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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 PI3K-Akt-mTOR signaling.A–B), S9 depresses EGF-triggered activation of PI3K-Akt-mTOR signaling pathway. Serum-deprived Rh30 cells (A) and SK-OV-3 cells (B) were treated with indicated concentrations of S9 for 1 h followed by EGF (50 ng/ml) stimulation for 10 min. Cells were harvested for Western blot analysis with antibodies specific for p-PDK1 (S241), PDK, p-Akt (T308), p-Akt (S473), Akt, p-mTOR (Ser2448), mTOR, p-p70S6K (T389), p70S6K, p-4E-BP1(T37/64), p-4E-BP1 (T70), 4E-BP1 and actin. Arrows indicate p70 isoform of S6 kinase protein. C) S9 blocks Akt membrane translocation and membrane ruffling. CHO (pCORON1000-EGFP-Akt) cells seeded on chamber were starved for 2 h then treated with 5 or 10 µM S9 for 1 h followed by IGF stimulation for 5 min. Fluorescent pictures were captured with confocal fluorescent microscopy. White arrows indicate cell membrane ruffling. Blue arrows indicate fluorescent foci. D) Total Akt granule intensity in each CHO (pCORON1000-EGFP-Akt) cell was counted with statistic module by IN Cell Analyzer 1000. Data shown are representative from two independent experiments.
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pone-0004881-g001: S9 inhibits PI3K-Akt-mTOR signaling.A–B), S9 depresses EGF-triggered activation of PI3K-Akt-mTOR signaling pathway. Serum-deprived Rh30 cells (A) and SK-OV-3 cells (B) were treated with indicated concentrations of S9 for 1 h followed by EGF (50 ng/ml) stimulation for 10 min. Cells were harvested for Western blot analysis with antibodies specific for p-PDK1 (S241), PDK, p-Akt (T308), p-Akt (S473), Akt, p-mTOR (Ser2448), mTOR, p-p70S6K (T389), p70S6K, p-4E-BP1(T37/64), p-4E-BP1 (T70), 4E-BP1 and actin. Arrows indicate p70 isoform of S6 kinase protein. C) S9 blocks Akt membrane translocation and membrane ruffling. CHO (pCORON1000-EGFP-Akt) cells seeded on chamber were starved for 2 h then treated with 5 or 10 µM S9 for 1 h followed by IGF stimulation for 5 min. Fluorescent pictures were captured with confocal fluorescent microscopy. White arrows indicate cell membrane ruffling. Blue arrows indicate fluorescent foci. D) Total Akt granule intensity in each CHO (pCORON1000-EGFP-Akt) cell was counted with statistic module by IN Cell Analyzer 1000. Data shown are representative from two independent experiments.

Mentions: We have previously reported that S9 potently down-regulated phosphorylation of Akt, mTOR, p70S6K and 4E-BP1 upon stimulation with EGF in Rh30 cells [8]. To gain insight of the effect of S9 on the PI3k-Akt-mTOR signaling, Rh30 cells were exposed to various concentrations of S9 followed by EGF stimulation. Although located in the upstream of this pathway, phosphorylation of PDK-1 at Ser 241 was insensitive to serum deprivation or EGF stimulation (Fig 1A), which is consistent with the observation reported elsewhere [9]. Phosphorylation of PDK-1 was slightly depressed in the presence of 40 µM S9. In contrast, S9 significantly inhibited phosphorylation of Akt at both Ser 473 and Thr 308 (Fig 1A). Compared with Ser473, Thr308 phosphorylation was more vulnerable to S9 treatment, which might be due to phosphorylation of Ser 473 prior to the phosphorylation at Thr 308 and is important for the recognition and activation of Akt by PDK-1 [10]. Similarly, S9 abrogated phosphorylation of mTOR at Ser 2448 and its downstream proteins p70S6K at Thr 389 and 4E-BP1 at Thr 37/64 and Thr 70 in a concentration-dependent manner. The inhibitory activity of S9 on the PI3K-Akt-mTOR signaling was also confirmed using ovary carcinoma SKOV-3 cells (Fig 1B). Inactivation of the PI3K-Akt-mTOR pathway was also reflected by decrease in the kinase activities of PI3K, Akt and mTOR in the RH30 cells after S9 treatment (Fig S1).


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 PI3K-Akt-mTOR signaling.A–B), S9 depresses EGF-triggered activation of PI3K-Akt-mTOR signaling pathway. Serum-deprived Rh30 cells (A) and SK-OV-3 cells (B) were treated with indicated concentrations of S9 for 1 h followed by EGF (50 ng/ml) stimulation for 10 min. Cells were harvested for Western blot analysis with antibodies specific for p-PDK1 (S241), PDK, p-Akt (T308), p-Akt (S473), Akt, p-mTOR (Ser2448), mTOR, p-p70S6K (T389), p70S6K, p-4E-BP1(T37/64), p-4E-BP1 (T70), 4E-BP1 and actin. Arrows indicate p70 isoform of S6 kinase protein. C) S9 blocks Akt membrane translocation and membrane ruffling. CHO (pCORON1000-EGFP-Akt) cells seeded on chamber were starved for 2 h then treated with 5 or 10 µM S9 for 1 h followed by IGF stimulation for 5 min. Fluorescent pictures were captured with confocal fluorescent microscopy. White arrows indicate cell membrane ruffling. Blue arrows indicate fluorescent foci. D) Total Akt granule intensity in each CHO (pCORON1000-EGFP-Akt) cell was counted with statistic module by IN Cell Analyzer 1000. Data shown are representative from two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654064&req=5

pone-0004881-g001: S9 inhibits PI3K-Akt-mTOR signaling.A–B), S9 depresses EGF-triggered activation of PI3K-Akt-mTOR signaling pathway. Serum-deprived Rh30 cells (A) and SK-OV-3 cells (B) were treated with indicated concentrations of S9 for 1 h followed by EGF (50 ng/ml) stimulation for 10 min. Cells were harvested for Western blot analysis with antibodies specific for p-PDK1 (S241), PDK, p-Akt (T308), p-Akt (S473), Akt, p-mTOR (Ser2448), mTOR, p-p70S6K (T389), p70S6K, p-4E-BP1(T37/64), p-4E-BP1 (T70), 4E-BP1 and actin. Arrows indicate p70 isoform of S6 kinase protein. C) S9 blocks Akt membrane translocation and membrane ruffling. CHO (pCORON1000-EGFP-Akt) cells seeded on chamber were starved for 2 h then treated with 5 or 10 µM S9 for 1 h followed by IGF stimulation for 5 min. Fluorescent pictures were captured with confocal fluorescent microscopy. White arrows indicate cell membrane ruffling. Blue arrows indicate fluorescent foci. D) Total Akt granule intensity in each CHO (pCORON1000-EGFP-Akt) cell was counted with statistic module by IN Cell Analyzer 1000. Data shown are representative from two independent experiments.
Mentions: We have previously reported that S9 potently down-regulated phosphorylation of Akt, mTOR, p70S6K and 4E-BP1 upon stimulation with EGF in Rh30 cells [8]. To gain insight of the effect of S9 on the PI3k-Akt-mTOR signaling, Rh30 cells were exposed to various concentrations of S9 followed by EGF stimulation. Although located in the upstream of this pathway, phosphorylation of PDK-1 at Ser 241 was insensitive to serum deprivation or EGF stimulation (Fig 1A), which is consistent with the observation reported elsewhere [9]. Phosphorylation of PDK-1 was slightly depressed in the presence of 40 µM S9. In contrast, S9 significantly inhibited phosphorylation of Akt at both Ser 473 and Thr 308 (Fig 1A). Compared with Ser473, Thr308 phosphorylation was more vulnerable to S9 treatment, which might be due to phosphorylation of Ser 473 prior to the phosphorylation at Thr 308 and is important for the recognition and activation of Akt by PDK-1 [10]. Similarly, S9 abrogated phosphorylation of mTOR at Ser 2448 and its downstream proteins p70S6K at Thr 389 and 4E-BP1 at Thr 37/64 and Thr 70 in a concentration-dependent manner. The inhibitory activity of S9 on the PI3K-Akt-mTOR signaling was also confirmed using ovary carcinoma SKOV-3 cells (Fig 1B). Inactivation of the PI3K-Akt-mTOR pathway was also reflected by decrease in the kinase activities of PI3K, Akt and mTOR in the RH30 cells after S9 treatment (Fig S1).

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