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Assessment of the chemosensitizing activity of TAT-RasGAP317-326 in childhood cancers.

Chevalier N, Gross N, Widmann C - PLoS ONE (2015)

Bottom Line: Although current anti-cancer protocols are reasonably effective, treatment-associated long-term side effects, induced by lack of specificity of the anti-cancer procedures, remain a challenging problem in pediatric oncology.The RasGAP-derived peptide did not increase cell death of normal lymphocytes, alone or in combination with the majority of the tested chemotherapies.Consequently, TAT-RasGAP317-326 may benefit children with tumors by increasing the efficacy of anti-cancer therapies notably by allowing reductions in anti-cancer drug dosage and the associated drug-induced side effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Lausanne, Lausanne, Switzerland.

ABSTRACT
Although current anti-cancer protocols are reasonably effective, treatment-associated long-term side effects, induced by lack of specificity of the anti-cancer procedures, remain a challenging problem in pediatric oncology. TAT-RasGAP317-326 is a RasGAP-derived cell-permeable peptide that acts as a sensitizer to various anti-cancer treatments in adult tumor cells. In the present study, we assessed the effect of TAT-RasGAP317-326 in several childhood cancer cell lines. The RasGAP-derived peptide-induced cell death was analyzed in several neuroblastoma, Ewing sarcoma and leukemia cell lines (as well as in normal lymphocytes). Cell death was evaluated using flow cytometry methods in the absence or in the presence of the peptide in combination with various genotoxins used in the clinics (4-hydroperoxycyclophosphamide, etoposide, vincristine and doxorubicin). All tested pediatric tumors, in response to at least one genotoxin, were sensitized by TAT-RasGAP317-326. The RasGAP-derived peptide did not increase cell death of normal lymphocytes, alone or in combination with the majority of the tested chemotherapies. Consequently, TAT-RasGAP317-326 may benefit children with tumors by increasing the efficacy of anti-cancer therapies notably by allowing reductions in anti-cancer drug dosage and the associated drug-induced side effects.

No MeSH data available.


Related in: MedlinePlus

The RasGAP moiety carries the tumor sensitizing activity of TAT-RasGAP317-326.A. CCRF-CEM cells were seeded in 6-well plates and directly treated with 10 μM TAT-RasGAP317-326, TAT, TAT-Scrambled or TAT-Mutated in the absence or in the presence of 4-HC, etoposide, vincristine or doxorubicin at the indicated concentrations. After 24 hours of drug incubation, 7-AAD or Annexin V-FITC staining was performed to evaluate cell death. B-C. The TC252 (B) and NB1-FBS (C) cell lines were treated similarly but in combination with 4-HC only. P10, TAT-RasGAP317-326; TAT-S, TAT-Scrambled; TAT-M, TAT-Mutated; 4-HC, 4-hydroperoxycyclophosphamide. Means with the same letter are not significantly different.
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pone.0120487.g005: The RasGAP moiety carries the tumor sensitizing activity of TAT-RasGAP317-326.A. CCRF-CEM cells were seeded in 6-well plates and directly treated with 10 μM TAT-RasGAP317-326, TAT, TAT-Scrambled or TAT-Mutated in the absence or in the presence of 4-HC, etoposide, vincristine or doxorubicin at the indicated concentrations. After 24 hours of drug incubation, 7-AAD or Annexin V-FITC staining was performed to evaluate cell death. B-C. The TC252 (B) and NB1-FBS (C) cell lines were treated similarly but in combination with 4-HC only. P10, TAT-RasGAP317-326; TAT-S, TAT-Scrambled; TAT-M, TAT-Mutated; 4-HC, 4-hydroperoxycyclophosphamide. Means with the same letter are not significantly different.

Mentions: To assess the specificity of the chemo-sensitizing activity of TAT-RasGAP317-326, three different control peptides were tested: a peptide composed of the TAT sequence only (TAT), a peptide in which the RasGAP sequence was scrambled (TAT-Scrambled), and a peptide in which the first tryptophan of the RasGAP sequence was substituted into an alanine residue (TAT-Mutated). This tryptophan was recently shown to be essential for the sensitizing activity of TAT-RasGAP317-326 in adult tumors [16]. The effect of TAT-RasGAP317-326 and the three control peptides was tested in CCRF-CEM cells in combination with various anti-cancer drugs at doses that allowed the greatest sensitization activity to be detected. CCRF-CEM cells were used because this leukemia cell line was the one most efficiently sensitized by the RasGAP-derived peptide. Fig 5A shows that all three control peptides had a tendency to slightly favor the death of CCRF-CEM cells when combined with the different anti-cancer drugs but in most cases this did not reach statistical significance. In contrast, TAT-RasGAP317-326 markedly, and always significantly, sensitized these cells to the drugs. The slight sensitization effect of the control peptides is most likely due to the cell penetrating activity of the TAT moiety, which has the potential to negatively affect cellular homeostasis [40]. Similar results were obtained when the TC252 Ewing sarcoma cell line and the NB1-FBS neuroblastoma cell line were used: TAT-RasGAP317-326 significantly increased their sensitivity to 4-HC, while the three control peptides did not, or only minimally (Fig 5B and 5C). These data indicate that the tumor sensitizing activity of TAT-RasGAP317-326 only marginally relies on its ability to penetrate cells via the TAT cell-permeable sequence. Therefore, it can be concluded that the sensitizing activity of TAT-RasGAP317-326 is mainly carried by the RasGAP-derived sequence.


Assessment of the chemosensitizing activity of TAT-RasGAP317-326 in childhood cancers.

Chevalier N, Gross N, Widmann C - PLoS ONE (2015)

The RasGAP moiety carries the tumor sensitizing activity of TAT-RasGAP317-326.A. CCRF-CEM cells were seeded in 6-well plates and directly treated with 10 μM TAT-RasGAP317-326, TAT, TAT-Scrambled or TAT-Mutated in the absence or in the presence of 4-HC, etoposide, vincristine or doxorubicin at the indicated concentrations. After 24 hours of drug incubation, 7-AAD or Annexin V-FITC staining was performed to evaluate cell death. B-C. The TC252 (B) and NB1-FBS (C) cell lines were treated similarly but in combination with 4-HC only. P10, TAT-RasGAP317-326; TAT-S, TAT-Scrambled; TAT-M, TAT-Mutated; 4-HC, 4-hydroperoxycyclophosphamide. Means with the same letter are not significantly different.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380404&req=5

pone.0120487.g005: The RasGAP moiety carries the tumor sensitizing activity of TAT-RasGAP317-326.A. CCRF-CEM cells were seeded in 6-well plates and directly treated with 10 μM TAT-RasGAP317-326, TAT, TAT-Scrambled or TAT-Mutated in the absence or in the presence of 4-HC, etoposide, vincristine or doxorubicin at the indicated concentrations. After 24 hours of drug incubation, 7-AAD or Annexin V-FITC staining was performed to evaluate cell death. B-C. The TC252 (B) and NB1-FBS (C) cell lines were treated similarly but in combination with 4-HC only. P10, TAT-RasGAP317-326; TAT-S, TAT-Scrambled; TAT-M, TAT-Mutated; 4-HC, 4-hydroperoxycyclophosphamide. Means with the same letter are not significantly different.
Mentions: To assess the specificity of the chemo-sensitizing activity of TAT-RasGAP317-326, three different control peptides were tested: a peptide composed of the TAT sequence only (TAT), a peptide in which the RasGAP sequence was scrambled (TAT-Scrambled), and a peptide in which the first tryptophan of the RasGAP sequence was substituted into an alanine residue (TAT-Mutated). This tryptophan was recently shown to be essential for the sensitizing activity of TAT-RasGAP317-326 in adult tumors [16]. The effect of TAT-RasGAP317-326 and the three control peptides was tested in CCRF-CEM cells in combination with various anti-cancer drugs at doses that allowed the greatest sensitization activity to be detected. CCRF-CEM cells were used because this leukemia cell line was the one most efficiently sensitized by the RasGAP-derived peptide. Fig 5A shows that all three control peptides had a tendency to slightly favor the death of CCRF-CEM cells when combined with the different anti-cancer drugs but in most cases this did not reach statistical significance. In contrast, TAT-RasGAP317-326 markedly, and always significantly, sensitized these cells to the drugs. The slight sensitization effect of the control peptides is most likely due to the cell penetrating activity of the TAT moiety, which has the potential to negatively affect cellular homeostasis [40]. Similar results were obtained when the TC252 Ewing sarcoma cell line and the NB1-FBS neuroblastoma cell line were used: TAT-RasGAP317-326 significantly increased their sensitivity to 4-HC, while the three control peptides did not, or only minimally (Fig 5B and 5C). These data indicate that the tumor sensitizing activity of TAT-RasGAP317-326 only marginally relies on its ability to penetrate cells via the TAT cell-permeable sequence. Therefore, it can be concluded that the sensitizing activity of TAT-RasGAP317-326 is mainly carried by the RasGAP-derived sequence.

Bottom Line: Although current anti-cancer protocols are reasonably effective, treatment-associated long-term side effects, induced by lack of specificity of the anti-cancer procedures, remain a challenging problem in pediatric oncology.The RasGAP-derived peptide did not increase cell death of normal lymphocytes, alone or in combination with the majority of the tested chemotherapies.Consequently, TAT-RasGAP317-326 may benefit children with tumors by increasing the efficacy of anti-cancer therapies notably by allowing reductions in anti-cancer drug dosage and the associated drug-induced side effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Lausanne, Lausanne, Switzerland.

ABSTRACT
Although current anti-cancer protocols are reasonably effective, treatment-associated long-term side effects, induced by lack of specificity of the anti-cancer procedures, remain a challenging problem in pediatric oncology. TAT-RasGAP317-326 is a RasGAP-derived cell-permeable peptide that acts as a sensitizer to various anti-cancer treatments in adult tumor cells. In the present study, we assessed the effect of TAT-RasGAP317-326 in several childhood cancer cell lines. The RasGAP-derived peptide-induced cell death was analyzed in several neuroblastoma, Ewing sarcoma and leukemia cell lines (as well as in normal lymphocytes). Cell death was evaluated using flow cytometry methods in the absence or in the presence of the peptide in combination with various genotoxins used in the clinics (4-hydroperoxycyclophosphamide, etoposide, vincristine and doxorubicin). All tested pediatric tumors, in response to at least one genotoxin, were sensitized by TAT-RasGAP317-326. The RasGAP-derived peptide did not increase cell death of normal lymphocytes, alone or in combination with the majority of the tested chemotherapies. Consequently, TAT-RasGAP317-326 may benefit children with tumors by increasing the efficacy of anti-cancer therapies notably by allowing reductions in anti-cancer drug dosage and the associated drug-induced side effects.

No MeSH data available.


Related in: MedlinePlus