基于肿瘤干细胞学说探究天花粉蛋白干预乳腺癌耐药株SP细胞的实验研究
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摘要
目的:研究天花粉蛋白(TCS)对乳腺癌紫杉醇耐药株侧群细胞(MCF-7/PTX-SP细胞)的诱导凋亡作用,从干细胞角度探讨其作用机理。方法:FACS分选MCF-7/PTX细胞中的SP细胞,流式细胞仪检测SP细胞、非SP细胞中CD~+_(44)CD~-_(24)细胞比例、软琼脂克隆形成实验对其进行鉴定;流式细胞仪检测不同浓度TCS对SP细胞的促凋亡作用;RT-PCR法检测不同浓度TCS对SP细胞内Bcl-2和Bax基因表达的影响。结果:非SP细胞及SP细胞中CD~+_(44)/CD~-_(24)细胞的比例分别为0.47%和59.14%;与非SP细胞相比,SP细胞具有更高的体外克隆形成能力;TCS对SP细胞具有凋亡诱导作用,随着药物浓度的增高,SP细胞的凋亡率也随之升高,差异有统计学意义(t1=-13.751,P<0.01;t2=-10.892,P<0.01;t3=-6.999,P<0.01);不同浓度TCS处理后SP细胞内Bax mRNA的表达量呈明显的剂量依赖性上升趋势(P<0.01),而Bcl-2 mRNA的表达变化无统计学差异(P>0.05)。各浓度组Bcl-2/Bax值呈减少趋势(P<0.01)。结论:天花粉蛋白对乳腺癌耐药株侧群细胞具有凋亡诱导作用,其机制可能与上调Bax基因的表达有关。
Objective:To study the apoptosis induction of TCS on MCF-7/PTX-SP cells and explore its action mechanism from the perspective of stem cell. Methods:SP cells were selected by FACS from MCF-7/PTX cells. The proportion of CD~+_(44)CD~-_(24) cells in SP cells and non-sp cells was detected by flow cytometry and the experiment of soft AGAR cloning was conducted to identify it. Flow cytometry detected the apoptosis of TCS on SP cells. RT-PCR detected the influence of TCS on Bcl-2 and Bax of SP cells. Results: The proportions of CD~+_(44)CD~-_(24) cells on NON-SP and SP cells were 0.47% and 59.14%. In vitro cloning of SP cells was significantly higher than that of non-sp cells. TCS had apoptotic induction effect on SP cells, and as the drug concentration increased, the apoptosis rate of SP cells increased, and the difference was statistically significant(t1=-13.751, P<0.01;t2=-10.892, P<0.01; t3=-6.999, P<0.01). The expression of Bax mRNA in SP cells was significantly increased in the TCS treated with different concentrations(P<0.01), while the expression changes of bcl-2 mRNA were not statistically significant(P>0.05). The bcl-2/Bax value of each concentration group decreased(P<0.01). Conclusion: TCS has apoptosis induction in the tumor resistant strain cells. The mechanism may be related to the increase of Bax gene expression.
Objective:To study the apoptosis induction of TCS on MCF-7/PTX-SP cells and explore its action mechanism from the perspective of stem cell. Methods:SP cells were selected by FACS from MCF-7/PTX cells. The proportion of CD~+_(44)CD~-_(24) cells in SP cells and non-sp cells was detected by flow cytometry and the experiment of soft AGAR cloning was conducted to identify it. Flow cytometry detected the apoptosis of TCS on SP cells. RT-PCR detected the influence of TCS on Bcl-2 and Bax of SP cells. Results: The proportions of CD~+_(44)CD~-_(24) cells on NON-SP and SP cells were 0.47% and 59.14%. In vitro cloning of SP cells was significantly higher than that of non-sp cells. TCS had apoptotic induction effect on SP cells, and as the drug concentration increased, the apoptosis rate of SP cells increased, and the difference was statistically significant(t1=-13.751, P<0.01;t2=-10.892, P<0.01; t3=-6.999, P<0.01). The expression of Bax mRNA in SP cells was significantly increased in the TCS treated with different concentrations(P<0.01), while the expression changes of bcl-2 mRNA were not statistically significant(P>0.05). The bcl-2/Bax value of each concentration group decreased(P<0.01). Conclusion: TCS has apoptosis induction in the tumor resistant strain cells. The mechanism may be related to the increase of Bax gene expression.
引文
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[20] 时百玲, 张玉柱, 周悦,等. 中药逆转乳腺癌多药耐药的研究概况及展望[J]. 上海中医药杂志, 2016(1):88-93.
[2] Tang Y, Liang J, Wu A, et al. Codelivery of Trichosanthin and Albendazole by Nano Self-Assembly for Overcoming Tumor Multidrug Resistance and Metastasis[M]. Acs Applied Materials & Interfaces, 2017.
[3] Chen Y, Zhang M, Jin H, et al. Prodrug-Like, PEGylated Protein Toxin Trichosanthin for Reversal of Chemoresistance[J]. Molecular Pharmaceutics, 2017, 14(5):1429.
[4] O’Brien SK, Chen L, Zhong W, et al.Breast cancer cells respond differentially to modulation of TGFβ2 signaling after exposure to chemotherapy or hypoxia[J]. Cancer Res, 2015, 75(21):4605-4616.
[5] Semenza GL. Regulation of the breast cancer stem cell phenotype by hypoxia-inducible factors[J]. Clin Sci(Lond),2015,129(12):1037-1045.
[6] Seo AN,Lee HJ,Kim EJ,et al.Expression of breast cancer stem cell markers as predictors of prognosis and response to trastuzumab in HER2-positive breast cancer[J]. Br J Cancer,2016(1):26.
[7] Lv X,Wang Y,Song Y, et al. Association between ALDH1+/CD133+ stem-like cells and tumor angiogenesis in invasive ductal breast carcinoma[J]. Oncol Lett, 2016, 11(3):1750-1756.
[8] Kida K,Ishikawa T,Yamada A,et al. Effect of ALDH1 on prognosis and chemoresistance by breast cancer subtype[J]. Breast Cancer Res Treat, 2016, 156(2): 261-269.
[9] Ruiz de Garibay G,Herranz C,Llorente A, et al. Lymphangioleiomyomatosis Biomarkers Linked to Lung Metastatic Potential and Cell Stemness[J]. PLoS One, 2015, 10(7):e0132546.
[10] 孙洪光, 杨振林, 程凯,等. CD+44CD-24/low乳腺癌干细胞分选鉴定及其多药耐药性研究[J]. 中华肿瘤防治杂志, 2013, 20(12):891-893.
[11] Zheng DW, Liao S, Zhu GC, et al. Side population cells and prog?ress in cancer stem cell research[J]. Zhong Nan Da Xue Xue Bao YiXue Ban,2014,39(5):525-531.
[12] Zhang HH, Xi HQ, Cai AZ, et al. Not all side population cells con?tain cancer stem-like cells in human gastric cancer cell lines[J].DigDis Sci,2013,58(1):132-139.
[13] Sun DX, Liao GJ, Liu KG,et al. Endosialin-expressing bone sarco?ma stem-like cells are highly tumor-initiating and invasive[J].MolMed Rep, 2015,12(4):5665-5670.
[14] Liang S, Sun K, Wang Y, et al. Role of Cyt-C/caspases-9,3, Bax/Bcl-2 and the FAS death receptor pathway inapoptosis induced by zinc oxide nanoparticles in humanaortic endothelial cells and the protective effect by alpha-lipoic acid[J]. Chem Biol Interact, 2016, 258(1):40-51.
[15] Li H, Lv B, Kong L, et al. Mediates resistance of rat pheochromocytoma cells to hypoxia-induced apoptosis via the Bax/Bcl-2/caspase-3 pathway[J]. Int J Mol Med, 2017,40(4): 1125-1133.
[16] 杨玉, 张蕾, 周海祥,等. 天花粉蛋白抗肿瘤机制研究进展[J]. 中医学报, 2017, 32(5):730-732.
[17] Zhang D, Chen B, Zhou J, et al. Low concentrations of trichosanthin induce apoptosis and cell cycle arrest via c-Jun N-terminal protein kinase/mitogen-activated protein kinase activation[J]. Molecular Medicine Reports, 2015, 11(1):349.
[18] 韩冰冰,李洁.天花粉蛋白对黑色素瘤细胞血管生成拟态及PI3K信号通路的影响[J].中国药理学通报,2013,29(10):1476-1477.
[19] 彭平平,黄利鸣,韩钰,等.重组天花粉蛋白高表达对人宫颈癌Caski细胞p73基因甲基化影响的机制研究[J].中国药理学通报,2013,29(9):1290-1293.
[20] 时百玲, 张玉柱, 周悦,等. 中药逆转乳腺癌多药耐药的研究概况及展望[J]. 上海中医药杂志, 2016(1):88-93.