In mammalian cells, non receptor-mediated apoptosis occurs via the cytochrome c-dependent assembly of a ϳ700-kd apoptotic protease-activating factor 1 (Apaf-1)/ caspase-9 containing apoptosome complex. This initiates the postmitochondrial-mediated effector caspase cascade. We now show that receptor mediated transforming growth factor ␤ 1 (TGF-␤ 1 )-induced apoptosis in rat hepatoma cells is accompanied by processing and activation of caspases-2, -3, -7, and -8. Furthermore, we show that caspase activation is mediated via the release of cytochrome c and the oligomerization of Apaf-1 into an ϳ700-kd apoptosome complex. Similarly, in vitro activation of hepatoma cell lysates with 2-deoxyadenosine 5-triphosphate (dATP) results in the formation of the ϳ700-kd apoptosome complex, which recruits and processes caspases-3 and -7. Z-VAD.FMK [benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone], the pan-caspase inhibitor totally inhibits dATP-stimulated caspase activation but does not block the assembly of the large Apaf-1 containing apoptosome complex. However, the recruitment and subsequent processing of caspases-3 and -7 to the apoptosome is blocked. Similarly, in intact cells, although Z-VAD.FMK blocked TGF-␤ 1 -induced apoptosis, it did not prevent the oligomerization of Apaf-1 into the apoptosome. However, recruitment and processing of caspases-3 and -7 were prevented by Z-VAD.FMK. These data show that TGF-␤ 1 induces apoptosis via release of cytochrome c and activation of the Apaf-1 apoptosome complex, which initiates the caspase cascade. (HEPATOLOGY 2000;32:750-760.)

In our continued effort to identify selective MRP1 modulators, we have developed two novel templates, 3 and 4, through rational drug design by identifying the key pharmacophore interaction at the 7-position of the pyrrolopyrimidine template 1. Further synthesis and SAR work on these novel templates gave a number of potent MRP1 modulators with great selectivity against Pgp. Additional studies to reduce the CYP3A4 inhibition are also reported. Several compounds of these classes were subjected to in vivo xenograft studies and in vivo efficacies were demonstrated.

XR5944 (MLN944), a novel bis-phenazine, has demonstrated potent cytotoxic activity against a variety of murine and human tumour models. In the present study, the antitumour activity of XR5944 was investigated in combination with 5-fluorouracil (5-FU) or irinotecan in human colon carcinoma cell lines and xenografts. In vitro cytotoxicity of the combinations following exposure to the drugs sequentially or simultaneously was evaluated by the sulphorhodamine-B assay and interactions were determined using medianeffect analysis. Antagonism was observed (CI41) following exposure of HT29 cells simultaneously to XR5944 and 5-FU or SN38 (active metabolite of irinotecan). In contrast, sequential exposure of either combination in either order demonstrated at least an additive response (CIp1). At least an additive response was also observed with these combinations in HCT116 cells regardless of schedule. Antitumour activity in HT29 xenografts in nude mice was enhanced by sequential administration of 5-FU (65 mg kg À1 ) or irinotecan (CPT-11) (35 mg kg À1 ) 48 h before XR5944 (5, 10, or 15 mg kg À1 ) compared to single agent treatment at the same or higher doses. Administration of irinotecan (35 mg kg À1 ) and XR5944 (15 mg kg À1 ) just 30 min apart yielded similar efficacy to sequential administration 48 h apart. All combinations were well tolerated. These data suggest that combinations of XR5944 with irinotecan or 5-FU are of significant interest in the treatment of colon cancer.

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