柚皮素通过Notch1/Hes1通路抑制肺癌干细胞增殖、迁移和分化
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摘要
为探讨柚皮素对肺癌干细胞增殖、迁移和分化的分子机制,本研究应用免疫磁珠法分选肺癌干细胞(A549-CSCs),并通过流式细胞术进行表面分子的鉴定;通过CCK8法检测不同浓度的柚皮素(25μg/m L,50μg/mL, 100μg/mL)对肺癌干细胞(A549-CSCs)活力的影响,Transwell检测柚皮素对A549-CSCs细胞迁移能力的影响,Q-PCR检测柚皮素对肺癌干细胞分化相关因子Sox2和Oct4 m RNA表达的影响,Western blotting法检测柚皮素对细胞内Notch1和Hes1蛋白表达的影响。流式细胞术检测结果显示,A549-CSCs细胞表面分子CD133呈阳性表达,符合肺癌干细胞特征。CCK8结果显示,与对照组(control)比较,25μg/m L、50μg/mL、100μg/mL柚皮素处理A549-CSCs 24 h,细胞活力显著降低(p<0.05);Transwell检测结果显示,与对照组比较,不同浓度柚皮素处理组A549-CSCs迁移能力显著降低(p<0.05);定量PCR (real-time polymerase chain reaction, Q-PCR)结果显示,与对照组比较,柚皮素处理组细胞Sox2和Oct4 m RNA表达水平显著降低(p<0.05);蛋白质印迹法(Western blotting)结果显示,与对照组相比柚皮素处理组细胞Notch1和Hes1蛋白表达水平均降低。本研究发现柚皮素可能通过抑制Notch1/Hes1通路抑制肺癌干细胞增殖、迁移和分化。这为柚皮素治疗肺癌提供临床依据。
In order to investigate the molecular mechanism of Naringenin on proliferation, migration and differentiation of lung cancer stem cells, in this research, the lung cancer stem cells(A549-CSCs) were sorted by immunomagnetic beads method and the surface molecules were identified by flow cytometry. The influence of naringenin in different concentrations(25 μg/m L, 50 μg/m L, 100 μg/mL) on the viability of A549-CSCs was evaluated by CCK8 assay. The effect of Naringenin on cell migration ability of A549-CSCs was detected by Transwell. The effect of Naringenin on mRNA expression level of Sox2 and Oct4 related to differentiation in lung cancer stem cells was measured by Q-PCR. The protein expression levels of Notch1 and Hes1 in cells treated with naringenin were examined by Western blotting. The results of flow cytometry showed that the cell surface molecule CD133 of A549-CSCs was positively expressed, which was consistent with the characteristics of lung cancer stem cells. The results of CCK8 showed that compared with the control group(Control), the cell viability of A549-CSCs treated with 25 μg/mL, 50 μg/mL, 100 μg/m L naringenin was significantly decreased after 24 h(p<0.05).The results of Transwell test showed tha t the migration ability of A549-CSCs in different concentrations of naringenin treatment group was significantly lower than that of the control group(p<0.05). Real-time polymerase chain reaction(Q-PCR) results showed that the expression levels of Sox2 and Oct4 mRNA in the naringenin-treated group were significantly lower than those in the control group(p<0.05). Western blotting results showed that the expression levels of Notch1 and Hes1 protein in the naringenin-treated group were both lower than those in the control group. This study suggested that naringin might inhibit the proliferation, migration and differentiation of lung cancer stem cells by inhibiting the Notch1/Hes1 pathway. This could provide a clinical basis for the treatment of lung cancer with Naringin.
In order to investigate the molecular mechanism of Naringenin on proliferation, migration and differentiation of lung cancer stem cells, in this research, the lung cancer stem cells(A549-CSCs) were sorted by immunomagnetic beads method and the surface molecules were identified by flow cytometry. The influence of naringenin in different concentrations(25 μg/m L, 50 μg/m L, 100 μg/mL) on the viability of A549-CSCs was evaluated by CCK8 assay. The effect of Naringenin on cell migration ability of A549-CSCs was detected by Transwell. The effect of Naringenin on mRNA expression level of Sox2 and Oct4 related to differentiation in lung cancer stem cells was measured by Q-PCR. The protein expression levels of Notch1 and Hes1 in cells treated with naringenin were examined by Western blotting. The results of flow cytometry showed that the cell surface molecule CD133 of A549-CSCs was positively expressed, which was consistent with the characteristics of lung cancer stem cells. The results of CCK8 showed that compared with the control group(Control), the cell viability of A549-CSCs treated with 25 μg/mL, 50 μg/mL, 100 μg/m L naringenin was significantly decreased after 24 h(p<0.05).The results of Transwell test showed tha t the migration ability of A549-CSCs in different concentrations of naringenin treatment group was significantly lower than that of the control group(p<0.05). Real-time polymerase chain reaction(Q-PCR) results showed that the expression levels of Sox2 and Oct4 mRNA in the naringenin-treated group were significantly lower than those in the control group(p<0.05). Western blotting results showed that the expression levels of Notch1 and Hes1 protein in the naringenin-treated group were both lower than those in the control group. This study suggested that naringin might inhibit the proliferation, migration and differentiation of lung cancer stem cells by inhibiting the Notch1/Hes1 pathway. This could provide a clinical basis for the treatment of lung cancer with Naringin.
引文
Alhdiri M.A.,Samat N.A.,and Mohamed Z.,2017,Risk estimation for lung cancer in libya:analysis based on standardized morbidity ratio,poisson-gamma model,BYM model and mixture model,Asian Pac.J.Cancer Prev.,18(3):673-679
Bawazeer N.A.,Choudary H.,Zamzami M.A.,Abdulaal W.H.,Zeyadi M.,ALbukhari A.,Middleton B.,and Moselhy S.S.,2017,Possible regulation of ldl-receptor by naringenin in Hepg2 hepatoma cell line,Afr.J.Tradit.Complement Altern.Med.,14(1):278-287
Boussios S.,Seraj E.,Zarkavelis G.,Petrakis D.,Kollas A.,Kafantari A.,Assi A.,Tatsi K.,Pavlidis N.,and Pentheroudakis G.,2016,Management of patients with recurrent/advanced cervical cancer beyond first line platinum regimens:Where do we stand?A literature review,Crit.Rev.Oncol.Hematol.,108:164-174
Chang H.L.,Chang Y.M.,Lai S.C.,Chen K.M.,Wang K.C.,Chiu T.T.,Chang F.H.,and Hsu L.S.,2017,Naringenin inhibits migration of lung cancer cells via the inhibition of matrix metalloproteinases-2 and-9,Exp.Ther.Med.,13(2):739-744
Holmes O.E.,MacRae R.,Cook G.,Cross P.,Nair V.,Marginean H.,and Pantarotto J.R.,2017,Age-not charlson co-morbidity index-predicts for mortality after stereotactic ablative radiotherapy for medically inoperable stageⅠnon-small cell lung cancer,Clin.Transl.Radiat.Oncol.,5:37-41
Hong S.W.,Hur W.,Choi J.E.,Kim J.H.,Hwang D.,and Yoon S.K.,2016,Role of ADAM17 in invasion and migration of CD133-expressing liver cancer stem cells after irradiation,Oncotarget,7(17):23482-23497
Huang X.,Huang J.,Leng D.,Yang S.,Yao Q.,Sun J.,and Hu J.,2017,Gefitinib-loaded DSPE-PEG2000 nanomicelles with CD133 aptamers target lung cancer stem cells,World J.Surg.Oncol.,15(1):167
Jang J.W.,Song Y.,Kim S.H.,Kim J.,and Seo H.R.,2017,Potential mechanisms of CD133 in cancer stem cells,Life Sci.,184:25-29
Johari B.,and Zargan J.,2017,Simultaneous targeted inhibition of Sox2-Oct4 transcription factors using decoy oligodeoxynucleotides to repress stemness properties in mouse embryonic stem cells,Cell Biol.Int.,41(12):1335-1344
Koren A.,Rijavec M.,Kern I.,Sodja E.,Korosec P.,and Cufer T.,2016,BMI1,ALDH1A1,and CD133 transcripts connect epithelial-mesenchymal transition to cancer stem cells in lung carcinoma,Stem Cells Int.,2016:9714315
Lee S.H.,Hyun S.K.,Kim H.B.,Kang C.D.,and Kim S.H.,2016,Potential role of CD133 expression in the susceptibility of human liver cancer stem-like cells to TRAIL,Oncol.Res.,24(6):495-509
Li J.,Chen J.N.,Zeng T.T.,He F.,Chen S.P.,Ma S.,Bi J.,Zhu X.F.,and Guan X.Y.,2016,CD133+liver cancer stem cells resist interferon-gamma-induced autophagy,BMC Cancer,16:15
Lopez-Pastorini A.,Riedel R.,Koryllos A.,Beckers F.,Ludwig C.,and Stoelben E.,2017,The impact of preoperative elevated serum C-reactive protein on postoperative morbidity and mortality after anatomic resection for lung cancer,Lung Cancer,109:68-73
Manchope M.F.,Casagrande R.,and Verri W.A.,2017,Jr.Naringenin:an analgesic and anti-inflammatory citrus flavanone,Oncotarget,8(3):3766-3767
Nosrati A.,Naghshvar F.,Maleki I.,and Salehi F.,2016,Cancer stem cells CD133 and CD24 in colorectal cancers in Northern Iran,Gastroenterol.Hepatol.Bed.Bench.,9(2):132-139
Rizzino A.,and Wuebben E.L.,2016,Sox2/Oct4:A delicately balanced partnership in pluripotent stem cells and embryogenesis,Biochim.Biophys.Acta,1859(6):780-791
Seo S.,Jeon H.Y.,and Kim H.,2017,Comparison of cellular transforming activity of OCT4,NANOG,and SOX2 in immortalized astrocytes,DNA Cell Biol.,36(11):1000-1009
Shi Y.,Shu B.,Yang R.,Xu Y.,Xing B.,Liu J.,Chen L.,Qi S.,Liu X.,Wang P.,Tang J.,and Xie J.,2015,Wnt and Notch signaling pathway involved in wound healing by targeting c-Myc and Hes1 separately,Stem.Cell Res.Ther.,6:120
Sodja E.,Rijavec M.,Koren A.,Sadikov A.,Koroёec P.,and Cufer T.,2016,The prognostic value of whole blood SOX2,NANOG and OCT4 m RNA expression in advanced small-cell lung cancer,Radiol.Oncol.,50(2):188-196
Yang L.,Lin Z.,Wang Y.,Gao S.,Li Q.,Li C.,Xu W.,Chen J.,Liu T.,Song Z.,and Liu G.,2018,Mi R-5100 increases the cisplatin resistance of the lung cancer stem cells by inhibiting the Rab6,Mol.Carcinog.,57(3):419-428
Zheng X.,Narayanan S.,Zheng X.,Luecke-Johansson S.,Gradin K.,Catrina S.B.,Poellinger L.,and Pereira T.S.,2017,ANotch-independent mechanism contributes to the induction of Hes1 gene expression in response to hypoxia in P19 cells,Exp.Cell Res.,358(2):129-139
Zimmerer R.M.,Matthiesen P.,Kreher F.,Kampmann A.,Spalthoff S.,Jehn P.,Bittermann G.,Gellrich N.C.,and Tavassol F.,2016,Putative CD133+melanoma cancer stem cells induce initial angiogenesis in vivo,Microvasc.Res.,104:46-54
Bawazeer N.A.,Choudary H.,Zamzami M.A.,Abdulaal W.H.,Zeyadi M.,ALbukhari A.,Middleton B.,and Moselhy S.S.,2017,Possible regulation of ldl-receptor by naringenin in Hepg2 hepatoma cell line,Afr.J.Tradit.Complement Altern.Med.,14(1):278-287
Boussios S.,Seraj E.,Zarkavelis G.,Petrakis D.,Kollas A.,Kafantari A.,Assi A.,Tatsi K.,Pavlidis N.,and Pentheroudakis G.,2016,Management of patients with recurrent/advanced cervical cancer beyond first line platinum regimens:Where do we stand?A literature review,Crit.Rev.Oncol.Hematol.,108:164-174
Chang H.L.,Chang Y.M.,Lai S.C.,Chen K.M.,Wang K.C.,Chiu T.T.,Chang F.H.,and Hsu L.S.,2017,Naringenin inhibits migration of lung cancer cells via the inhibition of matrix metalloproteinases-2 and-9,Exp.Ther.Med.,13(2):739-744
Holmes O.E.,MacRae R.,Cook G.,Cross P.,Nair V.,Marginean H.,and Pantarotto J.R.,2017,Age-not charlson co-morbidity index-predicts for mortality after stereotactic ablative radiotherapy for medically inoperable stageⅠnon-small cell lung cancer,Clin.Transl.Radiat.Oncol.,5:37-41
Hong S.W.,Hur W.,Choi J.E.,Kim J.H.,Hwang D.,and Yoon S.K.,2016,Role of ADAM17 in invasion and migration of CD133-expressing liver cancer stem cells after irradiation,Oncotarget,7(17):23482-23497
Huang X.,Huang J.,Leng D.,Yang S.,Yao Q.,Sun J.,and Hu J.,2017,Gefitinib-loaded DSPE-PEG2000 nanomicelles with CD133 aptamers target lung cancer stem cells,World J.Surg.Oncol.,15(1):167
Jang J.W.,Song Y.,Kim S.H.,Kim J.,and Seo H.R.,2017,Potential mechanisms of CD133 in cancer stem cells,Life Sci.,184:25-29
Johari B.,and Zargan J.,2017,Simultaneous targeted inhibition of Sox2-Oct4 transcription factors using decoy oligodeoxynucleotides to repress stemness properties in mouse embryonic stem cells,Cell Biol.Int.,41(12):1335-1344
Koren A.,Rijavec M.,Kern I.,Sodja E.,Korosec P.,and Cufer T.,2016,BMI1,ALDH1A1,and CD133 transcripts connect epithelial-mesenchymal transition to cancer stem cells in lung carcinoma,Stem Cells Int.,2016:9714315
Lee S.H.,Hyun S.K.,Kim H.B.,Kang C.D.,and Kim S.H.,2016,Potential role of CD133 expression in the susceptibility of human liver cancer stem-like cells to TRAIL,Oncol.Res.,24(6):495-509
Li J.,Chen J.N.,Zeng T.T.,He F.,Chen S.P.,Ma S.,Bi J.,Zhu X.F.,and Guan X.Y.,2016,CD133+liver cancer stem cells resist interferon-gamma-induced autophagy,BMC Cancer,16:15
Lopez-Pastorini A.,Riedel R.,Koryllos A.,Beckers F.,Ludwig C.,and Stoelben E.,2017,The impact of preoperative elevated serum C-reactive protein on postoperative morbidity and mortality after anatomic resection for lung cancer,Lung Cancer,109:68-73
Manchope M.F.,Casagrande R.,and Verri W.A.,2017,Jr.Naringenin:an analgesic and anti-inflammatory citrus flavanone,Oncotarget,8(3):3766-3767
Nosrati A.,Naghshvar F.,Maleki I.,and Salehi F.,2016,Cancer stem cells CD133 and CD24 in colorectal cancers in Northern Iran,Gastroenterol.Hepatol.Bed.Bench.,9(2):132-139
Rizzino A.,and Wuebben E.L.,2016,Sox2/Oct4:A delicately balanced partnership in pluripotent stem cells and embryogenesis,Biochim.Biophys.Acta,1859(6):780-791
Seo S.,Jeon H.Y.,and Kim H.,2017,Comparison of cellular transforming activity of OCT4,NANOG,and SOX2 in immortalized astrocytes,DNA Cell Biol.,36(11):1000-1009
Shi Y.,Shu B.,Yang R.,Xu Y.,Xing B.,Liu J.,Chen L.,Qi S.,Liu X.,Wang P.,Tang J.,and Xie J.,2015,Wnt and Notch signaling pathway involved in wound healing by targeting c-Myc and Hes1 separately,Stem.Cell Res.Ther.,6:120
Sodja E.,Rijavec M.,Koren A.,Sadikov A.,Koroёec P.,and Cufer T.,2016,The prognostic value of whole blood SOX2,NANOG and OCT4 m RNA expression in advanced small-cell lung cancer,Radiol.Oncol.,50(2):188-196
Yang L.,Lin Z.,Wang Y.,Gao S.,Li Q.,Li C.,Xu W.,Chen J.,Liu T.,Song Z.,and Liu G.,2018,Mi R-5100 increases the cisplatin resistance of the lung cancer stem cells by inhibiting the Rab6,Mol.Carcinog.,57(3):419-428
Zheng X.,Narayanan S.,Zheng X.,Luecke-Johansson S.,Gradin K.,Catrina S.B.,Poellinger L.,and Pereira T.S.,2017,ANotch-independent mechanism contributes to the induction of Hes1 gene expression in response to hypoxia in P19 cells,Exp.Cell Res.,358(2):129-139
Zimmerer R.M.,Matthiesen P.,Kreher F.,Kampmann A.,Spalthoff S.,Jehn P.,Bittermann G.,Gellrich N.C.,and Tavassol F.,2016,Putative CD133+melanoma cancer stem cells induce initial angiogenesis in vivo,Microvasc.Res.,104:46-54