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Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation.

Carson C, Raman P, Tullai J, Xu L, Henault M, Thomas E, Yeola S, Lao J, McPate M, Verkuyl JM, Marsh G, Sarber J, Amaral A, Bailey S, Lubicka D, Pham H, Miranda N, Ding J, Tang HM, Ju H, Tranter P, Ji N, Krastel P, Jain RK, Schumacher AM, Loureiro JJ, George E, Berellini G, Ross NT, Bushell SM, Erdemli G, Solomon JM - PLoS ONE (2015)

Bottom Line: In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel.This toxicity suggests that englerin A itself is probably unsuitable for further drug development.However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, United States of America.

ABSTRACT
Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.

No MeSH data available.


Related in: MedlinePlus

Englerin A agonizes the TRPC4/C5 ion channels and channel activation is needed for cell growth inhibition.(A) Calcium flux stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation): TRPC5 (closed diamonds), TRPC4beta (closed squares), TRPC4 (closed circles), TRPC6 (open squares), mock transfected cells (open circles). (B) Membrane depolarization stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation), markers as above. (C) TRPC4 current evoked by stimulation of 5 μM Englerin A, saline, or 5 μM Englerin A + 10 μM ML204 in 293T cells with Doxycyline-induced TRPC4. Currents were elicited by 200 ms voltage ramps from -100 to +100 mV, applied every 10 s from holding potential of 0 mV. (D) Summary of englerin A, englerin-B and ML-204 activity on membrane currents (mean +/- S.E.M.) (E) A-673 cell viability in the presence or absence of 50 nM englerin A and/or 50 μM ML204, a TRPC4/C5 channel blocker (mean +/- standard deviation).
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pone.0127498.g006: Englerin A agonizes the TRPC4/C5 ion channels and channel activation is needed for cell growth inhibition.(A) Calcium flux stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation): TRPC5 (closed diamonds), TRPC4beta (closed squares), TRPC4 (closed circles), TRPC6 (open squares), mock transfected cells (open circles). (B) Membrane depolarization stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation), markers as above. (C) TRPC4 current evoked by stimulation of 5 μM Englerin A, saline, or 5 μM Englerin A + 10 μM ML204 in 293T cells with Doxycyline-induced TRPC4. Currents were elicited by 200 ms voltage ramps from -100 to +100 mV, applied every 10 s from holding potential of 0 mV. (D) Summary of englerin A, englerin-B and ML-204 activity on membrane currents (mean +/- S.E.M.) (E) A-673 cell viability in the presence or absence of 50 nM englerin A and/or 50 μM ML204, a TRPC4/C5 channel blocker (mean +/- standard deviation).

Mentions: TRPC4 and TRPC5 are reported to be non-selective sodium and calcium ion channels[22, 23]. The ability of englerin A to induce calcium influx in TRPC4/C5 expressing cells was assessed using a calcium binding dye assay. Englerin A caused a dose-dependent increase in intracellular calcium in HEK293T cells expressing TRPC4 or TRPC5, but not in mock transfected cells or in cells expressing TRPC6 (Fig 6A). A dye detecting membrane depolarization shows a similar profile (Fig 6B). TRPC4beta is a splice variant of TRPC4(22) which is also activated by englerin A leading to an increase in intracellular calcium in TRPC4beta overexpressing cells. These data indicate that englerin A activates the TRPC4/C5 channels resulting in an increase in intracellular calcium and membrane depolarization.


Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation.

Carson C, Raman P, Tullai J, Xu L, Henault M, Thomas E, Yeola S, Lao J, McPate M, Verkuyl JM, Marsh G, Sarber J, Amaral A, Bailey S, Lubicka D, Pham H, Miranda N, Ding J, Tang HM, Ju H, Tranter P, Ji N, Krastel P, Jain RK, Schumacher AM, Loureiro JJ, George E, Berellini G, Ross NT, Bushell SM, Erdemli G, Solomon JM - PLoS ONE (2015)

Englerin A agonizes the TRPC4/C5 ion channels and channel activation is needed for cell growth inhibition.(A) Calcium flux stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation): TRPC5 (closed diamonds), TRPC4beta (closed squares), TRPC4 (closed circles), TRPC6 (open squares), mock transfected cells (open circles). (B) Membrane depolarization stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation), markers as above. (C) TRPC4 current evoked by stimulation of 5 μM Englerin A, saline, or 5 μM Englerin A + 10 μM ML204 in 293T cells with Doxycyline-induced TRPC4. Currents were elicited by 200 ms voltage ramps from -100 to +100 mV, applied every 10 s from holding potential of 0 mV. (D) Summary of englerin A, englerin-B and ML-204 activity on membrane currents (mean +/- S.E.M.) (E) A-673 cell viability in the presence or absence of 50 nM englerin A and/or 50 μM ML204, a TRPC4/C5 channel blocker (mean +/- standard deviation).
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pone.0127498.g006: Englerin A agonizes the TRPC4/C5 ion channels and channel activation is needed for cell growth inhibition.(A) Calcium flux stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation): TRPC5 (closed diamonds), TRPC4beta (closed squares), TRPC4 (closed circles), TRPC6 (open squares), mock transfected cells (open circles). (B) Membrane depolarization stimulated by englerin A in HEK293T cells overexpressing different TRPC proteins (mean +/- standard deviation), markers as above. (C) TRPC4 current evoked by stimulation of 5 μM Englerin A, saline, or 5 μM Englerin A + 10 μM ML204 in 293T cells with Doxycyline-induced TRPC4. Currents were elicited by 200 ms voltage ramps from -100 to +100 mV, applied every 10 s from holding potential of 0 mV. (D) Summary of englerin A, englerin-B and ML-204 activity on membrane currents (mean +/- S.E.M.) (E) A-673 cell viability in the presence or absence of 50 nM englerin A and/or 50 μM ML204, a TRPC4/C5 channel blocker (mean +/- standard deviation).
Mentions: TRPC4 and TRPC5 are reported to be non-selective sodium and calcium ion channels[22, 23]. The ability of englerin A to induce calcium influx in TRPC4/C5 expressing cells was assessed using a calcium binding dye assay. Englerin A caused a dose-dependent increase in intracellular calcium in HEK293T cells expressing TRPC4 or TRPC5, but not in mock transfected cells or in cells expressing TRPC6 (Fig 6A). A dye detecting membrane depolarization shows a similar profile (Fig 6B). TRPC4beta is a splice variant of TRPC4(22) which is also activated by englerin A leading to an increase in intracellular calcium in TRPC4beta overexpressing cells. These data indicate that englerin A activates the TRPC4/C5 channels resulting in an increase in intracellular calcium and membrane depolarization.

Bottom Line: In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel.This toxicity suggests that englerin A itself is probably unsuitable for further drug development.However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, United States of America.

ABSTRACT
Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.

No MeSH data available.


Related in: MedlinePlus