Antidepressant prescription patterns and polypharmacy in outpatient psychiatry: a cross-sectional study.

Background and aim: Prescription patterns of antidepressants have changed over the years with a shift towards newer antidepressants with better tolerability and safety. Polypharmacy is common in psychiatry settings. The study aimed to evaluate the antidepressant drug prescription pattern and polypharmacy in a psychiatry outpatient setting. Investigations: This prospective observational study was conducted in a psychiatric outpatient clinic. The medication use data of eligible patients were collected. In addition, the rationale of antidepressant medication prescription, the defined daily dosage (DDD), the prescribed daily dose (PDD), and the PDD to DDD ratio were assessed. The assessment of prescription polypharmacy was conducted utilizing the framework provided by the National Association of State Mental Health Program Directors. Results: Data from 131 patients was analyzed. Major depressive disorder (32.8%) was the most common disorder for which antidepressants were prescribed. The majority, 91 (69.4%), received monotherapy. Selective serotonin reuptake inhibitors were the most frequently prescribed drugs in 69 (52.7%). Mirtazapine was the most frequently 32(24.4%) prescribed drug. Escitalopram and mirtazapine were the most commonly prescribed combination therapy (4.6%). Antipsychotic medications (37.4%) were the most widely co-prescribed medications, along with antidepressants. The PDD to DDD ratio was less than 1 for mirtazapine and imipramine; they were ≥1 for others. Psychiatric polypharmacy was documented in 87.1% of prescriptions. The total polypharmacy was not significantly (p>0.05) associated with demographic, illness, and treatment-related variables. Conclusion: Selective serotonin reuptake inhibitors were the most commonly prescribed antidepressants, monotherapy, and combination therapy. A substantial amount of patients received concomitant administration of antidepressants or psychotropic drugs, warranting careful monitoring.

S-adenosylmethionine blocks tumorigenesis and with immune checkpoint inhibitor enhances anti-cancer efficacy against BRAF mutant and wildtype melanomas.

Despite marked success in treatment with immune checkpoint inhibitor (CPI), only a third of patients are responsive. Thus, melanoma still has one of the highest prevalence and mortality rates; which has led to a search for novel combination therapies that might complement CPI. Aberrant methylomes are one of the mechanisms of resistance to CPI therapy. S-adenosylmethionine (SAM), methyl donor of important epigenetic processes, has significant anti-cancer effects in several malignancies; however, SAM's effect has never been extensively investigated in melanoma. We demonstrate that SAM modulates phenotype switching of melanoma cells and directs the cells towards differentiation indicated by increased melanogenesis (melanin and melanosome synthesis), melanocyte-like morphology, elevated Mitf and Mitf activators' expression, increased antigen expression, reduced proliferation, and reduced stemness genes' expression. Consistently, providing SAM orally, reduced tumor growth and progression, and metastasis of syngeneic BRAF mutant and wild-type (WT) melanoma mouse models. Of note, SAM and anti-PD-1 antibody combination treatment had enhanced anti-cancer efficacy compared to monotherapies, showed significant reduction in tumor growth and progression, and increased survival. Furthermore, SAM and anti-PD-1 antibody combination triggered significantly higher immune cell infiltration, higher CD8+ T cells infiltration and effector functions, and polyfunctionality of CD8+ T cells in YUMMER1.7 tumors. Therefore, SAM combined with CPI provides a novel therapeutic strategy against BRAF mutant and WT melanomas and provides potential to be translated into clinic.

Regulation of parathyroid hormone-related peptide by estradiol: effect on tumor growth and metastasis in vitro and in vivo.

We evaluated the capacity of estradiol (E(2)) to regulate PTHrP production, cell growth, tumor growth, and metastasis to the skeleton in breast cancer. In estrogen receptor (ER)-negative human breast cancer cells, MDA-MB-231, and cells transfected with full-length cDNA encoding ER (S-30), E(2) caused a marked decrease in cell growth and PTHrP production, effects that were abrogated by anti-E(2) tamoxifen. E(2) also inhibited PTHrP promoter activity in S-30 cells. For in vivo studies, MDA-MB-231 and S-30 cells were inoculated into the mammary fat pad of female BALB/c nu.nu mice. Animals receiving S-30 cells developed tumors of significantly smaller volume compared with MDA-MB-231 tumor-bearing animals. This change in tumor volume was reversed when S-30 cells were inoculated into ovariectomized (OVX) hosts. Inoculation of MDA-MB-231 cells into the left ventricle resulted in the development of lesions in femora and tibia as determined by x-ray analysis. In contrast, these lesions were significantly smaller in volume and number in animals inoculated with S-30, and this lower incidence was reversed in OVX animals. Bone histological analysis showed that the tumor volume to tissue volume ratio was comparable with that seen by x-ray. Immunohistochemical analysis showed that PTHrP production was inhibited in S-30 group and restored to levels comparable to that seen in MDA-MB-231 tumor-bearing animals when S-30 cells were inoculated in OVX animals. Collectively these studies show that E(2) production is inversely correlated with PTHrP production and that the growth-promoting effect of PTHrP has a direct impact on tumor growth at both nonskeletal and skeletal sites.

Induction of osteoblast differentiation indexes by PTHrP in MG-63 cells involves multiple signaling pathways.

Parathyroid hormone (PTH)-related peptide (PTHrP) can modulate the proliferation and differentiation of a number of cell types including osteoblasts. PTHrP can activate a G protein-coupled PTH/PTHrP receptor, which can interface with several second-messenger systems. In the current study, we have examined the signaling pathways involved in stimulated type I collagen and alkaline phosphatase expression in the human osteoblast-derived osteosarcoma cells, MG-63. By use of Northern blotting and histochemical analysis, maximum induction of these two markers of osteoblast differentiation occurred after 8 h of treatment with 100 nM PTHrP-(1-34). Chemical inhibitors of adenylate cyclase (H-89) or of protein kinase C (chelerythrine chloride) each diminished PTHrP-mediated type I collagen and alkaline phosphatase stimulation in a dose-dependent manner. These effects of PTHrP could also be blocked by inhibiting the Ras-mitogen-activated protein kinase (MAPK) pathway with a Ras farnesylation inhibitor, B1086, or with a MAPK inhibitor, PD-98059. Transient transfection of MG-63 cells with a mutant form of Galpha, which can sequester betagamma-subunits, showed significant downregulation of PTHrP-stimulated type I collagen expression, as did inhibition of phosphatidylinositol 3-kinase (PI 3-kinase) by wortmannin. Consequently, the betagamma-PI 3-kinase pathway may be involved in PTHrP stimulation of Ras. Collectively, these results demonstrate that, acting via its G protein-coupled receptor, PTHrP can induce indexes of osteoblast differentiation by utilizing multiple, perhaps parallel, signaling pathways.

The role of the plasminogen activation system in angiogenesis and metastasis.

The urokinase plasminogen activator system (uPA) has been demonstrated to be required for the movement of cells through a matrix. These observations have been extended to the migration of endothelial cells during the process of angiogenesis, and recent data suggest that the uPA system is central to this process. uPA is a serine protease that is capable of initiating an extracellular cascade of proteolysis that involves the activation of plasminogen and matrix metalloproteases. These proteolytic cascades remodel extracellular matrix (ECM) and basement membrane (BM), allowing for the movement of cells across and through these barriers. In addition, these proteolytic cascades process and release various growth and differentiation factors that are sequestered on the cell surface or within the ECM, which contribute to the evolution of a migratory or invasive cell phenotype. uPA is also able to modulate signaling and cell adhesion through its specific cell surface receptor, uPAR. Recent data suggest that the nonproteolytic activities of the uPA system are coupled to adhesion, migration and signaling through various integrins. This article reviews the evidence for the role of this system in tumor angiogenesis and metastasis, which suggests that the uPA system initiates multiple cascades that contribute to these processes.

Urokinase/urokinase receptor and vitronectin/alpha(v)beta(3) integrin induce chemotaxis and cytoskeleton reorganization through different signaling pathways.

Vitronectin (VN) and pro-urokinase (pro-uPA) stimulated migration of rat smooth muscle cells in a dose-dependent and additive way, and induced motile-type changes in cell morphology together with a complete reorganization of the actin filaments and of the microtubules. All these effects were inhibited by pertussis toxin, or by antibodies directed against the urokinase receptor (uPAR) or against the VN receptor alpha(v)beta(3) suggesting that an association between the two receptors is required to mediate both signals. Investigation of the signaling pathways showed that increasing the intracellular cAMP resulted in a selective inhibition of VN-induced cell migration. On the other hand, PD 98059, an inhibitor of MEK, differentially inhibited the pro-uPA- but not the VN-induced cell migration. Phosphorylation and nuclear translocation of Erk by pro-uPA was directly observed. We conclude that the signaling pathways of pro-uPA and VN must be at least in part different.

Expression of the urokinase plasminogen activator receptor is transiently required during "priming" of PC12 cells in nerve growth factor-directed cellular differentiation.

We previously identified the urokinase plasminogen activator receptor (UPAR) as a gene induced by nerve growth factor (NGF), but not by epidermal growth factor (EGF), in PC12 cells (Farias-Eisner et al. [2000] J. Neurosci. 20:230-239). Antisense oligonucleotides for the UPAR mRNA or an antibody directed against UPAR protein, added simultaneously with NGF, block NGF-induced morphological and biochemical differentiation of PC12 cells. In this report, we show that anti-UPAR antibody blocks morphological differentiation and the expression of two NGF-specific secondary response genes, collagenase-1 and transin, in PC12 cells only during the first 2 hr following NGF exposure. These data suggest that induced UPAR expression is required only over a short period of time following exposure to NGF for the differentiation program in PC12 cells to proceed. For two models of "primed" PC12 cells, we found that UPAR expression and function are not required for NGF-induced differentiation. UPAR and the secondary response genes collagenase-1 and transin are not induced in "primed" PC12 cells in response to NGF, and anti-UPAR antibody does not block morphological differentiation in these cells. Our data suggests that UPAR is required only transiently during the "priming" of PC12 cells in NGF-induced PC12 cell differentiation.

Molecular basis of the spectrum of skeletal complications of neoplasia.

BACKGROUND: Neoplasia may produce a spectrum of dysregulatory effects on bone and mineral metabolism. The range of these effects and the known molecular mechanisms causing them are reviewed. METHODS: The current review is mainly based on previously published scientific reports from North America, Europe, and Japan that were identified from references in the literature. RESULTS: Osteolysis is the most common skeletal manifestation of neoplasia and may be focal or generalized. When tumors release abundant parathyroid hormone-related peptide (PTHrP) into the circulation, this may act as an endocrine substance to produce generalized osteopenia and, ultimately, hypercalcemia. PTHrP also may act in a paracrine manner to enhance focal osteolysis associated with metastasis and to generate hypercalcemia. The increased circulating PTHrP in tumor states also can augment serum calcium by renal mechanisms. PTHrP may contribute to focal osteolysis by tumor metastases, even in the absence of hypercalcemia. Strategies to reduce PTHrP production or PTHrP signaling, therefore, may be useful to treat the tumor-induced bone resorption induced both in hypercalcemic and nonhypercalcemic states. The most commonly used intervention, bisphosphonates, targets the osteoclast directly. Although osteolytic lesions generally occur with some degree of reactive new bone formation, osteoblastic lesions may be particularly abundant in association with certain tumors, such as prostate carcinoma. The mechanisms underlying these lesions remain unknown; however, a variety of osteoblast growth factors may contribute. These include the urokinase system, which may have growth factor activity as well as enzymatic activity. Finally, osteomalacia may be a manifestation of tumors either through accelerated bone formation with insufficient mineralization or through the production of a phosphaturic substance. CONCLUSIONS: Elucidation of the mechanisms underlying the spectrum of skeletal manifestations of neoplasia is yielding important insights into both tumor diagnosis and patient management.

A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo.

Urokinase plasminogen activator (uPA) plays an important role in the progression of several malignancies including breast cancer. We have identified a noncompetitive antagonist of the uPA-uPAR interaction derived from a nonreceptor binding region of uPA (amino acids 136-143). This 8-mer capped peptide (A6) inhibited breast cancer cell invasion and endothelial cell migration in a dose-dependent manner in vitro without altering cell doubling time. Intraperitoneal administration of A6 resulted in a significant inhibition of tumor growth and suppressed the development of lymph node metastases in several models of breast cancer cell growth and metastasis. Large areas of tumor necrosis and extensive positive staining by TUNEL were observed on histological and immunohistochemical analysis of experimental tumor sections from A6-treated animals. A6 treatment also resulted in a decrease in factor VIII-positive tumor microvessel hot-spots. These results identify a new epitope in uPA that is involved in the uPA-uPAR interaction and indicate that an antagonist based on this epitope is able to inhibit tumor progression by modulating the tumor microenvironment in the absence of direct cytotoxic effects in vivo.

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.

Role of mitogen-activated protein kinases in the induction of parathyroid hormone-related peptide.

Tumor production of parathyroid hormone-related protein (PTHRP) is responsible for most cases of hypercalcemia of malignancy. The transplantable rat Leydig tumor H-500 is known to cause hypercalcemia in rats by the release of abundant PTHRP and to closely reproduce the human syndrome. We have demonstrated recently that Ras oncogene can stimulate PTHRP gene expression in Fr3T3 fibroblasts in vitro and cause hypercalcemia in vivo. Using rat Leydig tumor H-500 cells, we have investigated the role of effector pathways downstream of Ras in serum-induced PTHRP expression. The Ras inhibitors B-1086 and Lovastatin decreased PTHRP mRNA expression. i.p. administration of B-1086 (50-100 mg/kg/day) into H-500 tumor-bearing male Fischer rats resulted in a dose-dependent reduction in tumor volume, serum calcium, plasma PTHRP, and tumoral PTHRP mRNA expression. Transient transfection of dominant-negative Ras (Ras N17) and Raf (Raf C4B) reduced, whereas activated Raf-1 (Raf BXB) increased, basal expression of PTHRP in H-500 cells. A similar decrease in PTHRP production was seen with a mitogen-activated protein kinase kinase (MEK) inhibitor (PD 098059), implicating the involvement of Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway. In addition, stimulation with UV light, which can activate c-Jun NH2-terminal kinase (JNK), or expression of an activated form of Rac (Rac V12) was sufficient to increase PTHRP mRNA. Moreover, a dominant-negative Rac (Rac N17) blocked serum-induced PTHRP gene expression. Collectively, these results demonstrate that PTHRP is induced via both Raf-ERK and Rac-JNK mediated pathways, effects which can be blocked by chemical inhibitors and dominant-negative mutants of these pathways in vitro and in vivo. Availability of selective inhibitors of Ras signaling molecules may therefore add to our existing armamentarium to control hypercalcemia of malignancy.

The urokinase plasminogen activator receptor (UPAR) is preferentially induced by nerve growth factor in PC12 pheochromocytoma cells and is required for NGF-driven differentiation.

Nerve growth factor (NGF)-driven differentiation of PC12 pheochromocytoma cells is a well studied model used both to identify molecular, biochemical, and physiological correlates of neurotrophin-driven neuronal differentiation and to determine the causal nature of specific events in this differentiation process. Although epidermal growth factor (EGF) elicits many of the same early biochemical and molecular changes in PC12 cells observed in response to NGF, EGF does not induce molecular or morphological differentiation of PC12 cells. The identification of genes whose expression is differentially regulated by NGF versus EGF in PC12 cells has, therefore, been considered a source of potential insight into the molecular specificity of neurotrophin-driven neuronal differentiation. A "second generation" representational difference analysis procedure now identifies the urokinase plasminogen activator receptor (UPAR) as a gene that is much more extensively induced by NGF than by EGF in PC12 cells. Both an antisense oligonucleotide for the UPAR mRNA and an antibody directed against UPAR protein block NGF-induced morphological and biochemical differentiation of PC12 cells; NGF-induced UPAR expression is required for subsequent NGF-driven differentiation.

Molecular mechanism of action of parathyroid hormone related peptide in hypercalcemia of malignancy: therapeutic strategies (review).

Parathyroid hormone related peptide (PTHRP) was identified and characterized from tumors manifesting the syndrome of hypercalcemia of malignancy (HM). These hypercalcemic effects are due to the strong sequence homology between parathyroid hormone (PTH) and PTHRP in the bioactive amino terminal region and its ability to interact with G-protein linked seven transmembrane PTH/PTHRP receptor. However, due to the expression of PTHRP in several fetal and adult tissues, it also has a variety of physiological actions including the ability to play an important role in cell growth and differentiation. Since the isolation and characterization of PTHRP gene, intense efforts have been made to study its circulating forms in health and disease, regulation of PTHRP gene expression and molecular mechanism of PTHRP action in normal and in tumor cells. Various in vivo models of HM were developed which mimicked the human syndrome. Mice homozygous for PTHRP deletion died at birth due to impaired chondrocyte differentiation resulting in skeletal deformities and respiratory failure. Since HM continues to be a major cause of cancer associated morbidity and mortality there is an urgent need to develop strategies to control the syndrome of HM. This review describes the biosynthesis and secretion of various molecular forms of PTHRP, regulation of PTHRP gene expression and discusses the molecular pathways involved in its actions. Based on this information, various therapeutic strategies which are currently under development are discussed. These studies will form the basis of future efforts aimed at optimization of these approaches for further clinical development.

Regulation of urokinase production by androgens in human prostate cancer cells: effect on tumor growth and metastases in vivo.

During the complex multistep process of tumor progression, prostate cancer is initiated as an androgen-sensitive, nonmetastatic cancer, followed by a gradual transition into a highly metastatic and androgen-insensitive variety that lacks the expression of functional androgen receptors (AR). Urokinase (uPA), a member of the serine protease family, has been implicated in the progression of various human malignancies, including prostate cancer. Although uPA production is regulated by various growth factors and cytokines, the role of sex steroids (androgens) in regulating uPA gene expression in prostate cancer is poorly understood. In the current study, we have examined the role of androgens in regulating uPA production and the invasive capacity of the androgen insensitive PC-3 cells transfected with the full-length human AR complementary DNA (PC-3T). Restoration of androgen responsiveness in PC-3T cells caused a marked decrease in cell doubling time. Treatment of PC-3T cells with dihydroxytestosterone (DHT) caused a dose-dependent decrease in uPA messenger RNA and protein production, resulting in their decreased ability to invade through the Matrigel. Nuclear runoff assays revealed that these effects were attributable to the ability of DHT to inhibit uPA gene transcription. AR antagonist flutamide (Flu) reversed the effect of DHT on proliferation and invasion of PC-3T cells. Both control (PC-3) and experimental (PC-3T) cells were injected into the right flank of male BALB/c nu/nu mice. Control animals developed palpable tumors and microscopic tumor metastases at lymph nodes, lungs, and liver at 6-week posttumor cell inoculation. In contrast to this, because of androgen sensitivity of PC-3T cells, palpable tumors were observed only at week 12, with occasional tumor metastases in lungs. Furthermore, inoculation of PC-3T cells into surgically castrated host animals resulted in the development of tumors at a much earlier time (week 10) and a high incidence of metastases, compared with regular animals receiving PC-3T cells. Collectively, these results demonstrate the ability of androgen to regulate uPA production, which may directly effect prostate cancer growth, invasion, and metastasis in vitro and in vivo.

Reversal of hypercalcemia with the vitamin D analogue EB1089 in a human model of squamous cancer.

EB1089, an analogue of 1,25 dihydroxyvitamin D with low calcemic activity is a potent inhibitor of parathyroid hormone-related peptide (PTHRP) production in vitro. The purpose of the present study was to determine whether EB1089 could reverse established hypercalcemia in BALB C nude mice implanted s.c. with a human epithelial cancer previously shown to produce high levels of PTHRP in vitro. Total plasma calcium was monitored before and after tumor development and increased steadily when the tumor reached > or =0.5 cm3. When total calcium was 22.85 mmol/liter, animals were treated with a constant infusion of EB1089 or vehicle alone for a period of 2 weeks. A significant and sustained reduction of plasma calcium from 3.2+/-0.1 to 2.7+/-0.08 (P < 0.01) mmol/liter was observed during infusion with EB1089. In contrast, calcium levels in vehicle-treated animals continued to rise during the infusion period. Tumor growth velocity also slowed significantly after the administration of EB1089 as compared with vehicle-treated animals. Plasma PTHRP levels measured at the end of the 2 weeks' infusion period were significantly lower in animals treated with EB1089 as compared with animals treated with vehicle alone (44+/-8 pg/ml versus 194+/-35 pg/ml, P < 0.001). These results, therefore, demonstrate that EB1089 can reverse established hypercalcemia in a human model of squamous cancer.

Src-dependence and pertussis-toxin sensitivity of urokinase receptor-dependent chemotaxis and cytoskeleton reorganization in rat smooth muscle cells.

The catalytically inactive precursor of urokinase-type plasminogen activator (pro-u-PA) induced a chemotactic response in rat smooth muscle cells (RSMC) through binding to the membrane receptor of urokinase (u-PA receptor [u-PAR]). A soluble form of u-PAR activated by chymotrypsin cleavage as well as a peptide located between domain 1 and 2 of u-PAR reproduced the effect of pro-u-PA on cell migration. The chemotactic pro-u-PA effect correlates with a dramatic reorganization of actin cytoskeleton, of adhesion plaques, and with major cell shape changes in RSMC. Pro-u-PA induced a decrease in stress fiber content, membrane ruffling, actin ring formation, and disruption leading to the characteristic elongated cell shape of motile cells with an actin semi-ring located close to the leading edge of cells. u-PAR effects on both chemotaxis and cytoskeleton were sensitive to pertussis toxin and, hence, possibly require G proteins. u-PAR effects are accompanied by a relocation of u-PAR, vitronectin receptor (VNR) alphavbeta3, beta1 integrin subunit, and Src tyrosine kinase to the leading membrane of migrating cells. In conclusion, our data show that pro-u-PA, via binding to u-PAR, controls a signaling pathway, regulated by tyrosine kinases and possibly G proteins, leading to cell cytoskeleton reorganization and cell migration.

Transcriptional regulation of urokinase (uPA) gene expression in breast cancer cells: role of DNA methylation.

Carcinoma of the breast is a leading hormone-dependent malignancy, resulting in a high rate of morbidity and mortality. During the complex multi-step process of tumor promotion, this common cancer is initiated as hormone-responsive (HR), non-metastatic cancer, followed by a gradual transition into a highly metastatic hormone-insensitive (HI) variety which lacks the functional estrogen receptor. This transition of cancer cells causes them to become refractory to hormonal treatment. Urokinase (uPA), a member of the serine protease family has been implicated in the progression of several malignancies including breast cancer. In the current study, we have examined the correlation between hormone sensitivity and uPA expression in HR normal mammary epithelial cells (HMEC) and in MCF-7 and T-47D breast cancer cell lines. Comparison was made with HI breast cancer cells MDA-231. uPA mRNA expression was seen only in the highly invasive, HI breast cancer cells MDA-231. Lack of uPA expression in HR normal (HMEC) and in minimally invasive, HR cells (MCF-7 and T-47D) was due to transcriptional suppression of uPA gene expression as determined by nuclear run-off assays. Since alteration of the DNA methylation status of CpG island in the 5' sequence of oncogenes and tumor suppressor genes has been demonstrated to change their expression, we examined DNA methylation as a potential molecular mechanism for regulating uPA gene transcription in these cancer cells. Southern blot analysis using methylation sensitive enzymes revealed that CpG island of uPA gene are methylated in HR, HMEC, MCF-7 and T-47D cells, whereas they are hypomethylated in HI and MDA-231 cells. Treatment of HR MCF-7 cells with cytosine DNA methyltransferase inhibitor 5' azacytidine caused a dose-dependent induction of uPA mRNA due to demethylation of the CpG island of the uPA gene which led to increased invasive ability of these HR cancer cells. Our results demonstrate that DNA methylation can regulate the transcription of the uPA gene to alter the invasive behaviour of these HR breast cancer cells.

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.

Metalloproteases and urokinase in angiogenesis and tumor progression.

Following malignant transformation, tumor cells and their surrounding stroma produce a variety of growth factors and proteolytic enzymes to induce new capillary formation (angiogenesis) and matrix degradation to promote tumor development. Two key families of proteases, matrix metalloproteases (MMPs) and urokinase (uPA) are now strongly implicated in this process of angiogenesis and matrix degradation. Both MMPs and uPA are abundantly produced by various tumors where their level of expression can serve as prognostic markers. Using gene transfer techniques, overexpression of these proteases and their receptors enhances the invasive and metastatic potential of tumor cells. Furthermore, blockage of actions of MMPs and uPA by molecular and chemical approaches results in a marked decrease in tumor growth to further validate the significance of these enzymes as targets for anti-cancer therapy.