姜黄素联合盐酸阿霉素的协同抗肿瘤效应及机制研究  

Synergistic antitumor effect of curcumin combined with doxorubicin hydrochloride on breast cancer cells and related mechanism

作　　者：陆慧毓 王凤玲 陈云娜[1] 王雷 周婷婷 陈卫东 LU Hui-yu;WANG Feng-ling;CHEN Yun-na;WANG Lei;ZHOU Ting-ting;CHEN Weidong(School of Pharmacy,Anhui University of Chinese Medicine,Hefei 230012;Ministry of Education Anhui Joint Collaborative Innovation Center for Quality Improvement of Anhui Genuine Chinese Medicinal Materials,Hefei 230012;Anhui Key Laboratory of Chinese Medicinal Formula,Hefei 230012;Hefei Hospital Affiliated to Anhui Medical University,Hefei 230012;Department of Pharmacy,Navy Military Medical University of People’s Liberation Army,Shanghai 200433)

机构地区：[1]安徽中医药大学药学院,合肥230012 [2]省部共建安徽道地中药材品质提升协同创新中心,合肥230012 [3]中药复方安徽省重点实验室,合肥230012 [4]安徽医科大学附属合肥医院,合肥230012 [5]中国人民解放军海军军医大学药学系,上海200433

出　　处：《中南药学》2023年第4期911-917,共7页Central South Pharmacy

基　　金：国家自然科学基金面上项目(No.82073923)。

摘　　要：目的探讨姜黄素是否能够与盐酸阿霉素协同诱导4T1乳腺癌细胞产生自噬性细胞死亡并促进盐酸阿霉素诱导的细胞凋亡。方法采用小鼠乳腺癌细胞系4T1为研究对象,分为对照组(PBS),姜黄素组(10μmol·L^(-1)姜黄素),盐酸阿霉素组(10μmol·L^(-1)盐酸阿霉素),联合应用组(10μmol·L^(-1)姜黄素+10μmol·L^(-1)盐酸阿霉素)。通过CCK-8法比较不同处理对4T1细胞的细胞毒性;采用凋亡检测试剂盒检测不同处理诱导产生的细胞凋亡;通过CCK-8法及自噬抑制策略[各组分别与3-甲基腺苷(3MA)共处理]探究联合应用组是否能够诱导4T1细胞产生自噬性细胞死亡;采用免疫荧光法研究不同处理组的自噬相关蛋白(LC3及P62)的表达及免疫原性死亡相关蛋白(CRT及HMGB1)的表达;采用三磷酸腺苷(ATP)检测试剂盒研究不同处理组ATP的释放情况,以评价免疫原性细胞死亡的程度;采用流式细胞仪检测不同处理组肿瘤细胞被骨髓原代巨噬细胞(BMDCs)吞噬摄取的程度以及对BMDCs表面MCH-Ⅱ表达的影响。结果CCK-8结果显示,姜黄素组本身不会产生明显的细胞毒性及诱导细胞凋亡,但与盐酸阿霉素联用后显著增强了盐酸阿霉素的细胞毒性及诱导细胞凋亡水平,经自噬抑制剂3MA共孵育后,这种协同效果消失,细胞毒性显著降低;与单独处理组相比,联合应用组的LC3表达水平显著上调,P62表达明显降低;免疫荧光结果显示姜黄素未能诱导免疫原性细胞死亡。与盐酸阿霉素组相比,联合应用组的CRT表达及HMGB1释放情况并无显著性差异,但ATP释放水平显著增加。流式细胞仪结果显示,与盐酸阿霉素组相比,联合应用组的肿瘤细胞能够更多的被BMDCs摄取吞噬,同时能够显著增强BMDCs表面MCH-Ⅱ的表达。结论姜黄素与盐酸阿霉素发挥协同抗肿瘤作用的机制与诱导肿瘤细胞产生自噬性细胞死亡,促进死亡肿瘤细胞释放ATP,增强死亡肿瘤细胞被BMDCs吞噬Objective To determine whether curcumin synergizes with doxorubicin hydrochloride to induce the death of autophagic cellsand promote doxorubicin hydrochloride-induced apoptosis in 4T1 breast cancer cells.Methods The 4T1 mouse breast cancer cell line was used as the study object.The mice were divided into a control group(PBS),a curcumin group(10μmol·L^(-1) curcumin),a doxorubicin hydrochloride group(10μmol·L^(-1) doxorubicin hydrochloride),and a combination group(10μmol·L^(-1) curcumin+10μmol·L^(-1) doxorubicin hydrochloride).The cytotoxicity of different treatments to 4T1 cells was compared with CCK-8 assay.Apoptosis induced by different treatments was detected with apoptosis detection kit.CCK-8 assay and autophagy inhibition strategy[co-treatment with 3-methyladenosine(3MA)]were used to determine the induction of autophagic cell death.Immunofluorescence technology was used to study the expression of autophagy-related proteins(LC3 and P62)and immunogenic death-related proteins(CRT and HMGB 1)in different treatment groups.To evaluate the extent of immunogenic cell death,ATP secretion of 4T1 cells in different groups was investigated with the ATP assay kit.The effect of different treatments on the phagocytosis and uptake of the dying tumor cells by bone marrow-derived macrophages(BMDCs)and the effect on MCH-Ⅱexpression of BMDCs were determined by flow cytometry.Results CCK-8 showed that curcumin did not obviously induce the cytotoxicity and apoptosis,but the combination of curcumin with doxorubicin hydrochloride significantly enhanced the level of cytotoxicity and cell apoptosis compared with that of doxorubicin hydrochloride alone.After co-incubation with autophagy inhibitor 3MA,the synergistic effect in the combination group disappeared and the cytotoxicity declined.Compared with the control group,the LC3 expression level was significantly increased and the P62 expression was decreased significantly in the combination group.The immunofluorescence showed that curcumin treatment failed to induce the death

关 键 词：抗肿瘤 姜黄素 盐酸阿霉素 自噬性细胞死亡 免疫原性细胞死亡 

分 类 号：R285[医药卫生—中药学]