RESUMO
Previous studies showed that P1G10, a proteolytic fraction from Vasconcellea cundinamarcensis latex, reduced the tumor mass in animals bearing melanoma, increased in vitro DNA fragmentation and decreased cell adhesion. Here, we present some molecular and cellular events related to the antimetastatic effect induced by the CMS-2 fraction derived from P1G10 in metastatic melanoma B16-F10 and melanocyte Melan-a. Using difference gel electrophoresis and mass spectrometry, we identified four proteins overexpressed in tumor cells, all of them related to proliferation, survival, migration and cell invasion, that had their expression normalized upon treatment with CMS-2: nucleophosmin 1, heat shock protein 65, calcyclin binding protein and eukaryotic translation initiation factor 4H. In addition, some antioxidant and glycolytic enzymes show increased expression after exposure to CMS-2, along with an induction of melanogenesis (differentiation marker). The down regulation of cofilin 1, a protein involved in cell motility, may explain the inhibition of cell migration and dendritic-like outgrowth in B16-F10 and Melan-a, observed after CMS-2 treatment. Taken together, it is argued that CMS-2 modulates the expression of proteins related to metastatic development, driving the cell to a more differentiated-like state. These effects support the CMS-2 antimetastatic activity and place this fraction in the category of anticancer agent.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Caricaceae/enzimologia , Cisteína Proteases/farmacologia , Melanoma/tratamento farmacológico , Metástase Neoplásica/tratamento farmacológico , Proteínas de Plantas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisteína Proteases/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma/patologia , Camundongos , Metástase Neoplásica/patologia , NucleofosminaRESUMO
Abstract Prior studies demonstrate that a proteinase fraction from Vasconcellea cundinamarcensis V.M. Badillo, Caricaceae, exhibits wound healing activity in gastric and cutaneous models and antitumoral/antimetastatic effects. Here, we present the toxicity, pharmacokinetics and biodistribution data for this proteinase fraction following a single dose into Swiss mice by i.v., s.c. or p.o. routes. The i.v. and s.c. toxicity assays demonstrate that proteinase fraction at ≤20 mg/kg is non-lethal after single injection, while parental administration (p.o.) of ≤300 mg/kg does not cause death. Based on p.o. acute toxicity dose using Organisation for Economic Cooperation and Development protocols, proteinase fraction ranks as Class IV “harmful” substance. Proteinase fraction shows high uptake determined as Kp (distribution tissue/blood) in organs linked to metabolism and excretion. Also, high bioavailability (≈100%) was observed by s.c. administration. The blood contents following i.v. dose fits into a pharmacokinetic bi-compartmental model, consisting of high removal constants – kel 0.22 h−1 and kd 2.32 h−1and a half-life – t½ = 3.13 h. The Ames test of proteinase fraction (0.01–1%) demonstrates absence of mutagenic activity. Likewise, genotoxic evaluation of proteinase fraction (5 or 10 mg/kg, i.p.) shows no influence in micronuclei frequency. In conclusion, the acute doses for proteinase fraction lack mutagenic and genotoxic activity, clearing the way for clinical assays.
RESUMO
The proteolytic enzymes from V. cundinamarcensis latex, (P1G10), display healing activity in animal models following various types of lesions. P1G10 or the purified isoforms act as mitogens on fibroblast and epithelial cells by stimulating angiogenesis and wound healing in gastric and cutaneous ulcers models. Based on evidence that plant proteinases act as antitumorals, we verified this effect on a murine melanoma model. The antitumoral effect analyzed mice survival and tumor development after subcutaneous administration of P1G10 into C57BL/6J mice bearing B16F1 low metastatic melanoma. Possible factors involved in the antitumoral action were assessed, i.e., cytotoxicity, cell adhesion and apoptosis in vitro, haemoglobin (Hb), vascular endothelial growth factor (VEGF), tumor growth factor-ß (TGF-ß), tumor necrosis factor-α (TNF-α) content and N-acetyl-glucosaminidase (NAG) activity. We observed that P1G10 inhibited angiogenesis measured by the decline of Hb and VEGF within the tumor, and TGF-ß displayed a non-significant increase and TNF-α showed a minor non-significant reduction. On the other hand, there was an increase in NAG activity. In treated B16F1 cells, apoptosis was induced along with decreased cell binding to extracellular matrix components (ECM) and anchorage, without impairing viability.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Antineoplásicos Fitogênicos/administração & dosagem , Carica/enzimologia , Melanoma Experimental/tratamento farmacológico , Peptídeo Hidrolases/administração & dosagem , Neoplasias Cutâneas/tratamento farmacológico , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Masculino , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Peptídeo Hidrolases/farmacologia , Proteínas de Plantas/administração & dosagem , Proteínas de Plantas/farmacologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Fator de Crescimento Transformador beta/metabolismo , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Liposome encapsulation of platinum (Pt) drugs has emerged as a promising strategy to overcome their toxicity and cellular Pt resistance. The aim of the present work was to examine the impact of liposome encapsulation of a novel antitumor lipophilic Pt complex, dichloro-(N-dodecyl)-propanediamine-platinum(II) complex (DDPP), on its pharmacological profile as an antitumor agent. Biological assays included acute toxicity and histopathological evaluations, pharmacokinetics, and growth inhibition of B16-F1 tumor cells in C57Bl/6 mice. Comparison was made with cisplatin and free DDPP dissolved in castor oil. DDPP encapsulated in pegylated liposomes showed reduced acute toxicity in mice following intraperitoneal administration, compared with the free complex. Free DDPP at 5 mg Pt/kg induced histopathological alterations in the liver, in contrast to liposomal DDPP and cisplatin. Interestingly, the marked loss of body weight following the treatment of mice with cisplatin was not observed after liposomal DDPP at the same Pt dose. Liposomal DDPP was found to inhibit tumor growth significantly, when administered at 5 mg Pt/kg/day for 3 days, similar to cisplatin, but in contrast to the free complex. Pharmacokinetic studies after intraperitoneal and intravenous administrations at 5 mg Pt/kg indicated greater and more prolonged Pt levels in the plasma, liver, spleen, and kidneys from liposomal DDPP, compared with free DDPP or cisplatin. The tumor concentration of Pt increased after liposomal DDPP over the 24-h period, whereas it decreased after cisplatin. In conclusion, the encapsulation of DDPP in pegylated liposomes reduced the drug toxicity and enhanced its antitumoral activity in mice, as a result of improved drug pharmacokinetics.
Assuntos
Antineoplásicos/administração & dosagem , Lipossomos/administração & dosagem , Compostos Organoplatínicos/administração & dosagem , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Cisplatino/farmacocinética , Cisplatino/farmacologia , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Injeções Intraperitoneais , Lipossomos/química , Masculino , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/tratamento farmacológico , Compostos Organoplatínicos/química , Compostos Organoplatínicos/farmacocinética , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Distribuição TecidualRESUMO
Previous studies demonstrated that proteinases from latex of C. candamarcensis act as mitogens on fibroblast and epithelial cells and a subsequent report showed their protective, angiogenic and wound healing effects on gastric ulcers. In this study, we present evidence of skin healing activity by the group of proteinases known as P1G10. By using a hairless mouse model, we compared the healing effect following topical application of various concentrations of P1G10. The data confirm that healing actions take place between 0.1 and 1%, without adverse local irritation or systemic toxicological action after a prolonged period of use. The wound healing effect is unaltered when P1G10 is previously inhibited with iodoacetamide. The low permeation of the hydrosoluble formulation Polawax(®) supports the maintenance of the drug at the site of application. These results extend the healing properties of these groups of enzymes in situations of dermatological trauma and open the way to future clinical applications.
Assuntos
Cisteína Endopeptidases/farmacologia , Glicoproteínas/farmacologia , Látex/química , Fitoterapia , Pele/efeitos dos fármacos , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia , Animais , Peso Corporal/efeitos dos fármacos , Carica/enzimologia , Cisteína Endopeptidases/uso terapêutico , Dextranos , Eletroforese em Gel de Poliacrilamida , Feminino , Géis , Glicoproteínas/uso terapêutico , Masculino , Camundongos , Camundongos PeladosRESUMO
Latex from Caricaceae contains proteolytic enzymes localized in the fruit, which are used ethnopharmacologically to treat digestive disorders. Some of these proteins display proliferative properties when probed with mammalian cells, suggesting a role in the reconstruction of wounded tissue. We tested the efficacy of a proteolytic fraction derived from Carica candamarcensis, designated as P1G10 in experimental rodent models, to protect and heal chemically induced gastric ulcers. The protective effect of oral administration of P1G10 fraction was analyzed in indomethacin-treated Wistar animals. The healing effect of P1G10 was studied following sub-serous injection of acetic acid in a Wistar rat model. The results show that P1G10 between 0.1 and 10 mg/kg protect indomethacin but not ethanol-induced gastric ulcers. The maximal protection attained was 67% with 10 mg/kg of P1G10. The healing rate by 10 mg/kg of P1G10 using the acetic acid ulcerogenic model is similar to that of omeprazole (10 mg/kg) or ranitidine (100 mg/kg). The effect of P1G10 at 10 mg/kg seems to be mediated by an increase in the mucus content by 25% and stimulation of angiogenesis by 64% in a manner similar to growth factors. These results confirm the protective and healing role of proteinases from C. candamarcensis.