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Heat shock protein 90 inhibitors repress latent membrane protein 1 (LMP1) expression and proliferation of Epstein-Barr virus-positive natural killer cell lymphoma.

Murata T, Iwata S, Siddiquey MN, Kanazawa T, Goshima F, Kawashima D, Kimura H, Tsurumi T - PLoS ONE (2013)

Bottom Line: We here screened small molecule inhibitors and isolated HSP90 inhibitors, Radicicol and 17-AAG, as candidates that suppress LMP1 expression and cell proliferation not only in EBV-positive SNK6 Natural Killer (NK) cell lymphoma cells, but also in B and T cells.Tumor formation in immuno-defficient NOD/Shi-scid/IL-2Rγ() (NOG) mice was also retarded.These results suggest that HSP90 inhibitors can be alternative treatments for patients with EBV-positive malignancies.

View Article: PubMed Central - PubMed

Affiliation: Division of Virology, Aichi Cancer Center Research Institute, Nagoya, Aichi, Japan.

ABSTRACT
Epstein-Barr virus (EBV) LMP1 is a major oncoprotein expressed in latent infection. It functions as a TNFR family member and constitutively activates cellular signals, such as NFκB, MAPK, JAK/STAT and AKT. We here screened small molecule inhibitors and isolated HSP90 inhibitors, Radicicol and 17-AAG, as candidates that suppress LMP1 expression and cell proliferation not only in EBV-positive SNK6 Natural Killer (NK) cell lymphoma cells, but also in B and T cells. Tumor formation in immuno-defficient NOD/Shi-scid/IL-2Rγ() (NOG) mice was also retarded. These results suggest that HSP90 inhibitors can be alternative treatments for patients with EBV-positive malignancies.

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Representative example of screening for small molecule inhibitors that repress LMP1 expression.The EBV-positive NK lymphoma cell line, SNK6, was seeded and small molecule inhibitors were added to the media at concentrations of 10 and 3 µM. After 72 h, cell RNA was collected and subjected to Real-Time RT-PCR using specific primers for LMP1 and GAPDH mRNAs. Relative LMP1 mRNA levels are shown after normalization to GAPDH mRNA levels.
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pone-0063566-g001: Representative example of screening for small molecule inhibitors that repress LMP1 expression.The EBV-positive NK lymphoma cell line, SNK6, was seeded and small molecule inhibitors were added to the media at concentrations of 10 and 3 µM. After 72 h, cell RNA was collected and subjected to Real-Time RT-PCR using specific primers for LMP1 and GAPDH mRNAs. Relative LMP1 mRNA levels are shown after normalization to GAPDH mRNA levels.

Mentions: CAEBV and ENKL committed to type II latency express the major EBV oncogene, LMP1. We therefore conducted a search for small molecular inhibitors that repress expression of LMP1. As an initial screen, the EBV-positive NK cell lymphoma, SNK6, was treated with chemicals or the vehicle DMSO at concentrations of 3 or 10 µM for 3 days. Cellular RNAs were harvested and subjected to Real-Time RT-PCR. Among 300 small molecule substances with identified targets, we found Radicicol and 17-AAG, inhibitors of HSP90, to decrease LMP1 transcripts (Fig. 1). In order to further evaluate the effect, SNK6 cells were administered 3, 1, 0.3, 0.1 µM of Radicicol or 17-AAG (Fig. 2A–C). LMP1 levels decreased to 41% and 48% of control (DMSO) with 3 µM of Radicicol and 17-AAG, respectively (Fig. 2A), with EBNA1 levels appearing relatively unaffected (Fig. 2B). We then examined protein levels of LMP1 in Fig. 2C. After 72 h with 3 or 1 µM of Radicicol or 17-AAG, LMP1 protein levels were decreased in SNK6 cells (Fig. 2C), in a similar fashion with the transcript levels. LMP1 expression in B95-8 or EBV-positive T cell line (SNT13) was also reduced by these HSP90 inhibitors (Fig. 2D, E). For unknown reasons, 17-AAG markedly suppressed LMP1 expression from SNT13 cells even at lower concentrations (Fig. 2E).


Heat shock protein 90 inhibitors repress latent membrane protein 1 (LMP1) expression and proliferation of Epstein-Barr virus-positive natural killer cell lymphoma.

Murata T, Iwata S, Siddiquey MN, Kanazawa T, Goshima F, Kawashima D, Kimura H, Tsurumi T - PLoS ONE (2013)

Representative example of screening for small molecule inhibitors that repress LMP1 expression.The EBV-positive NK lymphoma cell line, SNK6, was seeded and small molecule inhibitors were added to the media at concentrations of 10 and 3 µM. After 72 h, cell RNA was collected and subjected to Real-Time RT-PCR using specific primers for LMP1 and GAPDH mRNAs. Relative LMP1 mRNA levels are shown after normalization to GAPDH mRNA levels.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3643901&req=5

pone-0063566-g001: Representative example of screening for small molecule inhibitors that repress LMP1 expression.The EBV-positive NK lymphoma cell line, SNK6, was seeded and small molecule inhibitors were added to the media at concentrations of 10 and 3 µM. After 72 h, cell RNA was collected and subjected to Real-Time RT-PCR using specific primers for LMP1 and GAPDH mRNAs. Relative LMP1 mRNA levels are shown after normalization to GAPDH mRNA levels.
Mentions: CAEBV and ENKL committed to type II latency express the major EBV oncogene, LMP1. We therefore conducted a search for small molecular inhibitors that repress expression of LMP1. As an initial screen, the EBV-positive NK cell lymphoma, SNK6, was treated with chemicals or the vehicle DMSO at concentrations of 3 or 10 µM for 3 days. Cellular RNAs were harvested and subjected to Real-Time RT-PCR. Among 300 small molecule substances with identified targets, we found Radicicol and 17-AAG, inhibitors of HSP90, to decrease LMP1 transcripts (Fig. 1). In order to further evaluate the effect, SNK6 cells were administered 3, 1, 0.3, 0.1 µM of Radicicol or 17-AAG (Fig. 2A–C). LMP1 levels decreased to 41% and 48% of control (DMSO) with 3 µM of Radicicol and 17-AAG, respectively (Fig. 2A), with EBNA1 levels appearing relatively unaffected (Fig. 2B). We then examined protein levels of LMP1 in Fig. 2C. After 72 h with 3 or 1 µM of Radicicol or 17-AAG, LMP1 protein levels were decreased in SNK6 cells (Fig. 2C), in a similar fashion with the transcript levels. LMP1 expression in B95-8 or EBV-positive T cell line (SNT13) was also reduced by these HSP90 inhibitors (Fig. 2D, E). For unknown reasons, 17-AAG markedly suppressed LMP1 expression from SNT13 cells even at lower concentrations (Fig. 2E).

Bottom Line: We here screened small molecule inhibitors and isolated HSP90 inhibitors, Radicicol and 17-AAG, as candidates that suppress LMP1 expression and cell proliferation not only in EBV-positive SNK6 Natural Killer (NK) cell lymphoma cells, but also in B and T cells.Tumor formation in immuno-defficient NOD/Shi-scid/IL-2Rγ() (NOG) mice was also retarded.These results suggest that HSP90 inhibitors can be alternative treatments for patients with EBV-positive malignancies.

View Article: PubMed Central - PubMed

Affiliation: Division of Virology, Aichi Cancer Center Research Institute, Nagoya, Aichi, Japan.

ABSTRACT
Epstein-Barr virus (EBV) LMP1 is a major oncoprotein expressed in latent infection. It functions as a TNFR family member and constitutively activates cellular signals, such as NFκB, MAPK, JAK/STAT and AKT. We here screened small molecule inhibitors and isolated HSP90 inhibitors, Radicicol and 17-AAG, as candidates that suppress LMP1 expression and cell proliferation not only in EBV-positive SNK6 Natural Killer (NK) cell lymphoma cells, but also in B and T cells. Tumor formation in immuno-defficient NOD/Shi-scid/IL-2Rγ() (NOG) mice was also retarded. These results suggest that HSP90 inhibitors can be alternative treatments for patients with EBV-positive malignancies.

Show MeSH
Related in: MedlinePlus