Impact of the basic amine on the biological activity and intracellular distribution of an aza-anthrapyrazole: BBR 3422.

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.

VIP activates G(s) and G(i3) in rat alveolar macrophages and G(s) in HEK293 cells transfected with the human VPAC(1) receptor.

We have characterized vasoactive intestinal peptide (VIP) receptor/G-protein coupling in rat alveolar macrophage (AM) membranes and find that pertussis toxin treatment and antisera against G(alphai3) and G(alphas) reduce high-affinity (125)I-VIP binding, indicating that both G(alphas) and G(alphai3) couple to the VIP-receptor. The predominant VIP-receptor subtype in AM is VPAC(1) and we examined the G-protein interactions of the human VPAC(1) that had been transfected into HEK293 cells. VPAC(1) has a molecular mass of 56 kDa; GTP analogs reduced (125)I-VIP binding to this protein demonstrating that high-affinity binding of VIP to the receptor requires coupling to G-protein. Functional VIP/VPAC(1)/G-protein complexes were captured by covalent cross-linking and analyzed by Western blotting. The transfected human VPAC(1) receptor in HEK293 was found to be coupled to G(alphas) but not G(alphai) or G(alphaq). Furthermore, pertussis toxin treatment had no effect on VPAC(1)/G-protein coupling in these cells. These observations suggest that the G-proteins activated by VPAC(1) may be dependent upon species and cell type.

Synthesis and antitumor activities of 5-methyl-1- and 2-[[2-dimethylaminoethyl]amino]-aza-thiopyranoindazoles.

The synthesis of 1- and 2-substituted aza-benzothiopyranoindazoles has been accomplished. The comparisons of the in vitro antitumor activities of the 2-substituted analogues with the benzothiopyranoindazole chemotypes indicate that the positioning of the nitrogen atom at C-9 (9-aza analogue 4d) leads to a substrate with potent antitumor activity. The 1-substituted aza-benzothiopyranoindazoles, in comparison with the corresponding 2-substituted analogues, exhibit a much lower potency.

Collateral methotrexate resistance in cisplatin-selected murine leukemia cells.

Resistance to anticancer drugs is a major cause of failure of many therapeutic protocols. A variety of mechanisms have been proposed to explain this phenomenon. The exact mechanism depends upon the drug of interest as well as the tumor type treated. While studying a cell line selected for its resistance to cisplatin we noted that the cells expressed a > 25,000-fold collateral resistance to methotrexate. Given the magnitude of this resistance we elected to investigate this intriguing collateral resistance. From a series of investigations we have identified an alteration in a membrane protein of the resistant cell as compared to the sensitive cells that could be the primary mechanism of resistance. Our studies reviewed here indicate decreased tyrosine phosphorylation of a protein (molecular mass = 66) in the resistant cells, which results in little or no transfer of methotrexate from the medium into the cell. Since this is a relatively novel function for tyrosine phosphorylation, this information may provide insight into possible pharmacological approaches to modify therapeutic regimens by analyzing the status of this protein in tumor samples for a better survival of the cancer patients.

Collateral methotrexate resistance in cisplatin-selected murine leukemia cells

Resistance to anticancer drugs is a major cause of failure of many therapeutic protocols. A variety of mechanisms have been proposed to explain this phenomenon. The exact mechanism depends upon the drug of interest as well as the tumor type treated. While studying a cell line selected for its resistance to cisplatin we noted that the cells expressed a >25,000-fold collateral resistance to methotrexate. Given the magnitude of this resistance we elected to investigate this intriguing collateral resistance. From a series of investigations we have identified an alteration in a membrane protein of the resistant cell as compared to the sensitive cells that could be the primary mechanism of resistance. Our studies reviewed here indicate decreased tyrosine phosphorylation of a protein (molecular mass = 66) in the resistant cells, which results in little or no transfer of methotrexate from the medium into the cell. Since this is a relatively novel function for tyrosine phosphorylation, this information may provide insight into possible pharmacological approaches to modify therapeutic regimens by analyzing the status of this protein in tumor samples for a better survival of the cancer patients

Multiple mechanisms confer drug resistance to mitoxantrone in the human 8226 myeloma cell line.

Selection for in vitro drug resistance can result in a complex phenotype with more than one mechanism of resistance emerging concurrently or sequentially. We examined emerging mechanisms of drug resistance during selection with mitoxantrone in the human myeloma cell line 8226. A novel transport mechanism appeared early in the selection process that was associated with a 10-fold resistance to mitoxantrone in the 8226/MR4 cell line. The reduction in intracellular drug concentration was ATP-dependent and ouabain-insensitive. The 8226/MR4 cell line was 34-fold cross-resistant to the fluorescent aza-anthrapyrazole BBR 3390. The resistance to BBR 3390 coincided with a 50% reduction in intracellular drug concentration. Confocal microscopy using BBR 3390 revealed a 64% decrease in the nuclear:cytoplasmic ratio in the drug-resistant cell line. The reduction in intracellular drug concentration of both mitoxantrone and BBR 3390 was reversed by a novel chemosensitizing agent, fumitremorgin C. In contrast, fumitremorgin C had no effect on resistance to mitoxantrone or BBR 3390 in the P-glycoprotein-positive 8226/DOX6 cell line. Increasing the degree of resistance to mitoxantrone in the 8226 cell line from 10 to 37 times (8226/MR20) did not further reduce the intracellular drug concentration. However, the 8226/MR20 cell line exhibited 88 and 70% reductions in topoisomerase II beta and alpha expression, respectively, compared with the parental drug sensitive cell line. This decrease in topoisomerase expression and activity was not observed in the low-level drug-resistant, 8226/MR4 cell line. These data demonstrate that low-level mitoxantrone resistance is due to the presence of a novel, energy-dependent drug efflux pump similar to P-glycoprotein and multidrug resistance-associated protein. Reversal of resistance by blocking drug efflux with fumitremorgin C should allow for functional analysis of this novel transporter in cancer cell lines or clinical tumor samples. Increased resistance to mitoxantrone may result from reduced intracellular drug accumulation, altered nuclear/cytoplasmic drug distribution, and alterations in topoisomerase II activity.

Drug resistance results in alterations in expression of immune recognition molecules and failure to express Fas (CD95).

It is demonstrated that methotrexate/cisplatin-sensitive L1210 cells express low levels of major histocompatibility complex (MHC) class II relative to the high levels expressed on methotrexate (MTX)/cisplatin-resistant L1210/DDP cells. L1210 cells express cell-surface Fas, while the L1210/DDP cells express no cell-surface Fas. Expression of costimulatory molecules B7-1/B7-2 and Fas is increased on L1210 cells, but not L1210/DDP, in the presence of methotrexate or trimetrexate (TMTX). Therefore, a component of the mechanism of action of some anti-cancer agents may be to facilitate immune recognition and T cell-directed, Fas-induced cell death. Loss of cell-surface Fas expression and failure of Fas (CD95)-dependent apoptotic death has been observed when cells develop drug resistance. The defect in apoptosis can be overcome by anti-cancer agents or experimental manipulation that induce Fas expression on the drug-resistant cells.

Mechanisms of asbestos-induced nitric oxide production by rat alveolar macrophages in inhalation and in vitro models.

To evaluate the contribution of reactive nitrogen species to inflammation by asbestos, Fischer 344 rats were exposed to crocidolite or chrysotile asbestos by inhalation to determine whether increases occurred in nitric oxide (NO.) metabolites from alveolar macrophages (AMs). AMs from animals inhaling asbestos showed significant elevations (p < .05) in nitrite/nitrate levels which were ameliorated by NG-monomethyl-L-arginine (NMMA), an inhibitor of inducible nitric oxide synthase (iNOS) activity. Temporal patterns of NO. generation from AMs correlated with neutrophil influx in bronchoalveolar lavage samples after asbestos inhalation or bleomycin instillation, another model of pulmonary fibrosis. To determine the molecular mechanisms and specificity of iNOS promoter activation by asbestos, RAW 264.7 cells, a murine macrophage-like line, and AMs isolated from control rats were exposed to crocidolite asbestos in vitro. These cells showed increases in steady-state levels of iNOS mRNA in response to asbestos and more dramatic increases in both iNOS mRNA and immunoreactive protein after addition of lipopolysaccharide (LPS). After transfection of an iNOS promoter/luciferase reporter construct, RAW 264.7 cells exposed to LPS, crocidolite asbestos and its nonfibrous analog, riebeckite, revealed increases in luciferase activity whereas cristobalite silica had no effects. Studies suggest that NO. generation may be important in cell injury and inflammation by asbestos.

Modulation of methotrexate resistance by genistein in murine leukemia L1210 cells.

We have previously shown that methotrexate (MTX) transport is impaired in murine L1210 cells selected for cisplatin (DDP) resistance (L1210/DDP) and that the decreased MTX uptake may be due to an altered 66 kDa membrane protein. We have further hypothesized that tyrosine phosphorylation is necessary for the function of this protein. To determine the importance of tyrosine phosphorylation we studied the effect of genistein, a tyrosine kinase inhibitor, on methotrexate sensitivity, uptake and tyrosine phosphorylation of the 66 kDa protein in L1210/0 cells. After 5 h of treatment with 50 microM genistein, methotrexate uptake was decreased by nearly 50% and the cells were protected from methotrexate cytotoxicity. Immunoblotting of whole cell lysates with a phosphotyrosine monoclonal antibody demonstrated that genistein treatment decreased phosphorylation of the 66 kDa membrane protein. We concluded that phosphorylation of a 66 kDa protein may be critical for methotrexate transport and that genistein protects L1210/0 cells from methotrexate toxicity.

Synthesis and antitumor evaluation of 2,5-disubstituted-indazolo[4, 3-gh]isoquinolin-6(2H)-ones (9-aza-anthrapyrazoles).

The synthesis and antitumor evaluation of 2, 5-disubstituted-indazolo[4,3-gh]isoquinolin-6(2H)-ones (9-aza-APs) are described. The key intermediates in the synthesis are benz[g]isoquinoline-5,10-diones which are substituted at positions 6 and 9 with groups of different nucleofugacity for SNAr displacements. The initial displacement of fluoride by a substituted hydrazine leads to the pyrazole analogues. Substitution of the remaining leaving group by an amine or BOC-protected amines leads to the 9-aza-APs 12. These analogues were converted into their maleate or hydrochloride salts 13. In two cases, namely, 13x and 13z, sidearm buildup was also employed in the synthetic pathway. In vitro evaluation of 9-aza-APs against the human colon tumor cell line LoVo uncovered for most of the compounds a cytotoxic potency lower than that of DuP-941 or mitoxantrone and comparable to that of doxorubicin. Only analogues 13c, 13n, and 13ff were as cytotoxic as DuP-941. Interestingly, while DuP-941 was highly cross-resistant in the LoVo cell line resistant to doxorubicin (LoVo/Dx), the 9-aza-APs carrying a distal lipophilic tertiary amine moiety in both chains were capable of overcoming the MDR resistance induced in this cell line. The 9-aza-APs show outstanding in vivo antitumor activity against both systemic P388 murine leukemia and MX-1 human mammary carcinoma transplanted in nude mice. At their optimal dosages, congeners 13a-c, 13f, 13n, 13q, 13x, and 13dd were highly effective against P388 leukemia with T/C% of 200-381, while the T/C% value of DuP-941 was 147. In the MX-1 tumor model, 24 compounds elicited percentages of tumor weight inhibitions (TWI) ranging from 50% to 99%. Congeners 13d, 13k, 13l, 13x, 13z, and 13ee emerged as the most effective ones, with TWI% 96, simliar to that of DuP-941 (TWI% = 95). On the basis of their efficacy profile in additional experimental tumors and lack of cardiotoxicity in preclinical models, two congeners have surfaced as potential clinical candidates.

Increased oxidant resistance of alveolar macrophages isolated from rats repeatedly exposed to cadmium aerosols.

This study investigated potential mechanisms of oxidant resistance in alveolar macrophages (AM) isolated from Lewis rats exposed repeatedly to cadmium aerosols. Macrophages from Cd-adapted animals significantly greater resistance to oxidant-induced cytotoxicity than control cells when challenged with hydrogen peroxide in vitro. Elevations in glutathione peroxidase and glutathione reductase activities were associated with increased oxidant tolerance but catalase activity was unchanged. Metallothionein (MT) expression (protein and mRNA) was dramatically up-regulated in response to in vivo Cd exposure. A study using immunocytochemistry and in situ hybridization techniques revealed significantly heterogeneity in the expression of metallothionein by AMs. The percentage of AMs positive for MT (protein and mRNA) and the degree of MT expression within individual cells increased in response to additional Cd exposures. A putative state of activation was suggested by differences in size and number of inclusion bodies in macrophages from Cd-adapted animals and by secretion of a cytokine with interleukin-1-like characteristics. In summary, AMs from Cd-adapted animals are distinguished from control cells with respect to: (1) increased oxidant resistance, (2) secretion of cytokines, (3) elevations in enzymes associated with glutathione metabolism, and (4) up-regulation in metallothionein expression.

Correlation of altered tyrosine phosphorylation with methotrexate resistance in a cisplatin-resistant subline of L1210 cells.

Collateral resistance to cisplatin and methotrexate has been reported in several cell lines. A murine leukemia cell line (L1210/DDP) selected for cisplatin resistance also has been shown to be highly resistant to methotrexate. Of the mechanisms proposed for methotrexate resistance, only changes in methotrexate transport into the cells were found in an earlier report. Methotrexate enters mammalian cells via an active transport system. In the present study, we demonstrated that the transport into the cell may be impaired in the resistant cells due to altered tyrosine phosphorylation of a membrane protein with a molecular mass of 66 kDa. This alteration was manifested by altered tyrosine phosphorylation of the 66 kDa protein and may be an underlying modification that renders the cells resistant to methotrexate. These results suggest involvement of tyrosine phosphorylation in folate transport and methotrexate resistant in L1210/DDP cells.

Investigations on the mechanisms of methotrexate resistance in a cisplatin-resistant L1210 murine leukemia cell subline.

We report a murine leukemia cell variant (L1210/DDP), selected for cisplatin (DDP) resistance, to be cross-resistant to methotrexate (MTX). Cross-resistance of L1210 cells to DDP and MTX has been observed by others, and has also been recorded in P388 murine leukemia and SSC-25 human squamous carcinoma cells. We demonstrated that MTX resistance is not due to dihydrofolate reductase (DHFR) gene amplification, increased DHFR enzyme activity or decreased MTX binding to the target enzyme. Of the mechanisms commonly proposed for MTX resistance, only differences in transport were observed when comparing sensitive (L1210/0) and resistant (L1210/DDP) cells. Our results suggest that MTX resistance in L1210/DDP cells is due to altered methotrexate uptake.

Comparison of aza-anthracenedione-induced DNA damage and cytotoxicity in experimental tumor cells.

Aza-anthracenediones are a new class of anti-cancer drugs, which demonstrate promising in vitro and in vivo activity. Our laboratory has synthesized a variety of structural analogs in which we determined previously that the positioning of the nitrogen within the backbone, as well as sidearm modification, results in dramatic differences in the potency of cytotoxicity. We reported previously that although DNA reactivity appears to be a necessary component for mediating cell death, it is not sufficient for predicting cytotoxicity of the aza-anthracenediones. We have chosen three aza-anthracenediones (BBR 2828, BBR 2778 and BBR 2378) to investigate the importance of DNA strand breaks and/or protein-concealed DNA breaks induced by aza-anthracenediones. We determined in the present study that, while all three drugs cause DNA breaks as determined by alkaline and neutral elution, as well as KCl-SDS precipitation, these breaks do not correlate directly with their potency as cytotoxic compounds. Further, we found significant differences in the types of DNA breaks induced by these drugs. Finally, we report that the persistence of protein-DNA complexes induced by all three drugs was similar and, therefore, cannot account for differences in the potency of cytotoxicity of the aza-anthracenediones. Thus, we postulate that, while the total number of drug-induced protein-concealed DNA breaks is an important indicator of drug toxicity, it is possible that the actual nature of the breaks may differ among the aza-anthracenedione congeners, and it is these differences in the actual proteins present in the DNA breaks that differentiate between aza-anthracenediones.

Correlation of DNA reactivity and cytotoxicity of a new class of anticancer agents: aza-anthracenediones.

Doxorubicin and mitoxantrone are carboxyclic anti-cancer drugs that interact with DNA through intercalation. Our laboratory has synthesized a new series of anti-tumor agents, the aza-anthracenediones, which are structurally related to mitoxantrone but contain a heterocyclic, rather than a carbocyclic, chromophore. Both the in vivo and in vitro anti-tumor activities of these compounds were exquisitely sensitive to the positioning of the nitrogen atom within the heterocyclic backbone. Compounds having a 2-aza were 30- to 100-fold more potent than the 1-aza or the di-aza compounds against L1210 cells in vitro. When tested in vivo, the 2-aza-anthracenediones had marked anti-tumor activity, in some cases curative, whereas the 1-aza-anthracenediones had but minimal antitumor activity. To define the importance of the aza positioning on DNA reactivity, spectral shift and gel mobility assays were used. The spectral shift assay suggested that the 2-aza compounds reacted with DNA solely through intercalation whereas the 1-aza-anthracenediones, and mitoxantrone all reacted with DNA through intercalative and non-intercalative processes. The affinity of DNA binding was five to seven times greater for the 2-aza compounds compared to the 1-aza or the di-aza derivatives. The retardation of supercoiled pBR322 DNA mobility in agarose gel electrophoresis further suggested an intercalative type of DNA interaction. Differences in DNA interaction appear related to but can not completely account for differences in cytotoxicity of the aza anthracenediones.

Aza and diaza bioisosteric anthracene-9,10-diones as antitumor agents.

Synthetic routes to aza and diaza bioisosteres related to the anthracene-9,10-dione, mitoxantrone, have been developed. The antitumor properties of these chemotypes are compared with those exhibited by the corresponding carbocyclic analogues. The sensitivity of the expressed cytotoxicities on the position(s) of the nitrogen atom(s) are discussed in terms of potential cellular targets. Several analogues show potential for clinical evaluations.

A non-characteristic response of L1210 cells to lovastatin.

Isoprenylated proteins are believed to play an important role in DNA synthesis and subsequent cell cycle progression. Inhibition of the biosynthesis of these isoprenylated proteins results in a decrease in DNA synthesis and a characteristic G1 blockade of the cell cycle. This inhibition can be achieved by incubation of cells in the presence of a hydroxy-methylglutaryl-coenzyme A reductase inhibitor (lovastatin) and can be reversed by addition of exogenous mevalonate. When incubated in the presence of lovastatin, L1210 wild-type cells are not inhibited at G1 but rather progress to a G2/M blockade, which is reversible with exogenous mevalonate. Thymidine incorporation has also shown that DNA synthesis is occurring until this blockade is achieved. However, when L1210 cells resistant to cisplatin are incubated in the presence of lovastatin, the characteristic G1 cell cycle blockade and DNA synthesis inhibition occur. The understanding of this mechanism and the role that isoprenylated proteins play in the regulation of DNA synthesis and cell cycle progression may gain great insight into the abnormal control of the cancer cell.

6,9-Bis[(aminoalkyl)amino]benzo[g]isoquinoline-5,10-diones. A novel class of chromophore-modified antitumor anthracene-9,10-diones: synthesis and antitumor evaluations.

Synthetic procedures have been developed which lead to the 2-aza congeners 3 and several related N-oxides 4. The analogues 3 exhibited a wide range of in vitro cytotoxicity against L1210 leukemia, the human colon adenocarcinoma cell line LoVo, and the doxorubicin resistant LoVo/DX cell line. Selected analogues of 3 showed significant P388 antileukemic activity in mice with 3c exhibiting high activity. This activity was also retained in the related N-oxide 4a. These heterocyclic bioisosteric models are representative of the first anthracene-9,10-diones which display antileukemic activity comparable to mitoxantrone.

Increased systemic, but not regional, neopterin production following intraperitoneal administration of interleukin-2 and lack of effect of pterins upon the lymphokine-activated killer cell phenomenon.

Circulating neopterin is derived from monocytes and/or macrophages that produce it upon stimulation by interferon-gamma released from activated T cells. Neopterin production has been proposed as a marker of biological response in the clinical administration of a number of cytokines. Changes in neopterin production as indicated by urinary neopterin excretion were studied in four patients with ovarian carcinoma receiving intraperitoneal interleukin-2 and lymphokine-activated killer cells. Neopterin production increased approximately threefold during treatment with interleukin-2 at doses which represent or exceed the maximum tolerated dose by this route of administration. Increased neopterin apparently was derived from systemic, not regional, tissues. The physiologic role(s) of pterins in immune responses is uncertain. In an in vitro system, the presence of neopterin or tetrahydrobiopterin or the pterin synthesis inhibitor, N-acetyl serotonin, did not modulate cytotoxic effects of lymphokine-activated killer cells.