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Differential efficacy of caspase inhibitors on apoptosis markers during sepsis in rats and implication for fractional inhibition requirements for therapeutics.

Méthot N, Huang J, Coulombe N, Vaillancourt JP, Rasper D, Tam J, Han Y, Colucci J, Zamboni R, Xanthoudakis S, Toulmond S, Nicholson DW, Roy S - J. Exp. Med. (2004)

Bottom Line: Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers.These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase.Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

View Article: PubMed Central - PubMed

Affiliation: Merck Frosst Centre for Therapeutic Research, Merck Research Laboratories, Montreal, Quebec, Canada H9H 3L1. nathalie_methot@merck.com

ABSTRACT
A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers. Inhibition of DNA fragmentation required substantially higher levels of caspase-3 attenuation than that required for blockade of other apoptotic events such as spectrin proteolysis and phosphatidylserine externalization. These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase. These findings suggest that putative caspase-independent apoptosis may be overestimated in some systems since blockade of spectrin proteolysis and other cell death markers does not accurately reflect the high degrees of caspase-3 inhibition needed to prevent DNA fragmentation. Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

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Effect of M867 dose titration on caspase-3 cleavage, αII-spectrin cleavage, and DNA fragmentation in CLP-induced septic rats. Rats that underwent CLP surgery were dosed by continuous i.v. infusion of either vehicle (n = 6) or M867 (n = 1 for each dose) for 24 h. (A) Percentage of residual αII-spectrin cleavage (circles) and DNA fragmentation (triangles) in each animal after administration of various doses of M867. Percentages were calculated from the average αII-spectrin and DNA fragmentation values obtained with the vehicle-treated animals. (B) Caspase-3 Western blotting on thymus extracts of animals dosed with M867.
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fig4: Effect of M867 dose titration on caspase-3 cleavage, αII-spectrin cleavage, and DNA fragmentation in CLP-induced septic rats. Rats that underwent CLP surgery were dosed by continuous i.v. infusion of either vehicle (n = 6) or M867 (n = 1 for each dose) for 24 h. (A) Percentage of residual αII-spectrin cleavage (circles) and DNA fragmentation (triangles) in each animal after administration of various doses of M867. Percentages were calculated from the average αII-spectrin and DNA fragmentation values obtained with the vehicle-treated animals. (B) Caspase-3 Western blotting on thymus extracts of animals dosed with M867.

Mentions: DNA fragmentation in cultured thymocytes is almost completely caspase-3 and ICAD dependent. Given the shift in potency that was observed between αII-spectrin and DNA cleavage in vitro, we asked whether a similar shift would be present in vivo during CLP-induced thymocyte apoptosis. To that end, a dose–response curve for αII-spectrin cleavage and DNA fragmentation was performed in septic rats, at M867 doses ranging from 0 to 10 mg/kg/h. M867 reduced αII-spectrin cleavage in a dose-dependent manner, with 50% inhibition at ∼0.5 mg/kg/h and an almost complete inhibition at 2 mg/kg/h (Fig. 4 A). Inhibition of DNA fragmentation was dose dependent but required larger amounts of drug (50% inhibition at ∼2 mg/kg/h; Fig. 4 A). Importantly, DNA fragmentation was almost abrogated by M867, indicating caspase-3 (and possibly caspase-7) dependency in this model. At all M867 doses, caspase-3 was processed equally well, evidence that this inhibitor did not prevent upstream events leading to caspase-3 activation (Fig. 4 B). Hence, the process of DNA fragmentation during experimental peritonitis is largely caspase-3 dependent. As is the case with cultured thymocytes, drug levels must be fourfold higher to inhibit DNA cleavage compared with αII-spectrin cleavage.


Differential efficacy of caspase inhibitors on apoptosis markers during sepsis in rats and implication for fractional inhibition requirements for therapeutics.

Méthot N, Huang J, Coulombe N, Vaillancourt JP, Rasper D, Tam J, Han Y, Colucci J, Zamboni R, Xanthoudakis S, Toulmond S, Nicholson DW, Roy S - J. Exp. Med. (2004)

Effect of M867 dose titration on caspase-3 cleavage, αII-spectrin cleavage, and DNA fragmentation in CLP-induced septic rats. Rats that underwent CLP surgery were dosed by continuous i.v. infusion of either vehicle (n = 6) or M867 (n = 1 for each dose) for 24 h. (A) Percentage of residual αII-spectrin cleavage (circles) and DNA fragmentation (triangles) in each animal after administration of various doses of M867. Percentages were calculated from the average αII-spectrin and DNA fragmentation values obtained with the vehicle-treated animals. (B) Caspase-3 Western blotting on thymus extracts of animals dosed with M867.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211770&req=5

fig4: Effect of M867 dose titration on caspase-3 cleavage, αII-spectrin cleavage, and DNA fragmentation in CLP-induced septic rats. Rats that underwent CLP surgery were dosed by continuous i.v. infusion of either vehicle (n = 6) or M867 (n = 1 for each dose) for 24 h. (A) Percentage of residual αII-spectrin cleavage (circles) and DNA fragmentation (triangles) in each animal after administration of various doses of M867. Percentages were calculated from the average αII-spectrin and DNA fragmentation values obtained with the vehicle-treated animals. (B) Caspase-3 Western blotting on thymus extracts of animals dosed with M867.
Mentions: DNA fragmentation in cultured thymocytes is almost completely caspase-3 and ICAD dependent. Given the shift in potency that was observed between αII-spectrin and DNA cleavage in vitro, we asked whether a similar shift would be present in vivo during CLP-induced thymocyte apoptosis. To that end, a dose–response curve for αII-spectrin cleavage and DNA fragmentation was performed in septic rats, at M867 doses ranging from 0 to 10 mg/kg/h. M867 reduced αII-spectrin cleavage in a dose-dependent manner, with 50% inhibition at ∼0.5 mg/kg/h and an almost complete inhibition at 2 mg/kg/h (Fig. 4 A). Inhibition of DNA fragmentation was dose dependent but required larger amounts of drug (50% inhibition at ∼2 mg/kg/h; Fig. 4 A). Importantly, DNA fragmentation was almost abrogated by M867, indicating caspase-3 (and possibly caspase-7) dependency in this model. At all M867 doses, caspase-3 was processed equally well, evidence that this inhibitor did not prevent upstream events leading to caspase-3 activation (Fig. 4 B). Hence, the process of DNA fragmentation during experimental peritonitis is largely caspase-3 dependent. As is the case with cultured thymocytes, drug levels must be fourfold higher to inhibit DNA cleavage compared with αII-spectrin cleavage.

Bottom Line: Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers.These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase.Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

View Article: PubMed Central - PubMed

Affiliation: Merck Frosst Centre for Therapeutic Research, Merck Research Laboratories, Montreal, Quebec, Canada H9H 3L1. nathalie_methot@merck.com

ABSTRACT
A rodent model of sepsis was used to establish the relationship between caspase inhibition and inhibition of apoptotic cell death in vivo. In this model, thymocyte cell death was blocked by Bcl-2 transgene, indicating that apoptosis was predominantly dependent on the mitochondrial pathway that culminates in caspase-3 activation. Caspase inhibitors, including the selective caspase-3 inhibitor M867, were able to block apoptotic manifestations both in vitro and in vivo but with strikingly different efficacy for different cell death markers. Inhibition of DNA fragmentation required substantially higher levels of caspase-3 attenuation than that required for blockade of other apoptotic events such as spectrin proteolysis and phosphatidylserine externalization. These data indicate a direct relationship between caspase inhibition and some apoptotic manifestations but that small quantities of uninhibited caspase-3 suffice to initiate genomic DNA breakdown, presumably through the escape of catalytic quantities of caspase-activated DNase. These findings suggest that putative caspase-independent apoptosis may be overestimated in some systems since blockade of spectrin proteolysis and other cell death markers does not accurately reflect the high degrees of caspase-3 inhibition needed to prevent DNA fragmentation. Furthermore, this requirement presents substantial therapeutic challenges owing to the need for persistent and complete caspase blockade.

Show MeSH
Related in: MedlinePlus