Inhibitory Effect Of Lobaplatin On Cell Proliferation Of Bladder Cancer And Possible Mechanism Of Inducing Apoptosis

OBJECTIVE:In order to explore the proliferation and apoptosis of human bladder cancer cells,we have carried out in vitro inhibition of proliferation and induction of apoptosisexperiments on bladder cancer cells in this study,and hope to find some potential possible mechanism for the bladder Cancer in the future of clinical treatment to provide more treatment drugs.METHODS:1.Human bladder cancer cell line(5637)confirmed that the cell status was good after the passage of cells,after several passages,the choice of cell morphology and good growth of cells observed,and electron microscopy to record their pictures.The ideal bladder cancer cells 5637 cells for cryopreservation treatment,and ultimately from-80 degrees refrigerator to liquid nitrogen tank for long-term preservation,in order to spare future cells for resuscitation.2.MTT experiment first through the microscope to count,and then the bladder cancer cells diluted into cell suspension,inoculated in 96-well plate,waiting for a day after the incubation of the hole to remove the supernatant,adding the concentration of lobaplatin and set parallel samples and control groups.The 96-well plates were incubated in the incubator for three days and then passed through a laboratory microplate reader.The wavelength was set at 490 nm to detect the absorbance values of the cells in each group.3.The cells of lobaplatin were added into 6-well plates,and Annexin V and propidium iodide were added.The apoptosis of human bladder carcinoma 5637 cells was detected by flow cytometry.The morphology of the selected bladder cancer cells was observed by electron microscopy.The cells with good growth were selected for digestion.After the suspension was prepared,the cells were centrifuged and the cells were precipitated.The cells were ultrathin and then treated with double staining.The changes of organelles in human bladder cancer 5637 cells were observed by transmission electron microscopy.Electron microscopy to observe the cells,when cultured to 80%-90% of the state,the scoring experiment,under the electron microscope fixed and take pictures,through the healing of the situation to assess the migration of 5637 cells.The human bladder cancer 5637 cells were subjected to starvation overnight,serum-free medium containing bladder cancer cells was added to the upper chamber,and serum medium was added to the lower chamber to measure the cell migration ability.Transwell chamber was used to determine the invasive ability in the upper chamber by adding Matrigel matrix,and then the transferred cells were fixed in methanol,and then 1% crystal violet staining,the bladder cancer cell invasion ability.Protein imprinting experiments can extract proteins from bladder cancer cells and determine the effect of lobaplatin on the expression of protein in bladder cancer cells.RESULTS:1.MTT experiments can be seen: three time period of bladder cancer cell proliferation inhibition curve at the beginning with the amount of lobaplatin increased in proportion to increase its cell inhibition,but the curve at 10μg / mL concentration start gradually gentle,and ultimately no longer increase the inhibitory effect.2.When Lobaplatin-induced apoptosis,the viability of the cells can be seen from the picture obtained by flow cytometry,and it is proved that lobaplatin plays an important role in promoting cell apoptosis.It was observed by transmission electron microscopy that the change of organelles in the cells was mainly due to the increase of the mitochondrial ridge and even the lesion.Many of the cell vacuoles were observed in the cells,and the intracellular rough endoplasmic reticulum And so on.Starting from the concentration of 20μg / ml lobaplatin we can observe that most mitochondria have changed and the chromatin is reduced in the nucleus.Bax and Caspase-3 proteins extracted from bladder cancer cells were upregulated in a concentration-dependent manner,whereas Survivin and Bcl-2 proteins extracted from bladder cancer cells were down-regulated in a concentration-dependent manner.3.The migration and invasion of bladder cancer cells are the main way to transfer,compared to the control group,given the group of lobaplatin in varying degrees can inhibit the invasion and migration of bladder cancer cells.Scratch test is also known as wound healing test,given lobaplatin from 10μg / mL began to suppress the effect is more obvious.Similarly,in the Transwell chamber experiment,we can see that the number of bladder cancer cells in the control group was the largest,and as the concentration increased,the number of cells passing through the membrane was sufficient to demonstrate that bladder cancer migration and invasion.CONCLUSION:1.The inhibition rate of bladder cancer 5637 cells increased with the increase of the concentration,and the inhibition rate began to decrease at the concentration of 10μg / mL.The inhibition curve gradually became gentle,and it was proved that lupine was dose-dependent and time-dependent The way to inhibit the proliferation of bladder cancer cells.2.The percentage of apoptotic cells was positively correlated with the proliferation inhibition curve,and the intracellular organelles were damaged to varying degrees.The most important was the mitochondrial lesions.The apoptotic proteins Bax and Caspase-3 inhibited the expression of Survivin and Bcl-2 in a concentration-dependent manner by concentration-dependent expression.3.Lobaplatin can block the migration and invasion of bladder cancer cells in vitro,with the increase of drug concentration,the better the migration and invasion.