NEOCARZINOSTATIN

Description

Neocarzinostatin was found in the culture broth of Streptomyces carzinostaticus by Ishida et al. of Tohoku University in 1957. It showed strong cytotoxicity against sarcoma 180 ascites, tumor cells, and leukemia SN36. Neocarzinostatin was found to be an acidic peptide of Mr 10700, consisting of 109 amino acid residues. Recently the principal agent of anticancer and antibacterial activity in neocarzinostatin was found to be a small molecular chromophore with the peptide component playing a role by stabilizing the chromophore in vivo. Neocarzinostatin is used to treat leukemia and gastric and pancreatic cancer.

Originator

Zinostatin,Yamanouchi

Uses

Neocarzinostatin from Streptomyces carzinostaticus has been used:

• as a DNA damaging agent to treat human fibroblast GM0637 cells
• as a radiomimetic antibiotic to induce DNA damage
• in combination with Vγ2Vδ2 T cells to determine the cytotoxicity in ovarian cancer cells

Manufacturing Process

Neocarzinostatin is produced by the cultivation of a strain of Streptomyces carzinostaticus var. neocarzinostaticus under suitable conditions as the cultivation of other Actinomycetes.
An aqueous culture medium was prepared containing the following ingredients (%): starch 2.0, exoleated soybean powder 2.0, dry yeast 0,5, sodium chloride 0.25, manganese chloride 0.0005, copper sulfate 0.0005, zinc sulfate 0.0005, calcium carbonate 0.2.
After sterilizing, and adjusting to pH 7.0, 100 ml of the medium was placed in each of several test tubes, 500 ml capacity, and sterilized. Streptomyc carzinostaticus var.neocarzinostaticus strain F-41 was inoculated therein, and fermented with agitation for 24 h at 27°C and then used as the stock culture. Next, an aqueous production culture medium was prepared containing (%): glucose 3.0, peptone 0.5, meat extract 0.5, sodium chloride 0.5, calcium carbonate 0.2.
After sterilization, the medium was adjusted to pH 7.0. 100 ml of the production medium was placed in each of 70 test tubes, 500 ml capacity and sterilized. 5 % by volume of the above-mentioned stock culture was added to the production culture medium in each test tube and fermented with agitation at 27°C. The pH be came 6.6 after an incubation period of 36 h, and 6.8-7.2 after 48 h. After that, the pH showed no further change. When the amount of neocarzinostatin in the liquid was measured by its action on Sarcina lutea, it had reached 40 mkg/ml by 24 h culture, 73 mkg/ml by 36 h, 100 mkg/ml by 48 h, and 130 mkg/ ml by 64 h. Fermentation was suspended after 64 h, and solids containing the mycelium were separated by filtration. Filter paper was used and 5 L of culture liquid containing 130 mkg/ml of the active ingredient were obtained.
The culture liquid obtained above was adjusted to pH 3.0 with a saturated oxalic acid solution and the precipitate formed therein collected by filtration. The filtrate was added to 50.0 g each of kaolin and Celite 545 powder (diatomaceous earth), and stirred for 15 h at 4°C and after chromoprotein was allowed to adsorb as much as possible, it was filtered. The resulting filtrate was divided and placed in cellophane bags; dry air was blown on them at 27°C for 24 h condensing them to about 600 ml. This concentrated solution at 4°C was desalted by cellophane dialysis for 24 h in distilled water. The yield of desalted concentrated solution from the culture liquid was approximately 80% (867 mkg/ml, 600 ml).
The concentrated solution obtained above was thoroughly stirred at 4°C solid ammonium sulfate was added, amounting to 25% (150.0 g) by volume; the resulting brown precipitate was collected by centrifugation and thoroughly washed. Ammonium sulfate (150.0 g) was again added, and after leaving it for 15 h at 4°C the greyish white precipitate formed was isolated by a refrigeration-centrifugation method. The precipitate was washed several times with a cool aqueous ammonium sulfate solution, dissolved in 20 ml of distilled water and dialyzed overnight at 4°C against distilled water. After desalting, the liquid was passed through a column of Sephadex G-25. The passage solution was lyophilized and 660.0 mg of a light yellow coarse powder, neocarzinostatin, was produced (the yield from the culture liquor was 56%).

Therapeutic Function

Antineoplastic

Biochem/physiol Actions

Neocarzinostatin is a protein-small molecule complex composed of an enediyne chromophore tightly bound to a 113 amino acid single chain protein. The complex possesses antiproliferative and antitumor activity. The chromophore is the active compound, which is responsible for DNA cleavage; while the apoprotein stabilizes and regulates the availability of the labile chromophore. NCS chromophore is bound non-covalently in a cleft of the binding protein and is dissociable. Upon addition of a thiol, the chromophore forms a highly reactive biradical species that can induce sequence-specific single and double strand breaks in DNA. Neocarzinostatin inhibits DNA synthesis and possesses antitumor activity in various human and animal tumors. NCS inhibits cellular proliferation by inducing G2 cell cycle arrest and apoptosis in both human papillomavirus (HPV) positive and negative cell lines.