The gelatinolytic assays were carried out in two different ways to obtain complementary information: firstly, cells were treated or not with the test compound and samples from these were submitted to gelatinase zymography to detect the effects of the kahweol treatment on the expression of gelatinase activities; secondly, in some experiments, samples from control, untreated HT - 1080 fibrosarcoma cells were submitted to zymography and, after electrophoresis, different concentrations of kahweol were added to the substrate buffer to determine the potential direct effect of
kahweol on gelatinase activity.
Data obtained on the effects of
kahweol on endothelial cell invasion (as determined by a continuous fluorescent assay) clearly show that kahweol induces an anti-invasive effect in HUVEC in a dose - dependent manner (Figure 7).
We also demonstrate the inhibitory effect of
kahweol on the endothelial cell potential to remodel extracellular matrix by targeting two key molecules involved in the process, MMP - 2 and uPA.
On the other hand, these data suggest that the potential effects of
kahweol on apoptosis could exhibit certain cell specificity.
Furthermore, the inhibitory effects of
kahweol on COX - 2 and MCP - 1 reinforce the idea of kahweol being a multi-targeted natural compound with high pharmacological potential.
Since invasion is dependent on extracellular matrix remodeling capabilities, this inhibitory effect strongly suggested that the two key extracellular membrane remodeling enzymes expressed by endothelial cells, namely, MMP - 2 and uPA could be other main key targets of the pharmacological action of
kahweol on endothelial cells.
Therefore, we studied the effects of
kahweol on the growth of endothelial cells.
Therefore, it would be advisable to test the potential effects of
kahweol on endothelial cell apoptosis.
Figure 6 shows the effects of 75 µM
kahweol on endothelial cell migration, as determined by the «wound healing» assay, after 8 and 24 h of treatment.
Not exact matches
To get new, additional insights
on the features of
kahweol as an anti-angiogenic compound, we carried out a complete set of in vitro assays previously used by us to characterize the anti-angiogenic effects of other compounds from natural sources, including aeroplysinin - 1, homocysteine, ursolic acid, puupehenone, hypericin, hyperforin and aloe - emodin, among others [11], [12], [13], [19], [20], [21].
On the other hand, the anti-oxidant nature of
kahweol also points to its potential anti-inflammatory capabilities.
In all these in vitro assays,
kahweol treatments were carried out under conditions (
kahweol concentration and duration of treatment) that did produce no cytotoxic effect
on cells.
Photographs were taken
on untreated (control) and 75 µM
kahweol - treated HUVEC cells at 0, 8 and 24 h after «wounding».
On the other hand, the present research work shows a confirmatory evidence of the potential of
kahweol to inhibit in vivo angiogenesis, by using another completely independent model system, namely, that of genetically modified zebrafish.
The minimal inhibitory concentration for
kahweol in this assay of «tubule - like» structures formation
on Matrigel was 25 µM, in the range of concentrations at which other known antiangiogenic compounds produce this kind of effect [13], [27].
Different in vitro assays were carried out in order to test the specific effects of
kahweol treatment
on several key steps of the angiogenic process in both endothelial and tumor cells.
The CAM and zebrafish in vivo assays and the ex vivo mouse aortic ring assay clearly identify
kahweol as a new anti-angiogenic compound, but gives no information
on which specific steps of angiogenesis are targeted by this compound.
Video images of blood flow thru intersegmental vessels were taken
on the caudal region next to vitellus in 48 h larvae after 24 h of treatment in the absence (control, Video S1) or presence (Video S2) of 50 µM
kahweol.
This effect
on cell survival was not endothelial cell - specific, since IC50 values for
kahweol treatment of several human tumoral cell lines were similar to those obtained for HUVEC (results not shown).
We show for the first time that
kahweol is an anti-angiogenic compound with inhibitory effects in two in vivo and one ex vivo angiogenesis models, with effects
on specific steps of the angiogenic process: endothelial cell proliferation, migration, invasion and tube formation
on Matrigel.
B) In situ determination of
kahweol effects
on HT - 1080 gelatinases, as determined by gelatin zymography with the presence of
kahweol in the incubation substrate buffer.
It has been shown previously that
kahweol exerts a suppressive effect
on COX - 2 expression in macrophages [38].
Research
on the antioxidant activity of trigonelline, cafestol and
kahweol has been less extensively investigated in humans.