The IN VITRO STUDY OF APOPTOSIS INDUCTION BY L-ASPARAGINASE OF ASPERGILLUS FUMIGATUS (ASPF-6) ON HUMAN LEUKEMIA CELL LINES: On Human Leukemia cell lines,HL-60

VIPINA R; VIJAYA CHITRA A

doi:10.22159/ajpcr.2019.v12i7.32158

Authors

VIPINA R Department of Microbiology, See Narayana Guru College, Coimbatore, Tamil Nadu, India.
VIJAYA CHITRA A Department of Microbiology, See Narayana Guru College, Coimbatore, Tamil Nadu, India.

DOI:

https://doi.org/10.22159/ajpcr.2019.v12i7.32158

Keywords:

L-asparaginase,, A fumigatus,, 3-(4,5-dimethyl thiazol-2-)-2,5-diphenyl tetrazolium bromide assay,, Annexin VPI and JC-1 Flurocytometry

Abstract

Objective: L-Asparaginase is a relatively widespread enzyme used in the lymphoblastic leukemia chemotherapy. In the present investigation, we report new potential fungal L-asparaginase producer Aspergillus fumigatus from Kadalundi mangrove forest, Kerala. The aim of the present investigation was to evaluate the concentration-dependent cytotoxicity and apoptosis-inducing the effect of extracted enzyme and its ability to induce apoptosis against human leukemia cell lines-HL-60.

Methods: The fungal strain was isolated by serial dilution method from rhizosphere soil of mangrove forest. The isolated enzyme purified by 80% ammonium sulfate precipitation, dialysis, and ion exchange chromatography. To determine cytotoxicity and level of apoptosis, three different tests were performed: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, JC-1 flow cytometry, and Annexin V/propidium iodide (PI) flow cytometric DNA fragmentation. The test compound efficiency compared with positive drug doxorubicin against HL-60 cell lines.

Results: The test compound exhibited a concentration-dependent cytotoxic effect on proliferation of HL-60 cell lines (IC-50 12.39 U) with a different level of apoptosis induction (LR-11.3%).

Conclusion: A. fumigatus derived L-asparaginase may be clinically useful and results in better utilization for limited fungal-derived drug resources and improve financial feasibility as a therapeutic option for human leukemia.

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References

Marshall SA, Lazar GA, Chirino AJ, Desjarlais JR. Rational design and engineering of therapeutic proteins. Drug Discov Today 2003;8:212-21.

Sarquis MI, Basker GO, Renganathan S. Design of experiments and ANN linked genetic algorithem for modeling and optimization of l-asparaginase production by Aspergillus terreus, MTCC 1782. Res J Math Stat 2009;1:55.

Sarquis MI, Oliveira EM, Santos AS, Costa GL. Production of l-asparaginase by filamentous fungi. Mem Inst Oswaldo Cruz 2004;99:489-92.

Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.

Muharram MM, Abdulhamd AT, Mounir MS. Recombinant expression, purification of l-asparaginase -11 from thermotolerant E. coli strain and evaluation of its antiproliferative activity. Afr J Microbiol 2014;8:1610 9.

Loureiro C. Purification and biochemical characterisation of native and pegglated form of l-asparaginase from Aspergillus terreus and evaluation of its antiproliferative activity. J Adv Microbiol 2012;2:138 45.

Prakasham RS, Hymavathi M, Subba Rao Ch, Arepalli SK, Venkateswara Rao J, Kennady PK, et al. Evaluation of antineoplastic activity of extracellular asparaginase produced by isolated Bacillus circulans. Appl Biochem Biotechnol 2010;160:72-80.

Gulati R, Saxena RK, Gupta R. A rapid plate assay for screening of l-asparaginase producing microorganism. J Appl Microbiol 1997;24:23 6.

Kafkewitz D, Goodman D. L-asparaginase production by the rumen anaerobe Vibrio succinogenes. Appl Microbiol 1974;27:206-9.

Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-75.

Pallaga U, Rao CS, Ragulapati SB. Studies on l-aspraginase production by using Staphylococcus capitis. J Chem Biol Phys Sci 2013;3:201-9.

Loureiro CB, Borges KS, Andrade AF, Tom LG. Purification and biochemical characterization of native and pegylated form of l-asparaginase from Aspergillus terreus and evaluation of its anti-proliferative activity. J Adv Microbiol 2012;2:138-45.

Rajesh MJ, Rajesh L, Satyaveni VV, Thirumurugan G, Sivasubramanian R. Effect of inducers and physical parameters on the production of l-asparaginase using Aspergillus terreus. J Bioprocess Biotech 2011;1:4-8.

Amena S, Vishalakshi N, Prabhakar M, Dayanand A, Lingappa K. Production, purification and characterization of l-asparaginase from Streptomyces gulbargensis. Braz J Microbiol 2010;41:173-8.

Wade HE, Robinson HK, Philips BW. Asparaginase and glutaminase activities of bacteria. Journal of general microbiology 1971; 299-312.

Dhevangi P, Poorani E. Isolation and characterization of l-asparaginase from marine Actinomycetes. Int J Biotechnol 2006;5:48-52.

E-Moharam M, Gamal-Eldeen AM, El-Sayed ST. Production, immobilization and anti tumor activity of l-asparaginase of Bacillus sp R36. J Am Sci 2010;6:72-5.

Mohammed A, Mazini AL. L-asparaginase activity by various bacteria. J Missan Res 2008;4:12-5.

Mishra A. Production of l-asparaginase, an anticancer agent, from Aspergillus niger using agricultural waste in solid state fermentation. J Appl Biochem Biotechnol 2006;135:32-42.

Jha SK, Pasrija D, Sinha RK, Singh HR, Nigam VK, Vidyarthi AS. Microbial l-asparaginase: A review on current scenario and future prospects. Int J Pharm Sci Res 2012;3:3076-90.

Sirisha B, Haritha. R. Efficacy the antiproliferative effect of l-asparaginase from Streptomyces enissocaesilis on different tumour cell line. Indian J Curr Res Acad Rev 2017;5:95-102.

Poorani E, Saseethran MK, Dhevagi P. L-asparaginase production and molecular identification of marine Streptomyces sp strain EPD27. Int J Integr Biol 2009;7:150.

Hussain I, Sharma A, Kumar S, Malik F. Purification and characterization of glutaminase free l-asparaginase from Enterobacter cloacae. In vitro evaluation of cytotoxic potential against human myeloid leukemia cell lines.Hl-60. J PLoS One 2016;18:21-6.

Khamna S, Yokota A, Lumyong S. Actinomycetes isolated from medicinal plant rhizosphere soils: Diversity and screening of anti-fungal compounds, indole 3 acetic acid and siderophore production. Int J Integr Biol 2009;6:22.

Bhat M, Marar T. Cytotoxic activity of purified l-asparaginase from Salinicoccus species. Int J Curr Microbiol Appl Sci 2015;4:701-2.

Oza VP, Trivedi SD, Parmar PP, Subramanian RB. Withania somnifera (Ashwagantha): A novel source of l-asparaginase. J Integr Plant Biol 2009;51:201.

Raha SK, Roy SK, Dey SK, Chakrabarty SL. Purification and properties of an L-asparaginase from Cylindrocarpon obtusisporum MB-10. Biochem Int 1990;21:987-1000.

Nguyen HA, Su Y, Lavie A. Design and characterization of Erwinia chrysanthemi l-asparaginase variants with diminished l-glutaminase activity. J Biol Chem 2016;291:17664-76.

Ommurugan B, Priya A, Bairy LK. A complete review of migraine. Asian Journal of Pharmaceutical and Clinical Research 2017;10:57-62.

Kumarai PV, Sankar GG, Prabakhar T. Optimization of l-asparaginase production by Streptomyces griseoluteus WS3/1 using experimental methods. J Pharm Biomed Sci 2011;10:101-4.

Bell TL, Adams MA. Ecophysiology of ecto mycorrhizal fungi associated with Pinus spp. J Plant Ecol 2004;171:35-52.

Ebrahiminezhad A, Rasoul-Amini S, Ghasemi Y. L-asparaginase production by moderate halophilic bacteria isolated from maharloo salt lake. Indian J Microbiol 2011;51:307-11.

Naveena B, Narayani TG, Sakthiselvan P, Partha N. Antioxidant and antimicrobial efficacies of Amaranthus polygonoides and its impact on l-asparaginase production. Afr J Biotechnol 2012;11:12483-90.

Dhevagi P, Poorani E. Isolation and characterization of l-asparaginase from marine action mycetes. Indian J Biotechnol 2006;5:514.

Pradeep SM, Mahmood R, Jagadeesh KS. Screening and characterization of l-asparaginase producing microorganisms from tulsi (Ocimum sanctum L.). Karnataka J Agric Sci 2010;2:394-660.

Patil N, Rao K, Naaz H, Ommurugan B, Baiju G, Naik A, et al. Myxedema madness: An intriguing case of depression in hypothyroidism. Asian Journal of Pharmaceutical and Clinical Research 2017;10:8-9.

Ommurugan B, Bairy LK, Priya A, Thomas J, Patil N. A fatal case of bilateral interstitial pneumonia (Blip): Interferon alpha 2 a a inducced. Asian Journal of Pharmaceutical and Clinical Research 2017;10:15-6.

Gaffar SA, Shethna YI. Purification and some biological properties of asparaginase from Azotobacter vinelandii. Appl Environ Microbiol 1977;33:508-14.