Colon 26-bearing Mice Research Articles

Carrier proteins-based boron delivery to tumor

The conjugation of maleimide-functionalized closo-dodecaborate (MID) to transferrin (TF), which has no free SH cysteine residue, proceeded at room temperature for 12 h in PBS buffer (pH = 7.4). MID-TF conjugates were accumulated into the cells in the time- and concentration-dependent manners through the TF receptor mediated mechanism. In vivo biodistribution study of MID-TF conjugates in colon 26-bearing mice revealed the time-dependent selective accumulation of MID-TF conjugates into tumor very similar to that of MID-BSA. The tumor boron concentration of MID-BSA was higher than that of MID-TF conjugates (62 ppm vs. 20 ppm at a dose of 30 mg [B]/kg) 12 h after administration, suggesting that the EPR effect is not remarkably observed in the case of MID-TF conjugates because TF has smaller molecular weight than BSA (48 kDa vs. 68 kDa).

Investigation of anti-tumor effect of doxorubicin-loaded human serum albumin nanoparticles prepared by a desolvation technique.

Nanoparticles of human serum albumin (HSA) prepared using a desolvation method possess sizes suitable for tumor accumulation. Here, we report on an investigation of the anti-tumor effects and biodistribution of doxorubicin-HSA nanoparticles in vitro and in vivo. The cytotoxicity of nanoparticles was evaluated in 2D and 3D colon 26 cell cultures. Furthermore, the biodistribution and the anti-tumor activity of nanoparticles in colon 26-bearing mice were investigated. Assessments on the effect on metastasis and the toxicity were also carried out. Doxorubicin-HSA nanoparticles showed cytotoxicity in colon 26 cancer cell cultures, although the cytotoxicity was less in the case of nanoparticles than in free doxorubicin. In vivo anti-tumor activity was more pronounced in nanoparticles despite the fact that their accumulation in tumors was not superior to that of free doxorubicin, suggesting that factors other than accumulation contribute to the enhanced anti-tumor activity of these nanoparticles. The administration of nanoparticles also resulted in the suppression of metastasis. The prepared nanoparticles appear to be effective for cancer therapy although further studies will be needed to clarify the details of anti-tumor activity and the toxicity of these nanoparticles.

Enhanced Delivery of Radiolabeled Polyoxazoline into Tumors via Self-Aggregation under Hyperthermic Conditions.

In order to develop a radiopharmaceutical for internal radiotherapy that had a high anticancer effect while exposing normal tissues to low radiation levels, we synthesized a radiolabeled polyoxazoline (POZ), a thermoresponsive polymer, and established a novel drug delivery system for targeting tumors by accelerating the accumulation of the radiolabeled POZ via self-aggregation under hyperthermic (42-43 °C) conditions. By living-cationic polymerization using 2-ethyl-2-oxazoline and 2-isopropyl-2-oxazoline, POZ derivatives (Et-IspPOZ) (10, 20, and 30 kDa) with lower critical solution temperatures (LCSTs) of 37-38 °C were synthesized; the POZ derivatives were soluble at the body temperature but self-aggregated upon heat treatment (42-43 °C). Next, the indium-111 (111In)-labeled Et-IspPOZ was prepared, and the effect of molecular weight and injected POZ dose on the accumulation of radioactivity in the tumors was investigated upon intravenous injection of probes under hyperthermic conditions in colon 26-bearing mice. The uptake of radioactivity in tumors was increased when the molecular weight of POZ was greater than 20 kDa, while it was independent of the injected POZ dose (4-40 nmol). The amount of radioactivity retained in the tumor did not change for up to 3 h after exposure to heat treatment was stopped. Furthermore, the tumor uptake of the Et-IspPOZ derivative with an LCST greater than 42 °C was significantly lower than that of Et-IspPOZ, which had an LCST of 37-38 °C, suggesting the involvement of the self-aggregation of POZ on tumor uptake. Finally, the intratumoral localization of fluorescence-labeled Et-IspPOZ was evaluated using in vivo confocal laser microscopy. Many bright fluorescence spots were observed in the heat-treated tumors nearby and within blood vessels. In conclusion, the high tumor uptake of radiolabeled Et-IspPOZ was elucidated under hyperthermic conditions; thereby, the possibility of developing a novel internal radiotherapy using radiolabeled POZ derivatives was demonstrated.

Systemic delivery of chTNT-3/CpG immunoconjugates for immunotherapy in murine solid tumor models.

CpG oligodeoxynucleotides (CpG) potently activate the immune system by mimicking microbial DNA. Conjugation of CpG to chTNT-3, an antibody targeting the necrotic centers of tumors, enabled CpG to accumulate in tumors after systemic delivery, where it can activate the immune system in the presence of tumor antigens. CpG chemically conjugated to chTNT-3 (chTNT-3/CpG) were compared to free CpG in their ability to stimulate the immune system in vitro and reduce tumor burden in vivo. In subcutaneous Colon 26 adenocarcinoma and B16-F10 melanoma models in BALB/c and C57BL/6 mice, respectively, chTNT-3/CpG, free CpG, or several different control constructs were administered systemically. Intraperitoneal injections of chTNT-3/CpG delayed tumor growth and improved survival and were comparable to intratumorally administered CpG. Compared to saline-treated mice, chTNT-3/CpG-treated mice had smaller average tumor volumes by as much as 72% in Colon 26-bearing mice and 79% in B16-bearing mice. Systemically delivered free CpG and CpG conjugated to an isotype control antibody did not reduce tumor burden or improve survival. In this study, chTNT-3/CpG retained immunostimulatory activity of the CpG moiety and enabled delivery to tumors. Because systemically administered CpG rapidly clear the body and do not accumulate into tumors, chTNT-3/CpG provide a solution to the limitations observed in preclinical and clinical trials.

Metabolites of 3'-O-acetylhamaudol Isolated from Angelica japonica Roots, and their Antitumor Actions

Objective: We previously reported antitumor effects of 3'-O-acetylhamaudol isolated from Angelica japonica roots. However, 3'-O-acetylhamaudol could not be detected in blood after its oral administration. In this study, we examined the antitumor effects and mechanisms of action of metabolites of 3-O-acetylhamaudol. Methods: Metabolites of 3'-O-acetylhamaudol were isolated from blood plasma by HPLC. The effects of hamaudol on tumor growth and tumorinduced angiogenesis were examined in colon 26-bearing mice. Furthermore, the effects of the metabolites on VEGFinduced VEGFR-2 phosphorylation were investigated in HUVECs. Key Findings: Metabolites of 3'-O-acetylhamaudol were identified as hamaudol and 8-hydroxymethylhamaudol. Hamaudol (25 and 50 mg/kg) inhibited tumor growth, final tumor weight and metastasis to the abdomen in colon 26-bearing mice. Hamaudol (10 mg/kg) also inhibited tumor-induced angiogenesis in tumor-packed chamber-bearing mice. Blood plasma after the oral administration of 3-O-acetylhamaudol (containing hamaudol and 8-hydorxymethylhamaudol), hamaudol and 8-hydroxymethylhamaudol inhibited VEGF-induced angiogenesis and VEGF-induced VEGFR-2 phosphorylation in HUVECs, and enhanced the production of IL-12 and IFN-γ induced by Con A in splenocytes. Conclusions: The antitumor effects of 3'-O-acetylhamaudol were due to the anti-angiogenic actions of hamaudol and/or 8-hydroxymethylhamaudol.

Development of Anti-tumor Blood Vessel Antibodies by Phage Display Method

Tumor blood vessels are essential for tumor growth. Therefore, these blood vessels are potential targets for anti-cancer therapy. The purpose of this study is to develop anti-tumor endothelial cell (TEC) antibodies for delivering anti-cancer agents or drugs. To achieve this goal, we utilized the phage antibody display library method to create monoclonal antibodies in vitro. Accordingly, we developed anti-TEC antibodies from an single chain Fv fragment (scFv) phage display library prepared using the Fv genes amplified from the mRNAs isolated from the TEC-immunized mice. The size of the phage antibody library prepared from the mRNA of the TEC-immunized mice was approximately 1.3x10(7) CFU. To select and enrich for the phages displaying the anti-TEC antibodies, cell panning was performed first using the TEC followed by subtractive panning using the normal endothelial cell. After five cycles of panning, the affinity of bound phage clones increased approximately 10 000 folds. Subsequently, clones isolated from the post-panning output library were tested for their antigen-specificity by ELISA and western blotting. One of the scFv phage clones showing antigen-specificity recognized only TEC in vitro, and when injected into the Colon26 bearing mice, this clone accumulated more on the tumor tissue than the wild type phage. These results suggest that the isolated an antibody and this clone's target molecule could be potentially useful for novel anti-tumor therapies.

Antitumor and antimetastatic actions of anthrone‐C‐glucoside, cassialoin isolated from Cassia garrettiana heartwood in colon 26‐bearing mice

We examined the antitumor and antimetastatic actions of 10-hydroxy-anthrone-C-glucoside cassialoin isolated from Cassia garrettiana heartwood in colon 26-bearing mice. Cassialoin (5 and 10 mg/kg) inhibited tumor growth and metastasis to the abdomen and the expression of CD31 (angiogenesis marker) in the tumors, and it increased the numbers of the gamma-interferon (IFN-gamma)-positive, CD8(+) T and natural killer cells in the small intestine or spleen of colon 26-bearing mice. Furthermore, cassialoin inhibited tumor-induced angiogenesis in colon 26-packed chamber-bearing mice. We examined the metabolic activities in the blood, stomach and small intestine after p.o. administration of cassialoin to mice. These results suggest that cassialoin might be converted to chrysophanol through chrysophanol-9-anthrone and metabolized to aloe-emodin from chrysophanol. Chrysophanol-9-anthrone inhibited vascular endothelial growth factor (VEGF) and matrix metallopeptidase-9 expression in colon 26 cells at 5 and 10 microM, and it inhibited VEGF-induced angiogenesis and migration in human umbilical vein endothelial cells (HUVEC) at 0.5-10 microM. Furthermore, chrysophanol-9-anthrone inhibited VEGF receptor (VEGFR)-2 expression and VEGF-induced VEGFR-2 phosphorylation. Aloe-emodin also inhibited the VEGF-induced angiogenesis by HUVEC at 1-100 microM. Cassialoin, chrysophanol-9-anthrone and aloe-emodin enhanced concanavalin A-induced IFN-gamma production in splenocytes of colon 26-bearing mice at a low concentration of 0.1 microM. From these results, it is suggested that the antitumor and antimetastatic actions of p.o. administered cassialoin may be partly due to cassialoin and its metabolites such as chrysophanol-9-anthrone and aloe-emodin through their anti-angiogenic activities and/or the modulation of the immune systems in the spleen and small intestine in tumor-bearing mice.

Antitumor activities of synthetic and natural stilbenes through antiangiogenic action

We reported that the antitumor and antimetastatic actions of resveratrol might be due to the inhibition of tumor-induced angiogenesis. To search for anticancer agents with stronger activity than resveratrol, we examined the antiangiogenic effects of 21 synthetic and/or natural stilbenes. Among these 21 stilbenes, 2,3-, 3,4-, and 4,4'-dihydroxystilbene inhibited the pro-matrix metalloproteinase (pro-MMP)-9 production in colon 26 cells at 5-25 microM, vascular endothelial growth factor (VEGF)-induced human umbilical vein endothelial cell (HUVEC) migration at 10 and 25 microM, and VEGF-induced angiogenesis at 5-50 microM. Resvertarol inhibited the pro-MMP-9 production and VEGF-induced angiogenesis at 25 or 50 microM. Thus, the inhibition of pro-MMP-9 production in colon 26 cells and VEGF-induced angiogenesis by three dihydroxystilbenes were greater than those of resveratrol. The three dihydroxystilbenes (8 mg/kg, intraperitoneal injection) inhibited the tumor-induced neovascularization in colon 26-packed chamber-bearing mice and the tumor growth in colon 26-bearing mice. Furthermore, the three dihydroxystilbenes inhibited VEGF-induced VEGFR-2 phosphorylation. On the other hand, the three dihydroxystilbenes had no effect on VEGFR-1 and-2 expression, and VEGF-induced VEGFR-1 phosphorylation in HUVECs. These findings suggest that the inhibition of tumor-induced neovascularization by these three dihydroxystilbenes may be due to the inhibition of VEGF-induced endothelial cell migration and VEGF-induced angiogenesis through the inhibition of VEGF-induced VEGFR-2 phosphorylation in endothelial cells and pro-MMP-9 expression in colon 26 cells.

Antitumor actions of a chromone glucoside cnidimoside A isolated from Cnidium japonicum

In a series of studies on the search for new antitumor and antimetastatic substances from the natural medicinal plants of the Umbelliferae family, we previously reported that chalcone derivatives isolated from Angelica keisekei roots have antitumor and antimetastatic activities. In the present study, we examined the effects of a chromone glucoside cnidimoside A isolated from Cnidium japonicum whole plants on tumor growth and tumor metastasis in colon 26-bearing mice. Cnidimoside A (50 mg/kg, twice daily) significantly inhibited tumor growth and final tumor weight compared to the growth in vehicle-treated colon 26-bearing mice (control). Furthermore, the number of mice with abdominal invasion of tumors was also reduced by orally administered cnidimoside A (50 mg/kg, twice daily). In this study, the CD8(+) T Cell- and interferon (IFN)-gamma-positive cell numbers in the small intestine in the colon 26-bearing mice were significantly reduced compared with those in the normal mice, but the natural killer (NK)-positive cell number did not differ significantly between the normal and colon 26-bearing mice. The CD8(+) T-, NK and IFN-gamma-positive cell numbers in the small intestine were significantly increased by orally administered cnidimoside A (50 mg/kg, twice daily) compared to those in vehicle-treated colon 26-bearing mice. In conclusion, it seems likely that the antitumor and antimetastatic actions of cnidimoside A may be partly associated with the stimulation of immune response in the small intestine.

Anti-tumor actions of major component 3′- O-acetylhamaudol of Angelica japonica roots through dual actions, anti-angiogenesis and intestinal intraepithelial lymphocyte activation

We recently demonstrated that two chalcones isolated from Angelica keiskei roots have anti-tumor and anti-metastatic activities through the inhibition of tumor-induced angiogenesis, but the anti-tumor substances of Angelica japonica roots are unknown. We attempted to clarify the anti-tumor action and its mechanisms of a major component 3′- O-acetylhamaudol isolated from A. japonica roots. We first examined the effects of 3′- O-acetylhamaudol on tumor growth in colon 26-bearing mice. Furthermore, we examined the effects of 3′- O-acetylhamaudol on angiogenic factors (vascular endothelial growth factor receptor-2 (VEGFR-2) phosphorylation in human umbilical vein endothelial cells (HUVECs), and vascular endothelial growth factor (VEGF) production and hypoxia-inducible factor (HIF)-1α expression in tumors). 3′- O-Acetylhamaudol (25 and 50 mg/kg, twice daily) inhibited the tumor growth in colon 26-bearing mice. Although 3′- O-acetylhamudol had no effect on the VEGF production and HIF-1α in colon 26 cells, it (10 μM) inhibited the VEGF-induced angiogenesis and VEGF-induced VEGFR-2 phosphorylation in HUVECs. 3′- O-Acetylhamaudol has anti-tumor effects mediated through dual mechanisms, i.e., anti-angiogenic actions and the modulation of the immune system in the spleen and small intestine in tumor-bearing mice.

Antitumor effect of whole body hyperthermia with α-galactosylceramide in a subcutaneous tumor model of colon cancer

Aim: Whole body hyperthermia (WBH) has been used clinically as an adjunct to radio- and chemotherapy in patients with various cancers. Recently, it has been reported that an activation of the immune system has recently been reported as a possible contributor to the therapeutic effects of WBH. Conversely, the glycolipid α-galactosylceramide (α-GalCer) is recognized by natural killer (NK) T cells together with the monomorphic MHC-like antigen, CD1d, in mice and humans. This study investigated the antitumor effects of WBH combined with α-GalCer in a mouse subcutaneous tumor model of colon cancer.Methods: Colon26 cells were inoculated subcutaneously into male BALB/c mice to establish subcutaneous tumor. Colon26-bearing mice were treated with WBH using far infrared rays three times/week. Rectal temperature was maintained for 60 min at 41°C. In some experimental groups, α-GalCer was intraperitoneally injected before WBH. We investigated the therapeutic effects of WBH, α-GalCer and combined therapy.Results: (1) Compared with controls, WBH alone resulted in significant inhibition of tumor growth. (2) No inhibitory effect on tumor growth was seen with α-GalCer. (3) The combination of WBH and α-GalCer showed significant inhibition of tumor growth and prolongation of survival. (4) Serum IFN-γ increased after 3 h and returned to basal levels by 24 h after α-GalCer administration. (5) CTL activity was enhanced following combination therapy with WBH and α-GalCer.Conclusion: WBH showed antitumor effects in a mouse subcutaneous tumor model of colon cancer. Addition of α-GalCer increased the efficacy of WBH, probably via enhancement of immune response.

Indomethacin acts as an antitumor and anticachexic agent in colon 26-bearing CDF1 mice

Background This study uses Colon 26-bearing CDF1 mice to assess the pharmacologic actions of indomethacin on tumor growth and cancer cachexia. The effect on tumor growth was evaluated by tumor weight, and the effect on cachexia was screened by changes in body weight and food intake. Additional indices measured included serum levels of immunosuppressive acidic protein and interleukin-6, and prostaglandin E2 content in the tumor tissues.

Effects of natural human interleukin-6 on thrombopoiesis and tumor progression in tumor-bearing mice

The growth of inoculated colon 26 adenocarcinoma (colon 26) in mice gradually increased the platelet count owing to murine IL-6 secreted from the tumor, while Lewis lung carcinoma (LLC) decreased the platelet count in the hosts, depending on the tumor growth. Natural human IL-6 injections (hIL-6), 280μg/kg/day, stimulated the platelet production in both types of carcinoma-bearing mice. When the administration of mitomycin C or cisplatin decreased the platelet number as a side reaction with a concomitant of suppressing the growth of colon 26 and LLC, respectively, hIL-6 could also increase the platelet count without the augmentation of tumor growth. However, loss of carcass weight was observed in colon 26-bearing mice treated with hIL-6, suggesting the development of cachexia is associated with hIL-6 administration. Despite the possibility of inducing cachexia in some types of tumors, our results suggest that IL-6 could be a useful means of restoring the decreased platelet number in cancer patients after intensive chemotherapy.

Enhanced delivery and antitumor activity of doxorubicin using long-circulating thermosensitive liposomes containing amphipathic polyethylene glycol in combination with local hyperthermia.

Enhanced delivery of doxorubicin (DXR) to a solid tumor subjected to local hyperthermia was achieved by using long-circulating, thermosensitive liposomes (TSL) composed of dipalmitoyl phosphatidylcholine (DPPC)/distearoyl phosphatidylcholine (DSPC) (9:1, m/m) and 3 mol% amphipathic polyethylene glycol (PEG) in colon 26-bearing mice. Inclusion of 3 mol% of distearoyl phosphatidylethanolamine derivatives of PEG (DSPE-PEG, amphipathic PEG) with a mean molecular weight of 1000 or 5000 in DPPC/DSPC liposomes resulted in decreased reticuloendothelial system (RES) uptake and a concomitant prolongation of circulation time, affording sustained increased blood levels of the liposomes. Concomitantly, DXR levels in blood were also kept high over a long period. The presence of amphipathic PEG did not interfere with the encapsulation of DXR by the pH gradient method (> 90% trapping efficiency) or with the temperature-dependent drug release from the liposomes. The optimal size of these liposomes was 180-200 nm in mean diameter for thermosensitive drug release and prolonged circulation time. The DXR levels in the tumor after injection of long-circulating TSL (DXR-PEG1000TSL or DXR-PEG5000TSL, at a dose of 5 mg DXR/kg) with local hyperthermia were much higher than after treatment with DXR-TSL lacking PEG or with free DXR, reaching 7.0-8.5 DXR micrograms/g tumor (approximately 2 times or 6 times higher than that of DXR-TSL or free DXR, respectively). Furthermore, the combination of DXR-PEGTSL and hyperthermia effectively retarded tumor growth and increased survival time.(ABSTRACT TRUNCATED AT 250 WORDS)

5?-Deoxy-5-fluorouridine improves cachexia by a mechanism independent of its antiproliferative action in colon 26 adenocarcinoma-bearing mice

The cytostatic agent 5'-deoxy-5-fluorouridine (5'-dFUrd) improves cachexia and prolongs survival, suppressing tumor growth in mice bearing large burdens of colon 26 adenocarcinoma. To investigate the mode of this anticachectic action, we isolated colon 26 variants that were resistant to the anticachectic activity in vivo in tumor-bearing mice that initially responded to 5'-dFUrd in terms of tumor growth and cachexia but again became cachectic and refractory to the drug after prolonged treatment. The original line and variants were equally susceptible to the antiproliferative action of 5'-dFUrd, and their growth was stopped. However, 5'-dFUrd given to cachectic mice exhibiting large burdens of these variants could not reverse wasting and only slightly prolonged the survival period. These results indicate that the anticachectic activity of 5'-dFUrd is independent of its antiproliferative action and that the survival of colon 26-bearing mice is shorter when the size of the tumors is not reduced to levels below those that cause cachexia.

Activity of 3 beta-hydroxy-13 alpha-amino-13,17-seco-5 alpha-androstane-17-oic-13,17-lactam(p-(bis(2-chloroethyl)amino)-phenyl) acetate(NSC 290205) in murine solid tumors.

The modified steroidal alkylating agent, 3 beta-hydroxy-13 alpha-amino-13,17-seco-5 alpha-androstane-17-oic-13,17-lactam[p-[bis(2-chloroethyl)amino]-phenyl]acetate showed excellent activity in treatment of there murine solid tumors. Colon 26-bearing CD2F1 mice lived twice as long as controls with tumor free survivors at the end of the 70-day observation period. CD8F1 mammary tumor growth was suppressed greater than 90% compared to controls. B16 melanoma-bearing B6D2F1 mice lived 50% longer than controls. This agent had previously been shown to be active in treatment of the Theagenion-Bahner angiosarcoma, the T8 Guerin tumor, L1210 leukemia and P388 leukemia.