Purification and Characterization of Glutaminase Free Asparaginase from Enterobacter cloacae: In-Vitro Evaluation of Cytotoxic Potential against Human Myeloid Leukemia HL-60 Cells

Abstract

Asparaginase is an important antileukemic agent extensively used worldwide but the intrinsic glutaminase activity of this enzymatic drug is responsible for serious life threatening side effects. Hence, glutaminase free asparaginase is much needed for upgradation of therapeutic index of asparaginase therapy. In the present study, glutaminase free asparaginase produced from Enterobacter cloacae was purified to apparent homogeneity. The purified enzyme was found to be homodimer of approximately 106 kDa with monomeric size of approximately 52 kDa and pI 4.5. Purified enzyme showed optimum activity between pH 7–8 and temperature 35–40°C, which is close to the internal environment of human body. Monovalent cations such as Na+ and K+ enhanced asparaginase activity whereas divalent and trivalent cations, Ca2+, Mg2+, Zn2+, Mn2+, and Fe3+ inhibited the enzyme activity. Kinetic parameters Km, Vmax and Kcat of purified enzyme were found to be 1.58×10−3 M, 2.22 IU μg-1 and 5.3 × 104 S-1, respectively. Purified enzyme showed prolonged in vitro serum (T1/2 = ~ 39 h) and trypsin (T1/2 = ~ 32 min) half life, which is therapeutically remarkable feature. The cytotoxic activity of enzyme was examined against a panel of human cancer cell lines, HL-60, MOLT-4, MDA-MB-231 and T47D, and highest cytotoxicity observed against HL-60 cells (IC50 ~ 3.1 IU ml-1), which was comparable to commercial asparaginase. Cell and nuclear morphological studies of HL-60 cells showed that on treatment with purified asparaginase symptoms of apoptosis were increased in dose dependent manner. Cell cycle progression analysis indicates that enzyme induces apoptosis by cell cycle arrest in G0/G1 phase. Mitochondrial membrane potential loss showed that enzyme also triggers the mitochondrial pathway of apoptosis. Furthermore, the enzyme was found to be nontoxic for human noncancerous cells FR-2 and nonhemolytic for human erythrocytes.

Growth inhibitory and proapoptotic effects of l‐asparaginase from Fusarium culmorum ASP‐87 on human leukemia cells (Jurkat)

“…This frequently results in dreadful side effects in patients including a drastic drop in cell counts (WBC and RBC) and severe anemic conditions. Although erythrocytes are not good models for normal cells, to understand whether asparaginase is hemocompatible, hemolysis assay was performed on erythrocytes .The in vitro hemolysis assay results were in concordance with earlier reports of Husain et al and Mahajan et al which showed l ‐asparaginase purified from Enterobacter cloacae and Bacillus licheniformis RAM‐8 was not toxic to human erythrocytes at higher concentrations. This result may suggest that l ‐asparaginase may exert selective cytotoxicity only on leukemic cells, making it an ideal candidate for cancer chemotherapy especially for leukemia.…”

“…The DNA fragmentation results were in concordance with earlier reports by Story et al which showed characteristic fragmentation of DNA of mouse lymphoma cell line (LY‐TH) after l ‐asparaginase treatment indicating apoptosis as the mode of cell death. l ‐asparaginase purified from Enterobacter cloacae also showed characteristic laddering pattern of DNA of human myeloid leukemia HL‐60 cell line . The fragmentation of DNA may be due to the activation of caspase 3 which inactivates DFF‐45 (DNA fragmentation factor) and ICDA (Inhibitor of caspase‐activated DNase) which in turn release active DFF‐40 or CAD (caspase‐activated DNase) .…”

“…The cytotoxicity of purified l ‐asparaginase was tested against human leukemic cell lines and found decrease in the cell viability of Jurkat cells in a dose‐dependent manner. Several reports earlier suggest the in vitro cytotoxicity of purified l ‐asparaginase against different cell lines such as human myeloid leukemia HL‐60 , human breast cancer MCF‐7 and human myelogenous leukemia K‐562 , human colon cancer CACO‐2 and human cancer prostrate PC‐3 , gastric cancer (AGS) , hepato cellular carcinoma (Hep‐G2) and human gastric cancer cell line (MKN‐28) and human T lymphoblastic leukemia cell line (MOLT‐4) . l ‐asparaginase purified from Penicillium brevicompactum NRC 829 and Aspergillus flavus (KUFS20) was cytotoxic to hepatocellular carcinoma (Hep‐G2) and human breast cancer cells (MCF‐7) with IC 50 value of 43.3 and 120.87 μg/mL, respectively .…”

Screening of Bacteria Producing Asparaginase Free of Glutaminase and Urease from Hot Springs in West Sulawesi

“…Removing the presence of glutaminase activity in L-asparaginase preparation will increase the efficacy of ALL treatment. Husain et al (2016), reported the cytotoxicity of purified glutaminasefree asparaginase from Enterobacter cloacae. The purified asparaginase free of glutaminase suppresses the proliferation of cancer cell HL-60 and non-toxic to normal cells in vitro.…”

Antitumor activity and ability to prevent acrylamide formation in fried foods of asparaginase from soybean root nodules

“…In addition, most L‐asparaginases have low catalytic activity and low stability as well as are only active in a narrow pH range (Jr et al, ; Zuo, Zhang, Jiang, & Mu, ). Many enzymes with L‐asparaginase activity are quite specific for L‐asparagine, but also have a low level of intrinsic L‐glutaminase activity that hydrolyzes the amino acid L‐glutamine, which is an undesirable effect for the food industry (Husain, Sharma, Kumar, & Malik, ; Shrivastava et al, ). Hence, it is necessary to search for novel L‐asparaginases since drawbacks in the food industry are increasingly reported (Zhang et al, ).…”