Dados do Trabalho

Título

A new class of microtubule inhibitors: cyclopenta[b]indole exhibits anti-leukemic effects

Introdução

Acute leukemias are a group of hematologic malignancies that originate through mutations in bone marrow progenitor cells and present a poor prognosis with high rates of mortality and relapse. Therefore, the search for new therapeutic options becomes imperative. In previous studies, our research group identified a novel synthetic compound derived from the cyclopenta[b]indoles class that exhibited selective cytotoxicity in cellular models of acute promyelocytic leukemia by disrupting microtubule dynamics.

Objetivo

To expand investigation into the cellular and molecular effects of cyclopenta[b]indole C2 on the leukemic phenotype of a molecularly heterogeneous panel of leukemia cell lines.

Métodos

A panel containing myeloid (n=12) and lymphoid (n=13) neoplasms was used. Kasumi-1, MV4-11, Jurkat, and Namalwa cells were selected for further detailed analyses. Two enantiomers of C2 (C2E1 and C2EB) were used. Cell viability was evaluated by MTT and clonogenicity by colony formation assay. By flow cytometry, apoptosis (labeling with annexin V/propidium iodide [PI]) and cell cycle (labeling with PI) were evaluated. Cell morphology was assessed by H&E staining and optical microscopy. Molecular markers of proliferation (STMN1), apoptosis (PARP1), and DNA damage (γH2AX) were investigated by Western Blot. Molecular docking and re-docking experiments of colchicine, C2E1, and C2EB ligands were performed using α/β-tubulin dimers as receptor (PDB:4O2B) using Autodock Vina and Gold software. Statistical analyzes were performed by ANOVA and Bonferroni post-test. A p-value < 0.05 was considered significant.

Resultados

C2E1 showed high cytotoxicity in malignant blood cells and only 4 out of 25 cells were considered resistant to the compound (IC50 > 50 μM). C2EB did not show considerable cytotoxic activity in the tested models and 19 of the 25 cells were considered resistant. Molecular docking analyses indicate that only C2E1 has a greater overlap in the colchicine A-ring docking region, which may be the cause for the low activity of C2EB. Therefore, only compound C2E1 was used in subsequent tests. C2E1 showed a concentration and time-dependent cytotoxic effect (IC50 ranged from 1.8 to 31.5 μM in 72 h). In acute leukemia cells, compound C2E1 decreased clonogenicity, induced apoptosis, and resulted in subG1 cell accumulation and cell cycle arrest in the G2/M phase after 48 h of drug exposure (all p < 0.05). Morphological analyzes indicated mitotic aberrations in C2E1-treated cells. C2E1 increased phosphorylation and reduced expression of STMN1 and increased expression of cleaved PARP1 and γH2AX.

Conclusões

Our results showed that the novel cyclopenta[b]indol compound reduced acute leukemia cell viability. Molecularly, also reduced proliferation markers and induced markers of apoptosis and DNA damage. Therefore, C2E1 was able to reproduce its previously characterized mechanism of action, which is the mitotic collapse by microtubule dynamics disturbance. Our study presented preclinical evidence of a new class of microtubule inhibitors as a potential therapeutic option in hematologic malignancies.

Palavras-chave

microtubule inhibitors, cyclopenta[b]indole and leukemia.

Financiador do resumo

FAPESP (2021/01460-1), CNPq and CAPES.