Moderate exercise training since adolescence reduces Walker 256 tumour growth in adult rats.

KEY POINTS: Cancer growth, cell proliferation and cachexia index can be attenuated by the beneficial programming effect of moderate exercise training, especially if it begins in adolescence. Walker 256 tumour-bearing rats who started exercise training during adolescence did not revert the basal low glycaemia and insulinaemia observed before tumour cell inoculation. The moderate exercise training improved glucose tolerance and peripheral insulin sensitivity only in rats exercised early in adolescence. The chronic effects of our exercise protocol are be beneficial to prevent cancer cachexia and hold clear potential as a nonpharmacological therapy of insulin sensitization. ABSTRACT: We tested the hypothesis that moderate exercise training, performed early, starting during adolescence or later in life during adulthood, can inhibit tumour cell growth as a result of changes in biometric and metabolic markers. Male rats that were 30 and 70 days old performed a treadmill running protocol over 8 weeks for 3 days week-1 , 44 min day-1 and at 55-65% V̇O2max . After the end of training, a batch of rats was inoculated with Walker 256 carcinoma cells. At 15 days after carcinoma cell inoculation, the tumour was weighed and certain metabolic parameters were evaluated. The data demonstrated that physical performance was better in rats that started exercise training during adolescence according to the final workload and V̇O2max . Early or later moderate exercise training decreased the cachexia index, cell proliferation and tumour growth; however, the effects were more pronounced in rats that exercised during adolescence. Low glycaemia, insulinaemia and tissue insulin sensitivity was not reverted in Walker 256 tumour-bearing rats who trained during adolescence. Cancer growth can be attenuated by the beneficial programming effect of moderate exercise training, especially if it begins during adolescence. In addition, improvement in glucose-insulin homeostasis might be involved in this process.

Metabolic effects of Hedyotis diffusa on rats bearing Walker 256 tumor revealed by NMR-based metabolomics.

Hedyotis diffusa, a traditional Chinese herbal medicine, is widely used for oncotherapy and shows a positive effect in the clinical treatment. But its mechanism of anticancer activities is complicated and unclear. This study was undertaken to assess the therapeutic effects and reveal detailed mechanisms of H. diffusa for oncotherapy. A Walker 256 tumor-bearing rat model was established, and metabolomic profiles of plasma and urine were obtained from 1 H NMR technique. Multivariate statistical analysis methods were used to characterize the discriminating metabolites between control (C), Walker 256 tumor-bearing rats model (M), and H. diffusa treatment (H) groups. Finally, 13 and 10 metabolomic biomarkers in urine and plasma samples were further identified as characteristic metabolites in M group, whereas H group showed a partial metabolic balance recovered, such as ornithine, N-acetyl-l-aspartate, l-aspartate, and creatinine in urine samples, and acetate, lactate, choline, l-glutamine, and 3-hydroxybutyrate in plasma samples. On the basis of the methods above, we hypothesized H. diffusa treatment reduced the injury caused by Walker 256 tumor and maintained a metabolic balance. Our study demonstrated that this method provided new insights into metabolic alterations in tumor-bearing biosystems and researching on the effects of H. diffusa on the endogenous metabolism in tumor-bearing rats.

Effect of microbubble-enhanced ultrasound on percutaneous ethanol ablation of rat walker-256 tumour.

OBJECTIVES: Percutaneous ethanol ablation (PEA) is an effective method for treating small liver cancer. Microbubble-enhanced ultrasound (MEUS) can potentially promote PEA by disrupting the tumour's circulation. In this study, treatment combining MEUS and PEA was performed to find any synergistic effects in tumour ablation. METHODS: Ten rats bearing subcutaneous Walker-256 tumours were treated by MEUS combined with PEA. The other 18 tumour-bearing rats that were treated by MEUS or PEA served as the controls. MEUS was conducted by therapeutic ultrasound (TUS) and microbubble injection. TUS was operated at a frequency of 831 KHz with a pressure amplitude of 4.3 MPa. Tumour blood perfusion was assessed by contrast-enhanced ultrasound (CEUS), and the tumour necrosis rate was determined by histological examination. RESULTS: CEUS showed that the tumour blood perfusion almost vanished in all of the MEUS-treated tumours. The contrast peak intensity dropped 84.8 % in the MEUS + PEA-treated tumours when compared to 46.3 % (p < 0.05) in the PEA-treated tumours 24 h after treatment. The tumour necrosis rate of the combination therapy was 97.50 %, which is much higher than that of the MEUS- (66.2 %) and PEA-treated (81.0 %) tumours. CONCLUSION: PEA combined with MEUS can induce a much more complete tumour necrosis. KEY POINTS: • This experiment demonstrated a novel method for enhancing percutaneous ethanol ablation. • Microbubble-enhanced therapeutic ultrasound is capable of disrupting tumour circulation. • Combined therapy of MEUS and PEA can induce more complete necrosis of tumours.

Changes in lipid metabolism during Walker 256 tumor growth and the therapeutic effect of hyperthermia.

The dynamics of lipoprotein content during Walker 256 tumor growth in rats was studied. Moderate changes in HDL and LDL were paralleled by significant changes in VLDL level. A 2-fold increase of VLDL in comparison with the intact control was recorded on day 10 of tumor growth. Exposure to total hyperthermia additionally stimulated VLDL synthesis and this parameter increased by 4 times and more in rats with tumors in comparison with controls. This effect of hyperthermia correlated with significant subsequent decrease of rat mortality caused by the lethal effect of the tumor.

Proliferation of Walker 256 carcinosarcoma cells: effect of whole-body hyperthermia and antitumor agents.

We studied the main biological characteristics of spontaneous growth of Walker 256 carcinosarcoma and under conditions of antitumor therapy. A combination of whole-body hyperthermia and cytostatic treatment (cyclophosphamide and melatonin) produced maximum suppression of the tumor growth, inhibition of mitotic activity of tumor cells, and stimulation of their necrotic and apoptotic death. The maximum decrease in mitotic activity of tumor cells was observed after combined exposure to whole-body hyperthermia and both cytostatic preparations; enhancement of apoptotic cell death and the decrease in the tumor node weight were also most pronounced under these conditions and practically no body weight loss was recorded in this case.

Therapeutic effects of all trans-retinoic acid combined with transarterial chemoembolization on Walker-256 hepatoma in rats.

In order to investigate the inhibitory effects of all trans-retinoic 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 hepatocarcinoma, 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 (V(preoperation)) 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 (V(postoperation)). The expression of factor and Ki VIII -67 in the tumor tissues was detected by immunohistochemistry. 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 mumol/L as compared with the control group (P<0.05). After treatment with 10 mumol/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.

[Effect of whole-body controlled hyperthermia on lipid peroxidation products and lysosomal enzyme levels in blood serum from Wistar rats with Walker-256 carcinosarcoma].

Whole-body controlled hyperthermia (up to 43.5 degrees C) of Wistar rats with Walker-256 carcinosarcoma was followed by symptoms of enhanced lipid peroxidation for 3-14 days and release of lysosomal enzymes into blood on day 14. Our data, in totality, point to a potential to stimulate tumor cell apoptosis.

[Change in the content of lipid-soluble antioxidants in animals with Walker-256 carcinosarcoma exposed to different therapies].

The time course of changes in the content of lipid-soluble antioxidants in the sera of male Wistar rats with transplantable Walker-256 carcinosarcoma in the application of controlled systemic hyperthermia, cyclophosphan, and melatonin were studied. When controlled systemic hyperthermia and cyclophosphan were used, the rat serum levels of alpha-tocopherol and retinol reduced whereas the addition of melatonin to therapy unchanged their content. Therefore, one of the promising ways of increasing the efficiency of treatment for malignancies may be to maintain the high activity of the antioxidant regulation system in nonneoplastic cells and tissues.

[An experimental study of energy controllable steep pulse in the treatment of rat with subcutaneous transplantive tumor].

This experimental study was designed to investigate the effects and the expressions of microvessel density (MVD), vascular endothelial growth factor (VEGF) on the transplanted tumor in the rat model with Walker-256 after energy controllable steep pulse(ECSP). The experiment revealed that the steep pulse electrical field has better effect on tumor, compared with the control. The positive cell staining intensity of VEGF in the control group was significantly higher than that in ECSP group (P < 0.05). The number of MVD in the tumor tissues of ECSP group was significantly lower than that of tumor control group (P < 0.05). These results showed that ECSP could inhibit the growth and angiogenesis of tumor and its pathway is to down-regulate the expression of VEGF possibly.

[Effect of steep pulsed electric fields on the immune response of tumor-bearing Wistar mice].

This study sought to evaluate the effect of steep pulsed electric fields (SPEFs) on the immune response of Wistar mice inoculated with Walker256 sarcoma. Thirty mice were randomly divided into three groups: control group (group A, inoculated with Walker256 sarcoma, not treated), treatment group (group B, inoculated with Walker256 sarcoma, treated by SPEFs), and normal control group (group C, inoculated with normal saline, not treated). Tumor size was measured before and every 3 days after treatment by vernier caliper. MTT methods were used to assess the lymphocytes proliferation and the natural killer (NK) cells activity. TNF-a activity was measured by ELISA. Statistical analysis was performed utilizing the SPSS10.0 software package. The experiment results revealed that tumor growth was significantly inhibited in group B as compared with group A (P < 0.01), and that lymphocytes proliferation, NK cells activity and TNF-a activity in group B were not significantly different from those in group C (P = 0.953, P = 0.130, P = 0.080, respectively) but markedly higher than those in group A (P < 0.05). The results also showed that SPEFs could not only kill tumor cells but also induce antitumor immune response and improve the immune function of the host efficiently.

Resistance exercise attenuates skeletal muscle oxidative stress, systemic pro-inflammatory state, and cachexia in Walker-256 tumor-bearing rats.

The aim of this study was to investigate the effects of resistance exercise training (RET) on oxidative stress, systemic inflammatory markers, and muscle wasting in Walker-256 tumor-bearing rats. Male (Wistar) rats were divided into 4 groups: sedentary controls (n = 9), tumor-bearing (n = 9), exercised (n = 9), and tumor-bearing exercised (n = 10). Exercised and tumor-bearing exercised rats were exposed to resistance exercise of climbing a ladder apparatus with weights tied to their tails for 6 weeks. The physical activity of control and tumor-bearing rats was confined to the space of the cage. After this period, tumor-bearing and tumor-bearing exercised animals were inoculated subcutaneously with Walker-256 tumor cells (11.0 × 107 cells in 0.5 mL of phosphate-buffered saline) while control and exercised rats were injected with vehicle. Following inoculation, rats maintained resistance exercise training (exercised and tumor-bearing exercised) or sedentary behavior (control and tumor-bearing) for 12 more days, after which they were euthanized. Results showed muscle wasting in the tumor-bearing group, with body weight loss, increased systemic leukocytes, and inflammatory interleukins as well as muscular oxidative stress and reduced mTOR signaling. In contrast, RET in the tumor-bearing exercised group was able to mitigate the reduced body weight and muscle wasting with the attenuation of muscle oxidative stress and systemic inflammatory markers. RET also prevented loss of muscle strength associated with tumor development. RET, however, did not prevent the muscle proteolysis signaling via FBXO32 gene messenger RNA expression in the tumor-bearing group. In conclusion, RET performed prior tumor implantation prevents cachexia development by attenuating tumor-induced systemic pro-inflammatory condition with muscle oxidative stress and muscle damage.

Effect of immunisation with cancer procoagulant on the growth of Walker 256 carcinosarcoma cells in rats.

The effect of preimmunisation with cancer procoagulant (CP) on the growth of Walker 256 carcinosarcoma cells in Wistar Lew/Han/IMP rats in vivo has been analysed. One group of rats was immunised with CP purified from human amnion-chorion membranes. The rest of the animals (control groups) were injected with 0.9% NaCl in Freund's adjuvant or were not immunised at all. When the presence of polyclonal anti-CP antibody was detected in CP-immunised rats' serum, all (CP-immunised and control) animals were injected i.p. with 4.8 x 10(5) Walker 256 cells per rat. After 5 days ascitic fluid was collected and viable cells were counted. The complete lack of viable Walker 256 carcinosarcoma cells in 24% of the CP-immunised rats was observed. However, the viable neoplastic cells were present in all control (NaCl-injected and nonimmunised) animals. It seems possible that CP plays an important role in tumour progression, so immunisation with CP can reduce the risk of development of malignant disease.

Effect of hyperthermia on vascular functions of normal tissues and experimental tumors; brief communication.

The effect of hyperthermia on vascular volume and vascular permeability of the Walker carcinoma 256, skin, and muscle of inbred SD male rats was investigated. Whereas heating at 43 degrees C for 1 hour induced a significant increase in the functional vascular volume and vascular permeability in the skin and muscle, it did not change these vascular functions in the tumors. The increased vascular volume in the normal tissues suggested an increased circulation, which may enhance the dissipation of heat and result in a differential effect of hyperthermia on normal tissues and tumors.

Blood flow in normal tissues and tumors during hyperthermia.

The effect of hyperthermia on the blood flow was studied in skin, muscle, and Walker 256 carcinoma implanted in the legs of SD rats. The radioactive microsphere method was used to measure the blood flow. Hyperthermia for 1 hour with water at 43 degrees C increased the blood flow in skin and muscle by about threefold to fourfold. On the contrary, hyperthermia had no appreciable effect on the blood flow in tumors. Consequently, during hyperthermia blood flow in the skin and muscle surrounding the tumors was greater than that in tumors larger than about 2 g. Despite the apparent increase in heat dissipation by the increased blood flow, the temperature of the skin was 42.6-42.8 degrees C, and the temperature at the core of tumors larger than 2 cm in diameter was 42.3-42.7 degrees C during the hyperthermia. The lower temperature in the muscle could be attributed to an increase in heat dissipation as a result of the increased blood flow. This differential rise in temperature by heating may account in part for the differential effect of hyperthermia on tumors and normal tissues in vivo.

Induction of acute necrosis in Walker 256 tumors in rats.

The destructive effect on cells of locally injected hypertonic solutions, resorption of which is delayed by some vasoactive agents, was observed in the skin and subcutis of the rat by Selye and his coworkers who described it as acute conditioned necrosis. The same phenomenon was demonstrated in Walker 256 tumors implanted in the thighs of female Sprague-Dawley rats. A strongly hypertonic solution of glucose was injected once in and around the tumor (when it had reached a mean diameter of 1.5 cm), and 5-hydroxytryptamine was given s.c. at a distance. This resulted in disappearance of the growth in an average of 10 days and absence of metastases 2 months after treatment in 84% of animals as opposed to a spontnaeous regression of the tumor in 4% of control rats.

Interstitial pressure gradients in tissue-isolated and subcutaneous tumors: implications for therapy.

High interstitial fluid pressure (IFP) in solid tumors is associated with reduced blood flow as well as inadequate delivery of therapeutic agents such as monoclonal antibodies. In the present study, IFP was measured as a function of radial position within two rat tissue-isolated tumors (mammary adenocarcinoma R3230AC, 0.4-1.9 g, n = 9, and Walker 256 carcinoma, 0.5-5.0 g, n = 6) and a s.c. tumor (mammary adenocarcinoma R3230AC, 0.6-20.0 g, n = 7). Micropipettes (tip diameters 2 to 4 microns) connected to a servo-null pressure-monitoring system were introduced to depths of 2.5 to 3.5 mm from the tumor surface and IFP was measured while the micropipettes were retrieved to the surface. The majority (86%) of the pressure profiles demonstrated a large gradient in the periphery leading to a plateau of almost uniform pressure in the deeper layers of the tumors. Within isolated tumors, pressures reached plateau values at a distance of 0.2 to 1.1 mm from the surface. In s.c. tumors the sharp increase began in skin and levelled off at the skin-tumor interface. These results demonstrate for the first time that the IFP is elevated throughout the tumor and drops precipitously to normal values in the tumor's periphery or in the immediately surrounding tissue. These results confirm the predictions of our recently published mathematical model of interstitial fluid transport in tumors (Jain and Baxter, Cancer Res., 48: 7022-7032, 1988), offer novel insight into the etiology of interstitial hypertension, and suggest possible strategies for improved delivery of therapeutic agents.

Intratumoral pharmacokinetics and in vivo gene expression of naked plasmid DNA and its cationic liposome complexes after direct gene transfer.

The pharmacokinetic properties and gene expression of naked plasmid DNA and its cationic liposome complexes were studied after direct intratumoral injection. Using a Walker 256 tissue-isolated tumor perfusion system, we quantified the recovery of naked plasmid DNA and cationic liposome complexes in the tumor, leakage from the tumor surface, and the venous outflow after intratumoral injection. Approximately 50% of naked plasmid DNA had been eliminated from the tumor 2 h after injection, whereas more than 90% of plasmid DNA was retained in the tumor when it was complexed with cationic liposomes. However, the distribution of these complexes in the tumor was restricted to the tissue surrounding the injection site. Pharmacokinetic analysis of the venous outflow profiles suggested that the rate-limiting process that determines the retention of plasmid DNA in the tumor is transferred from the injection site in the tumor tissue and that complexation with cationic liposomes may retard this process. Furthermore, we examined the gene expression of chloramphenicol acetyltransferase DNA constructs (naked pCMV-CAT) and the corresponding cationic liposome [3-beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol] complexes. A similar level of gene expression was observed in vivo after direct intratumoral injection of naked DNA and its cationic liposome complexes. In both cases, a great variation was observed between tumors, and localization of gene-transduced cells in the tumor tissue was limited to the area in the vicinity of the injection site. Thus, these pharmacokinetic and gene expression studies have demonstrated that cationic liposomes can enhance the retention of injected DNA in the tumor model, whereas cationic liposome complex does not necessarily improve gene expression because of its poor dissemination in this tumor. The present study also suggested that there is a need to control the behavior of the injected naked plasmid DNA and its cationic liposome complexes to ensure better distribution throughout the tumor.

Increased thermal response to ultrasound in the Walker carcinosarcoma treated with vasoactive drugs.

In order to evaluate the potential of a highly selective Ca2+ entry blocker (nisoldipine) and of 5-hydroxytryptamine (5-HT) as adjuvant in hyperthermia treatment, we studied the differential flow response and time-course of tumor and normal tissue temperature following the administration of the two substances and during ultrasound heating. In 12 rats bearing Walker 256 carcinomas i.p. injection of 0.2-0.4 mg/kg nisoldipine caused a reduction in the tumor-to-muscle flow relationship of 4.4 +/- 1.9 (SD) to 1.74 +/- 0.86 as determined by intraarterial 133Xe injection; i.p. injection of 2-8 mg/kg 5-HT (N = 13) caused a respective reduction from 3.9 +/- 2.67 to 1.3 +/- 1.59. During a 20-min period of 41 degrees C normal tissue temperature-controlled ultrasound heating without drugs, tumor temperature attained 40.8 +/- 0.9 degrees C (N = 16). Nisoldipine or 5-HT injection at continuing 41 degrees C normal tissue temperature controlled energy delivery produced an instantaneous further increment of tumor temperature, eventually to 44.0 +/- 1.14 degrees C or 44.2 +/- 1.26 degrees C, respectively, after a period of 20 min. Injection of 0.9% NaCl (N = 4) solution caused only insignificant changes. Blood pressure and muscle perfusion were distinctly influenced by nisoldipine, but not by 5-HT. Since both drugs instantaneously increased the temperature differential between tumor and normal tissue, though by different vasoaction, they should be considered as adjuvants in hyperthermia.

Resistance exercise prevents impaired homocysteine metabolism and hepatic redox capacity in Walker-256 tumor-bearing male Wistar rats.

OBJECTIVE: The aim of this study was to investigate changes in homocysteine (Hcy) metabolism and redox balance in response to exercise treatment in a tumor-bearing rat model. METHODS: Male Wistar rats were exposed, or not, to a resistance exercise program 6 wk before inoculation with Walker-256 tumor cells or vehicle. After application, rats maintained their routine for 12 d and were then sacrificed for plasma and liver analyses. RESULTS: Impaired Hcy metabolism was evident after 12 d of tumor cell inoculation as demonstrated by significantly increased (P < 0.05) plasma total homocysteine (tHcy) concentration (53%) and decreased plasma cysteine, methionine, and vitamin B12 concentrations. Decreased hepatic cystathionine ß-synthase (CBS) and betaine-homocysteine S-methyltransferase mRNA levels were found in tumor-bearing rats but not in controls. Tumor inoculation also decreased levels of liver reduced glutathione (GSH) and increased hepatic oxidative stress compared with non-tumor controls. However, resistance exercise prevented the tumor-impaired transsulfuration pathway as demonstrated by the decreased plasma tHcy, hepatic CBS expression, and increased GSH in tumor-exercised versus tumor-sedentary rats. Remarkably, all measures of liver oxidative stress were suppressed by exercise training. Tumor weight was unchanged between groups. CONCLUSION: Resistance exercise prevented tHcy accumulation and liver oxidative damage caused by Walker-256 tumor cell inoculation; the modulatory effects of resistance exercise on Hcy metabolism appear to be at the level of transsulfuration pathway.

The alteration of protein profile of Walker 256 carinosarcoma cells during the apoptotic process induced by ultrasound.

The objective of this study was to investigate the alteration of the protein profile in cells after sonication and to identify the key proteins involved in the process of cell apoptosis. Walker 256 carinosarcoma cells were exposed to focused ultrasound (US) at the intensity of 2.0, 7.0, 10.2, 14.2 and 17.0 W/cm2 (I(spta)) for 10 min in vitro and the morphologic and functional changes of the cells were detected by hematoxylin & eosin staining and flow cytometry, with double staining of fluorescein isothiocyanate (FITC)-labeled Annexin V/propidium iodide (PI). The protein compositions in the cells after sonication were detected by 2-D SDS polyacrylamide gel electrophoresis. Our results showed that apoptosis of Walker 256 carinosarcoma cells could be induced by US. The percentage of early apoptosis and secondary necrosis increased with increasing intensity of US irradiation. Comparing with the protein patterns of cells before sonication, it was found that around 420 new protein spots were present in the gel after sonication. Among them, Hsp60 and Bcl-2 like protein 13 were found to be involved in the process of cell apoptosis and US-induced apoptosis of the cells was probably performed through the pathway of promoting the activation of caspase-3.