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EGFR inhibition in glioma cells modulates Rho signaling to inhibit cell motility and invasion and cooperates with temozolomide to reduce cell growth.

Ramis G, Thomàs-Moyà E, Fernández de Mattos S, Rodríguez J, Villalonga P - PLoS ONE (2012)

Bottom Line: Interestingly, erlotinib also prevents spontaneous multicellular tumour spheroid growth in U87MG cells and cooperates with sub-optimal doses of temozolomide (TMZ) to reduce multicellular tumour spheroid growth.This cooperation appears to be schedule-dependent, since pre-treatment with erlotinib protects against TMZ-induced cytotoxicity whereas concomitant treatment results in a cooperative effect.Cell cycle arrest in erlotinib-treated cells is associated with an inhibition of ERK and Akt signaling, resulting in cyclin D1 downregulation, an increase in p27(kip1) levels and pRB hypophosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Cancer Cell Biology Group, Institut Universitari d'Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears, Illes Balears, Spain.

ABSTRACT
Enforced EGFR activation upon gene amplification and/or mutation is a common hallmark of malignant glioma. Small molecule EGFR tyrosine kinase inhibitors, such as erlotinib (Tarceva), have shown some activity in a subset of glioma patients in recent trials, although the reported data on the cellular basis of glioma cell responsiveness to these compounds have been contradictory. Here we have used a panel of human glioma cell lines, including cells with amplified or mutant EGFR, to further characterize the cellular effects of EGFR inhibition with erlotinib. Dose-response and cellular growth assays indicate that erlotinib reduces cell proliferation in all tested cell lines without inducing cytotoxic effects. Flow cytometric analyses confirm that EGFR inhibition does not induce apoptosis in glioma cells, leading to cell cycle arrest in G(1). Interestingly, erlotinib also prevents spontaneous multicellular tumour spheroid growth in U87MG cells and cooperates with sub-optimal doses of temozolomide (TMZ) to reduce multicellular tumour spheroid growth. This cooperation appears to be schedule-dependent, since pre-treatment with erlotinib protects against TMZ-induced cytotoxicity whereas concomitant treatment results in a cooperative effect. Cell cycle arrest in erlotinib-treated cells is associated with an inhibition of ERK and Akt signaling, resulting in cyclin D1 downregulation, an increase in p27(kip1) levels and pRB hypophosphorylation. Interestingly, EGFR inhibition also perturbs Rho GTPase signaling and cellular morphology, leading to Rho/ROCK-dependent formation of actin stress fibres and the inhibition of glioma cell motility and invasion.

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Erlotinib inhibits glioma cell proliferation.(A) Representative phase-contrast micrographs of glioma cell lines left untreated (control) or treated for 48 h with 10 µM erlotinib (erlotinib). (B) Glioma cell lines were left untreated (untreated) or treated with 10 µM erlotinib (erlotinib) and the number of cells counted every 24 h. Data from a representative experiment out of three repetitions is shown, representing the total number of viable cells in untreated and erlotinib-treated conditions at the indicated time-points. (C) Glioma cell lines were treated for 72 h with the indicated concentrations of erlotinib. The mean ± SD values from three independent experiments, each conducted in duplicate, are shown in the graph, representing the percentage of viable cells relative to untreated conditions. The differences between control and erlotinib treatment are statistically significant (Student's t-test: *P<0.05, **P<0.01 and ***P<0.001, respectively). (D) Glioma cell lines were treated for 72 h with 10 µM erlotinib. Sensitivity to erlotinib of each cell line is expressed as the mean ± SD percentage of growth inhibitory activity from three independent experiments, each conducted in duplicate. The differences between control and erlotinib treatment are statistically significant (Student's t-test: *P<0.05) (E) Representative phase-contrast micrographs of U87MG cells left for 4–6 days to allow formation of multicellular tumour spheroids (MCTS), untreated (control) or treated with 10 µM erlotinib (erlotinib). The graph indicates the mean ± SD values of MCTS formation from three independent experiments, each conducted in duplicate, expressed as the percentage of MCTS relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's t-test: **P<0.01).
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pone-0038770-g001: Erlotinib inhibits glioma cell proliferation.(A) Representative phase-contrast micrographs of glioma cell lines left untreated (control) or treated for 48 h with 10 µM erlotinib (erlotinib). (B) Glioma cell lines were left untreated (untreated) or treated with 10 µM erlotinib (erlotinib) and the number of cells counted every 24 h. Data from a representative experiment out of three repetitions is shown, representing the total number of viable cells in untreated and erlotinib-treated conditions at the indicated time-points. (C) Glioma cell lines were treated for 72 h with the indicated concentrations of erlotinib. The mean ± SD values from three independent experiments, each conducted in duplicate, are shown in the graph, representing the percentage of viable cells relative to untreated conditions. The differences between control and erlotinib treatment are statistically significant (Student's t-test: *P<0.05, **P<0.01 and ***P<0.001, respectively). (D) Glioma cell lines were treated for 72 h with 10 µM erlotinib. Sensitivity to erlotinib of each cell line is expressed as the mean ± SD percentage of growth inhibitory activity from three independent experiments, each conducted in duplicate. The differences between control and erlotinib treatment are statistically significant (Student's t-test: *P<0.05) (E) Representative phase-contrast micrographs of U87MG cells left for 4–6 days to allow formation of multicellular tumour spheroids (MCTS), untreated (control) or treated with 10 µM erlotinib (erlotinib). The graph indicates the mean ± SD values of MCTS formation from three independent experiments, each conducted in duplicate, expressed as the percentage of MCTS relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's t-test: **P<0.01).

Mentions: In order to characterize the cellular effects of EGFR inhibition in glioma cells, we treated a panel of 6 human glioma cell lines (LN229, U87MG, HS683, T98G, U251, U373) with erlotinib. Erlotinib reduced cell proliferation in all cell lines tested (Figures 1A, 1B). Growth curve experiments upon long-term erlotinib treatment indicated that erlotinib decreased total cell number (Figure 1B), but did not affect cellular viability as indicated by trypan blue staining (data not shown). Dose-response experiments confirmed that 10 µM erlotinib exerted an inhibitory effect on glioma cell growth ranging from 30% (U373 cells) to 80% inhibition (LN229 cells) (Figures 1C, 1D). Since U87MG cells spontaneously form multicellular tumour spheroids in culture [14], we also investigated whether erlotinib could prevent multicellular tumour spheroid formation. Whereas control U87MG cells formed high numbers of large and dense multicellular tumour spheroids, erlotinib-treated cells were largely resistant to spheroid formation (Figures 1E). These observations confirm that EGFR inhibition with erlotinib severely reduces glioma cell proliferation.


EGFR inhibition in glioma cells modulates Rho signaling to inhibit cell motility and invasion and cooperates with temozolomide to reduce cell growth.

Ramis G, Thomàs-Moyà E, Fernández de Mattos S, Rodríguez J, Villalonga P - PLoS ONE (2012)

Erlotinib inhibits glioma cell proliferation.(A) Representative phase-contrast micrographs of glioma cell lines left untreated (control) or treated for 48 h with 10 µM erlotinib (erlotinib). (B) Glioma cell lines were left untreated (untreated) or treated with 10 µM erlotinib (erlotinib) and the number of cells counted every 24 h. Data from a representative experiment out of three repetitions is shown, representing the total number of viable cells in untreated and erlotinib-treated conditions at the indicated time-points. (C) Glioma cell lines were treated for 72 h with the indicated concentrations of erlotinib. The mean ± SD values from three independent experiments, each conducted in duplicate, are shown in the graph, representing the percentage of viable cells relative to untreated conditions. The differences between control and erlotinib treatment are statistically significant (Student's t-test: *P<0.05, **P<0.01 and ***P<0.001, respectively). (D) Glioma cell lines were treated for 72 h with 10 µM erlotinib. Sensitivity to erlotinib of each cell line is expressed as the mean ± SD percentage of growth inhibitory activity from three independent experiments, each conducted in duplicate. The differences between control and erlotinib treatment are statistically significant (Student's t-test: *P<0.05) (E) Representative phase-contrast micrographs of U87MG cells left for 4–6 days to allow formation of multicellular tumour spheroids (MCTS), untreated (control) or treated with 10 µM erlotinib (erlotinib). The graph indicates the mean ± SD values of MCTS formation from three independent experiments, each conducted in duplicate, expressed as the percentage of MCTS relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's t-test: **P<0.01).
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pone-0038770-g001: Erlotinib inhibits glioma cell proliferation.(A) Representative phase-contrast micrographs of glioma cell lines left untreated (control) or treated for 48 h with 10 µM erlotinib (erlotinib). (B) Glioma cell lines were left untreated (untreated) or treated with 10 µM erlotinib (erlotinib) and the number of cells counted every 24 h. Data from a representative experiment out of three repetitions is shown, representing the total number of viable cells in untreated and erlotinib-treated conditions at the indicated time-points. (C) Glioma cell lines were treated for 72 h with the indicated concentrations of erlotinib. The mean ± SD values from three independent experiments, each conducted in duplicate, are shown in the graph, representing the percentage of viable cells relative to untreated conditions. The differences between control and erlotinib treatment are statistically significant (Student's t-test: *P<0.05, **P<0.01 and ***P<0.001, respectively). (D) Glioma cell lines were treated for 72 h with 10 µM erlotinib. Sensitivity to erlotinib of each cell line is expressed as the mean ± SD percentage of growth inhibitory activity from three independent experiments, each conducted in duplicate. The differences between control and erlotinib treatment are statistically significant (Student's t-test: *P<0.05) (E) Representative phase-contrast micrographs of U87MG cells left for 4–6 days to allow formation of multicellular tumour spheroids (MCTS), untreated (control) or treated with 10 µM erlotinib (erlotinib). The graph indicates the mean ± SD values of MCTS formation from three independent experiments, each conducted in duplicate, expressed as the percentage of MCTS relative to untreated cells. The differences between control and erlotinib treatment are statistically significant (Student's t-test: **P<0.01).
Mentions: In order to characterize the cellular effects of EGFR inhibition in glioma cells, we treated a panel of 6 human glioma cell lines (LN229, U87MG, HS683, T98G, U251, U373) with erlotinib. Erlotinib reduced cell proliferation in all cell lines tested (Figures 1A, 1B). Growth curve experiments upon long-term erlotinib treatment indicated that erlotinib decreased total cell number (Figure 1B), but did not affect cellular viability as indicated by trypan blue staining (data not shown). Dose-response experiments confirmed that 10 µM erlotinib exerted an inhibitory effect on glioma cell growth ranging from 30% (U373 cells) to 80% inhibition (LN229 cells) (Figures 1C, 1D). Since U87MG cells spontaneously form multicellular tumour spheroids in culture [14], we also investigated whether erlotinib could prevent multicellular tumour spheroid formation. Whereas control U87MG cells formed high numbers of large and dense multicellular tumour spheroids, erlotinib-treated cells were largely resistant to spheroid formation (Figures 1E). These observations confirm that EGFR inhibition with erlotinib severely reduces glioma cell proliferation.

Bottom Line: Interestingly, erlotinib also prevents spontaneous multicellular tumour spheroid growth in U87MG cells and cooperates with sub-optimal doses of temozolomide (TMZ) to reduce multicellular tumour spheroid growth.This cooperation appears to be schedule-dependent, since pre-treatment with erlotinib protects against TMZ-induced cytotoxicity whereas concomitant treatment results in a cooperative effect.Cell cycle arrest in erlotinib-treated cells is associated with an inhibition of ERK and Akt signaling, resulting in cyclin D1 downregulation, an increase in p27(kip1) levels and pRB hypophosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Cancer Cell Biology Group, Institut Universitari d'Investigació en Ciències de la Salut (IUNICS), Universitat de les Illes Balears, Illes Balears, Spain.

ABSTRACT
Enforced EGFR activation upon gene amplification and/or mutation is a common hallmark of malignant glioma. Small molecule EGFR tyrosine kinase inhibitors, such as erlotinib (Tarceva), have shown some activity in a subset of glioma patients in recent trials, although the reported data on the cellular basis of glioma cell responsiveness to these compounds have been contradictory. Here we have used a panel of human glioma cell lines, including cells with amplified or mutant EGFR, to further characterize the cellular effects of EGFR inhibition with erlotinib. Dose-response and cellular growth assays indicate that erlotinib reduces cell proliferation in all tested cell lines without inducing cytotoxic effects. Flow cytometric analyses confirm that EGFR inhibition does not induce apoptosis in glioma cells, leading to cell cycle arrest in G(1). Interestingly, erlotinib also prevents spontaneous multicellular tumour spheroid growth in U87MG cells and cooperates with sub-optimal doses of temozolomide (TMZ) to reduce multicellular tumour spheroid growth. This cooperation appears to be schedule-dependent, since pre-treatment with erlotinib protects against TMZ-induced cytotoxicity whereas concomitant treatment results in a cooperative effect. Cell cycle arrest in erlotinib-treated cells is associated with an inhibition of ERK and Akt signaling, resulting in cyclin D1 downregulation, an increase in p27(kip1) levels and pRB hypophosphorylation. Interestingly, EGFR inhibition also perturbs Rho GTPase signaling and cellular morphology, leading to Rho/ROCK-dependent formation of actin stress fibres and the inhibition of glioma cell motility and invasion.

Show MeSH
Related in: MedlinePlus