Cytotoxic activity of L-asparaginase isolated from endophytic aspergillus nomius of Justicia adhatoda on A549 cell lines

Abstract

Introduction: L-Asparaginase belonging to a group of amidase possesses a broad spectrum of antitumor activity. It catalyzes the hydrolysis of L-asparagine to ammonia and L-aspartic acid. Objective: In the present study, L-asparaginase producing endophytic fungi were isolated from Justicia adhatoda and cytotoxicity studied on A549 cell line. Methodology: The screening modified Czapek-Dox media contained asparagine as the source and phenol red as the indicator. The positive zones were identified and sub cultured to obtain pure culture. Results: A quantitative assay of asparaginase was carried out and the level of production was found to be 1.8916 (μmol/ml). The molecular weight of the enzyme was 66 kDa. MTT assay and dual staining with acridine orange/ethidium bromide were done to assess the type of cell death induced by the enzyme in A549 cells. The percentage of apoptotic cells after treatment showed a drastic increase in apoptotic cells (P < 0.001) to 62% and 84%, respectively. Flow cytometry analysis was performed to evaluate the cell cycle arrest phase induced by different concentration of partially purified sample (80 μg/ml and 160 μg/ml). Lower concentration (80 μg/ml) showed arrest at S phase with 19% cells accumulated. At higher concentration (160 μg/ml), it showed an increased cell population at S phase with 30% with alterations in the other phase of cell cycle. Conclusion: The above observations show that the isolated asparaginase exhibited a marked cytotoxic activity against A549 cell lines. Further animal model studies, toxicity assays and pharmacological studies of this asparaginase from the endophyte would help to authenticate the use of this enzyme as an anticancer drug.