VCaP, a cell-based model system of human prostate cancer.

OBJECTIVES: We report the isolation and characterization of a novel prostate cancer cell line derived from a vertebral metastatic lesion, Vertebral-Cancer of the Prostate (VCaP). METHODS: Prostate cancer tissue was harvested at autopsy from a metastatic lesion to a lumbar vertebral body of a patient with hormone refractory prostate cancer. This tissue was aseptically xenografted into SCID mice and later harvested and plated on tissue culture dishes. For characterization, soft agar clonegenic assay, in vivo xenograft growth, in vitro doubling time, karyotype analysis, immunocytochemistry for cytokeratin-18 expression immunochemistry for PSA (prostate specific antigen), RT PCR for PAP (prostatic acid phosphatase) and northern blot and western blot analysis to determine expression of Rb and p53, were performed. Androgen receptor expression was measured by transient transfection with a luciferase reporter construct. RESULTS: VCaP cells are immortal in vitro and can be passaged serially in vivo. They express large quantities of prostate specific antigen (PSA). This cell line also expresses prostatic acid phosphatase (PAP), cytokeratin-18 and the androgen receptor, and is androgen sensitive in vitro and in vivo. CONCLUSIONS: This cell line was derived from a metastatic tumor to the vertebrae of a prostate cancer patient. It exhibits many of the characteristics of clinical prostate carcinoma, including expression of PSA, PAP, and AR. We believe that VCaP will be a useful addition to the existing models of prostate cancer, and enable more advanced study of the mechanisms of prostate cancer progression and metastasis.

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.

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.

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.

Preferential adhesion of prostate cancer cells to a human bone marrow endothelial cell line.

BACKGROUND: In virtually all patients with advanced prostate cancer, the disease metastasizes to bone and causes osteoblastic growth. However, the mechanisms that contribute to bone metastasis are poorly understood. It has been hypothesized that the bone provides a favorable growth environment for prostate cancer cells, which nonselectively seed the bone marrow from the bloodstream. Alternatively, prostate cancer cells may preferentially bind to bone marrow endothelial cells. We developed an in vitro model of bone endothelium and tested the hypothesis that prostate cancer cells adhere preferentially to bone marrow endothelial cells. METHODS: We isolated and characterized a human bone marrow endothelial (HBME-1) cell line. Cells were transfected with the simian virus 40 large T antigen for immortalization. Cell surface receptors were characterized by immunohistochemistry and flow cytometry. The adhesion of cancer cells to HBME-1 and to endothelial cell lines from other organs was tested in an in vitro binding assay as were inhibitors of adhesion. RESULTS: The immortalized HBME-1 cell line demonstrated many properties characteristic of endothelial cells, including positive staining for von Willibrand factor and rapid formation of tubule structures when exposed to extracellular matrices. In an in vitro assay, prostate cancer cells adhered preferentially to human bone marrow endothelium when compared with endothelium derived from other sources. Preferential adhesion was blocked, in part, by antibodies to galectin-3 and LFA-1. IMPLICATIONS: These data suggest that the propensity of prostate cancer cells to establish themselves in bone is due, at least in part, to their preferential adhesion to human bone marrow endothelial cells.

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.

The effect of castanospermine on the metastatic properties of prostate cancer cells.

BACKGROUND: Most deaths from prostate cancer result from the metastatic spread of the disease. Castanospermine has been shown to inhibit tumor growth and metastasis in mouse and rat models. We hypothesized that castanospermine might inhibit metastasis in the Dunning model of rat prostate adenocarcinoma by interfering with the metastatic properties of tumor cells. MATERIALS AND METHODS: We examined the cytotoxicity of castanospermine toward the metastatic MAT-LyLu and nonmetastatic AT. 1 cell lines and its effects on cell motility and adhesion to endothelial cells. We assessed castanospermine's effects on in vivo metastasis in Copenhagen rats. RESULTS: Castanospermine was not cytotoxic toward the MAT-LyLu and AT. 1 cell lines at concentrations through 10 micrograms/mL, nor did it significantly affect cell motility, adhesion to endothelial cells, or in vivo metastasis. CONCLUSIONS: Within the Dunning model, castanospermine did not appear to significantly affect cell characteristics related to metastatic potential.

Inhibition of prostate cancer growth by 9-aminocamptothecin and estramustine.

OBJECTIVES: Hormone-refractory prostate cancer continues to be associated with a very poor prognosis. Agents that interact with the nuclear matrix have been demonstrated to have activity against hormone-refractory prostate cancer. It was the aim of this study to assess the activity of estramustine, an estradiol-nitrogen mustard conjugate, and 9-aminocamptothecin (9-AC), a topoisomerase I inhibitor, in a preclinical model of hormone-refractory prostate cancer. METHODS: We used the Dunning rat prostatic adenocarcinoma model to demonstrate that the combination of estramustine and 9-AC interacts at the level of the nuclear matrix to inhibit the growth of prostate cancer cells. RESULTS: We demonstrate that the combination of these two agents at pharmacologically achievable doses are cytotoxic to rat and human prostate cancer cells in vitro and in vivo in the rat. CONCLUSIONS: The combination of the two drugs was significantly more cytotoxic than either drug alone. We have instituted a Phase II clinical trial in patients with hormone-refractory prostate cancer using 9-AC based on these preclinical findings.

Establishment of an immortalized Copenhagen rat bone marrow endothelial cell line.

Bone marrow endothelial cells are critical mediators in the processes of cell trafficking as well as cancer metastasis, however few established models exist. An immortal cell line of Copenhagen rat bone marrow endothelium was established after infection of primary cultured cells with Adenovirus-12 SV40 hybrid virus and designated YPBE-1. The established cell line has continued to proliferate more than 70 population doublings and has not undergone "crisis". It stains positively for SV40 T-antigen in its nuclei by immunohistochemistry and grows in a monolayer with a cobblestone appearance. It demonstrates Dil-Ac-LDL uptake as an endothelial marker. YPBE-1 does not express Integrin beta 3 or endothelin, but does express Integrin alpha 6 beta 1 on the plasma membrane and demonstrates tube formation in Matrigel. This cell line of rat bone marrow endothelial origin should be useful for studying mechanisms of bone metastasis and cell trafficking.

Unique nuclear matrix protein alterations in head and neck squamous cell carcinomas: intermediate biomarker candidates.

The progression of normal squamous epithelium to a malignant metastatic phenotype may depend on cellular genetic events and the failure of host mechanisms. Intermediate biomarkers are needed to more effectively identify and quantify malignant progression and develop the potential for specific treatments and prevention strategies. The nuclear matrix is the RNA-protein scaffold of the nucleus, which controls in part nuclear shape, DNA organization, and DNA function. Nuclear matrix proteins in all previously studied cell types show a common set of nuclear matrix proteins and a subset of tissue- and cell type-specific proteins. In every system studied to date, the nuclear matrix has been demonstrated to undergo quantifiable alterations in its protein composition with transformation to the malignant phenotype. The loss and gain of nuclear matrix proteins are being investigated as biomarkers for malignant transformation in breast, colon, and prostate carcinoma. We have investigated nuclear matrix protein composition in laryngeal and oral cavity primary squamous cell tumors and metastatic cervical lymph nodes. Laryngeal carcinoma demonstrated the gain of two specific nuclear matrix proteins in comparison with noncancerous squamous epithelium. Squamous cell carcinoma matrixes demonstrate greater heterogeneity than do previously studied adenocarcinoma matrixes, and yet they display specific matrix proteins that may represent important potential biomarkers.

Establishment of immortalized Copenhagen rat prostate endothelial cell lines.

Since the introduction of culture methods for umbilical vein endothelial cells, many successful attempts for culturing endothelial cells have been reported. The successful establishment of immortalized cell lines of organ specific endothelium, however, has been rare. Primary cultured endothelial cells isolated from the Copenhagen rat prostate were immortalized by infection with an Adenovirus-12 SV40 hybrid virus. Two immortal cell lines of Copenhagen rat prostate endothelium were established and designated YPEN-1 and YPEN-2. The established cell lines have continued to proliferate more than 80 population doublings and have not undergone senescence. They stain positively for but are nonproducers of SV4O0 T-antigen and grow in a monolayer with a cobblestone appearance. They demonstrate Dil-Ac-LDL uptake as an endothelial marker. YPEN-1 and YPEN-2 cells exhibit positive staining for endothelin and MRC OX-43 and express Integrin a6 beta 1 and Integrin beta 3 on their plasma membrane and demonstrate tube formation in Matrigel. Doubling times of YPEN-1 and YPEN-2 are 26 hours and 21 hours, respectively. Genetically, YPEN-1 and YPEN-2 are both diploid. These cell lines of rat prostate endothelial origin should be useful for studying angiogenesis and its inhibition.

Inhibition of prostate cancer growth by estramustine and colchicine.

Hormone-refractory prostate cancer continues to be associated with a very poor prognosis. Agents that inhibit microtubule function have been found to be cytotoxic to prostate cancer cells in preclinical and clinical settings. It was the aim of this study to assess the activity of estramustine and colchicine, two microtubule inhibitors, in hormone-refractory prostate cancer. In clinically achievable concentrations, the combination of estramustine and colchicine was cytotoxic to both the Dunning rat prostate adenocarcinoma cell line MAT-LyLu (MLL) and human prostate cancer cells (PC-3). Microtubule function was assessed in vitro to evaluate possible mechanisms of action. In motility and cell cycle analysis assays, estramustine and colchicine inhibited cellular motility but not cell cycle transit. In vivo, these two agents both inhibited the growth of implanted Dunning rat prostate adenocarcinoma MLL cells but did not appear to have additive effects. The use of oral colchicine in the treatment of hormone-refractory prostate cancer requires further investigation.

Inhibition of prostate cancer growth by vinblastine and tamoxifen.

Hormone refractory prostate cancer continues to be an aggressive and fatal disease. Agents which inhibit microtubule function have been found to be cytotoxic to prostate cancer cells in preclinical and clinical settings. It was the aim of this study to assess the activity of tamoxifen and vinblastine, two microtubule inhibitors, in hormone refractory prostate cancer. In clinically achievable concentrations, the combination of tamoxifen and vinblastine was cytotoxic to both Dunning rat prostate adenocarcinoma cell line MAT-LyLu (MLL) and human prostate cancer cells (PC-3). In cell cycle analysis assays, tamoxifen and vinblastine inhibited cell cycle transit. In vivo, these two agents inhibited the growth of implanted MLL cells. It appears that tamoxifen and vinblastine may be effective agents for the treatment of patients with hormone refractory prostate cancer.

Immunomagnetic isolation of endothelial cells from normal rat prostate tissue.

Angiogenesis, or the process of the formation of new blood vessels, is an essential event for the successful growth of tumors in vivo. The discovery of agents which inhibit angiogenesis has been limited by the lack of available experimental endothelial cell systems which mimic specific organ- or tumor-derived endothelial cells. It is well recognized that there are measurable functional and morphological differences between normal tissue endothelium and tumor vessels, however, the cause of these differences remains unclear. We report here a technique for tissue specific endothelial cell isolation from the rat prostate utilizing immunomagnetic techniques, providing a tool for the study of endothelial cells and their inhibition.

An in vitro and in vivo study of antitumor effects of genistein on hormone refractory prostate cancer.

Hormone refractory prostate cancer remains an incurable disease. Newer agents with more activity are required. Genistein is a flavone compound with anti-tumor activity against various tumor systems in vitro. This study is undertaken to assess the efficacy of genistein against hormone refractory prostate cancer. In vitro, genistein appears to be cytotoxic to both the rat prostate cancer cell line MAT-LyLu and the human prostate adenocarcinoma cell line, PC-3. In vivo, however, genistein failed to significantly inhibit the growth of subcutaneously implanted MAT-LyLu cells. More information regarding the pharmacokinetics and bio-availability of genistein is needed to determine if this is an active agent in human metastatic prostate cancer.

The effect of maltose tetrapalmitate (MTP) on prostate cancer growth in vivo and in vitro.

Maltose tetrapalmitate (MTP), a non-toxic synthetic glycolipid analog of lipid A, has been shown to have antitumor activity in tumor-transplanted animals. Its mode of action has been postulated to be as an immunoadjuvant or as an anti-angiogenesis agent. MTP has been shown to have antitumor properties in lung, bladder, mammary, colon, liver and soft tissue tumors, but its action on prostate cancer has not yet been investigated. The effect of MTP alone and in combination with hydrocortisone hemisuccinate on prostate cancer and the ability of MTP to inhibit angiogenesis were examined in this study. In vitro, MTP was minimally cytotoxic to rat prostate cancer cells and to bovine and human endothelial cells at high concentrations. In the angiogenesis inhibition assays, the MTP alone exhibited no anti-angiogenesis effect and significant anti-angiogenesis activity only when combined with hydrocortisone hemisuccinate at high doses. In vivo, however, MTP demonstrated significant inhibition of prostate cancer growth. These results suggest that MTP decreases prostate cancer growth in vivo but it is not an angiogenesis inhibitor in rat prostate cancer.

Pentosan inhibits angiogenesis in vitro and suppresses prostate tumor growth in vivo.

Pentosan polysulfate (PPS) is a highly negatively charged polysaccharide which has activity against multiple tumor types in the preclinical setting. We demonstrate here that Pentosan inhibits the growth of the anaplastic Dunning R3327 rat prostate adenocarcinoma MAT-LyLu when treatment was started when the tumor was not palpable but has little effect against established tumors. This inhibition may be mediated by the effect of Pentosan on endothelial cells. Pentosan, in combination with hydrocortisone, inhibits endothelial cell motility and tubule formation in vitro and inhibits capillary formation in the chicken chorioallantoic membrane (CAM) assay. These data suggest that Pentosan may be a potent inhibitor of tumor-associated angiogenesis and may be an effective agent for the prevention and/or suppression of prostate cancer growth.

Nuclear matrix proteins in normal and breast cancer cells.

The progression from normal breast epithelium to a malignant phenotype may depend on changes in genetic events as well as failure of host mechanisms. Intermediate biomarkers are needed to more effectively identify malignant progression as well as to develop the potential for more specific treatments and prevention strategies. The nuclear matrix is the RNA-protein network which forms the skeleton of the nucleus and participates in DNA organization as well as multiple cellular functions. Nuclear matrix proteins have been demonstrated to be tissue and cell type specific as well as to reflect the state of cell differentiation and/or transformation. We prepared nuclear matrices from normal and cancer breast tissue from 10 patients with infiltrating ductal carcinoma of the breast as well as the MCF-10 mortal, immortal, and transfected breast cell lines. Nuclear matrices derived from normal human breast tissue and tumor tissue share common nuclear matrix proteins as well as demonstrate specific changes which appear to occur with the acquisition of the cancer phenotype. The MCF-10 cell lines demonstrate a phenotype that is intermediate between the normal and cancer tissue. These data suggest that the nuclear matrix may be an important biomarker in the pathogenesis of breast cancer.

Inhibition of prostate cancer growth by estramustine and etoposide: evidence for interaction at the nuclear matrix.

Metastatic prostate cancer which is refractory to hormone therapy remains an incurable disease for which there is no effective therapy. We have begun to investigate the nuclear matrix, the RNA-protein network of the nucleus that plays an important role in DNA replication and gene expression, as a target for cancer chemotherapy. It was postulated that estramustine phosphate (EMP), an estradiol-nitrogen mustard conjugate that binds to the nuclear matrix, might enhance the cytotoxicity of etoposide (VP-16), a topoisomerase II inhibitor that acts at the level of the nuclear matrix. In a nascent DNA synthesis assay, EMP and etoposide interact to selectively inhibit new DNA synthesis on the nuclear matrix. In vitro, EMP and etoposide appeared to act synergistically to inhibit the growth of the metastatic Dunning rat prostate adenocarcinoma cell line Mat-LyLu as well as the metastatic human prostate adenocarcinoma cell line PC-3. In vivo, EMP and etoposide inhibited prostate adenocarcinoma growth in the Dunning Copenhagen rat model. These data have formed the basis of a Phase I/II clinical trial to examine the effect of EMP and etoposide in patients with stage D hormone-refractory prostate cancer.