Previous studies have shown that Arabidopsis equilibrative nucleoside transporters （AtENTs） possess transport activities when produced in yeast cells and are differentially expressed in Arabidopsis organs. Herein, w...Previous studies have shown that Arabidopsis equilibrative nucleoside transporters （AtENTs） possess transport activities when produced in yeast cells and are differentially expressed in Arabidopsis organs. Herein, we report further analysis on the nucleoside transport activities and transcriptional patterns of AtENT members. The recombinant proteins of AtENTs 3, 6, and 7, but not those of AtENTs 1, 2, 4, and 8, were found to transport thymidine with high affinity. Contrary to previ- ous suggestion that AtENT 1 may not transport uridine, this work showed that recombinant AtENT 1 was a pH-dependent and high-affinity transporter of uridine. When grown on MS plates, the AtENT3 knockout plants were more tolerant to the cytotoxic uridine analog 5-fluorouridine than wild-type plants and the knockout plants ofAtENT 1 or AtENT6. Con- sistent with this observation, the AtENT3 knockout line exhibited a significantly decreased ability to take up [^3H]uridine via the roots when compared with wild-type plants and the plants with mutated AtENT 1 or AtENT6. This indicates that AtENT3, but not AtENTs 1 and 6, is the main transporter for uridine uptake in Arabidopsis roots. The transcription of AtENTs 1, 3, 4, 6, 7, and 8 was regulated in a complex manner during leaf development and senescence. In contrast, the six AtENT members were coordinately induced during seed germination. This work provides new information on the transport properties of recombinant AtENT proteins and new clues for future studies of the in vivo transport activities and physiological functions of the different ENT proteins in Arabidopsis plants.展开更多
Resistance to gemcitabine in pancreatic cancer poses a significant clinical challenge.Further investigation is warranted to assess whether nano-formulation strategy can be employed to enhance the sensitivity of resist...Resistance to gemcitabine in pancreatic cancer poses a significant clinical challenge.Further investigation is warranted to assess whether nano-formulation strategy can be employed to enhance the sensitivity of resistant strains to gemcitabine therapy.In this study,using gemcitabine-resistant pancreatic cancer cell lines,we examined the therapeutic potential of a gemcitabine nanodelivery platform and assessed the ability to overcome drug resistance against resistant strains.Silencing of human equilibrative nucleoside transporter 1(hENT1)led to reduced cellular uptake of gemcitabine,resulting in chemoresistance in pancreatic cancer.Gemcitabine nanoparticles circumvented the entry blockade caused by hENT1 silencing through endocytosis.Nanoparticle entry via clathrin-mediated endocytosis increased intracellular gemcitabine accumulation in gemcitabine-resistant pancreatic cancer cells.Moreover,gemcitabine nanoparticles are preferential in vivo delivery to tumor tissues,likely due to the enhanced permeability and retention effect.In comparison to free gemcitabine,gemcitabine nanoparticles demonstrate a more pronounced cytotoxic effect on gemcitabine-resistant pancreatic cancer cells,with favorable biosafety.This study improved the efficacy of gemcitabine through nanotechnology,providing a novel strategy to address gemcitabine-resistant pancreatic cancer.展开更多
Aim:Gemcitabine is a frontline agent for locally-advanced and metastatic pancreatic ductal adenocarcinoma(PDAC),but neither gemcitabine alone nor in combination produces durable remissions of this tumor type.We develo...Aim:Gemcitabine is a frontline agent for locally-advanced and metastatic pancreatic ductal adenocarcinoma(PDAC),but neither gemcitabine alone nor in combination produces durable remissions of this tumor type.We developed three PDAC patient-derived xenograft(PDX)models with gemcitabine resistance(gemR)acquired in vivo,with which to identify mechanisms of resistance relevant to drug exposure in vivo and to evaluate novel therapies.Methods:Mice bearing independently-derived PDXs received 100 mg/kg gemcitabine once or twice weekly.Tumors initially responded,but regrew on treatment and were designated gemR.We used immunohistochemistry to compare expression of proteins previously associated with gemcitabine resistance[ribonucleotide reductase subunit M1(RRM1),RRM2,human concentrative nucleoside transporter 1(hCNT1),human equilibrative nucleoside transporter 1(hENT1),cytidine deaminase(CDA),and deoxycytidine kinase(dCK)]in gemR and respective gemcitabine-naïve parental tumors.Results:Parental and gemR tumors did not differ in tumor cell morphology,amount of tumor-associated stroma,or expression of stem cell markers.No consistent pattern of expression of the six gemR marker proteins was observed among the models.Increases in RRM1 and CDA were consistent with in vitro-derived gemR models.However,rather than the expected decreases of hCNT1,hENT1,and dCK,gemR tumors expressed no change in or higher levels of these gemR marker proteins than parental tumors.Conclusion:These models are the first PDAC PDX models with gemcitabine resistance acquired in vivo.The data indicate that mechanisms identified in models with resistance acquired in vitro are unlikely to be the predominant mechanisms when resistance is acquired in vivo.Ongoing work focuses on characterizing unidentified mechanisms of gemR and on identifying agents with anti-tumor efficacy in these gemR models。展开更多
Gemcitabine is a cytidine analogue frequently used in the treatment of various cancers.However,the development of chemoresistance limits its effectiveness.Gemcitabine resistance is regulated by various factors,includi...Gemcitabine is a cytidine analogue frequently used in the treatment of various cancers.However,the development of chemoresistance limits its effectiveness.Gemcitabine resistance is regulated by various factors,including aberrant genetic and epigenetic controls,metabolism of gemcitabine,the microenvironment,epithelial-to-mesenchymal transition,and acquisition of cancer stem cell properties.In many situations,results using cell lines offer valuable lessons leading to the first steps of important findings.In this review,we mainly discuss the factors involved in gemcitabine metabolism in association with chemoresistance,including nucleoside transporters,deoxycytidine kinase,cytidine deaminase,and ATP-binding cassette transporters,and outline new perspectives for enhancing the efficacy of gemcitabine to overcome acquired chemoresistance.展开更多
Pancreatic ductal adenocarcinoma(PDA) is one of the major causes for cancer death worldwide. Treatment of metastatic disease remains challenging as only certain patients benefit from advances made with the intensified...Pancreatic ductal adenocarcinoma(PDA) is one of the major causes for cancer death worldwide. Treatment of metastatic disease remains challenging as only certain patients benefit from advances made with the intensified chemotherapy regimen folinic acid, irinotecan and oxaliplatin, the epidermal growth factor receptor inhibitor erlotinib or the recently FDA-approved nab-paclitaxel. Up to date, no established approach for prediction of treatment response or specific treatment allocation exists. Translational research was able to identify a number of potential biomarkers that might help to improve the dismal prognosis of PDA by facilitating upfront treatment allocation. This topic highlight is focused on current evidence on potential biomarkers for tumor biology, prognosis and prediction of treatment efficacy.展开更多
: The plant phytohormone cytokinin plays an important role in many facets of plant growth and development by regulating cell division and differentiation. Recent studies have shed significant light into the mechanisms...: The plant phytohormone cytokinin plays an important role in many facets of plant growth and development by regulating cell division and differentiation. Recent studies have shed significant light into the mechanisms of cytokinin metabolism and signaling. However, little is known about how the hormone is transported in planta, although it has been proposed that the hormone is presumably transported in nucleoside-conjugated forms. Here, we report the identification and characterization of cytokinin transporters in Arabidopsis. We previously reported that a gain-of-function mutation in the PGA22/AtIPT8 gene caused overproduction of cytokinins in planta. In an effort to screen for suppressor of pga 22/atipt 8 (soi) mutants, we identified a mutant soi33-1. Molecular and genetic analyses indicated that SOI33 encodes a putative equilibrative nucleoside transporter (ENT), previously designated as AtENT8. Members of this small gene family are presumed to be involved in the transport of nucleosides in eukaryotic cells. Under conditions of nitrogen starvation, loss-of-function mutations in SOI33/AtENT8 or in a related gene AtENT3 cause a reduced sensitivity to the nucleoside-type cytokinins isopentenyladenine riboside (iPR) and transzeatin riboside (tZR), but display a normal response to the free base-type cytokinins isopentenyladenine (iP) and trans-zeatin (tZ). Conversely, overexpression of SOI33/AtENT8 renders transgenic plants hypersensitive to iPR but not to iP. An in planta measurement experiment indicated that uptake efficiency of 3H-labeled iPR was reduced more than 40% in soi33 and atent3 mutants. However, a mutation in AtENT1 had no substantial effect on the cytokinin response and iPR uptake efficiency. Our results suggest that SOI33/ AtENT8 and AtENT3 are involved in the transport of nucleoside-type cytokinins in Arabidopsis.展开更多
基金This work was supported by grants from the National Natural Science Foundation of China （39770491） the Chinese Academy of Sciences （KSCX2-SW-304）.
文摘Previous studies have shown that Arabidopsis equilibrative nucleoside transporters （AtENTs） possess transport activities when produced in yeast cells and are differentially expressed in Arabidopsis organs. Herein, we report further analysis on the nucleoside transport activities and transcriptional patterns of AtENT members. The recombinant proteins of AtENTs 3, 6, and 7, but not those of AtENTs 1, 2, 4, and 8, were found to transport thymidine with high affinity. Contrary to previ- ous suggestion that AtENT 1 may not transport uridine, this work showed that recombinant AtENT 1 was a pH-dependent and high-affinity transporter of uridine. When grown on MS plates, the AtENT3 knockout plants were more tolerant to the cytotoxic uridine analog 5-fluorouridine than wild-type plants and the knockout plants ofAtENT 1 or AtENT6. Con- sistent with this observation, the AtENT3 knockout line exhibited a significantly decreased ability to take up [^3H]uridine via the roots when compared with wild-type plants and the plants with mutated AtENT 1 or AtENT6. This indicates that AtENT3, but not AtENTs 1 and 6, is the main transporter for uridine uptake in Arabidopsis roots. The transcription of AtENTs 1, 3, 4, 6, 7, and 8 was regulated in a complex manner during leaf development and senescence. In contrast, the six AtENT members were coordinately induced during seed germination. This work provides new information on the transport properties of recombinant AtENT proteins and new clues for future studies of the in vivo transport activities and physiological functions of the different ENT proteins in Arabidopsis plants.
基金supported by the National Natural Science Foundation of China(Nos.82273490,82073296,and 81773193 to Hangxiang Wang,No.81721091 to Shusen Zheng)Research Project of Jinan Microecological Biomedicine Shandong Laboratory(No.JNL-2022010B to Hangxiang Wang)+1 种基金Natural Science Foundation of Shandong Province(No.ZR2023ZD59 to Hangxiang Wang)Zhejiang Provincial Natural Science Foundation of China(No.LR19H160002 to Hangxiang Wang).
文摘Resistance to gemcitabine in pancreatic cancer poses a significant clinical challenge.Further investigation is warranted to assess whether nano-formulation strategy can be employed to enhance the sensitivity of resistant strains to gemcitabine therapy.In this study,using gemcitabine-resistant pancreatic cancer cell lines,we examined the therapeutic potential of a gemcitabine nanodelivery platform and assessed the ability to overcome drug resistance against resistant strains.Silencing of human equilibrative nucleoside transporter 1(hENT1)led to reduced cellular uptake of gemcitabine,resulting in chemoresistance in pancreatic cancer.Gemcitabine nanoparticles circumvented the entry blockade caused by hENT1 silencing through endocytosis.Nanoparticle entry via clathrin-mediated endocytosis increased intracellular gemcitabine accumulation in gemcitabine-resistant pancreatic cancer cells.Moreover,gemcitabine nanoparticles are preferential in vivo delivery to tumor tissues,likely due to the enhanced permeability and retention effect.In comparison to free gemcitabine,gemcitabine nanoparticles demonstrate a more pronounced cytotoxic effect on gemcitabine-resistant pancreatic cancer cells,with favorable biosafety.This study improved the efficacy of gemcitabine through nanotechnology,providing a novel strategy to address gemcitabine-resistant pancreatic cancer.
文摘Aim:Gemcitabine is a frontline agent for locally-advanced and metastatic pancreatic ductal adenocarcinoma(PDAC),but neither gemcitabine alone nor in combination produces durable remissions of this tumor type.We developed three PDAC patient-derived xenograft(PDX)models with gemcitabine resistance(gemR)acquired in vivo,with which to identify mechanisms of resistance relevant to drug exposure in vivo and to evaluate novel therapies.Methods:Mice bearing independently-derived PDXs received 100 mg/kg gemcitabine once or twice weekly.Tumors initially responded,but regrew on treatment and were designated gemR.We used immunohistochemistry to compare expression of proteins previously associated with gemcitabine resistance[ribonucleotide reductase subunit M1(RRM1),RRM2,human concentrative nucleoside transporter 1(hCNT1),human equilibrative nucleoside transporter 1(hENT1),cytidine deaminase(CDA),and deoxycytidine kinase(dCK)]in gemR and respective gemcitabine-naïve parental tumors.Results:Parental and gemR tumors did not differ in tumor cell morphology,amount of tumor-associated stroma,or expression of stem cell markers.No consistent pattern of expression of the six gemR marker proteins was observed among the models.Increases in RRM1 and CDA were consistent with in vitro-derived gemR models.However,rather than the expected decreases of hCNT1,hENT1,and dCK,gemR tumors expressed no change in or higher levels of these gemR marker proteins than parental tumors.Conclusion:These models are the first PDAC PDX models with gemcitabine resistance acquired in vivo.The data indicate that mechanisms identified in models with resistance acquired in vitro are unlikely to be the predominant mechanisms when resistance is acquired in vivo.Ongoing work focuses on characterizing unidentified mechanisms of gemR and on identifying agents with anti-tumor efficacy in these gemR models。
文摘Gemcitabine is a cytidine analogue frequently used in the treatment of various cancers.However,the development of chemoresistance limits its effectiveness.Gemcitabine resistance is regulated by various factors,including aberrant genetic and epigenetic controls,metabolism of gemcitabine,the microenvironment,epithelial-to-mesenchymal transition,and acquisition of cancer stem cell properties.In many situations,results using cell lines offer valuable lessons leading to the first steps of important findings.In this review,we mainly discuss the factors involved in gemcitabine metabolism in association with chemoresistance,including nucleoside transporters,deoxycytidine kinase,cytidine deaminase,and ATP-binding cassette transporters,and outline new perspectives for enhancing the efficacy of gemcitabine to overcome acquired chemoresistance.
文摘Pancreatic ductal adenocarcinoma(PDA) is one of the major causes for cancer death worldwide. Treatment of metastatic disease remains challenging as only certain patients benefit from advances made with the intensified chemotherapy regimen folinic acid, irinotecan and oxaliplatin, the epidermal growth factor receptor inhibitor erlotinib or the recently FDA-approved nab-paclitaxel. Up to date, no established approach for prediction of treatment response or specific treatment allocation exists. Translational research was able to identify a number of potential biomarkers that might help to improve the dismal prognosis of PDA by facilitating upfront treatment allocation. This topic highlight is focused on current evidence on potential biomarkers for tumor biology, prognosis and prediction of treatment efficacy.
基金国家自然科学基金，中国科学院知识创新工程项目，科技部资助项目，the Ministry of Education, Culture, Sports, Science，Grants-in-Aids for Scientific Research on Functional Analysis of Genes Relevant to Agriculturally Important Traits in Rice Genome，中国科学院'百人计划'，国家自然科学基金
文摘: The plant phytohormone cytokinin plays an important role in many facets of plant growth and development by regulating cell division and differentiation. Recent studies have shed significant light into the mechanisms of cytokinin metabolism and signaling. However, little is known about how the hormone is transported in planta, although it has been proposed that the hormone is presumably transported in nucleoside-conjugated forms. Here, we report the identification and characterization of cytokinin transporters in Arabidopsis. We previously reported that a gain-of-function mutation in the PGA22/AtIPT8 gene caused overproduction of cytokinins in planta. In an effort to screen for suppressor of pga 22/atipt 8 (soi) mutants, we identified a mutant soi33-1. Molecular and genetic analyses indicated that SOI33 encodes a putative equilibrative nucleoside transporter (ENT), previously designated as AtENT8. Members of this small gene family are presumed to be involved in the transport of nucleosides in eukaryotic cells. Under conditions of nitrogen starvation, loss-of-function mutations in SOI33/AtENT8 or in a related gene AtENT3 cause a reduced sensitivity to the nucleoside-type cytokinins isopentenyladenine riboside (iPR) and transzeatin riboside (tZR), but display a normal response to the free base-type cytokinins isopentenyladenine (iP) and trans-zeatin (tZ). Conversely, overexpression of SOI33/AtENT8 renders transgenic plants hypersensitive to iPR but not to iP. An in planta measurement experiment indicated that uptake efficiency of 3H-labeled iPR was reduced more than 40% in soi33 and atent3 mutants. However, a mutation in AtENT1 had no substantial effect on the cytokinin response and iPR uptake efficiency. Our results suggest that SOI33/ AtENT8 and AtENT3 are involved in the transport of nucleoside-type cytokinins in Arabidopsis.
