A Synthetic Analogue of the Indigo Jeans Dye Tryptanthrin, NT1, Induces Apoptosis in Vitro, Ex Vivo and in Vivo in CD95-Dependent Manner in Pediatric Leukemia and Lymphoma Cells Sensitizing Malignant Cells to Cytostatic Drugs Via Downregulation of XIAP
Authors: Jesse Patrick , Riepl Herbert , Wieder Thomas , Henze Guenter , Prokop Aram 
Published: April 2020

Abstract
Despite advances in chemotherapeutic treatment of hematological diseases in childhood, there is significant rate of relapse with high death rates, so that research in new therapeutic molecules is on demand to combat resistance. Search for low molecular weight inhibitors in plant material therefore offers a wide spectrum of new anticancer compounds. Tryptanthrin is found in several plants like Isatis tinctoria or Polygonum tinctorium. Our experiments revealed that NT1, the modified 8-Nitro-3-chloro-derivative of tryptanthrin, specifically induced apoptosis in BJAB, Nalm6, Reh, Sup-B15 and p388 cells, as evidenced by DNA fragmentation, Annexin/PI staining, dissipation of the mitochondrial membrane potential measured via flow cytometry and processing of caspase-3 by Western Blot analysis. These events occurred in the absence of cellular lysis, i. e. in the absence of release of lactate dehydrogenase from the cells, thereby excluding NT1-induced necrosis. A dose-dependent inhibition of proliferation up to 96% hereby precedes the DNA fragmentation and apoptosis. In contrast, healthy leucocytes were not affected by treatment with NT1. NT1-induced cell death was furthermore functionally characterized by the use of different cellular model systems. Investigating control vector-(pcDNA3-mock-transfected) and pcDNA3-FADDdn-transfected BJAB cells, which were stably transfected with a dominant-negative FADD mutant lacking the N-terminal death effector domain, revealed that NT1-induced apoptosis is mainly executed via the CD95-dependent cascade. These findings were confirmed by real-time PCR, which unveiled an up to 8-fold increase of caspase-8, 20-fold upregulation of the FAS ligand and the members 9, 11B and 25 of the TNF receptor superfamily during NT1-induced apoptosis. The strong inhibitor of apoptosis Bcl-2 often leads to severe drug resistance in treatment of malignancies. Melanoma cells transfected with the pIRES plasmid (MelHO/pIRES) and the Bcl-2 cDNA in pIRES (Mel-HO/Bcl-2), which in consequence 30-fold overexpress the Bcl-2 protein, were incubated with NT1 for 72h. Apoptosis showed to be independent on increased Bcl-2 levels, which is in line with an upregulation of the anti-Bcl-2 protein Harakiri during NT1 treatment measured by real-time PCR. Apoptosis-related proteins are important molecules for predicting chemotherapy response and prognosis in pediatric leukemia. Recent studies have demonstrated that members of the inhibitor of apoptosis (IAP) family proteins like survivin or XIAP are upregulated in pediatric ALL or AML. XIAP mRNA expression was found to be 160-fold down regulated in BJAB cells treated with NT1 as compared to untreated cells. Thus, NT1 is able to sensitize resistant tumor cells to cytostatic therapy in combined treatment with other drugs as shown in further experiments. Analyzing primary leukemia cells from 24 children with de novo ALL, 4 with relapsed ALL and 6 with newly diagnosed AML, we could demonstrate that NT1 overcomes drug resistance against daunorubicin (p&lt;0.05) ex vivo. First in vivo experiments in a lymphoma NOD/SCID mouse model showed a significant decrease in tumor growth after two 5-day blocks of oral NT1 monotherapy (200mg/kg/BW) (p&lt;0.05). No bodyweight loss or severe side effects could be detected. Taken together, the tryptanthrin derivative NT1 comprises a totally new, very promising class of cytostatic agents for lymphoma and leukemia therapy. Future studies will aim to elucidate the target structures of this compound.