Clomiphene analogs with activity in vitro and in vivo against human breast cancer cells.

Six hundred triphenylethylenes were assayed for antiproliferative activity against MCF-7, LY2, and MDA-MB-231 breast cancer cells using sulforhodamine B dye to measure proliferation. Here we report on just 63 of the compounds, mostly clomiphene analogs, with substitutions on the alpha' or beta ring, at the vinyl position or in the side chain, of which 23 were active, as defined by antiproliferation IC50 values < or =1 microM. Activity profiles showed that 23 and 11 analogs were active toward MCF-7 and LY2, respectively, but none were active against MDA-MB-231. The IC50 values of tamoxifen were 2.0 microM against MCF-7 and 7.5 microM against LY2 and MDA-MB-231. Estradiol reversed antiproliferative activities of several E isomers but not their Z isomer counterparts. Clomiphene side chain analogs 46 [(E)-1-butanamine, 4-[4-(2-chloro-1,2-diphenylethenyl) phenoxy]-N,N-diethyl-dihydrogen citrate (MDL 103,323)] and 57 [(E)-N-[p-(2-chloro-1,2-diphenylvinyl) phenyl]-N,N-diethylethylenediamine dihydrogen citrate (MDL 101,986)] were 4- to 5-fold more effective than tamoxifen. Methylene additions up to (-CH2-)12 in the clomiphene side chain showed that analog 46 [(-CH2-)4 side chain] had maximal antiproliferative activity, binding affinity, and inhibition of transcription of an estrogen response element luciferase construct in transfected MCF-7 cells. Intraperitoneal administration of 46 or 57 inhibited progression of MCF-7 breast tumor xenografts in nude mice with ED50 values of <0.02 mg/mouse/day. Both analogs may hold promise for treating ER positive breast cancer and are of interest for further development.

Quantitation of vascular endothelial growth factor mRNA levels in human breast tumors and metastatic lymph nodes.

In situ hybridization analysis provides a means to qualitatively study the heterogeneity of primary tumors and metastases based on the types of genes transcribed. In this study, we have tested some parameters for quantitative analysis of in situ hybridizations with paraffin-embedded human breast tumors and measured mRNA levels for the angiogenic protein, vascular endothelial growth factor (VEGF). VEGF mRNAs were highly tumor specific, with the highest levels near necrotic regions within the tissues (0.1 to 2.7 dpm/mm2). Normal cells within the tissue sections did not have detectable levels of VEGF mRNA. For comparison, tumor levels of c-myc (4 to 46 dpm/mm2) and glyceraldehyde-3-phosphate dehydrogenase mRNAs (48 to 214 dpm/mm2) were measured. The mRNAs for both of these genes were more broadly expressed across the tissue sections. The hybridization pattern for VEGF mRNAs was consistent with hypoxia-induced VEGF mRNA steady-state levels and supports the hypothesis that oxidative stress regulates VEGF expression in breast tumors.

Decreased vascular endothelial growth factor expression associated with tumor regression induced by (E)-2'-deoxy-2'-(fluoromethylene)cytidine (MDL 101,731).

The effect of the antitumor drug MDL 101,731 [(E)-2'-deoxy-2'-(fluoromethylene)cytidine] on tumor growth and on steady-state vascular endothelial growth factor (VEGF) mRNA levels in MDA-MB-231, PC-3, MCF-7, and HT-29 human tumor xenografts grown in nude mice was examined, using quantitative in situ hybridization. MDL 101,731 caused regression of MDA-MB-231 and PC-3 tumor xenografts, but only inhibition of growth (without regression) of MCF-7 xenografts. The drug caused inhibition of growth of HT-29 xenografts at low doses, and regression at high doses. When treatment with MDL 101,731 led to tumor regression, VEGF mRNA levels were decreased. When treatment led only to inhibition of growth, there was no significant change in VEGF mRNA. Further examination of the tumor xenografts revealed that elevated VEGF mRNA was associated with hypoxic zones surrounding areas of necrosis in the tumors, and that the drop in VEGF mRNA observed in tumors from mice treated with MDL 101,731 correlated with a loss of zones of necrosis. In contrast, treatment with cisplatin led to either an increase (PC-3) or no change (MDA-MB-231) in VEGF mRNA levels, and no loss of necrotic zones. Quantitative analysis of changes in VEGF mRNA levels was supported by immunohistochemical analysis of VEGF protein in the same tumor specimens. In vitro, MDL 101,731 was a potent inhibitor of VEGF secretion in cells exposed to hypoxia, whereas there was no effect of cisplatin on VEGF secretion by three of the four cell lines tested. These findings suggest that inhibition of VEGF expression by MDL 101,731 may distinguish this compound from other classes of cytotoxic agents, such as cisplatin.

Progression of MCF-7 breast cancer cells to antiestrogen-resistant phenotype is accompanied by elevated levels of AP-1 DNA-binding activity.

We have isolated a variant of the MCF-7 human breast tumor that is characterized by a hormone-independent, yet hormone-responsive, phenotype. This tumor, designated MCF-WES, was derived from MCF-7 tumor cells implanted in the mammary fat pad of a nude mouse in the absence of estradiol supplementation. MCF-WES tumors remain responsive to estradiol; however, unlike the parental MCF-7 tumors, they are stimulated to grow by tamoxifen. Additionally, MCF-WES cells are resistant to the pure steroidal antiestrogen, ICI 182,780. To our knowledge, a tumor with this combination of properties has not yet been described. Nuclear estrogen receptor (ER) levels in MCF-WES cells were 10% of those for MCF-7 under steroid-depleted conditions. MCF-WES tumor ER levels were 32% of those in MCF-7 tumors. Similarly, in vivo expression of ER mRNA for MCF-WES was 20% of levels determined for MCF-7. Further characterization of MCF-WES cells showed that they have increased levels of AP-1 DNA-binding activity. The marked increase in AP-1 binding activity may act to bypass the hormone dependence that is a characteristic of MCF-7 cells. It is also probable that the increase in AP-1 binding activity is responsible for the finding that MCF-WES cells secrete greater quantities of metalloproteinase activity in comparison to parental MCF-7 cells, suggesting progression to a more invasive, malignant phenotype. More complete characterization of this new cell line will help elucidate hormone-independent breast cancer and possibly identify targets for therapy.

Regulation of ornithine decarboxylase mRNA levels in human breast cancer cells: pattern of expression and involvement of core enhancer promoter element.

Ornithine decarboxylase (ODC) expression is increased by growth factors and is obligatory for progression through the cell cycle in a wide variety of cell types. In this study, a variant human ODC cDNA was identified, sequenced, and used to probe mRNA levels in human breast tumor cell lines and xenografts. ODC mRNA was elevated about 3-fold in estrogen receptor-negative (ER-) tumors (MDA-MB-231) when compared with ER-positive (ER+) tumors (MCF-7), as assessed by quantitative autoradiographic analysis of in situ hybridization experiments. The pattern of ODC mRNA in MDA-MB-231 (ER-) xenografts was polarized to the extreme periphery of the tumor, whereas the distribution of ODC mRNA was more evenly distributed in MCF-7 (ER+) xenografts. This correlates with hematoxylin and eosin staining patterns, suggesting that ER+ and ER- xenografts have a differential dependence on host vasculature for growth factor supply. ODC mRNA was elevated 5-fold in MDA-MB-231 cells versus MCF-7 cells when analyzed in cell culture. These relative mRNA levels correlate with increased levels of "core" enhancer binding nuclear proteins in MDA-MB-231 cells over that detected in MCF-7 cells.

Measurement of mRNA levels in tumor xenografts with quantitative autoradiography and in situ hybridization.

In situ methodologies allow qualitative and semi-quantitative analysis of spatial gene expression in whole organisms or tissues. We have applied quantitative autoradiography to in situ hybridizations of sections from human breast tumor xenografts to measure mRNA levels for ornithine decarboxylase, estrogen receptor, transforming growth factor alpha, and glyceraldehyde-3-phosphate dehydrogenase. Comparisons of control and tamoxifen-treated animals show significant decreases in MCF-7 tumor estrogen receptor mRNA levels in the drug-treated animals. Combining quantitative autoradiography with in situ hybridization allows measurement of absolute rather than relative mRNA levels for genes of interest, and to monitor effector-induced changes in these mRNAs in vivo.

Regression of human breast tumor xenografts in response to (E)-2'-deoxy-2'-(fluoromethylene)cytidine, an inhibitor of ribonucleoside diphosphate reductase.

(E)-2'-Deoxy-2'-(fluoromethylene)cytidine (MDL 101,731) is a mechanism-based inhibitor of ribonucleoside diphosphate reductase (J. Stubbe, personal communication), an enzyme involved in DNA synthesis and therefore a potential target for cancer chemotherapy. In the present report, we show that MDL 101,731 inhibits the proliferation of several human breast cancer cell lines, including the estrogen-dependent cell line, MCF-7, and the estrogen-independent cell lines MDA-MB-231, MDA-MB-468, and MDA-MB-435 in vitro at nanomolar concentrations (50% inhibitory concentration, 15-26 nM). Administration of MDL 101,731 caused marked regression of tumors which formed after s.c. inoculation of all four of the cell lines in athymic (nude) mice. MDA-MB-231 tumors were found to be most sensitive to MDL 101,731 with a 90-100% cure rate at doses of MDL 101,731 between 2 and 20 mg/kg, given as once daily i.p. injections, 5 days/week for as little as 3 weeks. Almost complete cessation of MDA-MB-231 tumor growth was obtained with a dose of 0.5 mg/kg MDL 101,731 following the same dosing regimen. MDA-MB-468, MDA-MB-435, and MCF-7 tumors were not as sensitive as MDA-MB-231, but tumor regression of 50, 65, and 80%, respectively, was obtained after 5-6 weeks of treatment. The effects of MDL 101,731 on spontaneous metastasis of MDA-MB-435 cells from the mammary fat pad to the lung was also examined, and it was found that the number of lung metastases was significantly decreased if mice received MDL 101,731 while the primary tumors were growing and after primary tumors were surgically excised. Additionally, preliminary evidence raises the possibility that MDL 101,731 may induce apoptosis in MDA-MB-231 tumors. Our data suggest that the use of MDL 101,731 for the treatment of breast cancer and possibly other solid tumors should be pursued.

Irreversible inhibition of S-adenosylmethionine decarboxylase in Plasmodium falciparum-infected erythrocytes: growth inhibition in vitro.

Blocking spermidine and spermine synthesis in Plasmodium falciparum-infected erythrocytes with irreversible inhibitors of S-adenosylmethionine decarboxylase (AdoMet DC; EC 4.1.1.50), prevented the growth of the parasite in vitro. The most potent of these compounds, MDL 73811, inhibited growth of chloroquine-sensitive and -resistant strains of P. falciparum equally, with an IC50 of 2-3 microM. Other structurally related compounds also inhibited parasite proliferation, but to a lesser degree, determined apparently by their potency for inhibition of AdoMet DC. The growth inhibition by MDL 73811 could be alleviated by incubating infected erythrocytes with spermidine and spermine, but not putrescine. Parasites treated with the drug were arrested at the trophozoite stage of the erythrocytic cycle and had putrescine levels which were elevated by about 3- to 4-fold. Treatment of crude extracts of purified parasites with 1 microM MDL 73811 inhibited AdoMet DC activity by greater than 90%. These biochemical changes in P. falciparum-infected cells were consistent with AdoMet DC inhibition being the primary effect of MDL 73811 treatment.

Disruption of Plasmodium falciparum-infected erythrocyte cytoadherence to human melanoma cells with inhibitors of glycoprotein processing.

Adherence of Plasmodium falciparum-infected erythrocytes (IE) to the venular endothelium in brain and other organs is characteristic of cerebral malaria, an often fatal complication in infected individuals. It has been shown that cytoadherence may be mediated through interaction of IE with glycoproteins on host target cell surfaces, including CD36 (GPIV), intercellular adhesion molecule-1 (ICAM-1), and thrombospondin. Inhibitors of glycoprotein synthesis and processing were tested for their abilities to decrease IE adherence to C32 human melanoma cells. The alpha-glucosidase inhibitor, castanospermine, was effective in disrupting cytoadherence in vitro when incubated with C32 cells (IC50 = 600-700 microM). Castanospermine-6-butyrate was even more effective than the parent compound (IC50 = 9 microM) in disrupting cytoadherence. The mannosidase inhibitors, swainsonine and deoxymannojirimycin, had no effect on cytoadherence at concentrations up to 2 mM. No effect on cytoadherence was observed when the glucosidase and mannosidase inhibitors were incubated with IE rather than the C32 cell cultures. The level of CD36 on the C32 cell surface was decreased as measured by fluorescence-activated cell sorting (FACS) analysis with the same inhibitors which inhibited cytoadherence. Cells labeled with fluorescein isothiocyanate (FITC) OKM5 monoclonal antibody, which recognizes CD36 and disrupts cytoadherence, showed decreased fluorescence when treated with tunicamycin and castanospermine-6-butyrate but not when treated with swainsonine or deoxymannojirimycin. ICAM-1 levels, as measured by surface labeling of C32 cells with FITC CD54 monoclonal antibody, were decreased in cells treated with tunicamycin. However, incubation of cells with castanospermine-6-butyrate or deoxymannojirimycin decreased cell surface ICAM-1 levels only slightly. These findings suggest that (1) in C32 cells, levels of cell surface CD36, and not ICAM-1, change proportionally to the level of cytoadherence; (2) drugs which can affect the carbohydrate moiety of cellular glycoproteins decrease cytoadherence of IE to C32 cells; and (3) protection against the development of cerebral malaria may be possible with inhibitors of glycoprotein biosynthesis.

Effects of alpha-difluoromethylornithine on protein synthesis and synthesis of the variant-specific glycoprotein (VSG) in Trypanosoma brucei brucei.

Protein synthesis in Trypanosoma brucei brucei was rapidly inhibited during polyamine depletion by DL-alpha-difluoromethylornithine (DFMO) in vitro and in vivo. [3H]Leucine incorporation was depressed 30-40% by 24 h and 80-90% by 48 h of DFMO treatment. Concomitantly there was an apparent decrease in the synthesis of the variant-specific glycoprotein (VSG) in DFMO-treated trypanosomes, as measured by decreased incorporation of [3H]myristic acid into VSG. The discovery of decreased protein synthesis in T. b. brucei during DFMO treatment is noteworthy, because it was reported previously that protein synthesis was paradoxically stimulated 2-4-fold during DFMO treatment in these organisms. Decreased protein synthesis probably relates to the biochemical mechanism of action of DFMO on trypanosomes.