Isolation of a prostate carcinoma cell proliferation-inhibiting factor from human seminal plasma and its similarity to transforming growth factor beta.

Human seminal plasma (SP) has been known to contain both growth-inhibitory and -stimulatory factors. We attempted to identify a factor that inhibited DNA synthesis in some metastatic prostate cancer cell lines. The SP factor was sensitive to digestion by trypsin, but its activity increased after boiling or dialysis against 1 M acetic acid, by 3- to 4-fold. The SP factor was partially purified using a cation-exchange resin. Apparent molecular mass determination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed it to be a M(r) 25,000 protein, and M(r) 13,000 after reduction. This protein strongly inhibited DNA synthesis in two metastatic androgen-independent human prostatic carcinoma cell lines (PC3 and DU145) and the Dunning R3327G rat prostatic adenocarcinoma. It was ineffective on androgen-dependent LNCaP cells. The proliferation-inhibiting activity of this SP protein was specifically and completely abolished by a neutralizing anti-transforming growth factor beta (TGF-beta) antiserum. Furthermore, immunoblot analysis using the anti-TGF-beta antiserum showed the similarity of this protein to TGF-beta. The maximum concentration of this protein in SP was 165 +/- 11.7 ng/ml (mean +/- SD), of which only one-fourth may be present in active form under normal conditions. Identification of a TGF-beta-like protein in SP might also explain the variety of growth and immune modulation properties of human SP.

Secretion of matrix metalloproteinases and their inhibitors (tissue inhibitor of metalloproteinases) by human prostate in explant cultures: reduced tissue inhibitor of metalloproteinase secretion by malignant tissues.

Unregulated secretion of matrix metalloproteinases (MMPs) or their endogenous protein inhibitors (tissue inhibitor of metalloproteinases, TIMPs) has been implicated in tumor invasion and metastasis. Species of MMPs and TIMPs secreted by epithelial cultures of normal, benign, and malignant prostate were identified and their levels were compared. Fragments of fresh tissue were cultured in a serum-free medium that supported the outgrowth of prostatic epithelial cells. Biochemical analysis of the conditioned media by gelatin zymography and enzyme assays showed that both normal and neoplastic tissues secreted latent and active forms of both M(r) 72,000 type IV collagenase (MMP-2) and M(r) 92,000 gelatinase (MMP-9). However, conditioned media from malignant prostate explants contained a higher proportion of the active form of MMP-2. Significant amounts of free TIMPs were secreted by normal juvenile and adult prostates, but they were either markedly reduced or not detectable in conditioned media from neoplastic tissues. These findings suggest that there is an imbalance of secretion between MMPs and TIMPs in prostatic carcinoma.

Association of elevated levels of hyaluronidase, a matrix-degrading enzyme, with prostate cancer progression.

Components of extracellular matrix and the matrix-degrading enzymes are some of the key regulators of tumor metastasis and angiogenesis. Hyaluronic acid (HA), a matrix glycosaminoglycan, is known to promote tumor cell adhesion and migration, and its small fragments are angiogenic. We have compared levels of hyaluronidase, an enzyme that degrades HA, in normal adult prostate (NAP), benign prostate hyperplasia (BPH) and prostate cancer (CaP) tissues and in conditioned media from epithelial explant cultures, using a sensitive substrate(HA)-gel assay and an ELISA-like assay. The results show a significant elevation (3-10-fold) of this enzyme in tumor tissues compared to that in NAP and BPH tissues. Furthermore, the hyaluronidase levels in tissues correlates well with the tumor grade. For example, the concentrations in a locally extended CaP lesion (191 +/- 7.9 milliunits/mg protein), and low-grade tumors (9.4 +/- 1.4 milliunits/mg protein), respectively. Among the primary epithelial explant cultures, CaP cultures secrete at least 10-fold higher levels of hyaluronidase that those secreted by NAP and BPH cultures. Furthermore, among the established prostate cancer cell lines, DU145, an androgen-unresponsive metastatic line, secretes 4-fold more hyaluronidase than LNCaP, an androgen-responsive and relatively well-differentiated cell line. We also show that prostatic hyaluronidase has an apparent M(r) approximate to 55,000, a pH optimum of 4.6, and is distinct from other well-characterized hyaluronidases.

Human prostatic inhibin suppresses tumor growth and inhibits clonogenic cell survival of a model prostatic adenocarcinoma, the Dunning R3327G rat tumor.

Prostatic inhibin (PI) is a M(r) 10,700 protein found in human seminal plasma and is secreted by the prostate. Recognition of alteration of PI levels in prostatic diseases prompted us to investigate its effect on an animal prostatic adenocarcinoma model, the Dunning R3327G rat tumor. PI not only inhibited in vitro growth of tumor cells but also suppressed tumor growth in vivo. A dose-dependent inhibition of both the clonogenic cell growth and rate of proliferation (DNA synthesis) was observed in tumor cell cultures incubated with purified PI. These inhibitory activities were similar in both androgen-dependent and androgen-independent Dunning tumor cell lines. A functional decapeptide of PI was also found to inhibit Dunning tumor cell colonies in a dose-dependent manner. Daily injection of purified PI into tumor-bearing rats suppressed the tumor growth. A 58% reduction in tumor weight and a 2-fold reduction in tumor growth rate were observed over a 15-day treatment period. Continued treatment with PI significantly suppressed the tumor growth rate by nearly 3-fold. These findings clearly demonstrate a potential application of PI for treating human prostatic adenocarcinoma.

Flow cytometric analysis of androgen receptor expression in human prostate tumors and benign tissues.

Androgen receptor (AR) plays an important role in growth and hormonal therapy of human prostate tumors. Immunohistochemical analysis of AR expression, a nonquantitative technique, is currently used for screening of receptor expression in prostate tissues. The present report describes a laser flow cytometric method for monitoring AR expression in human cell lines and in archival formalin-fixed paraffin-embedded prostate tissues and tumors. Multiparametric flow analysis can be used for simultaneous detection of other cellular markers (e.g., DNA aneuploidy), and by gated analysis, AR expression in subpopulations of a tumor can be quantitatively determined.

The in vitro evaluation of 25-hydroxyvitamin D3 and 19-nor-1alpha,25-dihydroxyvitamin D2 as therapeutic agents for prostate cancer.

Prostate cancer cells contain specific receptors [vitamin D receptors (VDRs)] for 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), which is known to inhibit the proliferation and invasiveness of these cells. These findings support the use of 1alpha,25(OH)2D3 for prostate cancer therapy. However, because 1alpha,25(OH)2D3 can cause hypercalcemia, analogues of 1alpha,25(OH)2D3 that are less calcemic but that exhibit potent antiproliferative activity would be attractive as therapeutic agents. We investigated the effects of two different types of less calcemic vitamin D compounds, 25-hydroxyvitamin D3 [25(OH)D3] and 19-nor-1alpha,25-dihydroxyvitamin D2 [19-nor-1,25(OH)2D2], and compared their activity to 1alpha,25(OH)2D3 on (a) the proliferation of primary cultures and cell lines of human prostate cancer cells; and (b) the transactivation of the VDRs in the androgen-insensitive PC-3 cancer cell line stably transfected with VDR (PC-3/ VDR). 19-nor-1alpha,25(OH)2D2, an analogue of 1alpha,25(OH)2D3 that was originally developed for the treatment of parathyroid disease, has been shown to be less calcemic than 1alpha,25(OH)2D3 in clinical trials. Additionally, we recently showed that human prostate cells in primary culture possess 25(OH)D3-1alpha-hydroxylase, an enzyme that hydroxylates the inactive prohormone, 25(OH)D3, to the active hormone, 1alpha,25(OH)2D3, intracellularly. We reasoned that the hormone that is formed intracellularly would inhibit prostate cell proliferation in an autocrine fashion. We found that 1alpha,25(OH)2D3 and 19-nor-1alpha,25(OH)2D2 caused similar dose-dependent inhibition in the cell lines and primary cultures in the [3H]thymidine incorporation assay and that both compounds were significantly more active in the primary cultures than in LNCaP cells. Likewise, 25(OH)D3 had inhibitory effects comparable to those of 1alpha,25(OH)2D3 in the primary cultures. In the chloramphenicol acetyltransferase (CAT) reporter gene transactivation assay in PC-3/ VDR cells, 1alpha,25(OH)2D3 and 19-nor-1alpha,25(OH)2D2 caused similar increases in CAT activity between 10(-11)and 10(-9) M. Incubation of PC-3/VDR cells with 5 x 10(-8) M 25(OH)D3 induced a 29-fold increase in CAT activity, similar to that induced by 10(-8) M 1alpha,25(OH)2D3. In conclusion, our data indicate that 25(OH)D3 and 19-nor-1alpha,25(OH)2D2 represent two different solutions to the problem of hypercalcemia associated with vitamin D-based therapies: 25(OH)D3 requires the presence of 1alpha-hydroxylase, whereas 19-nor-1alpha,25(OH)2D2 does not. Both drugs are approved for human use and may be good candidates for human clinical trials in prostate cancer.

Human prostate cells synthesize 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3.

Epidemiological and laboratory data support a role for vitamin D in the growth and differentiation of human prostatic cells. These findings prompted us to ask whether prostatic cells could convert 25-hydroxyvitamin D3 (25-OH-D3), the major circulating metabolite of vitamin D3, to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the hormonally active metabolite, in a manner similar to cultured human keratinocytes. Therefore, we investigated three well-characterized human prostate cancer cell lines, LNCaP, DU 145, and PC-3; two primary cultures of cells derived from noncancerous human prostates (one normal and one benign prostatic hyperplasia); and primary cultures of normal human keratinocytes for their ability to synthesize 1,25(OH)2D3. Assays were performed in the presence of 25-OH-D3 as the enzyme substrate and 1,2-dianilinoethane, an antioxidant and free radical scavenger, and in the presence and absence of clotrimazole, a cytochrome P450 inhibitor. DU 145 and PC-3 cells produced 0.31 +/- 0.06 and 0.07 +/- 0.01 pmol of 1,25(OH)2D3/mg protein/h, respectively. No measurable 1,25(OH)2D3 was detected in LNCaP cells. The normal and benign prostatic hyperplasia primary cultures and keratinocyte cultures produced 3.08 +/- 1.56, 1.05 +/- 0.31, and 2.1 +/- 0.1 pmol of 1,25(OH)2D3/mg protein/h, respectively, using a calf thymus receptor binding assay to measure 1,25(OH)2D3 in the presence of 1,2-dianilinoethane. The identity of the analyte as 1,25(OH)2D3 was supported by high performance liquid chromatography using [3H]25-OH-D3 as the enzyme substrate and a solvent system that is specific for 1,25(OH)2D3. The production of 1,25(OH)2D3 in the prostate cancer cell lines and in the primary cultures was completely inhibited in the presence of clotrimazole. This report demonstrates that two of three human prostate cancer cell lines, as well as primary cultures of noncancerous prostatic cells, possess 1alpha-hydroxylase activity and can synthesize 1,25(OH)2D3 from 25-OH-D3. Together with recent data indicating that 1,25(OH)2D3 inhibits the invasiveness of human prostate cancer cells (G. G. Schwartz et al., Cancer Epidemiol. Biomark. Prev., 6: 727-732, 1997), these data suggest a potential role for 25-OH-D3 in the chemoprevention of invasive prostate cancer.

Inhibition of prostate cancer metastasis in vivo: a comparison of 1,23-dihydroxyvitamin D (calcitriol) and EB1089.

The steroid hormone 1,25-dihydroxyvitamin D [1,25(OH)2D, also known as calcitriol] is known to inhibit the proliferation and to promote the differentiation of human prostate cancer cells. Additionally, we showed that 1,25(OH)2D markedly inhibits the invasiveness of human prostate cancer cells in vitro (G. G. Schwartz et al., Cancer Epidemiol. Biomark. Prev., 6: 727-732, 1997). These properties support the use of 1,25(OH)2D as differentiation therapy in prostate cancer. However, the use of 1,25(OH)2D in vivo is limited by the risk of hypercalcemia. We therefore compared the effects of 1,25(OH)2D and of EB1089, an analogue of 1,25(OH)2D with reduced calcemic effects, in an in vivo model of androgen-insensitive metastatic prostate cancer, the rat Dunning MAT LyLu prostate cancer model. Tumor growth and metastasis were studied using Copenhagen rats given s.c. injections of MAT LyLu cells. Fifty male rats were divided into five groups of 10 rats each. Four experimental groups received i.p. injections of low and high doses of 1,25(OH)2D and EB1089 (0.5 and 1.0 microg/kg, low and high, respectively). A control group received injections of vehicle only. Tumor volumes were measured three times per week. Rats were weighed weekly. The number of metastases to the lungs and the extent of hypercalcemia were evaluated. Compared with controls, tumor volumes were significantly smaller in all experimental groups. Similarly, the number of lung metastases (number of foci/lung) was reduced markedly by both 1,25(OH)2D and EB1089. Control rats developed 22.7 (+/- 1.98 SE) tumor foci per lung. Rats treated with 1,25(OH)2D and with EB1089 (1.0 microg/kg) developed 10.4 (+/- 2.81) and 7.70 (+/- 1.29) tumor foci, respectively (P < 0.001 and P < 0.0001, respectively; drug versus control). Compared with controls (10.79 +/- 0.1 mg/dl), serum calcium levels were significantly elevated in both 1,25(OH)2D and EB1089-treated rats (P < 0.01). However, EB1089 was significantly less calcemic than 1,25(OH)2D (12.59 +/- 0.21 mg/dl versus 14.47 +/- 0.46 mg/dl; 1.0 microg/kg; P < 0.001). Rats treated with 1,25(OH)2D showed marked weight loss: 20.0 +/- 1.9% and 26.3 +/- 1.7% of their initial weight (low and high doses, respectively, P < 0.001). Weight loss was significantly lower in rats treated with EB1089 at the high dose 8.4 (+/- 2.9) %. Moreover, rats treated with low-dose EB1089 gained 5.2 (+/- 3.7) % of their initial weight. In conclusion, 1,25(OH)2D and EB1089 showed marked and equivalent inhibition of prostate cancer metastasis in vivo. EB1089 was significantly less calcemic than 1,25(OH)2D and did not induce severe weight loss. This is the first report of a vitamin D analogue that significantly inhibits prostate cancer metastasis in vivo and that does so without producing cachexia or unacceptable hypercalcemia.

Cytotoxic activity and inhibition of tumor cell invasion by derivatives of a chemically modified tetracycline CMT-3 (COL-3).

Tetracyclines such as chlortetracycline and doxycycline with antimicrobial activity were reported to possess cytostatic and cytotoxic activity against mammalian tumor cells, often at high doses. Non-antimicrobial chemically modified tetracyclines (CMTs), with limited systemic toxicity but with significant tumor cell toxicity and antimetastatic activity, are attractive for long term treatment for cancer. We recently reported one such CMT, 6-deoxy,6-demethyl 4-dedimethylamino tetracycline (CMT-3) is a potent anti-tumor and anti-metastatic drug. Here we report on the anti-cell proliferation and anti-invasive activity of five nitro derivatives of CMT-3 (CMT-3N). All the five CMT-3Ns (CMT-302, CMT-303, CMT-306, CMT-308 and CMT-316) inhibited in vitro cell proliferation of prostate cancer cells. The 50% growth inhibition concentration (IC(50)) of CMT-3Ns was similar to that of CMT-3. Although CMT-3 was by far the most potent anti-cell proliferation drug, all CMT-3Ns except CMT-303 and CMT-308 had similar anti-cell proliferation activity (IC(50): 2.5 -5.7 microg/ml). IC(50)s for CMT-303 and CMT-308 were approximately 8.1 and -12.4 microg/ml, respectively. Activity against tumor cell invasion was tested in vitro using the Matrigel invasion assay. All CMT-3Ns had similar anti- invasive activity. While cytotoxic activity of CMT-3 was strongly associated with cell death-effector caspase activation, mitochondrial permeablization and apoptosis, the CMT-3Ns weakly induced apoptosis and did not activate Caspase-3. However, the CMT-3Ns were able to induce mitochondrial permeabilization. This dichotomous mechanism of cytotoxic activity of CMTs may have significance in their selection for clinical application.

A sandwich enzyme-linked immunoadsorbent assay for detection of murine macrophage colony-stimulating factor (CSF-1).

A simple three-layer sandwich enzyme-linked immunoadsorbent assay (sandwich-ELISA) has been developed for murine macrophage colony-stimulating factor-1 (CSF-1) using the two monoclonal antibodies on which we recently reported (J. Immunol. (1988) 141, 483). The anti-CSF-1 monoclonal antibodies used in this assay recognize different epitopes of the same antigen, thereby permitting the detection of low amounts of CSF-1. This assay is specific to murine CSF-1. Recombinant human macrophage colony-stimulating factor, murine GM-CSF, or IL-3, either alone or together with CSF-1, does not interfere with the assay. The advantage of this assay over other reported immunoassays for CSF-1 is that radiolabeled or large quantities of purified CSF-1 are not required. This sandwich-ELISA compares favorably with other assays in its rapidity, simplicity, and sensitivity.

Tear fluid gelatinase B activity correlates with IL-1alpha concentration and fluorescein clearance in ocular rosacea.

PURPOSE: To correlate tear fluorescein clearance with interleukin-1alpha (IL-1alpha) concentration and gelatinase B (matrix metalloproteinase [MMP]-9) activity in the tear fluid of patients with ocular rosacea and normal control subjects. METHODS: Gelatinase activity was evaluated by gelatin zymography in tear fluid obtained from 13 patients with ocular rosacea (including 1 patient with recurrent epithelial erosion, 2 with recurrent peripheral corneal infiltrates and vascularization, and 2 patients with epithelial basement membrane dystrophy) and 13 normal subjects with normal aqueous tear production and no irritation symptoms. Tear fluorescein clearance was evaluated by measuring fluorescence in tear fluid collected from the inferior meniscus 15 minutes after instillation of 5 microl of 2% Na-fluorescein with a CytoFluor II fluorometer. Pro-MMP-9 and IL-1alpha concentrations in the tear fluid were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared with normal control subjects, patients with ocular rosacea had a greater delay of tear fluorescein clearance (P < 0.001), a higher tear IL-1alpha concentration (P < 0.001), and a greater pro-gelatinase B (92 kDa) activity (P < 0.001) in their tear fluid. The 84-kDa active form of gelatinase B was observed in 46% of the rosacea tear samples and none of the controls. The zymographic results were confirmed by ELISA that showed a significantly greater concentration of pro-MMP-9 (92 kDa) in the tear fluid of rosacea patients than controls. Delayed tear clearance was correlated with elevated tear IL-1alpha concentration (p=0.67, P < 0.001) and increased tear gelatinase B activity (p=0.84, P < 0.001). Tear IL-1alpha concentration was correlated with tear gelatinase B activity (p=0.58, P < 0.002). CONCLUSIONS: Gelatinase B (MMP-9) activity is greater in patients with ocular rosacea than in normal eyes. The majority of this activity is due to 92-kDa proform of this enzyme. This activity is correlated with delayed tear clearance and tear fluid concentration of interleukin-1alpha, a proinflammatory cytokine that has been reported to stimulate gelatinase B production. Elevated gelatinase B activity in ocular rosacea may be involved in the pathogenesis of the irritation symptoms, recurrent epithelial erosions, vascularization, and epithelial basement membrane dystrophy that develops in the corneas of patients with this condition.

Doxycycline inhibition of interleukin-1 in the corneal epithelium.

PURPOSE: To evaluate the effect of doxycycline on the regulation of interleukin (IL)-1 expression and activity in human cultured corneal epithelium. METHODS: Human corneal limbal epithelium (HLE) was cultured from explants prepared from limbal rings of donor corneas. Primary cultured limbal epithelial cells were treated with either 10 microg/ml lipopolysaccharide (LPS), LPS with 10 microg/ml doxycycline, or LPS with 0.1 mg/ml methylprednisolone (MP) for 24 hours. The intracellular and supernatant protein amounts of IL-1alpha, the precursor and mature forms of IL-1beta, IL-1 receptor antagonist (IL-1 RA), and the intracellular level of IL-1beta-converting enzyme (ICE) were measured with enzyme-linked immunosorbent assays (ELISAs). Western blot analysis was performed to evaluate IL-1 RA protein. mRNA steady state amounts were determined by RNase protection assay (RPA) for IL-1alpha, IL-1beta, IL-1 RA, and ICE. RESULTS: LPS increased the mRNA and protein amounts of intracellular and released IL-1alpha, mature IL-1beta, and IL-1 RA. Doxycycline inhibited the LPS-induced IL-1beta increase in the mRNA and protein amounts in the corneal epithelium and upregulated the expression of the anti-inflammatory IL-1 RA protein. In addition, doxycycline reduced the steady state level of the cellular ICE protein but did not affect the level of ICE transcripts. IL-1beta secreted to the conditioned media of HLE was functionally active in inducing matrix metalloproteinase (MMP)-1 and MMP-3 in cultured corneal fibroblasts. Doxycycline significantly decreased IL-1beta bioactivity in the supernatants from LPS-treated corneal epithelial cultures. These effects were comparable to those induced by the corticosteroid, MP. CONCLUSIONS: Doxycycline can suppress the steady state amounts of mRNA and protein of IL-beta and decrease the bioactivity of this major inflammatory cytokine. These data may partially explain the clinically observed anti-inflammatory properties of doxycycline. The observation that doxycycline was equally potent as a corticosteroid, combined with the relative absence of adverse effects, makes it a potent drug for a wide spectrum of ocular surface inflammatory diseases.

Regulation of MMP-9 activity in human tear fluid and corneal epithelial culture supernatant.

PURPOSE: To evaluate human corneal epithelial culture supernatant and tear fluid for the presence of activators and inhibitors of matrix metalloproteinase (MMP)-9, MMP-3, and tissue inhibitor of metalloproteinase (TIMP)-1, respectively, and to evaluate the effect of MMP-3 on the activation of MMP-9 in these specimens. METHODS: Unstimulated tear fluid was collected from patients with ocular rosacea and normal control subjects. Levels of MMP-9, MMP-3, and TIMP-1 were determined by enzyme-linked immunosorbent assay (ELISA) and/or immunoblot analysis. Supernatants from primary human corneal epithelial cultures and human tear fluid were incubated with MMP-3. Cultured epithelial cells and their supernatants were also treated with doxycycline before MMP-3 was added. Gelatin zymography was used to identify activated 82-kDa MMP-9. MMP-9 activity was assessed with a commercial MMP-9 activity assay system. RESULTS: MMP-9 and TIMP-1 were detected at significantly higher concentrations in rosacea-affected than in normal tear fluids. MMP-3 was detected exclusively in the tear fluid of patients with ocular rosacea who had corneal epithelial disease. Treatment of the supernatant and tear fluid with MMP-3 resulted in two bands with molecular weights of 92 kDa and 82 kDa, representing pro-MMP-9 and activated MMP-9, respectively. Doxycycline added to the conditioned media did not affect activation of MMP-9 by MMP-3. However, 24-hour treatment of corneal epithelial cultures with doxycycline resulted in a lower concentration and activity of MMP-9 in their supernatants. CONCLUSIONS: MMP-9 and TIMP-1 are produced by the human corneal epithelium and are present in tear fluid. MMP-3 alone is sufficient to activate MMP-9 on the ocular surface. Doxycycline does not directly inhibit this activation by MMP-3, but it decreases MMP-9 activity when added to corneal epithelial cultures.

Inhibition of aromatase activity and growth suppression by 4-methoxy-4-androstene-3,17-dione in an androgen sensitive human prostatic carcinoma cell line.

Aromatization of testosterone to estradiol was investigated in a human prostatic carcinoma cell line, LNCaP. A saturable, dose and time-dependent aromatization of testosterone was observed. Kinetic parameters, Km (201 nM) and V(max) (0.76 pmol/h) per mg) and also the inhibition constants (Ki) for various aromatase inhibitors were calculated from standard Lineweaver-Burke plots. The steroidal aromatase inhibitor 4-hydroxy-4-androstene-3,17-dione (4-OHA) and its derivative, 4-methoxy-4-androstene-3,17-dione (4-OMA), inhibited aromatization of testosterone in a competitive pattern of inhibition. The derivative 4-OMA is the stronger inhibitor of the two, with an apparent Ki of 1.12 microM, whereas the apparent Ki of 4-OHA is 3.28 microM. Long term incubation with 4-OMA suppressed proliferative activity of LNCaP cells in the presence of physiological levels of testosterone (10(-10) M to 10(-7) M). In contrast, 4-OHA was a growth promoter. These results suggest a potential role for aromatase in hormone responsive prostate cancer.

Modulation of aromatase activity by growth factors in an androgen sensitive human prostate cancer cell line, LNCaP.

The effects of steroids and peptide growth factors on aromatase activity in an androgen sensitive human prostate cancer cell line (LNCaP) were investigated. Factors were selected based on their observed modulation of the enzyme in other tissues. Incubation with epidermal growth factor and transforming growth factor-I decreased aromatase activity in LNCaP cells by 25-40%. Insulin like growth factor-1, dexamethasone, dibutyryl cAMP and phorbol 12-myristate 13-acetate, all of which are modulators of aromatase in other tissues, had no significant effect on aromatase activity in LNCaP cells. In addition, the cAMP-dependent protein kinase and protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2 methylpiperazine (H-7) had no effect on the enzyme activity. Factors affecting prostatic aromatase may be distinct from those for other known species.

MMP inhibition in prostate cancer.

Matrix metalloproteinases (MMPs) play a significant role during the development and metastasis of prostate cancer (CaP). CaP cells secrete high levels of MMPs and low levels of endogenous MMP inhibitors (TIMPs), thus creating an excess balance of MMPs. Established CaP cell lines that express high levels of MMPs frequently metastasize to the bone and the lungs. Drugs such as Taxol and alendronate that reduce cell motility and calcium metabolism reduce bony metastasis of xenografted CaP tumors. We tested several synthetic, nontoxic inhibitors of MMPs that can be administered orally, including doxycycline (DC) and chemically modified tetracyclines (CMTs) on CaP cells in vitro and on a rat CaP model in vivo. Among several anti-MMP agents tested, CMT-3 (6-deoxy, 6-demethyl,4-de-dimethylamino tetracycline) showed highest activity against CaP cell invasion and cell proliferation. Micromolar concentration of CMT-3 and DC inhibited both the secretion and activity of MMPs by CaP cells. When tested for in vivo efficacy in the Dunning rat CaP model by daily oral gavage, CMT-3 and DC both reduced the lung metastases (> 50%). CMT-3, but not DC, inhibited tumor incidence (55 +/- 9%) and also reduced the tumor growth rate (27 +/- 9.3%). More significantly, the drugs showed minimum systemic toxicity. Ongoing studies indicate that CMT-3 may inhibit the skeletal metastases of CaP cells and delay the onset of paraplegia due to lumbar metastases. These preclinical studies provide the basis for clinical trials of CMT-3 for the treatment of metastatic disease.

Neuroendocrine peptides stimulate adenyl cyclase in normal and malignant prostate cells.

Elevations of intracellular cAMP in human prostate cancer cells have been shown to increase invasiveness and to promote neuronal differentiation. Since neuroendocrine peptides capable of activating adenyl cyclase are present in prostatic nerves and epithelial neuroendocrine cells, we investigated normal and malignant human prostate cells for changes in intracellular cAMP in response to the prostatic peptides vasoactive intestinal peptide (VIP), calcitonin (CT), and calcitonin gene-related peptide (CGRP). Normal prostate epithelial cells and LNCaP prostate cancer cells exhibited, respectively, 6- and 30-fold increases in intracellular cAMP in response to VIP. ALVA-31 and PPC-1 prostate cancer cells demonstrated 20- to 200-fold increases in cAMP in response to CGRP, while normal epithelial cells and LNCaP cells exhibited smaller (2- to 6-fold) responses. Only DU-145 cells increased cAMP substantially in response to CT. VIP receptor mRNA was identified by Northern blot analysis only in those cells that responded to VIP. CT receptor mRNA was identified only in DU-145 cells by polymerase chain reaction and Southern blot analysis. These results suggest that VIP and possibly CGRP receptors are likely to be present in both normal and malignant prostate cells. VIP or CGRP may regulate secretion of proteases by normal or prostate cancer cells and may influence epithelial cell differentiation.

Enhancement of radiation response of prostatic carcinoma by lonidamine.

Potential application of lonidamine (LND) to enhance radiation toxicity in prostate cancer was investigated using human prostate cancer cell lines and a rat tumor model (Dunning MAT LyLu). LND alone was cytotoxic with 50% inhibition concentration (IC50) between 0.5 and 0.8 mM. Preincubation with LND increased clonogenic toxicity of radiation. The sensitizer enhancement ratio was 1.8 to 2.2, depending on the cell line tested and it was consistent with inhibition of repair from potentially lethal damage. LND had limited effect in vivo on the Dunning model, consistent with its in vitro effect on the same cell line. LND did not alter the primary growth of the tumor. Fractionated irradiation caused a 40% decrease in tumor growth. LND injection (50 mg/kg) before fractionation did not cause any further decrease in tumor growth. Radiation dose fractionation and the combination treatment significantly reduced tumor metastasis in lungs.

Expression of CD44 in prostate cancer cells: association with cell proliferation and invasive potential.

High level expression of the cell surface adhesion molecule CD44 standard form and its splice variants, have been causally linked to tumor metastasis. In this study, we investigated the significance of CD44 expression in human prostatic carcinoma cells. Immunocytochemistry showed high level expression of CD44 in cells from a high grade prostate tumor, and two androgen-independent, invasive prostatic carcinoma lines, PC-3 and TSU-Pr1. Normal prostatic epithelial cells and LNCaP, a low metastatic, androgen sensitive cell line, expressed none to a very low level of CD44, although mRNA transcripts were detected in all cell lines. Immunoprecipitation detected two proteins of M(r) approximately 140 kDa and 210 kDa in PC-3, and predominantly the M(r) approximately 95 kDa protein in TSU Pr1, but none in LNCaP. Most importantly, a neutralizing antibody to CD44 inhibited cell proliferation and basement membrane invasive activity, suggesting a definitive role of CD44 in prostate tumor growth and metastasis.

Enhancement of radiation response of prostatic carcinoma by taxol: therapeutic potential for late-stage malignancy.

Radiation therapy for advanced prostate cancer has dose-limiting complications and often results in limited tumor control. A combination of radiation and taxol, a potential radiation sensitizer, may enhance therapeutic efficacy at currently used individual doses. Human prostatic carcinoma lines in vitro, and Dunning rat prostatic adenocarcinoma in vivo, were treated with taxol and radiation individually, and in combination. Cytotoxicity of taxol was comparable between androgen sensitive and insensitive lines, with 50% growth inhibition at 9.6 to 12.7 nM. Combining agents increased cytotoxicity, with a dose modifying ratio of 1.8 at 0.1% survival. Flow cytometry showed an enhancement of radiation toxicity associated with taxol-induced cell cycle phase arrest at G2/M. Injection of taxol (4 mg/kg/day x 5), radiation dose fractionation (1.5 Gy/day x 5) and their combination significantly delayed Dunning tumor growth. Adverse side effects were minimal. The results imply that combination of these agents may have clinical potential in prostate cancer treatment.