2-脱氧-D-葡萄糖联合羟基喜树碱协同诱导乳腺癌细胞凋亡的作用
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摘要
目的探究2-脱氧-D-葡萄糖(2-deoxy-D-glucose, 2-DG)联合羟基喜树碱(hydroxycamptothecin, HCPT)对乳腺癌细胞抗肿瘤活性的影响及其机制。方法采用2-DG(0、1.25、2.5、5、10、20 mmol/L)、HCPT(0、5、10、20、40μmol/L)单独作用以及5 mmol/L 2-DG与各浓度HCPT联合作用于乳腺癌细胞MDA-MB-231与MCF-7,使用MTT法检测细胞增殖;溴化丙啶(propidium iodide,PI)检测5 mmol/L 2-DG、10μmol/L HCPT单独作用以及联合作用对MDA-MB-231细胞的凋亡作用;不同浓度2-DG作用于MDA-MB-231细胞6 h后,测定细胞内ATP含量变化;Western blot检测MDA-MB-231细胞Akt、P-Akt、Bcl-2/Bax、PARP、Caspase-8和Caspase-3蛋白的表达。结果 5 mmol/L 2-DG与HCPT联合具有协同作用,处理48 h结合指数(CI<1),合用组凋亡率均高于单用组(P<0.05),同时两者合用抑制Akt蛋白的磷酸化以及增加了Caspase-3蛋白的活化,增加了对PARP蛋白的剪切。结论 2-DG联合HCPT可以协同诱导乳腺癌细胞凋亡,其机制可能是抑制肿瘤细胞能量生成以及抑制Akt蛋白的磷酸化和增强Caspase-3蛋白的活性。
Objective To investigate the effect of 2-deoxy-d-glucose(2-DG) combined with hydroxycamptothecin(HCPT) on anti-tumor activity of breast cancer cells and its mechanism. Methods MDA-MB-231 and MCF-7 breast cancer cells were incubated with varying concentrations of 2-DG(0, 1.25, 2.5, 5, 10, 20 mmol/L), HCPT(0, 5, 10, 20, 40 μmol/L) and 2-DG(5 mmol/L) combined with HCPT. Cell viability was measured using the MTT assay; Propidium iodide(PI) detected the apoptosis of MDA-MB-231 cells by 5 mmol/L 2-DG, 10 μmol/L HCPT alone or in combination; MDA-MB-231 cells were treated with 2-DG(0, 2.5, 5, 10, 20 mmol/L) and the level of ATP was detected by ATP kit; the expression of Akt, p-Akt, Bcl-2/Bax, PARP, Caspase-8 and Caspase-3 proteins in MDA-MB-231 cells were measured by Western blot assay. Results The combination of 2-DG(5 mmol/L) and HCPT had a synergistic effect. The 48 h combination index(CI<1) was higher than that of the single-use group(P<0.05). At the same time, the combination of the two drugs inhibits the phosphorylation of Akt protein and increases the activation of Caspase-3 protein, thereby increasing the cleavage of PARP proteins. Conclusion The combination of 2-DG and HCPT can synergistically induce the apoptosis of breast cancer cells, which may be caused by inhibiting the energy generation of tumor cells, inhibiting the phosphorylation of Akt protein and enhancing the activity of caspase-3 protein.
Objective To investigate the effect of 2-deoxy-d-glucose(2-DG) combined with hydroxycamptothecin(HCPT) on anti-tumor activity of breast cancer cells and its mechanism. Methods MDA-MB-231 and MCF-7 breast cancer cells were incubated with varying concentrations of 2-DG(0, 1.25, 2.5, 5, 10, 20 mmol/L), HCPT(0, 5, 10, 20, 40 μmol/L) and 2-DG(5 mmol/L) combined with HCPT. Cell viability was measured using the MTT assay; Propidium iodide(PI) detected the apoptosis of MDA-MB-231 cells by 5 mmol/L 2-DG, 10 μmol/L HCPT alone or in combination; MDA-MB-231 cells were treated with 2-DG(0, 2.5, 5, 10, 20 mmol/L) and the level of ATP was detected by ATP kit; the expression of Akt, p-Akt, Bcl-2/Bax, PARP, Caspase-8 and Caspase-3 proteins in MDA-MB-231 cells were measured by Western blot assay. Results The combination of 2-DG(5 mmol/L) and HCPT had a synergistic effect. The 48 h combination index(CI<1) was higher than that of the single-use group(P<0.05). At the same time, the combination of the two drugs inhibits the phosphorylation of Akt protein and increases the activation of Caspase-3 protein, thereby increasing the cleavage of PARP proteins. Conclusion The combination of 2-DG and HCPT can synergistically induce the apoptosis of breast cancer cells, which may be caused by inhibiting the energy generation of tumor cells, inhibiting the phosphorylation of Akt protein and enhancing the activity of caspase-3 protein.
引文
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[2] MIN X,HENG H,YU HL,et al.Anticancer effects of 10-hydroxycamptothecin induce apoptosis of human osteosarcoma through activating caspase-3,p53 and cytochrome c pathways.Oncol Lett,2018,15(2):2459-2464.
[3] WU H,YU T,TIAN Y,et al.Enhanced liver-targeting via coadministration of 10-Hydroxycamptothecin polymeric micelles with vinegar baked Radix Bupleuri.Phytomedicine,2018,44:1-8.
[4] WEI Y,LI C,ZHANG Y,et al.Hydroxycamptothecin mediates antiproliferative effects through apoptosis and autophagy in A549 cells.Oncol Lett,2018,15(5):6322-6328.
[5] CHEN F,ZHAO Y,PAN Y,et al.Synergistically enhanced therapeutic effect of a carrier-free hcpt/dox nanodrug on breast cancer cells through improved cellular drug accumulation.Mol Pharm,2015,12(7):2237-2244.
[6] SINGH S,PANDEY S,BHATT AN,et al.Chronic dietary administration of the glycolytic inhibitor 2-deoxy-d-glucose (2-dg) inhibits the growth of implanted ehrlich’s ascites tumor in mice.PLoS One,2015,10(7):e132089[2019-04-19].https://doi.org/10.1371/journal.pone.0132089.
[7] ZHANG Y,HUANG F,WANG J,et al.2-DG-Regulated RIP and c-FLIP Effect on Liver Cancer Cell Apoptosis Induced by TRAIL.Med Sci Monit,2015,21:3442-3448.
[8] PICCOLO MT,MENALE C,CRISPI S.Combined anticancer therapies:an overview of the latest applications.Anticancer Agents Med Chem,2015,15(4):408-422.
[9] DWARKANATH BS,ZOLZER F,CHANDANA S,et al.Heterogeneity in 2-deoxy-D-glucose-induced modifications in energetics and radiation responses of human tumor cell lines.Int J Radiat Oncol Biol Phys,2001,50(4):1051-1061.
[10] JHA B,POHLIT W.Effect of 2-deoxy-D-glucose on DNA double strand break repair,cell survival and energy metabolism in euoxic Ehrlich ascites tumour cells.Int J Radiat Biol,1992,62(4):409-415.
[11] 李其响,张配,刘浩.肿瘤细胞能量代谢特点及其研究进展.中国药理学通报,2017,33(11):1499-1502.
[12] LIU Z,SUN Y,HONG H,et al.3-bromopyruvate enhanced daunorubicin-induced cytotoxicity involved in monocarboxylate transporter 1 in breast cancer cells.Am J Cancer Res,2015,5(9):2673-2685.
[13] OSHIKATA A,MATSUSHITA T,UEOKA R.Enhancement of drug efflux activity via MDR1 protein by spheroid culture of human hepatic cancer cells.J Biosci Bioeng,2011,111(5):590-593.
[14] HSIANG YH,HERTZBERG R,HECHT S,et al.Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I.J Biol Chem,1985,260(27):14873-14878.
[15] MENG G,WANG W,CHAI K,et al.Combination treatment with triptolide and hydroxycamptothecin synergistically enhances apoptosis in A549 lung adenocarcinoma cells through PP2A-regulated ERK,p38 MAPKs and Akt signaling pathways.Int J Oncol,2015,46(3):1007-1017.
[16] LIU F,LIU Y,LIU X,et al.Inhibition of IGF1R enhances 2-deoxyglucose in the treatment of non-small cell lung cancer.Lung Cancer,2018,123:36-43.
[17] AHMAD IM,MUSTAFA EH,MUSTAFA NH,et al.2DG enhances the susceptibility of breast cancer cells to doxorubicin.Open Life Sciences,2010,5(6):739-748.
[18] CIRUELOS GE.Targeting the PI3K/AKT/mTOR pathway in estrogen receptor-positive breast cancer.Cancer Treat Rev,2014,40(7):862-871.
[19] POLIVKA JJ,JANKU F.Molecular targets for cancer therapy in the PI3K/AKT/mTOR pathway.Pharmacol Ther,2014,142(2):164-175.
[20] JANKU F,KASEB AO,TSIMBERIDOU AM,et al.Identification of novel therapeutic targets in the PI3K/AKT/mTOR pathway in hepatocellular carcinoma using targeted next generation sequencing.Oncotarget,2014,5(10):3012-3022.
[21] SATHE A,NAWROTH R.Targeting the PI3K/AKT/mTOR Pathway in Bladder Cancer.Methods Mol Biol,2018,1655:335-350.