Prolyl Hydroxylase Domain 3 Influences The Radiosensitivity Of Pancreatic Cancer Cell Mia-paca2 In Hypoxia

ObjectiveProlyl hydroxylase domain 3（PHD3）is a rate-limiting enzyme which regulates the degradation of Hypoxia-inducible factors（HIFs）.Lack of PHD3 allows tumor to overcome hypoxic growth inhibition and sustain radioresistance in pancreatic cancer.Herein,we focused on the impact of PHD3 expression in radiosensitivity ofpancreatic cancer.We also investigated the underlying molecular mechanisms ofPHD3 overexpression on the radiotherapy efficacy under either normoxic or hypoxic conditions.We aim to develop a novel target for pancreatic cancer radiotherapy.MethodsHuman pancreatic adenocarcinoma Mia-paca2 cells were used in this study.PHD3 was overexpressed in pancreatic cancer Mia-paca2 cells via lentiviralexpression.We divide Mia paca2 cells into four groups:Negative control group（NC）,PHD3 overexpression group（PHD3）,Irradiation group（IR）,PHD3 overexpression+Irradiation group（PHD3+IR）.Cells were maintained in a humidified incubatorcontaining 10%CO₂,20%O₂ or 1%O₂ at 37°C respectively and irradiated with asingle fraction of 2 Gy with high-energy radiation X-ray.Cell cycle progression and apoptosis were assayed by flow cytometry.HIF-1α,EGFR and PHD3 proteinexpression were assessed by Western blotting.Cell survival was determined in a colony formation assay.ResultsAfter 2Gy irradiation,the expression of HIF-1αand p-EGFR were suppressed by overexpression of PHD3 under normoxic/hypoxic conditions.With overexpression of PHD3 in Mia-paca2 cells,the number of monoclonal cells were decreased.With the increasing of radiation dose,the number of monoclonal cells also decreased undernormoxic/hypoxic conditions.The results of flow cytometry showed that the cell apoptosis was raised by hypoxic conditions compared to normoxic conditions.When overexpressed PHD3 cells were treated with 2Gy irradiation,the cell apoptosis rate was further increased compared to the control group and the single irradiation group.This status was firmly related to the cell cycles.PHD3 overexpression with irradiation treatment caused a significant increase in the percentage of cells in the G2/M phase compared with the percentage of untreated normal cells.Compared with the control group,combination group had a shorter S phase and longer G2/M phase.ConclusionIn summary,PHD3 overexpression enhanced the radiation-induced inhibition of Mia-paca2 cell proliferation,apoptosis,and cell cycle arrest at the G2/M phase by suppressing the EGFR/HIF-1αsignaling axis.The PHD3 expression may serve as a novel biomarker for improving radiosensitivity in pancreatic cancer.