干细胞因子和缺氧诱导因子1α在胰腺癌细胞的表达和机制
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摘要
目的研究干细胞因子(SCF)和缺氧诱导因子1α(HIF-1α)在胰腺癌中表达的相关性,并探讨SCF对HIF-1α表达的调控机制。方法使用免疫组织化学染色检测胰腺癌标本SCF和HIF-1α的表达,分析两种分子表达的相关性。用(0、1、10、100)ng/m L SCF和5μmol/L c-KIT抑制剂格列卫(Gleevec)单独或联合处理胰腺癌PANC-1细胞,实时荧光定量PCR检测HIF-1αmRNA水平,Western blot法检测HIF-1α蛋白水平、胞外信号调节激酶1/2(ERK1/2)、AKT磷酸化水平。结果胰腺癌组织SCF和HIF-1α的表达上调,二者表达呈正相关。在PANC-1细胞中,SCF不能影响HIF-1α的mRNA表达,但能够上调HIF-1α的蛋白表达,且呈浓度依赖性。Gleevec不能影响HIF-1α的mRNA表达,但能够抑制SCF对HIF-1α蛋白的上调作用,下调ERK1/2、AKT的磷酸化水平。结论 SCF/c-KIT可激活胰腺癌PANC-1细胞AKT和ERK信号通路上调HIF-1α蛋白表达。
Objective To study the correlation between the expressions of stem cell factor( SCF) and hypoxia inducible factor 1 alpha( HIF-1α) in pancreatic cancer,and investigate the mechanism by which SCF regulates the expression of HIF-1α.Methods Immunohistochemistry was used to detect the expressions of SCF and HIF-1α in pancreatic cancer specimens and to analyze the correlation between SCF and HIF-1α expressions. Pancreatic cancer PANC-1 cells were treated with different doses of SCF( 0,1,10,100 ng/m L) alone or combined with c-KIT inhibitor Gleevec( 5 μmol/L). Real-time fluorescent quantitative PCR( qRT-PCR) was performed to detect the level of HIF-1α mRNA,and Western blotting to detect the HIF-1αprotein level,the phosphorylation levels of ERK1/2 and AKT. Results SCF and HIF-1α were up-regulated in pancreatic cancer samples and they had an obvious positive correlation. In PANC-1 cells,SCF didn't affect the expression of HIF-1αmRNA,but up-regulated the expression of HIF-1α protein in a dose-dependent manner. Gleevec inhibited the SCF-induced up-regulation of HIF-1α protein,but did not affect the mRNA. And Gleevec blocked the phosphorylation of AKT and ERK1/2.Conclusion SCF/c-KIT can up-regulate the protein expression of HIF-1α by activating AKT and ERK signaling pathways in pancreatic cancer cells.
Objective To study the correlation between the expressions of stem cell factor( SCF) and hypoxia inducible factor 1 alpha( HIF-1α) in pancreatic cancer,and investigate the mechanism by which SCF regulates the expression of HIF-1α.Methods Immunohistochemistry was used to detect the expressions of SCF and HIF-1α in pancreatic cancer specimens and to analyze the correlation between SCF and HIF-1α expressions. Pancreatic cancer PANC-1 cells were treated with different doses of SCF( 0,1,10,100 ng/m L) alone or combined with c-KIT inhibitor Gleevec( 5 μmol/L). Real-time fluorescent quantitative PCR( qRT-PCR) was performed to detect the level of HIF-1α mRNA,and Western blotting to detect the HIF-1αprotein level,the phosphorylation levels of ERK1/2 and AKT. Results SCF and HIF-1α were up-regulated in pancreatic cancer samples and they had an obvious positive correlation. In PANC-1 cells,SCF didn't affect the expression of HIF-1αmRNA,but up-regulated the expression of HIF-1α protein in a dose-dependent manner. Gleevec inhibited the SCF-induced up-regulation of HIF-1α protein,but did not affect the mRNA. And Gleevec blocked the phosphorylation of AKT and ERK1/2.Conclusion SCF/c-KIT can up-regulate the protein expression of HIF-1α by activating AKT and ERK signaling pathways in pancreatic cancer cells.
引文
[1]Hidalgo M.Pancreatic cancer[J].N Engl J Med,2010,362(17):1605-1617.
[2]Ercan G,Karlitepe A,Ozpolat B.Pancreatic cancer stem cells and therapeutic approaches[J].Anticancer Res,2017,37(6):2761-2775.
[3]Ma J,Siegel R,Jemal A.Pancreatic cancer death rates by race among US men and women,1970-2009[J].J Natl Cancer Inst,2013,105(22):1694-1700.
[4]Ilic M,Ilic I.Epidemiology of pancreatic cancer[J].World J Gastroenterol,2016,22(44):9694-9705.
[5]Kim H J,Yonezawa T,Teruya T,et al.Nobiletin,a polymethoxy flavonoid,reduced endothelin-1 plus SCF-induced pigmentation in human melanocytes[J].Photochem Photobiol,2015,91(2):379-386.
[6]Faber T W,Pullen N A,Fernando J F,et al.ADAM10 is required for SCF-induced mast cell migration[J].Cell Immunol,2014,290(1):80-88.
[7]Tan J,Zou Y,Wu X W,et al.Increased SCF in follicular fluid and granulosa cells positively correlates with oocyte maturation,fertilization,and embryo quality in humans[J/OL].Reprod Sci,2017:1933719117697125.doi:10.1177/1933719117697125.[Epub ahead of print].
[8]Williams D A,Majumdar M K.Analysis of steel factor(stem cell factor)isoforms in the hematopoietic microenvironment[J].Stem Cells,1994,12(Suppl 1):67-74;discussion 75-77.
[9]Zsebo K M,Williams D A,Geissler E N,et al.Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-KIT tyrosine kinase receptor[J].Cell,1990,63(1):213-224.
[10]Choudhary S,Pardo A,Rosinke R,et al.Targeting c-KIT receptor in neuroblastomas and colorectal cancers using stem cell factor(SCF)-based recombinant bacterial toxins[J].Appl Microbiol Biotechnol,2016,100(1):263-277.
[11]Attramadal C G,Kumar S,Gao J,et al.Low mast cell density predicts poor prognosis in oral squamous cell carcinoma and reduces survival in head and neck squamous cell carcinoma[J].Anticancer Res,2016,36(10):5499-5506.
[12]Ohi Y,Kosuge H,Tanese K.Case of syringomatous carcinoma:Positive immunohistochemical staining of c-KIT and phosphorylatedextracellular signal-regulated kinase 1/2[J].J Dermatol,2015,42(12):1191-1192.
[13]Masoud G N,Li W.HIF-1αpathway:role,regulation and intervention for cancer therapy[J].Acta Pharm Sin B,2015,5(5):378-389.
[14]Xie Y,Zhong D W.AEG-1 is associated with hypoxia-induced hepatocellular carcinoma chemoresistance via regulating PI3K/AKT/HIF-1alpha/MDR-1 pathway[J].EXCLI J,2016,15:745-757.
[15]Liu F,Lin B,Liu X,et al.ERK signaling pathway is involved in HPV-16E6 but not E7 oncoprotein-induced HIF-1αprotein accumulation in NSCLC cells[J].Oncol Res,2016,23(3):109-118.
[16]Zhang M,Ma Q,Hu H,et al.Stem cell factor/c-KIT signaling enhances invasion of pancreatic cancer cells via HIF-1αunder normoxic condition[J].Caner letters,2011,303(2):108-117.
[17]Abbaspour Babaei M,Kamalidehghan B,Saleem M,et al.Receptor tyrosine kinase(c-KIT)inhibitors:a potential therapeutic target in cancer cells[J].Drug Des Devel Ther,2016,10:2443-2459.
[18]Lau S T,Hansford L M,Chan W K,et al.Prokineticin signaling is required for the maintenance of a de novo population of c-KIT+cells to sustain neuroblastoma progression[J].Oncogene,2015,34(8):1019-1034.
[19]Kimura W,Ma J,Takeshita A,et al.Analysis of c-KIT protein expression in pancreatic neoplasms and its implication for prognosis[J].Hepatogastroenterology,2007,54(80):2203-2208.
[20]Remels A H,Gosker H R,Verhees K J,et al.TNF-α-induced NF-κB activation stimulates skeletal muscle glycolytic metabolism through activation of HIF-1α[J].Endocrinology,2015,156(5):1770-1781.
[21]Kuschel A,Simon P,Tug S.Functional regulation of HIF-1αunder normoxia--is there more than post-translational regulation?[J].J Cell Physiol,2012,227(2):514-524.
[22]Sch9del J,Grampp S,Maher E R,et al.Hypoxia,hypoxia-inducible transcription factors,and renal cancer[J].Eur Urol,2016,69(4):646-657.
[23]Mimeault M,Batra S K.Hypoxia-inducing factors as master regulators of stemness properties and altered metabolism of cancer-and metastasisinitiating cells[J].J Cell Mol Med,2013,17(1):30-54.
[24]Yu J,Li J,Zhang S,et al.IGF-1 induces hypoxia-inducible factor 1α-mediated GLUT3 expression through PI3K/AKT/m TOR dependent pathways in PC12 cells[J].Brain Res,2012,1430:18-24.
[25]Fleming I N,Andriu A,Smith T A.Early changes in[18F]FDG incorporation by breast cancer cells treated with trastuzumab in normoxic conditions:role of the AKT-pathway,glucose transport and HIF-1α[J].Breast Cancer Res Treat,2014,144(2):241-248.
[2]Ercan G,Karlitepe A,Ozpolat B.Pancreatic cancer stem cells and therapeutic approaches[J].Anticancer Res,2017,37(6):2761-2775.
[3]Ma J,Siegel R,Jemal A.Pancreatic cancer death rates by race among US men and women,1970-2009[J].J Natl Cancer Inst,2013,105(22):1694-1700.
[4]Ilic M,Ilic I.Epidemiology of pancreatic cancer[J].World J Gastroenterol,2016,22(44):9694-9705.
[5]Kim H J,Yonezawa T,Teruya T,et al.Nobiletin,a polymethoxy flavonoid,reduced endothelin-1 plus SCF-induced pigmentation in human melanocytes[J].Photochem Photobiol,2015,91(2):379-386.
[6]Faber T W,Pullen N A,Fernando J F,et al.ADAM10 is required for SCF-induced mast cell migration[J].Cell Immunol,2014,290(1):80-88.
[7]Tan J,Zou Y,Wu X W,et al.Increased SCF in follicular fluid and granulosa cells positively correlates with oocyte maturation,fertilization,and embryo quality in humans[J/OL].Reprod Sci,2017:1933719117697125.doi:10.1177/1933719117697125.[Epub ahead of print].
[8]Williams D A,Majumdar M K.Analysis of steel factor(stem cell factor)isoforms in the hematopoietic microenvironment[J].Stem Cells,1994,12(Suppl 1):67-74;discussion 75-77.
[9]Zsebo K M,Williams D A,Geissler E N,et al.Stem cell factor is encoded at the Sl locus of the mouse and is the ligand for the c-KIT tyrosine kinase receptor[J].Cell,1990,63(1):213-224.
[10]Choudhary S,Pardo A,Rosinke R,et al.Targeting c-KIT receptor in neuroblastomas and colorectal cancers using stem cell factor(SCF)-based recombinant bacterial toxins[J].Appl Microbiol Biotechnol,2016,100(1):263-277.
[11]Attramadal C G,Kumar S,Gao J,et al.Low mast cell density predicts poor prognosis in oral squamous cell carcinoma and reduces survival in head and neck squamous cell carcinoma[J].Anticancer Res,2016,36(10):5499-5506.
[12]Ohi Y,Kosuge H,Tanese K.Case of syringomatous carcinoma:Positive immunohistochemical staining of c-KIT and phosphorylatedextracellular signal-regulated kinase 1/2[J].J Dermatol,2015,42(12):1191-1192.
[13]Masoud G N,Li W.HIF-1αpathway:role,regulation and intervention for cancer therapy[J].Acta Pharm Sin B,2015,5(5):378-389.
[14]Xie Y,Zhong D W.AEG-1 is associated with hypoxia-induced hepatocellular carcinoma chemoresistance via regulating PI3K/AKT/HIF-1alpha/MDR-1 pathway[J].EXCLI J,2016,15:745-757.
[15]Liu F,Lin B,Liu X,et al.ERK signaling pathway is involved in HPV-16E6 but not E7 oncoprotein-induced HIF-1αprotein accumulation in NSCLC cells[J].Oncol Res,2016,23(3):109-118.
[16]Zhang M,Ma Q,Hu H,et al.Stem cell factor/c-KIT signaling enhances invasion of pancreatic cancer cells via HIF-1αunder normoxic condition[J].Caner letters,2011,303(2):108-117.
[17]Abbaspour Babaei M,Kamalidehghan B,Saleem M,et al.Receptor tyrosine kinase(c-KIT)inhibitors:a potential therapeutic target in cancer cells[J].Drug Des Devel Ther,2016,10:2443-2459.
[18]Lau S T,Hansford L M,Chan W K,et al.Prokineticin signaling is required for the maintenance of a de novo population of c-KIT+cells to sustain neuroblastoma progression[J].Oncogene,2015,34(8):1019-1034.
[19]Kimura W,Ma J,Takeshita A,et al.Analysis of c-KIT protein expression in pancreatic neoplasms and its implication for prognosis[J].Hepatogastroenterology,2007,54(80):2203-2208.
[20]Remels A H,Gosker H R,Verhees K J,et al.TNF-α-induced NF-κB activation stimulates skeletal muscle glycolytic metabolism through activation of HIF-1α[J].Endocrinology,2015,156(5):1770-1781.
[21]Kuschel A,Simon P,Tug S.Functional regulation of HIF-1αunder normoxia--is there more than post-translational regulation?[J].J Cell Physiol,2012,227(2):514-524.
[22]Sch9del J,Grampp S,Maher E R,et al.Hypoxia,hypoxia-inducible transcription factors,and renal cancer[J].Eur Urol,2016,69(4):646-657.
[23]Mimeault M,Batra S K.Hypoxia-inducing factors as master regulators of stemness properties and altered metabolism of cancer-and metastasisinitiating cells[J].J Cell Mol Med,2013,17(1):30-54.
[24]Yu J,Li J,Zhang S,et al.IGF-1 induces hypoxia-inducible factor 1α-mediated GLUT3 expression through PI3K/AKT/m TOR dependent pathways in PC12 cells[J].Brain Res,2012,1430:18-24.
[25]Fleming I N,Andriu A,Smith T A.Early changes in[18F]FDG incorporation by breast cancer cells treated with trastuzumab in normoxic conditions:role of the AKT-pathway,glucose transport and HIF-1α[J].Breast Cancer Res Treat,2014,144(2):241-248.