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S9, a novel anticancer agent, exerts its anti-proliferative activity by interfering with both PI3K-Akt-mTOR signaling and microtubule cytoskeleton.

Zhang C, Yang N, Yang CH, Ding HS, Luo C, Zhang Y, Wu MJ, Zhang XW, Shen X, Jiang HL, Meng LH, Ding J - PLoS ONE (2009)

Bottom Line: S9 possessed inhibitory activity against both PI3K and mTOR with little effect on other tested 30 kinases.Further study revealed that S9 inhibited tubulin polymerization via binding to colchicine-binding site of tubulin and resulted in microtubule disturbance.Taken together, S9 targets both PI3K-Akt-mTOR signaling and microtubule cytoskeleton, which combinatorially contributes its antitumor activity and provides new clues for anticancer drug design and development.

View Article: PubMed Central - PubMed

Affiliation: Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT

Background: Deregulation of the phosphatidylinositol 3-kinases (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway plays a central role in tumor formation and progression, providing validated targets for cancer therapy. S9, a hybrid of alpha-methylene-gamma-lactone and 2-phenyl indole compound, possessed potent activity against this pathway.

Methodology/principal findings: Effects of S9 on PI3K-Akt-mTOR pathway were determined by Western blot, immunofluorescence staining and in vitro kinas assay. The interactions between tubulin and S9 were investigated by polymerization assay, CD, and SPR assay. The potential binding modes between S9 and PI3K, mTOR or tubulin were analyzed by molecular modeling. Anti-tumor activity of S9 was evaluated in tumor cells and in nude mice bearing human cancer xenografts. S9 abrogated EGF-activated PI3K-Akt-mTOR signaling cascade and Akt translocation to cellular membrane in human tumor cells. S9 possessed inhibitory activity against both PI3K and mTOR with little effect on other tested 30 kinases. S9 also completely impeded hyper-phosphorylation of Akt as a feedback of inhibition of mTOR by rapamycin. S9 unexpectedly arrested cells in M phase other than G1 phase, which was distinct from compounds targeting PI3K-Akt-mTOR pathway. Further study revealed that S9 inhibited tubulin polymerization via binding to colchicine-binding site of tubulin and resulted in microtubule disturbance. Molecular modeling indicated that S9 could potentially bind to the kinase domains of PI3K p110alpha subunit and mTOR, and shared similar hydrophobic interactions with colchicines in the complex with tubulin. Moreover, S9 induced rapid apoptosis in tumor cell, which might reflect a synergistic cooperation between blockade of both PI3-Akt-mTOR signaling and tubulin cytoskeleton. Finally, S9 displayed potent antiproliferative activity in a panel of tumor cells originated from different tissue types including drug-resistant cells and in nude mice bearing human tumor xenografts.

Conclusions/significance: Taken together, S9 targets both PI3K-Akt-mTOR signaling and microtubule cytoskeleton, which combinatorially contributes its antitumor activity and provides new clues for anticancer drug design and development.

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Related in: MedlinePlus

S9 inhibits the kinase catalytic activity of PI3K and mTOR.A–B) Active kinases were immunoprecipitated from exponentially growing Rh30 cells and kinase assays were performed. In the presence of indicated concentrations of S9, phosphorylation PIP2 to PIP3 were measured by ELISA (A). Phosphorylation of 4E-BP1 (B) was detected by Western blot. Band intensity was quantitated by optical densitometric analysis and normalized to vehicle control. Representative images were presented and relative kinase activity was plotted as mean±SD of three independent experiments. C) S9 abrogates rapamycin-induced hyperphosphorylation of Akt (Ser 473). Rh30 cells were starved overnight and treated with 10 µM of S9 and/or rapamycin (Rapa) for 1.5 h on the next day, followed by stimulation with 50 ng/ml EGF for 10 min. Phosphorylated Akt at Serine 473 and Threonine 308 and total Akt were detected by Western blot. D) Rh30 cells were exposed to 10 µM camptothecin (CPT) for 1 h after pre-incubation with indicated concentrations of S9 or wortmannin (Wort) for 30 min. Cells were collected and γ-H2AX levels were detected with Western blot analysis. E–F) The binding mode of S9 within PI3K (E), mTOR (F). The protein is represented by cartoon. The residues interacting with the compounds are shown in sticks. All of the structural diagrams were prepared using PyMOL (http://pymol.sourceforge.net).
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pone-0004881-g002: S9 inhibits the kinase catalytic activity of PI3K and mTOR.A–B) Active kinases were immunoprecipitated from exponentially growing Rh30 cells and kinase assays were performed. In the presence of indicated concentrations of S9, phosphorylation PIP2 to PIP3 were measured by ELISA (A). Phosphorylation of 4E-BP1 (B) was detected by Western blot. Band intensity was quantitated by optical densitometric analysis and normalized to vehicle control. Representative images were presented and relative kinase activity was plotted as mean±SD of three independent experiments. C) S9 abrogates rapamycin-induced hyperphosphorylation of Akt (Ser 473). Rh30 cells were starved overnight and treated with 10 µM of S9 and/or rapamycin (Rapa) for 1.5 h on the next day, followed by stimulation with 50 ng/ml EGF for 10 min. Phosphorylated Akt at Serine 473 and Threonine 308 and total Akt were detected by Western blot. D) Rh30 cells were exposed to 10 µM camptothecin (CPT) for 1 h after pre-incubation with indicated concentrations of S9 or wortmannin (Wort) for 30 min. Cells were collected and γ-H2AX levels were detected with Western blot analysis. E–F) The binding mode of S9 within PI3K (E), mTOR (F). The protein is represented by cartoon. The residues interacting with the compounds are shown in sticks. All of the structural diagrams were prepared using PyMOL (http://pymol.sourceforge.net).

Mentions: As S9 inhibits the PI3K-Akt-mTOR pathway signaling cascade, PI3K kinase assays were first carried out to determine whether S9 inhibits the catalytic activity of PI3K. PI3K were activated with EGF and immunoprecipitated from Rh30 cells, and kinase assays were performed in the presence of S9. As shown in Figure 2A, the ability of PI3K to generate PIP3 decreased after S9 treatment in a concentration-dependent manner and the activity decreased to 53.2% in the presence of 1 µM S9. Since S9 is capable of inhibiting the activity of PI3K, we detected whether S9 inhibits the activity of mTOR, which bears a highly homologous COOH-terminal catalytic domain with PI3K. As shown in Figure 2B, phosphorylation of 4E-BP1 by mTOR was greatly down-regulated by S9 treatment. 1 µM S9 decreased mTOR kinase activity by 58.7%. Thus, S9 is a new dual inhibitor of PI3K and mTOR.


S9, a novel anticancer agent, exerts its anti-proliferative activity by interfering with both PI3K-Akt-mTOR signaling and microtubule cytoskeleton.

Zhang C, Yang N, Yang CH, Ding HS, Luo C, Zhang Y, Wu MJ, Zhang XW, Shen X, Jiang HL, Meng LH, Ding J - PLoS ONE (2009)

S9 inhibits the kinase catalytic activity of PI3K and mTOR.A–B) Active kinases were immunoprecipitated from exponentially growing Rh30 cells and kinase assays were performed. In the presence of indicated concentrations of S9, phosphorylation PIP2 to PIP3 were measured by ELISA (A). Phosphorylation of 4E-BP1 (B) was detected by Western blot. Band intensity was quantitated by optical densitometric analysis and normalized to vehicle control. Representative images were presented and relative kinase activity was plotted as mean±SD of three independent experiments. C) S9 abrogates rapamycin-induced hyperphosphorylation of Akt (Ser 473). Rh30 cells were starved overnight and treated with 10 µM of S9 and/or rapamycin (Rapa) for 1.5 h on the next day, followed by stimulation with 50 ng/ml EGF for 10 min. Phosphorylated Akt at Serine 473 and Threonine 308 and total Akt were detected by Western blot. D) Rh30 cells were exposed to 10 µM camptothecin (CPT) for 1 h after pre-incubation with indicated concentrations of S9 or wortmannin (Wort) for 30 min. Cells were collected and γ-H2AX levels were detected with Western blot analysis. E–F) The binding mode of S9 within PI3K (E), mTOR (F). The protein is represented by cartoon. The residues interacting with the compounds are shown in sticks. All of the structural diagrams were prepared using PyMOL (http://pymol.sourceforge.net).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2654064&req=5

pone-0004881-g002: S9 inhibits the kinase catalytic activity of PI3K and mTOR.A–B) Active kinases were immunoprecipitated from exponentially growing Rh30 cells and kinase assays were performed. In the presence of indicated concentrations of S9, phosphorylation PIP2 to PIP3 were measured by ELISA (A). Phosphorylation of 4E-BP1 (B) was detected by Western blot. Band intensity was quantitated by optical densitometric analysis and normalized to vehicle control. Representative images were presented and relative kinase activity was plotted as mean±SD of three independent experiments. C) S9 abrogates rapamycin-induced hyperphosphorylation of Akt (Ser 473). Rh30 cells were starved overnight and treated with 10 µM of S9 and/or rapamycin (Rapa) for 1.5 h on the next day, followed by stimulation with 50 ng/ml EGF for 10 min. Phosphorylated Akt at Serine 473 and Threonine 308 and total Akt were detected by Western blot. D) Rh30 cells were exposed to 10 µM camptothecin (CPT) for 1 h after pre-incubation with indicated concentrations of S9 or wortmannin (Wort) for 30 min. Cells were collected and γ-H2AX levels were detected with Western blot analysis. E–F) The binding mode of S9 within PI3K (E), mTOR (F). The protein is represented by cartoon. The residues interacting with the compounds are shown in sticks. All of the structural diagrams were prepared using PyMOL (http://pymol.sourceforge.net).
Mentions: As S9 inhibits the PI3K-Akt-mTOR pathway signaling cascade, PI3K kinase assays were first carried out to determine whether S9 inhibits the catalytic activity of PI3K. PI3K were activated with EGF and immunoprecipitated from Rh30 cells, and kinase assays were performed in the presence of S9. As shown in Figure 2A, the ability of PI3K to generate PIP3 decreased after S9 treatment in a concentration-dependent manner and the activity decreased to 53.2% in the presence of 1 µM S9. Since S9 is capable of inhibiting the activity of PI3K, we detected whether S9 inhibits the activity of mTOR, which bears a highly homologous COOH-terminal catalytic domain with PI3K. As shown in Figure 2B, phosphorylation of 4E-BP1 by mTOR was greatly down-regulated by S9 treatment. 1 µM S9 decreased mTOR kinase activity by 58.7%. Thus, S9 is a new dual inhibitor of PI3K and mTOR.

Bottom Line: S9 possessed inhibitory activity against both PI3K and mTOR with little effect on other tested 30 kinases.Further study revealed that S9 inhibited tubulin polymerization via binding to colchicine-binding site of tubulin and resulted in microtubule disturbance.Taken together, S9 targets both PI3K-Akt-mTOR signaling and microtubule cytoskeleton, which combinatorially contributes its antitumor activity and provides new clues for anticancer drug design and development.

View Article: PubMed Central - PubMed

Affiliation: Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.

ABSTRACT

Background: Deregulation of the phosphatidylinositol 3-kinases (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway plays a central role in tumor formation and progression, providing validated targets for cancer therapy. S9, a hybrid of alpha-methylene-gamma-lactone and 2-phenyl indole compound, possessed potent activity against this pathway.

Methodology/principal findings: Effects of S9 on PI3K-Akt-mTOR pathway were determined by Western blot, immunofluorescence staining and in vitro kinas assay. The interactions between tubulin and S9 were investigated by polymerization assay, CD, and SPR assay. The potential binding modes between S9 and PI3K, mTOR or tubulin were analyzed by molecular modeling. Anti-tumor activity of S9 was evaluated in tumor cells and in nude mice bearing human cancer xenografts. S9 abrogated EGF-activated PI3K-Akt-mTOR signaling cascade and Akt translocation to cellular membrane in human tumor cells. S9 possessed inhibitory activity against both PI3K and mTOR with little effect on other tested 30 kinases. S9 also completely impeded hyper-phosphorylation of Akt as a feedback of inhibition of mTOR by rapamycin. S9 unexpectedly arrested cells in M phase other than G1 phase, which was distinct from compounds targeting PI3K-Akt-mTOR pathway. Further study revealed that S9 inhibited tubulin polymerization via binding to colchicine-binding site of tubulin and resulted in microtubule disturbance. Molecular modeling indicated that S9 could potentially bind to the kinase domains of PI3K p110alpha subunit and mTOR, and shared similar hydrophobic interactions with colchicines in the complex with tubulin. Moreover, S9 induced rapid apoptosis in tumor cell, which might reflect a synergistic cooperation between blockade of both PI3-Akt-mTOR signaling and tubulin cytoskeleton. Finally, S9 displayed potent antiproliferative activity in a panel of tumor cells originated from different tissue types including drug-resistant cells and in nude mice bearing human tumor xenografts.

Conclusions/significance: Taken together, S9 targets both PI3K-Akt-mTOR signaling and microtubule cytoskeleton, which combinatorially contributes its antitumor activity and provides new clues for anticancer drug design and development.

Show MeSH
Related in: MedlinePlus