Hypoxia,as an important hallmark of the tumor microenvironment,is a major cause of oxidative stress and plays a central role in various malignant tumors,including glioblastoma.Elevated reactive oxygen species(ROS)in a...Hypoxia,as an important hallmark of the tumor microenvironment,is a major cause of oxidative stress and plays a central role in various malignant tumors,including glioblastoma.Elevated reactive oxygen species(ROS)in a hypoxic microenvironment promote glioblastoma progression;however,the underlying mechanism has not been clarified.Herein,we found that hypoxia promoted ROS production,and the proliferation,migration,and invasion of glioblastoma cells,while this promotion was restrained by ROS scavengers N-acetyl-L-cysteine(NAC)and diphenyleneiodonium chloride(DPI).Hypoxia-induced ROS activated hypoxia-inducible factor-1α(HIF-1α)signaling,which enhanced cell migration and invasion by epithelial-mesenchymal transition(EMT).Furthermore,the induction of serine protease inhibitor family E member 1(SERPINE1)was ROS-dependent under hypoxia,and HIF-1αmediated SERPINE1 increase induced by ROS via binding to the SERPINE1 promoter region,thereby facilitating glioblastoma migration and invasion.Taken together,our data revealed that hypoxia-induced ROS reinforce the hypoxic adaptation of glioblastoma by driving the HIF-1α-SERPINE1 signaling pathway,and that targeting ROS may be a promising therapeutic strategy for glioblastoma.展开更多
Objective:We aimed to develop a novel anti-HIF-1αintrabody to decrease gemcitabine resistance in pancreatic cancer patients.Methods:Surface plasmon resonance and glutathione S-transferase pull-down assays were conduc...Objective:We aimed to develop a novel anti-HIF-1αintrabody to decrease gemcitabine resistance in pancreatic cancer patients.Methods:Surface plasmon resonance and glutathione S-transferase pull-down assays were conducted to identify the binding affinity and specificity of anti-HIF-1αVHH212[a single-domain antibody(nanobody)].Molecular dynamics simulation was used to determine the protein-protein interactions between hypoxia-inducible factor-1α(HIF-1α)and VHH212.The real-time polymerase chain reaction(PCR)and Western blot analyses were performed to identify the expressions of HIF-1αand VEGF-A in pancreatic ductal adenocarcinoma cell lines.The efficiency of the VHH212 nanobody in inhibiting the HIF-1 signaling pathway was measured using a dual-luciferase reporter assay.Finally,a PANC-1 xenograft model was developed to evaluate the anti-tumor efficiency of combined treatment.Immunohistochemistry analysis was conducted to detect the expressions of HIF-1αand VEGF-A in tumor tissues.Results:VHH212 was stably expressed in tumor cells with low cytotoxicity,high affinity,specific subcellular localization,and neutralization of HIF-1αin the cytoplasm or nucleus.The binding affinity between VHH212 and the HIF-1αPAS-B domain was 42.7 n M.Intrabody competitive inhibition of the HIF-1αheterodimer with an aryl hydrocarbon receptor nuclear translocator was used to inhibit the HIF-1/VEGF pathway in vitro.Compared with single agent gemcitabine,co-treatment with gemcitabine and a VHH212-encoding adenovirus significantly suppressed tumor growth in the xenograft model with 80.44%tumor inhibition.Conclusions:We developed an anti-HIF-1αnanobody and showed the function of VHH212 in a preclinical murine model of PANC-1 pancreatic cancer.The combination of VHH212 and gemcitabine significantly inhibited tumor development.These results suggested that combined use of anti-HIF-1αnanobodies with first-line treatment may in the future be an effective treatment for pancreatic cancer.展开更多
基金This work was supported by the National Natural Science Foundation of China(Nos.81772688 and 81372698)the Priority Academic Program Development of Jiangsu Higher Education Institutions(PAPD)+1 种基金the Research Foundation for Talented Scholars of Xuzhou Medical University(No.RC20552223)the Postgraduate Research&Practice Innovation Program of Jiangsu Province(No.KYCX20_2463),China。
文摘Hypoxia,as an important hallmark of the tumor microenvironment,is a major cause of oxidative stress and plays a central role in various malignant tumors,including glioblastoma.Elevated reactive oxygen species(ROS)in a hypoxic microenvironment promote glioblastoma progression;however,the underlying mechanism has not been clarified.Herein,we found that hypoxia promoted ROS production,and the proliferation,migration,and invasion of glioblastoma cells,while this promotion was restrained by ROS scavengers N-acetyl-L-cysteine(NAC)and diphenyleneiodonium chloride(DPI).Hypoxia-induced ROS activated hypoxia-inducible factor-1α(HIF-1α)signaling,which enhanced cell migration and invasion by epithelial-mesenchymal transition(EMT).Furthermore,the induction of serine protease inhibitor family E member 1(SERPINE1)was ROS-dependent under hypoxia,and HIF-1αmediated SERPINE1 increase induced by ROS via binding to the SERPINE1 promoter region,thereby facilitating glioblastoma migration and invasion.Taken together,our data revealed that hypoxia-induced ROS reinforce the hypoxic adaptation of glioblastoma by driving the HIF-1α-SERPINE1 signaling pathway,and that targeting ROS may be a promising therapeutic strategy for glioblastoma.
基金supported by grants from the National Key Research and Development Project(Grant No.2019YFA0905600)the Major State Basic Research Development Program of the Natural Science Foundation of Shandong Province in China(Grant No.ZR2020ZD11)+2 种基金the National Natural Science Foundation of China(Grant Nos.81772633 and 31470967)the Science and Technology Program of Tianjin,China(Grant No.19YFSLQY00110)the Taishan Scholars Program of Shandong Province。
文摘Objective:We aimed to develop a novel anti-HIF-1αintrabody to decrease gemcitabine resistance in pancreatic cancer patients.Methods:Surface plasmon resonance and glutathione S-transferase pull-down assays were conducted to identify the binding affinity and specificity of anti-HIF-1αVHH212[a single-domain antibody(nanobody)].Molecular dynamics simulation was used to determine the protein-protein interactions between hypoxia-inducible factor-1α(HIF-1α)and VHH212.The real-time polymerase chain reaction(PCR)and Western blot analyses were performed to identify the expressions of HIF-1αand VEGF-A in pancreatic ductal adenocarcinoma cell lines.The efficiency of the VHH212 nanobody in inhibiting the HIF-1 signaling pathway was measured using a dual-luciferase reporter assay.Finally,a PANC-1 xenograft model was developed to evaluate the anti-tumor efficiency of combined treatment.Immunohistochemistry analysis was conducted to detect the expressions of HIF-1αand VEGF-A in tumor tissues.Results:VHH212 was stably expressed in tumor cells with low cytotoxicity,high affinity,specific subcellular localization,and neutralization of HIF-1αin the cytoplasm or nucleus.The binding affinity between VHH212 and the HIF-1αPAS-B domain was 42.7 n M.Intrabody competitive inhibition of the HIF-1αheterodimer with an aryl hydrocarbon receptor nuclear translocator was used to inhibit the HIF-1/VEGF pathway in vitro.Compared with single agent gemcitabine,co-treatment with gemcitabine and a VHH212-encoding adenovirus significantly suppressed tumor growth in the xenograft model with 80.44%tumor inhibition.Conclusions:We developed an anti-HIF-1αnanobody and showed the function of VHH212 in a preclinical murine model of PANC-1 pancreatic cancer.The combination of VHH212 and gemcitabine significantly inhibited tumor development.These results suggested that combined use of anti-HIF-1αnanobodies with first-line treatment may in the future be an effective treatment for pancreatic cancer.
