The Radiosensitivity-enchancing Effect Of Flavopiridol On Eca9706Cell Line Invitrostudy

There are400,000new cases of esophagus cancer in the world. Among thesepatients, over a half are in China. Meanwhile, the death rate of esophagus cancerpatients are the highest in our country,150,000people die of esophagus cancer everyyear, about a quarter of the people die of malignant cancers in our country, half of thepeople die of esophagus cancer in the world. Esophagus cancer has threatened humanhealth badly[1]. Radiation therapy is one of the main treatments for esophagus cancer[2],but the curative effect is dissatisfactory. After conventional radiotherapy, residual tumorrate is75-96%[3],5years survival rate is only8%-16%[4], rooting in the tolerances of thenormal tissues around the esophagus cancer are poor, regular exposure doses are notenough to kill the primary focal tumors. Conventional chemotherapy dose not havebeautiful effects, and with big side effects, difficult for patients to tolerate. Othertreatments, for example, gene therapy, biotherapy either have no beautiful effects[5].Therefore, looking for radiosensitizer that can improve the tumor cells in the sensitivityof radiotherapy become the new way of optimizing esophageal cancer treatments. In1974, Adams et al found that MIsO had certain radiosensitivity-enchancing effect, butits/large neural poisonous-sness limit the clinical application. So far there is no a kindof radio-sensitizer with clinical effective and low poisonousness[6].Flavopiridol is a new kind of anticarcinogen jointly developed by The AmericanNational Cancer Institute(NCI) and Hoechst Marion Roussel (HMR)[7], now in phase Iand II clinical trials[8]. It is a kind of powerful small molecular cyclin-dependentkinase(CDK) inhibitors, can block the cell cycle G1/S and G2/M control points by directlysuppressing the activity of CDK, make the cells can not turn into phaseS, so the cellsgrowth are inhibited[9,10]. Recent studies indicate that Flavopiridol can enhance theradiosensitivity of TE8, TE9(high/loew differentiation esophagus squamous cell carcinoma cell lines)[10], and with strong inhibitory effect on the growth of human Juvesarcoma cell line[11]. While, the most common esophageal cancer in China is squamouscell carcinoma, it is not yet reported that whether Flavopiridol haveradiosensitivity-enchancing effect on Eca9706cell line. Therefore, this experimentcultured Eca9706cells in vitro, treated the cells with Flavopiridol or/and radiotherapy,tested the cell survival rate, cell cycle and apoptosis related signal pathways, toinvestigate the radiosensitivity-enchancing effect of Flavopiridol on Eca9706cell lineand its related mechanism, provide new ideas and methods for clinical treatment ofesophageal cancers.Purpose: To investigate the radiosensitivity-enchancing effect of Flavopiridol(inhibitors of the cell cycle protein kinase-CDK) on Eca9706cell line (humanesophagus squamous cell carcinoma cell line)and its related mechanism.Methods: Assayed the inhibitory rate of growth and radiosensitivity-enchancingrate of the carcinoma cell line under Flavopiridol effect by MTT and cloningexperiment. Using immunocyto-chemistry to measure the expression of survivin,caspase-3, P53. Tested the apoptosis of the Eca9706cell line in different groups byTUNEL.Results:1.Results of MTT and cloning experiment showed that Flavopiridol could inhabitthe survival rate of Eca9706cells significantly, related with concentration and time, theIC50was2.56μmol/L.2.The differences between the survival fractions of cells treated with Flavopiridolcombined with radiotherapy and single radiotherapy had statistical significance(P<0.05).Many target click model, the survival curves fitting: the sensitization enhancement ratioof Eca9706cell line R+0.1×IC50, R+0.2×IC50were1.2375and1.3456.3. Results of immunocytochemistry showed that the positive rate of caspase-3inthe test group was67%, had differences with the other groups(P<0.05); the positive rateof survivin in the test group was24%, had differences with the other groups(P<0.05);the positive rate of P53in the test group was31%, had differences with the othergroups(P<0.05).4. Results of TUNEL showed that apoptosis index(AI) of drug group was(35.8±1.94)%, AI of radiation group was (47.1±1.71)%, AI of the test group was(68.3±2.24)%, AI of the control group was (5.1±0.71)%. Conclusion:1. Flavopiridol had proliferate inhibition effection on Eca9706cells, the IC50was2.56μmol/L.2. Flavopiridol at the concentration with low cytotoxicity had radiosensitivity-enchancing effect on Eca9706cell line, and the radiosensi-tivity-enchancing effectenhancing with the increasing concentration of Flavopiridol.3. The expression levels of survivin and P53dropped while the expression ofcaspase-3rised in the test group compared to the other groups (P<0.05).4. The cell number of apoptosis in the test group was higher than the othergroups(P=0.00). Low dose Flavopiridol could induce cell apoptosis, increase thesensitivity of radiation, may be the mechanism of blocking cell cycle and inducing cellapoptosis.