SEARCH

SEARCH BY CITATION

Keywords:

  • ACTIBIND;
  • actin;
  • antiangiogenic;
  • anticarcinogenic;
  • ribonuclease

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND

ACTIBIND is an Aspergillus niger extracellular ribonuclease (T2-ribonuclease [RNase]) that possesses actin-binding activity. In plants, ACTIBIND inhibits the elongation and alters the orientation of pollen tubes by interfering with the intracellular actin network. The question rose whether ACTIBIND can also affect mammalian cancer development.

METHODS

Cell colony formation was performed in human colon (HT-29, Caco-2, RSB), breast (ZR-75-1), and ovarian (2780) cancer cells in the presence or absence of 1 μM ACTIBIND. In HT-29 and ZR-75-1 cells, the effect of ACTIBIND on cell migration was studied by microscopic observations and by invasion assay through Matrigel. Tube formation was assessed in human umbilical vein endothelial cells (HUVEC) in the presence of angiogenin or basic fibroblast growth factor (bFGF) (1 μg/mL each) following overnight incubation with 1 or 10 μM ACTIBIND. In an athymic mouse xenograft model, HT-29 cells were injected subcutaneously, followed by subcutaneous (0.4-8 mg/mouse/injection) or intraperitoneal (0.001-1 mg/mouse/injection) injections of ACTIBIND. In a rat dimethylhydrazine (DMH)-colorectal carcinogenesis model, ACTIBIND was released directly into the colon via osmotic micropumps (250 μg/rat/day) or given orally via microcapsules (1.6 mg/rat/day). Aberrant crypt foci, tumors in the distal colon, and tumor blood vessels were examined.

RESULTS

ACTIBIND had an anticlonogenic effect unrelated to its ribonuclease activity. It also inhibited angiogenin-induced HUVEC tube formation in a dose-responsive manner. ACTIBIND was found to bind actin in vitro. It also bound to cancer cell surfaces, leading to disruption of the internal actin network and inhibiting cell motility and invasiveness through Matrigel-coated filters. In mice, ACTIBIND inhibited HT-29 xenograft tumor development, given either as a subcutaneous or intraperitoneal treatment. In rats, ACTIBIND exerted preventive and therapeutic effects on developing colonic tumors induced by DMH. It also reduced the degree of tumor observation.

CONCLUSIONS

This study indicated that ACTIBIND is an effective antiangiogenic and anticarcinogenic factor. Cancer 2006. © 2006 American Cancer Society.

In addition to their ability to degrade RNA, certain ribonucleases (RNases) display a variety of biological activities. A great deal of interest has been directed towards the members of the RNase A family. For example, human angiogenin and eosinophil cationic protein (ECP) exhibit angiogenic and cytostatic activities, respectively,1–3 and bovine seminal RNase has antispermatogenic, transplanted-bone-marrow-stimulating, immunosuppressive, and antitumor activities.4, 5 Onconase, an RNase isolated from Northern Leopard Frog (Rana pipiens) oocytes and early embryos, has been shown to possess antitumor properties and is currently being investigated in Phase III trials for use in cancer therapy.6, 7

Members of the RNase T2 family are widely distributed in living organisms, from viruses to mammals.8 Some T2-RNases in microorganisms and plants are capable of digesting extracellular polyribonucleotides, thereby accelerating phosphate uptake; others may protect against possible pathogens.8, 9 In self-incompatible plants, specific T2-RNases encoded by the S-locus are responsible for rejecting self-pollen, thus preventing self-fertilization.10, 11 In the human genome, deletion of a region of chromosome 6 (6q27) is known to be associated with several malignancies.12, 13 This region contains the putative tumor suppressor gene RNase6PL which shares homology with the RNase T2 family.14, 15

ACTIBIND is a T2-RNase produced by Aspergillus niger B1 (CMI CC 324626). Our previous studies have shown that ACTIBIND is an extracellular glycoprotein containing 32- and 36-kD isoforms, which share a common 29-kDa protein moiety.16–18 Unrelated to its RNase activity, ACTIBIND inhibits the growth and orientation of pollen tubes, thus decreasing fruit set in different plant species.16–18 ACTIBIND binds F-actin in vitro at a molar ratio of 1:2. Consequently, ACTIBIND induces cross-linkage between actin filaments in pollen tubes and arrests their elongation, interfering with normal cytoplasmic streaming at the leading edge (Roiz and Shoseyov, in preparation).

The elongation of plant pollen tubes as well as the motility of mammalian cells require the formation of actin-rich cell protrusions such as phyllopodia and lamellipodia.19, 20 Furthermore, the presence of actin-rich pseudopods has been described as a prerequisite for cancer-cell function.21 The ability of ACTIBIND to interfere with the intracellular actin network in plant cells raised the question of whether it can also affect mammalian cancer development. The aim of the present research was to study the anticarcinogenic and antiangiogenic effects of ACTIBIND.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Chemicals, Drugs, and Supplies

Eosin was from Acros Organics (Geel, Belgium); E. coli RNase I was from Ambion (Austin, TX); polyclonal rabbit-anti-ACTIBIND was prepared at Anilab (Rehovot, Israel); Matrigel was from Becton-Dickinson (Bedford, MA); Dulbecco minimum Eagle medium (DMEM), fetal calf serum (FCS), glutamine and antibiotic–antimycotic solution were from Biological Industries (Bet Haemek, Israel); ECGF was from Biomedical Technologies Inc. (Stoughton, MA); Diff-Quik stain was from Hamilton Thorne Research (Beverly, MA); bFGF was from Promega (Madison, MI); recombinant human angiogenin was from R&D Systems, Inc (Minneapolis, MN); Tetramethyl-rhodamine B isothiocyanate (TRITC)-labeled phalloidin, rabbit antiactin, fluorescein isothiocyanate (FITC)-conjugated goat antirabbit, Mayer hematoxylin and G-actin were from Sigma-Aldrich Co. (St Louis, MO); Dimethylhydrazine (DMH) was from Fluka (St. Gallen, Switzerland); monoclonal mouse antiactin IgM followed by FITC-conjugated goat antimouse IgM (Ab-1 kit) was from Oncogene (Cambridge, MA). The enhanced chemiluminescence (ECL) Western blot detection system was from Amersham Pharmacia Biotech, Ltd., (Buckinghamshire, UK); terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) Klenow-Frag-El kit was from Oncogene, (Cambridge, MA); CD31 antibody was from Pharmingen (San Diego, CA); PECAM-1 (H-300), sc-8306 antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Colony-formation Assay

The colony-formation assay was performed essentially as described7 with minor modifications. Human colon (HT-29, Caco-2, RSB), breast (ZR-75-1), and ovarian (2780) cancer cells were grown in 50-mL flasks (105 cells per flask). The medium contained 7 mL of DMEM supplemented with 10% fetal calf serum (FCS), 1% glutamine, and 1% antibiotic-antimycotic solution in the presence or absence of 1 μM ACTIBIND. The colon cancer cells were also exposed to the same concentration of enzymatically inactivated (EI)-ACTIBIND, which was preheated for 30 minutes in an autoclave (120°C, 120 kPa) until no RNase activity was detected. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. After 48 hours, 1000 cells/well were seeded in 96-well plates in 200 μL medium, in the presence or absence of 1 μM ACTIBIND. HT-29, Caco-2, and ZR-75-1 cell lines were grown for 7 days; 2780 and RSB cell lines were grown for 10 and 14 days, respectively. The cells were then fixed in 4% formaldehyde and stained with methylene blue. The number of colonies was counted (N = 3).

Human Umbilical Vein Endothelial Cell (HUVEC) Angiogenesis Assay

As previously described,22 freshly isolated HUVEC were maintained in M199 medium supplemented with 20% FCS, 1% glutamine, 1% antibiotic–antimycotic solution, 0.02% ECGF, and 50 units/100 mL heparin. They were then plated in a 96-well plate (14 × 103 cells/well) previously coated with growth factor-depleted Matrigel, in M199 medium containing 5% FCS and 0.005% ECGF supplemented with angiogenin or bFGF (1 μg/mL each), ACTIBIND (1 or 10 μM), or phosphate-buffered saline (PBS) were added, and the final volume in each well was 120 μL. After overnight incubation at 37°C, the plates were photographed, and the extent of tube formation was assessed (N = 3).

Membrane Blotting

Actin was bound to ACTIBIND or to other proteins as described.23 ACTIBIND, angiogenin and E. coli RNase I (1 μg of each protein) were loaded on 10% SDS-PAGE and transferred onto a Hybond ECL nitrocellulose membrane. G-actin (1 μg) was used as a positive control. The membrane was blocked at 4°C by overnight incubation in rinse buffer (10 mM Tris-HCl pH 7.5; 150 mM NaCl; 1 mM EDTA; 0.02% Triton X-100) containing 5% bovine serum albumin (BSA), under constant shaking, and then washed twice for 10 minutes with rinse buffer. The membrane was incubated in buffer G (2 mM Tris pH 8; 0.2 mM CaCl2; 0.2 mM ATP), followed by overnight incubation at 4°C with 5 μg/ml G-actin in buffer G under constant stirring. Following 3 washes in rinse buffer containing 0.2 mM CaCl2, the membrane was immunolabeld with antiactin IgM following by FITC-conjugated goat antimouse IgM (Ab-1 kit). Signals were detected with the ECL Western Blot Detection System.

Microscopic Observations

For actin observation, HT-29 cells were cultured for 3 days in DMEM in the presence or absence of 1 μM ACTIBIND or EI-ACTIBIND and fixed for 10 minutes in 4% formaldehyde in PBS on ice. Cell membrane was permeabilized with PBS containing 0.05% Tween 20 (PBST) (1 hour, room temperature), and then stained for 1 hour with TRITC-labeled phalloidin. For ACTIBIND immunostaining assay, we used rabbit anti-ACTIBIND followed by FITC-conjugated goat antirabbit. Cells were observed with a confocal laser scanning microscope, model 510 (Zeiss, Oberkochen, Baden-Wuttemberg, Germany).

For cytologic observations, HT-29, ZR-75-1, and 2780 cells were cultured as described above. Cells were fixed, stained with Mayer hematoxylin and eosin (H & E) and observed by light microscope (BX-40, Olympus, Hamburg, Germany).

Invasion Assay Through Matrigel

HT-29 and ZR-75-1 cells were cultured with 1 or 10 μM ACTIBIND or in PBS for 4 days as described above. Wells and Matrigel-coated inserts of a commercially available 24-well invasion chamber were rehydrated in 500 μL of serum-free medium overnight and processed according to the manufacturer's instructions; 500 μl of HT-29 and ZR-75-1 control or ACTIBIND-treated cell suspensions containing 2.5 × 104 cells each were added to the top of the chambers, and 750 μl of DMEM media containing 10% FCS was added to the lower chamber. The invasion chambers were incubated for 22 hours in a 37°C cell-culture incubator. The noninvading cells on the upper surface of the membrane of the insert were removed by scrubbing. The cells on the lower surface of the membrane were stained with Diff-Quik stain. The membranes were fixed, and the cells were counted under a light microscope (N = 3).

Animal Studies

Xenografts

The in vivo preventive and therapeutic effects of ACTIBIND on HT-29–derived xenografts were studied in athymic mice (CD-1 nu/nu; Charles River, Wilmington, MA) by using 2 modes of administration. In the subcutaneous/subcutaneous (sc/sc, N = 10) model, viable HT-29 cells (2 × 105/100 μl) were injected subcutaneously into the left hip, and ACTIBIND (0.4, 2, 4, 8 mg/100 μl) or PBS was injected subcutaneously into the area of the cell injection. ACTIBIND was administrated daily for 42 days, starting from the day of cell injection. In the subcutaneous/intraperitoneal (sc/ip, N = 6) model, HT-29 cells (106 cell/100 μL) were injected as described above. ACTIBIND (0.001, 0.01, 0.1, 0.5, 1.0 mg/mouse) or PBS was injected every other day for 30 days starting from 24 hours after cell injection (preventive treatment) or from when tumors were palpable (therapeutic treatment). At the end of this experiment, the tumors were excised for size measurements and for histopathological examinations. Each treatment was repeated twice.

Carcinogen-treated rats

The in vivo preventive and therapeutic effects of ACTIBIND on colorectal carcinogenesis induced with dimethylhydrazine (DMH)24–26 were studied in Charles River-derived outbred male rats. ACTIBIND or EI-ACTIBIND was given via subcutaneously implanted osmotic micropumps (ALZET, Cupertino, CA), allowing directed release of ACTIBIND (250 μg/rat/day) into the colon via a catheter.

ACTIBIND was also given orally, using enterocoated cellulose acetate-phthalate (CAP) microcapsules27 (1.6 mg/rat/day). During the experiment, feces were collected weekly to monitor ACTIBIND uptake by means of RNase activity assays.18 In the preventive regime, rats were sacrificed 8 or 11 weeks after direct or oral application of ACTIBIND. Aberrant crypt foci (ACF) in the distal 5 cm of the colons28 or tumors were examined. In the therapeutic regime, 11 weeks after the first DMH injection, ACTIBIND was administered (as described above) for an additional 6 weeks, and tumors were examined. Tumors were fixed, and 10-μm paraffin sections were stained with H & E for histology and with the TUNEL procedure for apoptosis according to the supplier's instructions. Blood vessels in median tumor cross-sections were localized using CD31 antibody followed by PECAM-1 (H-300), sc-8306 antibody. In each median tumor cross-section, the blood vessels were counted, their diameters were measured, and the total area was calculated. Relative area (percentage) was calculated as the ratio between total blood-vessel area and tumor-section area. The experiments were repeated twice, and each treatment was applied to 6 to 10 rats.

All animal experiments were approved by the Ethics Committee for Animal Experimentation, Faculty of Agricultural, Food and Environmental Quality Sciences, the Hebrew University of Jerusalem, Israel.

Statistical Analysis

Means were compared by analysis of variance (ANOVA) or Student t test. Differences were considered statistically significantly at P <.05. All statistical tests were two-sided.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Actibind Effects In Vitro

ACTIBIND solutions were tested for the presence of bacterial endotoxin by the limulus amebocyte lysate detection assay (Associates of Cape Cod Inc, East Falmouth, MA). The value measured in ACTIBIND solutions was below the detection limit of the assay (0.05 EU/mL) as obtained in standard curves using endotoxin as a control standard (from E. coli 0113:H10, Associates of Cape Cod Inc., East Falmouth, MA.).

We assessed the effect of ACTIBIND on the colony-forming ability of human cancer cell lines originating from colon, breast, and ovarian tissues. ACTIBIND significantly inhibited colony formation of all cancer cells tested (Fig. 1). The anticlonogenic effect was significant only in ACTIBIND-pretreated cells and was much less effective in cells not previously exposed to ACTIBIND (not shown). In HT-29, Caco-2, and RSB colon cancer cells (Fig. 1A), continuous exposure to ACTIBIND resulted in a substantial reduction in their clonogenic ability (56%, 71%, and 58%, respectively compared with controls P <.01). EI-ACTIBIND showed a similar effect in HT-29, Caco-2, and RSB cells (53%, 78%, and 80% of inhibition, respectively; P <.01). Similarly, ACTIBIND inhibited the clonogenic ability in breast ZR-75-1 and ovarian 2780 cancer cells (Fig 1B; 33% and 81%, respectively, compared with controls; P <.05). Colonies of HT-29 and ZR-75-1 cells were stained with H & E for microscopic observation. The peripheral cells of the control colonies displayed numerous cytoplasmic extensions (Fig. 2A,C), whereas in the ACTIBIND-treated colonies, the peripheral cell extensions were arrested (Fig. 2B,D). These results suggest that ACTIBIND's treatment effects on the morphology of the cells may impinge upon their motility.

thumbnail image

Figure 1. The effect of ACTIBIND on clonogenicity in A, colon cancer cell lines (HT-29, Caco-2 and RSB) and in B, breast (ZR-75-1) and ovarian (2780) cell lines (N = 3 for each cell line). The number of colonies in ACTIBIND-treated cells (open square, □) was significantly lower (P <.01) than in controls (solid square, ▪). In colon cancer cells, EI-ACTIBIND (hatched square, ░) also showed anticlonogenic effect (P <.01).

Download figure to PowerPoint

thumbnail image

Figure 2. The effect of ACTIBIND on the morphology and motility of HT-29 colon (A,B) and ZR-75-1 breast cancer cells (C,D). (A,C) In the control cells, fully developed cell extensions were observed (arrows). (B,D) In ACTIBIND-treated cells, the cell extensions were inhibited (arrowheads). Scale bar = 10 μm.

Download figure to PowerPoint

To assess the ability of ACTIBIND to inhibit angiogenesis, we examined its effect on tube formation of vascular structures in an in vitro HUVEC assay. ACTIBIND significantly inhibited angiogenin as well as bFGF-induced tube formation in a dose–dependent manner in comparison with controls (Fig. 3). ACTIBIND did not affect HUVEC proliferation or viability as assessed by MTT assay, either after exposure to angiogenin or bFGF (not shown).

thumbnail image

Figure 3. HUVEC tube formation on Matrigel induced by angiogenin (A–C) and bFGF (D–F). (A,D) control; (B,E) 1 μM ACTIBIND; (C,F) 10 μM ACTIBIND. ACTIBIND inhibits both angiogenin and bFGF-induced tube formation (N = 3 for each treatment).

Download figure to PowerPoint

The Western blot analysis using G-actin to bind to different proteins demonstrated that ACTIBIND, similar to angiogenin and to E. coli RNase I which is also a T2-RNase,29 binds actin (Fig. 4).

thumbnail image

Figure 4. Actin binding activity on SDS-PAGE. Proteins were blotted on a nitrocellulose membrane, and actin-binding capability was accomplished after overnight incubation with 5 μg/mL actin. Proteins bound to actin were in evidence subsequent to reaction with antiactin followed by HRP-conjugated secondary antibody. Lanes 1-6: SDS-PAGE protein staining. Lanes 7-11: Western blot analysis. Lane 1: Molecular weight markers. Lanes 2,7: actin (positive control); Lanes 3,8: ACTIBIND; Lanes 4,9: RNase I; Lanes 5,10: Angiogenin; Lanes 6,11: BSA (negative control).

Download figure to PowerPoint

Actibind Binds to the Cancer Cell Surface and Affects the Intracellular Actin Network

HT-29 cells grown in the presence or absence of ACTIBIND exhibited different patterns of specific anti-ACTIBIND immunostaining. Control cells exhibited very faint fluorescence (Fig. 5A), whereas ACTIBIND-treated cells showed intense fluorescence, particularly in the cell edges and extensions (Fig. 5B). Treatment with rabbit preimmune serum resulted in a very faint signal, demonstrating the specificity of anti-ACTIBIND (not shown). The effect of ACTIBIND on the organization of intracellular actin network was tested using TRITC-phalloidin staining. Control cells showed a fine actin network filling the cell cytoplasm (Fig. 5C). In contrast, ACTIBIND-treated cells demonstrated intense staining in the peripheral zone of the cytoplasm, whereas the inner area remained unstained (Fig. 5D). Similar results were obtained in cells treated with EI-ACTIBIND (not shown).

thumbnail image

Figure 5. ACTIBIND localization and actin staining in HT-29 colon cancer cells. Cells were grown for 72 hours in the absence or presence of ACTIBIND. (A,B) Immunostaining with rabbit anti-ACTIBIND followed by FITC-conjugated goat anti-rabbit. (A) In control cells only faint fluorescence appeared. (B) In ACTIBIND-treated cells, intense anti-ACTIBIND staining was observed on cell surface, specially on cell edges and extensions. (C,D) Staining for internal actin with TRITC-phalloidine following detergent permeabilization. (C) In control cells, fine actin network was observed. (D) In ACTIBIND-treated cells, the internal actin network was disrupted. Scale bar = 10 μm.

Download figure to PowerPoint

Inhibition of Tumor Cell Invasion by ACTIBIND

Because ACTIBIND was shown to affect cancer cell morphology and actin organization, we examined whether cell motility was also affected. Both HT-29 and ZR-75-1 cells were able to penetrate the Matrigel-coated filters (Table 1). ACTIBIND significantly and dose-dependently inhibited invasiveness of both cell lines. ACTIBIND (1 μM) reduced the invasiveness of HT-29 and ZR-75-1 cells by 40% (P<.05 and P<.01, respectively). At a concentration of 10 μM, the inhibitory effect of ACTIBIND was increased to 80% (P<.01 and P<.001, respectively). These findings further emphasize that the ability of ACTIBIND to inhibit cytoplasmic extensions (Fig. 2) impinges on the cancer cell's migration and invasion capabilities.

Table 1. Effect of ACTIBIND on HT-29-Colon and ZR-75-1-Breast Cancer Cell Invasiveness Through Matrigel-coated Filters (N = 3)
Cell LinesCell Counts, Mean ± SE
ControlACTIBIND
1 μM10 μM
  • *

    P <.05

  • P <.01

  • P <.001

HT-29963.7 ± 95.7*623.3 ± 104.2223.7 ± 13.1
ZR-75-11297.7 ± 62.5784.0 ± 51.5271.7 ± 16.6

Actibind Inhibits Colon Cancer Growth In Vivo

Mouse xenograft cancer model

By using the xenograft athymic mouse model, we found that ACTIBIND caused a significant reduction in the growth rate of HT-29-derived carcinoma in a saturation mode (Fig. 6). In the sc/sc model, inhibition of 50% relative to control was gained at a minimal dose of 2 mg/injection ACTIBIND (Fig. 6A). In the sc/ip model, the minimal ACTIBIND doses needed for 50% inhibition of tumor growth were 0.001 and 0.01 mg/injection in the preventive and therapeutic treatments, respectively (Fig. 6B,C).

thumbnail image

Figure 6. The effect of ACTIBIND on HT-29-derived cells implanted in athymic mouse xenograft models. (A) sc/sc model (N = 10). ACTIBIND (0.4, 2, 4 and 8 mg/injection) or PBS was administrated daily for 42 days, starting from the day of cell injection. Inhibition of 50% relative to control was gained at a minimal dose of 2 mg/injection. (B) sc/ip model—a preventive treatment (N = 6). ACTIBIND (0.001, 0.01, 0.1, 0.5 and 1 mg/injection) or PBS was injected every other day for 30 days starting from 24 hours after cell injection. (C) sc/ip model—a therapeutic treatment (N = 6). ACTIBIND (0.01 and 0.5 mg/injection) or PBS was started on 70-mm3 preexisting HT-29 human tumors and administrated every other day for 30 days. In the preventive and the therapeutic treatments, the minimal ACTIBIND doses needed for 50% inhibition of tumor growth were 0.001 and 0.01 mg/injection respectively.

Download figure to PowerPoint

Histologic analysis showed that in ACTIBIND-treated tumors, the cancer cells were isolated in compact capsules and were considerably smaller than those of the control (Fig. 7A,B). To monitor the presence of ACTIBIND and its targeting to blood vessels, we labeled tumor sections using rabbit anti-ACTIBIND and FITC-conjugated goat antirabbit IgG. Substantial accumulation of ACTIBIND was observed in the basal membrane of tumor blood vessels in the preventive (Fig. 7D,E) as well as in the therapeutic model (Fig. 7G,H).

thumbnail image

Figure 7. Paraffin sections of HT-29 derived xenografts in nude mice. (A) Contol xenografts. (B) ACTIBIND-treated xenografts. (C-E) Blood vessels from tumors treated with sc injections. (F-H) Blood vessels from tumors treated with ip injections. (C,F) In controls, H & E staining showed the basal membrane (arrow). (D, G) Immunostaining resulted in only faded image. (E,H) In ACTIBIND-treated tumors, ACTIBIND accumulated in the arterial basal membrane. H & E staining: A-C, F, H; FITC-labeled immunostaining with anti-ACTIBIND: D, E, G, H. Scale bars = 5 μm (E,H); 20 μm (B).

Download figure to PowerPoint

Rat cancer model

To assess additional anticancer properties of ACTIBIND, we induced in vivo colon cancer in male rats by administering the carcinogen DMH. ACTIBIND was administered orally in CAP-coated microcapsules or by direct intestinal application via microosmotic pumps. The latter mode of application was used to supply ACTIBIND as well as EI-ACTIBIND. We applied 2 different protocols: 1) a preventive treatment in which ACTIBIND or EI-ACTIBIND was administered concomitantly with the carcinogenic treatment, and 2) a therapeutic treatment in which we administered ACTIBIND or EI-ACTIBIND subsequent to adenocarcinoma formation. RNase activity and Western blot analysis were employed to monitor the concentration of ACTIBIND in feces samples. Fivefold higher RNase activity was measured in feces of ACTIBIND-treated rats than in the controls, and intact ACTIBIND was observed in Western blot analyses of all modes of application (not shown).

ACTIBIND significantly inhibited tumor development in both preventive and therapeutic treatment regimes (Table 2). The preventive treatment in which ACTIBIND was continuously administered for 8 weeks by osmotic pump, induced a 50% reduction in the number of ACF (P <.01), which are known as early markers of colon carcinogenesis. ACTIBIND administered orally in microcapsules led to a reduction of about 50% in the number (P <.05) and size (P <.05) of tumors as compared with the control following 11 weeks of treatment. In the same manner, ACTIBIND significantly reduced the number of adenocarcinomas compared with the control (P <.01). In the therapeutic treatment, we applied ACTIBIND directly via osmotic pumps, from Weeks 12 to 17, after the first DMH injection. ACTIBIND treatments resulted in a 50% reduction in the number of tumors per colon (P <.01). In all the above treatments, EI-ACTIBIND, like ACTIBIND, showed a similar inhibitory effect on tumor development.

Table 2. Effect of ACTIBIND on DMH-induced Colon Cancer in Rats: Pooled Data From 2 Subsequent Experiments (N = 16)
TreatmentParameterControlACTIBIND
  • DMH: dimethylhydrazine; ACF: aberrant crypt foci.

  • *

    P <.01

  • P <.05

Preventive*ACF from osmotic pumps117.8 ± 13.255.5 ± 3.5
 Tumor no. from microcapsules8.3 ± 2.04.5 ± 0.6
 *Tumor malignancy from microcapsules  
 Adenoma3.6 ± 1.13.1 ± 0.8
 Adenocarcinoma5.9 ± 0.82.4 ± 0.6
TherapeuticTumor no. from osmotic pumps11.3 ± 6.05.5 ± 1.5

The effect of ACTIBIND on angiogenesis was measured when the therapeutic treatment with osmotic pumps was completed (Table 3). ACTIBIND significantly reduced the number of blood vessels per tumor by 40% relative to the control (P <.01). In addition, a 70% reduction in total blood-vessel area per tumor section (P<.05) was observed. Tumor-associated neovascularization, as indicated by microvessel density (MVD), was determined by means of identifying blood vessels within the tumor (Fig. 8A,B). Concomitant with the decrease in MVD, a significant 16.5-fold increase (P<.0001) in the number of apoptotic cells was observed in ACTIBIND-treated relative to control tumors (Table 3, Fig. 8C,D).

Table 3. Effect of ACTIBIND on Angiogenesis (Control N = 12, ACTIBIND N = 10) and Apoptosis (N = 3, 10 Microscopic Fields of Each) in DMH-induced Tumors in Rats
 ParameterControlACTIBIND
  • DMH: dimethylhydrazine.

  • *

    P <.01

  • P <.05

AngiogenesisBlood vessels per tumor*56.8 ± 5.031.9 ± 3.6
 Relative area, %1.0 ± 0.30.34 ± 0.1
ApoptosisApoptotic cells per microscopic field2.0 ± 0.237.0 ± 5.0
thumbnail image

Figure 8. The effect of ACTIBIND on blood vessels and on apoptosis rate in DMH-induced colonic tumors. (A,C) Controls; (B,D) ACTIBIND-treated tumors. (A,B) Immunostaining for CD31. Red areas represent the numerous blood vessels in control tumor. (C,D) TUNEL assay. Brown nuclei represent the numerous apoptotic cells in ACTIBIND-treated tumors. Scale bar = 100 μm.

Download figure to PowerPoint

In none of the described treatments did ACTIBIND affect body weight, behavior, or liver histology. All of these parameters were comparable to those of healthy animals.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

In this study, we show that ACTIBIND directly affects cancer cells by altering their clonogenic ability as well as their morphology and intracellular actin organization. We also demonstrate that ACTIBIND is an effective antiangiogenic agent in vivo and in vitro, because it significantly affects neovascularization of tumors and HUVEC tube formation. In the above experiments, ACTIBIND and EI-ACTIBIND show similar effects, indicating that the antitumorigenic and antiangiogenic activities may not related to RNase activity.

Previous studies have shown that Northern Leopard Frog-derived RNase (onconase) inhibits clonogenicity and protein synthesis of 9L glioma cancer cells without affecting cell density. These studies claim that onconase activity depends on its ability to bind to a cell-surface receptor.30, 31 Our findings demonstrate that the novel RNase described herein, ACTIBIND, binds actin in vitro (Fig. 4), as well as on the cancer cell's surface (Fig.5A,B). Surface actin has been shown to act as a receptor for angiogenin in endothelial cells.23, 32–34 The actin-angiogenin complex activates plasminogen and plasmin, bearing essential proteolytic activities involved in angiogenesis.35–37 Our in vivo experiments demonstrate that ACTIBIND significantly inhibits angiogenesis and apoptosis (Fig. 8, Table 3) processes in DMH-induced colonic tumors in rats. Cumulatively, the evidence leads us to propose that cell-surface actin may be a target for ACTIBIND in cancer cells as well as in endothelial cells. Therefore, ACTIBIND may compete with angiogenin for cell-surface actin and, in this manner, blocks the formation of the actin-angiogenin complex required for cancer cell organization and angiogenesis in developing neoplastic tissue.

Angiogenin was first isolated from HT-29 cells conditioned medium.38 It was shown to be a key factor for tumor growth because antagonists of its activity inhibited the establishment and metastatic spread of human tumor cells39 Moreover, increased serum angiogenin concentration in colorectal cancer is correlated with cancer progression.40 Unlike angiogenin, bFGF was not reported as an actin-binding protein. However, the ability of ACTIBIND to inhibit bFGF-induced tube formation (Fig. 3) suggests that ACTIBIND may act via different metabolic pathways. A number of unanswered questions remain regarding aspects of cell binding.

ACTIBIND reduced the development of peripheral cytoplasmic extensions in different cancer cell lines (Fig. 2). These results correlated with ACTIBIND's ability to disrupt the intracellular cytoskeletal network and to generate F-actin accumulation towards the cell-membrane boundaries. In addition, ACTIBIND significantly and dose-dependently affected the invasiveness of HT-29 and ZR-75-1 cells through Matrigel (Table 1). The effect of ACTIBIND on the morphology of cancer cells mimicked our previous observations in lily pollen tubes where ACTIBIND bound F-actin induced massive F-actin accumulation at the growing tip, which led to growth arrest (Roiz and Shoseyov, in preparation). In migrating cells, the extension of leading edges depends upon actin assembly in response to signal-transduction pathways.20, 41 Furthermore, in cancer cells, the structure of the actin network is directly related to malignant potential, because it determines cell motility.21

We, therefore, suggest that ACTIBIND interferes with cancer cells' ability to communicate, organize into colonies, and motilize through extracellular matrix substrates by binding to cell-surface actin and disrupting the intracellular actin network.

The anticarcinogenic and antiangiogenic effects of ACTIBIND were demonstrated in vivo in different animal models. In athymic mice, treatment with ACTIBIND inhibited HT-29-derived xenograft development in both sc/sc and sc/ip models (Fig. 6). We also demonstrated that ACTIBIND accumulates in the mouse artery basement membrane (Fig. 7). Endothelial cells, pericytes, and their respective supporting extracellular matrix, all contain considerable concentrations of α-smooth–muscle actin.34, 42, 44–47 For this reason, targeting extracellular actin seems to be an appropriate approach to arresting angiogenesis. We can claim that actin residues provide epitopes for ACTIBIND binding in a fashion similar to the specific actin binding observed in vitro (Fig. 4). These results are supported by previous studies in which antibodies against either angiogenin or actin delayed or prevented the appearance of HT-29-inoculated tumors in athymic mice.39, 43

In rats, ACTIBIND inhibited DMH-induced colon cancer. ACTIBIND applied directly to the colon via osmotic pump was effective from the early stages of ACF-induced carcinogenesis to later stages of malignant tumor development for all parameters examined. Furthermore, a reduction in the number of blood vessels per tumor as well as blood-vessel size was observed at the end of the therapeutic treatment with osmotic pumps (Table 3). Therapeutic treatment with ACTIBIND administered via osmotic pumps not only diminished MVD, but concomitantly induced cell apoptosis in the colonic tumors (Fig. 8). Apoptosis could be affected directly by interaction between ACTIBIND and tumor cells, or, alternatively, it could be the result of hypoxia due to significant reduction in the number of blood vessels.48, 49 In this regard, cortical actin localization has been observed in cells undergoing apoptosis. The specific mechanism linking these two phenomena is still unclear.50–52 These results implicate ACTIBIND as a potential antiangiogenic substance.

ACTIBIND given orally exerted a clear preventive, but not therapeutic effect (Table 2). We followed the route of fluorescent, protein-loaded, CAP microcapsules along the rat gut and found that only traces of the initial fluorescence reached the colon. Encapsulated ACTIBIND most likely meets the same fate. These traces may be sufficient to prevent cancer initiation but are not sufficient to treat fully developed tumors. We believe that improving the encapsulation coating would allow for improved delivery of ACTIBIND to the colon.

The inactivated ACTIBIND maintained its actin-binding, anticlonogenic, and anticancer activities in vitro as well as in vivo. Unlike ACTIBIND, RNase activity is essential for onconase anticancer activity.30 We, thus, assume that the anticancer effects of ACTIBIND and onconase are mediated by different mechanisms.

The resistance of many tumors to conventional therapy has led to attempts to develop novel and more effective strategies to improve anticancer agents. Mobile cells (e.g., angiogenic and cancer cells) may be more sensitive to ACTIBIND because they display significantly more cell-surface actin than stationary cells.2, 45 RNases are now the focus of a great deal of attention because of their newly discovered activities and applications, particularly in cancer biology.1 Therefore, a better understanding of the mechanism(s) through which ACTIBIND, the fungal T2-RNase, exerts its antiangiogenic and anticarcinogenic properties is required.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES