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Cell Death & Disease Jan 2018
Topics: Animals; Antimetabolites, Antineoplastic; Humans; Mice; Protein Biosynthesis; Puromycin
PubMed: 29348556
DOI: 10.1038/s41419-017-0056-x -
Asian Pacific Journal of Cancer... May 2022Cancer is life-threatening disease and being global health problems. Chemotherapy is one of the most used therapy for cancer since many years ago. Chemotherapy is also...
UNLABELLED
Cancer is life-threatening disease and being global health problems. Chemotherapy is one of the most used therapy for cancer since many years ago. Chemotherapy is also toxic for normal cell, not specific to the target cells. Consequently, chemotherapy has various side effects. Monoclonal antibody (MAb) has been developed for specific therapy which only has killing effect in cancer cells, but the survival rate of most MAbs around 20%. Therefore, in clinical practice, MAbs administration should combine with chemotherapeutic agents. For effectiveness of therapy and to minimalize adverse effects, anticancer agent with selective cytotoxic effect on target cells is needed, the immunotoxin.
OBJECTIVE
This study introduces a novel approach to conjugate monoclonal antibody (Cetuximab) and toxin (Puromycin), in order to selectively inhibit proliferation of triple negative breast cancer (TNBC) and to enhance the efficacy of MAb in target cells killing.
METHODS
Cetuximab was conjugated with Puromycin using a linker, i.e SATP (Succinimidyl-acetylthiopropionate) and tested on triple negative breast cancer cell lines (MDA-MB-231) which expressed EGFR (epidermal growth factor receptor). Cetuximab is MAb which targets EGFR. MCF-7 was used as control cells since it has low or no EGFR expression. Cell counting were conducted as viability assay at 24 hours, 48 hours, and 72 hours after treatment.
RESULTS
The results showed significant reduction of live cells number in Conjugate 20 µg/mL cultured in MDA-MB-231 compared to MCF-7 after 24 hours, 48 hours, and 72 hours incubation. In all time period of incubation, significant reduction of MDA-MB-231 live cells number was also observed in Conjugate 20 µg/mL compared to Cetuximab 20 µg/mL.
CONCLUSION
Synthesized conjugate showed its target-specific effect in TNBC and improved the efficacy of Cetuximab on TNBC. In the future, this conjugate can be a potential anticancer therapy in treating triple-negative breast cancer.
Topics: Antibodies, Monoclonal; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cetuximab; ErbB Receptors; Humans; Puromycin; Triple Negative Breast Neoplasms
PubMed: 35633567
DOI: 10.31557/APJCP.2022.23.5.1803 -
The Journal of International Medical... Dec 2020To investigate the mechanism through which tacrolimus, often used to treat refractory nephropathy, protects against puromycin-induced podocyte injury.
OBJECTIVE
To investigate the mechanism through which tacrolimus, often used to treat refractory nephropathy, protects against puromycin-induced podocyte injury.
METHODS
An model of puromycin-induced podocyte injury was established by dividing podocytes into three groups: controls, puromycin only (PAN group), and puromycin plus tacrolimus (FK506 group). Podocyte morphology, number, apoptosis rate and microtubule associated protein 1 light chain 3 alpha () expression were compared.
RESULTS
Puromycin caused podocyte cell body shrinkage and loose intercellular connections, but podocyte morphology in the FK506 group was similar to controls. The apoptosis rate was lower in the FK506 group versus PAN group. The low level of LC3 mRNA observed in untreated podocytes was decreased by puromycin treatment; however, levels of LC3 mRNA were higher in the FK506 group versus PAN group. Although LC3-I and LC3-II protein levels were decreased by puromycin, levels in the FK506 group were higher than the PAN group. Fewer podocyte autophagosomes were observed in the control and FK506 groups versus the PAN group. Cytoplasmic LC3-related fluorescence intensity was stronger in control and FK506 podocytes versus the PAN group.
CONCLUSIONS
Tacrolimus inhibited puromycin-induced mouse podocyte damage by regulating expression and enhancing autophagy.
Topics: Animals; Apoptosis; Autophagy; Mice; Podocytes; Puromycin; Puromycin Aminonucleoside; Tacrolimus
PubMed: 33322998
DOI: 10.1177/0300060520971422 -
Cell Reports Methods Apr 2022The regulation of gene expression via protein translation is critical for growth, development, and stress response. While puromycin-based techniques have been used to...
The regulation of gene expression via protein translation is critical for growth, development, and stress response. While puromycin-based techniques have been used to quantify protein translation in , they have been limited to using lysate from whole worms. To achieve tissue-specific quantification of ribosome activity in intact , we report the application of O-propargyl-puromycin in a cuticle defective mutant followed by conjugation of an azide fluorophore for detection using fluorescent confocal microscopy. We apply this technique to quantify translation in response to heat shock, cycloheximide, or knockdown of translation factors Furthermore, we demonstrate that O-propargyl-puromycin can be used to quantify translation between tissues or within a tissue like the germline. This technique is expected to have a broad range of applications in determining how protein translation is altered in different tissues in response to stress or gene knockdowns or with age.
Topics: Animals; Caenorhabditis elegans; Protein Biosynthesis; Puromycin; Microscopy, Fluorescence
PubMed: 35497499
DOI: 10.1016/j.crmeth.2022.100203 -
A New Photocaged Puromycin for an Efficient Labeling of Newly Translated Proteins in Living Neurons.Chembiochem : a European Journal of... Dec 2018Monitoring newly synthesized proteins is becoming increasingly important to characterize proteome composition in regulatory networks. Puromycin is a peptidyl transfer...
Monitoring newly synthesized proteins is becoming increasingly important to characterize proteome composition in regulatory networks. Puromycin is a peptidyl transfer inhibitor, widely used in cell biology for tagging newly synthesized proteins. Here, we report synthesis and application of an optimized puromycin carrying a photolabile protecting group as a powerful tool for tagging nascent proteins with high spatiotemporal resolution. The photocaged 7-N,N-(diethylaminocumarin-4-yl)-methoxycarbonyl-puromycin (DEACM-puromycin) was synthesized and compared with the previously developed 6-nitroveratryloxycarbonyl puromycin (NVOC-puromycin). The photochemical behavior as well as the effectiveness in controlling puromycylation in living hippocampal neurons using two-photon excitation is superior to the previously used NVOCpuromycin. We further report on the application of light-controlled puromycylation to visualize new translated proteins in neurons.
Topics: Animals; Cell Survival; Coumarins; Hippocampus; Molecular Probes; Neurons; Proteins; Puromycin; Rats; Ultraviolet Rays
PubMed: 30311996
DOI: 10.1002/cbic.201800408 -
Science (New York, N.Y.) Dec 1967A first experiment compared the behavior of goldfish injected with puromycin immediately after each of a weekly series of brief discriminative training sessions in the...
A first experiment compared the behavior of goldfish injected with puromycin immediately after each of a weekly series of brief discriminative training sessions in the shuttlebox to that of appropriate controls. Discrimination was not prevented, nor was escape from shock impaired, but probability of response to the conditioned stimuli, both positive and negative, was reduced substantially. These results suggest that puromycin interferes with the consolidation of conditioned fear. The null outcome of a second experiment, in which all training was given in a single long session instead of a series of short sessions, suggests (contrary to recent indications) that consolidation begins in the training session. The conditioned-fear hypothesis is supported by the results of a third experiment in which the animals were shocked upon entering a goalbox to which they had previously learned to swim for food; animals injected with puromycin, immediately after the shock, entered the goalbox more readily 1 week later than did appropriate controls.
Topics: Animals; Behavior, Animal; Conditioning, Psychological; Cyprinidae; Discrimination Learning; Puromycin; Visual Perception
PubMed: 6060367
DOI: 10.1126/science.158.3808.1594 -
Angewandte Chemie (International Ed. in... Apr 2016Newly synthesized proteins constitute an important subset of the proteome involved in every cellular process, yet existing chemical tools used to study them have major...
Newly synthesized proteins constitute an important subset of the proteome involved in every cellular process, yet existing chemical tools used to study them have major shortcomings. Herein we report a suite of cell-permeable puromycin analogues capable of being metabolically incorporated into newly synthesized proteins in different mammalian cells, including neuronal cells. Subsequent labeling with suitable bioorthogonal reporters, in both fixed and live cells, enabled direct imaging and enrichment of these proteins. By taking advantage of the mutually orthogonal reactivity of these analogues, we showed multiplexed labeling of different protein populations, as well as quantitative measurements of protein dynamics by fluorescence correlation spectroscopy, could be achieved in live-cell environments.
Topics: HeLa Cells; Humans; Neurons; Protein Biosynthesis; Puromycin
PubMed: 26971527
DOI: 10.1002/anie.201511030 -
Science (New York, N.Y.) Feb 1966Mice injected bitemporally with puromycin 5 hours before training learned to escape or to avoid shock by choosing the correct limb of a Y-maze. When retested 15 minutes...
Mice injected bitemporally with puromycin 5 hours before training learned to escape or to avoid shock by choosing the correct limb of a Y-maze. When retested 15 minutes after training they had normal retention. In the ensuing 2(3/4) hours the animals injected with puromycin, unlike the controls, showed a progressive decrease of savings to less than 7 percent.
Topics: Animals; Avoidance Learning; Behavior, Animal; Memory; Mice; Psychopharmacology; Puromycin
PubMed: 5903589
DOI: 10.1126/science.151.3710.594 -
STAR Protocols Dec 2023Translation is a fundamental process of cellular behavior. Here, we present a protocol for measuring translation in Drosophila epithelial tissues using...
Translation is a fundamental process of cellular behavior. Here, we present a protocol for measuring translation in Drosophila epithelial tissues using O-propargyl-puromycin (OPP), a puromycin derivative. We detail steps for larval dissection, OPP incorporation, fixation, OPP labeling, immunostaining, and imaging. We also provide details of quantification analysis. Significantly, OPP addition to methionine-containing media enables polypeptide labeling in living cells. Here, we study wing imaginal discs, an excellent model system for investigating growth, proliferation, pattern formation, differentiation, and cell death. For complete details on the use and execution of this protocol, please refer to Lee et al. (2018), Ji et al. (2019), and Kiparaki et al. (2022)..
Topics: Animals; Drosophila; Imaginal Discs; Larva; Puromycin
PubMed: 37862174
DOI: 10.1016/j.xpro.2023.102653 -
Psychological Reports Apr 1972
Topics: Animals; Avoidance Learning; Bufonidae; Memory; Puromycin
PubMed: 5024944
DOI: 10.2466/pr0.1972.30.2.627