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Aberrant T-cell factor (TCF) transcription is implicated in the majority of colorectal cancers (CRCs). TCF transcription induces epithelial-mesenchymal transition (EMT), promoting a tumor-initiating cell (TIC) phenotype characterized by increased proliferation, multidrug resistance (MDR), invasion and metastasis. The data presented herein characterize topoisomerase IIα (TopoIIα) as a required component of TCF transcription promoting EMT. Using chromatin immunoprecipitation (ChIP) and protein co-immunoprecipitation (co-IP) studies, we show that TopoIIα forms protein-protein interactions with ß-catentin and TCF4 and interacts with Wnt response elements (WREs) and promoters of direct target genes of TCF transcription, including: MYC, vimentin, AXIN2 and LEF1. Moreover, both TopoIIα and TCF4 ChIP with the N-cadherin promoter, which is a new discovery indicating that TCF transcription may directly regulate N-cadherin expression. TopoIIα N-terminal ATP-competitive inhibitors, exemplified by the marine alkaloid neoamphimedine (neo), block TCF activity in vitro and in vivo. Neo effectively inhibits TopoIIα and TCF4 from binding WREs/promoter sites, whereas protein-protein interactions remain intact. Neo inhibition of TopoIIα-dependent TCF transcription also correlates with significant antitumor effects in vitro and in vivo, including the reversion of EMT, the loss of TIC-mediated clonogenic colony formation, and the loss of cell motility and invasion. Interestingly, non-ATP-competitive inhibitors of TopoIIα, etoposide and merbarone, were ineffective at preventing TopoIIα-dependent TCF transcription. Thus, we propose that TopoIIα participation in TCF transcription may convey a mechanism of MDR to conventional TopoIIα inhibitors. However, our results indicate that TopoIIα N-terminal ATP-binding sites remain conserved and available for drug targeting. This article defines a new strategy for targeted inhibition of TCF transcription that may lead to effective therapies for the treatment of CRC and potentially other Wnt-dependent cancers.

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A new tripeptide, pre-sclerotiotide F (3), was isolated from a marine sediment-derived fungus, Aspergillus insulicola, along with five known compounds, one of which was new at the time of isolation, scerotiotide F (4). The absolute configuration elucidation of the new compound was determined using a combination of NMR, HR-ESI-MS, and optical rotation analyses. Cytotoxicities were measured in vitro against selected cancer cells. The effects of pre-sclerotiotide F (3) and sclerotiotide F (4) on LPS-induced NF-κB and iNOS expression were also measured.

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A series of glaucarubinone analogues, obtained from natural sources as well as synthesized by us, were studied both in vitro and in vivo. The focus of the in vitro assessment was to define solid tumor-selective compounds by quantitating differential cytotoxic activity between murine and human solid tumor cells and either murine leukemia or normal cells. Subsequent in vivo studies were aimed at determining the therapeutic efficacy of these analogues against the murine models. Structure-activity analysis consequent to both the in vitro and in vivo studies demonstrated that few changes could be made in the parent glaucarubinone structure (outside of the C-15 position) without abrogating either cytotoxicity or potency. However, significant changes could be made at the C-15 position which modified, either enhanced or diminished, in vitro differential cytotoxicity, potency, human solid tumor selectively, and differential cytotoxicity to a MDR-expressing murine mammary tumor.

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Lyngbyastatin 1 (1a), a new cytotoxic analogue of dolastatins 12 (2a) and 11 (4), was isolated as an inseparable mixture with its C-15 epimer (1b) from extracts of a Lyngbya majuscula/Schizothrix calcicola assemblage and a L. majuscula strain collected near Guam. Dolastatin 12 (2a) was also encountered as an inseparable mixture with its C-15 epimer (2b) in L. majuscula/S. calcicola assemblages. At least one of the compounds in each mixture appeared to exist in solution as a mixture of slowly interconverting conformers resulting in broadened signals in 1H NMR spectra. Structure elucidation therefore relied principally on mass spectroscopy and chemical degradation studies. Both 1ab and 2ab proved toxic with only marginal or no antitumor activity when tested against colon adenocarcinoma #38 or mammary adenocarcinoma #16/C. Both 1ab and 2ab were shown to be potent disrupters of cellular microfilament networks.

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A new solid tumor selective cytotoxic analogue of dolastatin 10 (1) has been isolated from the marine cyanobacterium Symploca hydnoides, collected near Guam. This metabolite has been assigned the trivial name symplostatin 1 (2). This discovery supports the proposal that many compounds isolated from the seahare Dolabella auricularia, the original source of the dolastatins, are of dietary origin.

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Cryptophycin (CP) is a newly developed anticancer agent isolated from the terrestrial cyanobacteria of the genus Nostoc. CP is a mitotic inhibitor, causing cells to accumulate in mitosis with the disappearance of intracellular microtubules. In this report, we studied the interaction and uptake of a new synthetic CP analog, CP-52, with 2 human tumor cell lines, THP-1 and H-125. In vitro colony-forming assay showed that CP-52 has antiproliferative activity against THP-1 and H-125 cell lines with IC50 of 0.1 ng/ml and 20 microg/ml, respectively; i.e., THP-1 cells are 200,000 times more sensitive to CP-52 than H-125 cells. The uptake of CP-52 by the target cells was carried out using tritiated CP-52 (3H-CP-52). The uptake of 3H-CP-52 by both THP-1 and H-125 cells was rapid, reaching a maximum within 20 min. Dissociation experiments showed that CP-52 interacts with the target cells irreversibly, presumably by binding to specific cellular sites with high affinity. With increasing doses of 3H-CP-52, the uptake was found to be saturable, reaching a steady state as the concentrations of 3H-CP-52 were raised to about 20 microg/ml. Under this condition, the maximal values of CP-52 uptake by THP-1 and H-125 cells was estimated to be 27 and 136 ng/10(5) cells, respectively. The uptake and accumulation of 3H-CP-52 with the target cells was effectively inhibited by prior treatment with unlabeled CP-52 and, to a lesser extent, vinblastine and taxol but not adriamycin, colchicine or mitomycin. In addition, the binding of 3H-CP-52 to purified tubulin was inhibited by vinblastine but not taxol. This finding suggested that CP-52 and taxol interact and bind to distinct regions of tubulin molecules. Further, it suggests that, in addition to tubulin, other intracellular and/or membrane components are involved in mediating the binding of CP-52.

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This study was directed at defining the optimal in vitro screening methodology for the selection of new anti-myelogenous leukemia agents. Using thousands of samples of synthetic compounds from the Eastman Pharmaceutical, Inc. and Sterling Research Corporation (Eastman/Sterling) inventory, a number of sequential combinations of 8 cell lines were employed to optimize the model. The cells in these lines were of either murine (L1210 lymphocytic leukemia, C1498 myelogenous leukemia, and colony-forming unit-granulocyte macrophage [CFU-GM]) or human (HL-60, K-562, HEL, and THP-1 myelogenous leukemias, and CFU-GM) origin. The focus of the study was to find the most efficient and cost-effective manner in which to test compounds for both cytotoxicity against the tumor cells and for differential cytotoxicity against tumor as compared with normal cells.

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The marine sponge Diacarnus cf. spinopoculum has provided a series of norterpenes, including five new compounds (7-11), two new ent-compounds [(-)-1a and (+)-1b], and three known compounds (2a, 2b, and 12). Eight of these compounds represent additional examples of the muqubilin/sigmosceptrellin classes (norsesterterpene peroxides) or the nuapapuin class (norditerpene peroxides). Also isolated were dinorditerpenones 11 and 12, which are biosynthetically related to the muqubilin/sigmosceptrellin structure classes. In all, 11 compounds were evaluated for their cytotoxic properties using a soft agar assay system and the NCI's 60 cell-line screen. Compounds without peroxide functionality were inactive. Overall, the norsesterterpene peroxides were less selective as cytotoxins than norditerpene peroxide analogues. Two compounds, nuapapuin A methyl ester (3) and nuapapuin B (7), which were somewhat selective in their cytotoxic behavior, were selected for further in vivo evaluation.

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Cryptophycin 1 is a natural product that was initially isolated from blue-green algae which has shown potent broad spectrum antitumor activity in preclinical in vitro and in vivo models. The drug strongly binds to tubulin and disrupts microtubule assembly for more than 24 hours after its removal. We evaluated cell survival, intracellular levels and inhibition of macromolecular synthesis in L1210 cells following exposure to cryptophycin 1 in vitro. Cell survival was strongly inhibited following drug exposure for either 1 or 4 hours. Intracellular drug levels were minimally affected by temperature (4 degrees C versus 37 degrees C) or exposure times up to 1 hour. However, extracellular drug concentration in culture media and increasing cell numbers did affect the concentration of intracellular drug levels in a nearly proportional manner. The synthesis of DNA and RNA was inhibited less than 5%, while protein synthesis inhibition was near 30%. Thus, none of the macromolecules were inhibited enough to explain the inhibition of tumor cell growth.

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Historically, many new anticancer agents were first detected in a prescreen; usually consisting of a molecular/biochemical target or a cellular cytotoxicity assay. The agent then progressed to in vivo evaluation against transplanted human or mouse tumors. If the investigator had a large drug supply and ample resources, multiple tests were possible, with variations in tumor models, tumor and drug routes, dose-decrements, dose-schedules, number of groups, etc. However, in most large programs involving several hundred in vivo tests yearly, resource limitations and drug supply limitations have usually dictated a single trial. Under such restrictive conditions, we have implemented a flexible in vivo testing protocol. With this strategy, the tumor model is dictated by in vitro cellular sensitivity; drug route by water solubility (with water soluble agents injected intravenously); dosage decrement by drug supply, dose-schedule by toxicities encountered, etc. In this flexible design, many treatment parameters can be changed during the course of treatment (e.g., dose and schedule). The discovery of two active agents are presented (Cryptophycin-1, and Thioxanthone BCN 183577). Both were discovered by the intravenous route of administration. Both would have been missed if they were tested intraperitoneally, the usual drug route used in discovery protocols. It is also likely that they would have been missed with an easy to execute fixed protocol design, even if injected i.v.

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Both the PC-3 and the TSU-PR1 prostate tumor models were found to be satisfactory for chemotherapeutic investigations in ICR-SCID mice. The 30 to 60 mg fragments implanted took in all mice (as judged by 100% takes in the controls of all experiments as well as the passage mice). The tumor volume doubling time was 4.0 days for PC-3 and 2.5 days for TSU-PR1. Nine agents were evaluated IV against early stage subcutaneous PC-3 tumors, with Nano-piposulfan being the only agent highly active (4.9 log kill). Three other agents were moderately active: Taxol (1.5 log kill), Cryptophycin-8 (1.6 log kill), Vinblastine (1.0 log kill). Five agents were inactive: VP-16, Adriamycin, CisDDPt, 5-FUra, and Cyclophosphamide. Ten agents were evaluated IV against early stage subcutaneous TSU-PR1 tumors. Three agents were highly active, producing > 6 log kill and cures: Taxol (5/5 cures), Cryptophycin-8 (5/5 cures), Vinblastine (2/4 cures). Two other agents were moderately active: Nano-piposulfan (1.2 log kill), and Cyclophosphamide (1.1 log kill). Five agents were inactive: VP-16, Adriamycin, CisDDPt, 5-FUra, and BCNU. In part, activity was determined by the ability of the SCID mice to tolerate meaningful dosages of the agents. Agents producing granulocyte toxicity (e.g., Adriamycin) were poorly tolerated and appeared less active than expected. Vinblastine, producing little or no granulocyte toxicity was very well tolerated and appeared to be more active than expected.

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A randomized intervention trial of dietary fat reduction to 15% of total calories was initiated in 1987 for women at high risk for breast cancer to determine the feasibility of recruiting and maintaining them on a low-fat diet. The study has enrolled 194 women between the ages of 18 and 67 years who met at least one of three eligibility criteria: 1) a first-degree relative with breast cancer, 2) a P2 or DY Wolfe mammographic pattern, and 3) a prior breast biopsy demonstrating epithelial hyperplasia with or without atypia. Eligible women must also have had diets that contained > or = 30% of calories from fat at entry. Women were randomized to a nonintervention usual diet vs. a 15% low-fat diet. Recruitment was sought through physicians, personal mailings, breast cancer patients, and the news media. Two study sites participated: a large urban hospital affiliated with a university medical center and a community oncology private practice. The results from both institutions were similar and demonstrated that a low-fat dietary plan could be effectively conducted in private as well as academic settings with recruitment tailored to the community where the trial is being conducted. Reduction in dietary fat intake was maximal during the first three months of the dietary intervention and remained stable throughout 12 months of follow-up. Reductions in total calories, weight loss, and percent body fat were minimal. The nonintervention group experienced no major change in their diet. We conclude that it is feasible to recruit women who are at high risk for breast cancer into a dietary intervention trial and with sufficient dietary counseling and motivation on the part of participants, reduction in dietary fat intake can be achieved and maintained. More in-depth analyses of these data will be presented in subsequent reports.

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The testing of new human leukemia-specific drugs for activity against primary acute myeloid leukemia (AML) blasts is severely limited by the low and variable clonogenic potential of primary human leukemias in culture. To circumvent this problem, we have modified a previously described flow cytometric approach to permit the simultaneous determination of live/dead cells, and the quantitation of the surviving cell fraction as a ratio of viable cells in treatment and control groups. The method utilizes the combination of calceinAM as a probe for intracellular esterase activity (green fluorescence,) which has the advantage over carboxyFDA of pH insensitivity and superior signal-to-noise ratio, and propidium iodide (red fluorescence) as an indicator of plasma membrane integrity. Suspension cultures of AML blood and marrow samples from patients were treated with known active agents as well as several new agents arising from a clonogenic disk assay screen. Quantitative dose-response values obtained from surviving cell fractions assayed by flow cytometry at 24 h following drug exposure demonstrated the utility of this assay for quantitating drug-induced cytotoxic effects on primary human AML cells in short-term culture.

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Cryptophycin-8 was prepared by the conversion of the epoxide group on cryptophycin-1 to a chlorohydrin. In the studies reported here, cryptophycin-8 was evaluated for preclinical activity against subcutaneous tumors of both mouse and human origin. At the highest non-toxic single course treatment, the following results were obtained (Table A). Cryptophycin-8 was less potent than cryptophycin-1 by approximately 4-fold; however, it was both more water soluble and had greater therapeutic efficacy, as demonstrated by % T/C, tumor cell log kill values, range of dose effectiveness and host cures.

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AML-193 is a cytokine-dependent human leukemia cell line established from the bone marrow of an M5-type acute monocytic leukemia (AML) patient. The effect of recombinant human interleukin-6 (rhIL-6) on the proliferation of AML-193 cells was investigated. Both granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and rhIL-3 promoted the DNA synthesis and growth of AML-193 cells in vitro. rhIL-6 alone did not support the growth of AML-193 cells, yet pretreatment of AML-193 cells with rhIL-6 markedly enhanced their proliferative response to subsequent rhGM-CSF or rhIL-3 stimulation. The growth-promoting effect induced by rhIL-6 was attributable in part to the upregulation of GM-CSF receptors on AML-193 cells; treatment of AML-193 cells with rhIL-6 for 24 to 48 hours greatly increased their GM-CSF binding activity, which occurred in a dose-dependent manner. Both the growth-promoting and receptor-upregulating effects induced by rhIL-6 could be blocked by treating AML-193 cells with neutralizing anti-gp130 antibodies (GPX7). Treatment of AML-193 cells with anti-gp130 antibodies alone also led to a notable decline in GM-CSF binding activity, suggesting a possible role of gpl30 in regulating the expression of GM-CSF receptors. When AML-193 cells were starved in cytokine-free medium and then restimulated with rhGM-CSF, a rapid increase (5 minutes) in lyn kinase activity was observed. A similar upregulation of lyn kinase activity by rhIL-6 treatment also was noted in AML-193 cells, but only after a prolonged incubation of the cells with rhIL-6 (>24 hours). These findings show that the growth-promoting effects of rhIL-6 are mediated through the upregulation of GM-CSF receptors on AML-193 cells by mechanisms that appear to involve the activation of both gp130 and lyn kinase.

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The in vitro testing of antitumor drugs involves the use of mouse and human tumor cells. In particular, there is interest in developing agents active against human solid tumors. We examined several biochemical parameters that may contribute to the differential sensitivity of the cell lines used in our laboratory to the toxic effects of antitumor compounds. The tumor cell lines examined were of mouse (colon 38, L1210 leukemia, and C1498 leukemia) and human origin (CEM leukemia, CX1 colon, H116 colon, HCT8 colon and H125 lung). Quinone reductase activity was markedly different between leukemia and solid-tumor cell lines of either mouse or human origin, with increased activity being observed in the solid-tumor cell lines relative to the leukemia lines. GSH transferase activity also was generally increased in solid-tumor relative to leukemia cell lines. Superoxide dismutase activity and thiol levels were similar in leukemia and solid-tumor cell lines, except that thiol levels were very low in colon 38. Mouse cell lines from in vitro passage had somewhat higher activity of superoxide dismutase and thiol levels than did cells maintained in vivo, indicating relatively increased antioxidant defenses. The toxicity of 2,3-dimethoxy-1,4-naphthoquinone, a model quinone that exerts its toxic effects via production of reactive oxygen species, was significantly lower in mouse lines maintained in vitro than in those tested in vivo, whereas the toxicity of another quinone, menadione, was just slightly lower. Quinone reductase activity, GSH transferase activity, and thiol levels were significantly higher in the human lines than in the mouse lines. Accordingly, the toxicity of both quinones tended to be lower in the human lines than in the mouse lines.

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N-2-(Diethylaminoethyl)-9-hydroxyellipticinium chloride (DHE) is a structural analogue of ellipticine that is currently a leading compound for clinical trials. We have investigated the mechanism of DNA damage by this compound in murine L1210 leukemia cells using the method of alkaline elution. Although DHE was about 100-fold more cytotoxic than ellipticine, this increased cytotoxicity was not accompanied by greater amounts of DNA strand breakage or protein-DNA cross-linking. The single strand breaks caused by both compounds were protein associated and could be accounted for by the presence of double strand breaks. DNA damage by the compounds therefore was consistent with topoisomerase II inhibition. Unlike DHE, 80% of the DNA damage elicited by ellipticine was repaired within 1 h after removal of drug. For DHE, 20-h incubations in drug-free media were required to obtain 70% repair of single strand DNA breaks. These data indicated that although both ellipticine and DHE may inhibit topoisomerase II, the type of DNA damage which resulted in topoisomerase II inhibition by DHE was much more persistent than the DNA damage elicited by ellipticine.

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Fat in the diet has been associated with increased breast cancer risk. In this study, blood samples were obtained from 21 women at high risk for breast cancer who had been randomly assigned to either a nonintervention diet or a low-fat diet. Oxidative damage was examined in the DNA from nucleated peripheral blood cells. The levels of oxidized thymine, specifically 5-hydroxymethyluracil, were threefold higher in the nonintervention diet group than in the low-fat diet group. Without regard to diet arm, there also was a significant linear relationship between daily total fat intake and 5-hydroxymethyluracil level. These results suggest that oxidative damage to DNA may be a marker of dietary fat intake. In addition, oxidative DNA damage may be a mechanistic link between fat in the diet and cancer risk, since such damage is associated with the process of tumor promotion.

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Datelliptium acetate (NSC 311152) is a water soluble analogue of ellipticine. It is a solid tumor selective compound. In vitro, in a disk diffusion, soft agar colony formation assay (25 micrograms/disk), the compound demonstrated solid tumor selectivity (compared to leukemia L1210) against colon adenocarcinoma 38 and pancreas ductal carcinoma 03. Upon intravenous administration, NSC 311152 was effective in vivo against a variety of murine solid tumors. Responses at maximum tolerated doses were: colon #07/A (T/C = 33%); 0.60 log cell kill), #38 [T/C = 0%; 4.2 log cell kill), colon #51/A (T/C = 2%; 1.2 log cell kill), undifferentiated colon #26/A (T/C = 38%; 0.4 log kill), mammary #16/C (T/C = 10%; 1.7 log cell kill), and pancreatic ductal carcinoma #03 (T/C = 0%; 80% cures through day 38). It was ineffective against pancreas #02 (T/C = 45%), mammary 17/A (T/C = 53%), and 17/A/ADR (T/C = 52%). At efficacious doses acute neurotoxicity (i.e. stupor and lethargy) and weight loss were noted (with rapid recovery from both toxicities). There were no delayed toxicities. The agent was slightly necrotizing and produced pain on SC injections. In lieu of its preclinical efficacy and toxicity profiles, we recommend further clinical investigation of this agent.

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