Cyclodextrin curcumin formulation improves outcome in a preclinical pig model of marginal kidney transplantation.

Decreasing organ quality is prompting research toward new methods to alleviate ischemia reperfusion injury (IRI). Oxidative stress and nuclear factor kappa beta (NF-κB) activation are well-described elements of IRI. We added cyclodextrin-complexed curcumin (CDC), a potent antioxidant and NF-κB inhibitor, to University of Wisconsin (UW) solution (Belzer's Solution, Viaspan), one of the most effective clinically approved preservative solutions. The effects of CDC were evaluated on pig endothelial cells and in an autologous donation after circulatory death (DCD) kidney transplantation model in large white pigs. CDC allowed rapid and lasting uptake of curcumin into cells. In vitro, CDC decreased mitochondrial loss of function, improved viability and lowered endothelial activation. In vivo, CDC improved function recovery, lowered histological injury and doubled animal survival (83.3% vs. 41.7%). At 3 months, immunohistochemical staining for epithelial-to-mesenchymal transition (EMT) and fibrosis markers was intense in UW grafts while it remained limited in the UW + CDC group. Transcriptional analysis showed that CDC treatment protected against up-regulation of several pathophysiological pathways leading to inflammation, EMT and fibrosis. Thus, use of CDC in a preclinical transplantation model with stringent IRI rescued kidney grafts from an unfavorable prognosis. As curcumin has proved well tolerated and nontoxic, this strategy shows promise for translation to the clinic.

Acyl chain-dependent effect of lysophosphatidylcholine on human neutrophils.

Lysophosphatidylcholine (LPC) is the most abundant lysophospholipid in plasma and tissues, and its level increases in ischemia and inflammation. LPC induces various proinflammatory actions in leukocytes, endothelial cells, and smooth muscle cells, but its effects may vary, depending on the acyl chain. In the present study, we identified the molecular species of LPC in human plasma and studied their effects on human neutrophils. Unsaturated LPC species over a wide concentration range (5-200 microM) induced long-lasting superoxide production in neutrophils. The response was preceded by a >10-min lag time and lasted for 60-90 min. Superoxide production was prevented when albumin was added together with LPC at a molar ratio of 1:2 or higher, and significant inhibition was observed even when albumin was added 4-8 min after LPC. Saturation of albumin by fivefold molar excess of stearic acid reduced the inhibitory effect significantly. Saturated LPCs, particularly the most abundant 16:0 species, induced significantly less superoxide production than the unsaturated species and only at 5-10 microM concentrations. Saturated LPC species elicited a several-fold higher increase in cytoplasmic calcium and at >20 microM, increased plasma membrane permeability. A mixture of LPCs mimicking the plasma LPC composition induced nearly similar superoxide production as the most active LPC18:1 alone. These results indicate remarkable acyl chain-dependent differences in the cellular effects of LPC. Elevation of LPC level may increase inflammation through activation of neutrophil NADPH oxidase, particularly when the simultaneous increase of free fatty acids diminishes the ability of albumin to scavenge LPCs.

Curcumin as Treatment for Bladder Cancer: A Preclinical Study of Cyclodextrin-Curcumin Complex and BCG as Intravesical Treatment in an Orthotopic Bladder Cancer Rat Model.

OBJECTIVE: To evaluate the antitumor effect of cyclodextrin-curcumin complex (CDC) on human and rat urothelial carcinoma cells in vitro and to evaluate the effect of intravesical instillations of CDC, BCG, and the combination in vivo in the AY-F344 orthotopic bladder cancer rat model. Curcumin has anticarcinogenic activity on urothelial carcinoma and is therefore under investigation for the treatment of non-muscle invasive bladder cancer. Curcumin and BCG share immunomodulating pathways against urothelial carcinoma. METHODS: Curcumin was complexed with cyclodextrin to improve solubility. Four human urothelial carcinoma cell lines and the AY-27 rat cell line were exposed to various concentrations of CDC in vitro. For the in vivo experiment, the AY-27 orthotopic bladder cancer F344 rat model was used. Rats were treated with consecutive intravesical instillations of CDC, BCG, the combination of CDC+BCG, or NaCl as control. RESULTS: CDC showed a dose-dependent antiproliferative effect on all human urothelial carcinoma cell lines tested and the rat AY-27 urothelial carcinoma cell line. Moreover, intravesical treatment with CDC and CDC+BCG results in a lower percentage of tumors (60% and 68%, respectively) compared to BCG (75%) or control (85%). This difference with placebo was not statistically significant (p=0.078 and 0.199, respectively). However, tumors present in the placebo and BCG-treated rats were generally of higher stage. CONCLUSIONS: Cyclodextrin-curcumin complex showed an antiproliferative effect on human and rat urothelial carcinoma cell lines in vitro. In the aggressive orthotopic bladder cancer rat model, we observed a promising effect of CDC treatment and CDC in combination with BCG.

Binding sites in the rat brain for Escherichia coli S fimbriae associated with neonatal meningitis.

Escherichia coli strains that cause sepsis and meningitis in neonatal infants carry S fimbriae that bind to sialyl galactoside units of cell surface glycoproteins. To investigate the possible role of S fimbriae in determining the tissue tropism of neonatal meningitis, we have studied the presence of binding sites for S fimbriae in different tissues of the neonatal rat which is susceptible to meningitis caused by S-fimbriated E. coli. Purified S fimbriae were incubated on cryostat sections of different rat organs and their binding was assessed by indirect immunofluorescence. In the brain of the neonatal rat, S fimbriae specifically bound to the luminal surfaces of the vascular endothelium and of the epithelium lining the choroid plexuses and brain ventricles. The binding was completely inhibited by the trisaccharide NeuAc alpha 2-3Gal beta 1-4Glc, a receptor analogue of S fimbriae, and by a preceding neuraminidase treatment of the sections. A recombinant E. coli strain expressing S fimbriae adhered in large numbers to the same tissue sites in the neonatal brain sections as did the purified fimbriae, whereas the non-fimbriated host strain and a recombinant strain expressing P fimbriae did not adhere to brain tissues. The results suggest that adhesion of S-fimbriated bacteria to the binding sites observed in the neonatal brain has a pathogenetic role during bacterial invasion from circulation into the cerebrospinal fluid.

Tumor cell-associated hyaluronan as an unfavorable prognostic factor in colorectal cancer.

Hyaluronan (HA) is a linear high molecular weight extracellular polysaccharide. It is thought to be involved in mitosis and the enhancement of wound healing, tumor invasion, and metastasis. Because its clinical relevance in cancer has not been explored, we scored HA in colorectal adenocarcinoma and studied its relationship with patient survival. A specific probe prepared from cartilage proteoglycan aggregates was used to stain paraffin-embedded tumor samples from 202 colorectal adenocarcinoma patients treated in Kuopio University Hospital and followed up for a mean of 14 years. The hypothesis that the percentage of HA-positive carcinoma cells (HA%) and HA intensity in cancer cells correlated with survival was tested with the log-rank test, hazard ratios, and their confidence intervals. Ninety-three % of tumors had at least a proportion of carcinoma cells positive for HA. HA intensity in tumor epithelium was stronger in Dukes' stages C and D tumors and in high-grade tumors. The cancer-related survival rate was lower among patients with strong HA intensity in tumor epithelium (P < 0.001) and high HA% (P < 0.001). Recurrence-free survival was also shorter in patients with an intense signal for HA (P = 0.001) and high HA% in tumor epithelium (P = 0.04). HA intensity in tumor epithelium independently predicted survival and recurrence-free survival (Cox's analysis). We conclude that a high proportion of HA-positive cancer cells and high intensity of the HA-signal predicts a poor survival rate. The abnormal expression of HA in the neoplastic colon epithelial cells is suggested to provide a distinct advantage for invasive growth and metastasis.

Relation of skin polyamines to the hairless phenotype in transgenic mice overexpressing spermidine/spermine N-acetyltransferase.

We recently generated a transgenic mouse line with activated polyamine catabolism due to overexpression of spermidine/spermine N1-acetyltransferase. Phenotypic changes in these animals included permanent loss of hair at the age of 3 wk. We have now further explored development of hair loss during early postnatal life. The first hair cycle appeared to be completed normally in the transgenic animals. At postnatal day 15, although macroscopically indistinguishable from their syngenic littermates, the transgenic animals already showed microscopically signs of hair follicle degeneration. Wild-type mice started their second anagen phase at day 27, whereas the transgenic animals did not display functional hair follicles at that time. Hair follicles were replaced by dermal cysts and epidermal utriculi. Analysis of skin polyamines revealed that the transgenic animals continuously overaccumulated putrescine. The view that an overaccumulation of putrescine was related to the disturbed hair follicle development was strengthened by the finding that doubly transgenic mice overexpressing, both spermidine/spermine N1-acetyltransferase and ornithine decarboxylase and with extremely high levels of putrescine in the skin, showed distinctly more severe skin changes compared with the singly transgenic animals. Interest ingly, in spite of their hairless phenotype, the spermidine/spermine N1-acetyltransferase transgenic mice, were significantly more resistant to the development of papillomas in response to the two-stage skin carcinogenesis. Analysis of skin polyamines indicated that the syngenic mice tripled their spermidine content when exposed to promotion, whereas the transgenic animals showed only modest changes. These results suggest that putrescine plays a pivotal part in normal hair follicle development.

Characterization of a scyllo-inositol-containing sialyloligosaccharide from normal human urine.

Three inositol-containing sialyloligosaccharides were isolated from normal human urine. Structural studies including gas-liquid chromatography of mono- and disaccharide derivatives, methylation analysis, mass spectrometry and glycosidase treatments indicated the structure NeuAc (alpha 2-3)Gal(beta 1-0)scyllo-inositol for one of the oligosaccharides isolated. This provides the first evidence for the natural occurrence of a scyllo-inositol glycoside in biological material. The two other oligosaccharides isolated were identified as two isomers of NeuAc(alpha 2-3)Gal(beta 1-0)myo-inositol, which have not been identified in normal urine before.

Binding of plasminogen to Escherichia coli adhesion proteins.

Immobilization of plasminogen via its lysine-binding sites is regarded as a prerequisite for its activation and function in fibrinolysis and pericellular proteolysis. In the present study, the interaction of plasminogen with fimbriae found on Escherichia coli strains causing invasive human infections was studied. Plasminogen displayed concentration-dependent and saturable binding to immobilized type 1 fimbriae and, several fold lower binding to P and S fimbriae. The binding to fimbriae was effectively inhibited by epsilon-aminocaproic acid indicating that it was mediated by the lysine-binding sites of plasminogen. Binding studies with mutated fimbriae and inhibition tests indicated that the interaction was not dependent on the lectin subunit of the fimbriae. These results indicate the existence of a novel type of host-microbe interaction which may be important in the invasion by bacteria of host tissues.

A N-acetyllactosamine-specific cell-binding activity in a plant pathogen, Erwinia rhapontici.

A strain of the phytopathogenic bacterial species, Erwinia rhapontici, was found to cause hemagglutination of human erythrocytes that was specifically inhibited by beta-galactosides. Of the monosaccharides tested, N-acetyl galactosamine and galactose efficiently inhibited the hemagglutination. The most potent inhibitor identified was Ga1 beta 1-4GlcNAc that was 30-100-fold more potent than Ga1 beta 1-3GlcNac or Ga1 beta 1-3GalNAc. Fetuin had no effect on the hemagglutination whereas asialofetuin was inhibitory. No blood group specificity was found for the hemagglutinin. These findings indicate that the E. rhapontici strain possesses a novel bacterial cell-binding activity with specificity for terminal N-acetyllactosamine residues.

Development of a highly purified multicomponent leukocyte IFN-alpha product.

A purification process was developed to obtain a human interferon- alpha (IFN-alpha) product that contains all major IFN-alpha subtypes produced by human leukocytes. The purification was accomplished by immunoaffinity chromatography using two monoclonal antibodies (mAb) and gel filtration. The process comprised two effective virus inactivation steps, solvent detergent treatment, and incubation at low pH, and the purified product was filtered with a 15-nm pore size virus removal filter. The overall yield of IFN-alpha in the process was about 60% when starting from the culture supernatant of Sendai virus-induced human leukocytes. The specific activity was about 1.0 x 10(8) IU/mg. The level of DNA and protein impurities including mouse IgG was very low. The product contained seven main subtypes: IFN-alpha 1, IFN-alpha 2, IFN-alpha 8, IFN-alpha 10, IFN-alpha 14, IFN-alpha 17, and IFN-alpha 21. The subtypes IFN-alpha 4 and IFN-alpha 7 were minor components. Reverse-phase HPLC indicated a constant subtype composition for the product from batch to batch. Stabilization of the pure IFN-alpha solution with albumin and Tween 80 was compared. In virus filtration, a better yield and higher filtration capacity were obtained with Tween. The addition of albumin resulted in the formation of IFN-albumin aggregates. During long-term storage, IFN-alpha was stable in both solutions for 2 years at 2-8 degrees C. The new method makes it possible to extensively purify all major IFN-alpha subtypes and obtain a virus-safe and stable product with a constant subtype composition.

HSV-tk gene therapy for human renal cell carcinoma in nude mice.

We have treated Caki-2 human renal cell carcinoma in vivo using herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Both stably transduced Caki-2 tumors, generated using retrovirus-mediated ex vivo HSV-tk gene transfer and direct intratumoral adenovirus-mediated HSV-tk gene transfer of wild type tumors, were tested. Similar treatments with LacZ containing retro- and adenoviruses were used as controls. The outcome was evaluated by imaging the tumors before and after the treatment with magnetic resonance imaging, and using histology, immunocytochemistry, and survival analysis. When implanted orthotopically into nude mouse kidneys, Caki-2 cells formed reproducible cystic papillary kidney carcinomas. In vivo magnetic resonance imaging provided an important tool for the evaluation of tumor growth. Transduction efficiency of wild-type tumors in vivo with adeno-LacZ was 22+/-14%. Significant tumor regression was achieved with direct intratumoral adeno-HSV-tk transduction followed by intraperitoneal ganciclovir (GCV) (P<.001). Also, the treatment of stably transduced Caki-2 tumors with intraperitoneal GCV resulted in a significant treatment response in the HSV-tk group as compared to the LacZ group (P<.009). Increased apoptosis and macrophage infiltrations, reduced proliferation, and degenerative changes were observed in the tumors treated with HSV-tk and GCV. Also, significant prolongation in survival was achieved with adeno-HSV-tk- and GCV-treated mice as compared to the controls. It is concluded that adeno-HSV-tk gene therapy may be useful for the treatment of renal cell carcinoma in vivo.

Plasma ascorbate protects coagulation factors against photooxidation.

It has been suggested that proteins, unlike lipids, are not protected against oxidative damage by antioxidants in plasma. We have studied the effect of photodynamic virus inactivation treatment of fresh human plasma on coagulation factor activities. Photodynamic treatment generates singlet oxygen which causes inactivation of fibrinogen and factor VII. Other coagulation factors or anticoagulant proteins are clearly less affected. We found that there is an inverse correlation between the extent of coagulation factor inactivation during the treatment and the plasma ascorbate concentration. The inactivation of coagulation factors was prevented in a dose-dependent manner by adding ascorbate to the plasma before the treatment. Ascorbate was consumed during the treatment at an apparently linear rate. Oxidation of urate and coagulation factors was enhanced when ascorbate had disappeared from plasma. These results indicate that ascorbate is a primary antioxidant against photooxidation in plasma and that it effectively prevents oxidative damage to coagulation factors and other proteins.

Enhancement of tissue plasminogen activator-catalyzed plasminogen activation by Escherichia coli S fimbriae associated with neonatal septicaemia and meningitis.

The effect of Escherichia coli strains isolated from blood and cerebrospinal fluid of septic infants on plasminogen activation was studied. These strains typically carry a filamentous surface protein, S fimbria, that has formerly been shown to bind to endothelial cells and interact with plasminogen. The bacteria effectively promoted plasminogen activation by tissue plasminogen activator (t-PA) which was inhibited by epsilon-aminocaproic acid. A recombinant strain expressing S fimbriae accelerated t-PA-catalyzed plasminogen activation to a similar extent as did the wild-type strains whereas the nonfimbriate recipient strain had no effect. After incubation with t-PA and plasminogen, the S-fimbriate strain displayed bacterium-bound plasmin activity whereas the nonfimbriate strain did not. Bacterium-associated plasmin generation was also observed with a strain expressing mutagenized S fimbriae that lack the cell-binding subunit SfaS but not with a strain lacking the major subunit SfaA. Both t-PA and plasminogen bound to purified S fimbriae in a lysine-dependent manner and purified S fimbriae accelerated t-PA-catalyzed plasminogen activation. The results indicate that E. coli S fimbriae form a complex with t-PA and plasminogen which enhances the rate of plasminogen activation and generates bacterium-bound plasmin. This may promote bacterial invasion and persistence in tissues and contribute to the systemic activation of fibrinolysis in septicaemia.

Occurrence of amphoterin (HMG1) as an endogenous protein of human platelets that is exported to the cell surface upon platelet activation.

Amphoterin (HMG1) is a 30-kD heparin-binding protein which is functionally associated with the outgrowth of cytoplasmic processes in developing neurones. Amphoterin has been shown to mediate adhesive and proteolytic interactions at the leading edge of motile cells. Recently it was shown that inhibition of amphoterin interactions with its cell surface receptor (RAGE) suppresses tumour growth and metastasis. In this work we have identified amphoterin polypeptide and its mRNA in human platelets. Amphoterin had a cytoplasmic localisation in resting platelets according to subcellular fractionation studies and immunogold electronmicroscopy. After platelet activation, part of amphoterin was associated with the external surface of plasma membrane. Externalisation of amphoterin during platelet activation was also detected in immunofluorescence studies. Amphoterin was detectable in human serum (0.2 ng/ml) but not in plasma. Resting platelets treated with PGI2 and forskolin bound to immobilised recombinant amphoterin independently of divalent cations. The binding induced a spicular morphology in platelets, and was effectively inhibited by heparin. Amphoterin-binding protein components on the platelet surface were not identified, but amphoterin bound to phosphatidylserine and sulfatide in lipid binding assays. Our results suggest that amphoterin is an endogenous protein in human platelets, which is exported to the cell surface during platelet activation. Interaction of amphoterin with the platelet surface may be mediated by sulfoglycolipids and phospholipids.

Irregular expression of hyaluronan and its CD44 receptor is associated with metastatic phenotype in laryngeal squamous cell carcinoma.

The distributions of hyaluronan (HA) and its CD44 receptor were studied in 24 normal, 27 dysplastic samples of laryngeal epithelium and in 172 squamous cell carcinomas (LSCC), using a specific probe prepared from cartilage proteoglycan (bHABC, biotinylated hyaluronan binding complex) and a monoclonal antibody (Hermes 3). HA and CD44 were expressed similarly in all normal and about 90% of dysplastic and neoplastic laryngeal epithelia. In the normal epithelium HA and CD44 were homogeneously distributed throughout the epithelium, whereas the most superficial layers were negative. This was in contrast to the picture in dysplastic epithelium and well-differentiated invasive carcinomas, which were entirely HA and CD44 positive. Local areas with a low signal for HA and CD44 were present in 11% and 22% of the samples with dysplasia, and in 27% and 28% of those with carcinoma, respectively. The presence of this staining irregularity was associated with poor differentiation of the carcinoma, a significantly elevated mitotic index and a high frequency of nodal spreading and metastases. Furthermore, the irregular staining showed a trend towards poor disease-free survival, suggesting that an altered metabolism of HA is a common feature in LSCC and is associated with an aggressive growth pattern.

Dependence of Staphylococcus epidermidis on non-transferrin-bound iron for growth.

The ability of Staphylococcus epidermidis strains to grow in the presence of human transferrin and varying amounts of ferric iron was studied. At initial bacterial densities up to 10(4) cfu ml(-1), none of the three strains grew when transferrin iron saturation was below the full saturation point, whereas the bacteria grew consistently when transferrin was fully iron-saturated and there was non-transferrin-bound iron in the medium. Precultivation of the bacteria under iron-restricted conditions to induce siderophore production did not abolish the growth dependence on non-transferrin-bound iron. At initial bacterial densities of 10(6) cfu ml(-1), the bacteria proliferated consistently also in the presence of partially saturated transferrin. The results indicate that at low bacterial densities, S. epidermidis cannot utilise transferrin-bound iron for growth and that its proliferation is dependent on non-transferrin-bound iron.

Local stimulation of proteoglycan synthesis in articular cartilage explants by dynamic compression in vitro.

Cultured bovine articular cartilage was subjected to 50 ms, 0.5-1.0 MPa compressions repeated at intervals of 2-60 s for 1.5 h and simultaneously labeled with 35SO4. The compression was delivered with a 4-mm-diameter nonporous loading head on an 8-mm-diameter cartilage explant. This method created directly compressed (central) and uncompressed (border) areas within the tissue. Analysis of the whole explant under a 0.5 MPa load showed significantly increased 35SO4 incorporation by compression repeated at 2- and 4-s but not at 20- and 60-s intervals. When the incorporation was studied separately in the border and central areas, a statistically significant stimulation was noticed in the central area with a 4-s cycle, while the border area was stimulated with a 2-s cycle. Autoradiography of the central area showed that the stimulation with 0.5 MPa and a 4-s cycle occurred through the whole depth of the cartilage, while raising the pressure to 1 MPa or the frequency to 2 s reduced the stimulation, particularly in the superficial cartilage. In the border area the stimulation with 0.5 MPa and a 2-s cycle was noted in the superficial zone only. The stimulation of proteoglycan synthesis is thus limited to certain loading frequencies and pressures and occurs in specific areas under and around the loaded site. Its rapid appearance suggests enhanced glycosylation or sulfation of core proteins or enhanced speed of posttranslational processing.

Influence of short-term hydrostatic pressure on organization of stress fibers in cultured chondrocytes.

The present study describes changes in the organization of stress fibers that occur in articular cartilage chondrocytes subjected to hydrostatic pressure. Primary cultures of chondrocytes from bovine articular cartilage, grown on coverslips, were subjected to 5, 15, or 30 MPa hydrostatic pressure at 37 degrees C. The pressure was applied continuously or cyclically at two frequencies: 0.125 Hz (4 seconds of pressure and 4 seconds of no pressure) or 0.05 Hz (1 second of pressure and 19 seconds of no pressure) for a period of 2 hours. Control chondrocytes showed a polygonal form with prominent stress fibers extending across the cells. The exposure of cells to 30 MPa pressure caused a nearly total disappearance of stress fibers and retraction of the cells from each other. With pressure at 15 MPa or cyclic pressure, the number of cells with stress fibers was decreased. In cells subjected to 5 MPa pressure, the stress fibers resembled those in control chondrocytes. The pressure effects were reversible after 2 hours. Pressure had no effect on the staining pattern of vinculin, which suggests that microfilaments are more vulnerable to pressure than vinculin. The results indicate that cytoskeletal changes may be an integral part of the response of chondrocytes to hydrostatic pressure.

A mechanical apparatus with microprocessor controlled stress profile for cyclic compression of cultured articular cartilage explants.

An apparatus was designed for mechanical compression of cultured articular cartilage explants with acylindrical plain-ended loading head (diameter 2-5 mm) driven by a stepping motor. A load cell under the culture dish was applied for feedback regulation utilizing a microprocessor-based control unit. The operating programs allowed either continuous or cyclic loading, the latter with adjustable loading/resting ratio. The improvements in the present design compared with previously described apparatuses for similar purposes include: (1) the accurately controlled compression by a load cell and a rapid feedback circuit; (2) the wide range of selectable stresses (25 kPa-12.5 MPa) with both continuous and cyclic loading modes; (3) the ability to handle cycles as short as 1 s with 15 ms peak loading phase. Using a 4 s cycle and 0.5 MPa load for 1.5 h resulted in a significantly enhanced incorporation of radiosulphate in cultured bovine articular cartilage explants, suggesting a stimulation of proteoglycan synthesis. Light and scanning electron microscopic examinations revealed a slight depression and superficial alterations in cartilage structure at the impact site following high pressures. We expect that this apparatus will help in revealing how articular cartilage tissue and chondrocytes respond to external mechanical stimuli.

Characterization of a new animal model for human renal cell carcinoma.

BACKGROUND: Human renal cell carcinoma (RCC) is the most common kidney malignancy with significant mortality. Human tumor xenograft models are important tools for cancer research. MATERIALS AND METHODS: We have established and characterized a new animal model for human RCC using Caki-2 cells implanted into the renal subcapsule (RSC) of nude mice. Histology, immunocytochemistry, in situ hybridization and magnetic resonance imaging (MRI) were used to analyze the tumors. RESULTS: The implantations generated reproducible carcinomas which closely resemble human RCC. The tumors showed cystic-papillary structures, rich capillary network and fibro-septa formations. Proliferation varied from 0-5% and from 1-60% in cystic and solid areas, respectively. Apoptosis was less than 1%. Macrophages and other inflammatory cell infiltrations were detected in the tumors. VEGF-A and angiopoietin I were expressed in a small number of cells in large tumors. Tumors did not metastasize outside peritoneal cavity. Survival of the tumor bearing animals was 23 +/- 3 weeks. CONCLUSIONS: It is concluded that Caki-2 carcinomas implanted into renal subcapsule of nude mice resemble human RCC in several aspects and represent a good animal model for studies regarding the pathogenesis and treatment of human RCC.