blot.Results:1.The viability of HeLa cells were inhibited by deguelin in a time and dose dependent manner.2.Morphological observation showed that ketone body can inhibit the proliferation of HeLa cells.The inhibition of ketone body+low suger is more obvious.Ketone had no significant inhibitory effect on normal human umbilical cord cells.3.Trypan blue staining showed that ketone body can inhibit the proliferation of HeLa cells.The ketone body can replace glucose as an energy source when human umbilical cord cells in carbon source starvation,Cell counting results showed that at a certain ketone body concentration,the inhibitory effect of low sugar on HeLa cell proliferation was higher than that in normal glucose.4.AO/EB Double Staining shows that ketone body can inhibit the proliferation of HeLa cells.The inhibition of ketone body+ low suger is more obvious.SCEG(single cell gel electrophoresis)test results shows that the damage of HeLa cells were from the nuclear DNA.Ketone body can damage the nuclear DNA of HeLa cells,and the damage degree of nuclear DNA is more severe after the concentration of glucose+etone body.Mitochondrial staining showed that After HeLa cells were treated with ketone body,mitochondrial activity was enhanced.The flow cytometry results showed that ketone body causes a G1 arrest in HeLa cells.5.Western Blot test showed that compared with the control group,the expression of main band of Caspase-3 in the experimental group were decreased,the expression of bad and bim in the experimental group were increased,the expression of CyclinEl,CyclinD1,CDK2 and CDK4 in the experimental group were decreased,the expression of P27 and P21 in the experimental group were increased.Conclusions:Ketone body can inhibit the proliferation of HeLa cells.The inhibition of ketone body+low suger is more obvious.Ketone body had no significant inhibitory effect on normal human umbilical cord cells.ketone body causes a G1 arrest in HeLa cells.Ketone body inducing apoptosis was dependent on damagement of the nuclear DNA of HeLa cells,and the damage degree of nuclear DNA is more severe after the concentration of glucose+ketone body.HeLa cell apoptosis was induced by Caspase-dependent apoptosis pathway.Ketone body can prevent normal p27 control of cyclinE/CDK2.This study provides a theoretical basis for the treatment of ketone body in cancer.