The critical roles of oxygen homeostasis in metabolism are indisputable and hypoxic responses are correlated with the pathogenesis of gastrointestinal, pulmonary, renal diseases and cancers. Evaluating tissue hypoxia ...The critical roles of oxygen homeostasis in metabolism are indisputable and hypoxic responses are correlated with the pathogenesis of gastrointestinal, pulmonary, renal diseases and cancers. Evaluating tissue hypoxia to predict treatment outcome is challenging, however, due to the lack of rapid, accurate and non-invasive methods. Hypoxia enhances prolyl-4-hydroxylase a1(P4HA1) expression, which can convert bradykinin(BK) to hydroxyprolyl-BK(Hyp-BK), leading us to hypothesize that circulating Hyp-BK/BK ratios may reflect tissue hypoxia and predict treatment outcomes. Direct quantification of Hyp-BK peptides in serum or plasma by conventional MALDI-TOF MS analysis is technically challenging. In our study, a nanopore-based fractionation and enrichment protocol was utilized to allow the simple workflow for circulating Hyp-BK/BK analysis. Hypoxia is linked to poor prognosis due to its role in promoting pancreatic cancer progression and metastasis. Here we show that P4HA1 expression was increased in pancreatic tumors versus adjacent tissue, associated with poor survival, and corresponded with tumor expression of the hypoxia inducible factor 1a(HIF-1a) and carbonic anhydrase 9(CA9). Hypoxiainduced P4HA1 expression and BK conversion to Hyp-BK were found to be HIF-1 a dependent, pretreatment serum Hyp-BK/BK ratios corresponded with tissue HIF-1 a and P4HA1 expression, and high Hyp-BK/BK levels corresponded with poor response to therapy. These results suggest that pretreatment circulating Hyp-BK/BK ratios may have value as a non-invasive, surrogate indicator of tissue hypoxia and tumor responses to therapy.展开更多
Circulating peptide is a potential source of biomarkers for cancer detection.However,the existence of large molecular weight proteins in plasma have a disastrous effect on circulating peptides isolating and detecting....Circulating peptide is a potential source of biomarkers for cancer detection.However,the existence of large molecular weight proteins in plasma have a disastrous effect on circulating peptides isolating and detecting.Herein,nanotrap fractionation following by mass spectrometry have been applied to quantify the levels of bradykinin (BK) and hydroxylated bradykinin (Hyp-BK) as a relative measure of KRAS-regulated prolyl-4-hydroxylase alpha-1 (P4HA1) which may serve as early diagnosis marker for pancreatic ductal adenocarcinoma (PDAC).We found that P4HA1 can be upregulated by KRASG12v,which is a PDAC driver mutation,using HPNE/KRAS and HPNE cells.And we revealed that P4HA1 is overexpressed in PDAC tumors,compared to normal and inflamed pancreatic tissues.RNA interference revealed that P4HA1 activity was primarily responsible for Hyp-BK production.Mass spectrometry analysis revealed that plasma Hyp-BK/BK ratio was higher in PDAC than pancreatitis patients and healthy controls,while the area under the receiver operating characteristic (ROC) curve (AUC) is 0.8209 (95%Cl,0.7269-0.9149).The Hyp-BK/BK association with PDAC was reproduced in another cohort,where this ratio was found to increase with advancing tumor stage.These novel findings paved the way for wider applications of Nanotrap coupled mass spectrometry as a powerful tool for revealing biosignatures from plasma.展开更多
基金the Arizona Biomedical Research Commission(ABRC)young investigator awardthe Fred Hutchinson Cancer Research Center(0000917241)Tulane University Startup fund。
文摘The critical roles of oxygen homeostasis in metabolism are indisputable and hypoxic responses are correlated with the pathogenesis of gastrointestinal, pulmonary, renal diseases and cancers. Evaluating tissue hypoxia to predict treatment outcome is challenging, however, due to the lack of rapid, accurate and non-invasive methods. Hypoxia enhances prolyl-4-hydroxylase a1(P4HA1) expression, which can convert bradykinin(BK) to hydroxyprolyl-BK(Hyp-BK), leading us to hypothesize that circulating Hyp-BK/BK ratios may reflect tissue hypoxia and predict treatment outcomes. Direct quantification of Hyp-BK peptides in serum or plasma by conventional MALDI-TOF MS analysis is technically challenging. In our study, a nanopore-based fractionation and enrichment protocol was utilized to allow the simple workflow for circulating Hyp-BK/BK analysis. Hypoxia is linked to poor prognosis due to its role in promoting pancreatic cancer progression and metastasis. Here we show that P4HA1 expression was increased in pancreatic tumors versus adjacent tissue, associated with poor survival, and corresponded with tumor expression of the hypoxia inducible factor 1a(HIF-1a) and carbonic anhydrase 9(CA9). Hypoxiainduced P4HA1 expression and BK conversion to Hyp-BK were found to be HIF-1 a dependent, pretreatment serum Hyp-BK/BK ratios corresponded with tissue HIF-1 a and P4HA1 expression, and high Hyp-BK/BK levels corresponded with poor response to therapy. These results suggest that pretreatment circulating Hyp-BK/BK ratios may have value as a non-invasive, surrogate indicator of tissue hypoxia and tumor responses to therapy.
文摘Circulating peptide is a potential source of biomarkers for cancer detection.However,the existence of large molecular weight proteins in plasma have a disastrous effect on circulating peptides isolating and detecting.Herein,nanotrap fractionation following by mass spectrometry have been applied to quantify the levels of bradykinin (BK) and hydroxylated bradykinin (Hyp-BK) as a relative measure of KRAS-regulated prolyl-4-hydroxylase alpha-1 (P4HA1) which may serve as early diagnosis marker for pancreatic ductal adenocarcinoma (PDAC).We found that P4HA1 can be upregulated by KRASG12v,which is a PDAC driver mutation,using HPNE/KRAS and HPNE cells.And we revealed that P4HA1 is overexpressed in PDAC tumors,compared to normal and inflamed pancreatic tissues.RNA interference revealed that P4HA1 activity was primarily responsible for Hyp-BK production.Mass spectrometry analysis revealed that plasma Hyp-BK/BK ratio was higher in PDAC than pancreatitis patients and healthy controls,while the area under the receiver operating characteristic (ROC) curve (AUC) is 0.8209 (95%Cl,0.7269-0.9149).The Hyp-BK/BK association with PDAC was reproduced in another cohort,where this ratio was found to increase with advancing tumor stage.These novel findings paved the way for wider applications of Nanotrap coupled mass spectrometry as a powerful tool for revealing biosignatures from plasma.
