Synopsis The Phosphoinositide-3-kinase (PI3K) pathway regulates cell proliferation, survival and migration and is consequently of great interest for targeted cancer therapy. Using a panel of small molecule PI3K isoform-selective inhibitors in a diverse set of breast cancer cell lines, we demonstrate that the biochemical and biological responses were highly variable and dependent on the genetic alterations present. p110α inhibitors were generally effective in inhibiting the phosphorylation of Akt and S6, two downstream components of PI3K signaling, in most cell lines examined. In contrast, 110β selective inhibitors only reduced Akt phosphorylation in PTEN mutant cell lines, and was associated with a lesser decrease in S6 phosphorylation. PI3K inhibitors reduced cell viability by causing a cell cycle arrest in the G1 phase of the cell cycle, with multi-targeted inhibitors causing the most potent effects. Cells expressing mutant Ras were resistant to the cell cycle effects of PI3K inhibition, which could be reversed using inhibitors of Ras signaling pathways. Taken together our data indicates that these compounds, alone or in suitable combinations, may be useful as breast cancer therapeutics, when used in appropriate genetic contexts.
Azacitidine (AZA) and decitabine (DAC) are cytidine azanucleoside analogs with clinical activity in myelodysplastic syndromes (MDS) and potential activity in solid tumors. To better understand the mechanism of action of these drugs, we examined the effects of AZA and DAC in a panel of non-small cell lung cancer (NSCLC) cell lines. Of 5 NSCLC lines tested in a cell viability assay, all were sensitive to AZA (EC of 1.8-10.5 µM), while only H1299 cells were equally sensitive to DAC (EC of 5.1 µM). In the relatively DAC-insensitive cell line A549, both AZA and DAC caused DNA methyltransferase I depletion and DNA hypomethylation; however, only AZA significantly induced markers of DNA damage and apoptosis, suggesting that mechanisms in addition to, or other than, DNA hypomethylation are important for AZA-induced cell death. Cell cycle analysis indicated that AZA induced an accumulation of cells in sub-G1 phase, whereas DAC mainly caused an increase of cells in G2/M. Gene expression analysis of AZA- and DAC-treated cells revealed strikingly different profiles, with many genes distinctly regulated by each drug. In summary, while both AZA and DAC caused DNA hypomethylation, distinct effects were demonstrated on regulation of gene expression, cell cycle, DNA damage, and apoptosis.
Acute myeloid leukemia (AML) is characterized by blast cells that are unable to mature into functional, terminally-differentiated hematopoietic cells. Inducing leukemic cells to differentiate restores a natural cell death program and inhibits proliferation. Azacitidine (AZA; 5-azacytidine; Vidaza) is a cytidine nucleoside analog used clinically for the treatment of myelodysplastic syndromes (MDS) and AML. AZA therapy was recently shown to significantly increase median overall survival in higher-risk MDS and World Health Organization AML (20-30% bone marrow blasts) patients compared with conventional care regimens, and a phase III clinical trial of AZA in patients with more advanced AML has been activated. We have shown previously that AZA induces dose-dependent cytotoxicity to AML cell lines; however, at sub-micromolar AZA concentrations, complete cell kill is not achieved. To explore an additional anti-leukemic mechanism of AZA in AML, we assessed the effect of AZA on induction of AML cell differentiation in vitro. AML cell lines, encompassing several FAB classifications, were evaluated, using all-trans retinoic acid (ATRA) and 1,25-dihydroxyvitamin D3 (VD3), two potent inducers of AML cell differentiation, as control compounds. Differentiation along the granulocytic/monocytic lineage was assayed by CD11b expression (antigen detection by flow cytometry and mRNA by Luminex) and nitroblue tetrazolium (NBT) reduction. ATRA, VD3, and AZA induced CD11b RNA and protein expression and NBT reduction in HL-60 and AML-193 cell lines. Gene expression profiles (GEPs) in HL-60 and AML-193 cells revealed significant overlap in the genes regulated by AZA-treatment vs. VD3- or ATRA-treatment. GEPs of HL-60 and AML-193 cells treated with AZA strongly correlated with publicly-available gene sets representing differentiated eosinophils, neutrophils, and monocytes, but negatively correlated with those of differentiated erythrocytes and megakaryocytes. Similar studies in primary AML cells are ongoing. Our results demonstrate that AZA can induce cellular differentiation of AML cell lines along the granulocytic/monocytic lineage in vitro, and suggest that cellular differentiation may contribute as one of multiple mechanisms of AZA's anti-leukemic activity in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 191.
Background: Chemosensitization by azacitidine (AZA) priming has been demonstrated preclinically and is now supported by clinical data, but the underlying mechanism for the priming effect is not well understood. Previous in vitro studies of priming have focused on individual cell lines and specific epigenetically-silenced genes; however, we show that sensitization is not a universal effect of AZA pretreatment in all cancer cell lines or with all combination agents. Studying the molecular differences between cell lines that are sensitized and those that are not may yield insights into the mechanisms of priming. Purpose: To identify solid cancer cell lines that consistently show AZA sensitization to platin-induced cytotoxicity, for future molecular characterization of the priming mechanism. Methods: Ninety-two solid cancer cell lines, from seven indications, were treated with vehicle or 1μM AZA for 24h before being treated with carboplatin (CARB), cisplatin (CIS), or Abraxane (ABX) for 72h. CellTiter-Glo was used to measure cell viability. The fractional product method and normalized dose-response curves were used to characterize the drug combination interaction. In subsequent experiments, various AZA pretreatment durations (4-72h) were tested. DNA was prepared for DNA methylation (LINE-1) analysis, and cell lysates were harvested for DNMT1 Western blotting. Results: In a subset of cancer cell lines, AZA pretreatment resulted in enhanced sensitivity of cells to CARB and CIS. Seven cell lines across 5 cancer indications consistently showed a sensitization effect with AZA priming, decreasing the CARB IC50 by a mean of 59.6% (range 52.9-68.4%) and CIS IC50 by a mean of 44.6% (range 36.2-54.6%). Priming for CARB was concordant with priming for CIS, but not for ABX, indicating that the AZA pretreatment does not globally increase the susceptibility of cells to cytotoxic agents through non-specific toxic effects; rather, these results suggest that priming by AZA has specific pharmacodynamic effects unique to the combination agent. Follow-on experiments demonstrated that AZA pretreatment of cells for 18-24h was required for priming, whereas the magnitude of priming was not increased with longer (48-72h) AZA pretreatment durations. Effects of AZA on DNMT1 depletion and DNA hypomethylation were detected in all cell lines tested. The magnitude of these proximal PD effects did not correlate with priming, suggesting that differences in sensitization cannot be attributed to differential drug uptake and DNA incorporation. Conclusions: We identified a panel of solid cancer cell lines that consistently demonstrate sensitization to CARB and CIS with AZA priming. By comparing genetic and epigenetic profiles between these lines and a closely matched set of cell lines that does not show priming, we hope to generate clinically testable hypotheses for predictors of sensitization in patients. Citation Format: Aaron N. Nguyen, Manith Norng, Antonio Luna-Moran, Kyle J. MacBeth, Jorge F. DiMartino. Development of a robust preclinical model for studying the mechanism of azacitidine priming for platin-induced cytotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4656. doi:10.1158/1538-7445.AM2013-4656
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.