Puromycin – Anti-Tumor Effect and Activity

November 1, 2018 Categories AG Scientific Blog
Photo by Louis Reed on Unsplash

Puromycin – Anti-Tumor Effect and Activity

What is Puromycin?

puromycinPuromycin is an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. It inhibits protein synthesis by disrupting peptide transfer on ribosomes causing premature chain termination during translation. It is a potent translational inhibitor in both prokaryotic and eukaryotic cells. Resistance to puromycin is conferred by the puromycin N-acetyl-transferase gene (pac) from Streptomyces. Puromycin has a fast mode of action, causing rapid cell death at low antibiotic concentrations.

Antitumor Effect:

Puromycin has been tested against a variety of experimental tumors in various animals. As might be expected, a range of effects from the destructive or inhibitory to mild or no activity was obtained, depending upon the type of tumor and dosage. In some cases toxicity accompanied carcinostatis, since peak activity was only apparent at maximum tolerated doses. Puromycin showed significant cytotoxicity in vitro against a series of tissue cultures derived from normal and neoplastic human and animal cells.

In an agar plate assay procedure for anti-tumor agents, which employed four human cell lines and sarcoma 180 of mouse origin, it was found that the human cell lines were about 2-4 times more sensitive to puromycin than was sarcoma 180 (Schuurmans et al. 1961).

Mammalian cells:

The working concentrations of puromycin for mammalian cell lines range from 1 to 10 µg/ml. It is recommended that an initial experiment be done to determine the optimal concentration of puromycin required to kill your host cell line. Puromycin quickly kills eukaryotic cells that do not contain the pac gene. Dying cells detach from the plates allowing easy and early identification of transformant clones. Suggested working conditions for selection in some mammalian cells are listed below:

Cell line Species

Tissue

Culture medium

Puromycin (µg/ml)

293 Human

Tranformed primary embryonal kidney

DMEM

3

HeLa Human

Epitheloid carcinoma, cervix

DMEM

3

B16 Mouse

Melanoma

RPMI

1-3

PC1.0 Hamster

Pancreatic adenocarcinoma

RPMI

10

Escherichia coli:

Puromycin-resistant transformants are selected in LB agar medium supplemented with 125 µg/ml of puromycin. But use of puromycin for E. coli selection requires precise pH adjustment and also depends on which strain is selected. Plates containing puromycin are stable for 1 month when stored at 4°C.

puromycinecoli

 

Recognition of the puromycin by E. coli L/F-transferase. (A) Chemical structure of puromycin (left) and that of the 3′-ends of Leu-tRNALeu and Phe-tRNAPhe (middle and right, respectively). The amino-acid moiety and the base moiety are colored pink and blue, respectively. (B) |Fo-Fc| omit map of puromycin (contour level 3.0sigma). (C) Recognition of the p-methoxybenzyl group and the puromycin base by the hydrophobic pocket, as shown by a surface model. (D) Ribbon model of (C). The hydrophobic amino acid involved in the recognition of the p-methoxybenzyl group and the base moiety of puromycin are colored green and blue, respectively. (E) The C-shaped edge of the hydrophobic pocket is composed of continuous amino-acid residues (Gly155-Glu156-Ser157-Met158; colored yellow and highlighted). The alpha-, beta– and gamma-carbons of puromycin are also shown.

Neprosis:

 
There is evidence of kidney damage in rats after repeated injections of puromycin for one to four weeks (Hewitt et al. 1953, Sherman et al. 1954, Borowsky et al. 1958). Mephrosis was manifested by elevated serum cholesterol, decreased total serum proteins, hypoalbuminemia, progressive proteinuria, and casts. Weights of the treated rats were somewhat lower than the controls.

Antitrypansome Activity

Puromycin cured Trypanosoma equiperdum infections in mice and rabbits, and was partially effective against T. cruzi. Multiple doses were more effective and less toxic than single doses (Hewitt et al. 1953). These results were confirmed in T. gambiense and T. rhodesiense infections in mice and in vitro (Trincao et al. 1955 and 1956), and T. equiperdum infections in mice (Agolini 1957).

Puromycin was tested for its trypanocidal properties against six species in mice (Tobie 1954). When treatment was begun approximately four hours after inoculation with the trypanosomes, or even at the height of infection, puromycin had a strong suppressive effect against all species except T. congolense. Administration of the antibiotic four days prior to inoculation did not prevent infections from progressing.

In two experiments with white rats infected with T. rhodesiense it was found that best results with puromycin were obtained with total doses of 430-500 mg. per kg., given in ten equal daily intraperitoneal doses commencing one or more days after the trypanosomes were visible in the peripheral blood. Some of the treated rats died without parasitaemia, presumably due to the toxicity of the antibiotic (Baker 1957).

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