Differential structural features in soleus and gastrocnemius of carnitine-treated cancer cachectic rats.

Muscle wasting is associated with chronic diseases and cancer. Elucidation of the biological mechanism involved in the process of muscle mass loss and cachexia may help identify therapeutic targets. We hypothesized that l-carnitine treatment may differentially revert muscle fiber atrophy and other structural alterations in slow- and fast-twitch limb muscles of rats bearing the Yoshida ascites hepatoma. In soleus and gastrocnemius of tumor-bearing rats (108 AH-130 Yoshida ascites hepatoma cells inoculated intraperitoneally) with and without treatment with l-carnitine (1 g/kg body weight for 7 days, intragastric), food intake, body and muscle weights, fiber typing and morphometry, morphological features, redox balance, autophagy and proteolytic, and signaling markers were explored. Levels of carnitine palmitoyl transferase were also measured in all the study muscles. l-Carnitine treatment ameliorated the atrophy of both slow- and fast-twitch fibers (gastrocnemius particularly), muscle structural alterations (both muscles), and attenuated oxidative stress, proteolytic and signaling markers (gastrocnemius). Despite that carnitine palmitoyl transferase-1 levels increased in both muscle types in a similar fashion, l-carnitine ameliorated muscle atrophy and proteolysis in a muscle-specific manner in cancer-induced cachexia. These data reveal the need to study muscles of different fiber type composition and function to better understand whereby l-carnitine exerts its beneficial effects on the myofibers in muscle wasting processes. These findings also have potential clinical implications, since combinations of various exercise and muscle training modalities with l-carnitine should be specifically targeted for the muscle groups to be trained.

Dietary Methionine Restriction-Based Cancer Chemotherapy in Rodents.

The elevated methionine (MET) requirement for the growth of tumors, first observed by Sugimura in 1959, termed MET dependence, is a potentially highly effective therapeutic target. Proof of this principle is that when MET restriction (MR) was initially established in co-cultures of cancer and normal cells, MET dependence could be exploited to selectively kill cancer cells without killing co-cultured normal cells. MET-dependent cells become reversibly blocked in the late S/G2 phase of the cell cycle under MR enabling selective and effective S-phase chemotherapy against these blocked cancer cells. Subsequent MET repletion with an anti-mitotic drug was totally effective at selectively eliminating the MET-dependent cancer cells enabling the normal MET-dependent cells to take over the culture. We have also observed that the MET analog ethionine (ETH) is synergistic with MR in arresting the growth of the Yoshida sarcoma both in vitro and eliminating metastasis when transplanted to nude mice. MR increased the efficacy of cisplatinum (CDDP) against the MX-1 human breast carcinoma cell line when grown in nude mice. MR increased 5-fluorouracil (5-FU) efficacy on a human gastric cancer xenograft, SC-1-NU, in nude mice. MET-restricted total parenteral nutrition (MR TPN) was effective in Yoshida sarcoma-bearing rats. MR TPN with doxorubicin (DOX) and vincristine (VCR) resulted in significant tumor suppression and prolonged survival of Yoshida-sarcoma-bearing rats. These results were the basis of subsequent studies that used methioninase to effect MR for effective cancer therapy.

Chemical sympathectomy increases neutrophil-to-lymphocyte ratio in tumor-bearing rats but does not influence cancer progression.

The sympathetic nervous system regulates many immune functions and modulates the anti-tumor immune defense response, too. Therefore, we studied the effect of 6-hydroxydopamine induced sympathectomy on selected hematological parameters and inflammatory markers in rats with Yoshida AH130 ascites hepatoma. We found that chemically sympathectomized tumor-bearing rats had significantly increased neutrophil-to-lymphocyte ratio, leukocyte-to-lymphocyte ratio, and plasma levels of tumor necrosis factor alpha. Although our findings showed that sympathetic denervation in tumor-bearing rats led to increased neutrophil-to-lymphocyte ratio, that is an indicator of the disease progression, we found no significant changes in tumor growth and survival of sympathectomized tumor-bearing rats.

Prevention of cancer recurrence in tumor margins by stopping microcirculation in the tumor and tumor-host interface.

Combretastatins interrupt blood flow of solid tumor vascular networks and lead to necrosis by blocking nutrients. However, tumors recover from tumor blood flow interruption-induced damage and develop viable rims. To investigate why cancer recurs and its prevention, we used a combretastatin derivative, Cderiv (=AC7700), and analyzed changes in tumor-host interface (T-HI) vessels, which were closest to cancer cells in the tumor margin after tumor vessel disruption, and the microenvironment surrounding them. Treatment with Cderiv (10 mg/kg) interrupted tumor blood flow in all regions of LY80 (a variant of Yoshida sarcoma) tumor, but not T-HI vessel blood flow. The same Cderiv dose given 72 h after 5 Gy irradiation stopped T-HI vessel blood flow and prevented cancer recurrence. Treatment in the reverse order, however, did not affect T-HI vessel blood flow. The greatest difference between the two treatments was the occurrence of gradual T-HI edema with the former. Severe T-HI edema compressed T-HI blood vessels, so that circulation stopped. Thus, the distance between a tumor margin and its nearest functioning host vessel became much larger, and the tumor marginal region became a microenvironment that lacked a nutritional supply. Cancer cells in tumor margins received nutrients through two circulation routes: tumor vessels and T-HI vessels. Our starvation methods, which involved treatment with Cderiv 72 h after 5 Gy irradiation, blocked both circulation routes and may have great potential as a clinical strategy to prevent cancer recurrence.

Diet-induced obesity and insulin resistance spur tumor growth and cancer cachexia in rats bearing the Yoshida sarcoma.

Obesity and insulin resistance are associated with increased risk of cancer and cancer mortality. However, it is currently unknown whether they contribute to the development of cancer cachexia, a syndrome that contributes significantly to morbidity and mortality in individuals with cancer. The present experiment addresses the question of whether preexisting obesity and insulin resistance alter tumor growth and cancer cachexia symptoms in Yoshida sarcoma bearing male rats. Obesity and insulin resistance were induced through 5 weeks of high-fat (HF) diet feeding and insulin resistance was confirmed by intraperitoneal glucose tolerance testing. Chow-fed animals were used as a control group. Following the establishment of insulin resistance, HF- and chow-fed animals were implanted with fragments of the Yoshida sarcoma or received a sham surgery. Tumor growth rate was greater in HF-fed animals, resulting in larger tumors. In addition, cancer cachexia symptoms developed in HF-fed animals but not chow-fed animals during the 18-day experiment. These results support a stimulatory effect of obesity and insulin resistance on tumor growth and cancer cachexia development in Yoshida sarcoma-bearing rats. Future research should investigate the relationship between obesity, insulin resistance, and cancer cachexia in human subjects.

Chronic exendin-4 treatment prevents the development of cancer cachexia symptoms in male rats bearing the Yoshida sarcoma.

Cancer cachexia is the syndrome of weight loss, loss of appetite, and wasting of skeletal muscle and adipose tissue experienced by many individuals with cancer. Currently, few effective treatment and prevention strategies are available for these patients, due in part to a poor understanding of the mechanisms contributing to cachexia. Insulin resistance has been associated with cancer cachexia in epidemiological, human, and animal research. The present experiment was designed to examine the ability of Exendin-4, a GLP-1 agonist and insulin sensitizing agent, to prevent the development of cachexia symptoms in male Sprague Dawley rats bearing the Yoshida sarcoma. Following tumor implantation or sham surgery, rats were treated daily with saline or Exendin-4 (3 µg/kg body weight/day) and were monitored for tumor growth and cachexia symptoms for 21-23 days. As a result of large variability in treatment effects, data were analyzed separately for animals with large and small tumors. Exendin-4 treatment reduced tumor growth and prevented the development of cancer cachexia symptoms in animals with small, but not large, tumors. In addition, insulin levels were preserved in Exendin-4-treated tumor-bearing animals. The results of this experiment demonstrate a novel preventative therapy for cancer cachexia and a novel use of Exendin-4. Further research is necessary to determine the mechanisms through which Exendin-4 exerts these potent effects.

Characterization of the Yoshida sarcoma: a model of cancer cachexia.

PURPOSE: Cancer cachexia contributes significantly to morbidity and mortality in individuals with cancer. Currently, the mechanisms contributing to the development of cachexia are largely unknown, leading to a paucity of treatment and prevention options. Animal models are necessary in determining causal mechanisms and in testing potential treatments. While the Yoshida sarcoma has been utilized for more than 50 years, the cachexia syndrome produced by this model has not been well characterized in the literature. METHODS: Tumor allografts were subcutaneously implanted in male Sprague Dawley rats (n = 16) and allowed to grow for 23 days. Control animals (n = 16) received a sham surgery. All rats were monitored daily for the presence of hallmark cachexia symptoms. RESULTS: The results demonstrate the presence of decreased body weight gain, as well as lower levels of body adiposity and skeletal muscle mass, in tumor-bearing animals, as compared to controls. CONCLUSIONS: While a large tumor burden was reached, the extent of cachexia was similar to that which is observed in many individuals with cancer cachexia. Future experiments utilizing this model are encouraged to identify mechanisms and effective treatment and prevention strategies.

Mitochondrial and sarcoplasmic reticulum abnormalities in cancer cachexia: altered energetic efficiency?

BACKGROUND: Cachexia is a wasting condition that manifests in several types of cancer, and the main characteristic is the profound loss of muscle mass. METHODS: The Yoshida AH-130 tumor model has been used and the samples have been analyzed using transmission electronic microscopy, real-time PCR and Western blot techniques. RESULTS: Using in vivo cancer cachectic model in rats, here we show that skeletal muscle loss is accompanied by fiber morphologic alterations such as mitochondrial disruption, dilatation of sarcoplasmic reticulum and apoptotic nuclei. Analyzing the expression of some factors related to proteolytic and thermogenic processes, we observed in tumor-bearing animals an increased expression of genes involved in proteolysis such as ubiquitin ligases Muscle Ring Finger 1 (MuRF-1) and Muscle Atrophy F-box protein (MAFBx). Moreover, an overexpression of both sarco/endoplasmic Ca(2+)-ATPase (SERCA1) and adenine nucleotide translocator (ANT1), both factors related to cellular energetic efficiency, was observed. Tumor burden also leads to a marked decreased in muscle ATP content. CONCLUSIONS: In addition to muscle proteolysis, other ATP-related pathways may have a key role in muscle wasting, both directly by increasing energetic inefficiency, and indirectly, by affecting the sarcoplasmic reticulum-mitochondrial assembly that is essential for muscle function and homeostasis. GENERAL SIGNIFICANCE: The present study reports profound morphological changes in cancer cachectic muscle, which are visualized mainly in alterations in sarcoplasmic reticulum and mitochondria. These alterations are linked to pathways that can account for energy inefficiency associated with cancer cachexia.

Vital microscopic analysis of polymeric micelle extravasation from tumor vessels: macromolecular delivery according to tumor vascular growth stage.

Particles larger than a specific size have been thought to extravasate from tumor vessels but not from normal vessels. Therefore, various nanoparticles incorporating anticancer drugs have been developed to realize selective drug delivery to solid tumors. However, it is not yet clear whether nanoparticles extravasate readily from all tumor vessels including vessels of microtumors. To answer this question, we synthesized new polymeric micelles labeled with fluorescein isothiocyanate (FITC) and injected them into the tail vein of rats with implanted skinfold transparent chambers. We also analyzed, by means of time-lapse vital microscopy with image analysis, extravasation of FITC micelles from tumor vessels at different stages of growth of Yoshida ascites sarcoma LY80. Polymeric micelles readily leaked from vessels at the interface between normal and tumor tissues and those at the interface between tumor tissues and necrotic areas. The micelles showed negligible extravasation, however, from the vascular network of microtumors less than 1 mm in diameter and did not accumulate in the microtumor. Our results suggest that we must develop a novel therapeutic strategy that can deliver sufficient nanomedicine to microtumors.

Radiopharmaceutical tracking of particles injected into tumors: a model to study clearance kinetics.

OBJECTIVES: Success of selective drug therapies depends on the drug's depot time in the target to treat. Depot time is currently being prolonged, using drug-eluting beads or microspheres for selective internal radiation therapy. The purpose of this study was to establish a model for investigating the depot time of particles injected into tumors in relation to tumor vascularization and particle size. MATERIALS AND METHODS: An animal model with two different vascularized tumors (Walker Carcinoma 256 (hypervascularized) and Yoshida Sarcoma (hypovascularized)) was used. The tumors were implanted into the hind leg of Wistar rats. When the tumors reached 10-15mm, rhenium radiolabeled particles of 25microm and 0.3microm were percutaneously injected into the tumors: large particles (Re-188 microspheres) in 10 hypo- and 10 hypervascularized tumors and small particles (Re-186 sulfid colloid) in 4 hypo- and 16 hypervascularized tumors with the co-injection of the vasoconstrictor, adrenalin (0.01 mg), into 8 hypervascularized tumors. Tumor activity was measured with a gamma camera at 10 min, 1h, 3h, 6h, 14h and 48h p.i. In addition, activity in the lung, liver, spleen, kidneys, and lymph nodes was measured at 48 h p.i. Measurements were adjusted for decay times and then compared. RESULTS: Drainage of the injected particles is bi-phasic, characterized by a fast wash-out. At 10 min p.i., intratumoral activity decreases to 70% of the initially injected activity. This is followed by a slow decline at 48 h p.i in which intratumoral activity decreases to at least 60% of the initially injected activity. Slow decline is independent of particle size and vascularization, whereas fast leakage depends on both. Co-injecting adrenalin significantly reduced the wash-out of the small particles. Radiolabeled microspheres accumulated mainly in the lungs, smaller colloids in the liver. CONCLUSIONS: Particle stay time, biodistribution, and stability can be easily monitored as shown in this animal model. The hematogeneous wash-out can be reduced, using larger particles and vasoconstrictors. Prolonged retention is independent of vascularization and particle size and appears to be sufficient for therapy.

Transarterial nephrectomy: the current status of experimental and clinical studies.

INTRODUCTION: Open nephrectomy is associated with significant morbidity. For several years, minimally invasive alternatives have been developed such as laparoscopic nephrectomy or transarterial renal ablation. This paper focuses on the different principles of vaso-occlusion and further improvements of the technique such as capillary chemoembolization in experimental as well as clinical studies. MATERIALS AND METHODS: Based on own in vitro studies, the principle of capillary embolization with occlusion of the entire arterial system up to the capillaries by a precipitating corn protein (Ethibloc) has been developed in animal studies (i.e., rat and canine kidney model). The precipitation speed of Ethibloc can be prolonged by 40% glucose per injection. The organ-ablative efficacy was evaluated in models of unilateral hypertension and chemically induced renal tumors (i.e., dimethyl-nitrosamine). Further studies using the model of unilateral transarterial implantation of Yoshida-sarcoma cells compared capillary chemoembolization using Ethibloc/mitomycin C (MMC) versus chemoperfusion and capillary embolization. Before starting clinical trials, the optimal mixture of Ethibloc and MMC was determined in vitro and in vivo. Prior to the vaso-occlusion, the volume of the arterial system of the kidney is determined by perfusion of the kidney with contrast-dye via a blocked balloon-catheter. Then 25% of the determined volume of 40% glucose is pre-injected followed by Ethibloc/MMC being injected with 1-cm(3) syringes. Once the capillary bed and tumor sinusoids are reached, the balloon catheter is emptied by postinjection of 40% glucose. RESULTS: Capillary embolization proved to be significantly superior to a central (i.e., ligation of renal artery) or peripheral type of occlusion resulting in complete coagulation necrosis of the normal rat and canine kidney with reduction of the elevated blood pressure, similar to nephrectomy in the model of renal hypertension. In the model of chemically induced renal tumors, complete necrosis of T2 stages could be achieved in 83% using Ethibloc compared to only 63% with Gelfoam particles, and 17% after ligation. In T3/T4 stages, the response rate was only 60% versus 0% after central and peripheral occlusion. In the highly aggressive Yoshida-sarcoma model, capillary chemoembolization yielded an 80% complete response rate compared to only 75% after capillary embolization and 70% after chemoperfusion. The optimal mixture of Ethibloc and MMC ranged between 1 and 2 mg of MMC to 1 cm(3) of Ethibloc, therefore for clinical trials 10 mg MMC was added to the 7.5 cm(3) syringe of Ethibloc. Clinical studies included 68 preoperatively as well as 62 palliatively embolized patients with renal cell carcinoma. The procedure was relatively well tolerated and usually associated with a mild postembolization syndrome. After an interval of up to 28 days, complete necrosis of the renal tumor could be achieved in tumors up to 9 cm in diameter. Hematuria ceased in all cases, and in selected cases long-lasting responses of very large tumors (i.e., vena cava involvement) could be achieved. DISCUSSION: Capillary chemoembolization represents an effective concept for ablation of malignant renal tumors. It offers control of tumor growth in case of temporary inoperability as well as cessation of hematuria in a palliative situation. Because of the local ablative efficiency, it may still represent a minimally invasive option in advanced stages of renal carcinoma (i.e., in combination with immunochemotherapy or targeted therapy).

Resveratrol does not ameliorate muscle wasting in different types of cancer cachexia models.

BACKGROUND & AIMS: Resveratrol has been reported to have antitumoural effects and recently it has been demonstrated that resveratrol partially blocks skeletal muscle wasting by interfering with NF-kappaB activation. We decided to investigate the potential anti-wasting properties of resveratrol on different models of cancer cachexia in experimental animals. METHODS AND RESULTS: Incubations of isolated extensor digitorum longus muscles in the presence of 30 microM of resveratrol caused a significant decrease in the rate of protein degradation. However, administration of resveratrol in vivo to both rats bearing the Yoshida AH-130 ascites hepatoma (at the dose of 1 mg/kg body weight) and mice bearing the Lewis lung carcinoma (at two different doses, 5 and 25 mg/kg body weight) had no effect on skeletal muscle mass or body weight in tumour-bearing rodents. In addition, a combination of resveratrol (3 mg/kg body weight) and fish oil was also unable to induce any changes in skeletal muscle weights. CONCLUSIONS: It is therefore concluded from this study that resveratrol is unable to influence muscle mass in vivo and has no potential role as anticachectic agent for the treatment of muscle wasting associated with tumour growth.

[Antitumor activity and function of S-1, a new oral tegafur-based formulation].

TS-1 (S-1), developed by the scientific theory of both potentiating antitumor activity of 5-fluorouracil (5-FU) and reducing gastrointestinal toxicity induced by 5-FU, is a new oral formulation consisting of 1 M tegafur, 0.4 M gimeracil and 1 M oteracil potassium. We investigated the antitumor efficacy of S-1 alone and in combination with other cytotoxic anticancer drugs using subcutaneously or orthotopically implanted murine and human tumors in rodents. As a single agent, S-1 showed higher antitumor activity with its low intestinal toxicity compared to continuous venous infusion 5-FU, the most effective dosing method of 5-FU, and/or to clinically available oral fluoropyrimidines such as UFT, doxyfluridine and capecitabine on various murine tumors and human tumor xenografts. Especially, it was noteworthy that S-1 as a DPD-inhibitory fluoropyrimidine markedly affected human tumor xenografts with high expression levels of DPD on which other fluoropyrimidines showed a low antitumor activity. In combination with other anticancer drugs such as CPT-11 and taxanes, S-1 exercised synergistic antitumor efficacy not only on 5-FU-sensitive tumors with low expression levels of thymidylate synthase (TS) but also on 5-FU-resistant tumors with originally higher and/or elevated levels of TS expression. As one of the reasonable mechanism of antitumor synergism by the combination, CPT-11 and taxanes were found to reduce the expression of TS in human tumor resistant to 5-FU with high expression TS levels. Throughout our preclinical antitumor studies of S-1, alone and/or in combination with other anticancer drugs, it would be expected to contribute greatly to the treatment of cancer patients.

BARD1 content correlates with increased DNA fragmentation associated with muscle wasting in tumour-bearing rats.

Apoptotic events have been clearly associated with muscle wasting in different types of experimental cancer cachexia. In these conditions, cell death is triggered by cytokines or tumour-produced factors. BARD1 is a nuclear protein that is also involved in apoptosis both in vitro and in vivo. The results presented here demonstrate that BARD1 content in skeletal muscle correlates with increased DNA fragmentation during experimental cancer cachexia. It is suggested that BARD1 acts as a modulator of muscle apoptosis or, alternatively, that BARD1 participates in the protein degradation by functioning as ubiquitin ligase.

Anticachectic effects of formoterol: a drug for potential treatment of muscle wasting.

In cancer cachexia both cardiac and skeletal muscle suffer an important protein mobilization as a result of increased proteolysis. Administration of the beta2-agonist formoterol to both rats and mice bearing highly cachectic tumors resulted in an important reversal of the muscle-wasting process. The anti-wasting effects of the drug were based on both an activation of the rate of protein synthesis and an inhibition of the rate of muscle proteolysis. Northern blot analysis revealed that formoterol treatment resulted in a decrease in the mRNA content of ubiquitin and proteasome subunits in gastrocnemius muscles; this, together with the decreased proteasome activity observed, suggest that the main anti-proteolytic action of the drug may be based on an inhibition of the ATP-ubiquitin-dependent proteolytic system. Interestingly, the beta2-agonist was also able to diminish the increased rate of muscle apoptosis (measured as DNA laddering as well as caspase-3 activity) present in tumor-bearing animals. The present results indicate that formoterol exerted a selective, powerful protective action on heart and skeletal muscle by antagonizing the enhanced protein degradation that characterizes cancer cachexia, and it could be revealed as a potential therapeutic tool in pathologic states wherein muscle protein hypercatabolism is a critical feature such as cancer cachexia or other wasting diseases.

A comparative pharmacology study between the intracolonic and oral routes of 5-FU administration in a colon cancer-bearing Yoshida sarcoma rat model.

We prepared a colon cancer-bearing Yoshida sarcoma rat model to examine the dose-response relationship of antitumor activity of intracolonically or orally administered 5-fluorouracil (5-FU; 45, 30, 20, 13, and 8 mg/kg). At doses of > or =20 mg/kg and > or =30 mg/kg, the 5-FU intracolonic and oral administration groups each showed a statistically significant difference in antitumor activity against the control group (P<0.05, Williams' test). A statistically significant dose-response relationship was noted in the two routes of administration, with an ED(50) value of 29 mg/kg. White blood cell count tended to decrease at high doses when 5-FU was administered intracolonically and showed a statistically significant decrease at doses of > or =30 mg/kg when 5-FU was administered orally. Regarding the time-course of body weight, even the 5-FU highest dose (45 mg/kg) intracolonic administration group showed no inhibited body weight increase compared to the control group. However, the 5-FU (> or =20 mg/kg) oral administration groups showed a statistically significant difference in body weight increase against the control group. These facts suggested that the intracolonic administration of 5-FU, while exhibiting more potent antitumor activity than that observed in oral administration, allows an extensive reduction in its toxicities compared to oral administration.

Preliminary study of the optimal dosing schedule for oral UFT/leucovorin chemotherapy.

BACKGROUND: We evaluated the optimal dosage schedule for combined oral chemotherapy using uracil/tegafur (UFT) and leucovorin (LV) in Yoshida sarcoma-bearing rats. MATERIALS AND METHODS: The antitumor activity and survival effect were compared between two schedules, thrice daily administration on 5 days of the week followed by 2 drug-free days (schedule A) and twice daily on 7 days of the week (schedule B). RESULTS: Significant tumor growth inhibition and improved survival rate were seen with both schedules of the UFT/LV group as compared to their respective control groups. Tumor growth inhibition was significantly greater in schedule A than schedule B. The number of survivors on day 60 after the tumor inoculation was higher in schedule A although the survival rate did not differ significantly. CONCLUSION: Administration of UFT/LV for 5 days of the week seemed to exhibit superior antitumor activity, with no increase in the incidence of adverse effects, as compared with the consecutive daily dosing schedule.

Inhibition of induced chemoresistance by cotreatment with (E)-5-(2-bromovinyl)-2'-deoxyuridine (RP101).

Induced chemoresistance leads to the reduction of apoptotic responses. Although several drugs are in development that circumvent or decrease existing chemoresistance, none has the potential to prevent or reduce its induction. Here, we present data from a drug that could perhaps fill this gap. Cotreatment of chemotherapy with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU, RP101) prevented the decrease of apoptotic effects during the course of chemotherapy and reduced nonspecific toxicity. Amplification of chemoresistance genes (Mdr1 and Dhfr) and overexpression of gene products involved in proliferation (DDX1) or DNA repair (UBE2N and APEX) were inhibited, whereas activity of NAD(P)H: quinone oxidoreductase 1 (NQO1) was enhanced. During recovery, when treatment was with BVDU only, microfilamental proteins were up-regulated, and proteins involved in ATP generation or cell survival (STAT3 and JUN-D) were down-regulated. That way, in three different rat tumor models, the antitumor efficiency of chemotherapy was optimized, and toxic side effects were reduced. Because of these beneficial properties of BVDU, a clinical pilot Phase I/II study with five human tumor entities has been started at the University of Dresden (Dresden, Germany). So far, no unwanted side effects have been observed.

Microvascular mechanisms by which the combretastatin A-4 derivative AC7700 (AVE8062) induces tumour blood flow stasis.

We previously reported that a novel combretastatin A-4 derivative, AC7700, has remarkable antitumour effects because of an irreversible stasis of tumour blood flow (TBF) and subsequent loss of nutrient supply to tumour tissue. Since early 2002, under the new designation AVE8062, AC7700 has undergone clinical trials in Europe and the US. Questions remain, however, concerning how AC7700 blocks TBF and why the TBF stasis does not recover. In this study, using a rat tumour LY80, a variant of Yoshida sarcoma, we examined whether TBF cessation after AC7700 administration is due to a direct action of the agent on tumour blood vessels. We constructed electrodes that can drop a small quantity of the drug solution directly at the site of blood flow measurement and inserted them subcutaneously and into the tumour. We compared the blood flow responses of normal vessels and tumour vessels after administration of 10-microl doses of various concentrations (0.2, 1, 10, and 50 mg ml(-1)) of the AC7700 solution. In addition, we assessed TBF stasis after i.v. and intra-arterial 10 mg x kg(-1) AC7700 administration in an LY80-induced kidney tumour. To determine why the TBF stasis is irreversible, we observed AC7700-induced changes in host arterioles and the tumour vascular network of the Sato lung carcinoma using a vital microscopic rat transparent chamber. Since an increase in tumour interstitial fluid pressure brings about a decrease in TBF, we also measured 10 mg x kg(-1) AC7700-induced changes in this pressure. The sensitivity of the blood flow response after intratumoral application of AC7700 was markedly higher in normal vessels relative to tumour vessels. Intra-arterial administration of AC7700 did not have stronger effects on TBF stasis than did i.v. administration. Intravital microscopy showed that AC7700 induced a powerful and long-lasting constriction of host arterioles, so that complete stasis of blood flow occurred in downstream vessels, which supplied blood to tumours. Owing to this stasis, the lumens of numerous tumour vessels narrowed or completely disappeared, and numerous erythrocytes stagnated in drainage vessels of the tumour vascular network. Haemolysis of these erythrocytes occurred after 2-3 h, resulting in complete thrombosis. There was no indication of reperfusion in vessels showing haemolysis. This haemolysis is thought to be the main cause for the irreversibility of TBF stasis. Since the tumour interstitial fluid pressure decreased after i.v. AC7700 administration, the possibility of stasis of TBF being caused by tumour vascular compression was excluded. All these results strongly suggest that the main target of AC7700 is host arterioles and that the stasis of TBF induced by AC7700 is not triggered by a direct action of the drug on tumour vessels.

Long-lasting gene expression by particle-mediated intramuscular transfection modified with bupivacaine: combinatorial gene therapy with IL-12 and IL-18 cDNA against rat sarcoma at a distant site.

The immune response is modulated by genetic adjuvants using plasmid vectors expressing cytokines. Skeletal muscle can express a foreign gene intramuscularly administered via a needle injection, and the potential of muscle as a target tissue for somatic gene therapy in treating cancer has been explored. In the present study, we investigated the efficacy of particle-mediated intramuscular transfection modified with a local anesthetic agent, bupivacaine, on luciferase and green fluorescent protein. The results indicate that these proteins are more efficiently expressed and persist longer in muscle modified in this way compared with the needle-injection method. Using an established rat sarcoma model, particle-mediated intramuscular gene-gun therapy with a combination of IL-12 and IL-18 cDNA was conducted. Growth of the distant sarcoma was significantly inhibited by particle-mediated intramuscular combination gene therapy, and the survival rate was also improved. Furthermore, the combination gene-gun therapy maintained significant levels of interferon-gamma and induced a high activity of tumor-specific cytotoxic T lymphocytes. These results suggest that the sustained local delivery of IL-12 and IL-18 cDNA using intramuscular gene-gun therapy modified with bupivacaine can induce long-term antitumor immunity, and can provide the great advantage of inhibiting the disseminated tumor.