New Triazacycloalkane Derivatives as Cytotoxic Agents for CLL Treatment: From Proof of Concept to the Targeting Biomolecule

Abstract

The 1,4,7-tris-(2-pyridinylmethyl)-1,4,7-triazacyclononane ligand (no3py) and its bifunctional analogue no3pyCOOK were synthesized to investigate their action toward zinc(II) depletion related to the apoptosis phenomenon in chronic lymphocytic leukemia (CLL) cells. no3py was used as the "free" ligand, while its "graftable" derivative was conjugated on a newly synthesized bifunctional sialoglycan, 6′-SL-NH 2 , selected to specifically bind CD22 biomarker expressed on the B-CLL cell surface. Both compounds were produced with good yields thanks to a Sonogashira coupling reaction and an orthoester function, respectively, for the chelator and the targeting moiety. The newly reported bioconjugate 6′-SL-no3py was then obtained through a peptidic coupling reaction. Biological in vitro studies of no3py and 6′-SL-no3py consisting of real-time detection of cell health (cytotoxicity and proliferation) and caspases 3/7 activation (crucial enzymes whose activation triggers cell death signaling pathways) have been investigated. First, Ramos, Daudi, and Raji B-cell lines, which present different sensitivity to zinc(II) content variation, were incubated with no3py and 6′-SL-no3py. Then, a videomicroscope allowed the real-time monitoring of the morphological changes leading to cell death from the detection of the cytotoxicity, the antiproliferative effect, and the caspasic activity. In terms of mechanism, the Zn 2+ chelator cytotoxic effect of no3py has been evidenced by a culture medium ion supplementation study and by the decrease of intracellular fluorescence of Zn-specific fluorophore zinquin in the presence of no3py and 6′-SL-no3py chelators. Finally, flow cytometry analysis with classical Annexin V staining was conducted to detect no3py-and 6′-SL-no3py-induced apoptotic cell death in B-CLL cells. Time-course analysis, using the Incucyte Live-Cell Analysis System, demonstrated that no3py induced cell death in a time-and dose-dependent manner with variability across cell lines. 6′-SL-no3py exhibited the same dosedependent trend as no3py, showing the efficiency of the targeting moiety. In both cases, the chelators depicted proliferation curves that were inversely correlated with kinetic death. Morphological changes specific to apoptosis and caspase 3/7 activation were observed for the three cell lines treated with no3py and 6′-SL-no3py, highlighting their role as apoptotic agents. A higher concentration of 6′-SL-no3py is needed to reach 50% of the B-CLL mortality, confirming a targeting of the chelator to the cell membrane. Overall, our results proved that the biological properties of the triazamacrocyclic chelator still remain even after addition of the targeting moiety. The free chelator as well as the bioconjugate constitute promising cytotoxic agents for CLL therapy through apoptosis induction.

Chronic active Epstein-Barr virus infection as the basis of neoplasm development. Transformation of chronic lymphocytic leukaemia to NK-cell lymphoma/leukaemia was observed by flow cytometry – a diagnostic report

“…The most common type of adult leukaemia is chronic lymphocytic leukaemia (CLL), which is characterised by abnormal growth and expansion of mature B-lymphocytes that express CD5/CD19/CD20/CD23-positive in the peripheral blood, bone marrow and lymphoid tissues [1][2][3]. Atypical neoplastic cells show various survival mechanisms implemented at the cellular level, e.g., avoidance of caspase-dependent apoptosis, B-cell receptor (BCR) signalling, and interaction between CLL cells and the microenvironment [4,5]. The pathogenesis of CLL in most studies is not recognised; however, every case has a strong dependence on proliferation and the progressive accumulation of characteristic monoclonal B-lymphocytes.…”