N-alkylisatin-based microtubule destabilizers bind to the colchicine site on tubulin and retain efficacy in drug resistant acute lymphoblastic leukemia cell lines with less in vitro neurotoxicity

Abstract

Background: Drug resistance and chemotherapy-induced peripheral neuropathy continue to be significant problems in the successful treatment of acute lymphoblastic leukemia (ALL). 5,7-Dibromo-N-alkylisatins, a class of potent microtubule destabilizers, are a promising alternative to traditionally used antimitotics with previous demonstrated efficacy against solid tumours in vivo and ability to overcome P-glycoprotein (P-gp) mediated drug resistance in lymphoma and sarcoma cell lines in vitro. In this study, three di-brominated N-alkylisatins were assessed for their ability to retain potency in vincristine (VCR) and 2-methoxyestradiol (2ME2) resistant ALL cell lines. For the first time, in vitro neurotoxicity was also investigated in order to establish their suitability as candidate drugs for future use in ALL treatment. Methods: Vincristine resistant (CEM-VCR R) and 2-methoxyestradiol resistant (CEM/2ME2-28.8R) ALL cell lines were used to investigate the ability of N-alkylisatins to overcome chemoresistance. Interaction of N-alkylisatins with tubulin at the the colchicine-binding site was studied by competitive assay using the fluorescent colchicine analogue MTC. Human neuroblastoma SH-SY5Y cells differentiated into a morphological and functional dopaminergic-like neurotransmitter phenotype were used for neurotoxicity and neurofunctional assays. Two-way ANOVA followed by a Tukey's post hoc test or a two-tailed paired t test was used to determine statistical significance. Results: CEM-VCR R and CEM/2ME2-28.8R cells displayed resistance indices of > 100 to VCR and 2-ME2, respectively. CEM-VCR R cells additionally displayed a multi-drug resistant phenotype with significant cross resistance to vinblastine, 2ME2, colchicine and paclitaxel consistent with P-gp overexpression. Despite differences in resistance mechanisms observed between the two cell lines, the N-alkylisatins displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell line. The N-alkylisatins proved to be significantly less neurotoxic towards differentiated SH-SY5Y cells than VCR and vinblastine, evidenced by increased neurite length and number of neurite branch points. Neuronal cells treated with 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin showed significantly higher voltage-gated

Optimised techniques for high-throughput screening of differentiated SH-SY5Y cells and application for neurite outgrowth assays

“…A number of studies have differentiated the SH-SY5Y cell line with RA for a short duration of 2–4 days, at which point the cells are then treated with a chemical factor, to evaluate the inhibitory or stimulatory effect on neurite outgrowth over a specified period 13 , 29 , 30 . However, such short differentiation protocols would be insufficient to produce a population of terminally differentiated cells 31 .…”

Molecular and Functional Characterization of Neurogenin-2 Induced Human Sensory Neurons

“…Following application of paclitaxel, a common chemotherapeutic used for several types of cancers, rat DRG neurons manifest altered excitability due to higher expression of voltage-gated potassium, hyperpolarizationactivated cyclic nucleotide-gated (HCN), and voltage-gated sodium channels together with a decrease in the expression of inwardly-rectifying potassium channels (Zhang and Dougherty, 2014). Ion channels have a critical role in the development and progression of peripheral neuropathies hence, strides have been made towards the development of cancer therapies aimed at curtailing excitability remodeling and neurotoxicity (Keenan et al, 2020).…”

The Battlefield of Chemotherapy in Pediatric Cancers

“…VCR, known initially as Leurocristine, is the first-line chemotherapeutic medication often administered in the combination chemotherapeutic treatment of pediatric hematologic malignancies and solid tumors [ 53 ]. VCR acts as mitotic inhibitors by binding to the β-tubulin subunit of αβ-tubulin heterodimers, thus functionally destabilizing microtubule fibers, which ultimately leads to the termination of cancer cell division [ 54 ]. Previous studies have shown a cumulative effect for its neurotoxicity and overdosage may cause very serious or fatal outcomes [ 55 , 56 ].…”