Enzymatic mechanism of the tumoricidal action of 4-iodo-3-nitrobenzamide

Abstract

Abstract. activation of the prodrug 4-iodo-3-nitrobenzamide critically depends on the cellular reducing system specific to cancer cells. in non-malignant cells, reduction of this prodrug to the non-toxic amine occurs by the flavoprotein of complex I of mitochondria receiving Mg 2+ -aTP-dependent reducing equivalents from nadH to nadPH via pyridine nucleotide transhydrogenation. This hydride transfer is deficient in malignant cells; therefore, the lethal synthesis of 4-iodo-3-nitrosobenzamide takes place selectively. enzymatic evidence for this mechanism has been provided by cellular studies with lysolecithin-permeabilized cells and cell fractions, which have identified the defect in transhydrogenation in neoplastic cells to be located at the energy transfer site. Confirming previous results, the present study demonstrates the validity of the selective tumoricidal action of the prodrug in cell cultures.

Regulation of malignant phenotype and bioenergetics by a π-electron donor-inducible mitochondrial MgATPase

“…The MgATP-driven NADH➝NADP + transhydrogenation can be assayed by either added AcPyrNADH or metabolically generated cellular NADH as a H donor (14). In the latter assay glutamate (5 nmol/mg mitochondrial protein) (15) was found to be the universal substrate that reduced NAD + present in the Su fraction at a 1 μM concentration.…”

“…Each induced band was extracted following the protocol of the MS laboratory and analyzed by MALDI-TOF for amino acid sequences at the UCSF Sandler Moore Mass Spectrometry Core facility, acknowledging support from NIH/NCI Cancer Center support grant (P30CA82103), the Sandler Family Foundation and the Gordon and Betty Moore Foundation. Spectrophotometric assays were performed as previously reported (14). The 1% Brij 58 extract of the permeabilized cells was used; i.e.…”

“…Cellular proteins were analyzed as reported (4). CV-1 and E-ras 20 cells were cultured as referenced in this paper, and LL-permeabilization was performed as reported (14). For protein analyses of Eπ cells logarithmically growing E-ras 20 cells (10 6 cells exposed to 200 μM AI for 5 days) were fractionated into soluble (Su) and particulate (Mx) by the LL technique (14).…”

“…CV-1 and E-ras 20 cells were cultured as referenced in this paper, and LL-permeabilization was performed as reported (14). For protein analyses of Eπ cells logarithmically growing E-ras 20 cells (10 6 cells exposed to 200 μM AI for 5 days) were fractionated into soluble (Su) and particulate (Mx) by the LL technique (14). The Mx fraction was resuspended in LL buffer (500 μl) for 5 min, then washed by sedimentation at 3000 rpm (Eppendorf microfuge) at 4˚C for 5 min and resuspended in 200 μl LL buffer containing 1% Brij 58, kept on ice for 5 min, then sedimented at 12,000 rpm.…”

“…A comparison of anti-HIV effect of nitrosogroup containing molecules with their prodrug, i.e. the nitrosubstituted species, demonstrates that retroviral infection induces cellular reduction of nitro-to the nitroso-species (28) a reaction which specifically exists in tumor cells (14). Retroviral infection therefore, induces deletion of π-ATPase and converts cells to the malignant phenotype that is susceptible to the tumoricidal prodrug (28).…”