Effect Of Ray Cartilage Extract On Human Mammary Carcinoma Cell Line MCF-7Proliferation And Apoptosis

ObjectiveInvestigate the inhibitory proliferation action and proapoptotic effect onhuman mammary carcinoma cell line MCF-7of ray cartilage extract（RCE）andtry to find out an appropriate effective concentration and a suitable responsetime.Observe the relationship between its effect and the cellular cycle andinvestigate forward a single step the cyclin D1and p21. Research its possibleinternal molecule biological mechanism of the phenomenon.MethodsCultivate the cellula of MCF-7with nutrient solution which is mixed with10%fetal calf serum, RPMI-1640and100U/ml penicillin streptomycin.Place thecellula in5%CO2incubator and control the temperature at37℃.Exchange thenutrient solution and swash with PBS liquid each2or3days.Go down to thefuture generation with trypsase each3or5days. The MCF-7cells were treatedfor different duration by different concentrations of RCE.Cultivate with96orificeplate and every group is arranged with5repeated foraminas. Methabenzthiazu-ron(MTT) assay was used to evaluate the proliferation of the cells and to find outan appropriate effective concentration and a suitable response time.The effectof RCE on the cell cycle was detected by flow cytometry.Single dye with IP isused to get plans of cell cycle DNA allocation. Double dye withAnnexinV-FITC/IP is used to obtain charts of cell allocation among normal, died and apoptosis in order to investigate its proapoptotic effect. The expressions ofthe cyclin D1and p21in MCF-7cells were determined by western blot.ResultsWith the different concentration of RCE and different duration to treat,MTTassay showed that the proliferation rate of MCF-7cells was centrationdependent and time dependent on RCE (p<0.05). RCE significantly inhibitedMCF-7cell proliferation (p<0.05).And the biggest inhibition concentration ofRCE is10μmol/L and the best duration is48hours.The percentage of S phasecells significantly increased and that of G2/M phase cells significantly decreasedwith prolongation of the drug duration (p<0.05).And cellular apoptosis index ofMCF-7increased with prolongation of the drug duration.The western blotshowed that in MCF-7cells the expression level of cyclin D1significantlydecreased and the expression of p21significantly increased with prolongation ofthe duration of RCE (p<0.05).Conclusions1、 RCE possesses the proliferation inhibitory effect on MCF-7cell andits biggest inhibition concentration is10μmol/L and the best durationis48hours.2、 RCE possesses the proapoptotic effect on human mammarycarcinoma cell line MCF-7.3、 RCE blocks transformation of the cells from S phase to G2/M phase.4、 The effect of RCE may be related to the inhibition of the cyclin D1expression and to the increase of the cyclin p21expression.