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Ad-p53 enhances the sensitivity of triple-negative breast cancer MDA-MB-468 cells to the EGFR inhibitor gefitinib.

Wang X, Song H, Yu Q, Liu Q, Wang L, Liu Z, Yu Z - Oncol. Rep. (2014)

Bottom Line: Neither endocrine nor anti-HER2 molecular targeting treatment yield promising results.At present, epidermal growth factor receptor (EGFR) inhibitor, as a single agent, is unable to obtain encouraging results in the treatment of TNBC, even though most of these tumors overexpress EGFR.These results suggest that Ad-p53 may block the PI3K/Akt pathway rather than the Raf/MEK/ERK pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Shandong Cancer Hospital, Jinan, Shandong 250117, P.R. China.

ABSTRACT
Triple-negative breast cancer (TNBC) accounts for 20% of all molecular subtypes of breast cancer. Neither endocrine nor anti-HER2 molecular targeting treatment yield promising results. At present, epidermal growth factor receptor (EGFR) inhibitor, as a single agent, is unable to obtain encouraging results in the treatment of TNBC, even though most of these tumors overexpress EGFR. In the present study, we used recombinant human p53 adenovirus (Ad-p53) and EGFR inhibitor gefitinib to treat the TNBC cell line MDA-MB-468. The combined treatment of gefitinib and Ad-p53 synergistically inhibited the proliferation of MDA-MB-468 cells; it restrained colony formation, enhanced cellular apoptosis and arrested the cell cycle in vitro, and decreased tumor burden of xenografts in nude mice. Western blot analysis revealed that Ad-p53 and gefitinib in combination significantly downregulated the phosphorylation of protein kinase B (p-Akt) and upregulated caspase-9 and cleaved caspase-3, while there were minimal effects on the expression of extracellular signal-regulated kinase (ERK) and phosphorylation of ERK (p-ERK). These results suggest that Ad-p53 may block the PI3K/Akt pathway rather than the Raf/MEK/ERK pathway. Importantly, wild-type p53 was able to reverse the drug resistance of MDA-MB-468 cells to gefitinib through inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. The apoptotic activity induced by this combined treatment may be regulated by caspase cascade-dependent activation.

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Combination of Ad-p53 and gefitinib suppresses the Akt pathway in MDA-MB-468 cells and increases the activity of caspase cascade protein. Cells were treated with Ad-p53 (MOI of 100), gefitinib (3 μM), alone or a combination for 48 h. Cell lysates were analyzed via western blotting using the indicated antibodies. GAPDH was used as a loading control. (A) p53 and EGFR expression was detected in the MDA-MB-468 cells by western blotting. Ad-p53 and gefitinib in combination significantly downregulated p-Akt and upregulated caspase-9 and cleaved caspase-3. ERK and p-ERK showed little change among the four groups. (B) Relative expression of p-Akt, (C) caspase-9 and (D) cleaved caspase-3 was evaluated by ANOVA. Tukey’s multiple comparison was applied to compare two subsequent samples. Data represent means ± SEM from three independent experiments. **P<0.01. Ad-p53, recombinant human p53 adenovirus; MOI, multiplicity of infection; EGFR, epidermal growth factor receptor; p-Akt, phosphatidylinositol-3 kinase; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylation of ERK.
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f5-or-33-02-0526: Combination of Ad-p53 and gefitinib suppresses the Akt pathway in MDA-MB-468 cells and increases the activity of caspase cascade protein. Cells were treated with Ad-p53 (MOI of 100), gefitinib (3 μM), alone or a combination for 48 h. Cell lysates were analyzed via western blotting using the indicated antibodies. GAPDH was used as a loading control. (A) p53 and EGFR expression was detected in the MDA-MB-468 cells by western blotting. Ad-p53 and gefitinib in combination significantly downregulated p-Akt and upregulated caspase-9 and cleaved caspase-3. ERK and p-ERK showed little change among the four groups. (B) Relative expression of p-Akt, (C) caspase-9 and (D) cleaved caspase-3 was evaluated by ANOVA. Tukey’s multiple comparison was applied to compare two subsequent samples. Data represent means ± SEM from three independent experiments. **P<0.01. Ad-p53, recombinant human p53 adenovirus; MOI, multiplicity of infection; EGFR, epidermal growth factor receptor; p-Akt, phosphatidylinositol-3 kinase; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylation of ERK.

Mentions: Ad-p53 or gefitinib, as a single agent, produced a slight reduction in the levels of p-Akt (S473) in the MDA-MB-468 cells (Fig. 5). Surprisingly, combined treatment synergistically produced a significant reduction in p-Akt (S473) (Fig. 5). The expression levels of caspase-9 and cleaved caspase-3 were low in the vehicle-treated cells. In contrast, when Ad-p53 or gefitinib was used as a single agent, the levels of caspase-9 and cleaved caspase-3 increased (Fig. 5). Nevertheless, when they were administered in combination, the levels were markedly increased. However, none of the treatments led to an obvious change in ERK and p-ERK. These results suggest that Ad-p53 may enhance the sensitivity of MDA-MB-468 cells to gefitinib by blocking the PI3K/Akt pathway rather than the Raf/MEK/ERK pathway.


Ad-p53 enhances the sensitivity of triple-negative breast cancer MDA-MB-468 cells to the EGFR inhibitor gefitinib.

Wang X, Song H, Yu Q, Liu Q, Wang L, Liu Z, Yu Z - Oncol. Rep. (2014)

Combination of Ad-p53 and gefitinib suppresses the Akt pathway in MDA-MB-468 cells and increases the activity of caspase cascade protein. Cells were treated with Ad-p53 (MOI of 100), gefitinib (3 μM), alone or a combination for 48 h. Cell lysates were analyzed via western blotting using the indicated antibodies. GAPDH was used as a loading control. (A) p53 and EGFR expression was detected in the MDA-MB-468 cells by western blotting. Ad-p53 and gefitinib in combination significantly downregulated p-Akt and upregulated caspase-9 and cleaved caspase-3. ERK and p-ERK showed little change among the four groups. (B) Relative expression of p-Akt, (C) caspase-9 and (D) cleaved caspase-3 was evaluated by ANOVA. Tukey’s multiple comparison was applied to compare two subsequent samples. Data represent means ± SEM from three independent experiments. **P<0.01. Ad-p53, recombinant human p53 adenovirus; MOI, multiplicity of infection; EGFR, epidermal growth factor receptor; p-Akt, phosphatidylinositol-3 kinase; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylation of ERK.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4306269&req=5

f5-or-33-02-0526: Combination of Ad-p53 and gefitinib suppresses the Akt pathway in MDA-MB-468 cells and increases the activity of caspase cascade protein. Cells were treated with Ad-p53 (MOI of 100), gefitinib (3 μM), alone or a combination for 48 h. Cell lysates were analyzed via western blotting using the indicated antibodies. GAPDH was used as a loading control. (A) p53 and EGFR expression was detected in the MDA-MB-468 cells by western blotting. Ad-p53 and gefitinib in combination significantly downregulated p-Akt and upregulated caspase-9 and cleaved caspase-3. ERK and p-ERK showed little change among the four groups. (B) Relative expression of p-Akt, (C) caspase-9 and (D) cleaved caspase-3 was evaluated by ANOVA. Tukey’s multiple comparison was applied to compare two subsequent samples. Data represent means ± SEM from three independent experiments. **P<0.01. Ad-p53, recombinant human p53 adenovirus; MOI, multiplicity of infection; EGFR, epidermal growth factor receptor; p-Akt, phosphatidylinositol-3 kinase; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylation of ERK.
Mentions: Ad-p53 or gefitinib, as a single agent, produced a slight reduction in the levels of p-Akt (S473) in the MDA-MB-468 cells (Fig. 5). Surprisingly, combined treatment synergistically produced a significant reduction in p-Akt (S473) (Fig. 5). The expression levels of caspase-9 and cleaved caspase-3 were low in the vehicle-treated cells. In contrast, when Ad-p53 or gefitinib was used as a single agent, the levels of caspase-9 and cleaved caspase-3 increased (Fig. 5). Nevertheless, when they were administered in combination, the levels were markedly increased. However, none of the treatments led to an obvious change in ERK and p-ERK. These results suggest that Ad-p53 may enhance the sensitivity of MDA-MB-468 cells to gefitinib by blocking the PI3K/Akt pathway rather than the Raf/MEK/ERK pathway.

Bottom Line: Neither endocrine nor anti-HER2 molecular targeting treatment yield promising results.At present, epidermal growth factor receptor (EGFR) inhibitor, as a single agent, is unable to obtain encouraging results in the treatment of TNBC, even though most of these tumors overexpress EGFR.These results suggest that Ad-p53 may block the PI3K/Akt pathway rather than the Raf/MEK/ERK pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Shandong Cancer Hospital, Jinan, Shandong 250117, P.R. China.

ABSTRACT
Triple-negative breast cancer (TNBC) accounts for 20% of all molecular subtypes of breast cancer. Neither endocrine nor anti-HER2 molecular targeting treatment yield promising results. At present, epidermal growth factor receptor (EGFR) inhibitor, as a single agent, is unable to obtain encouraging results in the treatment of TNBC, even though most of these tumors overexpress EGFR. In the present study, we used recombinant human p53 adenovirus (Ad-p53) and EGFR inhibitor gefitinib to treat the TNBC cell line MDA-MB-468. The combined treatment of gefitinib and Ad-p53 synergistically inhibited the proliferation of MDA-MB-468 cells; it restrained colony formation, enhanced cellular apoptosis and arrested the cell cycle in vitro, and decreased tumor burden of xenografts in nude mice. Western blot analysis revealed that Ad-p53 and gefitinib in combination significantly downregulated the phosphorylation of protein kinase B (p-Akt) and upregulated caspase-9 and cleaved caspase-3, while there were minimal effects on the expression of extracellular signal-regulated kinase (ERK) and phosphorylation of ERK (p-ERK). These results suggest that Ad-p53 may block the PI3K/Akt pathway rather than the Raf/MEK/ERK pathway. Importantly, wild-type p53 was able to reverse the drug resistance of MDA-MB-468 cells to gefitinib through inactivation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. The apoptotic activity induced by this combined treatment may be regulated by caspase cascade-dependent activation.

Show MeSH
Related in: MedlinePlus