| Objective: Osteosarcoma is the most common primary bone tumor inchildhood and adolescence. It is highly malignant and aggressive and itmetastasizes primarily to the lung easily. With the improvement ofchemotherapy dose density the prognosis is gradually improved and the5-yearsurvival rate is about70%. It was thought traditionally osteosarcoma was notsensitive to radiotherapy. But with the deepening of the radiation biologyknowledge it is found that if the dose is high enough, a variety of tumors everthought not sensitive to irradiation can still obtain good curative effect throughirradiation. In recent years image-guided radiation therapy (IGRT) being themain form of precise radiotherapy technology offers hopes to improve theradiation dose. Moreover it is can not be ignored that discoveries of moreradiosensitizers also significantly improved the curative effect of radiotherapy.Paclitaxel (PTX) and therarubicin (THP), both commonly used in thetreatment of osteosarcoma as chemotherapy drugs, have the function ofblocking the cells in the state of specific cell cycle. Based on these we studygrowth inhibition on osteosarcoma cell MG-63of PTX and THP, theradiotherapy sensitization effect of both kinds of drug used lonely and jointlyon MG-63cells as well as the relationship between sensitization effect andexposure time, and preliminarily explore its mechanism of action, in order toprovide more theoretical basis for clinical application.Methods: MG-63cells were cultured in a incubator maintained at37℃in5%CO2and incubated in high-glucose DMEM media that wassupplemented with10%fetal bovine serum (FBS),100U/ml penicillin and100μg/ml streptomycin. Cells in the exponential phase of growth were usedin the experiment.1Observations of cell morphology: MG-63cells incubated in different concentrations of paclitaxel ortherarubicin for different time were observed and taken photos under invertedphase-contrast microscope.2Cell growth inhibition test:MG-63cells, digested by pancreatic enzyme, were adjusted to theconcentration of3×104/ml, and planted in96-well culture plate,100ml perwell. About12h later adherent cells were exposed in different concentrationsof paclitaxel (6,0.6,0.06,0.006,0.0006μg/ml) and therarubicin and (10,1,0.1,0.01,0.001μg/ml) and allowed to incubate for24h. Prior to harvesting,10μlof5mg/ml MTT was added to each well. After incubating for4h,0.1ml ofDMSO was added to stop the reactions. The absorbance value for each wellwas determined spectrophotometrically at520nm on a Microplate Reader3Colony forming cell assay:On one hand MG-63cells treated with paclitaxel and therarubicin for24hwere exposed in different doses (0,2,4,6,8Gy) irradiation. On the other handMG-63cells treated with paclitaxel and therarubicin for different time (0,12,24,36h) were exposed in irradiation (4Gy). After about2weeks, the numberof clones generated in each dish was counted and the lower limit of clone sizescored was50cells. Single hit multi-target model was used to determinesurvival curve parameters. According to the parameters the sensitivityenhancement ratio (SER) and the best exposure time are calculated.4Flow cytometry:MG-63cells were randomly divided into4groups: control group, PTXgroup, THP group and PTX plus THP group. Each treatment group was treatedwith corresponding drugs for24h and then radiated by X-ray (6Gy). After24h, the cells were harvested and washed twice with cold PBS. The cells werefixed in ice-cold70%ethanol and then stored at4°C overnight. Prior toanalysis, the cells were washed twice with PBS. For analyzing the cell cycle,cells were suspended in0.5ml of cold PI and then incubated at37°C for anadditional30min in the dark. Others were added with the desired antibodies(bcl-2, bax, caspase-3) and secondary antibodies. Results:1The cultured cells were observed using inverted phase-contrastmicroscope throughout the culture period as mentioned. When MG-63cellswere seeded at the beginning of the culture, they were triquetrous, polygonal,spindle, irregular cells and adherent to the plastic surface of the culture plate.Extended dendrites projecting in many directions from the cell body wereeasily found. With increasing concentration and extension of time cells werefound to be rounder, smaller in shape and semidetached from the plasticsurface of the culture plate generally. At last in the plate of the highestconcentration part of the cells suspending in the culture medium and part cellswere found sparse on the culture plate surface. Under high power microscope,these cultured cells were found not only with condensed cytoplasm but alsochipped membrane, incomplete morphology and significantly slow growth.2MTT assay showed that both paclitaxel and therarubicin significantlydecreased the growth rate of MG-63cells compared with control group, thehigher the drug concentration with the bigger the cell inhibition rate (P <0.05).The inhibiting effect of both paclitaxel and therarubicin on MG-63cellsgrowth was in a dose dependent manner. IC10was0.01μg/ml and0.004μg/mlrespectively.3According to D0and Dqvalue the sensitivity enhancement ratio (SER)of PTX,THP and PTX plus THP was1.14,1.49,0.97(D0rate)，1.17,1.06,40.33(Dqrate) and1.08,1.04,1.61（SF2rate）. Cell survival fractions werestatistically significant after the cells were treated with PTX, THP and PTXplus THP for different hours combined with irradiation (P<0.05).4Flow cytometry analysis showed that combined with irradiation PTXincreased G2/M phase to63.7%, THP increased G2/M phase to83.25%andPTX plus THP increased G2/M phase to92.4%, compared to11.15%inunexposed cells. Apoptosis-related proteins (bcl-2, bax, caspase-3) expressionincreased in the treatment group, whereas the bcl-2/bax ratio decrease. PTXplus THP combined with irradiation group expressed most. Conclusions:1Both paclitaxel and therarubicin can inhibit MG-63cell growth. Theinhibitory effect increases with the increase of drug concentration, whichshows a concentration dependence of the drug toxicity.2Both paclitaxel and therarubicin have radiotherapy sensitization effectfor MG-63cell. The drugs in combination can enhance the effect. Thesensitization effect is positively related to drug effect time.3The sensitization mechanism may be related to the G2/M phaseblocking effect of paclitaxel and therarubicin and enhancement ofapoptosis-related proteins (bcl-2, bax, caspase-3) expression. |
