5-Fluorocytosine combined with Fcy–hEGF fusion protein targets EGFR-expressing cancer cells

Lan, Keng Hsueh; Shih, Yi Sheng; Chang, C. Allen; Yen, Sang Hue; Lan, Keng Li

doi:10.1016/j.bbrc.2012.10.050

Cited by 4 publications

(7 citation statements)

References 17 publications

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“…Finally, the determined V max and K m values for CNGRC-yCD catalyzed 5-FC transformation to 5-FU reaction were 101.1 ± 5.2 μm/min/μg and 0.56 ± 0.09 mM, respectively, which were similar to those reported previously, i.e. the reported V max and K m values were 20 ~ 80 μm/min/μg and 0.4 ~ 0.8 mM, respectively [ 20 , 22 ]. These data together with the expression and purification method would provide useful guidance for further preclinical drug-candidate studies.…”

Section: Discussionsupporting

confidence: 87%

“…In the past, significant in vitro and in vivo studies on the inhibition of tumor growth and cell death have been reported after the mammalian cell transfection of CD gene therapy and administration of 5-FC [ 19 , 23 , 24 ]. On the other hand, our studies and those of others have demonstrated that the CD fusion protein conjugates of epidermal growth factor (EGF) [ 20 ] or endostatin [ 25 ] maintain high CD enzyme activity to convert 5-FC into 5-FU and have showed effective targeted antitumor potency.…”

Section: Introductionmentioning

confidence: 84%

“…Our previous studies and those of others have shown promising evidences about fusing CD with targeting molecules (e.g. EGF) for tumor treatment [ 17 – 20 , 25 ]. The results of this study showed that in the CNGRC-yCD/5-FC treatment group, the viabilities of high-APN-expressing human tumor cell lines and endothelial cells were significantly decreased compared to those of low-APN-expressing tumor cell lines.…”

Section: Discussionmentioning

confidence: 99%

“…The combined therapy employing cytosine deaminase (CD) and the nucleoside analog 5-fluorocytosine (5-FC) is an effective approach offered by the enzyme-prodrug system. CD was endogenously expressed in yeast and bacteria but not in mammalian cells and could efficiently convert the less-toxic 5-FC into the more-cytotoxic pyrimidine analog 5-fluorouracil (5-FU), which would lead to the inhibition of nucleotide and protein synthesis in tumor cells [ 17 – 20 ]. Moreover, it has been proposed that the transformation efficiency of 5-FC into 5-FU by yeast CD, i.e.…”

Section: Introductionmentioning

confidence: 99%

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Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine

Chang

Liau

et al. 2016

J Biomed Sci

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BackgroundThe enzyme-prodrug system is considered a promising tool for tumor treatment when conjugated with a targeting molecule. The asparagine-glycine-arginine (NGR) motif is a developing and interesting targeting peptide that could specifically bind to aminopeptidase N (APN), which is an NGR receptor expressed on the cell membrane of angiogenic endothelial cells and a number of tumor cells within the tumor tissues. The objective of this study was to develop a novel targeted enzyme-prodrug system using 5-fluorocytosine (5-FC) and an NGR-containing peptide fused with yeast cytosine deaminase (yCD), i.e. CNGRC-yCD fusion protein, to target APN-expressing cells within the tumor tissues and to convert 5-FC into 5-fluorouracil (5-FU) to kill tumors.ResultsBoth yCD and CNGRC-yCD proteins were cloned into the pET28a vector and expressed by an Escherichia coli host. Both yCD and CNGRC-yCD proteins were purified and the yields were approximately 20 mg/L with over 95 % purity. The binding assay demonstrated that the CNGRC-yCD fusion protein had specific binding affinity toward purified APN recombinant protein and high-APN-expressing cells, including human endothelial cells (HUVECs) and various types of human tumor cell lines, but not low-APN-expressing tumor cell lines. Moreover, the enzyme activity and cell viability assay showed that the CNGRC-yCD fusion protein could effectively convert 5-FC into 5-FU and resulted in significant cell death in both high-APN-expressing tumor cells and HUVECs.ConclusionsThis study successfully constructs a new targeting enzyme-prodrug system, CNGRC-yCD fusion protein/5-FC. Systematic experiments demonstrated that the CNGRC-yCD protein retained both the APN-binding affinity of NGR and the enzyme activity of yCD to convert 5-FC into 5-FU. The combined treatment of the CNGRC-yCD protein with 5-FC resulted in the significantly increased cell death of high-APN-expressing cells as compared to that of low-APN-expressing cells.

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Section: Discussionsupporting

confidence: 87%

Section: Introductionmentioning

confidence: 84%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine

Chang

Liau

et al. 2016

J Biomed Sci

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“…The authors showed that the viability of EGFR-overexpressing cells was significantly inhibited by Fcy-hEGF combined with 5-FC in a dose-dependent manner, and the values of IC50 for high EGFR-overexpressing cells were approximately 10-fold lower than those of low EGFR-overexpressing cells. This novel EGFR-targeting enzymatic prodrug system, Fcy-hEGF/5-FC, displayed potential to be a potent inhibitor for treatment of EGFR-overexpressing cancers, while reducing the severe side effects of 5-FU [24].…”

Section: Introductionmentioning

confidence: 99%

Bi-Functional Radiotheranostics of 188Re-Liposome-Fcy-hEGF for Radio- and Chemo-Therapy of EGFR-Overexpressing Cancer Cells

Huang

Hsu

Lin

et al. 2021

IJMS

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Epidermal growth factor receptor (EGFR) specific therapeutics is of great importance in cancer treatment. Fcy-hEGF fusion protein, composed of yeast cytosine deaminase (Fcy) and human EGF (hEGF), is capable of binding to EGFR and enzymatically convert 5-fluorocytosine (5-FC) to 1000-fold toxic 5-fluorocuracil (5-FU), thereby inhibiting the growth of EGFR-expressing tumor cells. To develop EGFR-specific therapy, 188Re-liposome-Fcy-hEGF was constructed by insertion of Fcy-hEGF fusion protein onto the surface of liposomes encapsulating of 188Re. Western blotting, MALDI-TOF, column size exclusion and flow cytometry were used to confirm the conjugation and bio-activity of 188Re-liposome-Fcy-hEGF. Cell lines with EGFR expression were subjected to treat with 188Re-liposome-Fcy-hEGF/5-FC in the presence of 5-FC. The 188Re-liposome-Fcy-hEGF/5-FC revealed a better cytotoxic effect for cancer cells than the treatment of liposome-Fcy-hEGF/5-FC or 188Re-liposome-Fcy-hEGF alone. The therapeutics has radio- and chemo-toxicity simultaneously and specifically target to EGFR-expression tumor cells, thereby achieving synergistic anticancer activity.

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Gene-Directed Enzyme/Prodrug Therapy of Rat Brain Tumor Mediated by Human Mesenchymal Stem Cell Suicide Gene Extracellular Vesicles In Vitro and In Vivo

Tibenský

Jakubechova

Altanerova³

et al. 2022

Cancers

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MSC-driven, gene-directed enzyme prodrug therapy (GDEPT) mediated by extracellular vesicles (EV) represents a new paradigm—cell-free GDEPT tumor therapy. In this study, we tested the efficacy of yeast cytosine deaminase::uracilphosphoribosyl transferase (yCD::UPRT-MSC)-exosomes, in the form of conditioned medium (CM) to inhibit the growth of C6 glioblastoma cells both in vitro and in vivo. MSCs isolated from human adipose tissue, umbilical cord, or dental pulp engineered to express the yCD::UPRT gene secreted yCD::UPRT-MSC-exosomes that in the presence of the prodrug 5-fluorocytosine (5-FC), inhibited the growth of rat C6 glioblastoma cells and human primary glioblastoma cells in vitro in a dose-dependent manner. CM from these cells injected repeatedly either intraperitoneally (i.p.) or subcutaneously (s.c.), applied intranasally (i.n.), or infused continuously by an ALZET osmotic pump, inhibited the growth of cerebral C6 glioblastomas in rats. A significant number of rats were cured when CM containing yCD::UPRT-MSC-exosomes conjugated with 5-FC was repeatedly injected i.p. or applied i.n. Cured rats were subsequently resistant to challenges with higher doses of C6 cells. Our data have shown that cell-free GDEPT tumor therapy mediated by the yCD::UPRT-MSC suicide gene EVs for high-grade glioblastomas represents a safer and more practical approach that is worthy of further investigation.

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5-Fluorocytosine combined with Fcy–hEGF fusion protein targets EGFR-expressing cancer cells

Cited by 4 publications

References 17 publications

Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine

Targeted antitumor prodrug therapy using CNGRC-yCD fusion protein in combination with 5-fluorocytosine

Bi-Functional Radiotheranostics of 188Re-Liposome-Fcy-hEGF for Radio- and Chemo-Therapy of EGFR-Overexpressing Cancer Cells

Gene-Directed Enzyme/Prodrug Therapy of Rat Brain Tumor Mediated by Human Mesenchymal Stem Cell Suicide Gene Extracellular Vesicles In Vitro and In Vivo

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