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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: Dose-response and cellular growth assays indicate that erlotinib reduces cell proliferation in all tested cell lines without inducing cytotoxic effects.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 prevents multicellular tumour spheroid formation and induces G1 arrest in glioma cells.(A) Glioma cell lines were left untreated (control) or were treated for 24 h with 10 µM erlotinib (erlotinib). Cells were harvested and their DNA content analyzed by flow cytometry as described in Materials and Methods. The cell cycle distribution is shown for each experimental condition. (B) The graph summarizes the flow cytometry data obtained in all glioma cell lines, indicating the cell cycle distribution in control and 24 h erlotinib-treated conditions for each cell line.
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pone-0038770-g002: Erlotinib prevents multicellular tumour spheroid formation and induces G1 arrest in glioma cells.(A) Glioma cell lines were left untreated (control) or were treated for 24 h with 10 µM erlotinib (erlotinib). Cells were harvested and their DNA content analyzed by flow cytometry as described in Materials and Methods. The cell cycle distribution is shown for each experimental condition. (B) The graph summarizes the flow cytometry data obtained in all glioma cell lines, indicating the cell cycle distribution in control and 24 h erlotinib-treated conditions for each cell line.

Mentions: In order to characterize the cell cycle arrest induced by erlotinib treatment in glioma cells, we performed flow cytometric analysis in a panel of control and erlotinib-treated glioma cell lines. Erlotinib treatment led to a significant accumulation of cells in G1 in all tested cell lines (Figure 2A), showing a sensitivity to erlotinib in correlation with our previous results (Figure 2B). Interestingly, erlotinib did not induce cell death as indicated by the absence of a detectable sub-G1 population (Figures 2A and 2B), in line with our previous data. Altogether, our results indicate that erlotinib inhibits glioma cell proliferation primarily by inhibiting S-phase entry.


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 prevents multicellular tumour spheroid formation and induces G1 arrest in glioma cells.(A) Glioma cell lines were left untreated (control) or were treated for 24 h with 10 µM erlotinib (erlotinib). Cells were harvested and their DNA content analyzed by flow cytometry as described in Materials and Methods. The cell cycle distribution is shown for each experimental condition. (B) The graph summarizes the flow cytometry data obtained in all glioma cell lines, indicating the cell cycle distribution in control and 24 h erlotinib-treated conditions for each cell line.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368887&req=5

pone-0038770-g002: Erlotinib prevents multicellular tumour spheroid formation and induces G1 arrest in glioma cells.(A) Glioma cell lines were left untreated (control) or were treated for 24 h with 10 µM erlotinib (erlotinib). Cells were harvested and their DNA content analyzed by flow cytometry as described in Materials and Methods. The cell cycle distribution is shown for each experimental condition. (B) The graph summarizes the flow cytometry data obtained in all glioma cell lines, indicating the cell cycle distribution in control and 24 h erlotinib-treated conditions for each cell line.
Mentions: In order to characterize the cell cycle arrest induced by erlotinib treatment in glioma cells, we performed flow cytometric analysis in a panel of control and erlotinib-treated glioma cell lines. Erlotinib treatment led to a significant accumulation of cells in G1 in all tested cell lines (Figure 2A), showing a sensitivity to erlotinib in correlation with our previous results (Figure 2B). Interestingly, erlotinib did not induce cell death as indicated by the absence of a detectable sub-G1 population (Figures 2A and 2B), in line with our previous data. Altogether, our results indicate that erlotinib inhibits glioma cell proliferation primarily by inhibiting S-phase entry.

Bottom Line: Dose-response and cellular growth assays indicate that erlotinib reduces cell proliferation in all tested cell lines without inducing cytotoxic effects.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