| ObjectiveBy cultivating human breast cancer cell line MCF-7and applying differentconcentrations of Pseudo-Ginsenoside GQ （PGQ） and Doxorubicin effects oncell lines, to observe cell morphology of regular human breast cancer cellline MCF-7and the effect on cell shape by PGQ and Doxorubicin. By using theinhibition rate determined by MTT method and cell apoptosis data detected byflow cytometry, to understand the growth trend of human breast cancer cellline MCF-7and draw the growth curve. To investigate the proliferationinhibition of Doxorubicin and PGQ in human breast cancer cell lines MCF-7.To investigate the effect of different concentrations and doses of PGQ onproliferation of human breast cancer cell lines MCF-7, finally to investigatethe influence of Doxorubicin cytotoxicity by Pseudo-Ginsenoside GQ in MCF-7human breast cancer cells.Method1.Cell morphological observation: Conventional culture human breast cancercell line MCF-7, and use inverted optical microscope to observe the normalgrowth of cells and the effect on cell shape by PGQ and Doxorubicin.2. Draw growth curve of the human breast cancer cell line MCF-7: Conventionalculture human breast cancer cell line MCF-7, then chose the exponentiallygrowing cells for experiment. Fully digest and prepare the cell suspension,count to1x104/mL, and then inoculate the cell suspension in96-well plates,all wells are divided into14groups, each group set three wells. After that,test the average absorbance value of the three wells in one group using theenzyme immune detector at570nm absorbance measurement by MTT colorimetricassay. Determine time as the abscissa, and the daily average absorbance valueas the ordinate, draw growth curve of human breast cancer cell line MCF-7. 3. To investigate the proliferation inhibition of Doxorubicin and PGQ in humanbreast cancer cell lines MCF-7by MTT colorimetry analysis: Routinelycollected logarithmic phase of growth cells, adjust cell concentration to4×104/mL, vaccinate100μL per well in96-well plates, culture one night,then add different concentrations of drug100μL per well after the cells arecompletely attached to the bottle well. High concentration PGQ single druggroup set up six concentration gradient, respectively1250μmol/L,1000μmol/L,750μmol/L,500μmol/L,250μmol/L,125μmol/L. Lowconcentration PGQ single drug group （close to animal experimental myocardialprotection optimal concentration） set up six concentration gradient,respectively125μmol/L,100μmol/L,75μmol/L,50μmol/L,25μmol/L,12.5μmol/L. Doxorubicin single drug group set up six concentration gradient,respectively20μmol/L,10μmol/L,5μmol/L,2.5μmol/L,1.25μmol/L,0.625μmol/L. Each concentration set three wells, and set up blank controlgroup, negative control group, each pore volume is200μL. Regular culture24h,48h and72h, then add20μL5mg/L tetramethyl azo saltsilicon solution（MTT） per well, incubate4h, after the violet crystals form completely,carefully discard the supernatant, add dimethyl phosphite alum （DMSO）150μL to each well, shake10minutes to make the crystals fully dissolved, detectthe absorbance value of each well in enzyme mark immune apparatus under570nm, calculate inhibition rate of different concentration of PGQ andDoxorubicin on MCF-7cells, calculation formula: proliferation inhibitionrate=1-（experimental well A value-blank control well A value）/（negativecontrol well A value-blank control well A value）. Repeat the experiment3times. According to Doxorubicin single-agent and PGQ single-agentexperiment result set doxorubicin concentration gradient of1μmol/L,0.5μmol/L and0.25μmol/L, combined the use of100μmol/L PGQ, in the sametime set the control group which is without PGQ. Culture72h, then detectinhibition rate by MTT method. 4. Flow cytometer detection: Routinely collected logarithmic phase of growthcells， adjust cell concentration to1×106/mL, apply PI single staining oncells, use flow cytometer to analyze the cell cycle.Results:1. Human breast cancer cell line MCF-7cell morphology: The normal shapeof MCF-7cells are usually irregular polygon, visible pseudopodia, grew singlelayer by static adherence, usually form into clusters, well differentiated,multinucleated cells are rare. Watch again after applied Doxorubicin andconcentrated PGQ，we can see a large number of visible cells debris, irregularadherent cell shape, loss of normal structure, increased floating cells. Wecan also find the typical morphological changes of cell death such as membraneshrivel, concentrated cytoplasm, nuclear pyknosis. The growth the cellsapplied of low concentration PGQ is roughly normal, we can see a small amountof floating cells, the cell membrane contraction of some cells, and the visiblevacuoles in cytoplasm.2. The growth curve of human breast cancer cell line MCF-7: The growth of MCFbegan to enter the logarithmic phase in3～4days, the cell proliferation ratesignificantly get faster, then since the7th day proliferation rate begin toslow down, after10days the cell growth gradually stop.3. The proliferation effect of Doxorubicin and PGQ single agent in human breastcancer cell line MCF-7: the effects of different concentration of Doxorubicinand different time to deal with human breast cancer cell line MCF–7on cellgrowth inhibition rate were statistically significant （P＜0.05）. Thecomparison between every two concentration of Doxorubicin group were bothstatistically significant, P＜0.05, the20μmol/L concentration ofDoxorubicin reached the highest inhibition of cell proliferation; Thecomparison between every two action time of Doxorubicin group were bothstatistically significant, P＜0.05,72hours reached the highest inhibitionof cell proliferation.IC50respectively:24h4.374μmol/L,48h1.311μmol/L, 72h0.749μmol/L. For high concentration PGQ, the growth inhibition rate ofdifferent concentration of human breast cancer cell line MCF-7cell wasstatistically significant （P＜0.05）, the comparison of effect on growthinhibition of the24h group and the48h group was statistically significant（P＜0.05）, the comparison of effect on growth inhibition of the48h groupand the72h group was not statistically significant（P＞0.05）. The comparisonbetween every two concentration of high concentration PGQ group were bothstatistically significant, P＜0.05, the1250μmol/L concentration of PGQreached the highest inhibition of cell proliferation.IC50respectively:24h862.401μmol/L,48h698.241μmol/L,72h654.096μmol/L. For lowconcentration PGQ, the growth inhibition rate of different concentration ofhuman breast cancer cell line MCF-7cell was not statistically significant（P＞0.05）, and the influence of different action time on the growthproliferation rate of breast cancer cells MCF-7was also not statisticallysignificant （P＞0.05）.4. The influence of Doxorubicin cytotoxicity by Pseudo-Ginsenoside GQ in MCF-7human breast cancer cells line: the results showed that the influence on growthand proliferation rate of the Doxorubicin in combination with lowconcentration PGQ （100μmol/L）on human breast cancer cell line MCF-7hasno statistical significance （P＞0.05）.5. Flow cytometer detection: the negative control group has a small amountof cell apoptosis, and the apoptosis ratio of high concentration PGQ group（1000μmol/L） in breast cancer cell line MCF-7is higher than the controlgroup （P＜0.05）. The apoptosis ratio of Doxorubicin single-agent（1.0μmol/L） group in breast cancer cell line MCF-7is higher than thecontrol group （P＜0.05）, at the same time the apoptosis ratio of thecombination group of low concentration PGQ （100μmol/L）and Doxorubicin（1.0μmol/L）has no statistical significance （P＞0.05） when compared withcontrol group. Conclusion:1. Doxorubicin and PGQ single-agent both has proliferation inhibition on humanbreast cancer cell line MCF-7.2. PGQ single-agent and PGQ+Doxorubicin combination did not promote humanbreast cancer cell line MCF-7proliferation, which meant PGQ did not clashwith the antitumor effect of doxorubicin, and prompt PGQ could become a newtype of myocardial protective agent for myocardial injury caused byanthracycline-based antitumor drugs. |
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