The Effects Of Cisplatin Ahd Etoposide On The Cell Cycle Of Human Ovarian Carcinoma Cell Line

Objective: Ovarian cancer is one of the three malignancyof female procreation system, which jeopardize women,s bodyand mind. Its incidence of disease and mortality rate are still riseyear after year, we still face great challenge in diagnose earlyand treatment early of ovarian cancer. Nearly, along with thedevelopment of cell molecule biology and tumor biology, wegradually realize that tumor is a kind of cell cycle disease, thecontrol of cell cycle is very important in tumors happen anddevelopment. Many chemotherapeutic agent aim at certainphase of cell cycle to play their effect, block the natural run ofcell cycle, so as to reach the intention of restrain cellproliferation and induce cell apoptosis. In terms of thedifference of cell cycle phase on which the chemotherapeuticagents act, they are divided into cell cycle specificchemotherapeutic agent and cell cycle nonspeclficchemotherapeutic agent .Cisplatin (DDP) is cell cyclenonspecific chemotherapeutic agent. In the therapy of ovariancancer, it is always the principal drug. But now, alone with theextensive apply, the phenomena of drug resistance stand out dayafter day, make the use of DDP have a lot of limit. So the key ofenhance its curative effect is to select reasonablechemotherapeutic agent to match DDP. Etoposide (VP-16) is akind of galenical and G2/M phase specific chemotherapeuticagent. The experiment would study DDP combined with VP-16affected SKOV3 cell, s growth through cell cycle dynamics onvitro: The cytotoxic effects of these agents on vitro ovariancancer cell line SKOV3 were measured by MTT assay;The cellcycle was analyzed by the flow cytometry ;Expression levelsof cyclinB₁ ,P34^cdc2 and survivin were determined byimmunocytochemistry method. These results were analyzed bystatistic methods in attempt to identify their relationship, whichmay find some possible mechanism. These could providetheoretical evidence to establish reasonable combinechemotherapy project of ovarian cancer.Methods: The ovarian cancer SKOV3 cell line wascultivated on vitro, then the cell of logarithm growth periodwere selected and divided into three groups: control group, DDPgroup and DDP match VP-16 group. The concentration of DDPwere 0.1 fold PSC,1 fold PSC and 10 PSC respective(the PSCof DDP is 3ug/ml), the concentration of VP-16 was 1 foldPSC(the PSC of VP-16 is 5ug/ml). The drugs with differentconcentration and SKOV3 cell were incubated for 24 hourtogether, then the cytotoxic effect that these agents act onSKOV3 cell line was measured by MTT assay, reckoned cellgrowth restrain rate, make use of the JIN's formula to determinecombination effect of DDP and VP-16; the distribution of cellcycle was examined by flow cytometry as well as analyzed thechange of cell cycle distribution; expression levels of cyclinB1,P34cdc2 and survivin were detected by immunocytochemistry s-pmethod.Statistic methods: The quantitative data were performedwith t-test and the categorical data were performed with ranksum test.Results:1. The inhibition to cell growth after DDP and VP-16treated SKOV3 cell. The result of MTT assay in the DDP groupshowed that the inhibition rate of DDP to cell was increasealong with the increase of drug concentration, combined withVP-16, when the concentrations of DDP were 0.1,1 and 10 foldPSC, the Q values were 1.05,1.3 and 1.06 respective. We couldmake out that DDP combined with VP-16 enhanced thecytotoxic effect, especially in 1 fold PSC of DDP.2. The change of cell cycle distribution after DDP andVP-16 treated SKOV3 cell. The distribution of cell cycle wasexamined by flow cytometry, well-balanced SKOV3 cell mainlylocated G0/G1 phase, DDP made this distribution take place greatchange, the proportion of G0/G1 phase decreased, and S phaseand G2/M phase increased. The difference was statisticalsignificant (t=20.83 and 9.78, p<0.05). DDP combined withVP-16 have been treating SKOV3 cell for 24 hour, after that, theproportion of G0/G1 phase decreased farther, the proportion of Sphase increased , compared with control group , the differencewas significant (t=28.54, p<0.05), but compared with DDPgroup, there had not statistical significant (t=0.08, p>0.05), theproportion of G2/M phase increased obviously, compared withcontrol group and DDP group, their differences both hadstatistical significant (p<0.05).3. The change of cyclinB1,P34cdc2 and survivin proteinafter DDP and VP-16 treated SKOV3 cell. The expression levelsof the three proteins were determined by immunocytochemistrys-p method. The cells were observed through microscope: thethree proteins all oriented in cell plasm in SKOV3 cell, took onbrown-yellow or sandy beige, while nucleolus was not stained.Every group and every protein was selected 30 visual fields, interms of the positive cell rate and degree of color in every field,we analyzed the results. From the examination, we discoveredthat the three proteins had high expression in SKOV3 cell, afterDDP treated the cells, The expression of cyclinB1and survivinprotein decreased obviously, compared with control group, thedifference was significant (p<0.05), while the expression ofP34^cdc2 had not obvious change; DDP combined with VP-16 hadbeen treating SKOV3 cell for 24 hour, after that their expressiondecreased farther, compared with DDP group, the difference hadstatistical significant (p<0.05). As well as the change and theblock of cell cycle were synchronization.Conclusion :1. Along with the augment of the concentration of DDP, itsexecution to ovarian cancer also strengthened, when DDPmatched VP-16, the inhibit rate to cell proliferation enhanced.From the result, we could conclude VP-16 strengthenedanti-tumor action of DDP, especially in the PSC of DDP, thecombination effect was best, we all know DDP can bring someharmful reaction to body when using it, and along with theincrease of concentration the reaction is aggravation. So thisstudy provided theoretical evidence to establish reasonablecombine chemotherapy project and quest best curative effect ofovarian cancer.2. After DDP and VP-16 treated SKOV3 cell, thedistribution of cell cycle took place obvious change, theproportion of G0/G1 phase decreased, and S phase and G2/Mphase increased. It showed that the natural run of cell cycle wasdisturbed, its continuity was breaked off, the drug made the runof cell cycle stagnate on S phase and G2/M phase, which madethe cell that came into next cell cycle through G2/M checkpointdecrease, then restrained the cell proliferation.3. Now about the mechanism that chemotherapeutic agentcause the block of cell cycle some period and time extend of cellcycle run is not very definitude. The study usedimmunocytochemistry to detect the expression of cell cycleproteins: cyclinB1,P34cdc2 and survivin, and analyzed therelationship between the expression level of three cell cycleproteins,cell proliferation and change of cell cycle distribution.The result showed that DDP combined with VP-16 enhanced theinhibit to cell growth and strengthened the block of cell cycle, atthe same time descended the expression of the three cell cycle...