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D-501036 [2,5-bis(5-hydroxymethyl-2-selenienyl)-3-hydroxymethyl-N-methylpyrrol], a novel selenophene derivative, is a highly potent cytotoxic agent with broad spectrum antitumor activity. The present study was undertaken to explore the mechanism(s) through which D-501036 exerts its action mode on the cancer cell death. D-501036 was found to suppress the growth of KB and HepG(2) cells in an irreversible manner. The results of annexin-V assays and PARP cleavage studies were consistent with the D-501036-induced apoptosis. Findings provided a strong support for the induction of mitochondria-mediated apoptosis by this drug. The examination of two canonical pathways of initiation caspases, those for caspases -8 and -9, revealed that caspase-9 protein and the activities of caspases -9 and -3 were increased in a dose- and time-dependent manner. The concentrations of Fas/Fas-L and procaspase-8 and the activity of caspase-8 were not altered. Furthermore, the mitochondrial membrane potential permeability and the release of cytochrome c to the cytosol were both increased by D-501036. The concentrations of the pro-apoptotic protein Bax and translocation of Bax from the cytosol to the mitochondria were increased in response to D-501036, whereas the concentrations of the anti-apoptotic protein Bcl-2 were decreased. Two DNA damage-related pro-apoptotic proteins, Puma and Noxa, were upregulated in a dose- and time-dependent manner. These pro-apoptotic and anti-apoptotic proteins are downstream effectors of p53. Accordingly, the phosphorylated and total forms of p53 were induced and p53 was translocated from the cytosol to the mitochondria in response to D-501036 treatment. Collectively, we conclude that D-501036 induces cellular apoptosis through the p53-associated mitochondrial pathway.

In an attempt to overcome the cardiotoxicity and cross-resistance problems caused by the anticancer drugs anthracyclines and anthracenediones during chemotherapy, we have developed a series of aza-anthracenedione compounds by modifying the chromophore and the side arms of anthracyclines and anthracenediones. One of these aza-anthracenediones, 6,9-bis[(2-aminoethyl)amino]benzo[g]isoquinoline-5,10-dione (BBR 2778), which is currently under phase II clinical trials, showed remarkable antitumor activity and appeared to lack a cardiotoxic effect in preclinical studies. However, it was still cross-resistant against multidrug resistance (MDR) cells expressing P-glycoprotein (P-gp). In contrast, another aza-anthracenedione, 6,9-bis[[2-(dimethylamino)ethyl]amino]benzo[g]isoquinoline-5,10-dione, which has side arm structures different from those of BBR 2778, was highly active against MDR cells. In this study, BBR 2778, BBR 2378, and an anthracenedione compound, 1,4-bis[(2-aminoethyl)amino]-5,8-dimethyl-9,10-anthracenedione, were used to assess the relationship between the chemical structures of these drugs and their interactions with DNA and P-gp. In addition, the biological and pharmacological influences of photoaffinity labeling were also studied for BBR 2778 and DEH. As the results indicate, the photolabeled analogs of BBR 2778 and DEH were less DNA-reactive and less cytotoxic. The more lipophilic compound, BBR 2378, and the photolabeled analogs of BBR 2778 and DEH inhibited P-gp labeling by azidopine better than did the more hydrophilic parental compounds. These studies suggested that the DNA binding affinity of BBR 2778 and DEH could be important in determining their cytotoxicity, and that the chemical structure of the side arms and the lipophilicity of these drugs are critical in determining their cross-resistance.

The anthrapyrazoles have entered clinical trials and show significant activity against breast cancer. However, these drugs are cardiotoxic and ineffective in multidrug-resistant (MDR) tumor cells. We have reported previously on the synthesis and antitumor characteristics of the 9-aza-anthrapyrazoles and their lack of cardiotoxicity; unfortunately, the leading candidates are cross-resistant in MDR-expressing cells. The results also indicated that the side arm structures of 9-aza-anthrapyrazole play a critical role in determining the drug resistance in MDR-expressing cells-only compounds that have a tertiary amine on both side arms are not cross-resistant. To further elucidate the biochemical and pharmacological impact of the side arm structures, one of the 9-aza-anthrapyrazole compounds, BBR 3422 [2-(2-aminoethyl)-5-(2-methylaminoethyl)indazolo[4,3-g,h]isoquinoline-6(2H)-one], was selected to be photolabeled with N-hydroxysuccinimidyl-4-azidosalicylic acid (NHS-ASA). In comparison to the parental compound, the photolabeled BBR 3422 was not as cytotoxic or DNA active, but it competed better than the parental compound against azidopine on P-glycoprotein labeling. In addition, confocal microscopic studies showed that BBR 3422 was clustered mainly in the cell nucleus, but its photolabeled analogue was located in the cytoplasm of the human breast cancer cell line MCF-7. Only a trace amount of both compounds was detected in the doxorubicin-derived resistant cell line MCF-7/ADR. The treatment of MCF-7/ADR cells with verapamil increased the intracellular amounts of both compounds.