Distinct single cell signal transduction signatures in leukocyte subsets stimulated with khat extract, amphetamine-like cathinone, cathine or norephedrine

Abstract

BackgroundAmphetamine and amphetamine derivatives are suggested to induce an immunosuppressive effect. However, knowledge of how amphetamines modulate intracellular signaling pathways in cells of the immune system is limited. We have studied phosphorylation of signal transduction proteins (Akt, CREB, ERK1/2, NF-κB, c-Cbl, STAT1/3/5/6) and stress sensors (p38 MAPK, p53) in human leukocyte subsets following in vitro treatment with the natural amphetamine cathinone, the cathinone derivatives cathine and norephedrine, in comparison with a defined extract of the psychostimulating herb khat (Catha edulis Forsk.). Intracellular protein modifications in single cells were studied using immunostaining and flow cytometry, cell viability was determined by Annexin V-FITC/Propidium Iodide staining, and T-lymphocyte proliferation was measured by 3H-thymidine incorporation.ResultsCathinone, cathine and norephedrine generally reduced post-translational modifications of intracellular signal transducers in T-lymphocytes, B-lymphocytes, natural killer cells and monocytes, most prominently affecting c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), p38 MAPK (p-Thr180/p-Tyr182) and p53 (both total p53 protein and p-Ser15). In contrast, the botanical khat-extract induced protein phosphorylation of STAT1 (p-Tyr701), STAT6 (p-Tyr641), c-Cbl (pTyr700), ERK1/2 (p-Thr202/p-Tyr204), NF-κB (p-Ser529), Akt (p-Ser473), p38 MAPK (p-Thr180/p-Tyr182), p53 (Ser15) as well as total p53 protein. Cathinone, cathine and norephedrine resulted in unique signaling profiles, with B-lymphocytes and natural killer cells more responsive compared to T-lymphocytes and monocytes. Treatment with norephedrine resulted in significantly increased T-lymphocyte proliferation, whereas khat-extract reduced proliferation and induced cell death.ConclusionsSingle-cell signal transduction analyses of leukocytes distinctively discriminated between stimulation with cathinone and the structurally similar derivatives cathine and norephedrine. Cathinone, cathine and norephedrine reduced phosphorylation of c-Cbl, ERK1/2, p38 MAPK and p53(Ser15), and norephedrine induced T-lymphocyte proliferation. Khat-extract induced protein phosphorylation of signal transducers, p38 MAPK and p53, followed by reduced cell proliferation and cell death. This study suggests that protein modification-specific single-cell analysis of immune cells could unravel pharmacologic effects of amphetamines and amphetamine-like agents, and further could represent a valuable tool in elucidation of mechanism(s) of action of complex botanical extracts.

The psychoactive drug of abuse mephedrone differentially disrupts blood-brain barrier properties

“…Activation of NF-κB and its contribution to BBB disruption occurs under a range of inflammatory insults and substance abuse [ 32 , 57 , 58 ]. Thus, cathinone and khat extract were reported to suppress (former) or induce (latter) phosphorylation of NF-κB in in vitro culture of leukocytes [ 59 ] supporting our findings in brain endothelial cells. To further understand how mephedrone may be activating NF-κB, we explored TLR4 signaling which has been reported to mediate METH-induced activation of NF-κB [ 37 ].…”

Early Response to the Plant Toxin Stenodactylin in Acute Myeloid Leukemia Cells Involves Inflammatory and Apoptotic Signaling

“…The apoptosis/necrosis rate in stenodactylin treated cells was evaluated through flow cytometry analysis. Briefly, cells were stained with Annexin V-fluorescein Isothiocyanate (FITC) and propidium iodide (PI), using the Apoptest ™ kit from Nexins Research (Hoeven, the Netherlands) according to the manufacturer's protocol (Bredholt et al, 2013). Data were collected on a Guava easyCyte ™ Flow Cytometer (Merck Millipore, Merck KGaA, Darmstadt, Germany) and analyzed with FlowJo 7.2.5 software (TreeStar Inc., Ashland, OR, USA).…”

Impact of khat (Catha edulis) and oral contraceptive use on telomerase levels and tumor suppressor genes p53 and p21 in normal subjects and breast cancer patients

“…On the other hand, khat non-significantly reduced the telomerase level in both the control group and BC patients, which may be attributed to its reported apoptotic effect 30 , 31 suggesting that khat induces caspase-dependent apoptotic cell death, mitochondria damage, reduced proliferation, and morphological features of autophagy in various leukemic cells 32 . Khat was also reported to inhibit cell proliferation in normal human keratinocytes and fibroblasts preceded by oxidative stress characterized by a rapid increase in intracellular ROS and depletion of GSH 33 , as well as having an inhibitory growth effect which is dependent on the concentration of khat, and the duration of exposure to khat.…”