SCCHN cell lines Tu212 (A) and Tu686 (B) were treated with 1:2 serial dilutions of erlotinib (0-40 μM) and celecoxib (0-20 μM) as single agents and in combination as described in the Methods section. After incubation for 72 hours, SRB assay was used to determine the percentage of survival relative to the untreated cells. CIs at effective doses which resulted in 50% (ED50), 75% (ED75), and 90% (ED90) inhibitory rates (1 - survival rate) were calculated using CalcuSyn software. A CI value of >1 is antagonism, = 1 is additivity, and
SCCHN cell lines Tu212 (A) and Tu686 (B) were treated with 1:2 serial dilutions of erlotinib (0-40 μM) and celecoxib (0-20 μM) as single agents and in combination as described in the Methods section. After incubation for 72 hours, SRB assay was used to determine the percentage of survival relative to the untreated cells. CIs at effective doses which resulted in 50% (ED50), 75% (ED75), and 90% (ED90) inhibitory rates (1 - survival rate) were calculated using CalcuSyn software. A CI value of >1 is antagonism, = 1 is additivity, and
Figure 2. Induction of G0/G1 arrest by…
Figure 2. Induction of G0/G1 arrest by erlotinib and celecoxib in SCCHN cells
Figure 2. Induction of G0/G1 arrest by erlotinib and celecoxib in SCCHN cells SCCHN cell lines Tu212 (A) and Tu686 (B) were treated with erlotinib (1 μM), celecoxib (10 μM), and their combination for 24, 48, and 72 hours. Cell cycle analysis was performed by flow cytometry as described in the Methods section. The average percentages of the cell population arrested at G0/G1 at each time point are presented with standard deviation from three repeated experiments. Significant differences (p ≤ 0.05) in comparison of the combination treatment with the control and each of the single treatments at all time points are shown. (C) Western blot analyses of cell cycle regulatory proteins in both Tu212 and Tu686 cells treated for 24, 48, and 72 hours. The experiments were repeated three times.
Figure 2. Induction of G0/G1 arrest by…
Figure 2. Induction of G0/G1 arrest by erlotinib and celecoxib in SCCHN cells
Figure 2. Induction of G0/G1 arrest by erlotinib and celecoxib in SCCHN cells SCCHN cell lines Tu212 (A) and Tu686 (B) were treated with erlotinib (1 μM), celecoxib (10 μM), and their combination for 24, 48, and 72 hours. Cell cycle analysis was performed by flow cytometry as described in the Methods section. The average percentages of the cell population arrested at G0/G1 at each time point are presented with standard deviation from three repeated experiments. Significant differences (p ≤ 0.05) in comparison of the combination treatment with the control and each of the single treatments at all time points are shown. (C) Western blot analyses of cell cycle regulatory proteins in both Tu212 and Tu686 cells treated for 24, 48, and 72 hours. The experiments were repeated three times.
Figure 2. Induction of G0/G1 arrest by…
Figure 2. Induction of G0/G1 arrest by erlotinib and celecoxib in SCCHN cells
Figure 2. Induction of G0/G1 arrest by erlotinib and celecoxib in SCCHN cells SCCHN cell lines Tu212 (A) and Tu686 (B) were treated with erlotinib (1 μM), celecoxib (10 μM), and their combination for 24, 48, and 72 hours. Cell cycle analysis was performed by flow cytometry as described in the Methods section. The average percentages of the cell population arrested at G0/G1 at each time point are presented with standard deviation from three repeated experiments. Significant differences (p ≤ 0.05) in comparison of the combination treatment with the control and each of the single treatments at all time points are shown. (C) Western blot analyses of cell cycle regulatory proteins in both Tu212 and Tu686 cells treated for 24, 48, and 72 hours. The experiments were repeated three times.
Figure 3. Signal transduction pathways affected by…
Figure 3. Signal transduction pathways affected by erlotinib and celecoxib in SCCHN cells
Figure 3. Signal transduction pathways affected by erlotinib and celecoxib in SCCHN cells Cell lysates were collected from SCCHN cell lines Tu212 and Tu686 after treatment with erlotinib (E: 1 μM), celecoxib (C: 10 μM), and their combination (EC) for 24, 48, and 72 hours. Untreated cells (NT) were used as a control at each time point. Western blot analyses were performed on total protein extracts from each of the time points to detect the expression levels of proteins involved in EGFR, AKT, mTOR, and COX-2 pathways. β-actin served as a loading control.
Figure 4. Inhibitory effects of erlotinib and…
Figure 4. Inhibitory effects of erlotinib and celecoxib on HUVEC tubular formation and Matrigel invasion
Figure 4. Inhibitory effects of erlotinib and celecoxib on HUVEC tubular formation and Matrigel invasion (A) Effects of erlotinib and celecoxib on capillary formation by HUVECs were examined in vitro. HUVECs were pretreated with vehicle DMSO (Control), celecoxib (CCB: 10 μM), erlotinib (ER: 1 μM), and their combination (Comb) for 12 hours, followed by inoculation in 24-well plates pre-coated with Matrigel and incubation overnight. A representative image is shown in the figure. The average number of HUVEC capillary tube branches in 10 fields was counted using an Olympus inverted microscope (CKX40; Olympus, New York, NY). (B) HUVECs suspended in serum-free medium containing 0.1% BSA with or without DMSO (control), celecoxib (CCB: 10 μM), erlotinib (ER: 1 μM), or combination of erlotinib and celecoxib (Comb: 1 μM and10 μM, respectively) in triplicate were seeded in the invasion chamber. After 36-40 hours of incubation, the number of invaded cells was counted and quantified as the sum of 10 random fields under the microscope with 200X magnification. Experiments were repeated twice. * indicates statistical significance (p<0.05) of the treatment compared with control.
Figure 5. Effect of erlotinib and celecoxib…
Figure 5. Effect of erlotinib and celecoxib on Tu212 xenograft tumor growth
Figure 5. Effect of erlotinib and celecoxib on Tu212 xenograft tumor growth Four groups of mice were orally gavaged with control (0.1% Tween 80 and 0.5% methylcellulose), erlotinib (75 mg/kg), celecoxib (50mg/kg), or the combination (n=8) of erlotinb (75 mg/kg) and celecoxib (50 mg/kg) for 6 days prior to a subcutaneous inoculation of 2 × 106 Tu212 cells. The animals were continuously gavaged with the agents 5 days a week for a total of 4 weeks. (A) Tumor volume was measured at the indicated time points. (B) Immunohistochemistry analyses shown as representative H&E staining (× 200) were performed for expression of proliferation marker Ki-67, mTOR substrate p-S6, and endothelium marker CD34. (C) Quantification of these biomarkers. * indicates statistical significance (p<0.05) of the treatment compared with control and ** indicates significant difference between the combination and either single agent.
Figure 6. Biomarker alterations upon treatment with…
Figure 6. Biomarker alterations upon treatment with the combination of erlotinib and celecoxib in patient…
Figure 6. Biomarker alterations upon treatment with the combination of erlotinib and celecoxib in patient tissue samples (A) Immunohistochemical staining of EGFR, pERK, and p-S6 before and after the combined treatment. (B) Correlation of the change in expression of EGFR, pERK, and p-S6 with patients’ responses at the last clinical time point. Seven patients were included in the analysis. (C) Heat map of significant proteins (p
Figure 6. Biomarker alterations upon treatment with…
Figure 6. Biomarker alterations upon treatment with the combination of erlotinib and celecoxib in patient…
Figure 6. Biomarker alterations upon treatment with the combination of erlotinib and celecoxib in patient tissue samples (A) Immunohistochemical staining of EGFR, pERK, and p-S6 before and after the combined treatment. (B) Correlation of the change in expression of EGFR, pERK, and p-S6 with patients’ responses at the last clinical time point. Seven patients were included in the analysis. (C) Heat map of significant proteins (p
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Publication types
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
MeSH terms
Antineoplastic Combined Chemotherapy Protocols
Carcinoma, Squamous Cell / metabolism
Carcinoma, Squamous Cell / pathology
Carcinoma, Squamous Cell / prevention & control*
Cell Cycle / drug effects
Cell Movement / drug effects
Cell Proliferation / drug effects
Cyclooxygenase Inhibitors / pharmacology*
ErbB Receptors / antagonists & inhibitors
Head and Neck Neoplasms / metabolism
Head and Neck Neoplasms / pathology
Head and Neck Neoplasms / prevention & control*
Pyrazoles / pharmacology*
Quinazolines / pharmacology*
Signal Transduction / drug effects*
Sulfonamides / pharmacology*
Substances
Cyclooxygenase Inhibitors
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