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Crucial Role of Reactive Oxygen Species (ROS) for the Proapoptotic Effects of Indirubin Derivatives in Cutaneous SCC Cells
Efficient drugs are needed for countering the worldwide high incidence of cutaneous squamous cell carcinoma (cSCC) and actinic keratosis. Indirubin derivatives represent promising candidates, but their effects in cSCC cells have not been reported before. Here, we investigated the efficacy of three indirubin derivatives (DKP-071,-073 and-184) in four cSCC cell lines. High efficacy was seen in SCL-I, SCL-II, SCC-12 and SCC-13, resulting in up to 80% loss of cell proliferation, 60% loss of cell viability and 30% induced apoptosis (10 µM). Apoptosis was further enhanced in com-binations with TNF-related apoptosis-inducing ligand (TRAIL). Induction of reactive oxygen species (ROS) appeared as critical for these effects. Thus, antioxidative pretreatment completely abol-ished apoptosis as well as restored cell proliferation and viability. Concerning the pathways, com-plete activation of caspases cascades (caspases-3,-4,-6,-7,-8 and-9), loss of mitochondrial membrane potential, activation of proapoptotic PKCδ (protein kinase C delta), inhibition of STAT3 (sig-nal transducer and activator of transcription 3), downregulation of antiapoptotic XIAP (X-linked inhibitor of apoptosis protein) and survivin as well as upregulation of the proapoptotic Bcl-2 protein Puma and the cell cycle inhibitor p21 were obtained. Importantly, all activation steps were pre-vented by antioxidants, thus proving ROS as a master regulator of indirubins’ antitumor effects. ROS induction presently develops as an important issue in anticancer therapy. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
The novel arylindolylmaleimide PDA-66 displays pronounced antiproliferative effects in acute lymphoblastic leukemia cells
Background: Prognosis of adult patients suffering from acute lymphoblastic leukemia (ALL) is still unsatisfactory. Targeted therapy via inhibition of deregulated signaling pathways appears to be a promising therapeutic option for the treatment of ALL. Herein, we evaluated the influence of a novel arylindolylmaleimide (PDA-66), a potential GSK3β inhibitor, on several ALL cell lines.Methods: ALL cell lines (SEM, RS4;11, Jurkat and MOLT4) were exposed to different concentrations of PDA-66. Subsequently, proliferation, metabolic activity, apoptosis and necrosis, cell cycle distribution and protein expression of Wnt and PI3K/Akt signaling pathways were analyzed at different time points.Results: PDA-66 inhibited the proliferation of ALL cells significantly by reduction of metabolic activity. The 72 h IC50 values ranged between 0.41 to 1.28 μM PDA-66. Additionally, caspase activated induction of apoptosis could be detected in the analyzed cell lines. PDA-66 influenced the cell cycle distribution of ALL cell lines differently. While RS4;11 and MOLT4 cells were found to be arrested in G2 phase, SEM cells showed an increased cell cycle in G0/1 phase.Conclusion: PDA-66 displays significant antileukemic activity in ALL cells and classifies as candidate for further evaluation as a potential drug in targeted therapy of ALL.
AMPA-15N - Synthesis and application as standard compound in traceable degradation studies of glyphosate
Stable isotope labeling of pollutants is a valuable tool to investigate their environmental transport and degradation. For the globally most frequently used herbicide glyphosate, such studies have, so far, been hampered by the absence of an analytical standard for its labeled metabolite AMPA-15N, which is formed during the degradation of all commercially available glyphosate isotopologues. Without such a standard, detection and quantitation of AMPA-15N, e.g. with LC-MS/MS, is not possible. Therefore, a synthetic pathway to AMPA-15N from benzamide-15N via the hemiaminal was developed. AMPA-15N was obtained in sufficient yield and purity to be used as a standard compound for LC-MS/MS analysis. Suitable MS-detection settings as well as a calibration using the internal standard (IS) approach were established for Fmoc-derivatized AMPA-15N. The use of different AMPA isotopologues as IS was complicated by the parallel formation of [M+H]+ and [M]+• AMPA-Fmoc precursor ions in ESI-positive mode, causing signal interferences between analyte and IS. We recommend the use of either AMPA-13C-15N, AMPA-13C-15N-D2 or a glyphosate isotopologue as IS, as they do not affect the linearity of the calibration curve. As a proof of concept, the developed analysis procedure for AMPA-15N was used to refine the results from a field lysimeter experiment investigating leaching and degradation of glyphosate-2-13C-15N. The newly enabled quantitation of AMPA-15N in soil extracts showed that similar amounts (0.05 - 0.22 mg·kg-1) of the parent herbicide glyphosate and its primary metabolite AMPA persisted in the topsoil over the study period of one year, while vertical transport through the soil column did not occur for either of the compounds. The herein developed analysis concepts will facilitate future design and execution of experiments on the environmental fate of the herbicide glyphosate.