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Sustained antiproliferative mechanisms by RB24, a targeted precursor of multiple inhibitors of epidermal growth factor receptor and a DNA alkylating agent in the A431 epidermal carcinoma of the vulva cell line.

Banerjee R, Rachid Z, Qiu Q, McNamee JP, Tari AM, Jean-Claude BJ - Br. J. Cancer (2004)

Bottom Line: Recently, with the purpose of enhancing the potency of epidermal growth factor receptor (EGFR)-based therapies, we designed a novel strategy termed 'Cascade-release targeting' that seeks to develop molecules capable of degrading to multiple tyrosine kinase (TK) inhibitors and highly reactive electrophiles, in a stepwise fashion.The cascade degradation of RB24 requires the generation of two reactive electrophiles: (a) an iminium ion and (b) a methyldiazonium ion.Using the EGFR-overexpressing human epidermoid carcinoma of the vulva cell line, A431, we demonstrate herein that (a) RB24 and its derived species (e.g. RB14, ZR08) irreversibly inhibit EGFR autophosphorylation, (b) RB24 induced significant levels of DNA strand breaks, (c) sustained inhibition of EGFR by RB24 was associated with blockade of MAPK activation and c-fos gene expression, (d) RB24 induced irreversible cell growth inhibition with a 100-fold greater potency than Temodaltrade mark, a clinical methyltriazene.

View Article: PubMed Central - PubMed

Affiliation: Cancer Drug Research Laboratory, Department of Medicine, Division of Medical Oncology, McGill University Health Center/Royal Victoria Hospital, 687 Pine Avenue West, Rm. M 7.15, Montreal, Quebec, Canada H3A 1A1.

ABSTRACT
Recently, with the purpose of enhancing the potency of epidermal growth factor receptor (EGFR)-based therapies, we designed a novel strategy termed 'Cascade-release targeting' that seeks to develop molecules capable of degrading to multiple tyrosine kinase (TK) inhibitors and highly reactive electrophiles, in a stepwise fashion. Here we report on the first prototype of this model, RB24, a masked methyltriazene, that in addition to being an inhibitor on its own was designed to degrade to RB14, ZR08, RB10+a DNA alkylating methyldiazonium species. The cascade degradation of RB24 requires the generation of two reactive electrophiles: (a) an iminium ion and (b) a methyldiazonium ion. Thus, we surmise that these species could alkylate the active site of EGFR, thereby irreversibly blocking its action and that DNA damage could be induced by the methyldiazonium. Using the EGFR-overexpressing human epidermoid carcinoma of the vulva cell line, A431, we demonstrate herein that (a) RB24 and its derived species (e.g. RB14, ZR08) irreversibly inhibit EGFR autophosphorylation, (b) RB24 induced significant levels of DNA strand breaks, (c) sustained inhibition of EGFR by RB24 was associated with blockade of MAPK activation and c-fos gene expression, (d) RB24 induced irreversible cell growth inhibition with a 100-fold greater potency than Temodaltrade mark, a clinical methyltriazene. The pronounced growth inhibitory potency of RB24 was attributed to its ability to simultaneously damage DNA and irreversibly block EGFR TK activity.

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Effect of RB24 on Erk1,2 activation in A431 cells. Serum-starved cells were preincubated for 2 h with the indicated concentrations of RB24 prior to stimulation with EGF for 15 min. Protein lysates were obtained and Western blot was performed as described by Tari and Lopez-Berestein (2000).
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fig3: Effect of RB24 on Erk1,2 activation in A431 cells. Serum-starved cells were preincubated for 2 h with the indicated concentrations of RB24 prior to stimulation with EGF for 15 min. Protein lysates were obtained and Western blot was performed as described by Tari and Lopez-Berestein (2000).

Mentions: Antiproliferative activity induced by RB24 requires the translation of inhibition of EGFR autophosphorylation into inhibition of downstream signalling. To determine whether blockade of EGFR autophosphorylation translates into inhibition of downstream signalling, we analysed the effect of the parent compound, RB24, on EGF-induced phosphorylation of Erk1,2 and c-fos expression in A431 cells. The results showed that RB24 induced complete inhibition of Erk1,2 phosphorylation at concentrations as low as 5 μM without affecting the levels of Erk1,2 (Figure 3Figure 3


Sustained antiproliferative mechanisms by RB24, a targeted precursor of multiple inhibitors of epidermal growth factor receptor and a DNA alkylating agent in the A431 epidermal carcinoma of the vulva cell line.

Banerjee R, Rachid Z, Qiu Q, McNamee JP, Tari AM, Jean-Claude BJ - Br. J. Cancer (2004)

Effect of RB24 on Erk1,2 activation in A431 cells. Serum-starved cells were preincubated for 2 h with the indicated concentrations of RB24 prior to stimulation with EGF for 15 min. Protein lysates were obtained and Western blot was performed as described by Tari and Lopez-Berestein (2000).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747684&req=5

fig3: Effect of RB24 on Erk1,2 activation in A431 cells. Serum-starved cells were preincubated for 2 h with the indicated concentrations of RB24 prior to stimulation with EGF for 15 min. Protein lysates were obtained and Western blot was performed as described by Tari and Lopez-Berestein (2000).
Mentions: Antiproliferative activity induced by RB24 requires the translation of inhibition of EGFR autophosphorylation into inhibition of downstream signalling. To determine whether blockade of EGFR autophosphorylation translates into inhibition of downstream signalling, we analysed the effect of the parent compound, RB24, on EGF-induced phosphorylation of Erk1,2 and c-fos expression in A431 cells. The results showed that RB24 induced complete inhibition of Erk1,2 phosphorylation at concentrations as low as 5 μM without affecting the levels of Erk1,2 (Figure 3Figure 3

Bottom Line: Recently, with the purpose of enhancing the potency of epidermal growth factor receptor (EGFR)-based therapies, we designed a novel strategy termed 'Cascade-release targeting' that seeks to develop molecules capable of degrading to multiple tyrosine kinase (TK) inhibitors and highly reactive electrophiles, in a stepwise fashion.The cascade degradation of RB24 requires the generation of two reactive electrophiles: (a) an iminium ion and (b) a methyldiazonium ion.Using the EGFR-overexpressing human epidermoid carcinoma of the vulva cell line, A431, we demonstrate herein that (a) RB24 and its derived species (e.g. RB14, ZR08) irreversibly inhibit EGFR autophosphorylation, (b) RB24 induced significant levels of DNA strand breaks, (c) sustained inhibition of EGFR by RB24 was associated with blockade of MAPK activation and c-fos gene expression, (d) RB24 induced irreversible cell growth inhibition with a 100-fold greater potency than Temodaltrade mark, a clinical methyltriazene.

View Article: PubMed Central - PubMed

Affiliation: Cancer Drug Research Laboratory, Department of Medicine, Division of Medical Oncology, McGill University Health Center/Royal Victoria Hospital, 687 Pine Avenue West, Rm. M 7.15, Montreal, Quebec, Canada H3A 1A1.

ABSTRACT
Recently, with the purpose of enhancing the potency of epidermal growth factor receptor (EGFR)-based therapies, we designed a novel strategy termed 'Cascade-release targeting' that seeks to develop molecules capable of degrading to multiple tyrosine kinase (TK) inhibitors and highly reactive electrophiles, in a stepwise fashion. Here we report on the first prototype of this model, RB24, a masked methyltriazene, that in addition to being an inhibitor on its own was designed to degrade to RB14, ZR08, RB10+a DNA alkylating methyldiazonium species. The cascade degradation of RB24 requires the generation of two reactive electrophiles: (a) an iminium ion and (b) a methyldiazonium ion. Thus, we surmise that these species could alkylate the active site of EGFR, thereby irreversibly blocking its action and that DNA damage could be induced by the methyldiazonium. Using the EGFR-overexpressing human epidermoid carcinoma of the vulva cell line, A431, we demonstrate herein that (a) RB24 and its derived species (e.g. RB14, ZR08) irreversibly inhibit EGFR autophosphorylation, (b) RB24 induced significant levels of DNA strand breaks, (c) sustained inhibition of EGFR by RB24 was associated with blockade of MAPK activation and c-fos gene expression, (d) RB24 induced irreversible cell growth inhibition with a 100-fold greater potency than Temodaltrade mark, a clinical methyltriazene. The pronounced growth inhibitory potency of RB24 was attributed to its ability to simultaneously damage DNA and irreversibly block EGFR TK activity.

Show MeSH
Related in: MedlinePlus