重组人乳铁蛋白对口腔癌KB细胞增殖及凋亡影响的研究
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摘要
目的研究重组人乳铁蛋白(recombinate human lactoferrin,rh LF)对人口腔KB细胞增殖及凋亡的影响,为口腔癌患者提供新的有效治疗途径提供参考。方法采用CCK-8法检测不同浓度(0、12.5、25、50、100、200μg/m L)的rh LF分别处理KB细胞24、48、72 h后对细胞增殖的影响;Immunofluorescence及Western blotting方法检测rh LF处理前后DNA损伤相关蛋白γ-H2AX的表达。并分别用0、50、200μg/m L的rh LF处理KB细胞,14 d后观察其对集落克隆形成的影响;0、200μg/m L rh LF处理KB细胞48 h后,收集细胞并使用线粒体膜电位检测试剂盒(JC-1)检测rh LF处理前后线粒体膜电位的变化。Annexin VPI染色检测rh LF对口腔癌KB细胞凋亡的影响;Western blotting检测0、50、200μg/m L的rh LF处理口腔癌KB细胞48 h后Parp、Caspase-3的表达情况。结果 rh LF处理KB细胞的增殖有显著的抑制作用。Immunofluorescence结果显示rh LF处理后γ-H2AX的表达明显增加,Western blotting结果也显示rh LF处理后γ-H2AX的表达成浓度依赖性增加。rh LF抑制KB细胞集落克隆的形成;JC-1荧光检测结果表明红绿荧光的相对比例降低,这种红绿荧光的相对比例降低提示膜电位下降;0、50、200μg/m L的rh LF处理口腔癌KB细胞48 h的凋亡率为2.02%、7.60%、48.07%;同时rh LF刺激口腔癌KB细胞能诱导Parp、Caspase-3的激活。结论 rh LF能显著抑制KB细胞的增殖并且诱导其细胞凋亡,其机制与线粒体凋亡途径的激活应激有关。
Objective To investigate the effects of rh LF on the proliferation and apoptosis in oral cancer KB cell line in order to achieve a new effective treatment for oral cancer patients. Methods KB cells were treated with different doses of rh LF( 0,12.5,25,50,100,200 μg/m L) for 24,48,72 h and cell viability was detected by CCK-8 assay. Immunofluorescence was used to evaluate the expression of DNA damage associated protein γ-H2 AX. Western blotting was performed to examine the expressions of γ-H2 AX. Colony formation of the cells was observed after KB cells had been treated with rh LF at 0,200 μg/m L for 14 days. JC-1 assay was used to measure the changes in mitochondrial membrane potential,Annexin V-PI staining was used to observe the cell apoptosis. Western blotting was employed to detect the expressions of Parp and Caspase-3. Results rh LF caused significant suppression of KB cells proliferation. Immunofluorescence showed the increase of γ-H2 AX after cells were incubated with rh LF. Western blotting displayed that the expressions of γ-H2 AX gradually increased in dose-dependent. Meanwhile,rh LF significantly inhibited KB cells colony formation. Exposure to rh LF( 0,50,200 μg/m L) after 48 h resulted in 2.02%,7.60% and 48.07% cell apoptotic rates. Exposure of the cells to increased concentrations of rh LF gradually increased the apoptotic rate and decreased mitochondrial membrane potential. rh LF increased the expression of activated Parp,Caspase-3. Conclusion rh LF can inhibit the proliferation and induce apoptosis of KB cells,the rh LF of which may be associated with the activation of the mitochondrial apoptotic pathway.
Objective To investigate the effects of rh LF on the proliferation and apoptosis in oral cancer KB cell line in order to achieve a new effective treatment for oral cancer patients. Methods KB cells were treated with different doses of rh LF( 0,12.5,25,50,100,200 μg/m L) for 24,48,72 h and cell viability was detected by CCK-8 assay. Immunofluorescence was used to evaluate the expression of DNA damage associated protein γ-H2 AX. Western blotting was performed to examine the expressions of γ-H2 AX. Colony formation of the cells was observed after KB cells had been treated with rh LF at 0,200 μg/m L for 14 days. JC-1 assay was used to measure the changes in mitochondrial membrane potential,Annexin V-PI staining was used to observe the cell apoptosis. Western blotting was employed to detect the expressions of Parp and Caspase-3. Results rh LF caused significant suppression of KB cells proliferation. Immunofluorescence showed the increase of γ-H2 AX after cells were incubated with rh LF. Western blotting displayed that the expressions of γ-H2 AX gradually increased in dose-dependent. Meanwhile,rh LF significantly inhibited KB cells colony formation. Exposure to rh LF( 0,50,200 μg/m L) after 48 h resulted in 2.02%,7.60% and 48.07% cell apoptotic rates. Exposure of the cells to increased concentrations of rh LF gradually increased the apoptotic rate and decreased mitochondrial membrane potential. rh LF increased the expression of activated Parp,Caspase-3. Conclusion rh LF can inhibit the proliferation and induce apoptosis of KB cells,the rh LF of which may be associated with the activation of the mitochondrial apoptotic pathway.
引文
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[20]Mc Ilwain DR,Berger T,Mak TW.Caspase functions in cell death and disease[J].CSH Perspect Biol,2013,5(2):a008656.
[2]Hirschey MD,De Berardinis RJ,Diehl AME,et al.Dysregulated metabolism contributes to oncogenesis[J].Semin Cancer Biol,2015,35(Suppl):S129-S150.
[3]Strohl WR.Fusionproteins for half-life extension of biologics as a strategy to make biobetters[J].Bio Drugs,2015,29(4):215-239.
[4]Yu HJ,Shin JA,Nam JS,et al.Apoptotic effect of dibenzylideneacetone on oral cancer cells via modulation of specificity protein 1and Bax[J].Oral Dis,2013,19(8):767-774.
[5]Zygogianni AG,Kyrgias G,Karakitsos P,et al.Oral squamous cell cancer:early detection and the role of alcohol and smoking[J].Head Neck Oncol,2011,3(6):2.
[6]Franceschi S,Levi F,La Vecchia C,et al.Comparison of the effect of smoking and alcohol drinking between oral and pharyngeal cancer[J].Int J Cancer,1999,83(1):1-4.
[7]Petersen PE.Oral cancer prevention and control—the approach of the World Health Organization[J].Oral Oncol,2009,45(5):454-460.
[8]Kademani D,Bell RB,Schmidt BL,et al.Oral and maxillofacial surgeons treating oral cancer:a preliminary report from the American Association of oral and maxillofacial surgeons task force on oral cancer[J].J Oral Maxillofac Surg,2008,66(10):2151-2157.
[9]Simons AL,Mattson DM,Dornfeld K,et al.Glucose deprivation-induced metabolic oxidative stress and cancer therapy[J].J Cancer Res Ther,2009,5(Suppl 1):S2-6.
[10]Pan MH,Ho CT.Chemopreventive effects of natural dietary compounds on cancer development[J].Chemical Soc Rev,2008,37(11):2558-2574.
[11]Baveye S,Elass E,Mazurier J,et al.Lactoferrin:a multifunctional glycoprotein involved in the modulation of the inflammatory process[J].Clin Chem Lab Med,1999,37(3):281-286.
[12]Sanchez L,Calvo M,Brock JH,et al.Biological role of lactoferrin[J].Arch Dis Child,1992,67(3):657-661.
[13]Kha DT,Wang G,Natrajan N,et al.Pathways for double-strand break repair in genetically unstable Z-DNA-forming sequences[J].J Biol,2010,398(4):471-480.
[14]Weiss CN,Ito K.DNA damage:a sensible mediator of the differentiation decision in hematopoietic stem cells and in leukemia[J].Int J Mol Sci,2015,16(3):6183-6201.
[15]Neumayer G,Helfricht A,Shim SY,et al.Targeting protein for xenopus kinesin-like protein 2(TPX2)regulates gamma-histone2AX(gamma-H2AX)levels upon ionizing radiation[J].J Biol Chem,2012,287(50):42206-42222.
[16]Solier S,Pommier Y.The nuclear gamma-H2AX apoptotic ring:implications for cancers and autoimmune diseases[J].Cell Mol Life Sci,2014,71(3):2289-2297.
[17]Visconti R,D'Adamio L.Functional cloning of genes regulating apoptosis in neuronal cells[J].Methods Mol Biol,2007,399(8):125-131.
[18]Kuribayashi K,Mayes PA,El-Deiry WS.What are caspases 3 and7 doing upstream of the mitochondria?[J].Cancer Biol Ther,2006,5(7):763-765.
[19]Dagbay K,Eron SJ,Serrano BP,et al.A multipronged approach for compiling a global map of allosteric regulation in the apoptotic caspases[J].Methods Enzymol,2014,544(16):215-249.
[20]Mc Ilwain DR,Berger T,Mak TW.Caspase functions in cell death and disease[J].CSH Perspect Biol,2013,5(2):a008656.