The Mechanism Of Protein Disulfide Isomerase PDI Regulating Autophagy And Radio/chemo-sensitivity

Tumor radiation resistance or chemotherapy resistance is one of the main challenges limiting the efficacy of tumor therapy.Radiation or chemotherapy drugs induced cellular regulatory processes such as DNA damage repair,autophagy,and metabolic reprogramming help cells survive,which is the fundamental reason for radiation resistance.Protein disulfide bond isomerase(PDI)is a sulfhydryl disulfide bond oxidoreductase mediated by the endoplasmic reticulum,which plays a key role in the occurrence and development of tumors.Our previous research found that PDI is a radiation therapy-resistant protein that blocks tumor cell apoptosis signals mainly by inducing mitochondrial dysfunction.Mitochondrial dysfunction is a major inducer of autophagy,but there is currently no research report on how PDI,as an autophagy protein,participates in tumor cell radiation resistance by regulating autophagy.Therefore,based on previous laboratory research,this study focuses on the regulation of tumor radiation sensitivity by PDI proteins.PDI has been proved to play an important role in various cancers,and high expression of PDI in tumor tissue is usually accompanied by the increase of tumor pathological grade.To clarify the regulatory role of PDI in colorectal cancer(CRC),we collected tumor samples from 30 patients with clinical colorectal cancer.Immunoblotting and immunohistochemical staining of the samples showed that the expression level of PDI protein in tumor tissue was significantly higher than that in adjacent tissues.We further analyzed the correlation between clinical pathological data and PDI expression levels in patients and found that the expression level of PDI was related to the survival status of patients.Next,we studied the effect of radiation on the expression level of PDI protein.The results showed that the expression level of PDI protein increased with the increase in the radiation dose,and the radiation induced a significant increase in the expression level of PDI in cell mitochondrial components.Specifically,knockdown of PDI significantly increased radiation or cisplatin induced cytotoxicity and apoptosis.Additionally,animal experiments also verified that knockdown of PDI combined with radiation therapy can significantly inhibit tumor growth in mice.This study preliminarily identified the important role of PDI as an autophagy regulatory factor in tumor radiosensitivity.Next,we continue to explore the molecular mechanism of PDI regulating cell death.As an endoplasmic reticulum resident protein,PDI plays a key role in the regulation of endoplasmic reticulum stress(ERS).On the one hand,this study found that PDI reduces the degradation of AKT by GRP78 by binding to GRP78,thereby relieving its inhibition of p-mTOR and inhibiting autophagy.These results indicate that knockdown of PDI expression can promote autophagy by regulating the classical AKT/mTOR pathway through GRP78.On the other hand,radiation or cisplatin can induce PDI protein translocation to mitochondria,while knocking down PDI can significantly increase radiation induced oxidative stress,downregulate mitochondrial membrane potential,and increase mitochondrial autophagy related protein expression.We found that mitophagy receptor PHB2 is the direct action protein of PDI through immunoprecipitation and mass spectrometry detection.PHB2,as a mitophagy receptor,mainly regulates autophagy by recruiting LC3Ⅱ.The results show that PDI can competitively bind PHB2 with LC3Ⅱ to inhibit the occurrence of mitophagy,and the thioredoxin-6 domain of PDI is the domain of interaction between PDI and PHB2.The above results suggest that knocking down the expression of PDI combined with radiation therapy can lead to excessive autophagy of cells,leading to autophagic cell death.The above results indicate that PDI can participate in the occurrence of autophagy by regulating the classic AKT/mTOR pathway,and can also participate in the regulation of autophagy through the PHB2 mediated mitochondrial autophagy pathway.This clarifies the new function of PDI in radio/chemotherapy induced cellular autophagy.Collectively,this study reveals the mechanism of PDI regulation of autophagy and mediates the radiosensitivity in tumor cells.This study provides a new insight into the understanding of mitochondria-targeted regulation of tumor radio/chemotherapy sensitivity,and provides a basis for the use of PDI inhibitors in clinical tumor adjuvant therapy.