新型重组杨梅素对三阴性乳腺癌的抑制作用及机制研究

陈付凉; 刘飞; 吴楠; 孙利; 李芮; 周杰; 黄银久; 薛伟; 钱中清

doi:10.13898/j.cnki.issn.1000-2200.2022.04.002

新型重组杨梅素对三阴性乳腺癌的抑制作用及机制研究

Inhibitory effect and mechanism of novel recombinant myricetin on triple negative breast cancer

摘要

摘要:
目的探讨新型重组杨梅素抑制4T1细胞的作用及机制。
方法通过对杨梅素化学式改造合成一种新的杨梅素衍生物；CCK-8细胞增殖力抑制实验，并计算半数抑制浓度(IC50)。划痕与Transwell实验观察新型杨梅素对4T1细胞迁移与侵袭能力的影响。流式细胞术观察4T1细胞在合成杨梅素干预后其周期与凋亡的变化情况，小鼠体内移植瘤模型评估新型杨梅素在模拟体内环境下对小鼠三阴性乳腺癌(TNBC)的抑制作用。HE和TUNEL染色观察小鼠移植肿瘤组织切片的细胞坏死与凋亡。最后，Western blotting检测4T1细胞干预后其相关蛋白(p53、Bcl2、Bax、Caspase 3)的表达。
结果IC50为5.5 μmol/mL。与DMSO组相比，5.5 μmol/mL新型重组杨梅素在作用于4T1细胞24 h，细胞计数和迁移率均降低，差异有统计学意义(P<0.01)。流式细胞术和肿瘤组织切片TUNEL染色结果显示，与DMSO组相比，5.5 μmol/mL新型重组杨梅素细胞凋亡细胞数增高，G₁期细胞比例较低，G₂期细胞比率增高，肿瘤生长体积和肿瘤重量均较小，p53和Bcl2蛋白下调，Caspase 3上调，差异有统计学意义(P<0.05~P<0.01)。
结论新型重组杨梅素可在体外和体内诱导4T1细胞凋亡，抑制其增殖。可能通过下调p53抑制4T1细胞增殖，通过Bcl2线粒体凋亡途径诱导4T1细胞凋亡。

Abstract:
ObjectiveTo investigate the inhibitory effect and mechanism of novel recombinant myricetin on 4T1 cells.
MethodsA new myricetin derivative was synthesized by modifying the chemical formula of myricetin.CCK-8 cell proliferation inhibition assay, and half inhibitory concentration(IC50) was calculated.The effects of novel myricetin on the migration and invasion of 4T1 cells were observed by scratch and Transwell assay.Flow cytometry was used to observe the changes of the cycle and apoptosis of 4T1 cells after the intervention of synthetic myricetin.The inhibitory effect of the novel myricetin on triple negative breast cancer(TNBC) was evaluated in the mouse tumor transplantation model in vivo.HE and TUNEL staining were used to observe the cell necrosis and apoptosis in transplanted tumor tissue sections of mice.Finally, the expression of related proteins(P53, Bcl2, Bax, Caspase 3) in 4T1 cells after intervention was detected by Western bloting.
ResultsThe IC50 was 5.5 μmol/mL.Compared with DMSO group, the cell count and migration rate of 4T1 cells were decreased after treated with 5.5 μmol/mL novel myricetin for 24 h(P<0.01).Flow cytometry and TUNEL staining of tumor tissue sections showed that compared with DMSO group, the number of apoptotic cells was increased, the proportion of G₁ phase cells was lower, the proportion of G₂ phase cells was higher, the tumor growth volume and tumor weight were smaller, p53 and Bcl2 proteins were down-regulated, Caspase 3 was up-regulated in 5.5 μmol/mL recombinant myricetin group(P<0.05 to P<0.01).
ConclusionsThe novel recombinant myricetin can induce the apoptosis and inhibit proliferation of 4T1 cells in vitro and in vivo, which may inhibit the proliferation of 4T1 cells by down-regulating p53 and induce the apoptosis of 4T1 cells through the Bcl2 mitochondrial apoptosis pathway.

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