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.

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

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