Non-anti-coagulant heparin inhibits metastasis but not primary tumor growth.

Experimental and clinical studies indicate that low molecular weight heparins (LMWH) may inhibit cancer and/or metastasis. The purpose of this study was to investigate whether it is possible to design non-anti-coagulant, anti-metastatic compounds based on heparin. The LMWH Tinzaparin and a series of non-anti-coagulant (NAC) heparin derivatives, varying in size from 2,500 to 10,000 Da, were tested for their anti-metastatic activity in an experimental B16F10 metastasis model. The most promising NAC heparin drug candidate and Tinzaparin were further evaluated in B16F10 model with spontaneous metastasis from a primary subcutaneous tumor. In the experimental model, Tinzaparin, NAC2500, and NAC6000 were inactive whereas both NAC8000 and NAC10000 significantly inhibited the number of induced experimental metastases by 69 and 73%, respectively. NAC8000 was chosen over NAC10000 for further studies because of its lower molecular weight with an expected better bioavailability. In the spontaneous model, Tinzaparin had no inhibitory effect on metastatic activity. In contrast, NAC8000 significantly inhibited the number of metastases by 58%. Neither Tinzaparin nor NAC8000 inhibited primary subcutaneous tumor growth. Together, these results indicate that the anti-metastatic effect of heparin derivatives is not a result of anti-coagulant activity. The non-anti-coagulant NAC8000 specifically inhibits early establishment of tumor cells, but not primary tumor growth. Therefore, NAC8000 is a promising non-anti-coagulant compound for preventing tumor metastasis.

Anticancer agent CHS 828 suppresses nuclear factor-kappa B activity in cancer cells through downregulation of IKK activity.

CHS 828, a pyridyl cyanoguanidine, has been shown to exert a significant antitumor effect in preclinical tests in vitro and in vivo, and CHS 828 is in phase I/II clinical trials. We have investigated the effect of CHS 828 on the nuclear factor-kappa B (NF-kappa B) because of its well-known role in the control of cell division and apoptosis. CHS 828 is able to inhibit the lipopolysaccharide (LPS)-induced nuclear localization as well as the transcriptional activity of NF-kappa B in human THP-1 leukemia cells. Moreover, CHS 828 has also been shown to inhibit the LPS-induced degradation of the I kappa B alpha and I kappa B beta in THP-1 cells, leading us to identify the I kappa B kinase complex as a molecular target of CHS 828. The IKK activity is inhibited by CHS 828 with an IC(50) of 8 nM. The inhibition of the IKK activity by different CHS 828 analogues correlates well with the inhibition of NYH small cell lung cancer cell proliferation in vitro and in vivo. Moreover, the inhibition of NF-kappa B transcriptional activity in different cancer cell lines by CHS 828 correlates to some extent with the reduction by CHS 828 of the size of the corresponding xenografts. Activation of NF-kappa B has been shown to induce expression of antiapoptotic proteins, and cancer cells have been shown to have high levels of constitutively active NF-kappa B. Therefore, we hypothesize that the anticancer activity of CHS 828 is due to inhibition of the IKK activity by which the antiapoptotic protection of NF-kappa B is removed, leading to the promotion of apoptosis.

A versatile in vivo chamber angiogenesis assay for measuring anti-angiogenic activity in mice.

We recently described an in vivo angiogenesis assay for rats--an optimized Matrigel plug assay based on subcutaneously implanted chambers with fixed volume and shape. Here we examine the possibility of switching the host animal from rat to mouse, thereby reducing the requirement for test compound an order of magnitude. The chambers consist of a plexiglas ring with a 0.2 ml volume and two nylon net filters. Chambers containing growth factor-reduced Matrigel supplemented with basic fibroblast growth factor (bFGF) were subcutaneously implanted in the right flank of three different strains of mice; BALB/c, C57BL/6J, and NMRI-nu. On day 10 post-implantation: i) each chamber was taken out, ii) a picture of the induced angiogenic response was taken, and iii) the redness of the chamber content was quantified by computer image analysis. The level of bFGF-induced angiogenesis in the mouse assay was lower than in the previously published rat assay. Importantly, the background angiogenesis in mice in chambers containing Matrigel alone was correspondingly decreased. Therefore, a more sensitive threshold for the computer image analysis was used. In all three strains of mice, bFGF-induced angiogenesis was significantly increased compared to Matrigel alone. Furthermore, the positive anti-angiogenic control compound TNP-470 (10 mg/kg/d s.c.) completely inhibited the bFGF-induced angiogenesis. The in vivo chamber angiogenesis assay allows quantitative analysis of angiogenic and anti-angiogenic activity in mice. The model is very robust and only little influenced by the choice of mouse strain.

In vivo chamber angiogenesis assay: an optimized Matrigel plug assay for fast assessment of anti-angiogenic activity.

Efficient in vitro and in vivo angiogenesis assays, to assess and compare anti-angiogenic activity are a prerequisite for the discovery and characterization of anti-angiogenic targets. Here we describe an optimized Matrigel plug assay based on subcutaneously implanted chambers and two fast and reproducible measuring techniques. Plexiglas ring/nylon net filter-chambers (0.2 ml) containing growth factor-reduced Matrigel and 300 ng basic fibroblast growth factor (bFGF) were subcutaneously implanted into the right flank of rats. Chamber angiogenesis was scored on day 5 and day 10 post-implantation by computer image analysis of the chamber, and by optical density reading at 415 nm of a PBS solution of the chamber content. bFGF significantly induced chamber angiogenesis and histological examination confirmed that numerous blood vessels were present in the bFGF-induced chambers. The anti-angiogenic control compound TNP-470 (10 mg/kg/d s.c.) completely inhibited the bFGF-induced angiogenesis. In contrast, the anti-inflammatory or immuno-suppressive compounds cyclosporin A (15 mg/kg/d p.o.), indomethacin (1 mg/kg/d p.o.), and prednisolone (5 mg/kg/d p.o.) showed no anti-angiogenic activity, indicating that the bFGF-induced angiogenesis was not driven by an inflammatory response or by a foreign body reaction. Finally, two candidate anti-angiogenic compounds were tested in the assay. Continuous low-dose therapy with cyclophosphamide (25 mg/kg/d p.o.) significantly inhibited bFGF-induced angiogenesis, whereas 1alpha,25-dihydroxyvitamin D3 (0.5 micro g/kg/d p.o.) showed no significant anti-angiogenic activity. In conclusion, this in vivo chamber angiogenesis assay is a useful new tool for drug evaluation as well as research into anti-angiogenesis.