Glucose Deprivation Induces Apoptosis In HeLa Cells

The occurrence of malignant tumor is associated with abnormal cellular metabolism.Tumor cells show an increased rate of glucose uptake,glycolysis metabolism and accumulation of extracellular lactic.Glycolysis provides tumor cells with enough ATP.The enough ATP is advantageous to proliferation and invasion of tumor cells.The glycolytic pathway shows low efficiency of productivity compared with the oxidative phosphorylation pathway.Glucose uptake from the extracellular environment in glycolytic pathway is roughly ten times more than normal cells.People pay more attention to the method of cancer treatment with glucose deprivation.In this study,we investigated HeLa cells treated with glucose deprivation to determine whether they can be driven to apoptosis and,if so,to define the pathway involved,which will provide theoretical basis for cell starvation treatment of cancer.The results are listed as follows:1.Prepare glucose-deprived culture medium at a concentration of 0 mmol/L,1 mmol/L,1.5 mmol/L,2 mmol/L with glucose-free DMEM culture medium power and treat HeLa cells for 24 h.MTT assay screened out the effective glucose deprivation concentration.The results showed that the inhibition rates were 56.16%,36.30%,25.00%and 16.44%.So we selected the 1 mmol/L glucose concentration as the glucose-deprived concentration and it did not count the glucose concentration of fetal bovine serum.2.With glucose deprivation working on HeLa cells for 24 h,48 h,72 h,the survival rate was determined by MTT assay.The results showed that the inhibition rates were 39.02%,56.07%,75.84%.Cell counting method was used to detect the rate of cell proliferation.The results showed that the proliferation number of HeLa cells treated with glucose deprivation was decreased significantly compared with controls.3.With glucose deprivation working on HeLa cells for 48 h,Cell morphology was observed under the inverted microscope.Cell surface ultrastructure was observed by scanning electron microscopy.Furthermore,we performed fluorescence analysis and confocal microscope to observe the distribution of microtubules(?-Tubulin)and microfilaments(F-actin).The results showed that the morphology changed,microvillus and pseudopodium on cell surfaces disappeared and nucleus structure was destroyed.4.With glucose deprivation working on HeLa cells for 48 h,the changes of the chromatins were observed under confocal microscopy with AO/EB staining.DNA damage and fracture were analyzed by single cell gel electrophoresis.Furthermore,we used fluorescence analysis and confocal microscope to observe the distribution of Lamin A/C.The results showed that it caused pycnosis and fragmentation in HeLa cells nucleus.DNA was damaged.Furthermore,fluorescence staining displayed the changes of the distribution of LaminA/C and the nuclear membranes folded.5.We observed the distribution of Cytochrome-c and AIF by fluorescence staining and the confocal microscopy.The results showed that the distribution of Cytochrome-c in nucleus increased obviously and there was no difference in the distribution of AIF.Western Blot detected expression changes in Bid,Bax,Bcl-xL,Puma,Bim,Bcl-2,Caspase-3,PARP,AIF and Mcl-1 proteins.The results showed that the increased expression of proapoptotic protein Bim and the decreased expression of antiapoptotic protein Bcl-2 and Mcl-1.However,proapoptotic protein Bid and Puma decreased and antiapoptotic protein Bcl-xL increased.The expression of Caspase-3 was decreased and PARP was cleaved.Glucose deprivation can obviously inhibit the growth of HeLa cells and induce the changes of morphology by destroying their skeletons.Furthermore,it can cause DNA damage and induce apoptosis.The mechanism might be related to the cleavage of PARP in mitochondrial apoptotic pathway by regulating the Bcl-2 family proteins.