| Research BackgroundAs the most common kind of cancer among the females,breast cancer is greatly affecting their life quality.There are about one million and ten thousand new cases reported each year in China,in which about four hundred and ten thousand will die.The methods for treating breast cancer include chemotherapy and endocrine therapy,however,such treatment appears to be non-selective due to development of multidrug resistance at the later stage and potent toxic side effects.In recent years,the application of Chinese medicine in breast cancer treatment,which shows advantages of multi-target,multi-effect,safety and development of less drug resistance,is attracting more and more attentions.Research Purpose1In vitro experimentFirst,determinimg the anti-proliferative effect of Tribuls terrestris and identifying its major effective component using different cancer cell lines;Second,evaluating its drug safety and the effect on tumor inhibition;Third,investigating the potential mechanisms of drug action based on the research about the effect of Tribuls terrestris L.on the DNA Methylation of breast cancer and the antagonistic effect of Tiliroside in breast cancer cells.2 In vivo experimentFirst,establishing the animal exnograft model of breast cancer which is estrogen dependent;Second,using Tribuls terrestris L.and its main effective component Tiliroside as intervention chemicals,finding its inhibiting effect on the exnograft model of nude mice;Third,based on the data such as tumor inhibition rate and viscera index,discussing its inhibiting effect and drug safety.Research Methods1 In vitro experimentResearch the effect of Tribuls terrestris L.and its main effective component Tiliroside on the cell proliferation among different cancer cell lines and normal cell line by MTS,comparing their cell proliferation inhibition ratios and calculating their IC50.Then test the effect of Tribuls terrestris L.on cancer cell’s Methylation with MethylFlash Methylated DNA Quantification Kit(Colorimetric),and the potential antagonistic effect of Tiliroside via estrogen receptor in breast cancer cells by E-Screen,and HD AC activity via HDAC Activity Colorimetric Kit.2In vivo experimentIn order to establish the estrogen-dependent exnograft model for breast cancer,athymic nude mice were first implanted with estrogen tablets under the skin.About 3 days after estrogen implantation,mice were subcutaneously injected with 0.2 ml breast cancer cell suspension which were equally mixed with Matrigel at a density of 5×107/ml.7 days after injection,15 nude mice were randomly separated into 3 groups as control group,Tribuls terrestris L.experimental group and Tiliroside experimental group.Every 2 days tumor diameters were recorded and gross tumor volume were calculated for each animal.Data were plotted and analyzed as the growth curves.By the end of three weeks,all mice were sacrificed.Tumor weight,organ index of liver and spleen were measured.Anti-tumor factor was calculated as well.Results1 In vitro experiment①Tribuls terrestris L.crude extract solution of each concentration showed significant growth inhibition on MCF-7,MDA-MB-231,HeLa,U-87,A-172 and FARAGE tumor cells.The growth inibition increased in a concentration-and time-dependent manner.No significant inhibitory effect were observed on normal MCF-10A mammary gland cells.Minor inhibition were found in normal END1/E6E7 cervical cells,and normal NHA astrocytes.Within the concentration range of IC50,there was a significant statistic difference between cancer cell lines and their normal counterparts(P<0.01).A weak statistical difference was observed between normal RPMI-1788 lymphocytes,and FARAGE lymphoma cells(P>0.05).②Tiliroside showed strong growth inhibition on ER(estrogen receptor)positive MCF-7 human mammary gland cells,with significant statistic difference(P<0.05);No inhibition were detected in ER negative MDA-MB-231 cells;minor inhibition against proliferation were found in MCF-10A cells,which showed notable lower anti-tumor factor compared to MCF-7.Statistic difference were found between these two cell lines(P<0.05).③Tribuls terrestris L.elevated the DNA methylation levels in MCF-7 and FARAGE tumor cells,with statistic difference compared with control group(P<0.05).④The inhibitory effect of Tiliroside on MCF-7 cells is influenced by 17(3-estradiol(E2).Tiliroside co-treatment or 2 hours’ pre-treatment with E2,resulted in significant lower growth inhibition ratio than Tiliroside alone experimental group.Statistic difference was found here as well(P<0.05).⑤When MCF-7 cells were treated with 40uM and 80uM Tiliroside solutions for 48 hours,the inhibitory ratio against HD AC activity were 15.26%and 45.93%respectively.We demonstrate that Tiliroside is able to inhibit the activity of histone deacetylase HDAC,with dose-dependent effect relationship between them.2In vivo experimentTumors in control group showed relatively fast proliferation,with significant statistic difference compared to both experimental groups(P<0.05);Tiliroside experimental group showed slightly higher anti-tumor ratio than Tribuls terrestris L.experimental group,without statistic difference between these two groups(P>0.05).No significant statistic difference were found between experimental groups and control group in terms of liver and spleen’s organ indexes(P>0.05).Conclusion1 The Chinese medicine Tribuls terrestris L.and its main effective component Tiliroside showed significant inhibitory effect on multiple tumor cell lines while with minor inhibition against normal cells proliferation,demonstrating its effective and safe anti-tumor performance.2 The anti-tumor mechanism of Tribuls terrestris L.is possibly related to the elevated tumor DNA methylation level.3 Tiliroside’s inhibitory effect against MCF-7 cells proliferation is influenced by 17β-estradiol(E2).The mechanism underlying the anti-proliferation effect towards mammary gland tumor cells possibly depends on competition against E2on ER within cell.4 HDAC activity in MCF-7 cells can be inhibited by Tiliroside,which potentially acts as HD AC inhibitor.5 Tribuls terrestris L.and Tiliroside are able to inhibit the growth of mammary gland tumor exnograft model,showing its effectiveness and safety in anti-tumor in vivo treatment. |
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