PP242对人晶状体上皮细胞凋亡的诱导及凋亡相关蛋白Bax表达的影响
|
摘要
目的研究PP242对人晶状体上皮细胞(HLECs)凋亡的诱导及凋亡蛋白Bax表达的影响。方法用不同浓度PP242处理人晶状体上皮细胞系SRA01/04,分别用浓度为0、250、500、750、1 000 nmol/L的PP242处理HLECs 48 h后,Annexin V/PI双染流式细胞仪检测细胞凋亡率;采用实时定量PCR及Western blotting方法检测Bax的m RNA及蛋白的表达。结果 250、500、750和1 000 nmol/L PP242对人晶状体上皮细胞系SRA01/04作用48 h后,早期凋亡率分别为7.20%±0.36%、13.77%±0.21%、16.31%±0.20%和18.67%±0.49%,与对照组(0 nmol/L)差异有统计学意义(P<0.05)。Western blotting结果显示,随着PP242浓度的增加,Bax蛋白表达量增加,与对照组(0 nmol/L)比较差异有统计学意义(P<0.05);实时定量PCR结果显示,Bax m RNA表达量随PP242药物浓度的增加而增加,相对定量m RNA结果分别为2.157±0.125、3.733±0.302、5.596±0.261和7.436±0.167,与对照组(0 nmol/L)比较差异有统计学意义(P<0.05)。结论 PP242可以诱导HLECs的凋亡,增加其凋亡蛋白Bax的表达。
Objective To study the effect of PP242 on apoptosis and the expression of the apoptosis protein Bax in human lens epithelial cells(HLECs). Methods Immortal HLECs SRA01/04 were cultured and treated with different concentrations(0,250,500,750,or 1 000 nmol/L) of PP242. Forty-eight hours after PP242 treatment,the apoptotic rate of cells in each group was examined with flow cytometry(FCA,Annexin V + PI staining). Using reverse transcription quantitative polymerase chain reaction and Western blotting,the m RNA and protein levels of Bax were examined. Results Forty-eight hours after treatment with 250,500,750,or 1 000 nmol/L PP242,the apoptotic rate of HLECs was 7.20%± 0.36%,13.77%± 0.21%,16.31%± 0.20%,or 18.67%± 0.49%,respectively. Apoptotic rate in the control group was significantly different(P < 0.05) than that in the treatment groups. Results of the Western blotting revealed that the level of Bax was significantly increased. Further,the results of reverse-transcription quantitative PCR showed that expression of Bax m RNA increased in the treatment groups. The relative quantification of Bax m RNA was 2.157±0.125,3.733±0.302,5.596±0.261,7.436±0.167. Compared with the control group,the difference was statistically significant(P < 0.05). Conclusion PP242 induces the expression of Bax and promotes apoptosis in cultured HLECs.
Objective To study the effect of PP242 on apoptosis and the expression of the apoptosis protein Bax in human lens epithelial cells(HLECs). Methods Immortal HLECs SRA01/04 were cultured and treated with different concentrations(0,250,500,750,or 1 000 nmol/L) of PP242. Forty-eight hours after PP242 treatment,the apoptotic rate of cells in each group was examined with flow cytometry(FCA,Annexin V + PI staining). Using reverse transcription quantitative polymerase chain reaction and Western blotting,the m RNA and protein levels of Bax were examined. Results Forty-eight hours after treatment with 250,500,750,or 1 000 nmol/L PP242,the apoptotic rate of HLECs was 7.20%± 0.36%,13.77%± 0.21%,16.31%± 0.20%,or 18.67%± 0.49%,respectively. Apoptotic rate in the control group was significantly different(P < 0.05) than that in the treatment groups. Results of the Western blotting revealed that the level of Bax was significantly increased. Further,the results of reverse-transcription quantitative PCR showed that expression of Bax m RNA increased in the treatment groups. The relative quantification of Bax m RNA was 2.157±0.125,3.733±0.302,5.596±0.261,7.436±0.167. Compared with the control group,the difference was statistically significant(P < 0.05). Conclusion PP242 induces the expression of Bax and promotes apoptosis in cultured HLECs.
引文
[1]LEISCHING GR,LOOS B,BOTHA MH,et al.The role of m TOR during cisplatin treatment in an in vitro and ex vivo model of cervical cancer[J].Toxicology,2015,33(5):72-78.DOI:10.1016/j.tox.2015.07.010.
[2]ONO A,OIKE R,OKUHASHI Y,et al.Comparative effects of PP242and rapamycin on m TOR signalling and NOTCH signalling in leukemia cells[J].Anticancer Res,2013,33(3):809-813.
[3]白雪,冯浩,张柳,等.PP242对人晶状体上皮细胞凋亡相关蛋白Bcl-2表达的影响[J].中国医科大学学报,2016,45(4):324-327.DOI:10.12007/j.issn.0258-4646.2016.04.009.
[4]MARTINEZ G,DE IONGH RU.The lens epithelium in ocular health and disease[J].Int J Biochem Cell Biol,2010,42(12):1945-1963.DOI:10.1016/j.biocel.2010.09.012.
[5]MILANI P,PIERRO L,PECE A,et al.Retinal photoreceptor focal disruption secondary to accidental Nd:YAG laser exposure[J].Int Ophthalmol,2011,31(5):409-412.DOI:10.1007/s10792-011-9469-1.
[6]POLIVKA J,JANKU F.Molecular targets for cancer therapy in the PI3K/AKT/m TOR pathway[J].Pharmacol Ther,2013,142(2):164-175.DOI:10.1016/j.pharmthera.2013.12.004.
[7]浦利军,杨勤.雷帕霉素对大鼠糖尿病性白内障晶状体上皮细胞凋亡蛋白Bcl-2/Bax表达的影响[J].中国生化药物杂志,2014,34(9):10-13.
[8]WANG BT,DUCKER GS,BARCZAK AJ,et al.The mammalian target of rapamycin regulates cholesterol biosynthetic gene expression and exhibits a rapamycin-resistant transcriptional profile[J].Proc Natl Acad Sci USA,2011,108(37):15201-15206.DOI:10.1073/pnas.1103746108.
[9]ZENG ZH,SHI YX,TSAO T,et al.Targeting of m TORC1/2 by the m TOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment[J].Blood,2012,120(13):2679-2689.DOI:10.1182/blood-2011-11-393934.
[10]贾松柏,石晶明,陈翾,等,紫外线对人晶状体上皮细胞凋亡的诱导剂Bcl-2,Bax基因的影响[J].中南大学学报,2012,37(7):730-735.
[11]LEE KH,PARK E,LEE HJ,et al.Effects of daily quercetin-rich supplementation on cardiometabolic risks in male smokers[J].Nutr Res Pract,2011,5(1):28-33.DOI:10.4162/nrp.2011.5.1.28.
[12]MILACKOVA I,PRNOVA MS,MAJEKOVA M,et al.2-Chloro-1,4-naphthpquinone derivative of quercetin as an inhibitoe of aldose reductase and anti-inflammatory agent[J].Enzyme Inhib Med Chem,2014,30(1):107-113.DOI:10.3109/14756366.2014.892935.
[13]ZHANG XA,ZHANG S,YIN Q,et al.Quercetin induces human colon cancer cells apoptosis by inhibiting the nuclear factor-kappa B pathway[J].Pharmacoqn Mag,2015,11(42):404-409.DOI:10.4103/0973-1296.153096.
[14]OZYURT H,CEVIK O,ZGEN Z,et al.Quercetin protects radiationinduced DNA damage and apoptosis in kidney and bladder tissues of rats[J].Free Radic Res,2014,48(10):1247-1255.DOI:10.1016/S0167-8140(15)31929-0.
[15]LI Y,JIA Y,ZHOU J,et al.Effect of methionine sulfoxide reductase B1 silencing on high-glucose-induced apoptosis of human lens epithelial cells[J].Life Sci,2013,92(3):193-201.DOI:10.1016/j.lfs.2012.11.021.
[16]刘思源,康刚劲.不同浓度17β-雌二醇对雌性大鼠晶状体上皮细胞凋亡的影响[J].眼科新进展,2011,31(8):725-729.
[2]ONO A,OIKE R,OKUHASHI Y,et al.Comparative effects of PP242and rapamycin on m TOR signalling and NOTCH signalling in leukemia cells[J].Anticancer Res,2013,33(3):809-813.
[3]白雪,冯浩,张柳,等.PP242对人晶状体上皮细胞凋亡相关蛋白Bcl-2表达的影响[J].中国医科大学学报,2016,45(4):324-327.DOI:10.12007/j.issn.0258-4646.2016.04.009.
[4]MARTINEZ G,DE IONGH RU.The lens epithelium in ocular health and disease[J].Int J Biochem Cell Biol,2010,42(12):1945-1963.DOI:10.1016/j.biocel.2010.09.012.
[5]MILANI P,PIERRO L,PECE A,et al.Retinal photoreceptor focal disruption secondary to accidental Nd:YAG laser exposure[J].Int Ophthalmol,2011,31(5):409-412.DOI:10.1007/s10792-011-9469-1.
[6]POLIVKA J,JANKU F.Molecular targets for cancer therapy in the PI3K/AKT/m TOR pathway[J].Pharmacol Ther,2013,142(2):164-175.DOI:10.1016/j.pharmthera.2013.12.004.
[7]浦利军,杨勤.雷帕霉素对大鼠糖尿病性白内障晶状体上皮细胞凋亡蛋白Bcl-2/Bax表达的影响[J].中国生化药物杂志,2014,34(9):10-13.
[8]WANG BT,DUCKER GS,BARCZAK AJ,et al.The mammalian target of rapamycin regulates cholesterol biosynthetic gene expression and exhibits a rapamycin-resistant transcriptional profile[J].Proc Natl Acad Sci USA,2011,108(37):15201-15206.DOI:10.1073/pnas.1103746108.
[9]ZENG ZH,SHI YX,TSAO T,et al.Targeting of m TORC1/2 by the m TOR kinase inhibitor PP242 induces apoptosis in AML cells under conditions mimicking the bone marrow microenvironment[J].Blood,2012,120(13):2679-2689.DOI:10.1182/blood-2011-11-393934.
[10]贾松柏,石晶明,陈翾,等,紫外线对人晶状体上皮细胞凋亡的诱导剂Bcl-2,Bax基因的影响[J].中南大学学报,2012,37(7):730-735.
[11]LEE KH,PARK E,LEE HJ,et al.Effects of daily quercetin-rich supplementation on cardiometabolic risks in male smokers[J].Nutr Res Pract,2011,5(1):28-33.DOI:10.4162/nrp.2011.5.1.28.
[12]MILACKOVA I,PRNOVA MS,MAJEKOVA M,et al.2-Chloro-1,4-naphthpquinone derivative of quercetin as an inhibitoe of aldose reductase and anti-inflammatory agent[J].Enzyme Inhib Med Chem,2014,30(1):107-113.DOI:10.3109/14756366.2014.892935.
[13]ZHANG XA,ZHANG S,YIN Q,et al.Quercetin induces human colon cancer cells apoptosis by inhibiting the nuclear factor-kappa B pathway[J].Pharmacoqn Mag,2015,11(42):404-409.DOI:10.4103/0973-1296.153096.
[14]OZYURT H,CEVIK O,ZGEN Z,et al.Quercetin protects radiationinduced DNA damage and apoptosis in kidney and bladder tissues of rats[J].Free Radic Res,2014,48(10):1247-1255.DOI:10.1016/S0167-8140(15)31929-0.
[15]LI Y,JIA Y,ZHOU J,et al.Effect of methionine sulfoxide reductase B1 silencing on high-glucose-induced apoptosis of human lens epithelial cells[J].Life Sci,2013,92(3):193-201.DOI:10.1016/j.lfs.2012.11.021.
[16]刘思源,康刚劲.不同浓度17β-雌二醇对雌性大鼠晶状体上皮细胞凋亡的影响[J].眼科新进展,2011,31(8):725-729.