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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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Suppression of LMP1 expression in EBV-positive NK cells by HSP90 inhibitors is likely dependent on IL-2.SNK6 cells, routinely cultured with IL-2, were washed extensively with PBS, and then cultured in the presence (black bars) or absence (gray bars) of IL-2 with Radicicol (R) or 17-AAG (A) at concentrations of 3, 1, 0.3 or 0.1 µM. After 72 h, cell RNAs were collected and subjected to Real-Time RT-PCR. Relative levels of LMP1 mRNA are shown after normalization to GAPDH mRNA levels. Each bar represents the mean and SD of three independent transfections.
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pone-0063566-g005: Suppression of LMP1 expression in EBV-positive NK cells by HSP90 inhibitors is likely dependent on IL-2.SNK6 cells, routinely cultured with IL-2, were washed extensively with PBS, and then cultured in the presence (black bars) or absence (gray bars) of IL-2 with Radicicol (R) or 17-AAG (A) at concentrations of 3, 1, 0.3 or 0.1 µM. After 72 h, cell RNAs were collected and subjected to Real-Time RT-PCR. Relative levels of LMP1 mRNA are shown after normalization to GAPDH mRNA levels. Each bar represents the mean and SD of three independent transfections.

Mentions: LMP1 gene transcription differs between type II and type III latency infection. In the latter, LMP1 transcription is turned on by EBNA2 [30]–[32], whereas LMP1 expression is independent of EBNA2 in type II. In the previous work of Jeon and others induction of apoptosis by HSP90 inhibitors was considered to be through AKT signaling inhibition [27]. The decreased expression of LMP1 gene we observed here (Fig. 1, 2) might have been brought about through suppression of the AKT pathway. In latency II, including the ENKL case, it has been frequently reported that cytokines, such as IL-4, IL-6, IL-10, IL-13 and IL-21, activate the JAK/STAT pathway, thereby inducing LMP1 gene expression through STAT [33]–[38]. Because JAK/STAT signaling can also be blocked by HSP90 inhibitors [39], it may also be involved. Actually, since our EBV-positive NK/T cells are cultured routinely with IL-2 and it is involved in LMP1 expression [40], the cytokine and its downstream signaling may also be implicated in the reduction by HSP90 inhibitors. To examine this, we cultured SNK6 cells with or without IL-2 and HSP90 inhibitors, and measured the levels of LMP1 mRNA (Fig. 5). As expected from the previous report [40], depletion of IL-2 down-regulated LMP1 levels to 5.5% of control, in DMSO-treated cells. Treatment with Radicicol (R) or 17-AAG (A) at 1 µM or higher caused reduction of LMP1 levels in the presence of IL-2 (black bars), but it did not significantly decrease the levels without IL-2 (gray bars). This result suggests that HSP90 inhibitors suppress LMP1 expression, which is activated by IL-2, and that the cell signalings elicited by IL-2, such as JAK/STAT, are likely be responsible for the LMP1 reduction by HSP90 inhibitors. Besides JAK/STAT pathways, NFκB signaling must also be notified, because it is known that IL-2 elicits NFκB signaling [41], and HSP90 inhibitors can repress NFκB [42], [43]. In addition, C/EBP contributes to LMP1 expression in type II [25], and may also be regulated by HSP90 inhibitors.


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)

Suppression of LMP1 expression in EBV-positive NK cells by HSP90 inhibitors is likely dependent on IL-2.SNK6 cells, routinely cultured with IL-2, were washed extensively with PBS, and then cultured in the presence (black bars) or absence (gray bars) of IL-2 with Radicicol (R) or 17-AAG (A) at concentrations of 3, 1, 0.3 or 0.1 µM. After 72 h, cell RNAs were collected and subjected to Real-Time RT-PCR. Relative levels of LMP1 mRNA are shown after normalization to GAPDH mRNA levels. Each bar represents the mean and SD of three independent transfections.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3643901&req=5

pone-0063566-g005: Suppression of LMP1 expression in EBV-positive NK cells by HSP90 inhibitors is likely dependent on IL-2.SNK6 cells, routinely cultured with IL-2, were washed extensively with PBS, and then cultured in the presence (black bars) or absence (gray bars) of IL-2 with Radicicol (R) or 17-AAG (A) at concentrations of 3, 1, 0.3 or 0.1 µM. After 72 h, cell RNAs were collected and subjected to Real-Time RT-PCR. Relative levels of LMP1 mRNA are shown after normalization to GAPDH mRNA levels. Each bar represents the mean and SD of three independent transfections.
Mentions: LMP1 gene transcription differs between type II and type III latency infection. In the latter, LMP1 transcription is turned on by EBNA2 [30]–[32], whereas LMP1 expression is independent of EBNA2 in type II. In the previous work of Jeon and others induction of apoptosis by HSP90 inhibitors was considered to be through AKT signaling inhibition [27]. The decreased expression of LMP1 gene we observed here (Fig. 1, 2) might have been brought about through suppression of the AKT pathway. In latency II, including the ENKL case, it has been frequently reported that cytokines, such as IL-4, IL-6, IL-10, IL-13 and IL-21, activate the JAK/STAT pathway, thereby inducing LMP1 gene expression through STAT [33]–[38]. Because JAK/STAT signaling can also be blocked by HSP90 inhibitors [39], it may also be involved. Actually, since our EBV-positive NK/T cells are cultured routinely with IL-2 and it is involved in LMP1 expression [40], the cytokine and its downstream signaling may also be implicated in the reduction by HSP90 inhibitors. To examine this, we cultured SNK6 cells with or without IL-2 and HSP90 inhibitors, and measured the levels of LMP1 mRNA (Fig. 5). As expected from the previous report [40], depletion of IL-2 down-regulated LMP1 levels to 5.5% of control, in DMSO-treated cells. Treatment with Radicicol (R) or 17-AAG (A) at 1 µM or higher caused reduction of LMP1 levels in the presence of IL-2 (black bars), but it did not significantly decrease the levels without IL-2 (gray bars). This result suggests that HSP90 inhibitors suppress LMP1 expression, which is activated by IL-2, and that the cell signalings elicited by IL-2, such as JAK/STAT, are likely be responsible for the LMP1 reduction by HSP90 inhibitors. Besides JAK/STAT pathways, NFκB signaling must also be notified, because it is known that IL-2 elicits NFκB signaling [41], and HSP90 inhibitors can repress NFκB [42], [43]. In addition, C/EBP contributes to LMP1 expression in type II [25], and may also be regulated by HSP90 inhibitors.

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