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Identification of Novel Small Organic Compounds with Diverse Structures for the Induction of Epstein-Barr Virus (EBV) Lytic Cycle in EBV-Positive Epithelial Malignancies.

Choi CK, Ho DN, Hui KF, Kao RY, Chiang AK - PLoS ONE (2015)

Bottom Line: Five hit compounds were selected after three successive rounds of increasingly stringent screening.They neither cause hyperacetylation of histone proteins nor significant PKC activation at their working concentrations, suggesting that their biological mode of action are distinct from that of the known chemical inducers.One of the two compounds bears structural resemblance to iron chelators and the other strongly activates the MAPK pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.

ABSTRACT
Phorbol esters, which are protein kinase C (PKC) activators, and histone deacetylase (HDAC) inhibitors, which cause enhanced acetylation of cellular proteins, are the main classes of chemical inducers of Epstein-Barr virus (EBV) lytic cycle in latently EBV-infected cells acting through the PKC pathway. Chemical inducers which induce EBV lytic cycle through alternative cellular pathways may aid in defining the mechanisms leading to lytic cycle reactivation and improve cells' responsiveness towards lytic induction. We performed a phenotypic screening on a chemical library of 50,240 novel small organic compounds to identify novel class(es) of strong inducer(s) of EBV lytic cycle in gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC) cells. Five hit compounds were selected after three successive rounds of increasingly stringent screening. All five compounds are structurally diverse from each other and distinct from phorbol esters or HDAC inhibitors. They neither cause hyperacetylation of histone proteins nor significant PKC activation at their working concentrations, suggesting that their biological mode of action are distinct from that of the known chemical inducers. Two of the five compounds with rapid lytic-inducing action were further studied for their mechanisms of induction of EBV lytic cycle. Unlike HDAC inhibitors, lytic induction by both compounds was not inhibited by rottlerin, a specific inhibitor of PKCδ. Interestingly, both compounds could cooperate with HDAC inhibitors to enhance EBV lytic cycle induction in EBV-positive epithelial cancer cells, paving way for the development of strategies to increase cells' responsiveness towards lytic reactivation. One of the two compounds bears structural resemblance to iron chelators and the other strongly activates the MAPK pathways. These structurally diverse novel organic compounds may represent potential new classes of chemicals that can be used to investigate any alternative mechanism(s) leading to EBV lytic cycle reactivation from latency.

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EBV lytic induction of the 5 hit compounds in the EBV-positive GC cells.AGS-BX1 cells were treated with (a) various concentrations of the hit compounds or (b) the hit compounds at the concentrations which maximally induced lytic cycle (optimal concentrations) at various time points. Expression of various EBV lytic proteins were analysed with Western blotting with cellular β-actin as a loading control. (c) AGS-BX1 cells were treated with the hit compounds at their optimal concentrations for 72h. The expression of Zta was visualized by immunofluorescent staining (red; middle panel) and the nuclei were visualized by DAPI (blue; top panel). (d) AGS-BX1 cells were treated with the hit compounds at their optimal concentrations for 72h. The percentage of cells induced into lytic cycle, i.e. expressing the IE protein Zta, was quantified by flow cytometry. (e) Production of infectious virions in AGS-BX1 cells after treatment with the hit compounds. AGS-Bx1 cells, which are capable of producing GFP-tagged EBV virions, were first treated by the hit compounds for 5 days. The supernatants containing the virions produced were then collected and incubated with Daudi cells, which are susceptible to superinfection of EBV. Percentage of superinfected Daudi cells, which were GFP-positive, were shown on the graph. The grey area represented unstained Daudi cells incubated with culture medium only.
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pone.0145994.g003: EBV lytic induction of the 5 hit compounds in the EBV-positive GC cells.AGS-BX1 cells were treated with (a) various concentrations of the hit compounds or (b) the hit compounds at the concentrations which maximally induced lytic cycle (optimal concentrations) at various time points. Expression of various EBV lytic proteins were analysed with Western blotting with cellular β-actin as a loading control. (c) AGS-BX1 cells were treated with the hit compounds at their optimal concentrations for 72h. The expression of Zta was visualized by immunofluorescent staining (red; middle panel) and the nuclei were visualized by DAPI (blue; top panel). (d) AGS-BX1 cells were treated with the hit compounds at their optimal concentrations for 72h. The percentage of cells induced into lytic cycle, i.e. expressing the IE protein Zta, was quantified by flow cytometry. (e) Production of infectious virions in AGS-BX1 cells after treatment with the hit compounds. AGS-Bx1 cells, which are capable of producing GFP-tagged EBV virions, were first treated by the hit compounds for 5 days. The supernatants containing the virions produced were then collected and incubated with Daudi cells, which are susceptible to superinfection of EBV. Percentage of superinfected Daudi cells, which were GFP-positive, were shown on the graph. The grey area represented unstained Daudi cells incubated with culture medium only.

Mentions: For detailed characterization of these newly identified compounds, we tested their cytotoxicity in EBV-positive and EBV-negative paired cell lines. We also tested the dose response, kinetics response of the compounds, the percentage of cells being induced into lytic cycle, and the ability to induce the production of infectious virus in AGS-BX1 cells upon treatment by each compound. Of the 5 compounds identified, 4 (compounds coded E11, C7, C8 and A10) displayed significantly higher toxicity to the EBV-positive cell line AGS-BX1 than the EBV-negative counterpart AGS at the lytic-inducing concentrations, demonstrating EBV-specific killing of these compounds (Fig 2). The 5 compounds could all induce dose-dependent expression of EBV viral lytic proteins at micromolar concentrations on AGS-BX1 cells, with maximal induction observed at doses ranging from 2.5 to 20μM (Fig 3A). The lytic induction kinetics varied between compounds, the fastest of which being the compounds coded E11 and C7, with the expression of IE proteins Zta, Rta, and early EBV lytic protein BMRF1 peaking at 24h post-treatment. Other compounds exhibited slower lytic induction kinetics, with the expression of Zta, Rta, and BMRF1 peaking at 48-72h post-treatment (Fig 3B). Interestingly, further time point experiments revealed that the expression of the master switch protein of lytic cycle, Zta, was detected even 15min after the incubation of E11 and C7 with the AGS-BX1 cells (S2 Fig). Such fast action and high potency make them attractive targets for investigation into their mechanism of action. Although all 5 compounds could potently induce the expression of IE and early EBV lytic proteins, not all induced the expression of late proteins. Among the 5, only E11 consistently induced the expression of late proteins, e.g. VCA-p18 and/or gp350/220 in AGS-BX1, HONE1-EBV and YCCEL1 cells (Fig 3B and S3 Fig). The expression of late proteins correlated closely with the production of infectious viral particles in the EBV infection assay. In particular, after a 5-day incubation with the compound triggered the most expression of late proteins E11, the filtered culture supernatant from AGS-BX1 cells infected 40% of the Daudi cells in the EBV infection assay (Fig 3F)–the most among the 5 hit compounds. Immunofluorescent staining of AGS-BX1 cells after 3-day incubation with the compounds revealed that most of the Zta proteins were localised within the nuclei of cells (Fig 3D), consistent with their role as a transcription transactivator and replication-associated factor [3, 48, 49]. We quantified the percentage of cells expressing Zta by flow cytometry to obtain an estimation of cells going into lytic cycle. On average the Zta-positive population approximately ranged from 20% to 40%, with E11 inducing the expression of Zta in up to 65% of cells (Fig 3E).


Identification of Novel Small Organic Compounds with Diverse Structures for the Induction of Epstein-Barr Virus (EBV) Lytic Cycle in EBV-Positive Epithelial Malignancies.

Choi CK, Ho DN, Hui KF, Kao RY, Chiang AK - PLoS ONE (2015)

EBV lytic induction of the 5 hit compounds in the EBV-positive GC cells.AGS-BX1 cells were treated with (a) various concentrations of the hit compounds or (b) the hit compounds at the concentrations which maximally induced lytic cycle (optimal concentrations) at various time points. Expression of various EBV lytic proteins were analysed with Western blotting with cellular β-actin as a loading control. (c) AGS-BX1 cells were treated with the hit compounds at their optimal concentrations for 72h. The expression of Zta was visualized by immunofluorescent staining (red; middle panel) and the nuclei were visualized by DAPI (blue; top panel). (d) AGS-BX1 cells were treated with the hit compounds at their optimal concentrations for 72h. The percentage of cells induced into lytic cycle, i.e. expressing the IE protein Zta, was quantified by flow cytometry. (e) Production of infectious virions in AGS-BX1 cells after treatment with the hit compounds. AGS-Bx1 cells, which are capable of producing GFP-tagged EBV virions, were first treated by the hit compounds for 5 days. The supernatants containing the virions produced were then collected and incubated with Daudi cells, which are susceptible to superinfection of EBV. Percentage of superinfected Daudi cells, which were GFP-positive, were shown on the graph. The grey area represented unstained Daudi cells incubated with culture medium only.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4696655&req=5

pone.0145994.g003: EBV lytic induction of the 5 hit compounds in the EBV-positive GC cells.AGS-BX1 cells were treated with (a) various concentrations of the hit compounds or (b) the hit compounds at the concentrations which maximally induced lytic cycle (optimal concentrations) at various time points. Expression of various EBV lytic proteins were analysed with Western blotting with cellular β-actin as a loading control. (c) AGS-BX1 cells were treated with the hit compounds at their optimal concentrations for 72h. The expression of Zta was visualized by immunofluorescent staining (red; middle panel) and the nuclei were visualized by DAPI (blue; top panel). (d) AGS-BX1 cells were treated with the hit compounds at their optimal concentrations for 72h. The percentage of cells induced into lytic cycle, i.e. expressing the IE protein Zta, was quantified by flow cytometry. (e) Production of infectious virions in AGS-BX1 cells after treatment with the hit compounds. AGS-Bx1 cells, which are capable of producing GFP-tagged EBV virions, were first treated by the hit compounds for 5 days. The supernatants containing the virions produced were then collected and incubated with Daudi cells, which are susceptible to superinfection of EBV. Percentage of superinfected Daudi cells, which were GFP-positive, were shown on the graph. The grey area represented unstained Daudi cells incubated with culture medium only.
Mentions: For detailed characterization of these newly identified compounds, we tested their cytotoxicity in EBV-positive and EBV-negative paired cell lines. We also tested the dose response, kinetics response of the compounds, the percentage of cells being induced into lytic cycle, and the ability to induce the production of infectious virus in AGS-BX1 cells upon treatment by each compound. Of the 5 compounds identified, 4 (compounds coded E11, C7, C8 and A10) displayed significantly higher toxicity to the EBV-positive cell line AGS-BX1 than the EBV-negative counterpart AGS at the lytic-inducing concentrations, demonstrating EBV-specific killing of these compounds (Fig 2). The 5 compounds could all induce dose-dependent expression of EBV viral lytic proteins at micromolar concentrations on AGS-BX1 cells, with maximal induction observed at doses ranging from 2.5 to 20μM (Fig 3A). The lytic induction kinetics varied between compounds, the fastest of which being the compounds coded E11 and C7, with the expression of IE proteins Zta, Rta, and early EBV lytic protein BMRF1 peaking at 24h post-treatment. Other compounds exhibited slower lytic induction kinetics, with the expression of Zta, Rta, and BMRF1 peaking at 48-72h post-treatment (Fig 3B). Interestingly, further time point experiments revealed that the expression of the master switch protein of lytic cycle, Zta, was detected even 15min after the incubation of E11 and C7 with the AGS-BX1 cells (S2 Fig). Such fast action and high potency make them attractive targets for investigation into their mechanism of action. Although all 5 compounds could potently induce the expression of IE and early EBV lytic proteins, not all induced the expression of late proteins. Among the 5, only E11 consistently induced the expression of late proteins, e.g. VCA-p18 and/or gp350/220 in AGS-BX1, HONE1-EBV and YCCEL1 cells (Fig 3B and S3 Fig). The expression of late proteins correlated closely with the production of infectious viral particles in the EBV infection assay. In particular, after a 5-day incubation with the compound triggered the most expression of late proteins E11, the filtered culture supernatant from AGS-BX1 cells infected 40% of the Daudi cells in the EBV infection assay (Fig 3F)–the most among the 5 hit compounds. Immunofluorescent staining of AGS-BX1 cells after 3-day incubation with the compounds revealed that most of the Zta proteins were localised within the nuclei of cells (Fig 3D), consistent with their role as a transcription transactivator and replication-associated factor [3, 48, 49]. We quantified the percentage of cells expressing Zta by flow cytometry to obtain an estimation of cells going into lytic cycle. On average the Zta-positive population approximately ranged from 20% to 40%, with E11 inducing the expression of Zta in up to 65% of cells (Fig 3E).

Bottom Line: Five hit compounds were selected after three successive rounds of increasingly stringent screening.They neither cause hyperacetylation of histone proteins nor significant PKC activation at their working concentrations, suggesting that their biological mode of action are distinct from that of the known chemical inducers.One of the two compounds bears structural resemblance to iron chelators and the other strongly activates the MAPK pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China.

ABSTRACT
Phorbol esters, which are protein kinase C (PKC) activators, and histone deacetylase (HDAC) inhibitors, which cause enhanced acetylation of cellular proteins, are the main classes of chemical inducers of Epstein-Barr virus (EBV) lytic cycle in latently EBV-infected cells acting through the PKC pathway. Chemical inducers which induce EBV lytic cycle through alternative cellular pathways may aid in defining the mechanisms leading to lytic cycle reactivation and improve cells' responsiveness towards lytic induction. We performed a phenotypic screening on a chemical library of 50,240 novel small organic compounds to identify novel class(es) of strong inducer(s) of EBV lytic cycle in gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC) cells. Five hit compounds were selected after three successive rounds of increasingly stringent screening. All five compounds are structurally diverse from each other and distinct from phorbol esters or HDAC inhibitors. They neither cause hyperacetylation of histone proteins nor significant PKC activation at their working concentrations, suggesting that their biological mode of action are distinct from that of the known chemical inducers. Two of the five compounds with rapid lytic-inducing action were further studied for their mechanisms of induction of EBV lytic cycle. Unlike HDAC inhibitors, lytic induction by both compounds was not inhibited by rottlerin, a specific inhibitor of PKCδ. Interestingly, both compounds could cooperate with HDAC inhibitors to enhance EBV lytic cycle induction in EBV-positive epithelial cancer cells, paving way for the development of strategies to increase cells' responsiveness towards lytic reactivation. One of the two compounds bears structural resemblance to iron chelators and the other strongly activates the MAPK pathways. These structurally diverse novel organic compounds may represent potential new classes of chemicals that can be used to investigate any alternative mechanism(s) leading to EBV lytic cycle reactivation from latency.

Show MeSH
Related in: MedlinePlus