Synthetic inhibitor of matrix metalloproteases decreases tumor growth and metastases in a syngeneic model of rat prostate cancer in vivo.

Members of the matrix metalloprotease (MMP) family are implicated in the progression of several malignancies including prostate cancer due to their ability to break down extracellular matrix (ECM) components. In this study, we have evaluated the ability of a synthetic MMP inhibitor (A-177430) to block tumor growth and metastases in a syngeneic model of rat prostate cancer. In an in vitro substrate assay, A-177430 exhibited nanomolar potency (IC(50) 2-6 nM) against the enzymatic activity of several MMPs. For in vivo studies, male Copenhagen rats were injected s.c. with Mat Ly Lu rat prostate cancer cells (1 x 10(6) cells ) into the right flank and animals were administered i.p.with different doses (10-100 mg/kg per day) of A-177430 for 16 days. Administration of A-177430 resulted in a dose-dependent decrease in tumor volume as compared to a control group of animals receiving vehicle alone. The maximum dose (100 mg/kg per day) of A-177430 exhibited complete arrest in tumor growth and prevented the development of macroscopic tumor metastases to lungs without exhibiting any noticeable side effects. Histologic examination of primary tumors from experimental animals showed extensive tumor necrosis and decreased tumor angiogenesis as determined by factor VIII staining of primary tumors following A-177430 treatment. These primary tumors from experimental animals also exhibited a significant increase in tumor cell DNA fragmentation as determined by TUNEL assay. Collectively, these results demonstrate the ability of MMP inhibitors to block tumor growth and metastases by blocking ECM degradation and by inhibiting tumor angiogenesis and promotion of prostate cancer cell apoptosis in vivo.

Regulated expression of HIV-1 Rev function in mammalian cell lines.

In order to facilitate further investigation of Rev function, we have generated two systems for the inducible expression of Rev in mammalian cell lines (HeLa and U937) using either a tetracycline-regulated promoter or fusion of Rev to a modified form of the hormone binding domain of the estrogen receptor. In the case of the fusion of Rev to the modified hormone binding domain of the estrogen receptor, we demonstrated induction of Rev function in response to tamoxifen administration to levels comparable to that of the unmodified Rev protein. Subsequently, U937 lines were generated that retained the observed pattern of hormone-dependent function of the Rev fusion protein. In the case of the tetracycline-regulated system, cell lines (both HeLa and U937) were generated that displayed tight regulation of Rev. In the case of the HeLa cell lines, they were used for the subsequent generation of stable cell lines expressing either HIV-1 env or chloramphenicol acetyl transferase (CAT) in a Rev-dependent fashion. Using the latter cell lines, we demonstrate the ability to control Rev expression over a broad concentration range and find that, as soon as Rev expression is detectable, induction of Rev-dependent gene expression is also observed.

Over-production of parathyroid hormone-related peptide results in increased osteolytic skeletal metastasis by prostate cancer cells in vivo.

Prostate carcinoma is one of the most common malignancies affecting males, resulting in a high rate of morbidity and mortality. This hormone-dependent malignancy is characteristically associated with a high incidence of osteoblastic skeletal lesions. However, osteolytic lesions invariably accompany blastic ones. In the current study, we assessed the role of parathyroid hormone-related peptide (PTHRP), a potent bone-resorbing agent, in contributing to bone breakdown and prostatic skeletal metastasis using a syngeneic rat prostate cancer model. The full-length cDNA encoding rat PTHRP was subcloned as a Hind III insert in the sense orientation into the mammalian expression vector pRc-CMV to generate the expression vector pRc-PTHRP-S. Both control and experimental plasmids were stably transfected into low PTHRP-producing Dunning R3227, Mat Ly Lu rat prostate cancer cells. Following antibiotic selection, monoclonal cell lines expressing the highest amount of PTHRP mRNA and immunoreactive PTHRP were selected as experimental tumor cells for further analysis. Increased PTHRP production by these cells had no significant effect in vitro on the invasive capacity of these cells. Control and experimental cells were inoculated s.c. into the right flank or by the intracardiac (i.c.) route into the left ventricle of inbred male Copenhagen rats. No skeletal metastases occurred after s.c. injection with either cells. In contrast, i.c. inoculation led to lumbar vertebra metastasis and consequent hind-limb paralysis. Furthermore, histological examination of skeletal metastases in experimental animals showed a marked increase in osteoclastic activity. Our results demonstrate that PTHRP can increase osteoclastic osteolysis in the presence of focal osseous prostate cancer metastases and may contribute to the lytic lesions which generally accompany osteoblastic lesions in prostate cancer.

Effect of nucleoside analogue BCH-4556 on prostate cancer growth and metastases in vitro and in vivo.

Prostate carcinoma is a common malignancy among males that results in high morbidity and mortality. Here, we have evaluated the capacity of nucleoside analogue BCH-4556 [beta-L-(-)-dioxolane-cytidine] to control prostate cancer progression in our syngeneic model of rat prostate cancer using the rat prostate cancer cell line Dunning R3227 Mat Ly Lu. Different concentrations (50 microM-1 mM) of BCH-4556 resulted in a marked decrease and, eventually, a complete arrest of Mat Ly Lu cell growth in vitro. Cells were inoculated via intracardiac (i.c.) route into the left ventricle or by s.c. injection into the right flank of male Copenhagen rats. Following i.c. inoculation, experimental animals were treated with 75 mg/kg BCH-4556 twice a day or with vehicle alone for 6 consecutive days, starting from day 1 or day 3 post-tumor cell inoculation. Control and experimental animals were monitored for the development of tumor metastases. Treatment with BCH-4556 did not significantly change the development of skeletal metastases and, hence, the time of development of hind limb paralysis. Experimental animals, however, did show a marked reduction in the incidence and size of tumor metastases at the adrenal glands. Following the development of palpable tumors after s.c. injection of Mat Ly Lu cells on day 8 post tumor cell inoculation, animals were treated i.p. with 25-75 mg/kg BCH-4556 twice a day or with vehicle alone for 6 consecutive days. Control animals developed large primary tumors and macroscopic metastasis to lungs, lymph nodes, kidneys, and spleen. In contrast, experimental animals receiving BCH-4556 showed a marked decrease in tumor volume and metastases after the last injection of BCH-4556. The maximum dose of BCH-4556 (75 mg/kg twice a day) caused a complete arrest in tumor growth that was maintained for up to 4-6 days without any evidence of cytotoxicity. These antitumor effects of BCH-4556 were more marked than those of doxorubicin in blocking tumor growth in this model of prostate cancer, and it continued to be effective following three cycles of treatment, without manifesting any signs of drug resistance.

Expression and characterization of recombinant rat parathyroid hormone-related peptide (1-141) and an amino-terminally-truncated analogue (38-141).

We have synthesized and purified recombinant parathyroid hormone related peptide (PTHrP (1-141)) and PTHrP (38-141) using an E. coli system that requires minimal purification. The cDNAs encoding PTHrP (1-141) and PTHrP (35-141) respectively were inserted into the multiple cloning site of the pTrcHis-B bacterial expression plasmid. The PTHrP encoded sequences were thereby fused at their NH2-termini to six histidine residues within the fusion protein. The recombinant plasmids were transfected into E. coli cells and PTHrP synthesis was induced by addition of 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C. The recombinant fusion proteins were purified by binding of the histidine residues to a nickel column followed by gradient elusion and dialysis. PTHrP (1-141) was released from its fusion protein by cyanogen bromide cleavage, whereas PTHrP (38-141) was released by enzymatic digestion with enterokinase. This rapid isolation method resulted in pure PTHrP (1-141) and (38-141) as judged by SDS-polyacrylamide gel electrophoresis and NH2-terminal sequence analysis. PTHrP (1-141) stimulated cAMP accumulation and mobilized intracellular calcium ([Ca2+]i) in UMR106 osteoblast-like cells, and stimulated phosphate transport in OK/E renal cells, whereas PTHrP (38-141) was inert in these bioassays. Availability of PTHrP and its NH2-terminally truncated analogue, which lacks the sequence necessary for its hypercalcemic actions, will enable their biological activities to be examined in greater detail.

Prevention of prostate-cancer metastasis in vivo by a novel synthetic inhibitor of urokinase-type plasminogen activator (uPA).

Urokinase-type plasminogen activator (uPA) is a serine protease associated with tissue remodeling, cellular invasiveness, matrix degradation and tumor growth. Over-expression of uPA by the rat prostate-cancer cell line Dunning R3227, Mat LyLu, results in increased tumor metastasis to several non-skeletal and skeletal sites. Histological examination of these skeletal lesions has shown them to be primarily osteoblastic. In the present study we examined the capacity of a selective inhibitor of uPA enzymatic activity, 4-iodo benzo[b]thiophene-2-carboxamidine (B-428), to prevent the development of tumor growth and invasiveness in a syngeneic model of rat prostate cancer using a Dunning R3227 cell line over-expressing rat uPA. Male Copenhagen rats were inoculated s.c. with experimental cells into the right flank and continuously infused i.p. with either vehicle alone or uPA inhibitor for 2 to 3 weeks. Animals were killed at timed intervals and evaluated for the development of tumor growth and metastasis. Serum from these animals was collected to examine any signs of nephrotoxicity. Control animals receiving vehicle alone developed large tumors at the site of inoculation as well as macroscopic metastases in the lungs, kidney, spleen and lymph nodes. In contrast, experimental animals receiving uPA inhibitor showed a marked decrease in primary tumor volume and weight as well as in the development of tumor metastases. The occasional tumor metastases observed after infusion of B-428 were significantly smaller than those observed in vehicle controls. These effects of B-428 were found to be dose-dependent without any adverse effects on the renal function of experimental animals. These studies demonstrate that uPA-specific inhibitors can decrease primary tumor volume and invasiveness as well as metastasis in a model of prostate cancer where uPA has been implicated as a major pathogenetic factor.