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Inhibition of NF-?B activation sensitizes U937 cells to 3'-azido-3'-deoxythymidine induced apoptosis.

ABSTRACT: In this study, we investigated molecular mechanisms underlying low susceptibility to apoptosis induced by the nucleoside analog azidothymidine (AZT) and the role of nuclear factor-?B (NF-?B) activation in these phenomena. A preliminary screening in different cell lines indicated U937 monocytic cell line as suitable to this purpose. Treatment of U937 cells even with suprapharmacological concentrations of AZT induced only moderate levels of apoptosis. Surprisingly, SuperArray analysis showed that AZT induced the transcriptional activity of both pro- and anti-apoptotic genes. Interestingly, moreover, several genes upregulated by AZT were NF-?B related. In fact, AZT, after an initial inhibition of NF-?B activation with respect to control, induced a transient, but consistent, increase in NF-?B-binding activity. Inhibition of NF-?B activation in U937 cells, stably transfected with a dominant-negative I?B? or by pharmacological treatment, sensitized them to apoptosis induced by AZT and impaired the upregulation of anti-apoptotic genes in response to AZT treatment, with respect to control cells. These results indicate that NF-?B activation by AZT has a role in protecting target cells from apoptotic cell death, improving our understanding of the toxicology and the therapeutic usage of this drug.

SUBMITTER: Matteucci C

PROVIDER: S-EPMC3035897 | biostudies-other | 2010

REPOSITORIES: biostudies-other

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Inhibition of NF-κB activation sensitizes U937 cells to 3'-azido-3'-deoxythymidine induced apoptosis.

Matteucci C C Minutolo A A Balestrieri E E Marino-Merlo F F Bramanti P P Garaci E E Macchi B B Mastino A A

Cell death & disease 20101007

In this study, we investigated molecular mechanisms underlying low susceptibility to apoptosis induced by the nucleoside analog azidothymidine (AZT) and the role of nuclear factor-κB (NF-κB) activation in these phenomena. A preliminary screening in different cell lines indicated U937 monocytic cell line as suitable to this purpose. Treatment of U937 cells even with suprapharmacological concentrations of AZT induced only moderate levels of apoptosis. Surprisingly, SuperArray analysis showed that ...[more]

PMID: 21368854

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Project description:AimsAcute myeloblastic leukemia (AML) is the most common type of acute leukemia in adults. Despite numerous treatment strategies including chemotherapy and radiotherapy, a large number of patients do not respond to treatment and experience relapse. The main problem of these patients is the development of resistance to anti-cancer drugs. Therefore, any endeavor to reduce drug resistance in these patients is of high priority. In general, several mechanisms such as changes in drug metabolic pathways, drug inactivation, drug target alterations and reduced drug accumulation in the cells contribute to drug resistance of cancer cells. In this context, evidence suggests that exosomes could reduce drug resistance by removing drugs from their parent cells. In the present study, we aimed to investigate the effects of exosome release inhibition on the resistance of U937 cells to PEGylated liposomal doxorubicin (PLD).Main methodsIn order to find a suitable ABCG2 (ATP-binding cassette sub-family G member 2) transporter substrate, virtual screening was performed among a list of drugs used in leukemia and PLD was selected. U937 cells were treated with PLD with/without co-treatment with the exosome release inhibitor, GW4869. Released exosomes within different study groups were isolated and characterized to determine the differences between groups. Doxorubicin presence in the isolated exosomes was also measured by high performance liquid chromatography (HPLC) to confirm drug export through the exosomes. Finally, the effect of exosome inhibition on the cytotoxicity of PLD on U937 cells was determined using different cytotoxicity assays including the standard lactate dehydrogenase (LDH) release assay and the flow cytometric analysis of apoptotic and non-apoptotic cell death.Key findingsGW4869 treatment caused a significant decrease in the exosome release of U937 cells compared to the untreated cells, as evidenced by the reduction of the protein content of the isolated exosomes (P<0.05). Co-treatment with GW4869 significantly increased cytotoxic cell death in the groups treated with 0.5 and 1 µM PLD, compared to the same groups without GW4869 co-treatment (P<0.05). Interestingly, co-treatment with GW4896 and 0.5 µM PLD was enough to induce the same cytotoxic effect as that of the sole 1 µM PLD group.SignificanceOur findings showed that U937 cells increase their resistance against the cytotoxic effects of PLD through the exosome-mediated expelling of the drug. Inhibition of exosome release could prevent PLD efflux and consequently increase the vulnerability of the U937 cells to the cytotoxic effects of PLD. Our results along with prior studies indicate that the integration of exosome release inhibitors into the common PLD-containing chemotherapy regimens could significantly lower the required concentrations of the drug and consequently reduce its associated side effects. Further studies are warranted to identify clinically safe inhibitors and investigate their clinical efficacy.

Dataset Information

Inhibition of NF-?B activation sensitizes U937 cells to 3'-azido-3'-deoxythymidine induced apoptosis.

Publications

Inhibition of NF-κB activation sensitizes U937 cells to 3'-azido-3'-deoxythymidine induced apoptosis.

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