Study On The Mechanical Changes Of Cells During Early Apoptosis Induced By Cytochalasin B

Cancer has become the second cause of death after cardiovascular and cerebrovascular diseases,and one of its main treatment methods is chemotherapy.However,anticancer efficacy is not significant due to the drug resistance and the poor inhibitory effects on certain tumor cells of existing chemotherapy drugs.It's urgent to need to develop new chemotherapy drugs.It was reported that the actin microfilament cytoskeleton of malignant cells had been very unstable in the literatures.Whereas,no specific drug for the actin microfilaments has yet been used clinically.The clinical chemotherapy drugs are mainly microtubule-targeting,which cannot effectively interfere with the microfilament polymerization of cancer cells to play an anticancer role.Cytochalasins,as a kind of microfilament depolymerizing agents,can be used to interrupt the microfilament polymerization of cancer cells to study its inhibitory effect on cancer cells and understand its killing mechanism to tumor cells.The studies on apoptosis have been focused on biochemistry,molecular biology,interregulation of apoptotic molecules,and biomechanics of late apoptotic cells.And very few researches have been done on the mechanical properties of early apoptotic cells.In particular,the correlation between the depolymerization of microfilament cytoskeleton and the increase of intracellular molecule crowding and mitochondrial damages and changes of cell surface ultrastructure is still a scientific problem that has not been recognized and studied.The solution to these problems has important research value for understanding the mechanism of apoptosis induced by microfilament-targeting agents and the development of new anticancer drugs.In view of the above problems,experimental models of apoptosis and intracellular molecular crowding were established on the basis of microfilament cytoskeleton depolymerization of cytochalasin B(CytB).The changes of cell mechanical properties and the correlation between cell mechanics and biological apoptosis were explored by using the methods of cell mechanics and nanomorphology as well as molecular biology techniques,such as immunofluorescence and western blotting.The mechanism of mechanical apoptosis was brought into the research on the development of microfilament-targeting anticancer drugs and their side effects.The innovative research conclusions obtained are as follows:1.By comparing the inhibitory effects of CytB on cervical cancer Hela cells and BMSCs with different stability of microfilament cytoskeleton,it was found that the growth inhibition rate and the apoptosis rate of Hela cells were higher than that of BMSCs at the same drug concentration.The apoptosis rate of BMSCs was still significantly lower than that of Hela cells,when the BMSCs were treated with 3 times drug concentration of the Hela cells.In addition,after treatment the microfilaments were depolymerized,young's modulus and volume of cells were decreased in different degrees.These results indicated that CytB has obvious killing effects on Hela cells,but it also has some side effects on BMSCs with normal microfilament cytoskeleton.The side effects led to cell damage and eventually apoptosis by destroying the mechanical structure and changing the mechanical properties of cells.2.Based on the killing effects of CytB on Hela cells,the mechanical properties and surface ultrastructure and intracellular molecular crowding of cells at early stage of apoptosis were quantitatively studied and qualitatively analyzed by using AFM,SEM and CLMS.It was found that the depolymerization of microfilaments led to the decrease of cell volume,increase of cell surface roughness and the aggravation of intracellular molecular crowding.Combining with the results of previous studies on mitochondrial apoptosis pathways of Hela cells induced by CytB and computer models of intracellular molecular crowding,this work is the first to cite the “molecular crowding model” to analyze and explain the relationship between intracellular molecular crowding and mitochondrial damage and increased cell surface roughness,which caused by microfilament depolymerization of CytB.It could be known that molecular crowding caused intracellular macromoleculars,such as microfilament fragments and mitochondria,to relocate and redistributed to the side of membrane,even be squeezed out of the cell membrane,resulting in mitochondrial damage and transport barrier,and increase of cell surface roughness,which led to cell apoptosis together.3.By applying single cell mechanics,Western blot,immunofluorescence and other experimental methods,the mechanical properties,nanomorphology and apoptosis related molecules of early apoptotic cells were quantitatively studied and qualitatively analyzed.It was found that the decrease of young's modulus and volume and the increase of surface roughness occurred before the PS exposure and Fas/CD95 activation.These results indicated that the mechanical properties of the cells,which has not experienced apoptosis in biology,had been changed and the cell activity had been decreased.It suggested that the decline of cellular young's modulus could be regarded as a mechanical index of cell damage and apoptosis induced by cytochalasin,which can be used to evaluate the anticancer effects of microfilament-targeting anticarcinogens.