Effect Of Curcumol Combined With Paclitaxel On The Proliferation And Apoptosis Of Triple-negative Breast Cancer Cells And Its Mechanism

Objective:To explore the effect of natural medicine monomer curcumol combined with paclitaxel on the proliferation and apoptosis of triple negative breast cancer MDA-MB-231 cells and the related molecular mechanisms.Methods:1.In order to determine the effect on cell proliferation,CCK-8 assays were used to measure the inhibitory rates of the viability of MDA-MB-231 cells which were treated with CC and PTX at different concentrations and different time points.2.According to the experimental data of CCK-8 assays,the half maximal inhibitory concentration(IC₅₀)corresponding to the effect of curcumol and paclitaxel on MDA-MB-231 cells for 48 hours was calculated.Based on this IC₅₀,the MDA-MB-231 cells of curcumol group,paclitaxel group and combined group were treated with different concentrations for 48 hours,and then CCK-8assay was applied to measure the cell viability.According to the above data,Compu Syn software was used to calculate the drug combination index（CI）to assess whether curcumol combined with paclitaxel was synergistic,additive or antagonistic in anti-MDA-MB-231 cells.According to the combined drug FA-CI curve,the subsequent experiments were divided into four groups:control group（Control）,curcumol group（CC,250μM）,paclitaxel group（PTX,2.5μM）and combined group（CC+PTX,250μM+2.5μM）.3.The cell clone formation assay was applied to detect the effects of cell proliferation in each group after 48 hours of drug action.4.Hochest 33258 immunofluorescence apoptosis staining kit and flow cytometry were applied to observe the morphological changes and analyze the differences in apoptosis rate of cells in each group after 48 hours of drug action.5.Western blot was applied to detect the protein expression of B-cell lymphoma-2（Bcl-2）,Bcl-2 associated X protein（Bax）,proliferating cell nuclear antigen（PCNA）,zinc finger and BTB domain containing 7A（ZBTB7A）,nuclear factor kapp B p65（NF-κB p65）.6.Establish a nude mice transplantation tumor model of breast cancer MDA-MB-231 cells,and divide them into control group（Control）,curcumol group（CC,100 mg/kg）,paclitaxel group（PTX,10 mg/kg）and combined drug group（CC+PTX,100 mg/kg+10 mg/kg）according to body mass using the random number table method 4 days later,7 nude mice in each group.All nude mice were subjected to intraperitoneal injection every two days,body mass,tumor length and short diameter were measured every four days.Nude mice were sacrificed after 7 consecutive drug treatments.The change of tumor growth and body mass of nude mice was analyzed.Immunohistochemical was applied to determine the localization and positive rate detection of PCNA,ZBTB7A and NF-κB p65.At the same time,Td T-mediated d UTP nick end labeling（Tunel）and 4’,6-diamidino-2-phenylindole（DAPI）apoptosis double staining method were applied to stain the tumor tissues in each group to analyze the difference in apoptosis rate.Results:1.Curcumol and paclitaxel inhibited the proliferation of MDA-MB-231cells in a time-and dose-dependent manner.2.The IC₅₀of curcumol and paclitaxel on MDA-MB-231 cells for 48 hours were 558.6μM and 4.4μM,respectively.There was a synergistic effect of curcumol combined with paclitaxel（CI<1）.The concentration of curcumol combined with paclitaxel on MDA-MB-231 cells with CI=0.509 and FA=0.593 for 48 hours was used as the basis for grouping in subsequent experiments.3.The colony formation assay showed that compared with Control group,the cell proliferation ability of the CC group,PTX group,and CC+PTX group decreased,and the lowest proliferation ability was found in the CC+PTX group（P<0.05）.4.Hochest 33258 immunofluorescence apoptosis staining showed that the nuclei of control group were evenly stained in blue,the nucleus of CC group,PTX group and CC+PTX group were unevenly stained,and some of the nuclei in these three groups were condensed,fragmented and dissolved.Hochest 33258immunofluorescence apoptosis staining and flow cytometry showed that compared with Control group,the apoptosis rate of the CC group,the PTX group,and the CC+PTX group increased,and the highest apoptosis was found in the CC+PTX group（P<0.05）.5.Western-blot showed that compared with the Control group,the protein expressions of Bcl-2,PCNA,ZBTB7A and NF-κB p65 were reduced,and the expression of Bax protein was increased in CC group,PTX group and CC+PTX group,which was most obvious in the CC+PTX group（P<0.05）.6.Nude mouse xenograft tumor experiment showed that compared with Control group,the volume and weight in CC group,PTX group,and CC+PTX group was significantly reduced,and the volume and weight in CC+PTX group was the lowest.There was no significant difference in body mass changes between the four groups of nude mice before and after the experiment.PCNA,ZBTB7A,and NF-κB p65 were positively localized in the nucleus,with brownish yellow particles.Compared with Control group,the positive expression rates of PCNA,ZBTB7A,NF-κB p65 in CC group,PTX group and CC+PTX group were significantly reduced,and the positive expression rates in CC+PTX group were most lowest（P<0.05）.Tunel and DAPI double staining methods showed that compared with Control group,the apoptosis rate of CC group,PTX group,and CC+PTX group increased significantly,and the apoptosis rate in CC+PTX group was the highest（P<0.05）.Conclusion:1.Curcumol and paclitaxel can significantly inhibit proliferation and promote apoptosis of triple-negative breast cancer cells MDA-MB-231,and the above effects show a synergistic effect in the combination of the two.2.The possible molecular mechanism of curcumol combined with paclitaxel affecting the proliferation and apoptosis of triple negative breast cancer MDA-MB-231 cells is to down-regulate the expression of ZBTB7A through the NF-κB signaling pathway.