可利霉素对胰腺癌细胞生物学功能的影响

白丽娜; 刘颖; 唐春晓; 朴红心; 林贞花; 杨万山; 金爱花

doi:10.3969/j.issn.1001-5256.2022.12.020

可利霉素对胰腺癌细胞生物学功能的影响

DOI: 10.3969/j.issn.1001-5256.2022.12.020

白丽娜¹,
刘颖¹,
唐春晓¹,
朴红心^2a,
林贞花¹,
杨万山¹,
金爱花^{1, 2b, , ,}

1.
延边大学医学院肿瘤研究中心，吉林延吉 133000
2a.
延边大学附属医院感染性疾病科，吉林延吉 133000
2b.
延边大学附属医院消化内科，吉林延吉 133000

基金项目:

吉林省教育厅科学技术研究项目 (JJKH20220554KJ);

吉林省卫生健康科技能力提升项目 (2021JC063)

利益冲突声明：本研究不存在研究者及与公开研究成果有关的利益冲突。

作者贡献声明：白丽娜负责实验设计、实施、论文撰写及数据分析；刘颖、唐春晓负责实验设计、实施及论文修改；杨万山负责实验设计与论文修改；金爱花、朴红心、林贞花负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
金爱花，drjinah@163.com

计量
- 文章访问数: 1401
- HTML全文浏览量: 935
- PDF下载量: 49
- 被引次数: 0
出版历程
- 收稿日期: 2022-05-26
- 录用日期: 2022-07-20
- 出版日期: 2022-12-20

Mechanism of carrimycin in regulating the biological function of pancreatic cancer cells

1.
Cancer Research Center, School of Medicine, Yanbian University, Yanji, Jilin 133000, China
2a.
Department of Infectious Diseases, The Affiliated Hospital of Yanbian University, Yanji, Jilin 133000, China
2b.
Department of Gastroenterology, The Affiliated Hospital of Yanbian University, Yanji, Jilin 133000, China

Research funding:

Science and Technology Research Project of Jilin Province Department of Education (JJKH20220554KJ);

Heatth Science and Technology Capacity Improvement Project of Jilin Province (2021JC063)

More Information

Corresponding author: JIN Aihua, drjinah@163.com (ORCID: 0000-0002-3405-8097)

摘要

摘要: 目的探讨可利霉素(Carrimycin)对胰腺癌细胞生物学功能的影响。方法胰腺癌细胞MIA PaCa-2、BxPC-3、Panc-1和PATU 8988以0(对照组)、2、4、8和16 μmol/L的可利霉素处理24、48和72 h后，MTT法检测细胞活性；EdU细胞增殖实验检测可利霉素对胰腺癌细胞DNA复制能力的影响；集落形成实验检测可利霉素对胰腺癌细胞增殖能力的影响；流式细胞术分析可利霉素对胰腺癌细胞周期的影响；划痕愈合实验分析可利霉素对胰腺癌细胞迁移能力的影响；免疫蛋白印迹实验检测上皮细胞-间充质转化和细胞周期依赖性蛋白激酶抑制因子1A(P21)等标志物的表达水平；免疫荧光实验检测上皮间质转化相关标志物的表达水平。多组间比较采用方差分析，进一步两两比较采用LSD-t检验。结果与对照组相比，可利霉素可显著抑制MIA PaCa-2、BxPC-3、Panc-1和PATU 8988细胞的增殖活性，且呈药物浓度与时间依赖性(P值均＜0.01)；4、8、16 μmol/L可利霉素可显著降低PC细胞MIA PaCa-2(t值分别为2.378、4.984、18.970，P值均＜0.05)及BxPC-3(t值分别4.879、6.089、9.521，P值均＜0.01)的DNA复制能力；4、8、16 μmol/L可利霉素处理PC细胞14 d后，MIA PaCa-2(t值分别为5.889、11.240、15.840，P值均＜0.001)和BxPC-3(t值分别为6.717、15.800、18.850，P值均＜0.001)细胞的集落形成能力随着药物浓度的增加而明显减少。4、8、16 μmol/L可利霉素处理后MIA PaCa-2细胞G1期比例(t值分别为9.071、12.280、19.360，P值均＜0.000 1)及BxPC-3细胞G1期比例(t值分别为3.061、4.962、8.868，P值均＜0.05)均增加，MIA PaCa-2细胞S期比例(t值分别为2.316、4.165、5.562，P值均＜0.05)及BxPC-3细胞S期比例(t值分别为2.424、3.264、5.744，P值均＜0.05)均降低。Western Blot实验进一步证实，与对照组比较，周期相关蛋白P21在MIA PaCa-2(t值分别为5.437、6.453、8.799，P值均＜0.001)和BxPC-3细胞(t值分别为25.130、44.750、52.960，P值均＜0.000 1)中的表达水平随可利霉素用药浓度增加而逐渐上调。划痕愈合实验结果显示，0和4、8、16 μmol/L可利霉素处理12、24和48 h PC细胞MIA PaCa-2(P值均＜0.05)及BxPC-3(P值均＜0.05)的横向迁移能力明显减弱。Western blot实验结果显示，与对照组比较，4、8、16 μmol/L可利霉素处理后MIA PaCa-2(t值分别为2.388、4.899、5.819，P值均＜0.05)及BxPC-3细胞(t值分别为2.533、5.836、6.774，P值均＜0.05)中上皮标志物E-cadherin的表达明显上调，4、8、16 μmol/L可利霉素处理后MIA PaCa-2(t值分别为12.440、14.830、16.800，P值均＜0.000 1)及BxPC-3细胞(t值分别为5.039、5.893、7.725，P值均＜0.01)中间质标志物Snail的表达水平显著下调，4、8、16 μmol/L可利霉素处理后MIA PaCa-2(t值分别为3.105、7.752、11.200，P值均＜0.05) 及BxPC-3细胞(t值分别为2.555、4.883、9.153，P值均＜0.05)中间质标志物Vimentin的表达水平也显著下调。结论可利霉素可有效抑制胰腺癌细胞的增殖、迁移及上皮细胞-间充质转化进程，从而发挥其抗肿瘤的生物学活性。
- 胰腺肿瘤 /
- 细胞增殖 /
- 细胞运动 /
- 上皮-间质转化
Abstract: Objective To investigate the effect of carrimycin on the biological function of pancreatic cancer cells. Methods Pancreatic cancer cell lines MIA PaCa-2, BxPC-3, Panc-1, and PATU 8988 were treated with carrimycin at concentrations of 0 (control group), 2, 4, 8, and 16 μmol/L for 24, 48, and 72 hours. MTT assay was used to measure cell viability; EdU cell proliferation assay was used to observe the effect of carrimycin on DNA replication of pancreatic cancer cells; colony formation assay was used to observe the effect of carrimycin on the proliferation of pancreatic cancer cells; flow cytometry was used to analyze the effect of carrimycin on the cell cycle of pancreatic cancer cells; wound healing assay was used to analyze the effect of carrimycin on the migration of pancreatic cancer cells; Western blot was used to measure the expression levels of the markers such as epithelial-mesenchymal transition (EMT) and cell cycle-dependent protein kinase inhibitor 1A (P21); immunofluorescence assay were used to measure the expression levels of EMT-related markers. An analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results Compared with the control group, carrimycin significantly inhibited the proliferative activity of MIA PaCa-2, BxPC-3, Panc-1, and PATU 8988 cells in a concentration- and time-dependent manner (all P < 0.01); carrimycin at concentrations of 4, 8, and 16 μmol/L significantly reduced DNA replication in MIA PaCa-2 cells (t=2.378, 4.984, and 18.970, all P < 0.05) and BxPC-3 cells (t=4.879, 6.089, and 9.521, all P < 0.01); after treatment with carrimycin at concentrations of 4, 8, and 16 μmol/L, colony formation ability significantly decreased with the increase in drug concentration in MIA PaCa-2 cells (t=5.889, 11.240, and 15.840, all P < 0.001) and BxPC-3 cells (t=6.717, 15.800, and 18.850, all P < 0.001). After treatment with carrimycin at concentrations of 4, 8, and 16 μmol/L, there was a significant increase in the proportion of cells in G1 phase in MIA PaCa-2 cells (t=9.071, 12.280, and 19.360, all P < 0.0001) and BxPC-3 cells (t=3.061, 4.962, and 8.868, all P < 0.05), and there was a significant reduction in the proportion of cells in S phase in MIA PaCa-2 cells (t=2.316, 4.165, and 5.562, all P < 0.05) and BxPC-3 cells (t=2.424, 3.264, and 5.744, all P < 0.05). Western blot further demonstrated that compared with the control group, the expression level of the cell cycle-related protein P21 gradually increased with the increase in the concentration of carrimycin in MIA PaCa-2 cells (t=5.437, 6.453, and 8.799, all P < 0.001) and BxPC-3 cells (t=25.130, 44.750, and 52.960, all P < 0.000 1). Wound healing assay showed that after treatment for 12, 24, and 48 hours, carrimycin at concentrations of 0, 4, 8, and 16 μmol/L significantly reduced the lateral migration of MIA PaCa-2 cells (all P < 0.05) and BxPC-3 cells (all P < 0.05). Western blot showed that compared with the control group, carrimycin treatment at concentrations of 4, 8, and 16 μmol/L significantly upregulated the expression of the epithelial marker E-cadherin in MIA PaCa-2 cells (t=2.388, 4.899, and 5.819, all P < 0.05) and BxPC-3 cells (t=2.533, 5.836, and 6.774, all P < 0.05) and significantly downregulated the expression of the interstitial marker Snail in MIA PaCa-2 cells (t=12.440, 14.830, and 16.800, all P < 0.000 1) and BxPC-3 cells (t=5.039, 5.893, and 7.725, all P < 0.01), and it also significantly downregulated the expression of the interstitial marker Vimentin in MIA PaCa-2 cells (t=3.105, 7.752, and 11.200, all P < 0.05) and BxPC-3 cells (t=2.555, 4.883, and 9.153, all P < 0.05). Conclusion Carrimycin can effectively inhibit the proliferation, migration, and EMT process of pancreatic cancer cells, thereby exerting an antitumor biological activity.
- Pancreatic Neoplasms /
- Cell Proliferation /
- Cell Movement /
- Epithelial-Mesenchymal Transition

HTML全文

图 1 MTT法检测0、2、4、8和16 μmol/L的可利霉素对PC细胞系的细胞增殖活性。

注：**P＜0.01;***P＜0.001;****P＜0.000 1。

Figure 1. Cell viabilityof PC cells treated by Carrimycin for 0, 2, 4, 8 and 16 μmol/L was measured by MTT assay

下载: 全尺寸图片幻灯片

图 2 EdU及集落形成实验检测不同浓度的可利霉素对MIA PaCa-2和BxPC-3细胞增殖的抑制作用

注：a，EdU实验检测0和4、8、16 μmol/L可利霉素对PC细胞MIA PaCa-2和BxPC-3的DNA复制能力的影响；b，集落形成实验检测0和4、8、16 μmol/L可利霉素对PC细胞MIA PaCa-2和BxPC-3克隆形成能力的影响。*P＜0.05，**P＜0.01，***P＜0.001，****P＜0.000 1。

Figure 2. The inhibitory effects of different concentrations of Carrimycin on the proliferation of MIA PaCa-2 and BxPC-3 cells were detected by EdU and colony formation assay

下载: 全尺寸图片幻灯片

图 3 细胞周期和Western Blot实验检测不同浓度的可利霉素对MIA PaCa-2和BxPC-3细胞周期的影响

注：a，细胞周期实验检测0和4、8、16 μmol/L可利霉素对PC细胞MIA PaCa-2和BxPC-3细胞周期的影响；b，Western blot实验检测0和4、8、16 μmol/L可利霉素处理后PC细胞MIA PaCa-2和BxPC-3中P21蛋白的表达量。*P＜0.05，**P＜0.01，***P＜0.001，****P＜0.0001。

Figure 3. Cell cycle and Western Blot experiments were performed to examine the effects of different concentrations of Carrimycin on the cell cycle of MIA PaCa-2 and BxPC-3 cells

下载: 全尺寸图片幻灯片

图 4 不同浓度的可利霉素对MIA PaCa-2和BxPC-3细胞迁移及EMT进程的影响

注：a，划痕愈合实验检测0和4、8、16 μmol/L可利霉素对PC细胞MIA PaCa-2和BxPC-3迁移能力的影响；b，0、4、8、16 μmol/L可利霉素处理PC细胞MIA PaCa-2和BxPc-3细胞伤口愈合率；c，Western blot实验检测0和4、8、16 μmol/L可利霉素处理后PC细胞MIA PaCa-2和BxPC-3中EMT相关蛋白E-cadherin、Snail和Vimentin的表达水平；d，免疫荧光实验检测0和4、8、16 μmol/L可利霉素处理后PC细胞MIA PaCa-2和BxPC-3中E-cadherin和Vimentin的表达水平。*P＜0.05，**P＜0.01，***P＜0.001，****P＜0.000 1。

Figure 4. Effects of different concentrations of Carrimycin on migration and EMT progression of MIA PaCa-2 and BxPC-3 cells

下载: 全尺寸图片幻灯片

表 1 不同浓度的可利霉素对MIA PaCa-2细胞和BxPC-3细胞迁移的影响

Table 1. Effect of carrimycin at different concentrations on the migration of MIA PaCa-2 cells and BxPC-3 cells migration

组别	对照组	4 μmol/L	8 μmol/L	16 μmol/L
MIA PaCa-2细胞
12 h	15.23±2.84	11.97±1.90	8.58±1.05	2.53±0.64
24 h	25.08±1.98	21.11±2.23	14.08±2.48	5.36±1.49
48 h	28.31±1.53	23.63±2.06	17.93±1.12	12.06±1.79
BxPC-3细胞
12 h	18.15±2.15	14.54±1.91	7.61±2.01	5.80±1.53
24 h	38.28±1.74	31.80±3.45	19.50±2.64	15.08±1.14
48 h	50.75±2.35	39.71±2.64	29.50±2.47	24.89±2.31

下载: 导出CSV

参考文献(18)

[1]	CHEN W, ZHENG R, BAADE PD, et al. Cancer statistics in China, 2015[J]. CA Cancer J Clin, 2016, 66(2): 115-132. DOI: 10.3322/caac.21338.
[2]	General Office of National Health Commission. Standard for diagnosis and treatment of pancreatic cancer (2022 edition)[J]. J Clin Hepatol, 2022, 38(5): 1006-1015. DOI: 10.3969/j.issn.1001-5256.2022.05.007. 国家卫生健康委办公厅. 胰腺癌诊疗指南(2022年版)[J]. 临床肝胆病杂志, 2022, 38(5): 1006-1015. DOI: 10.3969/j.issn.1001-5256.2022.05.007.
[3]	ZHU H, WEI M, XU J, et al. PARP inhibitors in pancreatic cancer: molecular mechanisms and clinical applications[J]. Mol Cancer, 2020, 19(1): 49. DOI: 10.1186/s12943-020-01167-9.
[4]	HAN A, XU R, LIU Y, et al. HSDL2 acts as a promoter in pancreatic cancer by regulating cell proliferation and lipid metabolism[J]. Onco Targets Ther, 2021, 14: 435-444. DOI: 10.2147/OTT.S287722.
[5]	NEOPTOLEMOS JP, KLEEFF J, MICHL P, et al. Therapeutic developments in pancreatic cancer: current and future perspectives[J]. Nat Rev Gastroenterol Hepatol, 2018, 15(6): 333-348. DOI: 10.1038/s41575-018-0005-x.
[6]	HAO TY, HE WQ. Advances in metabolic engineering of macrolide antibiotics[J]. Chin J Biotech, 2021, 37(5): 1737-1747. DOI: 10.13345/j.cjb.200686. 郝天怡, 赫卫清. 大环内酯类抗生素代谢工程的研究进展[J]. 生物工程学报, 2021, 37(5): 1737-1747. DOI: 10.13345/j.cjb.200686.
[7]	JIN Y, ZUO HX, LI MY, et al. Anti-tumor effects of carrimycin and monomeric isovalerylspiramycin I on hepatocellular carcinoma in vitro and in vivo[J]. Front Pharmacol, 2021, 12: 774231. DOI: 10.3389/fphar.2021.774231.
[8]	LIANG SY, ZHAO TC, ZHOU ZH, et al. Anti-tumor effect of carrimycin on oral squamous cell carcinoma cells in vitro and in vivo[J]. Transl Oncol, 2021, 14(6): 101074. DOI: 10.1016/j.tranon.2021.101074.
[9]	ZHANG XY, HAO RH, LIU F, et al. Progress in Application of Rapamycin in Tumor Treatment[J]. Food Drug, 2018, 20(1): 65-70. DOI: 10.3969/j.issn.1672-979X.2018.01.016. 张晓元, 郝荣华, 刘飞, 等. 雷帕霉素在肿瘤治疗方面研究进展[J]. 食品与药品, 2018, 20(1): 65-70. DOI: 10.3969/j.issn.1672-979X.2018.01.016.
[10]	EDELMAN MJ, SHVARTSBEYN M. Epothilones in development for non—small-cell lung cancer: novel anti-tubulin agents with the potential to overcome taxane resistance[J]. Clin Lung Cancer, 2012, 13(3): 171-180. DOI: 10.1016/j.cllc.2011.02.005.
[11]	GIANNAKAKOU P, GUSSIO R, NOGALES E, et al. A common pharmacophore for epothilone and taxanes: molecular basis for drug resistance conferred by tubulin mutations in human cancer cells[J]. Proc Natl Acad Sci U S A, 2000, 97(6): 2904-2909. DOI: 10.1073/pnas.040546297.
[12]	LI YL, SUN J, HU X, et al. Epothilone B induces apoptosis and enhances apoptotic effects of ABT-737 on human cancer cells via PI3K/AKT/mTOR pathway[J]. J Cancer Res Clin Oncol, 2016, 142(11): 2281-2289. DOI: 10.1007/s00432-016-2236-y.
[13]	CUI J, ZHOU J, HE W, et al. Targeting selenoprotein H in the nucleolus suppresses tumors and metastases by Isovalerylspiramycin I[J]. J Exp Clin Cancer Res, 2022, 41(1): 126. DOI: 10.1186/s13046-022-02350-0.
[14]	LIU Y, QUAN Y. Relationship between inflammatory microenvironment and epithelial-mesenchymal transition and its significance in pancreatic carcinoma[J]. Chin J Clin Res, 2018, 31(12): 1619-1623. DOI: 10.13429/j.cnki.cjcr.2018.12.006. 刘宇, 全颖. 胰腺癌炎性微环境与细胞上皮间质转化的关系及意义[J]. 中国临床研究, 2018, 31(12): 1619-1623. DOI: 10.13429/j.cnki.cjcr.2018.12.006.
[15]	BAKIR B, CHIARELLA AM, PITARRESI JR, et al. EMT, MET, plasticity, and tumor metastasis[J]. Trends Cell Biol, 2020, 30(10): 764-776. DOI: 10.1016/j.tcb.2020.07.003.
[16]	YEUNG KT, YANG J. Epithelial-mesenchymal transition in tumor metastasis[J]. Mol Oncol, 2017, 11(1): 28-39. DOI: 10.1002/1878-0261.12017.
[17]	KONG HR, DAI SJ, CHEN H, et al. α-solanine inhibits epithelial-mesenchymal transition in trichostatin A-resistant human pancreatic cancer cells through the WNT signaling pathway[J]. J Hepatopancreatobiliary Surg, 2020, 32(2): 89-96. DOI: 10.11952/j.issn.1007-1954.2020.02.006. 孔鸿儒, 戴胜杰, 陈豪, 等. α-龙葵碱通过WNT信号通路抑制曲古抑菌素A-耐药人胰腺癌细胞的上皮-间质转化进程[J]. 肝胆胰外科杂志, 2020, 32(2): 89-96. DOI: 10.11952/j.issn.1007-1954.2020.02.006.
[18]	LIU JB, QI M, LI QQ, et al. The mechanism of cordycepin in inhibition of pancreatic cancer stem cells proliferation and metastasis[J]. Acta Pharm Sin, 2017, 52(9): 1404-1409. DOI: 10.16438/j.0513-4870.2017-0353. 刘建兵, 戚梦, 李巧琪, 等. 虫草素抑制胰腺癌干细胞增殖及转移的机制研究[J]. 药学学报, 2017, 52(9): 1404-1409. DOI: 10.16438/j.0513-4870.2017-0353.