低强度超声对HeLa肿瘤细胞凋亡的诱导作用及其机制

doi:10.3877/cma.j.issn.2095-1221.2020.05.004

目的

探讨低强度超声在诱导HeLa肿瘤细胞凋亡中的作用及其机制。

方法

将体外培养的HeLa肿瘤细胞根据超声干预强度分为阴性对照组（切断电源后给予假的超声干预）和0.5、1.3、2.0 W/cm²超声干预组，分别通过MTT实验测定细胞活力、存活率，HE染色检测细胞形态，细胞凋亡实验检测细胞凋亡率，流式细胞术测定ROS含量及Western blot实验检测caspase-12、survivin和内参蛋白β-actin的表达情况。多组间比较采用单因素方差分析，各个实验组与对照组比较采用Dunnet-t检验。

结果

与阴性对照组比较，0.5、1.3、2.0 W/ cm²超声干预组的HeLa肿瘤细胞活力[（99.23±1.56）﹪比（80.52±1.72）﹪、（54.31±1.69）﹪（27.75±1.26）﹪]、细胞存活率[（99.91±1.51）﹪比（76.69±1.92）﹪、（52.57±1.63）﹪、（29.81±1.22）﹪]、survivin蛋白表达（51.19±0.21比43.46±0.34、25.28±0.29、18.32±0.3）均降低，ROS含量（11.21±0.45比24.34±1.23、38.26±2.47、52.18±1.56），细胞凋亡率[（4.23±1.21）﹪比（24.16±1.91）﹪、（48.34±1.66）﹪、（70.27±0.98）﹪]、caspase-12蛋白表达（13.05±0.21比20.23±0.19、33.17±0.32、41.52±0.21）均升高，差异具有统计学意义（P均< 0.05）。

结论

低强度超声可能通过诱导HeLa肿瘤细胞中caspase-12蛋白表达增加和survivin蛋白表达的减少而使HeLa肿瘤细胞发生凋亡。

关键词: Caspase-12, Survivin, 低强度超声, HeLa细胞, 细胞凋亡, 子宫颈癌

objective

To investigate the role and mechanism of low intensity ultrasound induced apoptosis of HeLa tumor cells.

Methods

According to the intensity of ultrasound intervention, HeLa tumor cells cultured in vitro were divided into blank control group (pseudo-ultrasound after power cut off) and setting up three ultrasound intervention groups (0.5, 1.3, 2.0 W/ cm², respectively) . Cell viability and survival rate were determined by MTT, and cell morphology was detected by HE staining. Apoptotic rate was detected by apoptosis experiment. ROS content was determined by flow cytometry and the expression of caspase-12, survivin and the internal reference protein β-actin was detected by Western blot. One-way ANOVA was used for comparison among multiple groups, and Dunnet-t test was used for multiple comparisons. Compared with blank control group, the cell viability (99.23﹪±1.56﹪vs 80.52﹪±1.72﹪, 54.31﹪±1.69﹪, 27.75﹪±1.26﹪, P < 0.05) , cell survival rates (99.91﹪±1.51﹪vs 76.69﹪±1.92﹪, 52.57﹪±1.63﹪, 29.81﹪±1.22﹪, P < 0.05) , ROS content (11.21±0.45 vs 24.34±1.23, 38.26±2.47, 52.18±1.56, P < 0.05) as well as the expression of surviving protein (51.19±0.21 vs 43.46±0.34, 25.28±0.29, 18.32±0.3, P < 0.05) were decreased in ultrasound intervention groups (0.5, 1.3, 2.0 W/cm², respectively) . While the apoptosis rate (4.23﹪±1.21﹪vs 24.16﹪±1.91﹪, 48.34﹪±1.66﹪, 70.27﹪±0.98﹪, P < 0.05) and the expression of caspase-12 protein (13.05±0.21 vs 20.23±0.19, 33.17±0.32, 41.52±0.21, P < 0.05) were increased. The difference was statistically significant (all P < 0.05) .

Conclusion

Low intensity ultrasound may induce the increase of caspase-12 protein expression and decrease of survivin protein expression in HeLa tumor cells, thus leading to apoptosis of HeLa tumor cells.

Key words: Caspase-12, Survivin, Low intensity ultrasound, HeLa cells, Apoptosis, Cervical cancer

表1 Hela肿瘤细胞活力、细胞存活率及凋亡率比较（﹪， ± s）

分组	实验重复次数	细胞活力	细胞存活率	细胞凋亡率
阴性对照组	3	99.23±1.56	99.91±1.51	4.23±1.21
0.5 W/cm²超声干预组	3	80.52±1.72^a	76.69±1.92^a	24.16±1.91^a
1.3 W/cm²超声干预组	3	54.31±1.69^a	52.57±1.63^a	48.34±1.66^a
2.0 W/cm²超声干预组	3	27.75±1.26^a	29.81±1.22^a	70.27±0.98^a
F值		1185.004	1087.433	1121.252
P值		< 0.001	< 0.001	< 0.001

表1 Hela肿瘤细胞活力、细胞存活率及凋亡率比较（﹪， ± s）

分组	实验重复次数	细胞活力	细胞存活率	细胞凋亡率
阴性对照组	3	99.23±1.56	99.91±1.51	4.23±1.21
0.5 W/cm²超声干预组	3	80.52±1.72^a	76.69±1.92^a	24.16±1.91^a
1.3 W/cm²超声干预组	3	54.31±1.69^a	52.57±1.63^a	48.34±1.66^a
2.0 W/cm²超声干预组	3	27.75±1.26^a	29.81±1.22^a	70.27±0.98^a
F值		1185.004	1087.433	1121.252
P值		< 0.001	< 0.001	< 0.001

图1 光学显微镜下观察超声干预对细胞形态的影响（苏木精-伊红染色，×40）

图2 透射电镜下观察超声干预对Hela肿瘤细胞结构（×600）

图3 低强度超声干预对Hela肿瘤细胞中caspase-12、survivin蛋白表达的影响

表2 不同超声强度干预对Hela肿瘤细胞中caspase-12蛋白和survivin蛋白表达水平的影响（ ± s）

分组	实验重复次数	ROS含量	caspase-12蛋白	survivin蛋白
阴性对照组	3	11.21±0.45	13.05±0.21	51.19±0.21
0.5 W/cm²超声干预组	3	24.34±1.23^a	20.23±0.19^a	43.46±0.34^a
1.3 W/cm²超声干预组	3	38.26±2.47^a	33.17±0.32^a	25.28±0.29^a
2.0 W/cm²超声干预组	3	52.18±1.56^a	41.52±0.21^a	18.32±0.31^a
F值		365.400	8634.002	8034.149
P值		< 0.001	< 0.001	< 0.001

表2 不同超声强度干预对Hela肿瘤细胞中caspase-12蛋白和survivin蛋白表达水平的影响（ ± s）

分组	实验重复次数	ROS含量	caspase-12蛋白	survivin蛋白
阴性对照组	3	11.21±0.45	13.05±0.21	51.19±0.21
0.5 W/cm²超声干预组	3	24.34±1.23^a	20.23±0.19^a	43.46±0.34^a
1.3 W/cm²超声干预组	3	38.26±2.47^a	33.17±0.32^a	25.28±0.29^a
2.0 W/cm²超声干预组	3	52.18±1.56^a	41.52±0.21^a	18.32±0.31^a
F值		365.400	8634.002	8034.149
P值		< 0.001	< 0.001	< 0.001

1	Feuerborn R, Brodde M, Schmidt H, et al. High density lipoprotein (HDL)-associated sphingosine 1-phosphate (S1P) inhibits macrophage apoptosis by stimulating STAT3 activity and survivin expression[J]. Atherosclerosis, 2017, 257:29-37.
2	Shi MF, Liu BZ, Liu GH, et al. Low intensity-pulsed ultrasound induced apoptosis of human hepatocellular carcinoma cells in vitro [J]. Ultrasonics, 2016, 64:43-53.
3	Ye QS, Meng CD, Shen YN, et al. Caveolin-1 mediates low-intensity ultrasound-induced apoptosis via downregulation of signal transducer and activator of transcription 3 phosphorylation in laryngeal carcinoma cells[J]. Ultrasound Med Biol, 2016, 42(9):2253-2260.
4	Jahagirdar D, Gore CR, Patel H, et al. Induction of apoptotic death and cell cycle arrest in HeLa cells by extracellular factors of breast cancer cells[J]. Asian Pac J Cancer Prev, 19(12):3307-3316.
5	Chen L, Liu LP, Li YH, et al. Melatonin increases human cervical cancer HeLa cells apoptosis induced by cisplatin via inhibition of JNK/Parkin/mitophagy axis[J]. In Vitro Cell DevBiolAnim, 2018, 54(1):1-10.
6	Jin XD, Li FF, Liu BT, et al. Different mitochondrial fragmentation after irradiation with X-rays and Carbon ions in HeLacells and its influence on cellular apoptosis[J]. Biochem Biophys Res Commun, 2018, 500(4):958-965.
7	Tang ZY, Sheng MJ, Qi YX, et al. Metformin enhances inhibitive effects of carboplatin on HeLa cell proliferation and increases sensitivity to carboplatin by activating mitochondrial associated apoptosis signaling pathway[J]. Eur Rev Med PharmacolSci, 2018, 22(23):8104-8112.
8	Khanam R, Kumar R, Hejazi II, et al. Piperazine clubbed with 2-azetidinone derivatives suppresses proliferation, migration and induces apoptosis in human cervical cancer HeLa cells through oxidative stress mediated intrinsic mitochondrial pathway[J]. Apoptosis, 2018, 23(2):113-131.
9	Li X, Wu Y, Cao Y, et al. Comparison of three common DNA concentration measurement methods[J]. Anal Biochem, 2014, 45(1):18-24.
10	Yan WL, Lu J, Li GT, et al. Amidated scolopin-2 inhibits proliferation and induces apoptosis of Hela cells in vitro and in vivo[J]. Biotechnol Appl Biochem, 2018, 65(5):672-679.
11	Li LW, Na C, Tian SY, et al. Ellagic acid induces HeLa cell apoptosis via regulating signal transducer and activator of transcription 3 signaling[J]. ExpTher Med, 2018, 16(1):29-36.
12	Wang DD, Wu QX, Pan WJ, et al. A novel polysaccharide from the Sarcodonaspratus triggers apoptosis in Hela cells via induction of mitochondrial dysfunction[J]. Food Nutr Res, 2018, 62.
13	Wong JH, Sze SC, Ng TB, et al. Apoptosis and anti-cancer drug discovery: the power of medicinal fungi and plants[J]. Curr Med Chem, 2018, 25(40):5613-5630.
14	Chen L, Wu LY, Yang WX. Nanoparticles induce apoptosis via mediating diverse cellular pathways[J]. Nanomedicine, 2018, 13(22): 2939-2955.
15	Zhang MZ, Du HX, Huang ZX, et al. Thymoquinone induces apoptosis in bladder cancer cell via endoplasmic reticulum stress-dependent mitochondrial pathway[J]. Chem Biol Interact, 2018, 292:65-75.
16	Abdullah A, Ravanan P. Kaempferol mitigates endoplasmic reticulum stress induced cell death by targeting caspase 3/7[J]. Sci Rep, 2018, 8(1):10.
17	Chen Q, Kang J, Fu CY. The independence of and associations among apoptosis, autophagy, and necrosis[J]. Signal Transduct Target Ther, 2018, 3:18.
18	Song JL, Yuan CM, Yang JE, et al. Novel flavagline-like compounds with potent Fli-1 inhibitory activity suppress diverse types of leukemia[J]. FEBS J, 2018, 285(24):4631-4645.
19	Hernández-Reséndiz S, Muñoz-Vega M, Contreras WE, et al. Responses of endothelial cells towards ischemic conditioning following acute myocardial infarction[J]. Cond Med, 2018, 1(5):247-258.
20	Solano-Gálvez S, Abadi-Chiriti J, Gutiérrez-Velez L, et al. Apoptosis: activation and inhibition in health and disease[J]. Med Sci (Basel), 2018, 6(3):54.

[1]	张刚, 秦勇, 黄超, 薛震, 吕松岑. 基于骨关节炎软骨细胞表型转化的新兴治疗靶点[J/OL]. 中华关节外科杂志(电子版), 2024, 18(03): 352-362.
[2]	王帆, 余辉, 谢佳乐, 许焕焕, 马瑞, 依日夏提·艾海提, 许珂, 许鹏. 成纤维样滑膜细胞在类风湿关节炎发病机制中的作用[J/OL]. 中华关节外科杂志(电子版), 2024, 18(02): 225-230.
[3]	钟雅雯, 王煜, 王海臻, 黄莉萍. 肌苷通过抑制线粒体通透性转换孔开放缓解缺氧/复氧诱导的人绒毛膜滋养层细胞凋亡[J/OL]. 中华妇幼临床医学杂志(电子版), 2024, 20(05): 525-533.
[4]	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/OL]. 中华妇幼临床医学杂志(电子版), 2023, 19(05): 540-549.
[5]	孙鸿坤, 艾虹, 陈正. 内质网应激介导的牙周炎骨改建失衡的研究进展[J/OL]. 中华口腔医学研究杂志(电子版), 2024, 18(04): 211-218.
[6]	罗远杰, 杨靖梅, 孟姝, 敖逸博, 申道南. 槲皮素防治口腔疾病的研究进展[J/OL]. 中华口腔医学研究杂志(电子版), 2024, 18(02): 117-122.
[7]	孟令凯, 李大勇, 王宁, 王桂明, 张炳南, 李若彤, 潘立峰. 袖状胃切除术对肥胖伴2型糖尿病大鼠的作用及机制研究[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(06): 638-642.
[8]	郑俊, 吴杰英, 谭海波, 郑安全, 李腾成. EGFR-MEK-TZ三联合分子的构建及其对去势抵抗性前列腺癌细胞增殖与凋亡的影响[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(05): 503-508.
[9]	黄程鑫, 陈莉, 刘伊楚, 王水良, 赖晓凤. OPA1 在乳腺癌组织的表达特征及在ER阳性乳腺癌细胞中的生物学功能研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(05): 275-284.
[10]	季加翠, 孙春斌, 罗恩丽. 姜黄素通过调节NF-κB/NLRP3通路减轻LPS诱导小胶质细胞神经炎症损伤[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(04): 193-203.
[11]	王颖, 吴德平, 刘煜, 刘国栋. miR-9-5p下调CXCR4减轻创伤性脑损伤大鼠的神经炎症和细胞凋亡[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(02): 65-72.
[12]	杜霞, 马梦青, 曹长春. 造影剂诱导的急性肾损伤的发病机制及干预靶点研究进展[J/OL]. 中华肾病研究电子杂志, 2024, 13(05): 279-282.
[13]	李景德, 张保艳, 卢培刚, 李博. 法舒地尔对大鼠急性脊髓损伤后神经细胞凋亡和BCL-2蛋白表达水平的影响[J/OL]. 中华神经创伤外科电子杂志, 2024, 10(02): 65-70.
[14]	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/OL]. 中华脑科疾病与康复杂志(电子版), 2023, 13(05): 264-270.
[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/OL]. 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

Effects and mechanisms of low intensity ultrasound-induced apoptosis of HeLa tumor 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

Effects and mechanisms of low intensity ultrasound-induced apoptosis of HeLa tumor cells