In vitro cytotoxicity of Artemisia vulgaris L. essential oil is mediated by a mitochondria-dependent apoptosis in HL-60 leukemic cell line

Abstract

BackgroundThe essential oil (EO) of Artemisia vulgaris L. has been traditionally used worldwide for treating a large number of diseases. Although major components in A. vulgaris EO have been shown to inhibit growth of different cancer cells, as pure compounds or part of other plants extracted oil, no information is known about its anti-proliferative activities. Therefore, the current investigation has evaluated the toxicity of the plant extracted oil from buds (AVO-b) and leaves (AVO-l) and characterized their growth inhibitory effects on cancer cells.MethodsAVO-b and AVO-l from A. vulgaris L. were extracted by hydrodistillation, and their effect on the viability of human HL-60 promyelocytic leukemia and various other cancer cell lines was tested using MTT assay. Flow cytometric analysis of apoptosis, DNA fragmentation assay, caspases enzymatic activities and Western blotting were used to determine the apoptotic pathway triggered by their action on HL-60 cells.ResultsLow concentrations of AVO-b and AVO-l inhibited the growth of HL-60 cells in a dose- and time-dependent manner. Employing flow cytometric, DNA fragmentation and caspase activation analyses, demonstrated that the cytotoxic effect of the oils is mediated by a caspase-dependent apoptosis. Kinetic studies in the presence and absence specific caspase inhibitors showed that activation of caspase-8 was dependent and subsequent to the activation of caspases-9 and -3. In addition, the essential oil caused a disruption of the mitochondrial transmembrane potential (ΔΨm), increased the release of cytochrome c to the cytosol, and altered the expression of certain members of Bcl-2 family (Bcl-2, Bax and Bid), Apaf-1 and XIAP. Interestingly, low doses of AVO-b and AVO-1 also induced apoptosis in various cancer cell lines, but not in noncancerous cells.ConclusionsThe results demonstrate that the EO-induced apoptosis in HL-60 cells is mediated by caspase-dependent pathways, involving caspases-3, -9, and -8, which are initiated by Bcl-2/Bax/Bid-dependent loss of ΔΨm leading to release of cytochrome c to the cytoplasm to activate the caspase cascade. The finding that AVO-b and AVO-l are more efficient to induce apoptosis in different cancer cell lines than noncancerous cells, suggests that A. vulgaris might be a promising source for new anticancer agents.

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“…Caspases play essential roles in apoptosis. Caspase 3 is regarded as an apoptosis marker, and caspase 9 is an initiator of caspase 3 activity (Hou et al, 2013;Saleh et al, 2014). As shown in Fig.…”

The Evaluation of Antimicrobial and Cytotoxic Activity of the Essential Oil Extracted From the Aerial Parts of Southernwood Herb (Artemisia Abrotanum L.) That Recently Grown in Iraq

“…The cytotoxic activity of A. abrotanum L. The result of cytotoxic activity of A.abrotanum is shown in Graph 1 below [22].…”

The Pyridone-Annelated Isoindigo (5‘-Cl) Induces Apoptosis, Dysregulation of Mitochondria and Formation of ROS in Leukemic HL-60 Cells

“…The assays were performed by incubating the cell lysates with 0.2 mM of the caspase-specific colorimetric tetrapeptide substrates, Ac-VDVAD-pNA (for caspase-2), Ac-IETD-pNA (for caspase-8), Ac-LEHD-pNA (for caspase-9), Ac-DEVD-pNA (for caspase-3/-7) or Ac-VEID-pNA (for caspase-6) for 1h at 37°C as described by [20]. The increase in the absorbance at 405 nm which corresponds to the amount of p-nitroaniline (pNA) liberated from the peptide substrates was converted into units of enzyme activity using a standard curve generated with free pNA.…”

“…Mitochondrial and cytosolic fractions from HL-60 cells treated with 5'-Cl were prepared by differential centrifugation at 4°C as described in our recent publication [20]. The release of cytochrome c from mitochondria into the cytoplasm of 5'-Cl-treated HL-60 cells was detected by probing the electrophoresed mitochondrial (30 µg) or cytosolic (50 µg) fractions, with an anti-cytochrome c antibody [(6H2): mAb 13561, Santa Cruz Biotechnology] as previously described [20].…”