Cytotoxicity of purified methionine γ- lyase produced by Pseudomonas putida on several cell lines
Many human malignant cell lines and primary tumours have absolute requirements for L-methionine (1). Under normal circumstances methionine comes from dietary proteins, most normal tissues can also synthesize methionine from either homocysteine or methylthioadenosine (2). Upon L-methionine depletion, L-methionine-dependent cancer cells are not able to divide and became arrested in the late S/ G2 phase of the cell cycle (3).Methionine cleaving enzyme (methionine γ- lyase) has been found to be an effective antitumour agent in vitro as well as in vivo (4). Methionine γ- lyase is a pyridoxal 5-phosphate dependent enzyme that catalyzes the α, γ- elimination of L-methionine to α-ketobutyrate, methanethiol and ammonia (5). Rhabdomyosarcoma (RD) cell line at passage 65, AhmedMohammed-Nahi-2003(AMN3) cell line at passage 180, AMGM (Ahmed – Majeed-Glioblastoma-Multiform) cell line at passage 65 and normal Rat Embryo Fibroblast (REF) cell line at passage 56, kindly provided by Iraqi center for cancer and medical genetics research (ICCMGR) were cultured in PRMI 1640 medium supplement with 10% heat inactivated fetal calf serum and incubated at 37 ºC with 5% CO2. Examination of cytotoxicity of methionine γ- lyase Cytotoxicity was determined with the crystal violet stain as previously described (7). In briefly , 1x105 cell/ml was seeded onto 96-well culture plates in 200 µl RPMI 1640 medium and incubated until the cell reached confluent monolayer(vary according to the kind of cell-line). After incubation period the medium was removed and 200 μl of various concentrations (1000, 500,250 and 125 μg/ml) from methionine γ- lyase were added to the plate by using 3- well replicates for each concentration. The plates were incubated at 37ºC for 96 hr. The control (cancer cell lines without treatment with enzyme) (3-well replicates) was treated with 200 μl of serum free medium and incubated at 37ºC for 96 hr. At the end of incubation period, the enzyme and medium was removed from plate and washed with PBS to remove unattached (dead) cells. Two hundred μl of crystal violet was added to each well and left for 20 min at 37ºC.The stain was removed by washing with tap water several times, and the plates were left to dry. After drying, each plate was read by using ELISA microplate spectrophotometer at 492nm wave length. The percentages of Inhibitory Rate (IR %) were estimated (8).