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Combination of mTOR and EGFR kinase inhibitors blocks mTORC1 and mTORC2 kinase activity and suppresses the progression of colorectal carcinoma.

Wang Q, Wei F, Li C, Lv G, Wang G, Liu T, Bellail AC, Hao C - PLoS ONE (2013)

Bottom Line: To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity.In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death.A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastrointestinal Surgery, First Hospital of Jilin University, Changchun, Jilin, China. wangquan-jlcc@hotmail.com

ABSTRACT
Mammalian target of rapamycin complex 1 and 2 (mTORC1/2) are overactive in colorectal carcinomas; however, the first generation of mTOR inhibitors such as rapamycin have failed to show clinical benefits in treating colorectal carcinoma in part due to their effects only on mTORC1. The second generation of mTOR inhibitors such as PP242 targets mTOR kinase; thus, they are capable of inhibiting both mTORC1 and mTORC2. To examine the therapeutic potential of the mTOR kinase inhibitors, we treated a panel of colorectal carcinoma cell lines with PP242. Western blotting showed that the PP242 inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT(S473)) was transient only in the first few hours of the PP242 treatment. Receptor tyrosine kinase arrays further revealed that PP242 treatment increased the phosphorylated epidermal growth factor receptor (EGFR) at Tyr 1068 (EGFR(T1068)). The parallel increase of AKT(S473) and EGFR(T1068) in the cells following PP242 treatment raised the possibility that EGFR phosphorylation might contribute to the PP242 incomplete inhibition of mTORC2. To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity. In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death. A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice. This study suggests that the combination of mTOR kinase and EGFR inhibitors may provide an effective treatment of colorectal carcinoma.

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PP242 and erlotinib treatment induce apoptotic cell death.(A) DLD-1 cells were treated with erlotinib (2 μM) and PP242 (1 μM), alone or in combination, for the indicated hours and then analyzed by flow cytometry. The percentage of sub-G1 apoptotic cells were presented as mean + SD. **, p < 0.01. (B) DLD-1 cells were treated with PP242 (1 μM) and/or erlotinib (2 μM) for the indicated hours and analyzed by western blotting for the cleavage of caspase-3, DFF45 and PARP with the molecular weights of the cleavage products indicated to the right of the panel.
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pone-0073175-g004: PP242 and erlotinib treatment induce apoptotic cell death.(A) DLD-1 cells were treated with erlotinib (2 μM) and PP242 (1 μM), alone or in combination, for the indicated hours and then analyzed by flow cytometry. The percentage of sub-G1 apoptotic cells were presented as mean + SD. **, p < 0.01. (B) DLD-1 cells were treated with PP242 (1 μM) and/or erlotinib (2 μM) for the indicated hours and analyzed by western blotting for the cleavage of caspase-3, DFF45 and PARP with the molecular weights of the cleavage products indicated to the right of the panel.

Mentions: PP242 has been reported to induce apoptosis in leukemia and breast cancer cells through inhibition of mTORC2 activity [42,43]. The combination of P242 and erlotinib can inhibit the mTORC2 activity, which suggests that the combination may induce apoptosis in colorectal carcinoma cells. To text this notion, we treated DLD-1 cells with PP242 (0.5 μM) or erlotinib (2 μM), alone and in combination in the presence of EGF (50 ng/ml). Flow cytometry detected approximately 5% sub-G1 apoptotic cells in the cells treated with PP242 or erlotinib alone (Figure 4A). In contrast, approximately 15-20% cells underwent apoptotic cell death under the combination treatment of PP242 and erlotinib (Figure 4A). To confirm the PP242 and erlotinib combination-induced apoptosis, we examined the treated cells on western blotting and thus revealed cleavage of caspase-3, DFF45 and PARP; the data confirmed the apoptotic cell death of the cells under the treatment of PP242 and erlotinib, in contrast, however, PP242 and erlotinib alone failed to induce apoptosis in the cells (Figure 4B). These results suggest that the combination of PP242 and erlotinib synergistically inhibits the growth of colorectal carcinoma cells in part through induction of apoptosis.


Combination of mTOR and EGFR kinase inhibitors blocks mTORC1 and mTORC2 kinase activity and suppresses the progression of colorectal carcinoma.

Wang Q, Wei F, Li C, Lv G, Wang G, Liu T, Bellail AC, Hao C - PLoS ONE (2013)

PP242 and erlotinib treatment induce apoptotic cell death.(A) DLD-1 cells were treated with erlotinib (2 μM) and PP242 (1 μM), alone or in combination, for the indicated hours and then analyzed by flow cytometry. The percentage of sub-G1 apoptotic cells were presented as mean + SD. **, p < 0.01. (B) DLD-1 cells were treated with PP242 (1 μM) and/or erlotinib (2 μM) for the indicated hours and analyzed by western blotting for the cleavage of caspase-3, DFF45 and PARP with the molecular weights of the cleavage products indicated to the right of the panel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750018&req=5

pone-0073175-g004: PP242 and erlotinib treatment induce apoptotic cell death.(A) DLD-1 cells were treated with erlotinib (2 μM) and PP242 (1 μM), alone or in combination, for the indicated hours and then analyzed by flow cytometry. The percentage of sub-G1 apoptotic cells were presented as mean + SD. **, p < 0.01. (B) DLD-1 cells were treated with PP242 (1 μM) and/or erlotinib (2 μM) for the indicated hours and analyzed by western blotting for the cleavage of caspase-3, DFF45 and PARP with the molecular weights of the cleavage products indicated to the right of the panel.
Mentions: PP242 has been reported to induce apoptosis in leukemia and breast cancer cells through inhibition of mTORC2 activity [42,43]. The combination of P242 and erlotinib can inhibit the mTORC2 activity, which suggests that the combination may induce apoptosis in colorectal carcinoma cells. To text this notion, we treated DLD-1 cells with PP242 (0.5 μM) or erlotinib (2 μM), alone and in combination in the presence of EGF (50 ng/ml). Flow cytometry detected approximately 5% sub-G1 apoptotic cells in the cells treated with PP242 or erlotinib alone (Figure 4A). In contrast, approximately 15-20% cells underwent apoptotic cell death under the combination treatment of PP242 and erlotinib (Figure 4A). To confirm the PP242 and erlotinib combination-induced apoptosis, we examined the treated cells on western blotting and thus revealed cleavage of caspase-3, DFF45 and PARP; the data confirmed the apoptotic cell death of the cells under the treatment of PP242 and erlotinib, in contrast, however, PP242 and erlotinib alone failed to induce apoptosis in the cells (Figure 4B). These results suggest that the combination of PP242 and erlotinib synergistically inhibits the growth of colorectal carcinoma cells in part through induction of apoptosis.

Bottom Line: To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity.In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death.A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastrointestinal Surgery, First Hospital of Jilin University, Changchun, Jilin, China. wangquan-jlcc@hotmail.com

ABSTRACT
Mammalian target of rapamycin complex 1 and 2 (mTORC1/2) are overactive in colorectal carcinomas; however, the first generation of mTOR inhibitors such as rapamycin have failed to show clinical benefits in treating colorectal carcinoma in part due to their effects only on mTORC1. The second generation of mTOR inhibitors such as PP242 targets mTOR kinase; thus, they are capable of inhibiting both mTORC1 and mTORC2. To examine the therapeutic potential of the mTOR kinase inhibitors, we treated a panel of colorectal carcinoma cell lines with PP242. Western blotting showed that the PP242 inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT(S473)) was transient only in the first few hours of the PP242 treatment. Receptor tyrosine kinase arrays further revealed that PP242 treatment increased the phosphorylated epidermal growth factor receptor (EGFR) at Tyr 1068 (EGFR(T1068)). The parallel increase of AKT(S473) and EGFR(T1068) in the cells following PP242 treatment raised the possibility that EGFR phosphorylation might contribute to the PP242 incomplete inhibition of mTORC2. To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity. In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death. A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice. This study suggests that the combination of mTOR kinase and EGFR inhibitors may provide an effective treatment of colorectal carcinoma.

Show MeSH
Related in: MedlinePlus