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Coenzyme A-independent transacylase (CoA-IT) mediates the transfer of polyunsaturated fatty acids from the sn-2 position of a donor phospholipid to the sn-2 position of an acceptor lyso-phospholipid. We have characterized this activity in U937 cells, a human monocytic cell line. The microsomes of these cells contained CoA-IT activity which demonstrated a fatty acid preference for transferring arachidonic acid into exogenously added 1-alkyl-2-lyso-GPC. This enzymatic activity was optimum between pH 6.5 and 9, was heat labile and displayed an apparent Km for 1-alkyl-2-lyso-GPC of 0.4 microM. This activity was not dependent on Ca2+, Mg2+, CoA or ATP, was not inhibited by 2-mercaptoethanol nor by addition of product, 1-alkyl-2-acyl-GPC. The activity of this enzyme was not altered by differentiation of U937 cells towards the macrophage with Me2SO. Treatment of U937 cells with dexamethasone had no effect on transacylase activity. The activity of this enzyme was decreased by the serine esterase inhibitors phenylmethyl-sulfonyl fluoride and N-tosyl-L-phenylalanine chloromethyl ketone and by the histidine modifier diethyl pyrocarbonate, suggesting that CoA-IT may belong to a family of acyltransferase enzymes typified by LCAT. CoA-IT activity was not affected by compounds that affect PLA2 activity, such as quinacrine, aristolochic acid and arachidonic acid, suggesting a mechanism of action for CoA-IT different from classical, low molecular weight PLA2 enzymes. In conclusion, U937 cells contain CoA-IT activity and this study extends our previous knowledge of this enzyme by demonstrating the differences between CoA-IT and PLA2 enzymes and suggesting similarities between CoA-IT and LCAT.

A series of gold(I) coordination complexes including analogues of the antiarthritic agent auranofin 1 were evaluated for in vitro cytotoxic potency against both B16 melanoma cells and P388 leukemia cells and in vivo antitumor activity against P388 leukemia in mice. A number of the complexes showed potent cytotoxic activity in vitro and antitumor activity in vivo, with the phosphine-coordinated gold(I) thiosugar complexes demonstrating the greatest in vitro and in vivo activity. The data compiled for 63 complexes of the general structural formula LAuX provide the basis for the following observations: potent in vitro cytotoxic activity is observed for substituted (phosphine) gold complexes, lack of potency in vitro correlates well with lack of antitumor activity, potent cytotoxicity in vitro is not necessarily predictive of activity in vivo, in vivo antitumor activity is generally optimized by ligation of Au(I) with a substituted phosphine and a thiosugar.

Spirogermanium is a germanium containing azaspirane which has been shown to have activity in experimental models of cancer and immune dysfunction. A series of analogs of the parent compound were synthesized and evaluated in a number of in vitro and in vivo biological assays to define the structure-activity relationships of this class of compounds relative to their potential therapeutic activities. In a colony-forming assay using HT-29 human colon carcinoma cells various analogs in which carbon replaced germanium (e.g. carbon) retained the potent cytotoxic activity in vitro seen with spirogermanium. Increased cytotoxic potency within the group of carbon containing analogs was directly related to increase in the length of the alkyl group(s) attached to the carbon atom opposite the azaspirane ring structure. DNA and protein synthesis by HT-29 cells was inhibited by these compounds. However, inhibition occurred only at supralethal concentrations or after long exposure times with the drug. None of the azaspiranes demonstrated in vivo anti-tumor activity against P388 leukemia or ADJ-PC6 plasmacytoma. The effect of these compounds on macrophage cell function was evaluated in vitro by their ability to modulate superoxide (O2-) production by macrophages. Spirogermanium inhibited the production of O2- by activated macrophages with an IC50 of 5 microM. Although macrophage viability did not appear to be decreased at the respective IC50 concentrations, the rank order potency for the analogs in the O2- production assay was directly proportional to that measured for their cytotoxic potency in the HT-29 colony formation assay. The results demonstrate that, within this class of compounds, (1) potent biological activity does not require the presence of germanium in the structure; (2) in vitro cytotoxic activity does not appear to be a direct result of the inhibition of macromolecular synthesis, and (3) macrophage function can be modulated in vitro at non-cytotoxic concentrations. These results are discussed in context with the reported anti-tumor activity of spirogermanium and the potential anti-arthritic and immunomodulatory activity of this class of compounds.

We have previously reported the cytotoxicity and antitumor activity of bis(diphenylphosphino)ethane (DPPE) and a variety of its transition metal complexes. During studies of the chemistry of a gold complex of this group [(AuCl)2(DPPE)], it was observed that this complex readily underwent ring closure on reaction with DPPE to form the tetrahedral complex [Au(DPPE)2]+. Various counterion forms (e.g., Cl-) of this cation were isolated and were found to exhibit a remarkably high stability in solution. Evaluation of [Au(DPPE)2]Cl in mice bearing i.p. P388 leukemia demonstrated that the compound produced an average of 87% increase in life span at its maximally tolerated dose (2-3 mumol/kg/day for 5 days). Activity was also seen in i.p. M5076 reticulum cell sarcoma (60% increase in life span) and s.c. mammary adenocarcinoma 16/c. Modest activity was evident in i.p. B16 melanoma and L1210 leukemia. A subline of P388 leukemia resistant to cisplatin was not cross-resistant to [Au(DPPE)2]Cl. In addition, combination therapy of [Au(DPPE)2]Cl and cisplatin against i.p. P388 demonstrated an advantage over single-agent therapy. In vitro studies of [Au(DPPE)2]Cl showed that the compound: is cytotoxic to tumor cell lines; is only minimally inhibited in its cytotoxic activity by the presence of serum; produces DNA protein cross-links and DNA strand breaks in cells; and inhibits macromolecular synthesis with a preferential inhibitory effect on protein synthesis relative to DNA and RNA synthesis. 31P nuclear magnetic resonance spectroscopy indicated that the compound is stable in the presence of serum proteins, thiols, or disulfides and that it reacts with Cu(II) resulting in the formation of a Cu(I)DPPE complex. The results of these in vivo and in vitro experiments suggest that the contrasting pharmacological profile of [Au(DPPE)2]Cl with respect to other gold(I) phosphine complexes may be related to both the kinetic stability of the complex and its stability in the presence of thiols.

Bis(diphenylphosphine)ethane (DPPE) and its bis[chlorogold(I)] [DPPE(Au2Cl2)], and bis[trichlorogold(III)] [DPPE(Au2Cl6)], complexes have in vivo antitumor activity. To determine if interaction with metals in situ can play a role in the antitumor activity of DPPE, we have studied the effects of DPPE, DPPE(Au2Cl2), DPPE(Au2Cl6) and mixtures of DPPE with metal salts on in vitro and in vivo biological systems. The in vitro cytotoxic potencies of the two DPPE-gold complexes were approximately 10-fold greater than that of DPPE. In addition, the cytotoxic potency of DPPE was increased when incubated with cells in the presence of Au(III) and Cu(II) salts, whereas Mg(II), Zn(II), Mn(II), Fe(II), Co(II), and Cd(II) had no effect. The effects of DPPE, DPPE(Au2Cl2) and mixtures of DPPE and metal salts on the activity of a model enzyme system, DNA polymerase alpha were measured. While DPPE did not inhibit the activity of DNA polymerase alpha, the DPPE(Au2Cl2) complex and mixtures of DPPE and Cu(II) salts inhibited the activity of the enzyme. Consistent with the effects observed in vitro, coadministration of Cu(II) or Au(III) increased the in vivo potency of DPPE in mice bearing i.p. P388 leukemia. Fifteen other DPPE analogues were evaluated for in vivo antitumor activity and for the effect of Cu(II) on their in vitro cytotoxic potency; there was a relationship between the ability of Cu(II) to potentiate the cytotoxic activities of DPPE analogues and their having in vivo antitumor activity.

The ability of gold coordination complexes to bind to DNA and produce inter-strand cross-links in DNA was assessed in an assay system based on the fluorescence properties of the DNA intercalative dye, ethidium bromide. Results from these studies using a variety of gold(I) and gold(III) complexes suggest that the ability of gold complexes to bind to and produce inter-strand cross-links in DNA is not dependent on the oxidation state of gold in the complex but is influenced by the nature of the coordinating ligands. Those complexes in which the gold was ligated through one or more weakly coordinating ligands showed evidence for DNA binding. However, only those complexes with two or more of these relatively weak coordinating ligands produced inter-strand cross-links. Both the amount of binding to and cross-linking of DNA by these compounds were decreased by treatment of the gold-DNA complex with 2-mercaptoethanol and other thiol containing agents. As shown by agarose gel electrophoresis, 2-mercaptoethanol caused a dissociation of the gold-DNA complexes and a regeneration of closed circular superhelical pBR322 DNA. DNA strand breakage also resulted from treatment of a number of gold-DNA complexes with 2-mercaptoethanol; this was observed with the gold compounds which were shown to produce inter-strand cross-links in DNA. The amount of DNA strand breakage produced by treatment of gold-DNA complexes with 2-mercaptoethanol was influenced by the initial conformation of the DNA; gold-DNA complexes which resulted from the binding of gold compounds to covalently closed superhelical DNA were more sensitive to the breakage induced by 2-mercaptoethanol treatment than those complexes in which closed circular, relaxed DNA was used as substrate. The DNA breakage was not reduced in partially anaerobic conditions or by free-radical scavengers, suggesting that it is not mediated by oxygen. The results are discussed with respect to the potential for the interaction of gold complexes with intracellular DNA and chromatin and their biological implications.

The use of the human tumor cloning assay as a predictor of clinical response of human tumors to drugs is predicated on the hypothesis that the in vivo response of a tumor to a drug can be correlated with the in vitro response of cells derived from the tumor. To test this hypothesis, we utilized a murine tumor model in which the in vivo and in vitro responses of a tumor can be accurately and reproducibly compared. Drug activity was assessed in P388 leukemia with the standard in vivo antitumor assay (i.p. tumor/i.p. drug administration) and an in vitro assay wherein the ascites tumor cells are removed from mice, treated with a drug, and directly cloned in soft agar to measure clonogenic capacity. The response of P388 cells to analogues within four separate classes of antitumor agents, anthracyclines, anthraquinones, platinum(II) coordination complexes, and phosphinogold(I) complexes was evaluated. The clonogenic assay failed to discriminate between highly active in vivo antitumor agents and analogues with only marginal in vivo efficacy (i.e., doxorubicin and daunorubicin versus rhodomycins A and B, ametantrone versus NSC 276740, cisplatin versus transplatin, [Au(dppe)2]Cl versus [Au(depe)2]PF6. Furthermore, the in vitro clonogenic assay failed to detect carboplatin which was a highly active agent in vivo. The basis for these discrepancies was explored by a more detailed comparison of doxorubicin and rhodomycin B. In vivo or in vitro drug exposure with subsequent measurement of cell kill by the in vitro clonogenic and in vivo tumorigenic assay demonstrated that the in vitro assay overestimated the cytotoxic potency of the drugs relative to the tumorigenic assay. Treatment of tumors in vivo with doxorubicin at doses below the maximally tolerated dose in mice resulted in multiple log cell kill as measured in vitro or in vivo, whereas rhodomycin B was cytotoxic only at dose levels exceeding its maximally tolerated dose. The results indicate that a subset of tumor stem cells capable of forming colonies in soft agar are significantly more sensitive to the cytotoxic effects of anthracyclines than are in vivo tumorigenic stem cells. Cytotoxic potency as measured by an in vitro soft agar clonogenic assay is not an accurate predictor of in vivo antitumor efficacy even in a model in which ascites tumor cells are directly exposed to i.p. drug. The in vitro cytotoxicity assay is useful only as a nonselective prescreen and must be used in combination with other indicators of tumor cell selectivity and dose-limiting organ toxicity.

SK&F 102912 (mu-[1,2-bis(diphenylphosphino)ethane]bis[(1-thio-beta-D- glucopyranosato-S)gold(I)], [(Autg)2(dppe)]) has shown reproducible and significant activity in transplantable murine tumor models and represents a structurally unique class of antineoplastic agents. A number of in vitro studies were performed to elucidate the cellular pharmacology of this gold-containing complex. [(Autg)2(dppe)] is a potent cytotoxic agent in vitro as demonstrated by its ability to inhibit the clonogenic capacity of a variety of tumor cell lines following a brief exposure to the drug. Cell-cycle analysis using HL-60 cells showed that low concentrations (2 microM) of [(Autg)2(dppe)] induced an S-phase block and higher concentrations induced a secondary block at the G1/S boundary. [(Autg)2(dppe)] had several effects on DNA metabolism and structure including preferential inhibition in cells of DNA synthesis (relative to RNA and protein synthesis) and the production of DNA single- and double-strand breaks as measured by alkaline elution. The cytoxic mechanism of this gold complex appears to be distinct from that of the monophosphine-gold complex auranofin.

The interactions of certain gold(I) and gold(III) complexes with isolated plasmid pBR322 DNA were defined and compared to those of cis-diamminedichloroplatinum(II), CDDP, using an agarose gel electrophoresis assay. Trichloro(pyridine)gold(III) appeared to bind to DNA as evidenced by its ability to produce dose-dependent changes in the electrophoretic mobilities of closed circular, supercoiled, closed circular, relaxed, and open circular plasmid DNAs. These effects suggest that the gold containing complex induces conformational changes in the plasmid as a result of the compound binding to the DNA and the subsequent unwinding of the double helix and shorting of the DNA. Auranofin [(2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S)-triethyl phosphine gold(I)] did not appear to interact with DNA under any conditions. However, its analog chloro(triethylphosphine) gold(I) interacted with DNA at pH 9.5 in borate buffer and produced electrophoretic mobility changes in pBR322 DNA which were different from those produced by the gold(III) complexes that were evaluated. Binding of chloro(triethylphosphine) gold(I) was inhibited by the co-addition of the thiosugar portion of auranofin suggesting preferential binding of the gold moiety to thiosugar, which results in the production of auranofin (or a sugar containing) gold complex and inhibition of gold binding to DNA. The interactions of a number of gold compounds with DNA were also evidenced by their abilities to inhibit the binding of ethidium bromide to DNA. The results from these studies indicate that: gold containing complexes can bind to, and produce conformational changes in, DNA; gold(I) and gold(III) complexes may interact with DNA via different chemical mechanisms to produce different conformational changes in DNA; and certain coordinating ligands in gold complexes (e.g. Cl, Br and SCN) can be exchanged for binding sites on DNA by gold.

The coordinated gold compound, 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S-triethyl phosphine gold (auranofin; Ridaura), was evaluated for antitumor activity in a variety of mouse tumor models. Of the 15 tumor models evaluated, auranofin was found to be active only against i.p. P388 leukemia. A number of dose schedules was used to measure activity against P388 with optimal activity observed at 12 mg/kg given daily, i.p., on Days 1 to 5. Auranofin was active against i.p. P388 leukemia only when administered i.p.; the drug was completely inactive when administered i.v., s.c., or p.o. on Days 1 to 5. Evaluation of the effects of auranofin in vitro demonstrated that survival curves for B16 melanoma cells as measured by the clongenic and dye exclusion assays were exponential and monophasic; cell cycle distribution was not altered, and auranofin displayed no preferential cytotoxicity to logarithmic or plateau growth phase cell populations; auranofin inhibited DNA, RNA, and protein synthesis at cytotoxic concentrations but showed no selective effect; the cytotoxic activity and cellular association of gold from auranofin were dose, time, and temperature dependent; and binding of auranofin gold to serum proteins markedly decreased cellular uptake of gold and cytotoxicity of auranofin in vitro.

S49 cyc- lymphoma cells contain a mutation resulting in loss of a functional guanine nucleotide regulatory protein rendering their adenylate cyclase refractory to most stimuli. S49 wild-type and cyc- clones were used in the present study to investigate the possible association of altered cAMP metabolism with tumorigenicity and metastatic potential. The S49 clones were implanted i.v., i.p., and intracerebrally in both athymic nude mice and syngeneic, immunocompetent BALB/c mice. Both S49 clones gave rise to tumors when inoculated into athymic mice, and no differences were observed in the tumorigenicity or metastatic potential of S49 wild-type and cyc- cells. Implantation of S49 clones in syngeneic BALB/c mice gave rise to few tumors except when administered intracerebrally, where wild-type cells were more tumorigenic than cyc- cells. This raises the possibility of differences in immunogenicity between the S49 clones. Analysis of cell lines derived from tumors grown in athymic mice showed that they retained the phenotype of the S49 clones used for inoculations. The results indicate that, despite differences in adenylate cyclase responsiveness, S49 wild-type and cyc- cells are both highly tumorigenic and metastatic.