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2020年,WHO国际癌症研究机构(IARC)发布全球最新癌症数据显示,乳腺癌新增人数达226万,肺癌为220万,乳腺癌正式取代肺癌,成为全球第一大癌症[1]。三阴性乳腺癌(TNBC)是该疾病的一种亚型,其雌激素受体(ER)、孕激素受体(PR)和人表皮生长因子受体2(Her-2)不表达,占所有乳腺癌的15%~20%。由于TNBC对内分泌和靶向治疗不敏感, 化疗一直是TNBC的最合适的医疗选择。尽管已经有几种化疗药物可用,但是化疗药物的耐药性,非选择性细胞毒性,使得TNBC复发转移率高,毒性反应较重, 部分病人无法耐受,且预后较差, 亟需探索新的方法来改善TNBC的临床治疗效果。近年来,许多研究致力于发现具有低成本效益且不良反应小的新型天然抗癌药物[2]。因此,杨梅素及其衍生物的抗肿瘤作用引起学者们的极大兴趣,研究主要集中在杨梅素诱导细胞凋亡,抑制肿瘤细胞增殖、迁移与侵袭等。PHILLIPS等[3]研究表明杨梅素通过抑制磷脂酰肌醇3-激酶(PI3K)信号通路诱导胰腺癌细胞凋亡。SUN等[4]发现,杨梅素在人类膀胱癌细胞中也显示出抗癌活性,SHIH等[5]研究表明,杨梅素可以通过抑制细胞外信号调控激酶ERK信号通路,抑制A549细胞的迁移和侵袭。2017年,苏丽娟等[6]研究发现,杨梅素通过上调PHLPP1的表达抑制A549细胞迁移。有趣的是杨梅素对肿瘤细胞杀伤性具有浓度依懒性,对正常细胞却没有毒害作用[7]。研究[8]发现,不同化学结构的杨梅素衍生物其抗癌活性不同。因此,笔者设想能否对杨梅素母体化学式进行改造,使用化学合成这种容易获得的方法得到具有抗癌活性的杨梅素衍生物,通过对天然化合物杨梅素化学式进行改造,获得可用于TNBC治疗作用的化学物质,为临床提供作为TNBC化疗药物的可能选择。
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不同时间节点,与DMSO组比较,1、3、5 μmol/mL浓度重组杨梅素组4T1乳腺癌细胞划痕率差异均无统计学意义(P>0.05),5.5、6、8、10 μmol/mL浓度重组杨梅素组4T1乳腺癌细胞划痕率降低,差异均有统计学意义(P<0.05~P<0.01);重组杨梅素对4T1乳腺癌细胞抑制存在浓度依赖性,可在5.5 μmol/mL浓度对TNBC细胞增殖有较好的抑制作用(见表 1)。
分组 n 24 h 48 h 72 h DMSO 6 0.566±0.054 1.906±0.302 2.126±0.233 1 μmol/mL 6 0.531±0.096 1.879±0.226 2.220±0.086 3 μmol/mL 6 0.476±0.101 1.592±0.201 2.097±0.237 5 μmol/mL 6 0.425±0.074 1.020±0.160** 1.707±0.221 5.5 μmol/mL 6 0.276±0.051 0.354±0.078** 0.564±0.178** 6 μmol/mL 6 0.238±0.044** 0.251±0.024** 0.468±0.094** 8 μmol/mL 6 0.178±0.083** 0.193±0.032** 0.183±0.035** 10 μmol/mL 6 0.163±0.039** 0.200±0.032** 0.191±0.030** F — 31.33 131.47 194.94 P — < 0.01 < 0.01 < 0.01 MS组内 — 0.005 0.027 0.026 q检验:不同时间节点与DMSO组比较**P<0.01 表 1 不同时间节点及浓度重组杨梅素对4T1细胞的抑制作用(x±s)
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与DMSO组相比,5.5 μmol/mL的新型重组杨梅素组细胞计数和迁移率均降低,差异有统计学意义(P<0.01),5.5 μmol/mL的新型重组杨梅素有抑制4T1细胞迁移与侵袭能力(见图 3、表 2)。
分组 细胞计数
(Transwell,n=5)迁移率/%
(划痕,n=4)DMSO 109.00±5.60 0.99±0.000 6 5.5 μmol/mL 30.00±4.79 0.97±0.000 9 t 23.97 36.98 P < 0.01 < 0.01 表 2 5.5 μmol/mL新型重组杨梅素衍生物对4T1细胞侵袭和迁移能力的影响(x±s)
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与DMSO组相比,5.5 μmol/mL新型重组杨梅素干预4T1细胞凋亡细胞数增高,细胞周期显示,G1期细胞比例较低,G2期细胞比率增高,差异有统计学意义(P<0.01);TNBC细胞在5.5 μmol/mL的新型重组杨梅素干预后24 h,4T1细胞早期凋亡显著,与DMSO组相比G1期、G2期细胞所占比例显著增加(见图 4、表 3)。
分组 n 凋亡 G1期 S期 G2期 DMSO 3 7.21±0.45 56.87±4.09 22.62±4.71 18.52±1.92 5.5 μmol/mL 3 53.3±1.45 30.79±1.69 12.44±0.84 53.18±4.90 t — 30.34 5.90 2.13 6.59 P — < 0.01 < 0.01 >0.05 < 0.01 表 3 5.5 μmol/mL重组杨梅素对4T1细胞凋亡和周期的影响(x±s)
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与DMSO组相比,5.5 μmol/mL新型重组杨梅素组肿瘤生长体积和肿瘤重量均较小,差异有统计学意义(P<0.05),新型重组杨梅素可抑制荷瘤小鼠乳腺癌肿瘤细胞增殖,并诱导肿瘤细胞凋亡(见图 5、6及表 4)。
分组 肿瘤成长体积/mm3
(n=8)肿瘤重量/g
(n=3)DMSO 545.00±38.73 1.205±0.109 5.5 μmol/mL 447.40±18.47 0.837±0.047 t 2.28 3.10 P < 0.05 < 0.05 表 4 5.5 μmol/mL新型重组杨梅素衍生物对小鼠移植肿瘤生长的影响(x±s)
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与DMSO组相比,重组杨梅素组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),重组杨梅素可通过下调突变型p53基因从而引起4T1细胞周期阻滞,并通过改变Bax和Bcl2基因比值变化,上调Caspase 3基因蛋白表达诱导乳腺癌细胞凋亡(见图 7、表 5)。
分组 n p53 Bcl2 Bax Caspase 3 DMSO 3 0.952±0.022 0.940 5±0.091 6 0.903 1±0.068 5 0.514±0.0403 5.5 μmol/mL 3 0.370±0.026 0.624 1±0.058 2 0.743 2±0.067 3 0.725±0.043 3 t — 16.91 2.93 1.67 3.57 P — < 0.01 < 0.05 >0.05 < 0.05 表 5 5.5 μmol/mL新型重组杨梅素对凋亡相关基因蛋白表达的影响(x±s)
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
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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. -
Key words:
- triple negative breast cancer /
- novel myricetin /
- 4T1 cells /
- apoptosis
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表 1 不同时间节点及浓度重组杨梅素对4T1细胞的抑制作用(x±s)
分组 n 24 h 48 h 72 h DMSO 6 0.566±0.054 1.906±0.302 2.126±0.233 1 μmol/mL 6 0.531±0.096 1.879±0.226 2.220±0.086 3 μmol/mL 6 0.476±0.101 1.592±0.201 2.097±0.237 5 μmol/mL 6 0.425±0.074 1.020±0.160** 1.707±0.221 5.5 μmol/mL 6 0.276±0.051 0.354±0.078** 0.564±0.178** 6 μmol/mL 6 0.238±0.044** 0.251±0.024** 0.468±0.094** 8 μmol/mL 6 0.178±0.083** 0.193±0.032** 0.183±0.035** 10 μmol/mL 6 0.163±0.039** 0.200±0.032** 0.191±0.030** F — 31.33 131.47 194.94 P — < 0.01 < 0.01 < 0.01 MS组内 — 0.005 0.027 0.026 q检验:不同时间节点与DMSO组比较**P<0.01 表 2 5.5 μmol/mL新型重组杨梅素衍生物对4T1细胞侵袭和迁移能力的影响(x±s)
分组 细胞计数
(Transwell,n=5)迁移率/%
(划痕,n=4)DMSO 109.00±5.60 0.99±0.000 6 5.5 μmol/mL 30.00±4.79 0.97±0.000 9 t 23.97 36.98 P < 0.01 < 0.01 表 3 5.5 μmol/mL重组杨梅素对4T1细胞凋亡和周期的影响(x±s)
分组 n 凋亡 G1期 S期 G2期 DMSO 3 7.21±0.45 56.87±4.09 22.62±4.71 18.52±1.92 5.5 μmol/mL 3 53.3±1.45 30.79±1.69 12.44±0.84 53.18±4.90 t — 30.34 5.90 2.13 6.59 P — < 0.01 < 0.01 >0.05 < 0.01 表 4 5.5 μmol/mL新型重组杨梅素衍生物对小鼠移植肿瘤生长的影响(x±s)
分组 肿瘤成长体积/mm3
(n=8)肿瘤重量/g
(n=3)DMSO 545.00±38.73 1.205±0.109 5.5 μmol/mL 447.40±18.47 0.837±0.047 t 2.28 3.10 P < 0.05 < 0.05 表 5 5.5 μmol/mL新型重组杨梅素对凋亡相关基因蛋白表达的影响(x±s)
分组 n p53 Bcl2 Bax Caspase 3 DMSO 3 0.952±0.022 0.940 5±0.091 6 0.903 1±0.068 5 0.514±0.0403 5.5 μmol/mL 3 0.370±0.026 0.624 1±0.058 2 0.743 2±0.067 3 0.725±0.043 3 t — 16.91 2.93 1.67 3.57 P — < 0.01 < 0.05 >0.05 < 0.05 -
[1] World Health Organization. World Cancer Report: Cancer Research for Cancer Prevention[R]. Geneva: WHO, 2020. [2] LEE MM, CHAN BD, WONG WY, et al. Synthesis and evaluation of novel anticancer compounds derived from the natural product brevilin A[J]. ACS Omega, 2020, 5(24): 14586. doi: 10.1021/acsomega.0c01276 [3] PHILLIPS PA, SANGWAN V, BORJA-CACHO D, et al. Myricetin induces pancreatic cancer cell death via the induction of apoptosis and inhibition of the phosphatidylinositol 3-kinase(PI3K) signaling pathway[J]. Cancer Lett, 2011, 308(2): 181. doi: 10.1016/j.canlet.2011.05.002 [4] SUN F, ZHENG XY, YE J, et al. Potential anticancer activity of myricetin in human T24 bladder cancer cells both in vitro and in vivo[J]. Nutr Cancer, 2012, (64): 599. [5] SHIH YW, WU PF, LEE YC, et al. Myricetin suppresses invasion and migration of human lung adenocarcinoma A549 cells: possible mediation by blocking the ERK signaling pathway[J]. J Agric Food Chem, 2009, 57(9): 3490. doi: 10.1021/jf900124r [6] 苏丽娟, 付有荣, 田莉, 等. 杨梅素通过上调PHLPP1的表达抑制A549细胞迁移[J]. 临床和实验医学杂志, 2017, 16(2): 131 doi: 10.3969/j.issn.1671-4695.2017.02.009 [7] JAYAKUMAR JK, NIRMALA P, PRAVEEN KUMAR, BA, et al. Evaluation of protective effect of myricetin, a bioflavonoid in dimethyl benzanthracene-induced breast cancer in female Wistar rats[J]. South Asian J Cancer, 2014, 3: 107. doi: 10.4103/2278-330X.130443 [8] SEMWAL DK, SEMWAL RB, COMBRINCK S, et al. Myricetin: A dietary molecule with diverse biological activities[J]. Nutrients, 2016, 8(2): 90. doi: 10.3390/nu8020090 [9] XUE W, SONG BA, ZHAO HJ, et al. Novel myricetin derivatives: Design, synthesis and anticancer activity[J]. Eur J Med Chem, 2015, 97: 155. doi: 10.1016/j.ejmech.2015.04.063 [10] HA TK, JUNG I, KIM ME, et al. Anti-cancer activity of myricetin against human papillary thyroid cancer cells involves mitochondrial dysfunc-tion-mediated apoptosis[J]. Biomed Pharmacother, 2017, 91: 378. doi: 10.1016/j.biopha.2017.04.100 [11] SHIOMI K, KURIYAMA I, YOSHIDA H, et al. Inhibitory effects of myricetin on mammalian DNA polymerase, topoisomerase, and human cancer cell proliferation[J]. Food Chem Toxicol, 2013, 139(1/4): 910. [12] HANG QY, LI R, ZENG GF, et al. Dihydromyricetin inhibits migration and invasion of hepatoma cells through regulation of MMP-9 expression[J]. World J Gastroenterol, 2014, 20(29): 10082. doi: 10.3748/wjg.v20.i29.10082 [13] LIU C, ZHAO P, YANG Y, et al. Ampelopsin suppresses TNF-α induced migration and invasion of U2OS osteosarcoma cells[J]. Mol Med Rep, 2016, 13(6): 4729. doi: 10.3892/mmr.2016.5124 [14] DU P, LI SJ, OJCIUS DM, et al. A novel Fas-binding outer membrane protein and lipopolysaccharide of Leptospira interrogans induce macrophage apoptosis through the Fas/FasL-caspase-8/-3 pathway[J]. Emerg Microbes Infect, 2018, 7(1): 135. [15] HAO Q, CHEN J, LIAO J, et al. p53 induces ARTS to promote mitochondrial apoptosis[J]. Cell Death Dis, 2021, 12(2): 204. doi: 10.1038/s41419-021-03463-8 [16] 时宗芬, 鲁星月, 张配, 等. 3-溴丙酮酸增强人鼻咽癌细胞对顺铂敏感性的作用及机制研究[J]. 蚌埠医学院学报, 2020, 45(9): 1147. [17] FENG J, CHEN X, WANG Y, et al. Myricetin inhibit-sproliferation and induces apoptosis and cell cycle arrest in gastric cancer cells[J]. Mol Cell Biochem, 2015, 408(1/2): 163. [18] LI HG, CHEN JX, XIONG JH, et al. Myricetin exhibits anti-glioma potential by inducing mitochondrial-mediated apoptosis, cell cycle arrest, inhibition of cell migration and ROS generation[J]. J BUON, 2016, 21(1): 182. [19] 董正远, 许河南, 王文锐, 等. 紫杉醇长循环纳米胶束联合vMIP-ⅡN端肽逆转乳腺癌细胞耐药的研究[J]. 蚌埠医学院学报, 2020, 45(9): 1141. [20] ADAMKOV M. Logical complexity of Bcl-2 family proteins function in the intrinsic apoptosis[J]. SPR ARK CELOK LEK, 2019, 147(1/2): 99. [21] HUANG H, CHEN AY, YE X, et al. Myricetin inhibits proliferation of cisplatin-resistant cancer cells through a p53-dependent apoptotic pathway[J]. Int J Oncol, 2015, 47(4): 1494. doi: 10.3892/ijo.2015.3133 [22] ABOTALEB M, SAMUEL SM, VARGHESE E, et al. Flavonoids in Cancer and Apoptosis[J]. Cancers(Basel), 2018, 11(1): 28. [23] ZHENG AW, CHEN YQ, ZHAO LQ, et al. Myricetin induces apoptosis and enhances chemosensitivity in ovarian cancer cells[J]. Oncol Lett, 2017, 13(6): 4974. doi: 10.3892/ol.2017.6031