A "protective umbrella" nanoplatform for loading ICG and multi-modal imaging-guided phototherapy.

In order to prevent the aggregation of ICG and enhance its stability, a novel nanoplatform (TiO2:Yb,Ho,F-ß-CD@ICG/HA) was designed for NIR-induced phototherapy along with multi-mode imaging(UCL/MRI/Flu). In this nanosysytem: TiO2:Yb,Ho,F was used as upconversion materials and applied in vivo for the first time; ß-CD acted as a "protective umbrella" to load separated ICG and avoid the low phototherapy efficiency because of its aggregation; HA was the capping agent of ß-CD to prevent ICG unexpected leaking and a target to recognize CD44 receptor. The nanosystem exhibited excellent size (~200 nm) and photo- and thermal-stability, preferable reactive oxygen yield and temperature response (50.4 °C) under 808 nm laser. It could efficiently target and suppress tumor growth. The imaging ability (UCL/MRI) of TiO2:Yb,Ho,F could facilitate diagnosis of the tumor, especially for deep tissues. Altogether, our work successfully improved the phototherapy efficacy through incorporating the ICG into the cavity of ß-CD and applied TiO2:Yb,Ho,F for upconversion imaging in vivo.

Biocompatibility and therapeutic evaluation of magnetic liposomes designed for self-controlled cancer hyperthermia and chemotherapy.

Magnetic liposome-mediated combined chemotherapy and hyperthermia is gaining importance as an effective therapeutic modality for cancer. However, control and maintenance of optimum hyperthermia are major challenges in clinical settings due to the overheating of tissues. To overcome this problem, we developed a novel magnetic liposomes formulation co-entrapping a dextran coated biphasic suspension of La0.75Sr0.25MnO3 (LSMO) and iron oxide (Fe3O4) nanoparticles for self-controlled hyperthermia and chemotherapy. However, the general apprehension about biocompatibility and safety of the newly developed formulation needs to be addressed. In this work, in vitro and in vivo biocompatibility and therapeutic evaluation studies of the novel magnetic liposomes are reported. Biocompatibility study of the magnetic liposomes formulation was carried out to evaluate the signs of preliminary systemic toxicity, if any, following intravenous administration of the magnetic liposomes in Swiss mice. Therapeutic efficacy of the magnetic liposomes formulation was evaluated in the fibrosarcoma tumour bearing mouse model. Fibrosarcoma tumour-bearing mice were subjected to hyperthermia following intratumoral injection of single or double doses of the magnetic liposomes with or without chemotherapeutic drug paclitaxel. Hyperthermia (three spurts, each at 3 days interval) with drug loaded magnetic liposomes following single dose administration reduced the growth of tumours by 2.5 fold (mean tumour volume 2356 ± 550 mm3) whereas the double dose treatment reduced the tumour growth by 3.6 fold (mean tumour volume 1045 ± 440 mm3) compared to their corresponding control (mean tumour volume 3782 ± 515 mm3). At the end of the tumour efficacy studies, the presence of MNPs was studied in the remnant tumour tissues and vital organs of the mice. No significant leaching or drainage of the magnetic liposomes during the study was observed from the tumour site to the other vital organs of the body, suggesting again the potential of the novel magnetic liposomes formulation for possibility of developing as an effective modality for treatment of drug resistant or physiologically vulnerable cancer.

Adaptations of Arginine's Intestinal-Renal Axis in Cachectic Tumor-Bearing Rats.

Malignancies induce disposal of arginine, an important substrate for the immune system. To sustain immune function, the tumor-bearing host accelerates arginine's intestinal-renal axis by glutamine mobilization from skeletal muscle and this may promote cachexia. Glutamine supplementation stimulates argi-nine production in healthy subjects. Arginine's intestinal-renal axis and the effect of glutamine supplementation in cancer cach-exia have not been investigated. This study evaluated the long-term adaptations of the interorgan pathway for arginine production following the onset of cachexia and the metabolic effect of glutamine supplementation in the cachectic state. Fischer-344 rats were randomly divided into a tumor-bearing group (n = 12), control group (n = 7) and tumor-bearing group receiving a glutamine-enriched diet (n = 9). Amino acid fluxes and net fractional extractions across intestine, kidneys, and liver were studied. Compared to controls, the portal-drained viscera of tumor-bearing rats took up significantly more glutamine and released significantly less citrulline. Renal metabolism was unchanged in the cachectic tumor-bearing rats compared with controls. Glutamine supplementation had no effects on intestinal and renal adaptations. In conclusion, in the cachectic state, an increase in intestinal glutamine uptake is not accompanied by an increase in renal arginine production. The adaptations found in the cachectic, tumor-bearing rat do not depend on glutamine availability.

Dose painting based on tumor uptake of Cu-ATSM and FDG: a comparative study.

BACKGROUND: Hypoxia and increased glycolytic activity of tumors are associated with poor prognosis. The purpose of this study was to investigate differences in radiotherapy (RT) dose painting based on the uptake of 2-deoxy-2-[(18) F]-fluorodeoxyglucose (FDG) and the proposed hypoxia tracer, copper(II)diacetyl-bis(N(4))-methylsemithiocarbazone (Cu-ATSM) using spontaneous clinical canine tumor models. METHODS: Positron emission tomography/computed tomography scans of five spontaneous canine sarcomas and carcinomas were obtained; FDG on day 1 and (64)Cu-ATSM on day 2 and 3 (approx. 3 and 24 hours pi.). Sub-volumes for dose escalation were defined by a threshold-based method for both tracers and five dose escalation levels were formed in each sub-volume. Volumetric modulated arc therapy plans were optimized based on the dose escalation regions for each scan for a total of three dose plans for each dog. The prescription dose for the GTV was 45 Gy (100%) and it was linearly escalated to a maximum of 150%. The correlations between dose painting plans were analyzed with construction of dose distribution density maps and quality volume histograms (QVH). Correlation between high-dose regions was investigated with Dice correlation coefficients. RESULTS: Comparison of dose plans revealed varying degree of correlation between cases. Some cases displayed a separation of high-dose regions in the comparison of FDG vs. (64)Cu-ATSM dose plans at both time points. Among the Dice correlation coefficients, the high dose regions showed the lowest degree of agreement, indicating potential benefit of using multiple tracers for dose painting. QVH analysis revealed that FDG-based dose painting plans adequately covered approximately 50% of the hypoxic regions. CONCLUSION: Radiotherapy plans optimized with the current approach for cut-off values and dose region definitions based on FDG, (64)Cu-ATSM 3 h and 24 h uptake in canine tumors had different localization of the regional dose escalation levels. This indicates that (64)Cu-ATSM at two different time-points and FDG provide different biological information that has to be taken into account when using the dose painting strategy in radiotherapy treatment planning.

Proteomic profiling of the hypothalamus in a mouse model of cancer-induced anorexia-cachexia.

BACKGROUND: Anorexia-cachexia is a common and severe cancer-related complication but the underlying mechanisms are largely unknown. Here, using a mouse model for tumour-induced anorexia-cachexia, we screened for proteins that are differentially expressed in the hypothalamus, the brain's metabolic control centre. METHODS: The hypothalamus of tumour-bearing mice with implanted methylcholanthrene-induced sarcoma (MCG 101) displaying anorexia and their sham-implanted pair-fed or free-fed littermates was examined using two-dimensional electrophoresis (2-DE)-based comparative proteomics. Differentially expressed proteins were identified by liquid chromatography-tandem mass spectrometry. RESULTS: The 2-DE data showed an increased expression of dynamin 1, hexokinase, pyruvate carboxylase, oxoglutarate dehydrogenase, and N-ethylmaleimide-sensitive factor in tumour-bearing mice, whereas heat-shock 70 kDa cognate protein, selenium-binding protein 1, and guanine nucleotide-binding protein Gα0 were downregulated. The expression of several of the identified proteins was similarly altered also in the caloric-restricted pair-fed mice, suggesting an involvement of these proteins in brain metabolic adaptation to restricted nutrient availability. However, the expression of dynamin 1, which is required for receptor internalisation, and of hexokinase, and pyruvate carboxylase were specifically changed in tumour-bearing mice with anorexia. CONCLUSION: The identified differentially expressed proteins may be new candidate molecules involved in the pathophysiology of tumour-induced anorexia-cachexia.

Impact of acetylsalicylic acid on tumor angiogenesis and lymphangiogenesis through inhibition of VEGF signaling in a murine sarcoma model.

Aspirin is a salicylate drug that is widely used, and recently it has been shown to influence the development of various types of cancers. Our previous study revealed that aspirin had an inhibitory effect on the growth of S180 sarcoma and 3AO human ovarian cancer cells. The present study utilized a murine S180 sarcoma model to investigate the molecular mechanisms involved in aspirin-induced tumor growth inhibition. Tumor-bearing mice were randomly divided into five groups with 10 mice in each group: i) control; ii) 5-fluorouracil (5-FU); iii) high-dose aspirin (250 mg/kg); iv) low-dose aspirin (50 mg/kg); and v) combination of 5-FU and aspirin (50 mg/kg). The effect of aspirin on tumor growth was observed by measuring tumor volume and evaluating the antitumor effect. Tumor histology and immunohistochemistry were performed to detect the microvessel density (MVD), lymphatic vessel density (LVD), and the expression levels of vascular endothelial growth factor A (VEGF-A) and VEGF-C. The expression of VEGF-A and VEGF-C was also confirmed and quantified by western blotting. We discovered significant growth delay in murine S180 sarcoma as a result of aspirin treatment. The inhibition rate of tumor growth induced by high-dose and low-dose aspirin was 33.5 and 22.2%, respectively (P<0.05). The expression of VEGF-A and VEGF-C in tumor tissues inhibited by aspirin was demonstrated by immunohistochemistry, and the MVD was decreased in a dose-dependent manner (p<0.05). Reduced LVD was particularly apparent in the high-dose aspirin group (p<0.05). Western blot data showed that the expression of both VEGF-A and VEGF-C was reduced after treatment with aspirin. In conclusion, the impact of aspirin-induced tumor growth delay of murine S180 sarcoma may correlate with the inhibition of angiogenesis and lymphangiogenesis by reducing VEGF-A and VEGF-C expression in tumor tissues.

Anti-tumor and anti-metastatic actions of wogonin isolated from Scutellaria baicalensis roots through anti-lymphangiogenesis.

Tumor growth and metastasis are associated with angiogenesis and lymphangiogenesis through the production of vascular endothelial growth factor (VEGF) or VEGF-C in tumors, and the phosphorylation of VEGF receptor (VEGFR)-2 or VEGFR-3 in vascular endothelial cells or lymphatic endothelial cells (LECs). Tumor-associated macrophages (TAMs) play an important role in tumor lymphangiogenesis, and consequently stimulate metastasis through the lymphatic system to lymph nodes. We examined the effects of wogonin isolated from Scutellaria baicalensis roots on tumor growth and metastasis using a highly metastatic model in osteosarcoma LM8-bearing mice. Wogonin (25 and 50 mg/kg, twice daily) reduced tumor growth and metastasis to the lung, liver and kidney, angiogenesis (CD31-positive cells), lymphangiogenesis (LYVE-1-positive cells), and TAM (F4/80-positive cell) numbers in the tumors of LM8-bearing mice. Wogonin (10-100 µM) also inhibited increases in IL-1ß production and cyclooxygenase (COX)-2 expression induced by lipopolysaccharide in THP-1 macrophages. Wogonin had no effect on VEGF-C production in LM8 cells, or VEGFR-3 expression in human lymphatic endothelial cells (HLECs), however, it inhibited VEGF-C-induced VEGFR-3 phosphorylation in HLECs. The anti-tumor and anti-metastatic actions of wogonin may be associated with the inhibition of VEGF-C-induced lymphangiogenesis through a reduction in VEGF-C-induced VEGFR-3 phosphorylation by the inhibition of COX-2 expression and IL-1ß production in TAMs.

Effects of dissolved oxygen tension and agitation rate on the production of heat-shock protein glycoprotein 96 by MethA tumor cell suspension culture in stirred-tank bioreactors.

Heat-shock protein glycoprotein (gp96) serves as a natural adjuvant for chaperoning antigenic peptide into the immune surveillance pathway. In our laboratory, MethA tumor cell suspension culture process has been recently developed for gp96 production in spinner flask. In this work, effects of dissolved oxygen tension (DOT) and agitation rate on this process were studied in stirred-tank bioreactor. The optimal conditions for gp96 production were different with those for MethA tumor cell growth. MethA tumor cell growth pattern was not much changed by various levels of DOT and agitation rate, while gp96 biosynthesis was more sensitive to DOT and agitation rate. Compared with 50% of DOT, the production and specific productivity of gp96 was increased by 27 and 66% at 10% of DOT, respectively. Compared with the agitation rate of 100 rpm, the production and volumetric productivity of gp96 was increased by 48 and 144% at the agitation rate of 200 rpm, respectively. Low DOT (i.e., 10% of air saturation) and high agitation rate (i.e., 200 rpm) were identified to be favorable for gp96 biosynthesis. The results of this work might be useful to scale-up the bioprocess into the pilot scale.

Stimulation of expression of the intestinal glutamine transporter ATB0 in tumor-bearing rats.

BACKGROUND: Glutamine supplementation ameliorates host catabolic response in tumor bearing states. The purpose of this in vivo study was to investigate intestinal glutamine transport and expression of glutamine transporter ATB(0) in methyl-cholanthrene (MCA)-sarcoma bearing rats. METHODS: Fisher-344 rats underwent subcutaneous flank implantation of MCA-sarcoma cells (saline as control) and were pair-fed an equal quantity of chow as controls, to account for tumor-induced anorexia, until tumors reached 10 or 20% body weight. Intestinal mucosal brush border membrane [3H]-Glutamine transport was measured. Glutamine transporter ATB(0) mRNA and protein levels were measured by real-time PCR and western blot techniques, respectively. RESULTS: Glutamine transport activity across the intestinal brush border membrane (BBM) was 3.7-fold higher in tumor-bearing rats (TBR) than in controls (TBR 153 +/- 22.6 vs. Control 41.9 +/- 9.7 pmol/mg protein/10s, P < .01). Transporter ATB(0) mRNA levels were 1.4-fold higher in tumor-bearing rats (Relative value TBR .61 +/- .12 vs. Control .43 +/- .1, P < .05). A 1.4-fold increase in transporter ATB(0) protein levels was observed in the tumor-bearing rats (Relative value TBR .52 +/- .07 vs. Control .37 +/- .04, P < .05). Circulating aortic plasma glutamine levels were 1.3-fold higher in tumor bearing rats ([Glutamine] = .63 +/- .02 Control vs. [Glutamine] = .74 +/- .01 mmol/l TBR, P < .0001). Portal venous plasma glutamine levels were also higher in tumor bearing rats ([Glutamine] = .47 +/- .01 Control vs. [Glutamine] = .60 +/- .02 mmol/l TBR, P < .0001). CONCLUSION: Intestinal brush border membrane glutamine transport activity, transporter ATB(0) mRNA and protein levels are up-regulate in tumor-bearing rats.

Effects of omega-3 fatty acids on orexigenic and anorexigenic modulators at the onset of anorexia.

In cancer anorexia, a decrease in food intake (FI) occurs concomitant with changes in orexigenic peptides such as neuropeptide Y (NPY) and anorexigenic peptides such as alpha-melanocyte-stimulating hormone (alpha-MSH) and anorexigenic neurotransmitter serotonin. omega-3 Fatty acid (omega-3FA) inhibits cytokine synthesis, and delays tumor appearance, tumor growth, and onset of anorexia in tumor-bearing rats. We hypothesize that, in cancer anorexia, omega-3FA is associated with quantitative reversal of hypothalamic NPY, alpha-MSH, and serotonin receptor (5-HT(1B)-receptor) enhancing FI. Fischer rats were divided into: MCA tumor bearing fed chow (TB-Chow) or omega-3FA diet (TB-omega-3FA) and controls: non-tumor bearing fed chow (NTB-Chow) or omega-3FA diet (NTB-omega-3FA). Rats were euthanized at anorexia and brains were removed for hypothalamic immunohistochemical study, using NPY, alpha-MSH, and 5-HT(1B)-receptor-specific antibodies and slides assessed by image analysis. Immunostaining specificity was controlled by omission of primary or secondary antibodies and pre-absorption test. At anorexia, FI decreased (P < 0.05) in TB-Chow but did not change in TB-omega-3FA rats. In TB-omega-3FA vs. TB-Chow, NPY immunoreactivity increased 38% in arcuate nucleus (ARC; P < 0.05), and 50% in magnocellular paraventricular nucleus (mPVN; P < 0.05). alpha-MSH decreased 64% in ARC and 29% in mPVN (P < 0.05). 5-HT(1B)-receptor immunoreactivity decreased 13% only in supraoptic nucleus (P < 0.05). No immunoreactivity was found in the control sections. omega-3FA modified hypothalamic peptides and 5-HT-(1B)-receptor immunoreactivity at anorexia, concomitant with an increase in FI, were probably mediated by omega-3FA inhibition of tumor-induced cytokines.

Normalization of hypothalamic serotonin (5-HT 1B) receptor and NPY in cancer anorexia after tumor resection: an immunocytochemical study.

Tumor growth leads to anorexia and decreased food intake, the regulation of which is via the integrated hypothalamic peptidergic and monoaminergic system. Serotonin (5-HT), an anorectic monoamine acts primarily via 5-HT 1B-receptors in hypothalamic nuclei while neuropeptide Y (NPY) acts an orexigenic peptide. We previously reported that 5-HT 1B-receptors are up regulated while NPY is down regulated in tumor-bearing (TB)-related anorexia, contributing to food intake reduction. In anorectic TB rats we hypothesize that after tumor resection when food intake has reverted to normal, normalization of 5-HT 1B-receptor and NPY will occur. The aim of this study was to demonstrate normalization of these hypothalamic changes compared to Controls. In anorectic tumor-bearing rats after tumor resection (TB-R) and in sham-operated (Control) rats, distribution of 5-HT 1B-receptors and NPY in hypothalamic nuclei was analyzed using peroxidase antiperoxidase immunocytochemical methods. Image analysis of immunostaining was performed and the data were statistically analyzed. Immunostaining specificity was controlled by omission of primary or secondary antibodies and pre-absorption test. Our results show that after TB-R versus Controls a normalization of food intake, 5-H-1B-receptor and NPY expression in the hypothalamus occurs. These data, discussed in context with our previous studies, support the hypothesis that tumor resection results not only in normalization of food intake but also in reversible changes of anorectic and orexigenic hypothalamic modulators.

Fungal polysaccharopeptide inhibits tumor angiogenesis and tumor growth in mice.

Angiogenesis is crucial to tumor growth and metastasis, and interruption of this process is a prime avenue for therapeutic intervention of tumor proliferation. The present study has made use of the S180 tumor-bearing mouse model to investigate the polysaccharopeptide, PSP, isolated from the edible mushroom Coriolus versicolor, a herbal medicine known for its anti-angiogenesis properties. Quantitative analysis of microcorrosion casting of the tumor tissue showed more angiogenic features such as dense sinusoids and hot spots, in control (untreated) than in PSP-treated animals. Immunostaining of tumor tissues with antibody against the endothelial cell marker (Factor VIII) demonstrated a positive correlation in that both the vascular density and tumor weight were lower in mice treated with PSP. Morphometric analysis of corrosion casts revealed that, even though the total amount of new vessel production was reduced, the basic tumor type-specific vascular architecture was retained. However, the expression of vascular endothelial cell growth factor (VEGF) in these tumors was suppressed. In conclusion, anti-angiogenesis should be one of the pathways through which PSP mediated its anti-tumor activity.

Tumor anorexia: effects on neuropeptide Y and monoamines in paraventricular nucleus.

Paraventricular (PVN) concentrations of neuropeptide Y (NPY), serotonin (5-HT) and dopamine (DA) in anorectic tumor-bearing (TB) rats were measured before and after tumor resection. At onset of anorexia in TB versus non-tumor bearing (NTB) Controls 5-HT increased from 12.19+/-0.49 pg/microg to 14.89+/-0.81 pg/microg ( P<0.05 ) while DA and NPY decreased from 7.34+/-0.42 pg/microg to 4.97+/-0.56 pg/microg and 23.47+/-4.27 pg/microg to 13.64+/-1.44 pg/microg, respectively ( P<0.05 ). After tumor resection, these neuromediators normalized when compared to sham-operated NTB rats. NTB pair-fed Controls were also studied. We conclude that the increased 5-HT and the decreased DA and NPY concentrations in PVN are associated with cancer anorexia and that the NPY food stimulatory effect is linked to serotoninergic and dopaminergic systems in hypothalamus.

[Glutamate transporter mediated increase of antitumor activity by theanine, an amino acid in green tea].

We have confirmed that theanine, a major amino acid in green tea, enhances the antitumor activity of doxorubicin (DOX) without an increase in DOX-induced side effects. We believe that the action of theanine is due to decreases in glutamate uptake via inhibition of the glutamate transporter, intracellular glutathione (GSH) synthesis, GS-DOX conjugate level, and subsequent extracellular transport of GS-DOX by the MRP5/GS-X pump. To increase the clinical usefulness of theanine, we examined its effects on the antitumor activity of cisplatin and irinotecan (CPT-11), which a known to be transported by the efflux system related to MRP. Cisplatin decreased tumor volume in M5076 tumor-bearing mice. Furthermore, the combination of theanine with cisplatin increased the decrease in tumor volume as compared with the cisplatin-alone group. Tumor volume in the CPT-11-alone group did not show a decrease, but the combination of theanine with CPT-11 significantly reduced tumor volume. The concentration of cisplatin in the tumor was significantly increased by combination with theanine, and thus we assume that it correlated with the enhancement on the antitumor activity of theanine. On the other hand, changes in drug concentrations with theanine were not observed in normal tissues, but rather it is indicated that theanine tends to reduce their concentrations. Therefore theanine enhances the antitumor activity not only of DOX but also of cisplatin or CPT-11.

Effects of hyperglycemia on oxygenation, radiosensitivity and bioenergetic status of subcutaneous RIF-1 tumors.

Since tissue oxygen tension is a balance between delivery and consumption of oxygen, considerable effort has been directed at increasing the former and/or decreasing the latter. Techniques to decrease the rate of cellular oxygen consumption (increasing the distance oxygen can diffuse into tissues) include increasing glycolysis by administering supra-physiologic levels of glucose. We have examined the effect of hyperglycemia produced by intravenous glucose infusion on the tissue oxygenation and radiation response of subcutaneously implanted murine radiation induced fibrosarcomas (RIF-1). A 0.3 M glucose solution was delivered via tail vein injection according to a protocol that maintained glucose at a plasma concentration of 17+/-1 mM. The effect of this treatment on radiation response (clonogenic and growth delay studies), tumor oxygenation (needle electrode pO2 and 2-[2-nitro-1H-imidazol-1-yl]-N-(2,2,3,3,3-pentafluoropropyl) acetamide (EF5) binding), and tumor bioenergetics and pH (31P NMR spectroscopy) was examined. Systemic measurements included hematocrit and blood glucose and lactate concentrations. The results of these studies suggest that these subcutaneously implanted RIF-1 tumors are both radiobiologically and metabolically hypoxic and that intravenous glucose infusion is not an effective method of modifying this metabolic state.

Temperature-dependent changes in physiologic parameters of spontaneous canine soft tissue sarcomas after combined radiotherapy and hyperthermia treatment.

PURPOSE: The objectives of this study were to evaluate effects of hyperthermia on tumor oxygenation, extracellular pH (pHe), and blood flow in 13 dogs with spontaneous soft tissue sarcomas prior to and after local hyperthermia. METHODS AND MATERIALS: Tumor pO2 was measured using an Eppendorf polarographic device, pHe using interstitial electrodes, and blood flow using contrast-enhanced magnetic resonance imaging (MRI). RESULTS: There was an overall improvement in tumor oxygenation observed as an increase in median pO2 and decrease in hypoxic fraction (% of pO2 measurements <5 mm Hg) at 24-h post hyperthermia. These changes were most pronounced when the median temperature (T50) during hyperthermia treatment was less than 44 degrees C. Tumors with T50 > 44 degrees C were characterized by a decrease in median PO2 and an increase in hypoxic fraction. Similar thermal dose-related changes were observed in tumor perfusion. Perfusion was significantly higher after hyperthermia. Increases in perfusion were most evident in tumors with T50 < 44 degrees C. With T50 > 44 degrees C, there was no change in perfusion after hyperthermia. On average, pHe values declined in all animals after hyperthermia, with the greatest reduction seen for larger T50 values. CONCLUSION: This study suggests that hyperthermia has biphasic effects on tumor physiologic parameters. Lower temperatures tend to favor improved perfusion and oxygenation, whereas higher temperatures are more likely to cause vascular damage, thus leading to greater hypoxia. While it has long been recognized that such effects occur in rodent tumors, this is the first report to tie such changes to temperatures achieved during hyperthermia in the clinical setting. Furthermore, it suggests that the thermal threshold for vascular damage is higher in spontaneous tumors than in more rapidly growing rodent tumors.

Membrane transport and antitumor activity of pirarubicin, and comparison with those of doxorubicin.

We have compared the membrane transport and antitumor activity of pirarubicin with those of doxorubicin in M5076 ovarian sarcoma, which exhibits low sensitivity to doxorubicin. Pirarubicin was rapidly taken up by M5076 cells and the intracellular concentration of pirarubicin reached more than 2.5-fold that of doxorubicin. In terms of the 50% cell growth-inhibitory concentration in vitro, pirarubicin was more effective than doxorubicin. Thus, the intracellular concentration influenced the cytotoxicity of these anthracycline agents. On comparison of the nuclear uptake of pirarubicin and doxorubicin, the nucleus/cell ratio of pirarubicin was found to be about 40%, whereas that of doxorubicin reached more than 80%. As the intranuclear concentration of pirarubicin is dependent on nuclear transport, the increases in not only cell membrane transport, but also nuclear membrane transport contributed to the enhancement of the efficacy of pirarubicin. In M5076 solid tumor-bearing mice, pirarubicin reduced the tumor weight to 60% of the control level, although doxorubicin had no effect. These results were supported by the intracellular uptake of pirarubicin. Moreover, theanine, which inhibited the pirarubicin efflux from M5076 cells, increased by 1.3-fold the pirarubicin concentration in the tumor and enhanced the therapeutic efficacy of pirarubicin 1.7-fold. In conclusion, our results suggest that an increase in the concentration of an anthracycline derivative in tumor cells due to alteration of cell membrane transport results in enhancement of the antitumor activity.

Reciprocal changes in hypothalamic receptor binding and circulating leptin in anorectic tumor-bearing rats.

Although reduced biological activity of the obese gene product, leptin, has been associated with obesity, little information is available concerning leptin alterations during anorexia. Therefore, we measured circulating leptin concentrations and hypothalamic leptin binding in anorectic tumor-bearing and pair-fed control rats. Plasma concentrations of leptin decreased in tumor-bearing rats early in the course of tumor growth, and fell to nearly non-detectable levels during severe anorexia. The pair-fed control rats that ate the same amount of food as did the anorectic tumor-bearing rats exhibited a 50% decrease in plasma leptin concentration. Concentrations of free fatty acids were elevated in both tumor-bearing and pair-fed groups, while circulating levels of triglycerides were increased only in anorectic tumor-bearing rats. Leptin receptor density was doubled in the hypothalamus of tumor bearing rats, while binding affinity was decreased by 50%. These results suggest that peripheral leptin production is down-regulated, perhaps due to increased lipolysis in tumor-bearing rats. It appears that hypothalamic leptin systems up-regulate receptor numbers in response to decreased blood leptin level, however, the decrease in binding affinity may compensate for these alterations. Therefore, the influence of leptin on hypothalamic neuropeptide Y feeding systems may be minimal in anorectic tumor-bearing rats.

Enhancement of oxidative cell injury and antitumor effects of localized 44 degrees C hyperthermia upon combination with respiratory hyperoxia and xanthine oxidase.

The effects of respiratory hyperoxia (RH) and xanthine oxidase (XO) during localized hyperthermia (HT) were investigated by determining markers of oxidative damage to lipids and proteins and tumor growth. Anesthetized rats with s.c. DS-sarcomas underwent one of the following treatments: (a) localized saline-bath HT (60 min, 44 degrees C); (b) HT + RH (100% O2); and (c) HT + RH + XO (15 units/kg i.v.). Sham-treated animals served as controls. Tumors were investigated for: (a) thiobarbituric acid-reactive substance formation and protein-bound 4-hydroxynonenal, as indicators of lipid peroxidation; (b) reactive oxygen-mediated protein modifications; (c) apoptosis; and (d) tumor volume growth. Upon treatment, increases in thiobarbituric acid-reactive substances, protein-bound 4-hydroxynonenal, protein-associated carbonyl functions, and number of cells undergoing apoptosis were found in tumor tissue, together with an inhibition of tumor growth. When treatment groups were compared, effects in the group HT + RH + XO were generally most pronounced. These findings indicate that the antitumor effect of HT is at least partially mediated through the selective induction of lipid peroxidation and oxidative injury in tumor cells, leading to apoptosis. This effect was enhanced by adding RH or RH + XO, presumably due to enhanced tissue damage following an increased formation of reactive oxygen species, with higher levels of lipid peroxidation and protein oxidation.

Localized hypothermia: impact on oxygenation, microregional perfusion, metabolic and bioenergetic status of subcutaneous rat tumours.

The effect of localized hypothermia on microcirculatory and metabolic parameters in s.c. DS sarcomas on the hind foot dorsum of Sprague-Dawley rats was investigated. Tumours were cooled by superfusion of the tumour surface with cooled saline solution to 25 degrees C or 15 degrees C. Control tumours remained at 35 degrees C. These temperatures were maintained for 30 min. In tumour oxygenation measurements, hypothermia at 25 degrees C and 15 degrees C caused progressive decreases in the size of the fraction of pO2 measurements between 0 and 2.5 mmHg together with a reduction in pO2 variability. No significant changes in median or mean pO2 or in the fraction of pO2 measurements between 0 and 5 mmHg, and 0 and 10 mmHg were observed. Using laser Doppler flowmetry, red blood cell flux was found to decrease significantly upon 25 degrees C or 15 degrees C hypothermia treatment to 67% and 37% of starting values respectively, whereas no significant changes were seen in control tumours over the whole observation period. Viscosity was measured in blood and plasma samples over a range of temperatures and was found to increase with decreasing temperature. Assessment of tumour glucose levels showed an increased concentration of glucose following 15 degrees C hypothermia, an observation consistent with a 'slowing down' of glycolysis. No changes in lactate or adenylate phosphate levels were observed. As a way of improving tumour oxygenation, localized hypothermia may therefore be a useful means of radiosensitization.