An in vitro model that can distinguish between effects on angiogenesis and on established vasculature: actions of TNP-470, marimastat and the tubulin-binding agent Ang-510.

In anti-cancer therapy, current investigations explore the possibility of two different strategies to target tumor vasculature; one aims at interfering with angiogenesis, the process involving the outgrowth of new blood vessels from pre-existing vessels, while the other directs at affecting the already established tumor vasculature. However, the majority of in vitro model systems currently available examine the process of angiogenesis, while the current focus in anti-vascular therapies moves towards exploring the benefit of targeting established vasculature as well. This urges the need for in vitro systems that are able to differentiate between the effects of compounds on angiogenesis as well as on established vasculature. To achieve this, we developed an in vitro model in which effects of compounds on different vascular targets can be studied specifically. Using this model, we examined the actions of the fumagillin derivate TNP-470, the MMP-inhibitor marimastat and the recently developed tubulin-binding agent Ang-510. We show that TNP-470 and marimastat solely inhibited angiogenesis, whereas Ang-510 potently inhibited angiogenesis and caused massive disruption of newly established vasculature. We show that the use of this in vitro model allows for specific and efficient screening of the effects of compounds on different vascular targets, which may facilitate the identification of agents with potential clinical benefit. The indicated differences in the mode of action between marimastat, TNP-470 and Ang-510 to target vasculature are illustrative for this approach.

[Dutch Medicines Act also applicable to repurposing]. / Ook bij 'repurposing' is Geneesmiddelenwet van kracht.

In this issue of the Dutch Journal of Medicine (NTvG), Strous and Van den Brink argue that Article 68 of the Dutch Medicines Act should be applied to repurposed drugs in a more liberal manner. Medicines prescribed on-label have been authorised by the relevant regulatory authority and are therefore guaranteed to have been substantiated by the necessary evidence and assessed for a positive benefit-risk balance. Article 68 states that medicines may be prescribed off-label only if they are described in the relevant professional treatment standards or guidelines. The more liberal application of Article 68 would allow the prescription of drugs that have not been adequately assessed for evidence substantiating their efficacy and safety in the field for which they are being prescribed. This is unwarranted, for repurposed drugs and for any other drug.

Celecoxib enhances doxorubicin-induced cytotoxicity in MDA-MB231 cells by NF-kappaB-mediated increase of intracellular doxorubicin accumulation.

Non-steroidal anti-inflammatory drugs (NSAIDs) and cyclo-oxygenase (COX) inhibitors are anti-inflammatory agents that have also shown to be useful in anticancer therapy. In the present study, we show that the specific COX-2 inhibitor celecoxib enhances the inhibitory effect of doxorubicin (dox) on human MDA-MB231 breast tumour growth in vivo and in vitro. We also found that celecoxib increased the intracellular accumulation and retention of dox in vitro. Since the NSAID indomethacin and the specific COX-2 inhibitor NS398 did not affect the in vitro actions of dox, these effects are likely to be mediated via a COX-independent mechanism. It has been suggested that some COX-inhibitors can enhance the actions of cytostatics by overcoming multidrug resistance through the inhibition of ABC-transporter proteins. However, we found that the three main ATP-binding cassette (ABC)-transporter proteins, implicated in dox transport, were inactive in MDA-MB231 cells. Therefore, the finding that the P-glycoprotein (P-gp) blocker PSC833 also increased cellular accumulation of dox was unexpected. In order to unravel the molecular mechanisms involved in dox accumulation, we examined the involvement of NF-kappaB, as this transcription factor has been implicated in celecoxib action as well as in chemoresistance. We found that celecoxib and PSC833, but not indomethacin or NS398, almost completely inhibited basal- and dox induced NF-kappaB gene-reporter activity and p65 subunit nuclear translocation. Furthermore, the NF-kappaB inhibitor PDTC mimicked the actions of celecoxib and PSC833 on cell growth and on intracellular accumulation of dox, suggesting that NF-kappaB is functionally involved in the actions of these compounds. In conclusion, we show that structurally different compounds, among which are celecoxib and PSC833, increase the intracellular accumulation of dox and enhance dox induced cytotoxicity in MDA-MB231 breast cancer cells most likely via the modulation of NF-kappaB activity.

Identification of differentially expressed genes in a renal cell carcinoma tumor model after endostatin-treatment.

Endostatin is a cleavage product of collagen XVIII that has shown to inhibit tumor-angiogenesis in experimental tumor models. At present, the exact molecular mechanism of action of endostatin is not completely elucidated. In this study, we wanted to identify specific target genes of endostatin. For this purpose, the human renal cell carcinoma RC-9 was subcutaneously implanted in nude mice and treated with endostatin. Tumor growth was inhibited by endostatin after 4 days of treatment. Using immunohistochemistry and the hypoxia marker pimonidazole, we demonstrate disintegration of blood vessels and hypoxia and anoxia as a result of the treatment. Hereafter, we applied the polymerase chain reaction (PCR)-based subtractive suppression hybridization (SSH) method, together with the mirror orientation selection (MOS) technique to identify specifically induced and suppressed genes after endostatin-treatment. We found eight genes to be specifically induced and 11 to be suppressed by the endostatin-treatment. Among other genes, core binding factor a-1/osteoblast-specific factor-2 (cbfa1/osf2) was found to be specifically suppressed by endostatin. Unexpectedly, cbfa1/osf2 was found to be specifically expressed in granulocytes in the tumor, not only in the experimental RC-9 tumor model, but in sections of human breast cancer as well. Since an effect of antiangiogenic therapy on granulocytes has been reported before, this might lead to new insights in the role of granulocytes in antiangiogenic therapy in general. In conclusion, the SSH-PCR implemented with the MOS-technique is a powerful tool to identify differentially expressed genes. Using these techniques, we have identified several target genes of endostatin, of which cbfa1/osf2 was found to be specifically expressed in granulocytes in the tumor.