Preclinical file (Studies of biological system models)

PRECLINICAL FILE (STUDIES OF BIOLOGICAL SYSTEM MODELS)

Cytostimulatories and cytoprotect!ves effects of peptides from chicken embryo on human fibroblasts

Gh. Mihaescu1, O. Mihaescu1, Doina Petrasincu2, M. Leabu3, F.Oancea4, R.OIinescu1

1- Research and Production Laboratory “Hipocrate” Bucharest

2- “Ion Cantacuzino” Institute, Bucharest 3 – Medicine and Pharmacy University “Carol Davila” Bucharest

4 – Research Institute for Plant Protection, Bucharest

5 – “Victor Babes” Institute, Bucharest

Key words: cytostimulatories effects, cytoprotective effects, human fibroblast embryonic peptides

Introduction The action of peptides as biosignal is an increased area of interest, both from academic and pharmaceutical field. An inpressive number of papers and patents related to biosignal peptides were issued in the last five years. For example, it was recently demonstrated that peptides acting as fibroblast growth factors protect the tissues against free radicals, generated by radiation [1] or during the reperfusion after ischaemia [2]. Also, our group proved that the treatment with drugs containing chicken embryo peptides decrease the oxidative stress in elderly patients [3].

Embryotherapy (the use of embryonic or foetal tissues or cells for the treatment of human diseases) was used, in an attempt to slow or even to reverse the aging processes, from the beginning of modern clinical medicine [4]. Immunologica! reactions (from allergy to anaphylactic shock) and virus and (possible) prionic infections were the most common side effects associated with the embryonic greffes or to injectio with embryonic cell suspensions. In order to avoid such dangerous secondary reactions, extracts from embryonic tissues were prepared and used in therapy [3,5]; the active ingredient of such extracts proved to be peptides acting as bioactive signals [5,6].

In this paper we present the effects of peptides from a chicken embryo extract (used as the active ingredient on two Romanian drugs, Humanofort and Gerontofort) on human fibroblast in vitro cultivated.

Materials and Methods.

Chicken embryo extracts. The chicken embryo was homogenised with distillated water, defatted by counter current extraction with n-hexane and freeze-dried. The resulting powder was resuspended in double distillated water, centrifuged at 10,000 g for 20 min (to avoid any further gel-chromatographic bed warping) and gel-chromatographied on a preparative columns (Sephadex G-50). The compounds from fractions included in chromatographic gel (with molecular weight under 50 kD) were unified and freeze-dried once again. The freeze-dried powder was solved in ultrapure water (obtained from MilliQ-Plus apparatus), centrifuged for 15 min at 15,000 g and purified on high performance gel chromatography on a Pharmacia-LKB apparatus, using a TSK G2000 SW column. The column was previously calibrated with bovine serum albumin (67kD), ovalbumin (42 kD), hemoglobin (18 kD) and citochrome c (12,7 kD). The gel-filtration was done during 30 min, with ultrapure water as mobile phase, at a flow of 0.5 ml per min. The separation process was UV monitorised, at two different wave length (220 nm and 280 nm). Several fractions was separated: FI …Fn. The protein concentration in peptidicfraction was measured with a modified Lowry method, using a Sigma kit (P5656). The tested concentrations on fibroblast were the following: 100 ^g for the initial extract and equivalent concentration for tested fractions(1ng/ml).

Human fibroblast cell strain used. A Romanian diploid human cell strain, ICP-23, was used , growth on usual Eagle BME culture medium supplemented with 10% foetal calf serum.

Embryonic extracts testing procedures. The tests was done on fibroblast growth in Barsky tube of 2 ml, in the presence or, respectively, in the absence of tested embryonic extracts. The cell proliferation in vitro was estimated using the following methods: measurement of optical density at 24 hours during a 96 hours cycle of multiplication and cell numbering after trypsin elution and stained with tripan blue 0,4%. The mortality was estimated by counting the cell with tripan blue exclusion. Two kind of experiments was made. In the first one, the embryonic extract was tested on young cells (passage 20 … 23) and senescent cells (passage 40 … 43) in complete culture media, including the foetal calf serum. The second type of experiments, the chicken embryo extract (GEE) was tested on incomplete culture media (without foetal calf serum) in an attempt to substitute the sources of growth factors.

Tritiated thymidine incorporation tests. In order to extent the studies on embryonic extracts effects on fibroblast cell an tritiated thymidine incorporation experiment was done. The culture medium was radiolabelled with tritiated thymidine in dose of 1 ji Ci/ml, after 48 hour of growth of fibroblast cell (approx. 105 cells/ml) on monolayer in Kole dish (treated or not treated with embryonic extract, according to the experimental procedure) The cell was recovered from Kole dish monolayer after other 24 hour of growth in radiolabelled medium,  centrifuged 5 min at 1,000g, washed twice with culture medium (without foetai calf serum) and resuspended in 5% trichloroacetic (TCA) solution in water (w/v). The suspension of cells in 5%TCA was kept 24 h at 4°C and centrifuged at 2,500 g. In supernatant the radioactivity of nucleic acids incorporating the radiolabelled thymidine was measured using liquid scintillation techniques (with a Liquid Scintillation Counter Berthold 5000/300). The same experimental variances as mentioned above (complete culture media and GEE substitute culture media) was studied. Results and Discussion.

The chicken embryo extract total extract (CCE) prove to exert an : cytostimulatory effects on young fibroblast cells (passage 20 in vitro) – Fig. 1a, After 96 hour the number of cells are greater with 36% (control – 17,27. 104 cells/ml, CCE treated samples – 23,60 .104 cells/ml). In the same time the v 1 mortality at 96 h of CCE treated samples (1,50%) are with 50,8% smaller than in control (2,95%) – Fig. 1b.

The same effects are observed on CCE treated fibroblast senescent cells (passage 40 in vitro) – Fig 2. The stimulatory activity (Fig. 2 a) is less important (23%) and also the protective effect seems to be smaller (40,11% at 48h) – Fig. 2b. However, if we consider the absolute values for mortality, we will find that the protective effect is very significant at 96 h (control – 7,0% mortality; CCE treated samples – 2,86% mortality, less even than the mortality in control for young cells!).

The data from Fig. 3a and 3b show that the stimulatory activity seems to be located in the F2 fraction and only some of the cytoprotective action remain in F1 fraction. After 96 hour the number of cells (passage 22 in vitro) are greater with 10% in F2 treated medium (control -14,9. 104 cells/ml, CCE treated samples – 16,41.104 cells/ml). In the same time the mortality of F2 treated samples (1,67%) are with 63,46% smaller than in control (4,58%). In F1 treated group the stimulatory activity is not present, but an significant cytoprotective action was established (control mortality – 4,58%; F1 mortality – 2,60%).

The same effects are observed on F1 and F2 treated fibroblast senescent cells (passage 42 in vitro) – Fig 4a and Fig. 4b. The stimulatory activity (Fig. 4 a) is located in F2 fraction (control – 8,8. 104 cells/ml at 96 h; F2 -11.0.104 cells/ml at 96 h) and the protective effect seems to be located in both F1 and F2 (control mortality – 5,32%; F1 mortality -1,21; F2 mortality -2,43%).

The results presented in fig. 1b, as percentage, may could be explained in other wise: as the ratio between cellular mortality of the embrionary extract F 2 , against control. At 24 hours, the mortality provided by F2 this 1/4 times tower then control; 5.7 times lower then control: 5.7 times at 48 h; 2.6 times at 72 h and 1.9 times at 96 hours.

Obviously, by comparing these indications, the peak of protection was obtained at 24 h, then it slowly decreases as smaller at 96 h, due to the consumption of active peptides involved in the stimulated cellular division.

The same phenomenon was noticed in the figs, 2b; 3b 4b, no more discussed for a concise interpretation.

Data obtained for tritiated thymidine incorporation correlate with data obtained with classical cells counting (tab 1). The chicken embryo extract (GEE) stimulate the radiolabelled thymidine incorporation (tab.2), and this stimulatory activity seem to be located predominantly in F2 fraction (tab.3). Interesting, the F2 fraction was established to be more stimulating than the total extract for senescent fibroblast cells (passage 40 … 42 in vitro). The substitution of foetal serum with CCE or with its components do not promote the tritiated thymidine incorporation (tab.4).

Tab.1. Cell growth, as cell mortality for ICP-23 human fibroblast cell (passage 23 in vitro) cultivated on medium with foetal calf serum substitute with chicken embryo peptides (F2 fraction)

1

Value followed with the same letter do not differ significantly for P<0.05% Tab. 2. Tritiated thymidine incorporation by ICP-23 human fibroblast cell treated with chicken embryo extract

2

Value followed with the same letter do not differ significantly for P<0.05%

Tab. 3. Radiolabelled thymidine incorporation by ICP-23 human fibroblast cell treated with the component of chicken embryo extract

3

Value followed with the same letter do not differ significantly for P<0.05%

Tab.4. Radiolabelled thymidine incorporation by ICP-23 human fibrobalst cell (passage 23 in vitro) cultivated on medium with foetal calf serum substitute with chicken embryo peptides (F2 fraction)

4

Value followed with the same letter do not differ significantly for P<0.05%

Cytostimulatory and cytoprotective effect were demonstrated for total embryonic extract used as active ingredient in Romanian drugs Humanofort and Gerontofort. This effects seem to be located predominantly in F2 (peptide) fraction and consist first-of-all in an improved response to growth factors from

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cells culture media. In media without foetal serum, the cytostimulatory activity is
not present, so only a increased responsiveness effect to other growth factors
is suggested.
The same increase of responsiveness to growth factors was established
for another embryonic extract [6], the CLP extract, made from sheep foetus
The experiment was done in senescent fibroblast cultures of hamster which
have approached the postmitotic state in vitro; responsiveness to growth factors
is restored upon exposure to CLP embryonic sheep cell extracts. It was
suggested that a senescent cell activating factor(s) SCAF(s) is present in
embryonic tissues and that embryonic substance(s) restore the growth factor
receptors.

Our data support the same interpretation. In embryonic tissues, factor(s)
acting on specific receptors rend them more sensitive and amplify the response
of cells. It is possible to speculate that these hyphothetic embryonic factors will
act by maintaining / restoring the most adequate spatial structures of receptors.
Further work to enrich, purify and characterise the responsible factors from
embryonic tissues are in course.

The cytoprotective activity of embryonic extracts could be in relation with
the protection against free radicals exerted by the different (peptides) growth
factors. As mentioned already, it was recently demonstrated that peptides
acting as fibroblast growth factors protect the tissues against free radicals,
generated by radiation [1] or during the reperfusion after ischaemia [2]. Our
group proved that the treatment with drugs containing chicken embryo peptides
decrease the oxidative stress in elderly patients [3]. The same protection against free
radicals can be suggested in cell culture, but future work are necessary to be done.
Beside that, oxygen free radicals are proved to be involved in modulation of fibroblast
profilation [7]. A possible effect of embryonic peptides on biological system acting on
free radical formulation or acting as free radical scavenger will (indirectly) influence the
fibroblast profilation. In conclusion our data presented here supports the idea that the
embryonic extract used as active ingredient has a cytostimulatories and cytoprotective
effects.