Today, cancer is understood as an epigenetic as well as a genetic disease. The main epigenetic hallmarks of the cancer cell are DNA methylation and histone modifications. The latter changes may be an optimal target for novel anticancer agents. The main goal of using histone deacetylase inhibitors (HDACIs) would be restoration of gene expression of those tumor-suppressor genes that have been transcriptionally silenced by promoter-associated histone deacetylation. However, HDACIs have pleiotropic effects that we are only just starting to understand. These may also be responsible for the induction of differentiation, cell-cycle arrest and pro-apoptotic effects.
There are now so many HDACIs available, with such different chemical structures and biological and biochemical properties, that it is hopeful that at least some of them will succeed, probably in combination with other agents or therapies.
In our studies we focussed ourselves on studies some new HDACIs, that can be useful for treating cancers, including leukemia and epithelial cancer. To do that, we used novel HDACIs, like BML-210, and their combination with the differentiation inducer all-trans retinoic acid (ATRA). Cell differentiation and proliferation in general, and specific gene expression require de novo protein synthesis and/or post-translational protein modifications. So, we tried to identify proteins in general and specifically the proteins that could be important for the cell differentiation process, and when and where in the cell the proteins appear.
We delineated that HDACIs inhibited leukemia (NB4 and HL-60) cell growth in a time- and dose-dependent way. Moreover, BML-210 blocked HeLa cell growth and promoted apoptosis in a time-dependent way. Combining of BML-210 with ATRA induced a differentiation process in leukemia cell lines that lead to apoptosis. This correlated with cell cycle arrest in G0/G1 stage and changes in expression of cell cycle proteins (p21, p53), transcription factors (NF-κB, Sp1) and their binding activity to consensus or specific promoter sequences. We also assessed histone modifications, i.e. H3 phosphorylation and H4 hyperacetylation due to HDACI, leading to chromatin remodeling and changes in gene transcriptions.
We have also studied changes in protein maps caused by HDACIs and differentiation agents, identifying differences for a few proteins due to growth inhibition and induction of differentiation in NB4 cells using BML-210 alone or in combination with ATRA. These proteins are involved in cell proliferation and signal transduction, like Rab, actin and calpain. One of them was alpha-dystrobrevin (α-DB). To further study possible roles of the latter, we determined changes of α-DB protein isoform expression that correlated with induction of differentiation. We thus identified a novel ensemble of α-DB interacting proteins in promyelocytic leukemia cells, including tropomyosin 3, actin, tubulin, RIBA, STAT and others, being important in cytoskeleton reorganization and signal transduction. Using confocal microscopy, we determined that α-DB co-localizes with HSP90 and F-actin in NB4 and HeLa cells. We also revealed that it changes sub-cellular compartment after treatment with ATRA and/or BML-210. α-DB silencing affected F-actin expression in HeLa cells, further supporting the idea that α-DB is involved in cytoskeleton reorganization in cells. Altogether, our results suggest that α−DB may work as a structural protein during proliferation and differentiation processes of human cancer cells.
Based on our findings, we suggest that HDACIs, like BML-210, can be promising anticancer agents, especially in leukemia treatment, by inducing apoptosis and regulating proliferation and differentiation through the modulation of histone acetylations and gene expression.
Linköping University Electronic Press, 2008.
leukemia, histone deacetylase inhibitors, retinoic acid, differentiation, apoptosis
2008-05-14, Linden, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 09:00 (English)