Induction of apoptosis in leukemic cells by the alkaloid extract of garden cress(Lepidium sativum L.)

Objective: Garden cress(Lepidium sativum L.) is an important herb in traditional medicine used to improve production of breast milk in women and semen in men. In the present research the authors evaluated its ability to destroy leukemic cancer(Jurkat E6-1) cells, using the alkaloid extract of this plant.Methods: Constituents of the alkaloid extract were analyzed by gas chromatography–mass spectrometry(GC–MS) and their cytotoxicity in leukemic cancer cells and healthy peripheral blood mononuclear cells(PBMCs) was assessed. Cell death via apoptosis was confirmed by DNA laddering, caspase-3 activity,annexin V-fluorescein isothiocyanate and mitochondrial toxicity assays. The specific course of gene activation in treated cells was determined through quantitative polymerase chain reaction(qPCR).Results: GC–MS analysis identified six alkaloids and proto-alkaloids, namely, benzyl isothiocyanate(1),2-ethoxy-4 H-3,1-benzoxazin-4-one(2),(4 R)-2-(2-aminophenyl)-4-phenyloxazoline(3), 5-acetyl-1,2-di hydro-6-methyl-2-oxo-4-phenyl-3-pyridinecarbonitrile(4), benzo[b][1,8]-naphthyridin-5(10 H)-on e,2,4,7-trimethyl(5) and 1,4-diaminoanthraquinone(6), in the alkaloid extract of L. sativum. Of these,compound 1 was previously identified in the seeds of L. sativum. Exposure to the alkaloid extract caused death of Jurkat E6-1 cells, with median lethal concentration(LC50) of 75.25 mg/mL. However, the alkaloid extract also showed a nontoxic and proliferative(1.6-fold) effect in healthy PBMCs. Further experiments performed with Jurkat cells at LC50 and sub-LC50 doses demonstrated DNA fragmentation, activation of caspase-3 and time-dependant phosphatidylserine translocation(apoptosis) from inner to outer cell membranes. Cell toxicity and assessment of adenosine triphosphate level, together with using qPCR to evaluate expression profile of major apoptosis genes, revealed that apoptosis may be induced by disruption in the mitochondrial outer membrane potential, through activation of extrinsic and intrinsic apoptosis pathways in Jurkat cells.Conclusion: The ability of the alkaloid extract of L. sativum seeds to induce apoptosis indicates a potential pharmacological use in cancer chemotherapy. The separation of individual active compounds and further in-depth exploration of the molecular mechanism of apoptosis may lead to novel chemotherapeutic compounds in our future antineoplastic research.

Cytochrome P450 BM3 of Bacillus megaterium-A possible endosulfan biotransforming gene

Computing chemistry was applied to understand biotransforrnation mechanism of an organochlorine pesticide, endosulfan. The stereo specific metabolic activity of human CYP-2B6 （cytochrome P450） on endosulfan has been well demonstrated. Sequence and structural similarity search revealed that the bacterium Bacillus megaterium encodes CYP-BM3, which is similar to CYP-2B6. The functional similarity was studied at organism level by batch-scale studies and it was proved that B. megaterium could metabolize endosulfan to endosulfan sulfate, as CYP-2B6 does in human system. The gene expression analyses also confirmed the possible role of CYP-BM3 in endosulfan metabolism. Thus, our results show that the protein structure based in-silico approach can help us to understand and identify microbes for remediation strategy development. To the best of our knowledge this is the first report which has extrapolated the bacterial gene for endosulfan biotransformation through in silico prediction approach for metabolic gene identification.