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P7170, a novel inhibitor of mTORC1/mTORC2 and Activin receptor-like Kinase 1 (ALK1) inhibits the growth of non small cell lung cancer.

Venkatesha VA, Joshi A, Venkataraman M, Sonawane V, Bhatia D, Tannu P, Bose J, Choudhari S, Srivastava A, Pandey PK, Lad VJ, Sangana R, Ahmed T, Damre A, Deore V, Sahu B, Kumar S, Sharma S, Agarwal VR - Mol. Cancer (2014)

Bottom Line: P7170 at a well-tolerated daily dose of 20 mg/kg significantly inhibited the growth of NSCLC xenografts independent of different mutations (EGFR, KRAS, or PIK3CA) or sensitivity to erlotinib.Pharmacokinetic-pharmacodynamic (PK-PD) analysis showed sub-micro molar tumor concentrations along with mTORC1/C2 inhibition.Our results provide evidence of antitumor activity of P7170 in the erlotinib -sensitive and -insensitive models of NSCLC.

View Article: PubMed Central - PubMed

Affiliation: Piramal Life Sciences Ltd, # 1 Nirlon Complex, Off: Western Express Highway, Goregaon (East), Mumbai, Maharashtra 400063, India. veena_828@yahoo.com.

ABSTRACT

Background: Lung cancer is the major cause of cancer-related deaths and many cases of Non Small Cell Lung Cancer (NSCLC), a common type of lung cancer, have frequent genetic/oncogenic activation of EGFR, KRAS, PIK3CA, BRAF, and others that drive tumor growth. Some patients though initially respond, but later develop resistance to erlotinib/gefitinib with no option except for cytotoxic therapy. Therefore, development of novel targeted therapeutics is imperative to provide improved survival benefit for NSCLC patients. The mTOR cell survival pathway is activated in naïve, or in response to targeted therapies in NSCLC.

Methods: We have discovered P7170, a small molecule inhibitor of mTORC1/mTORC2/ALK1 and investigated its antitumor efficacy using various in vitro and in vivo models of human NSCLC.

Results: P7170 inhibited the phosphorylation of AKT, S6 and 4EBP1 (substrates for mTORC2 and mTORC1) levels by 80-100% and growth of NSCLC cells. P7170 inhibited anchorage-independent colony formation of NSCLC patient tumor-derived cells subsistent of disease sub-types. The compound also induced apoptosis in NSCLC cell lines. P7170 at a well-tolerated daily dose of 20 mg/kg significantly inhibited the growth of NSCLC xenografts independent of different mutations (EGFR, KRAS, or PIK3CA) or sensitivity to erlotinib. Pharmacokinetic-pharmacodynamic (PK-PD) analysis showed sub-micro molar tumor concentrations along with mTORC1/C2 inhibition.

Conclusions: Our results provide evidence of antitumor activity of P7170 in the erlotinib -sensitive and -insensitive models of NSCLC.

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P7170 induced apoptosis and decreased STAT3 activity in NSCLC cells. (A) Cell growth inhibition of exponentially growing H460 NSCLC cells by P7170. (B) Annexin V and propidium iodide positive cells analyzed by flow cytometry after 16 h of treatment. (C) H460 cells were seeded in 90 mm Petri dishes (1 × 106 cells/ plate). The cells were serum starved for 16 h. Fresh medium was added to the plates and treated with P7170 (0.1, 0.3, and1 μM) for 24 h; total cellular proteins were processed for western blotting for cleaved PARP; (D) Inhibition of STAT3 activity performed using Panomics STAT3 luciferase kit, corresponding cell toxicity at same concentrations of P7170 was determined by PI-based assay (plotted on the secondary axis on right side of the panel). (E) Immunofluorescence staining for STAT3 phosphorylation in A549 NSCLC cells: pSTAT3 (red) and nucleus (blue). See Methods for details.
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Fig2: P7170 induced apoptosis and decreased STAT3 activity in NSCLC cells. (A) Cell growth inhibition of exponentially growing H460 NSCLC cells by P7170. (B) Annexin V and propidium iodide positive cells analyzed by flow cytometry after 16 h of treatment. (C) H460 cells were seeded in 90 mm Petri dishes (1 × 106 cells/ plate). The cells were serum starved for 16 h. Fresh medium was added to the plates and treated with P7170 (0.1, 0.3, and1 μM) for 24 h; total cellular proteins were processed for western blotting for cleaved PARP; (D) Inhibition of STAT3 activity performed using Panomics STAT3 luciferase kit, corresponding cell toxicity at same concentrations of P7170 was determined by PI-based assay (plotted on the secondary axis on right side of the panel). (E) Immunofluorescence staining for STAT3 phosphorylation in A549 NSCLC cells: pSTAT3 (red) and nucleus (blue). See Methods for details.

Mentions: To further understand the mechanism of cell growth inhibition, H460 cells were treated with P7170 for 24 h and analyzed by flow cytometry for propidium positive dead cells. A significant increase in dead cells upon treatment with P7170 was observed at 10 nM compared to untreated control (p < 0.05; Figure 2A). In the Annexin V-propidium iodide apoptosis assay, we found that treatment with 300 nM P7170 resulted in a 35% loss of cell viability with 22% cells in late apoptosis/necrosis, and 11% cells in early apoptosis indicating the onset of cell death (Figure 2B; Additional file 3: Figure S3). The onset of apoptosis correlated with cell growth inhibition at similar concentrations (100 – 300 nM) and at the same incubation time of 24 h (Figure 2A). To ascertain whether apoptotic response by P7170 involves DNA damage or elicited DNA damage-repair response, PARP cleavage, a widely used marker for measuring DNA-damage repair response was examined. In a separate experiment, treatment of H460 cells with an increasing concentration of P7170 was found to induce PARP cleavage (Figure 2C).Figure 2


P7170, a novel inhibitor of mTORC1/mTORC2 and Activin receptor-like Kinase 1 (ALK1) inhibits the growth of non small cell lung cancer.

Venkatesha VA, Joshi A, Venkataraman M, Sonawane V, Bhatia D, Tannu P, Bose J, Choudhari S, Srivastava A, Pandey PK, Lad VJ, Sangana R, Ahmed T, Damre A, Deore V, Sahu B, Kumar S, Sharma S, Agarwal VR - Mol. Cancer (2014)

P7170 induced apoptosis and decreased STAT3 activity in NSCLC cells. (A) Cell growth inhibition of exponentially growing H460 NSCLC cells by P7170. (B) Annexin V and propidium iodide positive cells analyzed by flow cytometry after 16 h of treatment. (C) H460 cells were seeded in 90 mm Petri dishes (1 × 106 cells/ plate). The cells were serum starved for 16 h. Fresh medium was added to the plates and treated with P7170 (0.1, 0.3, and1 μM) for 24 h; total cellular proteins were processed for western blotting for cleaved PARP; (D) Inhibition of STAT3 activity performed using Panomics STAT3 luciferase kit, corresponding cell toxicity at same concentrations of P7170 was determined by PI-based assay (plotted on the secondary axis on right side of the panel). (E) Immunofluorescence staining for STAT3 phosphorylation in A549 NSCLC cells: pSTAT3 (red) and nucleus (blue). See Methods for details.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4289333&req=5

Fig2: P7170 induced apoptosis and decreased STAT3 activity in NSCLC cells. (A) Cell growth inhibition of exponentially growing H460 NSCLC cells by P7170. (B) Annexin V and propidium iodide positive cells analyzed by flow cytometry after 16 h of treatment. (C) H460 cells were seeded in 90 mm Petri dishes (1 × 106 cells/ plate). The cells were serum starved for 16 h. Fresh medium was added to the plates and treated with P7170 (0.1, 0.3, and1 μM) for 24 h; total cellular proteins were processed for western blotting for cleaved PARP; (D) Inhibition of STAT3 activity performed using Panomics STAT3 luciferase kit, corresponding cell toxicity at same concentrations of P7170 was determined by PI-based assay (plotted on the secondary axis on right side of the panel). (E) Immunofluorescence staining for STAT3 phosphorylation in A549 NSCLC cells: pSTAT3 (red) and nucleus (blue). See Methods for details.
Mentions: To further understand the mechanism of cell growth inhibition, H460 cells were treated with P7170 for 24 h and analyzed by flow cytometry for propidium positive dead cells. A significant increase in dead cells upon treatment with P7170 was observed at 10 nM compared to untreated control (p < 0.05; Figure 2A). In the Annexin V-propidium iodide apoptosis assay, we found that treatment with 300 nM P7170 resulted in a 35% loss of cell viability with 22% cells in late apoptosis/necrosis, and 11% cells in early apoptosis indicating the onset of cell death (Figure 2B; Additional file 3: Figure S3). The onset of apoptosis correlated with cell growth inhibition at similar concentrations (100 – 300 nM) and at the same incubation time of 24 h (Figure 2A). To ascertain whether apoptotic response by P7170 involves DNA damage or elicited DNA damage-repair response, PARP cleavage, a widely used marker for measuring DNA-damage repair response was examined. In a separate experiment, treatment of H460 cells with an increasing concentration of P7170 was found to induce PARP cleavage (Figure 2C).Figure 2

Bottom Line: P7170 at a well-tolerated daily dose of 20 mg/kg significantly inhibited the growth of NSCLC xenografts independent of different mutations (EGFR, KRAS, or PIK3CA) or sensitivity to erlotinib.Pharmacokinetic-pharmacodynamic (PK-PD) analysis showed sub-micro molar tumor concentrations along with mTORC1/C2 inhibition.Our results provide evidence of antitumor activity of P7170 in the erlotinib -sensitive and -insensitive models of NSCLC.

View Article: PubMed Central - PubMed

Affiliation: Piramal Life Sciences Ltd, # 1 Nirlon Complex, Off: Western Express Highway, Goregaon (East), Mumbai, Maharashtra 400063, India. veena_828@yahoo.com.

ABSTRACT

Background: Lung cancer is the major cause of cancer-related deaths and many cases of Non Small Cell Lung Cancer (NSCLC), a common type of lung cancer, have frequent genetic/oncogenic activation of EGFR, KRAS, PIK3CA, BRAF, and others that drive tumor growth. Some patients though initially respond, but later develop resistance to erlotinib/gefitinib with no option except for cytotoxic therapy. Therefore, development of novel targeted therapeutics is imperative to provide improved survival benefit for NSCLC patients. The mTOR cell survival pathway is activated in naïve, or in response to targeted therapies in NSCLC.

Methods: We have discovered P7170, a small molecule inhibitor of mTORC1/mTORC2/ALK1 and investigated its antitumor efficacy using various in vitro and in vivo models of human NSCLC.

Results: P7170 inhibited the phosphorylation of AKT, S6 and 4EBP1 (substrates for mTORC2 and mTORC1) levels by 80-100% and growth of NSCLC cells. P7170 inhibited anchorage-independent colony formation of NSCLC patient tumor-derived cells subsistent of disease sub-types. The compound also induced apoptosis in NSCLC cell lines. P7170 at a well-tolerated daily dose of 20 mg/kg significantly inhibited the growth of NSCLC xenografts independent of different mutations (EGFR, KRAS, or PIK3CA) or sensitivity to erlotinib. Pharmacokinetic-pharmacodynamic (PK-PD) analysis showed sub-micro molar tumor concentrations along with mTORC1/C2 inhibition.

Conclusions: Our results provide evidence of antitumor activity of P7170 in the erlotinib -sensitive and -insensitive models of NSCLC.

Show MeSH
Related in: MedlinePlus