全反式维甲酸对小鼠黑色素瘤B16F10细胞生物学行为的影响

郑先珍; 姚秋娥; 石三军; 李欣; 卢来春; 赵语

doi:10.16016/j.1000-5404.201707065

您当前的位置：

首页 >

文章列表页 >

全反式维甲酸对小鼠黑色素瘤B16F10细胞生物学行为的影响

基础医学 | 更新时间：2026-01-09

- 全反式维甲酸对小鼠黑色素瘤B16F10细胞生物学行为的影响
- Effects of all-trans retinoic acid on biological behaviors in murine melanoma cell line B16F10
- 第三军医大学学报 2017年39卷第22期页码：2169-2175
- 作者机构：
  
  1.401331 重庆，重庆医科大学附属大学城医院药学部
  2.400042 重庆，第三军医大学大坪医院野战外科研究所药剂科
  3.400038 重庆，第三军医大学药学院教学实验中心
- 作者简介：
  
  [ 赵语, E-mail: 630175584@qq.com ZHAO Yu, E-mail: 630175584@qq.com
- 基金信息：
- DOI：10.16016/j.1000-5404.201707065
  中图分类号： R730.23;R739.5;R986
- 收稿：2017-07-08，
  
  修回：2017-08-09，
  
  纸质出版：2017-11-30
- 稿件说明：
移动端阅览
郑先珍, 姚秋娥, 石三军, 等. 全反式维甲酸对小鼠黑色素瘤B16F10细胞生物学行为的影响[J]. 第三军医大学学报, 2017,39(22):2169-2175.

Xianzhen ZHENG, Qiu'e YAO, Sanjun SHI, et al. Effects of all-trans retinoic acid on biological behaviors in murine melanoma cell line B16F10[J]. Journal of Third Military Medical University, 2017, 39(22): 2169-2175.
郑先珍, 姚秋娥, 石三军, 等. 全反式维甲酸对小鼠黑色素瘤B16F10细胞生物学行为的影响[J]. 第三军医大学学报, 2017,39(22):2169-2175. DOI： 10.16016/j.1000-5404.201707065.

Xianzhen ZHENG, Qiu'e YAO, Sanjun SHI, et al. Effects of all-trans retinoic acid on biological behaviors in murine melanoma cell line B16F10[J]. Journal of Third Military Medical University, 2017, 39(22): 2169-2175. DOI： 10.16016/j.1000-5404.201707065.

摘要

目的

探讨全反式维甲酸(all-trans retinoic acid

ATRA)对小鼠黑色素瘤B16F10细胞体外增殖、迁移、凋亡、致瘤性的影响及其初步作用机制。

方法

采用MTT法检测ATRA对小鼠黑色素瘤细胞B16F10和小鼠成纤维细胞L929增殖能力的影响。显微镜下观察给药后两种细胞的形态变化，应用划痕实验和Transwell实验检测ATRA对B16F10细胞迁移能力的影响，分别采用DAPI染色和Annexin V-FITC/PI染色观察B16F10细胞凋亡情况，JC-1染色实验检测B16F10细胞线粒体膜电位，荧光分光光度计检测B16F10细胞中Caspase-9、Caspase-3的活化情况，利用体内成瘤实验观察ATRA对B16F10细胞致瘤性的影响。

结果

MTT结果显示ATRA呈浓度依赖性地抑制B16F10细胞和L929细胞增殖，但B16F10细胞的存活率明显低于L929细胞的存活率(

0.05)；分别以低、中、高3个浓度(6.25、16、40 μmol/L)的ATRA处理细胞24 h后，显微镜下可见B16F10细胞在中浓度时呈凋亡样改变，高浓度时细胞基本死亡，丧失正常形态；而L929细胞形态仅在高浓度时发生显著变化；划痕实验和Transwell实验提示B16F10细胞的体外迁移能力在中浓度即受到明显抑制(

0.05)；继续以中浓度(16 μmol/L)的ATRA作用B16F10细胞24 h，DAPI染色可见细胞核裂解，甚至可见凋亡小体；Annexin V-FITC/PI染色结合流式细胞仪测得细胞凋亡率为(37.27±4.42)%；JC-1染色实验提示线粒体膜电位明显降低；荧光分光光度计测得给药后Caspase-9、Caspase-3显著被活化，活性分别增加了(1.54±0.00)、(3.09±0.01)倍(

0.05)；成瘤实验结果显示ATRA处理后的B16F10细胞致瘤性显著降低(

0.01)，甚至消失。

结论

ATRA能显著抑制B16F10细胞体外增殖、迁移能力，并能通过降低线粒体膜电位，活化Caspase-9、Caspase-3蛋白诱导细胞凋亡，进而抑制B16F10致瘤性。

Abstract

Objective

To investigate the inhibitory effects of all-trans retinoic acid (ATRA) on the proliferation

migration

apoptosis and tumorigenicity of B16F10 cells and the preliminary mechanism

in vitro

and

in vivo

Methods

The effect of ATRA on the proliferation of murine melanoma cell line B16F10 and mouse fibroblast cell line L929 was detected with MTT assay. The morphological changes were observed in the 2 cell lines after treatment under an inverted microscope. Migration ability of B16F10 cells after ATRA treatment was measured by wound healing assay and Transwell chamber assay. The apoptosis of B16F10 cells was analyzed with the use of DAPI staining and Annexin V-FITC/PI staining. The effect of ATRA on the mitochondrial membrane potential and the expression of Caspase-9 and Caspase-3 in the B16F10 cells were detected by JC-1 staining and fluorescence spectrophotometry respectively. Tumorigenicity

in vivo

was evaluated by tumor formation in C57BL

/6 mice.

Results

ATRA exerted inhibitory effects on the proliferation in B16F10 and L929 cells in dose-dependent manners

but the survival rate was significantly lower in the B16F10 cells than the L929 cells (

0.05). After the 2 cell lines were treated with ATRA at low

moderate and high doses (6.25

16 and 40 μmol/L) respectively for 24 h

the B16F10 cells displayed apoptosis-like changes under moderate dose

and almost all died under high dose

without normal cellular morphology

while

the L929 cells only had morphological changes under high dose of ATRA. Wound healing and Transwell chamber test showed that moderate dose of ATRA (16 μmol/L) significantly inhibited the migration and invasion abilities in B16F10 cells

and the treatment for 24 h induced cell apoptosis

confirmed by nuclear cleavage and apoptotic bodies after DAPI staining

with a apoptotic rate of (37.27±4.42)%. The mitochondrial membrane potential was reduced while the expression levels of Caspase-9 and Caspase-3 were increased by 1.54±0.00 and 3.09±0.01 times respectively (

0.05). ATRA treatment also resulted in decreased and even no tumorigenicity of B16F10 cells (

0.001).

Conclusion

ATRA treatment significantly decreases the proliferation and migration of B16F10 cells

and further inhibits tumorigenicity through decreasing mitochondrial potential and activating Caspase-9 and Caspase-3 to promote cell apoptosis.

关键词

Keywords

references

李欣, 石三军, 周敏, 等.酪氨酸激酶抑制剂达沙替尼抗黑色素瘤的体外研究[J].第三军医大学学报, 2016, 38(23):2479-2485.DOI: 10.16016/j.1000-5404.20160 6059.

LI X, SHI S J, ZHOU M, et al . Effect of Tyrosine kinase inhibitor dasatinib on melanoma cell lines in vitro [J ] . J Third Mil Med Univ, 2016, 38(23): 2479-2485. DOI: 10.16016/j.1000-5404.201606059..

SHI S, ZHOU M, LI X, et al . Synergistic active targeting of dually integrin αvβ3/CD44-targeted nanoparticles to B16F10 tumors located at different sites of mouse bodies[J ] . J Control Release, 2016, 235: 1-13. DOI: 10.1016/j.jconrel.2016.05.050.

DOU J, PAN M, WEN P, et al . Isolation and identification of cancer stem-like cells from murine melanoma cell lines[J ] .Cell Mol Immunol, 2007, 4(6): 467-472.

JOHNSON D E, REDNER R L. An atractive future for differentiation therapy in AML[J]. Blood Rev, 2015, 29(4): 263-268. DOI: 10.1016/j.blre.2015.01.002.

PAN J,WANG Q, TU P. A Topical medication of All-Trans Retinoic acid reduces sebum excretion rate in patients with forehead acne[J]. Am J Ther, 2017, 24(2): e207-e212. DOI:10.1097/MJT.0000000000000390.

曾维根, 胡攀, 王佳妮, 等.全反式维甲酸对乳腺癌干细胞的抑制作用[J].中华肿瘤杂志, 2013, 35(2): 89-93. DOI:10.3760/cma.j.issn.0253-3766.2013.02.003.

ZENG W G, HU P, WANG J N, et al . All-trans retinoic acid effectively inhibits breast cancer stem cells growth in vitro [J ] . Chin J Oncol, 2013, 35(2): 89-93. DOI:10.3760/cma.j.issn.0253-3766.2013.02.003..

LIM Y C, KANG H J, KIM Y S, et al . All-trans-retinoic acid inhibits growth of head and neck cancer stem cells by suppression of Wnt/β-catenin pathway[J ] . Eur J Cancer, 2012, 48(17): 3310-3318. DOI:10.1016/j.ejca.2012.04.013.

REJI R M, SIDDIKUZZAMAN. ATRA entrapped in DSPC liposome enhances anti-metastasis effect on lung and liver during B16F10 cell line metastasis in C57BL6 mice[J]. Anti Cancer Agents Med Chem, 2017, 17(6): 875-884. DOI: 10.2174/1871520616666160927103458.

DOU J, HE X, LIU Y, et al . Effect of downregulation of ZEB1 on vimentin expression, tumour migration and tumourigenicity of melanoma B16F10 cells and CSCs[J ] . Cell Biol Int, 2014, 38(4): 452-461. DOI: 10.1002/cbin.10223.

FRIEDMAN M D, JEEVAN D S, TOBIAS M, et al . Targeting cancer stem cells in glioblastoma multiformeusing mTOR inhibitors and the differentiating agent all-trans retinoic acid[J ] . Oncol Rep, 2013, 30(4): 1645-1650. DOI:10.3892/or.2013.2625.

ASGARI M M, BRASKY T M, WHITE E. Association of vitamin A and carotenoid intake with melanoma risk in a large prospective cohort[J]. J Invest Dermatol, 2012, 132(6): 1573-1582. DOI:10.1038/jid.2012.21.

YIN W, SONG Y, LIU Q, et al . Topical treatment of all-trans retinoic acid inhibits murinemelanoma partly by promoting CD8+ T-cell immunity[J ] . Immunology, 2017.[Epub ahead of print ] . DOI: 10.1111/imm.12768.

SENGUPTA S, RAY S, CHATTOPADHYAY N, et al . Effect of retinoic acid on integrin receptors of B16F10 melanoma cells[J ] . J Exp Clin Cancer Res, 2000, 19(1): 81-87.

周勇, 杨洁, 赵华, 等.全反式维甲酸对恶性黑色素瘤A375细胞增殖及Fas蛋白表达的影响[J].四川大学学报(医学版), 2010, 41(3): 464-466.

ZHOU Y, YANG J, ZHAO H, et al . Effect of all-trans retinoic acid on the proliferation of and Fas protein expression in human malignant melanoma A375 cells[J ] . J Sichuan Univ (Med Sci Edi), 2010, 41(3): 464-466.

ZHANG H, SATYAMOORTHY K, HERLYN M, et al . All-trans retinoic acid (atRA) differentially induces apoptosis in matched primary and metastatic melanoma cells-a speculation on damage effect of atRA via mitochondrial dysfunction andcell cycle redistribution[J ] . Carcinogenesis, 2003, 24(2): 185-191..

潘猛, 文萍, 窦骏, 等.从鼠黑色素瘤细胞系中分离和鉴定癌干细胞样细胞[J].生物技术通讯, 2008, 19(4): 500-502.DOI: 10.3969/j.issn.1009-0002.2008.04.006.

PAN M, WEN P, DOU J, et al . Isolation and identification of cancer stem like cells from murine melanoma cell lines[J ] . Lett Biotech, 2008, 19(4): 500-502. DOI: 10.3969/j.issn.1009-0002.2008.04.006.

浏览量

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

LINC00525靶向miR-338-3p促进胃癌细胞凋亡和降低其耐药性

TCEAL7基因对结直肠癌Caco2细胞的增殖、迁移、侵袭及凋亡的影响

下调Notch3表达对骨肉瘤细胞生物学特性的影响

SOX15基因对Caco2结肠癌细胞增殖、凋亡及迁移侵袭的影响

SERPINE1对结肠癌细胞增殖、迁移、侵袭及凋亡的影响