A phase I, open-label, single-arm, dose-escalation study of E7107, a precursor messenger ribonucleic acid (pre-mRNA) splicesome inhibitor administered intravenously on days 1 and 8 every 21 days to patients with solid tumors.

The aim of this study was to determine the maximum tolerated dose, dose-limiting toxicities, and pharmacokinetic profile of E7107 in patients with advanced solid tumors. Patients in this phase I, open-label, single-arm, dose-escalation study had metastatic or locally advanced solid tumors and received E7107 as a 30-minute intravenous infusion at doses of 0.6, 1.2, 1.8, 2.4, 3.2, 4.3, and 5.7 mg/m(2). Twenty-six patients were enrolled in the study. At 5.7 mg/m(2), two patients experienced dose-limiting toxicities including diarrhea, vomiting, dehydration, and myocardial infarction on Days 1-3 following E7107 administration. Three additional patients were recruited at the lower dose and all six patients tolerated E7107 4.3 mg/m(2) with no dose-limiting toxicities. The maximum tolerated dose of E7107 was therefore 4.3 mg/m(2). The most common drug-related adverse events were nausea, vomiting, and diarrhea. Vision loss was experienced by two patients at Cycles 2 and 7, each patient receiving 3.2 mg/m(2) and 4.3 mg/m(2), respectively. This resulted in the study being put on clinical hold. Pharmacokinetic analysis showed that E7107 was rapidly distributed with a moderate elimination half-life (6-13 h) and high clearance. Exposure to E7107 was dose-related. The best tumor response was stable disease in eight patients. E7107 is a unique first-in-class molecule. The incidence of two cases of vision loss probably related to E7107 led to study discontinuation.

Vascular disrupting agents.

It has been well established that a functioning vascular supply is essential for solid tumor growth and metastases. In the absence of a viable vascular network, tumors are unable to grow beyond a few millimeters and therefore remain dormant. Initiation of angiogenesis allows for continued tumor growth and progression. Targeting tumor vasculature, either by inhibiting growth of new tumor blood vessels (antiangiogenic agents) or by destroying the already present tumor vessels (vascular disrupting agents; VDA), is an area of extensive research in the development of new antitumor agents. These two groups differ in their direct physiological target, the type or extent of disease that is likely to be susceptible, and the treatment schedule. VDAs target the established tumor blood vessels, resulting in tumor ischemia and necrosis. These agents show more immediate effects compared to antiangiogenic agents and may have more efficacy against advanced bulky disease. VDAs can be divided into two groups--ligand-bound and small molecule agents. Both VDA groups have demonstrated antitumor effects and tumor core necrosis, but consistently leave a thin rim of viable tumor cells at the periphery of the tumor. More evidence suggests VDAs will have their greatest effect in combination with conventional chemotherapy or other modes of treatment that attack this outer rim of cells.

Decreased galectin-3 expression in prostate cancer.

BACKGROUND: Galectin-3 is a carbohydrate-binding protein whose level of expression has been shown to be correlated with metastatic potential in a number of different tumor types. The purpose of this investigation was to examine galectin-3 expression in several tumorigenic and nontumorigenic prostate cell lines and prostate tissue samples. METHODS: The expression of galectin-3 in cell lines and tissue samples was evaluated by tissue immunohistochemistry and Western blot analysis. RESULTS: Human cell lines PC-3M, PC-3, DU-145, PrEC-1, and MCF10A demonstrated the presence of galectin-3. Galectin-3 was not detected in TSU-pr1 and LNCaP by Western blot analysis. We furthered our studies by examining a series of human prostate tissue samples for expression of galectin-3. Overall, approximately 60-70% of the normal tissue examined demonstrated heterogenous expression of galectin-3. In stage II tumors, however, there was a dramatic decrease in galectin-3 expression in both PIN and tumor sections, with only 10.5% (2/19) of these samples expressing this protein. Stage III tumors also demonstrated a decreased expression of galectin-3, although this downregulation was not as dramatic, with 35% of PIN samples and 52% of tumor tissue expressing galectin-3 (P < 0.01). CONCLUSIONS: These data demonstrate that galectin-3 is downregulated in prostate cancer. The altered downregulation pattern of galectin-3 observed between tumor stages suggests different roles for galectin-3 in the progression of prostate cancer.

The study of gemcitabine in combination with other chemotherapeutic agents as an effective treatment for prostate cancer.

BACKGROUND: Gemcitabine has demonstrated clinical activity against several common cancers. Our studies examine the ability of gemcitabine, both alone and in combination with other chemotherapeutic agents, to inhibit the in vitro and in vivo growth of several prostate cancer cell lines. MATERIALS AND METHODS: Cultures of LNCaP, PC-3 or MLL cells were exposed to either gemcitabine or other appropriate agents for specified amounts of time. Cells were lysed and nuclei counted utilizing a Coulter Counter. For in vivo experiments, animals were injected with 1 x 10(5) MLL cells subcutaneously into the right flank. Animals were treated as indicated for 14 days. Tumors were then excised, weighed and measured. RESULTS: In both human (PC-3 and LNCaP) and rat prostate (MLL) cancer cell lines our studies demonstrated gemcitabine had a strong effect in vitro, with an IC50 of approximately 500 nM in the human lines and 10 nM in MLL cells. In vivo, studies using the Dunning prostate cancer model in Copenhagen rats resulted in a dose response inhibition of tumor growth, with an 80% decrease in tumor size in rats treated with gemcitabine at 10 mg/kg. CONCLUSIONS: Our results demonstrated the potent activity of gemcitabine against prostate cancer in the Dunning rat model and suggest the addition of paclitaxel may not aid in this activity.

Signaling network of paclitaxel-induced apoptosis in the LNCaP prostate cancer cell line.

OBJECTIVES: To attempt to identify the relationship of the key regulator molecules in paclitaxel-induced apoptosis using two metastatic cell lines: the human prostate carcinoma LNCaP line and the cervical carcinoma HeLa cell line. METHODS: Both LNCaP and HeLa cells were continuously exposed to clinically achievable concentrations of paclitaxel and observed for activation of programmed cell death as measured by cytotoxic dose-response curves, poly(adenosine diphosphate-ribose) polymerase cleavage, bcl-2 phosphorylation, and the activation of caspase-7 (interleukin-1 beta converting enzyme (ICE)-LAP3). RESULTS: Initially, we asked whether paclitaxel-induced bcl-2 phosphorylation is triggered by the spindle assembly checkpoint via an active cdc2 kinase-dependent pathway and whether phosphorylation of endogenous bcl-2 is the signal that activates cell death machinery. Paclitaxel-induced G2/M cell cycle arrest correlated with cdc2 kinase activity and bcl-2 phosphorylation. Olomoucin, a specific inhibitor of cyclin-dependent kinases, inhibited bcl-2 phosphorylation. On the basis of these studies, we then investigated whether bcl-2 was phosphorylated in a cell cycle-dependent fashion. Analysis of synchronized HeLa cells demonstrated that endogenous bcl-2 is phosphorylated in a G2/M cell cycle-dependent manner without apoptosis. CONCLUSIONS: Our results indicate that the events associated with paclitaxel-induced cytotoxicity are connected to each other and represent the signaling network of paclitaxel-induced mitotic arrest and cell death. In addition, we confirmed that the death-decision of paclitaxel-induced apoptosis is not mediated by bcl-2 phosphorylation and believe that this decision may be mediated by the activated spindle assembly checkpoint.

Paclitaxel-associated multimininucleation is permitted by the inhibition of caspase activation: a potential early step in drug resistance.

The chemotherapeutic agent paclitaxel disrupts microtubule dynamics causing mitotic arrest, which leads to cell death. However, in paclitaxel-resistant tumor cells, treatment with paclitaxel induces abnormal progression through prophase resulting in a multimininucleated phenotype. Multimininucleation and subsequent polyploidization have been correlated with paclitaxel resistance. Paclitaxel treatment of HeLa cells resulted in cell death via typical activation of the apoptotic machinery, whereas treatment of the relative paclitaxel-resistant prostate cancer cell line PC-3 induced an attenuated caspase activation and multimininucleation. The multimininucleated phenotype could be mimicked in HeLa cells treated with paclitaxel and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), a peptide caspase inhibitor. Interestingly, we observed no discernible difference in the pattern of cdc-2 kinase activation or phosphorylation of bcl-2-like proteins in PC-3 and HeLa cells treated with paclitaxel, which demonstrated that these molecules could not be used as indicators for the degree of caspase activation. In this study, we establish a connection between relative paclitaxel resistance, caspase attenuation/inhibition, and the multimininucleated phenotype.

A model to study c-myc and v-H-ras induced prostate cancer progression in the Copenhagen rat.

Normal rat prostate epithelial cells (EPYP-1) were isolated and immortalized with the Simian Virus-40 (SV40) large T-antigen, and transfected with the v-H-ras (EPYP-1-ras) and the c-myc oncogenes (EPYP-1-myc; EPYP-1-ras-myc) to serially create a step-wise model of tumor development in the rat prostate. Pronounced morphological differences were observed between EPYP-1 and the transfected sublines. The immortal epithelial cells (EPYP-1) maintained a cuboidal shape with orderly, contact mediated inhibition of growth. Oncogene transfected clones displayed a spindle shaped structure with multiple overlapping pseudopodia. Transfected cells also exhibited a greater degree of dysplasia, loss of contact inhibition growth and the upregulation of an epithelial tumor marker, cytokeratin-18. All cells exhibited anchorage independent and androgen independent growth. In vivo, EPYP-1 cells and EPYP-1-myc and formed slowly growing non-metastatic, benign tumors in immune compromised mice, while EPYP-1-ras and EPYP-1-ras-myc transfected cells produced rapidly growing, malignant tumors in similar animals. This model augments the hypothesis that tumor initiation and progression can be caused by as few as two discrete genetic events. In addition, the "normal" rat prostate epithelium and transfected daughter cell lines represent a tumor model system with distinct, well understood genetic alterations: activation of ras and myc. This model will be a valuable addition to the current cell lines used in the investigation of prostate cancer carcinogenesis.

Examination of the DNA methylation properties in nontumorigenic and tumorigenic breast epithelial cell lines.

Molecular changes in the progressive state of tumorigenesis often include altered patterns of DNA methylation. Utilizing a series of breast epithelial cell lines, the overall 5-methylcytosine content in genomic DNA demonstrated an overall decrease when comparing two malignant cell lines (MCF-7 and T47D) with a mortal cell line (MCF 1 2M) and several derivative cell lines of the immortalized MCF10 cultures (MCF10A,-2A, -5A, A1neoT2, and 139B6). Further investigation on the methylation status of these cells lines indicated no difference in DNA methyltransferase activity, both at a protein and mRNA levels, in the nontumorigenic cell lines examined while activity was 3-10 fold higher in the tumorigenic lines (MCF7, T47D, SkBr3, MB-MDA-231, -468). Examination of the CpG island in the 5' promoter region of the estrogen receptor gene indicates that this region is unmethylated in the mortal and immortal nontumorigenic lines as well as the tumorigenic lines examined, with the exception of the estrogen receptor negative breast cell line MB-MDA-468 which appears to be partially methylated at this site. These results indicate methylation of this CpG island does not account for the inactivation of the estrogen receptor gene in immortalized nontumorigenic breast cells, suggesting another mechanism of transcriptional inactivation of ER in this environment.

The effect of amiloride on the metastatic properties of prostate cancer in the Dunning rat model.

Most deaths from cancer result from the metastatic spread of the disease. The antidiuretic amiloride has been shown to inhibit tumor growth and metastasis in several tumor systems. The object of these studies was to examine the effect on the in vitro and in vivo tumor growth and metastasis in the MatLyLu subline of the Dunning model of rat prostate cancer. In vitro, amiloride was found to have cytotoxic effects only at high concentrations, with an IC50 of 100 microg/ml. In vitro analysis of the ability of amiloride to inhibit invasion of MLL cells demonstrated that this drug was ineffective at all concentrations examined. In vivo, amiloride did not inhibit tumor growth or metastases development. Our studies demonstrate that amiloride does not have activity in this model of prostate cancer and suggest it may not be an appropriate therapy for the treatment of prostate cancer.

The isolation and characterization of epithelial cells from canine prostate.

BACKGROUND: Prostate cancer causes approximately 40,000 deaths in the United States annually (1,2). Adenocarcinoma of the prostate occurs primarily in two species: human and dog (3,4). Although less common in dogs, the etiologic factors responsible for spontaneous canine prostate cancer are presumably the same as for humans. Given the similar etiology and epidemiology of the disease among the two species, a model of canine prostate epithelial cells would be a powerful tool to study the disease. METHODS: Prostate epithelial cells were isolated from a sexually intact, adult beagle, and primary cultures established. Epithelial clones were immortalized by transfection with the Simian Virus 40 large T-antigen cDNA. Cells were characterized using immunohistochemical techniques. RESULTS: The immortal prostate epithelial cell line expresses cytokeratin-18, and prostatic acid phosphatase, markers specific for prostate epithelial cells. K-9PE-I, a stable, fast growing, canine prostate epithelial cell line is available for further study. CONCLUSIONS: A cell based model of canine prostate epithelium was isolated, immortalized, and characterized. The cell line will be available for further study of prostate disease.

Crystal structure, receptor binding, and gene regulation of 2- and 4-nitroestradiols.

Crystal structures of 2-nitroestradiol and 4-nitroestradiol showed two different molecular conformations for each compound. The crystal structure of 4-nitroestradiol, as well as that of 4-nitroestrone-3-methyl ether, displayed a nitro group in which the oxygens were perpendicular to the aromatic ring and were this nonconjugating. On the other hand, the nitro-oxygens in 2-nitroestradiol were periplanar, with the aromatic ring permitting conjugating. This latter structure bound to estrogen receptor with 1/1000th the affinity of estradiol and was inefficient in gene stimulation. 4-Nitroestradiol possessed a relative binding affinity 40-fold greater than that of the 2-nitro derivative and actively induced responsive genes at a concentration of 10(-8) M. Whereas binding affinity can be explained primarily by polar groups and skeletal structure, gene induction may be linked to electronic induction in ring A that causes a requisite electronegative isopotential around the molecule. This electronegative characteristic also produces conformational changes in the alicyclic backbone of the estrogen, specially ring B, which could interfere with the molecular fit of the nitroestradiols with estrogen receptor.

The effects of omega-3 and omega-6 fatty acids on in vitro prostate cancer growth.

Dietary intake of essential fatty acids (EFA) may play a role in prostate cancer cell proliferation. Epidemiological studies have demonstrated that men whose dietary intake is high in omega-3 fatty acid (FA) composition have a lower incidence of clinical prostate cancer, suggesting that external factors such as diet may play an important role in development and growth of prostate cancer. Furthermore, in prostate cancer cell lines, omega-6 and omega-3 FAs have demonstrated promotional and inhibitory effects respectively. To investigate the effects of dietary fats on nontumorigenic prostate cell growth we conducted in vitro studies with human metastatic PC-3, LNCaP and TSU prostate cell lines, the rat metastatic Mat-Ly-Lu cell line and rat non-metastatic epithelial cell lines EPYP1, EPYP2 and EPYP3. Cell lines were treated with linoleic acid (LA), an omega-6 FA (n-6), as well as linolenic (LLA) and eicosapentaenoic (EPA) acids, which are both omega-3 FAs (n-3). All cell lines were treated with 10% and 0.5% serum supplemented media plus fatty acid for comparison. Our results demonstrate that linoleic acid(n-6) has promotional effects at doses of 1-100ng/ml in all cell lines with the exception of EPYPl. Experiments with linolenic acid (n-3) demonstrated consistent growth promotion in all cell lines examined with the exception of the EPYP2 cell line in which there was no significant effect. EPA had no effect in culture media supplemented with 10% serum, while in media containing 0.5% serum this FA demonstrated significant promotion in all human lines. Previous studies have indicated that EPA should inhibit human prostate cancer growth in vitro, however our results demonstrated promotion at low concentrations (lng/ml). At higher concentrations, EPA did inhibit prostate cell growth. These data indicate low levels of dietary fat, regardless of composition, may play a role in prostate cancer proliferation and could be an avenue for therapeutic intervention.

Characterization of the estrogen receptor transfected MCF10A breast cell line 139B6.

There has been increasing evidence which suggests that abnormal expression of the estrogen receptor (ER) protein in nonmalignant breast tissue may be important in the carcinogenic process. To examine the effects of ER expression in immortalized nonmalignant mammary epithelial cells, an expression vector containing human ER cDNA was transfected into the ER negative human breast cells, MCF10A. Characterization of a clone stably expressing ER, 139B6, provided evidence for the regulated synthesis of a functional ER capable of binding estradiol-17 beta (E2) and undergoing processing. Expression of the ER gene did not enable E2 to stimulate endogenous genes [progesterone receptor (PgR), pS2, cathepsin D and TGF alpha] which normally respond to estrogens in breast cancer cells. The ER in 139B6 cells was, however, capable of inducing expression of an ERE-regulated reporter gene, indicating its ability to interact with transcriptional machinery. Furthermore, cultures in log growth displayed a slight increase in doubling time in the presence of E2. These results indicate that ER expression alone is not sufficient to induce a transformed phenotype. Thus, the 139B6 cell line should provide a new model for determining what additional changes lead to increased growth potential in response to E2 and for exploring how E2 itself may help bring about changes leading to progression of preneoplastic breast epithelial cells.

Differential induction of pS2 and cathepsin D mRNAs by structurally altered estrogens.

The influence of structural alterations to the estradiol-17 beta (E2) molecule on the induction of pS2 and Cathepsin D (Cath D) mRNAs has been examined by Northern analysis of RNA extracted from MCF-7 cells. Exposure of cultures to estratriene did not affect the level of expression of these estrogen-responsive genes. Addition of one hydroxyl group to estratriene at either of the hydroxylated positions of E2 (3-phenolic or 17 beta) yielded monohydroxyestrogens which stimulated the synthesis of Cath D and pS2 mRNAs to a level comparable to that induced by 10(-10) M E2 and displayed a decrease in activity at the higher concentrations (10(-8) - 10(-7) M) similar to that of the parent estrogen. Both of these genes were induced maximally by estrogens with D-ring alterations. Movement of the phenolic hydroxyl group of E2 to other positions on the A-ring yielded ligands which were highly discriminatory in the induction of these messages. 1-Hydroxyestratrien-17 beta-ol was capable of stimulating maximal synthesis of both pS2 and Cath D mRNAs when added to cultures of MCF-7 cells at a concentration of 10(-8) M. Placement of the phenolic hydroxyl at position 4 greatly diminished the induction of these two estrogen-responsive genes. On the other hand, positioning the A-ring hydroxyl group on carbon 2 yielded a ligand which brought about the induction of one gene (pS2) but was marginally effective in the induction of Cath D mRNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)