RESUMEN
A series of pyridoxine hydroxamic acid analog bearing a 5-aryl-spacers were synthesized. Evaluation of these novel HIV integrase complex inhibitors revealed compounds with high potency against wild-type HIV virus.
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Inhibidores de Integrasa VIH/química , Integrasa de VIH/química , VIH-1/fisiología , Ácidos Hidroxámicos/química , Animales , Dominio Catalítico , Femenino , Integrasa de VIH/metabolismo , Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/farmacocinética , Semivida , Humanos , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/farmacocinética , Concentración 50 Inhibidora , Piridoxina/química , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
PURPOSE: In vivo evaluation of good manufacturing practice-grade targeted liposomal doxorubicin (TVT-DOX), bound to a CD13 isoform expressed on the vasculature of solid tumors, in human tumor xenografts of neuroblastoma, ovarian cancer, and lung cancer. EXPERIMENTAL DESIGN: Mice were implanted with lung, ovarian, or neuroblastoma tumor cells via the pulmonary, peritoneal, or orthotopic (adrenal gland) routes, respectively, and treated, at different days post inoculation, with multiple doses of doxorubicin, administered either free or encapsulated in untargeted liposomes (Caelyx) or in TVT-DOX. The effect of TVT-DOX treatment on tumor cell proliferation, viability, apoptosis, and angiogenesis was studied by immunohistochemical analyses of neoplastic tissues and using the chick embryo chorioallantoic membrane assay. RESULTS: Compared with the three control groups (no doxorubicin, free doxorubicin, or Caelyx), statistically significant improvements in survival was seen in all three animal models following treatment with 5 mg/kg (maximum tolerated dose) of TVT-DOX, with long-term survivors occurring in the neuroblastoma group; increased survival was also seen at a dose of 1.7 mg/kg in mice bearing neuroblastoma or ovarian cancer. Minimal residual disease after surgical removal of neuroblastoma primary mass, and the enhanced response to TVT-DOX, was visualized and quantified by bioluminescence imaging and with magnetic resonance imaging. When treated with TVT-DOX, compared with Caelyx, all three tumor models, as assayed by immunohistochemistry and chorioallantoic membrane, showed statistically significant reductions in cell proliferation, blood vessel density, and microvessel area, showing increased cell apoptosis. CONCLUSION: TVT-DOX should be evaluated as a novel angiostatic strategy for adjuvant therapy of solid tumors.
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Antibióticos Antineoplásicos/administración & dosificación , Doxorrubicina/administración & dosificación , Neoplasias Experimentales/tratamiento farmacológico , Neovascularización Patológica/tratamiento farmacológico , Animales , Antígenos CD13/metabolismo , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Inmunohistoquímica , Liposomas , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In an effort to develop new agents and molecular targets for the treatment of cancer, aspargine-glycine-arginine (NGR)-targeted liposomal doxorubicin (TVT-DOX) is being studied. The NGR peptide on the surface of liposomal doxorubicin (DOX) targets an aminopeptidase N (CD13) isoform, specific to the tumor neovasculature, making it a promising strategy. To further understand the molecular mechanisms of action, we investigated cell binding, kinetics of internalization as well as cytotoxicity of TVT-DOX in vitro. We demonstrate the specific binding of TVT-DOX to CD13-expressing endothelial [human umbilical vein endothelial cells (HUVEC) and Kaposi sarcoma-derived endothelial cells (SLK)] and tumor (fibrosarcoma, HT-1080) cells in vitro. Following binding, the drug was shown to internalize through the endosomal pathway, eventually leading to the localization of doxorubicin in cell nuclei. TVT-DOX showed selective toxicity toward CD13-expressing HUVEC, sparing the CD13-negative colon-cancer cells, HT-29. Additionally, the nontargeted counterpart of TVT-DOX, Caelyx, was less cytotoxic to the CD13-positive HUVECs demonstrating the advantages of NGR targeting in vitro. The antitumor activity of TVT-DOX was tested in nude mice bearing human prostate-cancer xenografts (PC3). A significant growth inhibition (up to 60%) of PC3 tumors in vivo was observed. Reduction of tumor vasculature following treatment with TVT-DOX was also apparent. We further compared the efficacies of TVT-DOX and free doxorubicin in the DOX-resistant colon-cancer model, HCT-116, and observed the more pronounced antitumor effects of the TVT-DOX formulation over free DOX. The potential utility of TVT-DOX in a variety of vascularized solid tumors is promising.
Asunto(s)
Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Doxorrubicina/administración & dosificación , Doxorrubicina/farmacocinética , Oligopéptidos/metabolismo , Animales , Antineoplásicos/farmacología , Transporte Biológico , Antígenos CD13/metabolismo , Línea Celular Tumoral , Neoplasias del Colon , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Endocitosis , Endosomas/metabolismo , Humanos , Técnicas In Vitro , Liposomas , Masculino , Ratones , Ratones Desnudos , Neoplasias de la Próstata , Unión ProteicaRESUMEN
Human PSP94 (prostate secretory protein of 94 amino acids) is a major protein synthesized by the prostate gland and secreted in large quantities in seminal fluid. Previous studies have suggested a potential biomedical utility of PSP94 in applications such as diagnosis/prognosis and in treatment of human prostate cancer (PCa). This study was designed to produce a recombinant human PSP94 (rPSP94) to evaluate its clinical and functional role in PCa. We cloned PSP94 cDNA and successfully expressed an active recombinant protein in yeast using Pichia pastoris expression system. A simple purification strategy was established that incorporated combination of membrane ultrafiltration (Pellicon tangential-flow system) and anion exchange chromatography using DE52 resin. The method minimized the technical level of expertise for the production of high quality functional protein. The purified rPSP94 (>98% purity) showed a single band with SDS-PAGE analysis and a peak with a molecular mass (M(r)) of 11,495 kDa using MALDI TOF mass spectrometry (MS). The in vitro competitive binding assays indicated high functional similarity of the rPSP94 with that of its native counterpart. Furthermore, in vivo administration of rPSP94 caused a significant growth inhibition of hormone refractory Mat LyLu tumors in Dunning rat model. Taken together, our data provides evidence for high suitability of the purified rPSP94 for evaluation of its potential diagnostic and therapeutic role in PCa and as a valuable analytical reference standard for clinical studies.
Asunto(s)
Proteínas de Secreción Prostática/genética , Proteínas de Secreción Prostática/uso terapéutico , Animales , Antineoplásicos/uso terapéutico , Cromatografía por Intercambio Iónico , Clonación Molecular , Humanos , Masculino , Pichia/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Proteínas de Secreción Prostática/aislamiento & purificación , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéutico , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
PURPOSE: PCK3145 is an anti-metastatic synthetic peptide with promising therapeutic efficacy against hormone-refractory prostate cancer. The characterization of the PCK3145 peptide cell surface binding/internalization mechanisms and of the receptors involved remained to be explored. RESULTS: [(14)C]PCK3145 cell surface binding assays showed rapid and transient kinetic profile, that was inhibited by RGD peptides, laminin, hyaluronan, and type-I collagen. RGD peptides were however unable to inhibit PCK3145 intracellular uptake. Far-Western ligand binding studies enabled the identification of the 37-kDa laminin receptor precursor (37LRP) as a potential ligand for PCK3145. Overexpression of the recombinant 37LRP indeed led to an increase in PCK3145 binding but unexpectedly not to its uptake. CONCLUSIONS: Our data support the implication of laminin receptors in cell surface binding and in transducing PCK3145 anti-metastatic effects, and provide a rational for targeting cancers that express high levels of such laminin receptors.
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Fragmentos de Péptidos/metabolismo , Proteínas de Secreción Prostática/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Laminina/metabolismo , Catequina/análogos & derivados , Catequina/farmacología , Comunicación Celular , Línea Celular Tumoral , Colágeno Tipo I/farmacología , Sinergismo Farmacológico , Humanos , Cinética , Metaloproteinasa 9 de la Matriz/metabolismo , Metástasis de la Neoplasia/tratamiento farmacológico , Oligopéptidos/farmacología , Receptores de Superficie Celular/metabolismoRESUMEN
PCK3145 is a synthetic peptide corresponding to amino acids 31-45 of prostate secretory protein 94 which can reduce experimental skeletal metastases and prostate tumor growth. These anti-metastatic and anti-tumoral effects of PCK3145 are partially explained by the in-vivo and in-vitro decrease in matrix metalloproteinase (MMP)-9 extracellular levels through as yet unidentified molecular mechanisms of action. Gelatin zymography and immunoblots were used to monitor the levels of secreted MMP-9 from HT-1080 cells. Flow cytometry was used to monitor HT-1080 cell surface binding of FITC-labeled PCK3145 and biotin-labeled laminin. PCK3145-coated cell culture dishes were used to monitor cell adhesion. HT-1080 cell lysates were used for immunoblotting of HuR, extracellular signal-regulated protein kinase (ERK) and phospho-ERK. Total RNA was isolated and RT-PCR used to monitor HuR gene expression. We found that PCK3145 bound to the HT-1080 cell surface and that this binding rapidly triggered ERK phosphorylation that, ultimately, led to a reduction of secreted MMP-9. Laminin inhibited both cell surface binding and ERK phosphorylation by PCK3145. Overexpression of the 67-kDa laminin receptor led to an increased binding of the cells to PCK3145. HuR, a protein that can bind to and stabilize MMP-9 mRNA, was found to be downregulated by PCK3145. The mitogen-activated protein kinase/ERK (MEK) inhibitor PD98059 as well as native laminin and SIKVAV laminin-derived peptide prevented that downregulation. Our data suggest that PCK3145 rapidly triggers intracellular signaling through cell surface laminin receptors. This leads to decreased HuR expression and subsequent destabilization of MMP-9 transcripts. This is the first molecular evidence demonstrating the intracellular signaling and anti-metastatic mechanism of action of PCK3145 that leads to the inhibition of MMP-9 secretion.
Asunto(s)
Inhibidores de la Metaloproteinasa de la Matriz , Fragmentos de Péptidos/farmacología , Proteínas de Secreción Prostática/farmacología , Receptores de Laminina/metabolismo , Transducción de Señal/efectos de los fármacos , Antígenos de Superficie/metabolismo , Neoplasias Óseas/enzimología , Neoplasias Óseas/prevención & control , Neoplasias Óseas/secundario , Línea Celular Tumoral , Proteínas ELAV , Proteína 1 Similar a ELAV , Flavonoides/farmacología , Humanos , Laminina/farmacología , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Oligopéptidos/farmacología , Fragmentos de Péptidos/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/patología , Proteínas de Secreción Prostática/química , Proteínas de Secreción Prostática/metabolismo , Unión Proteica , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Unión al ARN/metabolismo , Receptores de Laminina/efectos de los fármacosRESUMEN
We have previously observed that the synthetic peptide corresponding to amino acids 31-45 (PCK3145) of PSP94 can reduce prostate tumor growth in vivo. Moreover, a recently concluded phase IIa clinical trial with patients with hormone refractory prostate cancer indicated that PCK3145 down-regulates the levels of plasma matrix metalloproteinase (MMP)-9, a MMP involved in metastasis and tumor angiogenesis. The purpose of our study was to investigate the molecular mechanisms of action of PCK3145 and whether this peptide could antagonize tumor neovascularization. We show that, in a syngeneic in vivo model of rat prostate cancer, the expression of endothelial cell (EC) specific CD31, a marker of tumor vessel density, was decreased by 43% in PCK3145-treated animals. In vitro, PCK3145 specifically antagonized in a dose-dependent manner the VEGF-induced ERK phosphorylation as well as the phosphorylation of the VEGFR-2 in cultured EC (HUVEC). These anti-VEGF effects were partly reproduced by pharmacological inhibitors such as PD98059 and PTK787, suggesting that PCK3145 inhibits the tyrosine kinase activity associated to VEGFR-2, which in turn prevents intracellular signalling through the MAPK cascade. Moreover, PCK3145 was also found to inhibit the PDGF-induced phosphorylation of PDGFR in smooth muscle cells. Finally, PCK3145 inhibited in vitro EC tubulogenesis and VEGF-induced MMP-2 secretion suggesting its potential implication as an antiangiogenic agent. Our study demonstrates that PCK3145 interferes with the tyrosine kinase activity associated with VEGF signalling axis in EC. The antiangiogenic properties of this peptide could be highly beneficial and exploited in novel antiangiogenic therapies, for patients with various cancers.
Asunto(s)
Neovascularización Patológica/fisiopatología , Fragmentos de Péptidos/farmacología , Neoplasias de la Próstata/irrigación sanguínea , Factor A de Crecimiento Endotelial Vascular/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/fisiología , Animales , Técnicas de Cultivo de Célula , Células Endoteliales , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Músculo Liso , Fosforilación , Neoplasias de la Próstata/tratamiento farmacológico , Proteínas de Secreción Prostática , Ratas , Transducción de Señal , Células Tumorales CultivadasRESUMEN
PURPOSE: PCK3145 is a synthetic peptide corresponding to amino acids 31-45 of prostate secretory protein 94, which can reduce experimental skeletal metastases and prostate tumor growth in vivo. Part of its biological action involves the reduction of circulating plasma matrix metalloproteinase (MMP)-9, a crucial mediator in extracellular matrix (ECM) degradation during tumor metastasis and cancer cell invasion. The antimetastatic mechanism of action of PCK3145 is however, not understood. EXPERIMENTAL DESIGN: HT-1080 fibrosarcoma cells were treated with PCK3145, and cell lysates used for immunoblot analysis of small GTPase RhoA and membrane type (MT)1-MMP protein expression. Conditioned media was used to monitor soluble MMP-9 gelatinolytic activity by zymography and protein expression by immunoblotting. RT-PCR was used to assess RhoA, MT1-MMP, MMP-9, RECK, and CD44 gene expression. Flow cytometry was used to monitor cell surface expression of CD44 and of membrane-bound MMP-9. Cell adhesion was performed on different purified ECM proteins, while cell migration was specifically performed on hyaluronic acid (HA). RESULTS: We found that PCK3145 inhibited HT-1080 cell adhesion onto HA, laminin-1, and type-I collagen suggesting the common implication of the cell surface receptor CD44. In fact, PCK3145 triggered the shedding of CD44 from the cell surface into the conditioned media. PCK3145 also inhibited MMP-9 secretion and binding to the cell surface. This effect was correlated to increased RhoA and MT1-MMP gene and protein expression. CONCLUSIONS: Our data suggest that PCK3145 may antagonize tumor cell metastatic processes by inhibiting both MMP-9 secretion and its potential binding to its cell surface docking receptor CD44. Such mechanism may involve RhoA signaling and increase in MT1-MMP-mediated CD44 shedding. Together with its beneficial effects in clinical trials, this is the first demonstration of PCK3145 acting as a MMP secretion inhibitor.
Asunto(s)
Receptores de Hialuranos/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Metástasis de la Neoplasia/fisiopatología , Fragmentos de Péptidos/farmacología , Proteínas de Secreción Prostática/química , Fibrosarcoma/patología , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Receptores de Hialuranos/biosíntesis , Poliésteres , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
This review focuses on the promising roles of prostate secretory protein of 94 amino acids (PSP-94) and one of its derived peptides (PCK3145) as potential therapeutic modalities for prostate cancer and its associated complications. Evaluation of these compounds was carried out in vitro and in vivo using syngeneic models of rat prostate cancer. Overproduction of parathyroid hormone-related protein (PTHrP) results in the development of hypercalcemia of malignancy in several malignancies including prostate cancer. In order to evaluate the effect of PSP-94 and PCK3145 on prostate cancer progression, the rat Dunning R3227 MatLyLu cell line transfected with full-length cDNA encoding PTHrP (MatLyLu-PTHrP) was used. As the main pathogenetic factor of hypercalcemia of malignancy, overexpression of PTHrP was aimed at mimicking the hypercalcemic nature seen in patients suffering from late-stage cancer. In vitro studies showed that PSP-94 and PCK3145 can cause a dose-dependent inhibition in the growth of MatLyLu-PTHrP cells. For in vivo studies, male Copenhagen rats were inoculated either s.c. into the right flank or directly into the left ventricle via intracardiac (i.c.) inoculation with MatLyLu-PTHrP cells. In these models, s.c. injection of MatLyLu cells results in the development of primary tumor growth, whereas i.c. inoculation routinely results in the development of experimental skeletal metastases in the lumbar vertebrae causing hind-limb paralysis. Administration of PSP-94 and PCK3145 into tumor-bearing animals resulted in a dose-dependent inhibition of primary tumor growth, and tumoral and plasma PTHrP levels, and in the reduction of plasma calcium levels. Additionally, treatment with PSP-94 or PCK3145 caused an inhibition of skeletal metastases resulting in a significant delay in the development of hind-limb paralysis. Interestingly, equimolar concentrations of PCK3145 were shown to be more effective in delaying the development of experimental skeletal metastases as compared to PSP-94. One of the possible mechanisms of action of these modalities is through the induction of apoptosis which was observed by both in-vitro and in-vivo analyses of MatLyLu-PTHrP cells and tumors. Several intracellular mechanisms can also be involved in inhibiting PTHrP production and anti-tumor effects of PSP-94 and PCK3145. Collectively, these studies warrant the continued clinical development of these agents as therapeutic agents for patients with hormone-refractory prostate cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Óseas/prevención & control , Fragmentos de Péptidos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Proteínas de Secreción Prostática/uso terapéutico , Animales , Neoplasias Óseas/secundario , Calcio/sangre , Línea Celular Tumoral , Humanos , Masculino , Proteína Relacionada con la Hormona Paratiroidea/sangre , Neoplasias de la Próstata/patología , RatasRESUMEN
PSP94 (prostate secretory protein of 94 amino acids), an abundant protein within semen, has reported local functions within the reproductive tract and reported systemic functions. Mechanisms of action remain poorly understood, but binding to undefined molecules within the prostate, pituitary, testis and blood may initiate some of these actions. PSP94 serum measurements, especially of bound and free forms, have potential clinical utility in prostate cancer management. Identification of the binding molecules will help in the understanding of PSP94's action, and enable further development of PSP94 serum assays. PSPBP (PSP94-binding protein) was purified from human serum by ammonium sulphate fractionation, ion-exchange and affinity chromatography. The glycosylated protein ran as two bands on SDS/PAGE (70 and 95 kDa). N-terminal sequencing yielded a 30-amino-acid sequence, identical with the translated N-terminal region of a previously published cDNA (GenBank accession number AX136261). Reverse transcriptase PCR and plaque hybridization demonstrated PSPBP mRNA in peripheral blood leucocytes and in a prostate cDNA library. Northern blotting showed 2 kb mRNA species in prostate, testis, ovary and intestine. Immunohistochemistry demonstrated PSPBP in tissues, including pituitary and Leydig cells, supporting a role for PSP94 in hormonal control at the pituitary gonadal axis. ELISA demonstrated that PSPBP levels were significantly lower (P=0.0014) in the serum of a prostate cancer population (n=65) compared with a control population (n=70). PSPBP identification will help the understanding of PSP94's functions and facilitate ELISA development to address the clinical value of PSP94 serum assays.
Asunto(s)
Proteínas Sanguíneas/aislamiento & purificación , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/aislamiento & purificación , Proteínas Portadoras/metabolismo , Perfilación de la Expresión Génica , Glicoproteínas/aislamiento & purificación , Glicoproteínas/metabolismo , Proteínas de Secreción Prostática/metabolismo , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Aminoácidos , Anticuerpos Monoclonales/inmunología , Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Western Blotting , Proteínas Portadoras/química , Proteínas Portadoras/genética , Clonación Molecular , Glicoproteínas/química , Glicoproteínas/genética , Glicosilación , Humanos , Inmunohistoquímica , Inmunoprecipitación , Cinética , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Peso Molecular , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/sangre , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
In previous studies, we have shown that prostate secretory protein (PSP-94) can reduce prostate cancer growth in vivo. In the current study, we identified the amino acid sequence of PSP-94 that is required for eliciting this response. For these studies, we used rat prostate cancer Mat Ly Lu cells overexpressing parathyroid hormone-related protein (PTHrP), which is the main pathogenetic factor responsible for hypercalcemia of malignancy. Synthetic peptides corresponding to amino acids 7-21 (PCK721), 31-45 (PCK3145), and 76-94 (PCK7694) of PSP-94 were synthesized. Only PCK3145 showed a significant reduction in tumor cell proliferation. For in vivo studies, syngenic male Copenhagen rats were inoculated s.c. with Mat Ly Lu cells overexpressing PTHrP into the right flank or into the left ventricle via intracardiac injection, which results in experimental metastases to the lumbar vertebrae causing hind-limb paralysis. Animals were infused with different doses (1, 10, and 100 microg/kg/day) of peptides for 15 days, and the effect of these treatments on tumor volume, skeletal metastases, or development of hind-limb paralysis was determined. Treatment with PCK3145 resulted in a dose-dependent decrease in tumor volume and delay in the development of skeletal metastases. Bone histomorphometry showed that after intracardiac inoculation of tumor cells, the highest dose of PCK3145 (100 microg/kg/day) resulted in reducing skeletal tumor burden, which delayed the development of hind-limb paralysis. Treatment with PCK3145 led to reduction of plasma calcium and PTHrP levels and a significant decrease in PTHrP levels in the primary tumors and in vertebrae of experimental animals. These effects of PCK3145 were due to its ability to promote tumor cell apoptosis. Collectively, the results of these studies have demonstrated the ability of a small peptide derived from PSP-94 to reduce tumor volume and experimental skeletal metastases-results that will be highly beneficial in the continued development of this peptide as a novel therapeutic agent for patients with hormone refractory, late-stage prostate cancer.
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Neoplasias Óseas/prevención & control , Miembro Posterior/patología , Hipercalcemia/tratamiento farmacológico , Fragmentos de Péptidos/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Proteínas de Secreción Prostática/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Neoplasias Óseas/secundario , Calcio/metabolismo , División Celular/efectos de los fármacos , Humanos , Hipercalcemia/sangre , Masculino , Proteína Relacionada con la Hormona Paratiroidea/metabolismo , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/patología , Ratas , Células Tumorales CultivadasRESUMEN
Prostate cancer is a common malignancy affecting men, which is often associated with skeletal metastases resulting in significant morbidity and mortality. In this hormone-dependent cancer, low levels of a prostate secretory protein of 94 amino acids (PSP-94) are associated with advanced disease stage. In the current study, we have examined the effect of PSP-94 on prostate cancer growth and experimental metastases to the skeleton. For these studies, MatLyLu rat prostate cancer cells were transfected with full-length cDNA encoding parathyroid hormone-related protein [PTHrP (MatLyLu-PTHrP cells)], which is known to be the major pathogenetic factor for malignancy-associated hypercalcemia. MatLyLu-PTHrP cells were inoculated s.c. into the right flank or via intracardiac route into the left ventricle of syngeneic male Copenhagen rats. Intracardiac inoculation of MatLyLu cells routinely results in the development of tumors in the lumbar vertebrae, resulting in hind-limb paralysis. Animals were infused with different doses of PSP-94 (0.1, 1.0, and 10.0 micro g/kg/day) starting on the day of tumor cell inoculation. Time of hind-limb paralysis and tumor volume were determined, and comparison was made between PSP-94-treated animals and control animals receiving vehicle alone. At the end of the study, animals were sacrificed, and plasma calcium, plasma PTHrP, and tumor PTHrP levels were determined. Whereas the highest dose of PSP-94 caused a modest but statistically significant delay in the development of hind-limb paralysis, a marked dose-dependent decrease in primary tumor volume was seen in experimental animals receiving PSP-94 due to its ability to promote tumor cell apoptosis. Furthermore, whereas control animals routinely developed hypercalcemia due to PTHrP production, treatment with PSP-94 led to a near normalization of plasma calcium and a marked reduction in PTHrP production as determined by radioimmunoassay and immunohistochemistry. Collectively, these results demonstrate the ability of PSP-94 to be an effective treatment modality for prostate cancer, where decrease in plasma PTHrP and calcium levels can serve as useful biochemical markers for monitoring the efficacy of this novel antitumor agent.