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Experiment 2.: Inhibition of cell proliferation measured by cell counting
Introduction :
Proliferation of vascular endothelial cells is one of the decisive steps in the angiogenesis process. Inhibition of endothelial cell proliferation as judged by labeled Thymidine incorporation could not clearly distinguish between cell dead and inhibition of proliferation (see experiment: Inhibition of cell proliferation measured by 3H-thymidine incorporation )
The total polar extract from refined de-oiled jojoba meal and some specific simmondsin derivates purified from this extract were evaluated in their ability to inhibit the bFGF (basic Fibroblast Growth factor) induced proliferation of confluent HUVEC (Human Umbilical Vein Endothelial Cells) monolayer. Upon induction, monolayer will proliferate towards 100% confluence; and arresting at 100% due to contact inhibition. Cell proliferation is measured by cell counting after microscopic image analysis
Material & Methods :
Cell culture
Human Umbilical Vein Endothelial Cell (HUVEC) isolations will be obtained as described by Jaffe et al (1973), and cultured on fibronectin-coated dishes in M199 supplemented with 20 mM HEPES (pH 7.3), 10% human serum, 10% heat-inactivated new born calve serum (NBCS), 150 µg/ml crude endothelial cell growth factor (ECGF), 2 mM L-glutamine, 5 U/ml heparin, 100 IU/ml penicillin and 100 µg/ml streptomycin at 37 oC under 5% CO2/ 95% air atmosphere to confluence. The confluent HUVEC cultures (passage 0) will be detached by trypsin/EDTA treatment, pooled and cultured after a split ratio of 1:3 till confluence (passage 1). Then the pooled HUVEC will be frozen in 10 cm2 aliquots in vials in liquid nitrogen. One week before the proliferation, vials of HUVEC (passage 1) will be thawed and cultured (after a split ratio of 1:3) to confluence (passage 2).
bFGF stimulation
A confluent monolayer of HUVEC (passage 2) will be detached by trypsin/EDTA solution, and allowed to adhere and spread at a cell density of 15% confluence in gelatine-coated flasks in M199-HEPES medium supplemented with 10% heat-inactivated NBCS and penicillin/streptomycin. These cells can normally further proliferate until 100% confluence is reached. After 18 h the HUVEC will be stimulated with 2.5 ng/ml basic fibroblast growth factor (bFGF) in M199-HEPES, penicillin/streptomycin, 10% NBCS and 0.1% DMSO in triplicate wells for 6 days, with a refreshment of the medium with and without jojoba test compounds in 0.1% DMSO at day 4.
Another confluent monolayer of HUVEC (passage 2) will be detached by trypsin/EDTA solution, and allowed to spread at a cell density of 100% confluence in gelatine-coated flasks in M199-HEPES medium supplemented with 10% heat-inactivated NBCS and penicillin/streptomycin. These cells cannot further proliferate due to contact inhibition. After 18 h the HUVEC will be stimulated with 2.5 ng/ml basic fibroblast growth factor (bFGF) in M199-HEPES, penicillin/streptomycin, 10% NBCS and 0.1% DMSO in triplicate wells for 3 days.
Both confluent monolayer (15 & 100%) were pre-incubated during 4 h or continuous-ly incubated with the test compounds at different concentrations (1, 0.5 en 0.25%).
Tyrphostin A47, a thyrosine kinase inhibitor that inhibits the bFGF stimulation was used as a positive control.
Cell counting
The morphology of the cells will be microscopically evaluated (pictures taken) and the cell number will be determined by image analysis.
Results :
Non-confluent (15%) and confluent (100%) monolayer of HUVEC were subsequently pre-incubated (left panels) or continuously incubated (right panels) with or without the indicated concentrations (w/v) of the jojoba compounds in the continuous presence of bFGF (See figure). After 3 (100%) or 6 days (15%) pictures were taken and the number of cells/cm2 was determined and expressed as mean ± std (as indicated by error bars) of triplicate wells.
15% confluent cells :
Cells were 4h pre-incubation with the test compounds, subsequently washed and again stimulated with bFGF. The thyrosine kinase inhibitor showed no negative effect on the further cell proliferation, nor did test compounds A1, A2, A4 and A5. However, after 4h of pre-incubation with 1% A3, washing and re-stimulation, the cells stopped proliferating suggesting fraction A3 to be toxic for proliferating cells at this concentration.
When incubating the cells continuously, the thyrosine kinase inhibitor decreased cell proliferation. Even so, test compounds A3 and A4 decreased cell proliferation in a concentration dependent way, whereby A3 was much stronger than A4.
100% confluent cells :
Compounds A1, A2, A4 and A5 nor the thyrosine kinase inhibitor showed any significant effect on either pre-incubated or continuously incubated cells. However, continuous incubation with compound A3 significantly decreases bFGF induced cell proliferation significantly.
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Legend :
Control = Vehicle (0.1% DMSO)
A1 = total polar extract from refined, de-oiled jojoba meal
A2 = partial purified fraction containing mainly dimethylsimmondsin, desmethylsimmondsin
(2 isomers) and didesmethylsimmondsin (all hydroxylated)
A3 = partial purified fraction (+/- 65%) containing mainly ferulates of all simmondsins
described in A2 (hydroxylated)
A4 = dimethylsimmondsin (> 90% pure, hydroxylated)
A5 = dimethylsimmondsin ferulate (> 90% pure, hydroxylated)
Tyr A47 = tyrokinase inhibitor
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Conclusions :
Fifteen % confluent cells are inhibited in their bFGF induced proliferation by the jojoba compounds similar to the inhibition of the thyrosine kinase inhibitor. However, the tested jojoba compounds do not necessarily interfere with the cell proliferation using the same mechanism.
Since 100% confluent cells show no significant mortality upon continuous treatment with jojoba compounds A1, A2, A4 and A5, we can conclude the non-cytotoxicity of these compounds. On the contrary, compound A3 has to be considered cytoxic to bFGF stimlated HUVEC under the given concentrations.
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References :
Jaffe et al (1973) J. Clin. Invest. 52, 2745-2746
Free translation of internal communication :
Dear Pieter & Paul
We have some more small questions/remarks regarding the last results.
The experiment with the confluent HUVEC cells shows us that mainly A3 (and to a lesser extent also A4) has a pernicious influence on the cell proliferation. The stimulation of the 15% confluent cells is considerably suppressed. On the contrary, the 100% confluent cells seem not to be impaired at all by these products, even not under continuous incubation.
Regardless the fact this phenomenon should be related to angiogenesis inhibition, a product blocking only the fast-dividing cells but not affecting the resting cells, seems very interesting as an anti-cancer agent.
Please your opinion about this.
Answer:
For sure, this implicates the product should not interfere negatively with normal existing blood vessels.
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