Inophyllin A (INO-A), a pyranoxanthone isolated from the roots of Calophyllum inophyllum represents a new xanthone with potential chemotherapeutic activity. In this study, the molecular mechanism of INO-A-induced cell death was investigated in Jurkat T lymphoblastic leukemia cells. Assessment of phosphatidylserine exposure confirmed apoptosis as the primary mode of cell death in INO-A-treated Jurkat cells. INO-A treatment for only 30 min resulted in a significant increase of tail moment which suggests that DNA damage is an early apoptotic signal. Further flow cytometric assessment of the superoxide anion level confirmed that INO-A induced DNA damage was mediated with a concomitant generation of reactive oxygen species (ROS). Investigation on the thiols revealed an early decrease of free thiols in 30 min after 50 μM INO-A treatment. Using tetramethylrhodamine ethyl ester, a potentiometric dye, the loss of mitochondrial membrane potential (MPP) was observed in INO-A-treated cells as early as 30 min. The INO-A-induced apoptosis progressed with the simultaneous activation of caspases-2 and -9 which then led to the processing of caspase-3. Taken together, these data demonstrate that INO-A induced early oxidative stress, DNA damage and loss of MMP which subsequently led to the activation of an intrinsic pathway of apoptosis in Jurkat cells.
This investigation evaluated the antileukemia properties of a zerumbone (ZER)-loaded nanostructured lipid carrier (NLC) prepared by hot high-pressure homogenization techniques in an acute human lymphoblastic leukemia (Jurkat) cell line in vitro. The apoptogenic effect of the ZER-NLC on Jurkat cells was determined by fluorescent and electron microscopy, Annexin V-fluorescein isothiocyanate, Tdt-mediated dUTP nick-end labeling assay, cell cycle analysis, and caspase activity. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay showed that ZER-NLC did not have adverse effects on normal human peripheral blood mononuclear cells. ZER-NLC arrested the Jurkat cells at G2/M phase with inactivation of cyclin B1 protein. The study also showed that the antiproliferative effect of ZER-NLC on Jurkat cells is through the intrinsic apoptotic pathway via activation of caspase-3 and caspase-9, release of cytochrome c from the mitochondria into the cytosol, and subsequent cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP). These findings show that the ZER-NLC is a potentially useful treatment for acute lymphoblastic leukemia in humans.
The cathepsin B inhibitor benzyloxycarbonyl-phenylalanine-alanine-chloromethyl ketone (z-FA-CMK) was recently found to induce apoptosis at low concentrations in Jurkat T cells, while at higher concentrations, the cells die of necrosis. In the present study, we showed that z-FA-CMK readily depletes intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) generation. The toxicity of z-FA-CMK in Jurkat T cells was completely abrogated by N-acetylcysteine (NAC), suggesting that the toxicity mediated by z-FA-CMK is due to oxidative stress. We found that L-buthionine sulfoximine (BSO) which depletes intracellular GSH through the inhibition of GSH biosynthesis in Jurkat T cells did not promote ROS increase or induce cell death. However, NAC was still able to block z-FA-CMK toxicity in Jurkat T cells in the presence of BSO, indicating that the protective effect of NAC does not involve GSH biosynthesis. This is further corroborated by the protective effect of the non-metabolically active D-cysteine on z-FA-CMK toxicity. Furthermore, in BSO-treated cells, z-FA-CMK-induced ROS increased which remains unchanged, suggesting that the depletion of GSH and increase in ROS generation mediated by z-FA-CMK may be two separate events. Collectively, our results demonstrated that z-FA-CMK toxicity is mediated by oxidative stress through the increase in ROS generation.