VM formed by highly aggressive melanoma cells is a novel form of tumor microcirculation pattern, which differs from classically described endothelium-dependent angiogenesis. Previous studies demonstrated the prognostic value of VM in primary melanomas, using metastatic recurrence as an endpoint 
. It should be emphasized that all of specimens in this study were already derived from metastases, and indeed VM was abundantly identified in almost all of the specimens (, Table S1
). There were no clinical associations with additional parameters such as age, gender, site of metastasis, size of tumor etc. It is therefore hypothesized that VM enables resistance to conventional anti-angiogenic medicaments. Taking into consideration that VM is an example of tumor cell plasticity displaying highly dedifferentiated phenotype, targeting this phenomenon is a complex task. It has been demonstrate that several drugs could inhibit VM (reviewed in 
). In this study, we found that NA could inhibit VM formation of the highly aggressive (HAG) melanoma cell line C8161 in vitro
. NA also reduced proliferation and increased invasion capacity, as well as cause apoptosis to a certain degree. It is possible that these effects could contribute indirectly to the inhibition of VM. Notably, it was previously reported that VM is associated with invasion 
, which was increased here by NA, thus arguing against a significant indirect mechanism. In addition, VM was still inhibited even one month after washout of NA (). Defining the molecular mechanisms underlying VM will further enable development of specific anti-VM strategies.
It was suggested that vascular channels in general, including VM channels, play a role not only in supplying oxygen and nutrients required for tumor growth, but also in enhancing tumor metastasis 
. We found in a series of clinical metastatic melanoma specimens both CD31-positive endothelial channels and CD31-negative PAS-positive VM channels. Importantly, the proportion of VM was, on average, similar to that of endothelial blood vessels, supporting a significant role for VM. In addition, we observed the unique architectures that were described by Folberg et al 
based on PAS staining, which are: straight channels, parallel straight pattern, parallel straight pattern with cross link, arcs (not closed), arcs with branching, closed loops, and networks.
Most of the literature data on VM in human cutaneous melanoma emerges from investigations on the model of C8161 (HAG) and C81-61 (PAG) isogenic, homogenous, cell lines. We were able to demonstrate tube formation activity of low-passage primary cultures of metastatic melanoma however it was not correlative to the percentage of VM channels observed in the paraffin embedded biopsies. It was previously reported that VM activity is associated with the expression of VE-cadherin, based on studies with HAG (VE-cadherin-High) and PAG (VE-cadherin -Negative) cells 
, jointly with the tyrosine kinase receptor EphA2, which was proposed as initiator of the VM signaling cascade 
. An association between the expression of VE-cadherin and VM activity in vitro
was mainly evident among established cell lines, and less among low-passage primary cultures (). The latter might be explained by the phenotypic heterogeneity and sensitivity to environmental manipulation of low-passage primary melanoma cultures 
. While in vivo
a small proportion of VE-cadherin positive cells might suffice for generation of capillary-like structures, the in vitro
conditions are probably more restrictive. This might also explain why there was no correlation between VM in situ
and tube formation activity in vitro
. Therefore, mechanistic VM studies in vitro
are limited to selected cell lines with an extreme phenotype, such as HAG, evident by the bright VE-cadherin expression. Nevertheless, targeting VM is still attractive. With the challenge of finding new drugs which could inhibit VM, the soybeans isoflavone Genistein 
was found to be able to inhibit VM formation of uveal melanoma through down-regulation of VE-cadherin in vitro
. In the present study, we show that treatment with NA downregulates VE-cadherin expression to inhibit VM activity. It could be proposed that NA abrogate channel-like structures directly by downregulation the most essential molecule for VM network formation. Indeed, VE-cadherin was downregulated significantly in both RNA (Table S2
) and protein () levels, whereas VEGF-A was downregulated at the RNA level (Table S2
), but not in the protein level ().
Hypoxia was found to encourage tube formation in vitro
and expression of genes associated with VM 
. Consequently, VM is mostly resistant to anti-angiogenic therapy 
, and is therefore probably involved metastasis through VM endorsement 
. NA was found to act by decreasing perfusion-limited tumor hypoxia through prevention of intermittent vascular shut-down 
. Another possible mechanism of action in vivo
for NA could be inhibition of VM formation through prevention of local hypoxia. Indeed, the VE-cadherin gene contains genomic binding sites for HIF-1α and HIF-2α transcription factors that are stabilized during condition of hypoxia 
. It was proposed that HIF-2α expression in aggressive tumor cells is associated with de-differentiation towards the endothelial lineage by transcriptional induction of VE-cadherin 
The ability of plastic tumor cells to present an endothelial phenotype is probably due to the reactivation of embryonic signaling cascades causing aggressive tumor cell to dedifferentiate 
. In addition, melanoma cells appear to reversibly generate stem-like cells through “phenotype switching”. By this process, melanoma cells can switch between migratory, stem-like state and proliferative state in response to changes in microenvironmental conditions 
. In the present study we observed that NA inhibited proliferation and enhanced invasiveness of HAG cells in vitro
(). It could imply that NA has a potential role in phenotype switching, but this must be thoroughly investigated in primary cultures in future studies (). In a recent review, Girouard and Murphy 
proposed that the undifferentiated, primitive, embryonic like stem cells profile of aggressive melanoma cells engage in VM, implying that melanoma stem cell may give rise to the patterned networks that characterize VM. Monzani et al 
showed that a stem cell population that potentially increases tumor progression, is found in melanoma biopsies. Thus, it is suggested that cancer stem cell (CSC) subpopulation inside the tumor are capable of organizing VM networks, depending on the environmental condition. All of these contribute to the failure of current therapeutic regimens by masking malignant target populations.
It has been observed in a mouse model that invasive phenotype cells escape melanoma allograft in large numbers 
. Hoek and Goding 
suggested that cells with invasive phenotype possibly submitted to senescence or apoptosis and only a fraction survive to switch back to the proliferative state. Alternatively, they proposed that the switch from invasive to proliferative is very low, which may lead to dormancy over long periods. The complexity of the metastatic phenotype, including heterogeneity and phenotype-switching, with plasticity close to that of embryonic cells, must be taken into account when designing new therapies. NA addresses the need for designing such strategies combined with other modalities, including the molecular based approach aimed to suppress the aggressive melanoma phenotype in individual patients 
. It was shown that NA had an effect of the differentiation of leukemia cells through its histone deacetylase inhibitory activity 
. Recent studies in experimental human tumors showed antiproliferative proapoptotic activities by NA 
and pronounced inhibition of growth and progression 
. It is highly likely that the broad alterations in gene expression we observed in melanoma following exposure to NA and the subsequent functional effects are due to the epigenetic regulation exerted by NA, e.g. by histone deacetylase inhibition. Thus, NA could be quilted as VM-targeted strategy in addition to its wide range of biological activities and its different effects mediated by different concentration. The combination of anti-VM agents like NA with other therapeutic strategies is expected to yield the best results.
In conclusion, this study shows that NA could successfully inhibit the VM formation of C8161 human cutaneous melanoma. One mechanism in which NA inhibits VM is associated with downregulation of VE-cadherin. Moreover, NA inhibits proliferation and increases invasiveness and apoptosis in melanoma cells. This study may provide preliminary evidence for future and wider research to elucidate the mechanisms underlies VM inhibition by NA and its mode of action.