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Objective:To investigate the effects of ligustrazine combined with emodin on angiogenesis of ascites carcinoma Walker-256 cells by observing their inhibition against nuclear factor-<italic>κ</italic>B （NF-<italic>κ</italic>B）， hypoxia-inducible factor-1<italic>α</italic> （HIF-1<italic>α</italic>）， and vascular endothelial growth factor-C （VEGF-C） in HIF signaling pathway. Method:Fifty SD rats were randomly divided into sham operation group， model group， ligustrazine group， emodin group and ligustrazine combined with emodin group. Following the in situ injection of rat ascites carcinoma Walker-256 cells into the liver of normal rats， they were grouped and administered with ligustrazine （10 mg·kg<sup>-1</sup>）， emodin （10 mg·kg<sup>-1</sup>）， and ligustrazine （10 mg·kg<sup>-1</sup>） plus emodin （10 mg·kg<sup>-1</sup>） for seven days. Afterwards， the tumor-inoculated liver tissue was sampled from the experimental group and prepared into pathological sections for investigating tumor cell survival and VEGF expression. The <italic>in vitro</italic> hypoxia and hypoglycemia model （oxygen-glucose deprivation model）， hypoxia model， and hypoglycemia model of Walker-256 cells were constructed respectively. In the ligustrazine group， emodin group， and ligustrazine combined with emodin group， three consecutive concentrations that did not affect the proliferation of Walker-256 cells were selected for investigation. The drugs were administered before modeling， and the model treatment lasted for 4 h. The levels of HIF-1<italic>α</italic>， VEGF-C， and NF-<italic>κ</italic>B in the cell culture supernatant of each group were tested. Result:After the rat liver was inoculated with Walker-256 cells， the total liver mass was significantly increased（<italic>P</italic><0.05）， higher than that in the ligustrazine group， the emodin group， or the ligustrazine combined with emodin group（<italic>P</italic><0.05）. Histopathological examination showed that the response of VEGF expression in the liver tissue of each administration group was lower than that of the model group. At the cellular level， the levels of HIF-1<italic>α</italic>， VEGF-C， and NF-<italic>κ</italic>B in oxygen-glucose deprivation model of the ligustrazine group and the ligustrazine combined with emodin group were significantly reduced（<italic>P</italic><0.05）， exhibiting a certain dose-dependent response， followed by the reduction in the hypoxia model. The levels of HIF-1<italic>α</italic> and NF-<italic>κ</italic>B in the oxygen-glucose deprivation model and the hypoglycemia model of the emodin（1×10<sup>-2</sup>，1×10<sup>-3</sup> mol·L<sup>-1</sup>） group and the ligustrazine combined with emodin（1×10<sup>-2</sup>，1×10<sup>-3</sup> mol·L<sup>-1</sup>） group were significantly reduced， but there was no significant change in VEGF-C level of the hypoxia model of all the administration groups. Conclusion:Ligustrazine or emodin alone or their combination inhibits the abnormal increase in the weight of rat liver after inoculation with Walker-256 cells and the expression of VEGF in the liver tissue. Ligustrazine and emodin inhibit the protein expression of NF-<italic>κ</italic>B and HIF-1<italic>α</italic>， thereby reducing the gene and protein expression of metastasis-related target VEGF-A activated by HIF-1<italic>α</italic> transcription， restricting tumor cell neovascularization， and inhibiting the invasion and spread of ascites carcinoma cells. Among them， ligustrazine has the most significant effect against hypoxia. Glucose interferes with the effect of ligustrazine. The combination of ligustrazine with emodin is conducive to diminishing the intervention of glucose and stabilizing the inhibition against tumor cells.
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Abstract The Walker-256 tumor is an important experimental model that allow the development of therapies as the biological behavior of this tumor is similar that occur in humans. In front of the above considerations, the aim of this study was to describe the experimental model of Walker-256 tumor, identify the implantations sites as well as define a usual quantity of tumoral cells to induce the ascitic and solid tumor, according to the specialized literature. Were selected 45 articles using the keyword "Walker-256 tumor", free available. Were possible to observe that 58% (n=26) of the studies inoculate the tumor cells in the animals flank 33% (n = 15) in the tibia bone, 7% (n = 3) in the femur and 2% (n = 1) in the paw. The major quantitates of cells used were 8 x 107 (20%), 1 x 105 (13%), 1 x 106 (11%) and 2 x 107 (11%). After that, the site commonly used to inoculate was the flank and quantitate still a controversy, being 1x105 and 8x107 the concentrations more used.
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Animales , Ratas , Carcinoma 256 de Walker/inducido químicamente , Modelos Animales , Carcinoma de Ehrlich , Línea Celular TumoralRESUMEN
This study was aimed to explore the antitumor effect of volatile oil of X ihuang pill and its immune mecha-nism in order to screen the antitumor active site of Xihuang pill. Among 70 female Wistar rats, 10 rats were random-ly selected as the blank control group; and the other 60 rats was used in the establishment of walker 256 breast can-cer cell tumor-bearing rat models. The model rats were randomly divided into the negative control group (model group), high-dose volatile oil group, middle-dose volatile oil group, low-dose volatile oil group, high-dose Xihuang pill group, and lentinan group (positive control group), with 10 rats in each group. The intragastric administration was given twice a day for 14 days. Blood was taken from the abdominal aorta. Tumor tissues was removed and weighed to calculate the tumor inhibitory rate. ELISA method was used to detect the level of IL-2, IL-6, IL-10, IFN-γ and TGF-β in peripheral blood. The flow cytometry was used to detect the content of CD3+ T cell, CD4+ T cell, CD8+ T cell, and B7-1 cell (CD80). The results showed that the tumor inhibitory rates of volatile oil of high-dose group and middle-dose group were 28.4% and 24.1%, respectively. Compared with the model group, the average level of IL-2 and IFN-γ of volatile oil of high-dose group and middle-dose group and CD3+ T cell, CD8+ T cell, B7-1 cell con-tent were obviously increased (P< 0.05). It was concluded that volatile oil of Xihuang pill had certain antitumor ef-fect, which was one of the antitumor active sites of Xihuang pill. The volatile oil of Xihuang pill upregulates the lev-els of IL-2 and IFN-γ, as well as the contents of CD3+ T cell, CD8+ T cell, B7-1 cell in order to increase the im-mune clearance function of tumor-bearing rats.
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Objective To observe the functional changes of T lymphocytes in the spleen of rats with bone cancer pain.Methods forty-one healthy female Sprague-Dawley rats were used in this study, and were divided into blank control, PBS and Walker-256 tumor groups.Bone cancer pain model was established by inoculation of Walker 256 cancer cells into the tibial cavity.The paw withdrawal threshold (PWT), paw withdrawal thermal latency (PWL), and spontaneous pain (SP) were all measured before modelling (as base) and at 4, 6, 8, 10, 12, 14, 16, 18, and 20 days after modelling. The function of T lymphocyte proliferation, and the content of T lymphocytes and their subgroups in the spleen were detected by cell counting kit-8 method and flow cytometry, respectively, on day 20 after modelling.Results Before modellng, there were no differences of PWT, PWL, and SP between the PBS and model groups.After modelling, the PWT and SP of model group were significantly decreased on day 4, and were always lower than that of PBS group during the experiment.Statistical analysis revealed that Walker-256 cancer cell inoculation in the tibia induced a significant decrease in PWL on day 8, 10 and 12 after modellng.Compared with the control group, T lymphocyte proliferation, content of T lymphocyte (CD3) and subgroups ( CD4 and CD8) in the PBS group were not significantly decreased.However, T lymphocyte proliferation and the content of CD3 lymphocytes in the model group were significantly lower than those in the blank control group and/or PBS group.Conclusions The bone cancer pain rat model may appear obvious mechanical allodynia and spontaneous pain.Its thermal pain hyperalgesiaonly occurred in the intermediate stage of bone cancer pain. The content of T lymphocytes and its subgroups, and the function of T lymphocyte proliferation are weakened to some extent in the bone cancer pain rat model.
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Objetivo: verificar os efeitos do óleo de copaíba por via intravaginal no tumor de Walker 256inoculado na vagina e útero de ratas. Método: o tumor de Walker 256 foi inoculado na vagina eútero de 20 ratas, e estas distribuídas em dois grupos (n=10), no grupo controle (GC) os animaisforam tratados com água destilada intravaginal na dose de 0,3mL e o grupo copaíba (GCO),tratados com óleo de copaíba na dose de 0,3mL intravaginal. Analisou-se a variação de peso doanimal, peso e volume tumoral, potencial de inibição (PI), além da análise histológica da vagina,útero e reto dos animais. Resultados: a média da variação de peso no grupo controle foi 11,44±8,03g e no grupo copaíba 10,88 ±3,48g, não houve diferença estatisticamente significante(p=0,83). No grupo controle o peso médio do tumor foi de 2,4 ±1,22 g e o volume de 2,52 ±1,41mL e no grupo copaíba o peso médio de 2,31 ±0,88g e o volume médio de 2,36 ±1.13 mL. Opotencial de inibição do óleo de copaíba por via intravaginal foi de 4,74%. Não houve diferençaem relação ao estudo histológico. Conclusão: o óleo de copaíba por via intravaginal nãoapresentou efeitos sobre o tumor de Walker 256, inoculado na vagina e útero de ratas, emrelação ao peso do animal, peso e volume tumoral e características histológicas
Objective: Evaluate the effects of copaiba oil intravaginally on the Walker 256 tumorinoculated into the vagina and uterus of rats. Methods: Was inoculated Walker 256 tumor in thevagina and uterus of 20 rats, they were divided into two groups (n = 10) in control groupanimals were treated intravaginally with distilled water at a dose of 0.3 ml and the groupcopaiba, the animals was treated with copaiba oil at a dose of 0.3 ml intravaginally. Wasanalyzed the variation of body weight, and tumor?s volume and weight and potential forinhibition of the oil. Results: The mean weight change in the control group was 11.44 ± 8.03 gand group copaiba 10.88 ± 3.48 g, there was no statistical difference (p = 0.83). In the controlgroup the mean tumor weight was 2.4 ± 1.22g and the volume of 2.52 ± 1.41 mL in groupcopaiba the average weight was 2.31 ± 0.88g and the mean volume was 2.36 ± 1.13 mL. Thepotential inhibition of copaiba oil intravaginally was 4.74%. Conclusion: The copaiba oilintravaginally had no effect on the Walker 256 tumor inoculated into the vagina and uterus ofrats, relative to the weight of the animal and the tumor volume and weight, and histologycalcaracteristy.
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The present study aimed at characterizing the subcutaneous development of the Walker 256 (W256) AR tumor, a regressive variant of the rat W256 A tumor. Wistar rats were injected subcutaneously with 4x10(6) W256 A or W256 AR tumor cells. The development of tumors was evaluated daily by percutaneous measurements. None of the W256 A tumors (n=20) regressed, but 62 percent of the W256 AR tumor-bearing rats (n=21) underwent complete tumor regression within 35 days. Continuous growth of AR tumors was characterized by an increase of the tumor growth rate from day 12, which reached values above 1.0 g/day, and were significantly higher (p<0.05) than those of the regressive AR tumors. Immunosuppression by irradiation before subcutaneous injection of AR cells completely abrogated tumor regression and was associated with severe metastatic dissemination. Daily evaluation of the tumor growth rate enabled the discrimination, in advance, between continuously growing tumors and those that regressed later on.
O objetivo neste estudo foi caracterizar o desenvolvimento subcutâneo do tumor de Walker 256 (W256) AR, uma variante regressiva do tumor de W256 A de rato. Ratos Wistar foram injetados com 4x10(6) células tumorais de W256 A ou W256 AR. O desenvolvimento tumoral foi avaliado diariamente. Nenhum dos tumores W256 A (n=20) regrediu, mas 62 por cento dos ratos com tumor W256 AR apresentaram regressão completa dos tumores em até 35 dias. O crescimento contínuo dos tumores AR foi caracterizado pelo aumento da taxa de crescimento tumoral a partir do dia 12, alcançando valores maiores que 1,0g/dia, que foram significativamente superiores (p<0,05) aos valores de taxa de crescimento dos tumores regressivos AR. A imunossupressão por irradiação precedendo a injeção das células tumorais AR eliminou completamente a regressão tumoral e favoreceu disseminação metastática severa. Este estudo caracterizou o desenvolvimento do tumor de W256 AR em condições específicas, documentando a regressão espontânea deste tumor após a injeção subcutânea de altas doses de células tumorais em ratos Wistar. A avaliação diária da taxa de crescimento tumoral permite discriminar precocemente os tumores com crescimento continuo daqueles que são regressivos. A taxa de crescimento tumoral é um parâmetro útil para a avaliação dos animais experimentais, particularmente no período que precede a regressão dos tumores.
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In order to investigate the inhibitory effects of all trans-retinoitc acid (ATRA) on differentiation and apoptosis of Walker-256 hepatocellular carcinoma cells and the therapeutic effects of ATRA combined with transarterial chemoembolization (TACE) on rat Walker-256 transplanted hepa-tocarcinoma, Walker-256 hepatocarcinoma cell lines were treated with ATRA at different concentrations. After culture for 48 h, the inhibitory rate of cell proliferation was determined by MTT assay; the changes of Fas and Bcl-2 mRNA expression were determined by RT-PCR, and the expression levels of Caspase3 and Caspase8 proteins were detected by Western blot. Twenty-seven Wistar rat models of hepatocarcinoma were set up successfully by implanting Walker-256 cell lines. The tumor volume at the 11th day after implantation (Vpreoperatioi) was measured by magnetic resonance imaging (MRI). The 27 rats were randomly and equally divided into three groups, and the therapy scheme was performed as follows: group A (ATRA 0.1 mg+mitomycin 0.05 mL+lipiodol 0.05 mL+gelfoam powder 0.025 mg); group B (mitomycin 0.05 mg+lipiodol 0.05 ml+gelfoam 0.025 mg; group C (0.9% NaCl 0.2 mL). After another 11 days, MRI was performed once again to measure the tumor volume (Vpostoperation)- The expression of factor VIII and Ki-67 in the tumor tissues was detected by immuno-histochemistry. The results showed that ATRA could suppress proliferation of Walker-256 cell lines. After treatment of Walker-256 cell lines with ATRA, the expression of Fas mRNA was significantly up-regulated and the Bcl-2 mRNA was significantly down-regulated by ATRA at the concentration of 10 umol/L as compared with the control group (P<0.05). After treatment with 10 umol/L ATRA for 48 h, the Caspase3 and Caspase8 were significantly activated as compared with the control group (P<0.05). Significant difference existed in growth rate among the three groups (P<0.01) and between either two groups (P<0.05). The expression rate of factor VIII and Ki-67 was gradually increased from group A, group B to group C. The study suggests that ATRA could inhibit the proliferation of Walker-256 cells and the effectiveness of the combined therapy (ATRA+TACE) for treating transplanted hepatoma of rats is superior to that of TACE alone.
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PURPOSE: The objective of this study was to develop a rat lung tumor model for anticancer drug testing. METHODS: Sixty-two female Wistar rats weighing 208 ± 20 g were anesthetized intraperitoneally with 2.5 percent tribromoethanol (1 ml/100 g live weight), tracheotomized and intubated with an ultrafine catheter for inoculation with Walker's tumor cells. In the first step of the experiment, a technique was established for intrabronchial implantation of 10(5) to 5×10(5) tumor cells, and the tumor take rate was determined. The second stage consisted of determining tumor volume, correlating findings from high-resolution computed tomography (HRCT) with findings from necropsia and determining time of survival. RESULTS: The tumor take rate was 94.7 percent for implants with 4×10(5) tumor cells, HRCT and necropsia findings matched closely (r=0.953; p<0.0001), the median time of survival was 11 days, and surgical mortality was 4.8 percent. CONCLUSION: The present rat lung tumor model was shown to be feasible: the take rate was high, surgical mortality was negligible and the procedure was simple to perform and easily reproduced. HRCT was found to be a highly accurate tool for tumor diagnosis, localization and measurement and may be recommended for monitoring tumor growth in this model.
OBJETIVO: O objetivo foi desenvolver um modelo de tumor de pulmão em rato que permita o teste de fármacos no tratamento deste câncer. MÉTODOS: Sessenta e dois ratos Wistar fêmeas, peso médio de 208±20 g, foram anestesiados com tribromo-etanol 2,5 por cento IP (1ml/100g de rato), traqueostomizados e intubados com cateter ultrafino para injetar células do tumor de Walker. Na 1ª etapa, estabeleceu-se a técnica do implante de células tumorais por via intrabrônquica e o índice de pega tumoral, usando-se de 10(5) a 5×10(5) células. Na 2ª, avaliou-se o volume tumoral e a correlação dos achados obtidos na tomografia computadorizada de alta resolução (TCAR) de tórax com os da necropsia e verificou-se a sobrevida. RESULTADOS: O índice de pega foi de 94,7, com o implante de 4×10(5) células do tumor; as medidas do tumor feitas na TCAR e comparadas com as da necropsia foram semelhantes (r=0, 953, p<0,0001); a sobrevida mediana foi de 11 dias; e a mortalidade cirúrgica de 4,8 por cento. CONCLUSÃO: O modelo mostrou-se viável, com alto índice de pega, mortalidade cirúrgica desprezível, de execução simples e fácil reprodutibilidade. A TCAR revelou alta acurácia no diagnóstico, localização e mensuração das lesões tumorais, credenciando-se para a monitorização de crescimento tumoral nesse modelo.
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Animales , Femenino , Ratas , /patología , Neoplasias Pulmonares/patología , Modelos Animales de Enfermedad , Modelos Lineales , Neoplasias Pulmonares/secundario , Células Tumorales Cultivadas , Tomografía Computarizada por Rayos X/métodos , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
O uso de hipertermoterapia isolada ou associada à quimioterapia ou radioterapia vem sendo empregada com sucesso no tratamento de vários tipos de tumores malignos. inclusive no câncer gástrico. A elevada incidência desse tipo de neoplasia no Ceará e a pouca eficiência dos atuais métodos terapêuticas empregados no seu tratamento nos levou a procurar novas opções. mais eficazes, que pudessem aumentar a sobrevida e a qualidade de vida dos pacientes portadores do referido tumor. O presente trabalho desenvolveu um modelo de tumor gástrico experimental e avaliou os efeitos da hipertermia (Ht) de corpo inteiro, obtida através da perfusão peritoneal hipertérmica contínua (PPCH), associada à quimioterapia (Qt) com Carboplatina (25mg/Kg de peso), administrada em dose única por via intraperitoneal, 60 minutos antes do início da PPCH. O modelo de tumor mostrou-se eficiente. com um índice de 100% de pega. Os animais inoculados apresentaram uma sobrevida de 13,2 ± 0.6 dias. Aqueles submetidos ao tratamento foram divididos em 3 grupos diferentes, de acordo com o tempo de evolução do tumor, ou seja, 3, 7 e 10 dias após a inoculação. A técnica de PPCH foi testada com sucesso em um grupo de 12 animais e proporcionou a obtenção de uma temperatura segura (41.8 ± 0,6) para avaliação da hipertermoterapia. Embora os efeitos da hipertermia isoladamente não tenha aumentado significativamente a sobrevida dos animais portadores do tumor gástrico experimental, a sua associação com a quimioterapia mostrou-se sinérgica e bastante eficiente, tendo aumentado significativamente (p<0,001) o percentual de sobrevida nas três fases de evolução tumoral, para 160,5, 107,8 78,0%, respectivamente nas feses inicial (3 dias), intermediária (7 dias) e avançada (10 dias) do tumor. Portanto, podemos concluir que a hipertermoquimioterapia. associando temperautras entre 41.5 e 42°C com Carboplatina (25mg/Kg de peso), são eficientes em tratar esse tipo de tumor experimental, mesmo na fase terminal do hospedeiro
The use of hyperthermotherapy, isolated or associated with chemotherapy or radiotherapy, has been employed with success on the treatment of many types of malign tumours, including gastric cancer. The high incidence of this type of cancer in Ceará and the low efficiency of the actual therapeutic methods used on its treatment made us look for new options, more efficient, that could raise the survival and the quality of life of the patients that have this cancer. This work developed an experimental model of gastric tumour and evaluated the effects of full body hyperthermia (Ht), obtained by continuous peritoneal hyperthermic perfusion (CPHP), associated to chemotherapy (Qt) with Carboplatin (25mg/Kg), administrated on one dose by intraperitoneal method, 60 min before the beginning of CPHP. The tumour model showed efficiency, with 100% rate of tumour growth. The inoculated animal showed an survival of 13.2 +0.6 days. Those that were submitted to the treatment were divided in 3 different groups, according to the tumour evolution time, which was 3,7 and 10 days after the inoculation. The CPHP technique was tested with success on a group of 12 animals and proportioned the obtaining of a secure temperature (41.8 +0.6°C) for the evaluation of the hyperthermotherapy. Although the effects of isolated hyperthermia didn't raise significantly the survival of the animals with the experimental gastric tumour, its association with chemotherapy showed synergism and was very efficient, having raised significantly (p<0,00l) the rate of survival of the three phases of tumour evolution to 160.5, 107.8 and 78% respectively on the initial (3days), intermediate (7 days) and advanced (l0 days) phase of the tumour. Therefore, we can conclude that hyperthermotherapy, associating temperatures between 41.5 and 42.0°C with Carboplatin (25mg/Kg), is efficient in treating this type of experimental tumour, even at host's terminal phase
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Animales , Ratas , Carboplatino , Fiebre , Ratas , Neoplasias GástricasRESUMEN
For evaluation of biological effect of p+(50.5 MeV) Be neutron beam produced by Korea Cancer Center Hospital(KCCH) cyclotron the RBE had been measured in experimental tumor Walker 256 carcinosarcoma as well as normal tissue, mouse intestine and bone marrow, in single and fractionated irradiation. As pilot study, the RBE had been measured for the mouse jejunal crypt cells in single whole body irradiation of which the result was 2.8. The obtained RBE values of TCD 50 of Walker 256 tumor, bone marrow and intestine in single irraiation were 1.9, 1.9 and 1.5 respectively. In fractionated irradiation, the RBE value of tumor Walker 256 was decreased as increasing of fraction number and increased as increaing of fraction size.