紫草素促进caspase-9活化诱导大肠癌细胞凋亡的分子机制研究

doi:10.3877/cmj.j.issn.2095-1221.2020.01.003

目的

探讨紫草素对大肠癌（CRC）细胞LoVo的抗肿瘤作用及其机制。

方法

以不同紫草素浓度梯度（0、2、4、6 μmol/L）处理CRC细胞LoVo 24 h，以4 μmol/L紫草素处理不同时间梯度（0、12、24、48 h）的CRC细胞LoVo。流式细胞技术结合Annexin V-FITC/PI双染色测定细胞凋亡率，Western blot检测细胞中caspase-9蛋白的表达及切割情况。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。

结果

与0 μmol/L处理CRC细胞LoVo 24 h比较，2、4、6 μmol/L的细胞凋亡率[（6.94±1.02）﹪比（10.61±1.12）﹪、（15.55± 1.35）﹪、（36.51±1.46）﹪]均升高；与4 μmol/L紫草素处理0 h的CRC细胞LoVo比较，12、24、48 h的细胞凋亡率[（1.33±0.59）﹪比（19.23±1.24）﹪、（22.24±1.41）﹪、（28.41±1.52）﹪]均升高，差异具有统计学意义（P均< 0.001）。当紫草素剂量≥2 μmol/L，处理时间≥12 h时，caspase-9蛋白表达上调并被诱导活化，而caspase-9抑制剂（Z-LEHD-FMK）预处理后，LoVo细胞凋亡率下降38.7﹪，差异有统计学意义（P < 0.05）。

结论

紫草素可以通过caspase-9蛋白的表达及其切割活性诱导CRC细胞凋亡。

关键词: 大肠癌, 细胞凋亡, caspase-9, 紫草素

Objective

To investigate the anti-tumor effect and mechanism of shikonin on colorectal cancer cell LoVo.

Methods

The colorectal cancer cell LoVo was treated with different concentration gradients (0, 2, 4, 6 μmol/L) for 24 hours. Besides, the other colorectal cancer cell LoVo was treated with 4μmol/L of shikonin according to different time gradients (0, 12, 24, 48 h) . The apoptosis rate was determined by flow cytometry combined with Annexin V-FITC/PI double staining. The expression and cleavage of caspase-9 protein were detected by Western blot. ANOVA and LSD-t tests were conducted to compare the average values between the control and experimental groups.

Results

Compared with the colorectal cancer cell LoVo treated with 0 μmol/L for 24 hours, the apoptosis rate of colorectal cancer cell LoVo treated with 2, 4, 6 μmol/L for 24 hours were increased [ (6.94±1.02) ﹪ vs (10.61±1.12) ﹪, (15.55±1.35) ﹪ and (36.51±1.46) ﹪]. Compared with the colorectal cancer cell LoVo treated with 4 μmol/L for 0 hours, the apoptosis rate of colorectal cancer cell LoVo treated with 4 μmol/L for 12 hours, 24 hours and 48 hours were increased [ (1.33±0.59) ﹪vs (19.23±1.24) ﹪, (22.24±1.41) ﹪ and (28.41±1.52) ﹪], differences were statistically significant (P< 0.001) . In the mean time, caspase-9 protein was up-regulated and activated when shikonin dose was≥2 μmol/L and treatment time was≥12 h, while caspase inhibitor (Z-LEHD-FMK) pretreatment could reduced the apoptosis rate significantly (P< 0.001) , by approximately 38.7﹪ (P< 0.05) .

Conclusion

Shikonin could induce apoptosis of colorectal cancer cells by regulating the expression of caspase-9 protein and its cleavage activity.

Key words: Colon cancer, Apoptosis, Caspase-9, Shikonin

图1 倒置荧光显微镜下观察紫草素剂量依赖性诱导大肠癌细胞LoVo凋亡（Annexin V和PI染色，×100）

图2 流式细胞技术检测紫草素剂量依赖性诱导大肠癌细胞LoVo凋亡

图3 倒置荧光显微镜观察紫草素时间依赖性诱导大肠癌细胞LoVo凋亡（Annexin V和PI染色，×100）

图4 流式细胞技术检测紫草素时间依赖性诱导大肠癌细胞LoVo凋亡

图5 紫草素剂量依赖性诱导CRC细胞LoVo中Cleaved caspase-9蛋白表达

图6 紫草素时间依赖性诱导大肠癌细胞LoVo中Cleaved caspase-9蛋白表达

图7 倒置荧光显微镜下观察caspase-9抑制剂Z-LEHD-FMK拯救紫草素诱导的大肠癌细胞LoVo凋亡（Annexin V和PI染色，×100）

图8 流式细胞技术检测caspase-9抑制剂Z-LEHD-FMK拯救紫草素诱导的大肠癌细胞LoVo凋亡情况

1	Pagès F, Mlecnik B, Marliot F, et al. International validation of the consensus Immunoscore for the classification of colon cancer: a prognostic and accuracy study[J]. Lancet, 2018, 391(10135):2128-2139.
2	Islami F, Goding Sauer A, Miller KD, et al. Proportion and number of cancer cases and deaths attributable to potentially modifiable risk factors in the United States[J]. Ca Cancer J Clin, 2018, 68(1):31-54.
3	Guglielmo A, Staropoli N, Giancotti M, et al. Personalized medicine in colorectal cancer diagnosis and treatment: a systematic review of health economic evaluations[J]. Cost EffResourAlloc, 2018, 16:2.
4	Gupta B, Chakraborty S, Saha S, et al. Antinociceptive properties of shikonin: in vitro and in vivo studies[J]. Can J Physiol Pharmacol, 2016, 94(7):788-796.
5	Yang H, Zhou P, Huang H, et al. Shikonin exerts antitumor activity via proteasome inhibition and cell death induction in vitro and in vivo[J]. Int J Cancer, 2009, 124(10):2450-2459.
6	Svandova EB, Vesela B, Lesot H, et al. Expression of Fas, FasL, caspase-8 and other factors of the extrinsic apoptotic pathway during the onset of interdigital tissue elimination[J]. Histochem Cell Biol, 2017, 147(4):497-510.
7	Zhao Y, Jing Z, Lv J, et al. Berberine activates caspase-9/cytochrome c-mediated apoptosis to suppress triple-negative breast cancer cells in vitro and in vivo [J]. Biomed Pharmacother, 2017, 95:18-24.
8	Leadsham JE, Gourlay CW. cAMP/PKA signaling balances respiratory activity with mitochondria dependent apoptosis via transcriptional regulation[J]. BMC Cell Biol, 2010, 11:92.
9	Zhai TY, Hei ZY, Ma Q, et al. Shikonin induces apoptosis and G0/G1 phase arrest of gallbladder cancer cells via the JNK signaling pathway[J]. Oncol Rep, 2017, 38(6):3473-3480.
10	阮敏, 杨雯君, 周晓健,等. 紫草素对口腔鳞癌Tca8113细胞增殖与凋亡的作用[J]. 中国口腔颌面外科杂志, 2010, 8(1):66-72.
11	Li Y, Zhang Z, Zhang X, et al. A dual PI3K/AKT/mTOR signaling inhibitor miR-99a suppresses endometrial carcinoma[J]. Am J Transl Res, 2016, 8(2):719-731.
12	彭雨. 紫草素及其衍生物抗肿瘤作用及其应用的研究进展[J]. 人人健康, 2019, (4):289-290.
13	Yang Q, Li S, Fu Z, et al. Shikonin promotes adriamycin-induced apoptosis by upregulating caspase-3 and caspase-8 in osteosarcoma[J]. Mol Med Rep, 2017, 16(2):1347-1352.
14	王晓兰, 吕和平, 康跃军. 肿瘤坏死因子α与炎症性肠病研究进展[J]. 河南科技大学学报(医学版), 2002, 20(3):264-266.
15	Blumenthal GM, Kluetz PG, Schneider J, et al. Oncology drug approvals: evaluating endpoints and evidence in an era of breakthrough therapies[J]. Oncologist, 2017, 22(7):762-767.
16	Zhang X, Cui JH, Meng QQ, et al. Advance in anti-tumor mechanisms of shikonin, alkannin and their derivatives[J]. Mini Rev Med Chem, 2018, 18(2):164-172.
17	辛国松, 季宇彬, 李先明,等. 几种天然植物中萘醌类成分抗肿瘤作用研究进展[J]. 科学技术创新, 2014(24):37-37.
18	Jorgensen I, Rayamajhi M, Miao EA. Programmed cell death as a defence against infection[J]. Nat Rev Immunol, 2017, 17(3):151-164.
19	Scheer R, Baidoshvili A, Zoidze S, et al. Tumor-stroma ratio as prognostic factor for survival in rectal adenocarcinoma: A retrospective cohort study[J]. World J GastrointestOncol, 2017, 9(12):466-474.
20	Zhang X, Chen Y, Cai G, et al. Carnosic acid induces apoptosis of hepatocellular carcinoma cells via ROS-mediated mitochondrial pathway[J]. Chem Biol Interact, 2017, 277:91-100.
21	高飞, 郭文. 线粒体促凋亡蛋白Smac/DIABLO及其与肿瘤的关系[J]. 肿瘤, 2007, 27(10):844-846.

[1]	孔莹莹, 谢璐涛, 卢晓驰, 徐杰丰, 周光居, 张茂. 丁酸钠对猪心脏骤停复苏后心脑损伤的保护作用及机制研究[J]. 中华危重症医学杂志(电子版), 2023, 16(05): 355-362.
[2]	张晓燕, 肖东琼, 高沪, 陈琳, 唐发娟, 李熙鸿. 转录因子12过表达对脓毒症相关性脑病大鼠大脑皮质的保护作用及其机制[J]. 中华妇幼临床医学杂志(电子版), 2023, 19(05): 540-549.
[3]	周伟, 蔡恒, 范海迪, 李惠中, 王传霞, 顾茂胜. cblC型甲基丙二酸血症MMACHC基因新突变对小鼠神经细胞凋亡及Wnt/β-catenin信号通路的作用机制[J]. 中华妇幼临床医学杂志(电子版), 2022, 18(05): 528-539.
[4]	徐煜琛, 李璐, 薛冬令, 赵德伟. 外泌体介导股骨头坏死机制与治疗的研究进展[J]. 中华损伤与修复杂志(电子版), 2022, 17(03): 247-252.
[5]	刘硕儒, 王功炜, 张斌, 李书豪, 胡成. 新型溶瘤病毒M1激活内质网应激致前列腺癌细胞凋亡的机制[J]. 中华腔镜泌尿外科杂志(电子版), 2023, 17(04): 388-393.
[6]	邓春文, 陈嵩, 钟裴, 闵师强, 万健. LncRNA CRNDE通过miR-181a-5p/SOX6轴调节脂多糖诱导人肺泡上皮细胞的炎症反应和细胞凋亡[J]. 中华细胞与干细胞杂志(电子版), 2023, 13(03): 129-136.
[7]	田鹏飞, 王丽娟, 肖圣超. 黄芪总黄酮通过调控miR-190a-5p对缺氧/复氧诱导的心肌细胞损伤的影响[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(06): 346-352.
[8]	郭周威, 屈艳玲, 李永慧. 类叶升麻苷通过上调miR-204对缺氧/复氧诱导H9C2细胞凋亡的抑制作用[J]. 中华细胞与干细胞杂志(电子版), 2022, 12(03): 145-152.
[9]	张师垚, 徐岩岩, 张琦, 李春强, 赵智成, 刘刚. m⁶A结合蛋白YTHDC2调节p38MAPK信号通路影响结直肠癌细胞凋亡[J]. 中华结直肠疾病电子杂志, 2023, 12(02): 117-124.
[10]	陈钰澜, 陈健文, 朱飞, 王田田, 张妍, 刘娇娜, 黄梦杰, 吴玲玲, 陈香美. 紫草素抑制缺血再灌注肾损伤后肾小管细胞的增殖和迁移[J]. 中华肾病研究电子杂志, 2022, 11(01): 15-21.
[11]	姚尧, 杨新明, 杜雅坤, 朱宁, 阴彦林, 贾永利, 张瑛, 张培楠, 田野, 陈丽星. 雷公藤甲素与甲泼尼龙调节细胞自噬和凋亡促进脊髓损伤修复的比较研究[J]. 中华神经创伤外科电子杂志, 2022, 08(03): 132-140.
[12]	于迪, 于海波, 吴焕成, 李玉明, 苏彬, 陈馨. 发状分裂相关增强子1差异表达对胆固醇刺激下血管内皮细胞的影响[J]. 中华脑科疾病与康复杂志(电子版), 2023, 13(05): 264-270.
[13]	陈文静, 唐乙厶, 赵蓓, 徐敏燕, 李涛. 表皮生长因子受体在黑色素瘤紫杉醇耐药性中的机制研究[J]. 中华临床医师杂志(电子版), 2022, 16(01): 94-99.
[14]	郭德华, 贺迎坤, 白卫星, 何艳艳, 李天晓. 脑动静脉畸形部分栓塞术后血管组织增殖与凋亡的变化[J]. 中华介入放射学电子杂志, 2022, 10(02): 152-157.
[15]	邱甜, 杨苗娟, 胡波, 郭毅, 何奕涛. 亚低温治疗脑梗死机制的研究进展[J]. 中华脑血管病杂志(电子版), 2023, 17(05): 518-521.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

The molecular mechanism of shikonin in promoting caspase-9 activation and inducing apoptosis in colorectal cancer cells

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

The molecular mechanism of shikonin in promoting caspase-9 activation and inducing apoptosis in colorectal cancer cells