Transducible Peptide Therapy for Uveal Melanoma and Retinoblastoma

Abstract

Objective:To determine whether transducible peptides that inhibit the oncoproteins HDM2 and Bcl-2 may selectively kill uveal melanoma and retinoblastoma cells. Methods: Peptides were tested by viability assay, flow cytometry, TUNEL (terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labeling) assay, Western blot analysis, and reverse transcriptionpolymerase chain reaction in cultured eye tumor cells and normal cells. Preclinical studies were performed in a rabbit xenograft model of retinoblastoma. Main Outcome Measures: Cell survival, apoptosis, gene expression, and tumor regression.

PNC‐28, a p53‐derived peptide that is cytotoxic to cancer cells, blocks pancreatic cancer cell growth in vivo

“…Surprisingly, in contrast to the results of other studies using p53 peptides and peptidomimetics,5, 7, 8, 9, 10, 11 BMRPA1.Tuc3 cells treated with PNC‐28 do not express high levels of any of the protein markers for p53‐induced apoptosis and cell cycle arrest, including BAX, waf‐p21 and caspases 12, 13. The DNA from these cells is fragmented and does not form the “ladder pattern” typical of DNA from cells undergoing apoptosis 12.…”

“…Using 2D NMR, we have found that this peptide forms an amphipathic structure consisting of 2 alpha‐helices in the p53 peptide segment and the penetratin segment, separated by a loop, a motif that has been described for several membrane‐lytic peptides such as melittin and maganin 14, 15. These results all suggest that our peptides, in contrast to the agents that block p53–hdm‐2 interactions,5, 7, 8, 9, 10, 11 induce necrosis of cancer cells selectively using mechanisms that are largely independent of classical apoptotic pathways induced by activated p53 protein and its “surrogates” such as p63 and p73 proteins1 and that involve, at least in part, damage to cancer cell membranes.…”

PNC‐28, a p53‐derived peptide that is cytotoxic to cancer cells, blocks pancreatic cancer cell growth in vivo

“…Surprisingly, in contrast to the results of other studies using p53 peptides and peptidomimetics,5, 7, 8, 9, 10, 11 BMRPA1.Tuc3 cells treated with PNC‐28 do not express high levels of any of the protein markers for p53‐induced apoptosis and cell cycle arrest, including BAX, waf‐p21 and caspases 12, 13. The DNA from these cells is fragmented and does not form the “ladder pattern” typical of DNA from cells undergoing apoptosis 12.…”

“…Using 2D NMR, we have found that this peptide forms an amphipathic structure consisting of 2 alpha‐helices in the p53 peptide segment and the penetratin segment, separated by a loop, a motif that has been described for several membrane‐lytic peptides such as melittin and maganin 14, 15. These results all suggest that our peptides, in contrast to the agents that block p53–hdm‐2 interactions,5, 7, 8, 9, 10, 11 induce necrosis of cancer cells selectively using mechanisms that are largely independent of classical apoptotic pathways induced by activated p53 protein and its “surrogates” such as p63 and p73 proteins1 and that involve, at least in part, damage to cancer cell membranes.…”

Inhibition of the p53-hdm2 Interaction with Low Molecular Weight Compounds

“…Second, Vassilev et al findings reveal that p53-hdm2 inhibitors prevent tumor growth in vivo. Data obtained with peptides already suggested that p53-hdm2 inhibitors are active in vivo 9 and these new data reinforce these initial evidences. Third, the lack of toxicity of imidazolines even at high doses (200 mg/kg twice a day) suggests that the activation of the p53 pathway in healthy tissues does not lead to major toxicity.…”

MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation

“… 21 – 23 In the McEvoy et al . studies, 22 , 23 the failure to detect MDM2 protein was inconsistent with prior reports, 3 , 18 , 19 including the same group’s analysis of a representative tumor. 21 Furthermore, the failure to detect MDM2 RNA in microarray analyses 22 could be explained by an initially incorrect MDM2 3′ untranslated sequence designation in the NCBI RefSeq database (data not shown).…”

PNC‐28, a p53‐derived peptide that is cytotoxic to cancer cells, blocks pancreatic cancer cell growth in vivo

“…Surprisingly, in contrast to the results of other studies using p53 peptides and peptidomimetics,5, 7, 8, 9, 10, 11 BMRPA1.Tuc3 cells treated with PNC‐28 do not express high levels of any of the protein markers for p53‐induced apoptosis and cell cycle arrest, including BAX, waf‐p21 and caspases 12, 13. The DNA from these cells is fragmented and does not form the “ladder pattern” typical of DNA from cells undergoing apoptosis 12.…”

“…Using 2D NMR, we have found that this peptide forms an amphipathic structure consisting of 2 alpha‐helices in the p53 peptide segment and the penetratin segment, separated by a loop, a motif that has been described for several membrane‐lytic peptides such as melittin and maganin 14, 15. These results all suggest that our peptides, in contrast to the agents that block p53–hdm‐2 interactions,5, 7, 8, 9, 10, 11 induce necrosis of cancer cells selectively using mechanisms that are largely independent of classical apoptotic pathways induced by activated p53 protein and its “surrogates” such as p63 and p73 proteins1 and that involve, at least in part, damage to cancer cell membranes.…”

Inhibition of the p53-hdm2 Interaction with Low Molecular Weight Compounds

“…Second, Vassilev et al findings reveal that p53-hdm2 inhibitors prevent tumor growth in vivo. Data obtained with peptides already suggested that p53-hdm2 inhibitors are active in vivo 9 and these new data reinforce these initial evidences. Third, the lack of toxicity of imidazolines even at high doses (200 mg/kg twice a day) suggests that the activation of the p53 pathway in healthy tissues does not lead to major toxicity.…”

MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation

“… 21 – 23 In the McEvoy et al . studies, 22 , 23 the failure to detect MDM2 protein was inconsistent with prior reports, 3 , 18 , 19 including the same group’s analysis of a representative tumor. 21 Furthermore, the failure to detect MDM2 RNA in microarray analyses 22 could be explained by an initially incorrect MDM2 3′ untranslated sequence designation in the NCBI RefSeq database (data not shown).…”

PNC‐28, a p53‐derived peptide that is cytotoxic to cancer cells, blocks pancreatic cancer cell growth in vivo

“…Surprisingly, in contrast to the results of other studies using p53 peptides and peptidomimetics,5, 7, 8, 9, 10, 11 BMRPA1.Tuc3 cells treated with PNC‐28 do not express high levels of any of the protein markers for p53‐induced apoptosis and cell cycle arrest, including BAX, waf‐p21 and caspases 12, 13. The DNA from these cells is fragmented and does not form the “ladder pattern” typical of DNA from cells undergoing apoptosis 12.…”

“…Using 2D NMR, we have found that this peptide forms an amphipathic structure consisting of 2 alpha‐helices in the p53 peptide segment and the penetratin segment, separated by a loop, a motif that has been described for several membrane‐lytic peptides such as melittin and maganin 14, 15. These results all suggest that our peptides, in contrast to the agents that block p53–hdm‐2 interactions,5, 7, 8, 9, 10, 11 induce necrosis of cancer cells selectively using mechanisms that are largely independent of classical apoptotic pathways induced by activated p53 protein and its “surrogates” such as p63 and p73 proteins1 and that involve, at least in part, damage to cancer cell membranes.…”

Inhibition of the p53-hdm2 Interaction with Low Molecular Weight Compounds

“…Second, Vassilev et al findings reveal that p53-hdm2 inhibitors prevent tumor growth in vivo. Data obtained with peptides already suggested that p53-hdm2 inhibitors are active in vivo 9 and these new data reinforce these initial evidences. Third, the lack of toxicity of imidazolines even at high doses (200 mg/kg twice a day) suggests that the activation of the p53 pathway in healthy tissues does not lead to major toxicity.…”

MDM2 but not MDM4 promotes retinoblastoma cell proliferation through p53-independent regulation of MYCN translation

“… 21 – 23 In the McEvoy et al . studies, 22 , 23 the failure to detect MDM2 protein was inconsistent with prior reports, 3 , 18 , 19 including the same group’s analysis of a representative tumor. 21 Furthermore, the failure to detect MDM2 RNA in microarray analyses 22 could be explained by an initially incorrect MDM2 3′ untranslated sequence designation in the NCBI RefSeq database (data not shown).…”