Limits...
Small Molecule Inhibitors of Plasminogen Activator Inhibitor-1 Elicit Anti-Tumorigenic and Anti-Angiogenic Activity.

Placencio VR, Ichimura A, Miyata T, DeClerck YA - PLoS ONE (2015)

Bottom Line: In vivo, oral administration of TM5441 (20 mg/kg daily) to HT1080 and HCT116 xenotransplanted mice increased tumor cell apoptosis and had a significant disruptive effect on the tumor vasculature that was associated with a decrease in tumor growth and an increase in survival that, however, were not statistically significant.The effect on tumor vasculature in vivo was further examined in endothelial cells (EC) in vitro and this analysis indicated that both TM5275 and TM5441 inhibited EC branching in a 3D Matrigel assay at concentrations where they had little effect on EC apoptosis.These studies bring novel insight on the activity of PAI-1 inhibitors and provide important information for the future design of inhibitors targeting PAI-1 as therapeutic agents in cancer.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Oncology and Blood and Bone Marrow Transplantation, Department of Pediatrics, University of Southern California, Los Angeles, California, United States of America; The Saban Research Institute of Children's Hospital, Los Angeles, California, 90027, United States of America.

ABSTRACT
Numerous studies have shown a paradoxical positive correlation between elevated levels of plasminogen activator inhibitior-1 (PAI-1) in tumors and blood of cancer patients with poor clinical outcome, suggesting that PAI-1 could be a therapeutic target. Here we tested two orally bioavailable small molecule inhibitors of PAI-1 (TM5275 and TM5441) for their efficacy in pre-clinical models of cancer. We demonstrated that these inhibitors decreased cell viability in several human cancer cell lines with an IC50 in the 9.7 to 60.3 μM range and induced intrinsic apoptosis at concentrations of 50 μM. In vivo, oral administration of TM5441 (20 mg/kg daily) to HT1080 and HCT116 xenotransplanted mice increased tumor cell apoptosis and had a significant disruptive effect on the tumor vasculature that was associated with a decrease in tumor growth and an increase in survival that, however, were not statistically significant. Pharmacokinetics studies indicated an average peak plasma concentration of 11.4 μM one hour after oral administration and undetectable levels 23 hours after administration. The effect on tumor vasculature in vivo was further examined in endothelial cells (EC) in vitro and this analysis indicated that both TM5275 and TM5441 inhibited EC branching in a 3D Matrigel assay at concentrations where they had little effect on EC apoptosis. These studies bring novel insight on the activity of PAI-1 inhibitors and provide important information for the future design of inhibitors targeting PAI-1 as therapeutic agents in cancer.

No MeSH data available.


Related in: MedlinePlus

Treatment with TM5275 or TM5441 increases intrinsic apoptosis.A. Representative immunoblots for HT1080 and HCT116 cells treated with 25 μM or 50 μM TM5275 or TM5441 as indicated for caspase 8, 9, 3, PARP, and their cleavage products, n = 3. The numbers over the cleaved protein bands indicate the ratio of the cleaved protein pixel density to the corresponding tubulin control band pixel density. B. Representative JC-1 FACS plots of cells treated for 48 hours with 50 μM TM5275 or TM5441. The graphs show the average % (± SD) of JC-1 membrane depolarization in control DMSO-treated and TM inhibitor-treated cells n = 3. * indicates P values compared to DMSO controls < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4514594&req=5

pone.0133786.g004: Treatment with TM5275 or TM5441 increases intrinsic apoptosis.A. Representative immunoblots for HT1080 and HCT116 cells treated with 25 μM or 50 μM TM5275 or TM5441 as indicated for caspase 8, 9, 3, PARP, and their cleavage products, n = 3. The numbers over the cleaved protein bands indicate the ratio of the cleaved protein pixel density to the corresponding tubulin control band pixel density. B. Representative JC-1 FACS plots of cells treated for 48 hours with 50 μM TM5275 or TM5441. The graphs show the average % (± SD) of JC-1 membrane depolarization in control DMSO-treated and TM inhibitor-treated cells n = 3. * indicates P values compared to DMSO controls < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

Mentions: To explore the relative contribution of the extrinsic and intrinsic apoptotic pathways in TM inhibitor-induced apoptosis, we examined the effect of these inhibitors on the cleavage (activation) of caspase 3, 8 and 9 by Western blot. The data (Fig 4A) revealed an absence of caspase 8 activation in both cell lines upon treatment with TM5275 or TM5441. In contrast they indicated activation of caspase 9 and 3 and a corresponding cleavage of Poly adipo-ribose polymerase (PARP), a substrate for caspase 3, which was consistent with an activation of the intrinsic apoptotic pathway by TM inhibitors. This was confirmed by the examination of mitochondrial membrane depolarization in HT1080 and HCT116 cells treated with TM inhibitors (50 μM) (Fig 4B). The data indicated an increase in mitochondrial depolarization in cells treated with TM5275 and TM5441 with a much more pronounced effect of TM5441 as previously observed in caspase 3/7 activity assays (Fig 3A). These results indicated that TM5275, and in particular TM5441, are potent stimulators of intrinsic apoptosis in tumor cells.


Small Molecule Inhibitors of Plasminogen Activator Inhibitor-1 Elicit Anti-Tumorigenic and Anti-Angiogenic Activity.

Placencio VR, Ichimura A, Miyata T, DeClerck YA - PLoS ONE (2015)

Treatment with TM5275 or TM5441 increases intrinsic apoptosis.A. Representative immunoblots for HT1080 and HCT116 cells treated with 25 μM or 50 μM TM5275 or TM5441 as indicated for caspase 8, 9, 3, PARP, and their cleavage products, n = 3. The numbers over the cleaved protein bands indicate the ratio of the cleaved protein pixel density to the corresponding tubulin control band pixel density. B. Representative JC-1 FACS plots of cells treated for 48 hours with 50 μM TM5275 or TM5441. The graphs show the average % (± SD) of JC-1 membrane depolarization in control DMSO-treated and TM inhibitor-treated cells n = 3. * indicates P values compared to DMSO controls < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4514594&req=5

pone.0133786.g004: Treatment with TM5275 or TM5441 increases intrinsic apoptosis.A. Representative immunoblots for HT1080 and HCT116 cells treated with 25 μM or 50 μM TM5275 or TM5441 as indicated for caspase 8, 9, 3, PARP, and their cleavage products, n = 3. The numbers over the cleaved protein bands indicate the ratio of the cleaved protein pixel density to the corresponding tubulin control band pixel density. B. Representative JC-1 FACS plots of cells treated for 48 hours with 50 μM TM5275 or TM5441. The graphs show the average % (± SD) of JC-1 membrane depolarization in control DMSO-treated and TM inhibitor-treated cells n = 3. * indicates P values compared to DMSO controls < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.
Mentions: To explore the relative contribution of the extrinsic and intrinsic apoptotic pathways in TM inhibitor-induced apoptosis, we examined the effect of these inhibitors on the cleavage (activation) of caspase 3, 8 and 9 by Western blot. The data (Fig 4A) revealed an absence of caspase 8 activation in both cell lines upon treatment with TM5275 or TM5441. In contrast they indicated activation of caspase 9 and 3 and a corresponding cleavage of Poly adipo-ribose polymerase (PARP), a substrate for caspase 3, which was consistent with an activation of the intrinsic apoptotic pathway by TM inhibitors. This was confirmed by the examination of mitochondrial membrane depolarization in HT1080 and HCT116 cells treated with TM inhibitors (50 μM) (Fig 4B). The data indicated an increase in mitochondrial depolarization in cells treated with TM5275 and TM5441 with a much more pronounced effect of TM5441 as previously observed in caspase 3/7 activity assays (Fig 3A). These results indicated that TM5275, and in particular TM5441, are potent stimulators of intrinsic apoptosis in tumor cells.

Bottom Line: In vivo, oral administration of TM5441 (20 mg/kg daily) to HT1080 and HCT116 xenotransplanted mice increased tumor cell apoptosis and had a significant disruptive effect on the tumor vasculature that was associated with a decrease in tumor growth and an increase in survival that, however, were not statistically significant.The effect on tumor vasculature in vivo was further examined in endothelial cells (EC) in vitro and this analysis indicated that both TM5275 and TM5441 inhibited EC branching in a 3D Matrigel assay at concentrations where they had little effect on EC apoptosis.These studies bring novel insight on the activity of PAI-1 inhibitors and provide important information for the future design of inhibitors targeting PAI-1 as therapeutic agents in cancer.

View Article: PubMed Central - PubMed

Affiliation: Division of Hematology, Oncology and Blood and Bone Marrow Transplantation, Department of Pediatrics, University of Southern California, Los Angeles, California, United States of America; The Saban Research Institute of Children's Hospital, Los Angeles, California, 90027, United States of America.

ABSTRACT
Numerous studies have shown a paradoxical positive correlation between elevated levels of plasminogen activator inhibitior-1 (PAI-1) in tumors and blood of cancer patients with poor clinical outcome, suggesting that PAI-1 could be a therapeutic target. Here we tested two orally bioavailable small molecule inhibitors of PAI-1 (TM5275 and TM5441) for their efficacy in pre-clinical models of cancer. We demonstrated that these inhibitors decreased cell viability in several human cancer cell lines with an IC50 in the 9.7 to 60.3 μM range and induced intrinsic apoptosis at concentrations of 50 μM. In vivo, oral administration of TM5441 (20 mg/kg daily) to HT1080 and HCT116 xenotransplanted mice increased tumor cell apoptosis and had a significant disruptive effect on the tumor vasculature that was associated with a decrease in tumor growth and an increase in survival that, however, were not statistically significant. Pharmacokinetics studies indicated an average peak plasma concentration of 11.4 μM one hour after oral administration and undetectable levels 23 hours after administration. The effect on tumor vasculature in vivo was further examined in endothelial cells (EC) in vitro and this analysis indicated that both TM5275 and TM5441 inhibited EC branching in a 3D Matrigel assay at concentrations where they had little effect on EC apoptosis. These studies bring novel insight on the activity of PAI-1 inhibitors and provide important information for the future design of inhibitors targeting PAI-1 as therapeutic agents in cancer.

No MeSH data available.


Related in: MedlinePlus