Improvement of insulin resistance by removal of systemic hydrogen peroxide by PEGylated catalase in obese mice.

Insulin resistance, a condition in which insulin action is impaired, is one of the characteristic features of type 2 diabetes. Excessive amounts of reactive oxygen species (ROS) interfere with the insulin signaling pathway, which leads to the progression of insulin resistance. To examine whether removal of systemic hydrogen peroxide is effective in improving insulin resistance, polyethylene glycol-conjugated catalase (PEG-catalase), a derivative with a long circulation half-life, was repeatedly injected into leptin-deficient ob/ob or high fat diet-induced obese mice for 16 or 10 consecutive weeks, respectively. Although ob/ob mice gradually gained weight with time irrespective of the treatment, repeated intraperitoneal injections of PEG-catalase significantly reduced glucose levels in the fed state. Glucose and insulin tolerance tests also showed PEG-catalase significantly improved glucose tolerance and insulin sensitivity in ob/ob mice, respectively. Similar but less marked results were obtained in the diet-induced obese mice. Treatment of 3T3-L1 adipocytes with glucose oxidase (GO) increased lipid hydroperoxide formation and reduced insulin-stimulated Akt phosphorylation. Addition of catalase or PEG-catalase significantly inhibited the GO-induced changes in adipocytes. These findings indicate that systemic removal of hydrogen peroxide by PEG-catalase activates the insulin signaling pathway and improves insulin resistance in obese mice.

Prevention of pulmonary metastasis from subcutaneous tumors by binary system-based sustained delivery of catalase.

Catalase delivery can be effective in inhibiting reactive oxygen species (ROS)-mediated acceleration of tumor metastasis. Our previous studies have demonstrated that increasing the plasma half-life of catalase by pegylation (PEG-catalase) significantly increases its potency of inhibiting experimental pulmonary metastasis in mice. In the present study, a biodegradable gelatin hydrogel formulation was used to further increase the circulation time of PEG-catalase. Implantation of (111)In-PEG-catalase/hydrogel into subcutaneous tissues maintained the radioactivity in plasma for more than 14 days. Then, the effect of the PEG-catalase/hydrogel on spontaneous pulmonary metastasis of tumor cells was evaluated in mice with subcutaneous tumor of B16-BL6/Luc cells, a murine melanoma cell line stably expressing luciferase. Measuring luciferase activity in the lung revealed that the PEG-catalase/hydrogel significantly (P<0.05) inhibited the pulmonary metastasis compared with PEG-catalase solution. These findings indicate that sustaining catalase activity in the blood circulation achieved by the use of pegylation and gelatin hydrogel can reduce the incidence of tumor cell metastasis.

Hydrogen peroxide-mediated nuclear factor kappaB activation in both liver and tumor cells during initial stages of hepatic metastasis.

Various factors involved in tumor metastasis are regulated by the transcription factor nuclear factor kappaB (NF-kappaB). Because NF-kappaB activation may contribute to establishment of hepatic metastasis, its activation in liver cells and tumor cells was separately evaluated in a mouse model of hepatic metastasis. pNF-kappaB-Luc, a firefly luciferase-expressing plasmid DNA depending on the NF-kappaB activity, was injected into the tail vein of mice by the hydrodynamics-based procedure, a well-established method for gene transfer to BALB/c male mouse liver. The luciferase activity in the liver was significantly increased by an intraportal inoculation of murine adenocarcinoma colon26 cells, but not of peritoneal macrophages, suggesting that the NF-kappaB in liver cells is activated when tumor cells enter the hepatic circulation. Then, colon26 cells stably transfected with pNF-kappaB-Luc were inoculated. The firefly luciferase activity, an indicator of NF-kappaB activity in tumor cells, was significantly increased when colon26/NFkappaB-Luc cells were inoculated into the portal vein of BALB/c male mice. The NF-kappaB activation in both liver and tumor cells was significantly inhibited by injection of catalase derivatives, which have been reported to inhibit hepatic metastasis of tumor cells. These findings indicate for the first time that NF-kappaB, a key agent regulating the expression of various molecules involved in tumor metastasis, is activated in both liver and tumor cells during the initial stages of tumor metastasis through a hydrogen peroxide mediated pathway. Thus, the removal of hydrogen peroxide will be a promising approach to treating hepatic metastasis.

SOD derivatives prevent metastatic tumor growth aggravated by tumor removal.

Although surgical removal is the most aggressive strategy to treat removable tumors, it sometimes aggravates tumor growth in metastatic sites. Because surgical procedures generate reactive oxygen species (ROS), known promoters of tumor metastasis and growth, we examined whether the growth of micrometastasis is inhibited by superoxide dismutase (SOD) derivatives after surgical removal of tumors in mice. Murine melanoma B16-BL6 cells were inoculated into the footpad to establish spontaneous pulmonary metastasis. The removal of the footpad tumor significantly (P < 0.05) increased the level of plasma lipoperoxides and the number of tumor cells in the lung. An intravenous injection of SOD or its pegylated-SOD derivative significantly (P < 0.05) inhibited the peroxidation and metastatic tumor growth. It also extended the survival period of mice undergoing removal of the footpad tumor. These findings indicate that the removal of tumor produces ROS, which then aggravates the growth of tumor cells in micrometastases. SOD derivatives can effectively prevent this metastatic tumor growth by detoxifying ROS.

Cationized catalase-loaded hydrogel for growth inhibition of peritoneally disseminated tumor cells.

A previous study demonstrated that ethylenediamine-conjugated catalase (ED-catalase) inhibits peritoneal dissemination of tumor cells in mice. To increase its inhibitory effects by sustained release, a hydrogel formulation of ED-catalase was prepared using a biodegradable hydrogel consisting of an acidic gelatin with an isoelectric point of 5.0. Although intraperitoneally injected ED-catalase solution rapidly disappeared from the cavity, more than 10% of ED-catalase remained even at 14 days after implantation of ED-catalase/hydrogel into the cavity. Then, the effect of ED-catalase/hydrogel on peritoneal dissemination of tumor cells was evaluated by measuring the luciferase activity of abdominal organs after intraperitoneal inoculation of colon26/Luc, a colon adenocarcinoma stably expressing luciferase. ED-catalase/hydrogel showed a significantly (P<0.05) greater effect on inhibiting the growth of tumor cells than ED-catalase solution, demonstrating the importance of the retention of ED-catalase within the cavity as far as inhibition is concerned. Serial in vivo images of luciferase activity revealed that the ED-catalase/hydrogel significantly (P<0.05) retarded the growth rate of tumor cells. Survival of tumor-bearing mice supported the findings obtained with the luminescence-based analyses. These findings indicate that the sustained release of ED-catalase from hydrogels into the cavity is highly effective in inhibiting the growth of peritoneally disseminated tumor cells.

Inhibition of peritoneal dissemination of tumor cells by cationized catalase in mice.

To inhibit peritoneal dissemination of tumor cells by destroying hydrogen peroxide, ethylenediamine-conjugated catalase (ED-catalase), a cationized derivative, was injected into the peritoneal cavity of mice. ED-catalase had about a 6-fold longer retention time within the cavity than unmodified catalase. Peritoneal dissemination was evaluated after intraperitoneal inoculation of B16-BL6/Luc, a melanoma clone stably expressing firefly luciferase, by measuring luciferase activity. An intraperitoneal injection of ED-catalase just before tumor inoculation significantly reduced the number of tumor cells in peritoneal organs. Catalase was less effective, confirming the importance of the retention of the enzyme within the cavity for the inhibition. ED-catalase injected 3 days after tumor inoculation was also effective in inhibiting tumor growth. A real-time quantitative PCR analysis revealed that ED-catalase significantly suppressed the expression of intercellular adhesion molecule-1. Daily dosing of ED-catalase for 7 days significantly prolonged the survival of tumor-bearing mice. These findings indicate that ED-catalase, which is retained for a long time within the peritoneal cavity, is highly effective in inhibiting the adhesion and proliferation of peritoneally disseminated tumor cells, and in increasing the survival of tumor-bearing mice.

Analysis of in vivo nuclear factor-kappaB activation during liver inflammation in mice: prevention by catalase delivery.

Nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays crucial roles in inflammation, immunity, cell proliferation, and apoptosis. Until now, there have been few studies of NF-kappaB activation in whole animals because of experimental difficulties. Here, we show that mice receiving a simple injection of plasmid vectors can be used to examine NF-kappaB activation in the liver. Two plasmid vectors, pNF-kappaB-Luc (firefly luciferase gene) and pRL-SV40 (Renilla reniformis luciferase gene), were injected into the tail vein of mice by the hydrodynamics-based procedure, an established method of gene transfer to mouse liver. Then, the ratio of the firefly and R. reniformis luciferase activities (F/R) was used as an indicator of the NF-kappaB activity in the liver. Injection of thioacetamide or lipopolysaccharide plus d-galactosamine increased the F/R ratio in the liver, and this was significantly (P<0.001) inhibited by an intravenous injection of catalase derivatives targeting liver nonparenchymal cells. Imaging the firefly luciferase expression in live mice clearly demonstrated that the catalase derivatives efficiently prevented the NF-kappaB-mediated expression of the firefly luciferase gene. Plasma transaminases and the survival rate of mice supported the findings obtained by the luminescence-based analyses. Thus, this method, which requires no genetic recombination techniques, is highly sensitive to the activation of NF-kappaB and allows us to continuously examine the activation in live animals. In conclusion, this novel, simple, and sensitive method can be used not only for analyzing the NF-kappaB activation in the organ under different inflammatory conditions but also for screening drug candidates for the prevention of liver inflammation.

Inhibition of adhesion and proliferation of peritoneally disseminated tumor cells by pegylated catalase.

Hydrogen peroxide may aggravate the peritoneal dissemination of tumor cells by activating the expression of a variety of genes. In this study, we used pegylated catalase (PEG-catalase) to examine whether prolonged retention of catalase activity within the peritoneal cavity is effective in inhibiting peritoneal dissemination in mouse models. Murine B16-BL6 cells or colon 26 cells labeled with firefly luciferase gene were inoculated intraperitoneally into syngeneic mice. Compared with unmodified catalase, PEG-catalase was retained in the peritoneal cavity for a long period after intraperitoneal injection. A single injection of PEG-catalase just before tumor inoculation significantly reduced the number of the tumor cells at 1 and 7 days. The changes in the expression of molecules involved in the metastasis were evaluated by real time quantitative PCR analysis. Inoculation of the tumor cells increased the expression of intercellular adhesion molecule (ICAM)-1 in the greater omentum, which was inhibited by PEG-catalase. An injection of PEG-catalase at 3 days after tumor inoculation also reduced the number of the tumor cells, suggesting that processes other than the adhesion of tumor cells to peritoneal organs are also inhibited. Daily doses of PEG-catalase significantly prolonged the survival time of tumor-bearing mice. These results indicate that intraperitoneal injection of PEG-catalase inhibits the multiple processes of peritoneal dissemination of tumor cells by scavenging hydrogen peroxide in the peritoneal cavity.

PEGylated catalase prevents metastatic tumor growth aggravated by tumor removal.

Although surgical removal is a primary option for treating tumors, it can lead to the increased growth of metastatic tumors. Because surgical procedures may generate reactive oxygen species (ROS), known promoters of tumor metastasis and growth, we investigated whether PEGylated catalase (PEG-catalase, plasma half-life of 13.6 h) was able to prevent this after surgical removal of a footpad tumor in mice. Murine melanoma cells labeled with the firefly luciferase gene were used to monitor the distribution of tumor cells. After inoculation into the footpad, tumor cells were found in the lung, and the number increased with time. The surgical removal of the footpad tumor significantly (p < 0.05) increased the number of metastatic tumor cells and the level of plasma lipoperoxides. An intravenous injection of PEG-catalase significantly (p < 0.05) suppressed the metastatic tumor growth as well as the peroxidation. Quantitative RT-PCR and Western blot analyses indicated that PEG-catalase markedly reduced the increase in the expression of epidermal growth factor receptor. These findings indicate that the removal of tumor produces ROS, which then aggravate metastatic tumor growth by activating several growth factors. PEG-catalase can effectively prevent this metastatic tumor growth by detoxifying the ROS.

Inhibition of peritoneal dissemination of tumor cells by single dosing of phosphodiester CpG oligonucleotide/cationic liposome complex.

Although unmethylated CpG dinucleotide-containing oligodeoxynucleotides (CpG ODN) are able to inhibit tumor metastasis through the induction of antitumor immunity, their stability and delivery to antigen presenting cells needs to be improved. In this study, we formulated a CpG ODN complex with cationic liposomes (CpG ODN-lipoplex) and its antitumor activity was evaluated in peritoneal dissemination models of tumor cells stably labeled with firefly luciferase gene. A single intraperitoneal administration of CpG ODN-lipoplex greatly reduced the number of tumor cells to 0.01% or lower compared with that detected in untreated mice, which may be associated with increased production of TNF-alpha and IL-12. CpG ODN-lipoplex increased the survival time of the tumor-bearing mice, and most long-term survivors rejected rechallenged tumor cells. These results indicate that a single dosing of CpG ODN-lipoplex is effective in inhibiting peritoneal dissemination and inducing long-lasting antitumor immunity.

Inhibition of metastatic tumor growth by targeted delivery of antioxidant enzymes.

To develop effective anti-metastatic therapy, targeted or sustained delivery of catalase was examined in mice. We found that mouse lung with metastatic colonies of adenocarcinoma colon26 cells exhibited reduced catalase activity. The interaction of the tumor cells with macrophages or hepatocytes generated detectable amounts of ROS, and increased the activity of matrix metalloproteinases. Hepatocyte-targeted delivery of catalase was successfully achieved by galactosylation, which was highly effective in inhibiting the hepatic metastasis of colon26 cells. PEGylation, which increased the retention of catalase in the circulation, effectively inhibited the pulmonary metastasis of the cells. To examine which processes in tumor metastasis are inhibited by catalase derivatives, the tissue distribution and proliferation of tumor cells in mice was quantitatively analyzed using firefly luciferase-expressing tumor cells. An injection of PEG-catalase just before the inoculation of melanoma B16-BL6/Luc cells significantly reduced the number of the tumor cells in the lung at 24 h. Daily dosing of PEG-catalase greatly inhibited the proliferation of the tumor cells, and increased the survival rate of the tumor-bearing mice. These results indicate that targeted or sustained delivery of catalase to sites where tumor cells metastasize is a promising approach for inhibiting metastatic tumor growth.

Inhibition of metastatic tumor growth in mouse lung by repeated administration of polyethylene glycol-conjugated catalase: quantitative analysis with firefly luciferase-expressing melanoma cells.

PURPOSE: To develop a novel and effective approach to inhibit tumor metastasis based on controlled delivery of catalase, we first evaluated the characteristics of the disposition and proliferation of tumor cells. Then, we examined the effects of polyethylene glycol-conjugated catalase (PEG-catalase) on tumor metastasis. On the basis of the results obtained, PEG-catalase was repetitively administered to completely suppress the growth of tumor cells. EXPERIMENTAL DESIGN: Murine melanoma B16-BL6 cells were stably transfected with firefly luciferase gene to obtain B16-BL6/Luc cells. These cells were injected intravenously into syngeneic C57BL/6 mice. PEG-catalase was injected intravenously, and the effect was evaluated by measuring the luciferase activity as the indicator of the number of tumor cells. RESULTS: At 1 hour after injection of B16-BL6/Luc cells, 60 to 90% of the injected cells were recovered in the lung. The numbers decreased to 2 to 4% at 24 hours, then increased. An injection of PEG-catalase just before inoculation significantly reduced the number of tumor cells at 24 hours. Injection of PEG-catalase at 1 or 3 days after inoculation was also effective in reducing the cell numbers. Daily dosing of PEG-catalase greatly inhibited the proliferation and the number assayed at 14 days after inoculation was not significantly different from the minimal number observed at 1 day, suggesting that the growth had been markedly suppressed by the treatment. CONCLUSIONS: These findings indicate that sustained catalase activity in the blood circulation can prevent the multiple processes of tumor metastasis in the lung, which could lead to a state of tumor dormancy.

Inhibition of experimental hepatic metastasis by targeted delivery of catalase in mice.

Bovine liver catalase derivatives possessing diverse tissue distribution properties were synthesized, and their effects on hepatic metastasis of colon carcinoma cells were examined in mice. An intraportal injection of 1 x 10(5) colon 26 cells resulted in the formation of more than 50 metastatic colonies on the surface of the liver at 14 days after injection. An intravenous injection of catalase (CAT; 35000 units/kg of body weight) significantly (P < 0.001) reduced the number of the colonies in the liver. Galactosylated (Gal-), mannosylated (Man-) and succinylated (Suc-) CAT were also tested in the same system. Of these derivatives, Gal-CAT showed the greatest inhibitory effect on hepatic metastasis, and the number of colonies was significantly (P < 0.001) smaller than following treatment with catalase. High activities of matrix metalloproteinases (MMPs), especially MMP-9, were detected in the liver of mice bearing metastatic tumor tissues, which was significantly (P < 0.05) reduced by Gal-CAT. These results, combined with our previous finding that Gal-CAT can be efficiently delivered to hepatocytes, indicate that the targeted delivery of catalase to the liver by galactosylation is a promising approach to suppress hepatic metastasis. Decreased MMP activity by catalase delivery seems to be involved in its anti-metastatic effect.