Abstract Topic: 5. Chronic lymphocytic leukemia and related disorders - Biology & Translational Research
Background: Chronic lymphocytic leukemia (CLL) is the most common leukemia in Western Countries, being more prevalent in men. Various genetic alterations can be found in this pathology, including chromosomic alterations, miRNA alterations, epigenetic changes, and somatic mutations. Oxidative stress, which results from the disequilibrium between reactive oxygen species (ROS) production and antioxidant defenses, seems also to play a crucial role in the development of this disease. The NRF2-KEAP1 pathway is a redox regulator being the principal mechanism of antioxidant and cytoprotective response against ROS. However, this pathway presents a dual role in cancer – can prevent tumor development and, after tumor establishment, can protect the tumor cells from therapeutic agents leading to treatment resistance.
Aims: The aim of this work was to evaluate the therapeutic potential of an NRF2 inhibitor, Brusatol (BRU), in monotherapy and in association with the BTK inhibitor Ibrutinib (IBR), in an in vitro CLL model, the HG-3 cell line.
Methods: The expression levels of NFE2L2 (which encodes NRF2) and KEAP1 genes were initially evaluated in HG-3 cells, using the qPCR. Then, the HG-3 cells were incubated, for 72 hours, in the absence and presence of increasing concentrations of Brusatol in monotherapy and in association with Ibrutinib. The metabolic activity was determined by resazurin assay. The type of cell death was analyzed by optical microscopy (May-Grünwald-Giemsa staining) and by flow cytometry (FC), using the annexin-V (AV) and 7- AAD double staining. The cell cycle was evaluated by flow cytometry using the propidium iodide (PI)/RNAse solution. The mitochondrial membrane potential was quantified by FC using JC-1probe. Intracellular peroxides, peroxides and peroxynitrite, superoxide anion, nitric oxide, and reduced glutathione levels were measured by fluorimetry using DCFH2-DA, DHR-123, DHE, DAF-FM DA, and MO fluorescent probes, respectively. Finally, the levels of gene expression of KEAP1, NF-κB1, NQO1, TXNRD1, HMOX1, and GPX1, which are regulated by NRF2 protein, were assessed using qPCR. Results were analyzed statistically considering a 95% significance level (p<0.05).
Results: The results showed that HG-3 cells express NFE2L2 and KEAP-1 genes. The exposure to increasing concentrations of BRU induced a reduction in metabolic activity which is dose and time-dependent. The IC50 of BRU at 48 hours was 110 nM. The association of BRU (50 nM) with Ibrutinib (0.5 µM) showed a synergic effect at 48 and 72 hours, leading to a higher reduction of cellular metabolic activity than drugs alone. BRU in monotherapy and in association with IBR induce a cytostatic and cytotoxic effect, mediated by cell cycle arrest in phase G0/G1 and apoptosis, respectively. Additionally, cells treated with both drug conditions showed a decrease in the mitochondrial membrane potential. By the analysis of reactive oxygen species (peroxides, peroxides and peroxynitrite, and superoxide anion), nitric oxide, and reduced glutathione it was determined that BRU treatment led to an increase in free radicals and a decrease in GSH. This result indicates that the cell death induced by BRU in different therapeutic schemes was mediated by inducing oxidative stress. However, BRU treatment does not seem to influence the expression of genes involved in the NRF2 signaling pathway.
Summary/Conclusion: Our results suggest that BRU could be a potential pharmacological agent for the therapeutic approach of CLL, in monotherapy and as an adjuvant to treatment with IBR.
Keywords: Chronic lymphocytic leukemia, Apoptosis, ibrutinib, Transcription factor