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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 effect of TAT-RasGAP317-326 as a chemosensitizer of leukemia cells and non-tumor lymphocytes.A. Two acute myeloid leukemia cell lines (THP-1 and M-07e) and one T acute lymphoblastic leukemia cell line (CCRF-CEM) were seeded in 6-well plates and directly treated with 4-HC, etoposide, vincristine or doxorubicin at the indicated concentrations in the presence or in the absence of 10 μM TAT-RasGAP317-326. After 24 hours of drug incubation, 7-AAD or Annexin V-FITC staining was performed to evaluate cell death (last four columns). Alternatively (first column), the cells were treated with increasing concentrations of TAT or TAT-RasGAP317-326 alone. After 24 hours, the evaluation of cell death was carried out using 7-AAD staining. B. Isolated lymphocytes from three distinct healthy subjects were treated as described in Fig. 2A. The dosages of chemotherapeutic agents used to treat the healthy lymphocytes from the three subjects are similar to those used to treat the T-ALL CCRF-CEM cells. Note that the graphs are derived from single experiments where lymphocytes are immediately used after their isolation (if cultured in vitro, they would experience high levels of spontaneous apoptosis that would prevent accurate measurement of anti-cancer drug- and peptide-induced death). T-ALL, T-acute lymphoblastic leukemia; AML, acute myeloid leukemia; 4-HC, 4-hydroperoxycyclophosphamide. * p<0.05 t-test after Bonferroni correction.
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pone.0120487.g002: The effect of TAT-RasGAP317-326 as a chemosensitizer of leukemia cells and non-tumor lymphocytes.A. Two acute myeloid leukemia cell lines (THP-1 and M-07e) and one T acute lymphoblastic leukemia cell line (CCRF-CEM) were seeded in 6-well plates and directly treated with 4-HC, etoposide, vincristine or doxorubicin at the indicated concentrations in the presence or in the absence of 10 μM TAT-RasGAP317-326. After 24 hours of drug incubation, 7-AAD or Annexin V-FITC staining was performed to evaluate cell death (last four columns). Alternatively (first column), the cells were treated with increasing concentrations of TAT or TAT-RasGAP317-326 alone. After 24 hours, the evaluation of cell death was carried out using 7-AAD staining. B. Isolated lymphocytes from three distinct healthy subjects were treated as described in Fig. 2A. The dosages of chemotherapeutic agents used to treat the healthy lymphocytes from the three subjects are similar to those used to treat the T-ALL CCRF-CEM cells. Note that the graphs are derived from single experiments where lymphocytes are immediately used after their isolation (if cultured in vitro, they would experience high levels of spontaneous apoptosis that would prevent accurate measurement of anti-cancer drug- and peptide-induced death). T-ALL, T-acute lymphoblastic leukemia; AML, acute myeloid leukemia; 4-HC, 4-hydroperoxycyclophosphamide. * p<0.05 t-test after Bonferroni correction.

Mentions: To investigate the sensitization effect of TAT-RasGAP317-326 in leukemia cells, three different cell lines were subjected to increasing concentrations of cytostatic agents in the absence or in the presence of 10 μM TAT-RasGAP317-326. Etoposide, vincristine, doxorubicin, and 4-hydroperoxycyclophosphamide (4-HC), the active form of cyclophosphamide, were the drugs used here and are each currently employed in the clinic. Cell death was assessed using 7-AAD that labels dead cells that have permeabilized plasma membrane and with Annexin V that binds to phosphatidylserine exposed on dead cells. Some of the anti-cancer drugs used here induced a concomitant appearance of the 7-AAD and Annexin V signals (Fig 1A shows the representative case of 4-HC). In other words, as soon as a cell became Annexin V-positive it also picked up the 7-AAD dye. Hence, in our hands, anti-cancer drugs such as 4-HC induced a necrosis-like type of death. In the next figures, the 7-AAD data are reported as we found them to be associated with a lower variance than those obtained with Annexin V staining (Fig 1B). The only exception was when doxorubicin was used. Indeed, this dye fluoresces at similar wavelengths as 7-ADD. In this case therefore, the Annexin V data are presented. These experiments revealed that TAT-RasGAP317-326 significantly sensitizes leukemia cells to almost all tested drugs (Fig 2A). However, in some conditions (for example when THP-1 cells are treated with vincristine) tumor cells did not respond well to TAT-RasGAP317-326. A limited sensitization effect of the RasGAP-derived peptide is not necessarily a consequence of intrinsic resistance to a drug as the CCRF-CEM cell line, which is vincristine-resistant, was efficiently sensitized by TAT-RasGAP317-326. This point is of particular clinical relevance as it indicates that TAT-RasGAP317-326 can exert a sensitization effect in chemo-resistant cells.


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

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

The effect of TAT-RasGAP317-326 as a chemosensitizer of leukemia cells and non-tumor lymphocytes.A. Two acute myeloid leukemia cell lines (THP-1 and M-07e) and one T acute lymphoblastic leukemia cell line (CCRF-CEM) were seeded in 6-well plates and directly treated with 4-HC, etoposide, vincristine or doxorubicin at the indicated concentrations in the presence or in the absence of 10 μM TAT-RasGAP317-326. After 24 hours of drug incubation, 7-AAD or Annexin V-FITC staining was performed to evaluate cell death (last four columns). Alternatively (first column), the cells were treated with increasing concentrations of TAT or TAT-RasGAP317-326 alone. After 24 hours, the evaluation of cell death was carried out using 7-AAD staining. B. Isolated lymphocytes from three distinct healthy subjects were treated as described in Fig. 2A. The dosages of chemotherapeutic agents used to treat the healthy lymphocytes from the three subjects are similar to those used to treat the T-ALL CCRF-CEM cells. Note that the graphs are derived from single experiments where lymphocytes are immediately used after their isolation (if cultured in vitro, they would experience high levels of spontaneous apoptosis that would prevent accurate measurement of anti-cancer drug- and peptide-induced death). T-ALL, T-acute lymphoblastic leukemia; AML, acute myeloid leukemia; 4-HC, 4-hydroperoxycyclophosphamide. * p<0.05 t-test after Bonferroni correction.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120487.g002: The effect of TAT-RasGAP317-326 as a chemosensitizer of leukemia cells and non-tumor lymphocytes.A. Two acute myeloid leukemia cell lines (THP-1 and M-07e) and one T acute lymphoblastic leukemia cell line (CCRF-CEM) were seeded in 6-well plates and directly treated with 4-HC, etoposide, vincristine or doxorubicin at the indicated concentrations in the presence or in the absence of 10 μM TAT-RasGAP317-326. After 24 hours of drug incubation, 7-AAD or Annexin V-FITC staining was performed to evaluate cell death (last four columns). Alternatively (first column), the cells were treated with increasing concentrations of TAT or TAT-RasGAP317-326 alone. After 24 hours, the evaluation of cell death was carried out using 7-AAD staining. B. Isolated lymphocytes from three distinct healthy subjects were treated as described in Fig. 2A. The dosages of chemotherapeutic agents used to treat the healthy lymphocytes from the three subjects are similar to those used to treat the T-ALL CCRF-CEM cells. Note that the graphs are derived from single experiments where lymphocytes are immediately used after their isolation (if cultured in vitro, they would experience high levels of spontaneous apoptosis that would prevent accurate measurement of anti-cancer drug- and peptide-induced death). T-ALL, T-acute lymphoblastic leukemia; AML, acute myeloid leukemia; 4-HC, 4-hydroperoxycyclophosphamide. * p<0.05 t-test after Bonferroni correction.
Mentions: To investigate the sensitization effect of TAT-RasGAP317-326 in leukemia cells, three different cell lines were subjected to increasing concentrations of cytostatic agents in the absence or in the presence of 10 μM TAT-RasGAP317-326. Etoposide, vincristine, doxorubicin, and 4-hydroperoxycyclophosphamide (4-HC), the active form of cyclophosphamide, were the drugs used here and are each currently employed in the clinic. Cell death was assessed using 7-AAD that labels dead cells that have permeabilized plasma membrane and with Annexin V that binds to phosphatidylserine exposed on dead cells. Some of the anti-cancer drugs used here induced a concomitant appearance of the 7-AAD and Annexin V signals (Fig 1A shows the representative case of 4-HC). In other words, as soon as a cell became Annexin V-positive it also picked up the 7-AAD dye. Hence, in our hands, anti-cancer drugs such as 4-HC induced a necrosis-like type of death. In the next figures, the 7-AAD data are reported as we found them to be associated with a lower variance than those obtained with Annexin V staining (Fig 1B). The only exception was when doxorubicin was used. Indeed, this dye fluoresces at similar wavelengths as 7-ADD. In this case therefore, the Annexin V data are presented. These experiments revealed that TAT-RasGAP317-326 significantly sensitizes leukemia cells to almost all tested drugs (Fig 2A). However, in some conditions (for example when THP-1 cells are treated with vincristine) tumor cells did not respond well to TAT-RasGAP317-326. A limited sensitization effect of the RasGAP-derived peptide is not necessarily a consequence of intrinsic resistance to a drug as the CCRF-CEM cell line, which is vincristine-resistant, was efficiently sensitized by TAT-RasGAP317-326. This point is of particular clinical relevance as it indicates that TAT-RasGAP317-326 can exert a sensitization effect in chemo-resistant cells.

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