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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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Related in: MedlinePlus

Difference in structures and biological activities between hit compounds and classical lytic inducers.(a) AGS-BX1 cells were treated with the hit compounds for 24h. Hyperacetylation of histone proteins and PKCδ phosphorylation were detected by Western blotting. No significant increase in the level of acetylated histone 3 (Acetyl-H3) and phosphorylated PKCδ was observed. (b) AGS-BX1 cells were treated by compound E11 or C7 for 1, 2, 4, 8, 12 24h. Changes in the phosphorylation of JNK and p38 MAPK were examined in conjunction with changes in the expression level of EBV proteins. JNK phosphorylation increased and sustained during the treatment period while level of phosphorylated p38 MAPK fluctuated across the treatment period. (c) AGS-BX1 cells were pre-treated with specific inhibitors of PI3K (LY294002, 15 μM, LY), MEK (PD98059, 50μM, PD), JNK (SP600125, 50μM, SP), p38 MAPK (SB202190, 20μM, SB), PKCδ (Rottlerin, 10μM) and ATM (KU-55933, 10μM, Ku) kinases for 1h before the addition of E11 and C7. Cells were harvest after 24h for examination of lytic induction by Western blotting. Lytic induction by E11 was significantly inhibited by SP, the specific JNK inhibitor while both PD (MEK inhibitor) and SP affected the induction by C7. (d) Structure of common HDAC inhibitors, SAHA and romidepsin, and the phorbol ester TPA, used for lytic induction of EBV. The structure of the newly identified compounds differs greatly from the known lytic inducers.
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pone.0145994.g005: Difference in structures and biological activities between hit compounds and classical lytic inducers.(a) AGS-BX1 cells were treated with the hit compounds for 24h. Hyperacetylation of histone proteins and PKCδ phosphorylation were detected by Western blotting. No significant increase in the level of acetylated histone 3 (Acetyl-H3) and phosphorylated PKCδ was observed. (b) AGS-BX1 cells were treated by compound E11 or C7 for 1, 2, 4, 8, 12 24h. Changes in the phosphorylation of JNK and p38 MAPK were examined in conjunction with changes in the expression level of EBV proteins. JNK phosphorylation increased and sustained during the treatment period while level of phosphorylated p38 MAPK fluctuated across the treatment period. (c) AGS-BX1 cells were pre-treated with specific inhibitors of PI3K (LY294002, 15 μM, LY), MEK (PD98059, 50μM, PD), JNK (SP600125, 50μM, SP), p38 MAPK (SB202190, 20μM, SB), PKCδ (Rottlerin, 10μM) and ATM (KU-55933, 10μM, Ku) kinases for 1h before the addition of E11 and C7. Cells were harvest after 24h for examination of lytic induction by Western blotting. Lytic induction by E11 was significantly inhibited by SP, the specific JNK inhibitor while both PD (MEK inhibitor) and SP affected the induction by C7. (d) Structure of common HDAC inhibitors, SAHA and romidepsin, and the phorbol ester TPA, used for lytic induction of EBV. The structure of the newly identified compounds differs greatly from the known lytic inducers.

Mentions: We compared the structures of the 5 novel compounds (Fig 1E) with each other and with those of the classical lytic inducers, the HDAC inhibitors and the phorbol ester TPA (Fig 5D). Except for the common possession of a hydrazone bridge for C7 and C8, the other hit compounds all possess distinct chemical structures. They are, too, structurally different from the HDAC inhibitors and phorbol esters. We also tried to compare the biological activities of the new inducers to HDAC inhibitors and phorbol ester. HDAC inhibitors, upon administration to the cells, caused rapid and sustained hyperacetylation of histone proteins [23]. As shown in Fig 5A, the newly identified compounds did not cause hyperacetylation of the histone 3 protein (Acetyl-H3), which is a marker for global histone hyperacetylation, after 24h incubation with AGS-BX1 cells as by SAHA. They did not lead to significant increase in protein kinase C δ (PKCδ) phosphorylation. We further investigated the biological effects of 2 of the 5 compounds, E11, which induced the largest proportion of AGS-BX1 cells into lytic cycle, and C7, which could induce all the cell lines tested into lytic cycle, in greater detail. We incubated these two compounds with AGS-BX1 cells for 1h, 2h, 4h, 8h, 12h and 24h respectively at the respective concentrations that maximally induce lytic cycle, and probed for the changes in several major kinase signalling pathways previously associated with EBV lytic cycle reactivation (Fig 5B). We observed strong and sustained phosphorylation of c-Jun N-terminal kinase (JNK) after treatment with E11 from 1h to 24h post-treatment, which was concurrent with the increase in protein level of Zta. After we treated the cells with C7, there was also an increase in phosphorylation of JNK at a level lower than that caused by E11 throughout the same treatment period. However, romidepsin, an HDAC inhibitor, did not increase the phosphorylation of JNK after 24h of treatment (S4 Fig). Apart from sustained increase in JNK phosphorylation, we also observed fluctuations in the level of phosphorylation of p38 MAPK kinase. Upon treatment of both compounds at 1h post-treatment, the level of p38 MAPK phosphorylation increased. It then went down at 2h post-treatment and remained low afterwards for E11, while the level went down from 2h to 12h then increased again at 24h post-treatment for C7. To test if the activation of these kinase pathways mediated EBV lytic reactivation, we tried to block these pathways with specific chemical pathway blockers of PI3K, MAPK/Erk kinase (MEK), JNK, p38 MAPK, PKCδ and ATM, which have been previously associated with EBV lytic reactivation [5, 8, 14, 29–33] (Fig 5C). We observed that only SP600125, a specific chemical inhibitor of JNK, weakened the expression of Zta, Rta, and EA-D (BMRF1) significantly upon the treatment by compound E11. For compound C7, more than one inhibitor appeared to be able to counteract the induction, though the effect being more partial. PD98095, the specific inhibitor of MEK, consistently reduced the expression of Rta, and SP600125 constantly reduced the expression of Zta and EA-D (BMRF1), suggesting that the induction of C7 might not be mainly mediated through a single pathway but two or more pathways. We have separately shown that only rottlerin, the specific inhibitor of PKCδ, was able to abrogate the lytic induction by the HDAC inhibitors romidepsin [23] and SAHA (S5 Fig). In comparison, compounds E11 and C7 produced substantially different response patterns upon the co-incubation with these specific inhibitors, further substantiating their difference in mechanism of action with the typical lytic inducers like HDAC inhibitors.


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)

Difference in structures and biological activities between hit compounds and classical lytic inducers.(a) AGS-BX1 cells were treated with the hit compounds for 24h. Hyperacetylation of histone proteins and PKCδ phosphorylation were detected by Western blotting. No significant increase in the level of acetylated histone 3 (Acetyl-H3) and phosphorylated PKCδ was observed. (b) AGS-BX1 cells were treated by compound E11 or C7 for 1, 2, 4, 8, 12 24h. Changes in the phosphorylation of JNK and p38 MAPK were examined in conjunction with changes in the expression level of EBV proteins. JNK phosphorylation increased and sustained during the treatment period while level of phosphorylated p38 MAPK fluctuated across the treatment period. (c) AGS-BX1 cells were pre-treated with specific inhibitors of PI3K (LY294002, 15 μM, LY), MEK (PD98059, 50μM, PD), JNK (SP600125, 50μM, SP), p38 MAPK (SB202190, 20μM, SB), PKCδ (Rottlerin, 10μM) and ATM (KU-55933, 10μM, Ku) kinases for 1h before the addition of E11 and C7. Cells were harvest after 24h for examination of lytic induction by Western blotting. Lytic induction by E11 was significantly inhibited by SP, the specific JNK inhibitor while both PD (MEK inhibitor) and SP affected the induction by C7. (d) Structure of common HDAC inhibitors, SAHA and romidepsin, and the phorbol ester TPA, used for lytic induction of EBV. The structure of the newly identified compounds differs greatly from the known lytic inducers.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145994.g005: Difference in structures and biological activities between hit compounds and classical lytic inducers.(a) AGS-BX1 cells were treated with the hit compounds for 24h. Hyperacetylation of histone proteins and PKCδ phosphorylation were detected by Western blotting. No significant increase in the level of acetylated histone 3 (Acetyl-H3) and phosphorylated PKCδ was observed. (b) AGS-BX1 cells were treated by compound E11 or C7 for 1, 2, 4, 8, 12 24h. Changes in the phosphorylation of JNK and p38 MAPK were examined in conjunction with changes in the expression level of EBV proteins. JNK phosphorylation increased and sustained during the treatment period while level of phosphorylated p38 MAPK fluctuated across the treatment period. (c) AGS-BX1 cells were pre-treated with specific inhibitors of PI3K (LY294002, 15 μM, LY), MEK (PD98059, 50μM, PD), JNK (SP600125, 50μM, SP), p38 MAPK (SB202190, 20μM, SB), PKCδ (Rottlerin, 10μM) and ATM (KU-55933, 10μM, Ku) kinases for 1h before the addition of E11 and C7. Cells were harvest after 24h for examination of lytic induction by Western blotting. Lytic induction by E11 was significantly inhibited by SP, the specific JNK inhibitor while both PD (MEK inhibitor) and SP affected the induction by C7. (d) Structure of common HDAC inhibitors, SAHA and romidepsin, and the phorbol ester TPA, used for lytic induction of EBV. The structure of the newly identified compounds differs greatly from the known lytic inducers.
Mentions: We compared the structures of the 5 novel compounds (Fig 1E) with each other and with those of the classical lytic inducers, the HDAC inhibitors and the phorbol ester TPA (Fig 5D). Except for the common possession of a hydrazone bridge for C7 and C8, the other hit compounds all possess distinct chemical structures. They are, too, structurally different from the HDAC inhibitors and phorbol esters. We also tried to compare the biological activities of the new inducers to HDAC inhibitors and phorbol ester. HDAC inhibitors, upon administration to the cells, caused rapid and sustained hyperacetylation of histone proteins [23]. As shown in Fig 5A, the newly identified compounds did not cause hyperacetylation of the histone 3 protein (Acetyl-H3), which is a marker for global histone hyperacetylation, after 24h incubation with AGS-BX1 cells as by SAHA. They did not lead to significant increase in protein kinase C δ (PKCδ) phosphorylation. We further investigated the biological effects of 2 of the 5 compounds, E11, which induced the largest proportion of AGS-BX1 cells into lytic cycle, and C7, which could induce all the cell lines tested into lytic cycle, in greater detail. We incubated these two compounds with AGS-BX1 cells for 1h, 2h, 4h, 8h, 12h and 24h respectively at the respective concentrations that maximally induce lytic cycle, and probed for the changes in several major kinase signalling pathways previously associated with EBV lytic cycle reactivation (Fig 5B). We observed strong and sustained phosphorylation of c-Jun N-terminal kinase (JNK) after treatment with E11 from 1h to 24h post-treatment, which was concurrent with the increase in protein level of Zta. After we treated the cells with C7, there was also an increase in phosphorylation of JNK at a level lower than that caused by E11 throughout the same treatment period. However, romidepsin, an HDAC inhibitor, did not increase the phosphorylation of JNK after 24h of treatment (S4 Fig). Apart from sustained increase in JNK phosphorylation, we also observed fluctuations in the level of phosphorylation of p38 MAPK kinase. Upon treatment of both compounds at 1h post-treatment, the level of p38 MAPK phosphorylation increased. It then went down at 2h post-treatment and remained low afterwards for E11, while the level went down from 2h to 12h then increased again at 24h post-treatment for C7. To test if the activation of these kinase pathways mediated EBV lytic reactivation, we tried to block these pathways with specific chemical pathway blockers of PI3K, MAPK/Erk kinase (MEK), JNK, p38 MAPK, PKCδ and ATM, which have been previously associated with EBV lytic reactivation [5, 8, 14, 29–33] (Fig 5C). We observed that only SP600125, a specific chemical inhibitor of JNK, weakened the expression of Zta, Rta, and EA-D (BMRF1) significantly upon the treatment by compound E11. For compound C7, more than one inhibitor appeared to be able to counteract the induction, though the effect being more partial. PD98095, the specific inhibitor of MEK, consistently reduced the expression of Rta, and SP600125 constantly reduced the expression of Zta and EA-D (BMRF1), suggesting that the induction of C7 might not be mainly mediated through a single pathway but two or more pathways. We have separately shown that only rottlerin, the specific inhibitor of PKCδ, was able to abrogate the lytic induction by the HDAC inhibitors romidepsin [23] and SAHA (S5 Fig). In comparison, compounds E11 and C7 produced substantially different response patterns upon the co-incubation with these specific inhibitors, further substantiating their difference in mechanism of action with the typical lytic inducers like HDAC inhibitors.

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