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Application of embryonic models for elaboration of anti-carcinogenic preparations of desired action

Aminov AV

Department of Molecular Basis of Integrative Activity, A.I. Karayev Institute of Physiology, Azerbaijan NAS 78 Sharif-zadeh St. Baku, AZ1100 Azerbaijan, Baku

E-mail : bhuvaneswari.bibleraaj@uhsm.nhs.uk

Mekhtiev AA

Department of Molecular Basis of Integrative Activity, A.I. Karayev Institute of Physiology, Azerbaijan NAS 78 Sharif-zadeh St. Baku, AZ1100 Azerbaijan, Baku

DOI: 10.15761/HMO.1000133

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Abstract

The article concerns application of embryonic model of Xenopus laevis for elaborating preparations directed either to suppression of cell proli­fe­ration, or, in opposite, to promoting forced cell differentiation. In the 1st series of studies the dynamics of the serotonin-modulating anticonsolidation protein (SMAP), being in linear rela­tions with serotonin, in the embryos of Xenopus laevis throughout the stages of embryo­genesis and metamorphosis was pursued with application of indirect ELISA-test. Beginning from the blastula stage till the end of the neurula its level remained unchanged. Thereafter continuous downregulation of the SMAP level, inter­rupted as a slight upregulation between 37th stage and onset of the 39th stage, was observed. In the 2nd series of studies incubation of the embryos of Xenopus laevis on the blastula and gastrula stages in fresh water containing  SMAP at a dose of 50 and 100 μg/ml led to delay in development and, finally, to death of all the em­bryos within 4 days of observation. In the 3rd series of studies blocking of SMAP activity with the anti-SMAP poly­clonal antibodies at a dose of 50 μg/ml in the embryos of Xenopus laevis, being on the 37th stage of deve­lopment, resulted in their passing ahead (by two stages earlier) of the meta­morphosis stage relatively to the rate of passing of this stage by the animals of the intact and control groups. So, if on the initial stages of embryogenesis SMAP realizes cytostatic activity, on the meta­morphosis stage blocking its activity with antibodies, conversely, leads to passing ahead cell differen­tiation. 

Key words

serotonin-modulating anticonsolidation protein (SMAP), antibodies, embryo¬genesis, metamorphosis, Xenopus laevis

Introduction

Certain stages of embryonic processes have high level of resemblance with carci­nogenesis processes, particularly in high proliferative activity of their com­posing cellular elements. Along with it, the advantage of studying embryonic stages rela­tively to cancer processes concludes in that that processes of cell proli­feration and diffe­ren­tiation are diverged over time, mostly within different embry­onic stages. So, utili­zation of embryonic models makes possible to elaborating the approaches directed purposefully and separately on the processes of cell prolife­ration and diffe­ren­tiation. Issuing from high proliferative potential of the cancer cells, it is clear why suppressive effects on cell proli­feration and the remedies pos­ses­sing with such activities are under meticulous attention of the most researches dealing with the studies of carci­nogenesis and seeking ways of combating with tumor. Along with it, from the first glance, the unusual interest to the processes of cell differentiation and study of the underlying molecular mechanisms, coming from the cancer-absorbed researchers, is related to a problem of the cancer stem cells constituting serious obstacle in treat­ment of malig­nant tumors [1-3]. The problem of their eradication is complicated by the fact that broadly applied chemothe­rapeutic preparations are ineffective against these cells. Sometimes the struggle with cancer stem cells is likened to the struggle with weeds: you can chop off the weeds’ stalks many times, but they will grow again and again, so far, their roots still remain in the soil. The same is referred to the cancer stem cells in the sense that the role of woods’ stalk in this case belongs to mature malignant tumor cells, while the role of woods’ roots – to the cancer stem cells themselves.

The goal of the present study was application of different stages of embryonic and early stages of development of Xenopus laevis for the purpose of elabo­rating preparations directed either to suppression of cell prolife­ration, or, in opposite, to promoting forced cell differentiation. This goal was achieved through application of serotonin-modulating anticonsolidation protein (SMAP) and anti-SMAP poly­clonal antibodies for the purpose of blocking its activity in different stages of the embryonic and early stages of development of Xenopus laevis.

Materials and methods

Biochemical techniques

SMAP, being in linear rela­tions with serotonin, was purified from the cow brains with application of two-step purification proce­dure as had been described earlier [4]: 1) partial preci­pitation with sodium sulfate in the range of 0-40% concentration; 2) gel-chroma­tography on the column (3.0 X 60.0 cm) of Sephadex G-150. SMAP purifi­cation was carried out under the screening control of the indirect ELISA-test [5] with appli­ca­tion of anti-SMAP rabbit immunoglobulins to selecting the SMAP-enriched protein fractions. The protein purity was checked by elec­tro­­phoresis in poly­acryl­amide gel.

The anti-SMAP poly­clonal immunoglobulins were produced through immu­nization of the rabbits with SMAP, using 300 μg of the protein always in mixture with the complete Freund adjuvant. SMAP and anti-SMAP immunoglobulins were frozen and kept under -70C.

The content of SMAP was determined in the embryos and tadpoles of Xeno­pus laevis by the indirect ELISA-test realized on the polystyrene plates of mode­rate adsorption [5]. The samples were homogenized and water-soluble pro­­teins were extracted and used as antigens in the ELISA-test. Specific po­lyclonal rabbit anti-SMAP anti­bo­dies were used as the first anti­bo­dies, while the anti-rabbit goat immu­no­glo­bulins, coupled with covalent bonds with horseradish peroxidase, were used as the second antibo­dies. Ortho­pheny­len­diamine was used as a substrate for per­oxi­­dase to visua­lize the results of the reaction. The reaction was stopped by adding of 3 M NaOH and the results were tran­s­formed into digital form by the ELISA-test reader of the model of “Molecular Devices Spectra Max 250” (MTX Lab Systems, Inc., USA) at the wavelength of 492 nm (wavelength of reference 630 nm) and analyzed with application of the t-Student criterion.

Embryonic technique

The embryonic studies were conducted on Xenopus laevis. The eggs from the sexually mature animals were obtained after injecting them with human gona­do­tropin at a dose of 150 units for males and 350 units for females. 

In the 1st series of studies after several hours since gonadotropin admini­stra­tion, females released roe which was fertilized by males. Simultaneously after roe fertilization samples of the embryos were taken from different stages of em­bryonic development (stages 1-2, 7, 9, 11, 13, 20, 22, 28), pre-metamorphosis, meta­mor­pho­sis and post-meta­morphosis (stages 37, 42, 44, 45, 49, 53) for evalu­ation of the level of SMAP with application of the indirect ELISA-test and anti-SMAP poly­clonal immunoglobulins. For evaluation of SMAP 10 samples of specimens within each stage were taken. The results were averaged within each group and diffe­rences between adjacent stages were evaluated on t-Student’s criterion.

In the 2nd series of studies the embryos, being on the blastula and gastrula stages, were placed into the Petri dish with fresh water containing different pre­pa­rations. The embryos were culled into three groups: 1) intact group (n = 17), 2) con­trol group – SMAP and anti-SMAP antibodies – both at a concentration of 50 μg/ml (n = 15), and 3) experimental group – SMAP at a concentration of 50 and 100 μg/ml (n = 17).

In the 3rd series of studies the animals, being on the 37th stage of development (beginning from the late tailbud stage), were placed into the Petri dish with fresh water containing different preparations. The embryos were culled into four groups: 1) intact group (n =13), 2) control group – SMAP and anti-SMAP antibodies - both at a concentration of 50 μg/ml (n = 13), 3) 1st experimental group – SMAP at a con­centration of 50 μg/ml (n = 13), 4) 2nd experimental group – SMAP at a concen­tration of 100 μg/ml (n = 13), and 5) 3rd experimental group – anti-SMAP antibodies – at a concentration of 50 μg/ml (n = 13). After 24 h the animals were transferred into the tanks with fresh water with regulated temperature and air supply. On the basis of elaborated tables of early stages of development of Xenopus laevis [6] registration of pas­sing metamorphosis stage by the animals of different groups was carried out. Particularly, definition of passing of this stage by the animals was realized on the basis of degree of the tail resorption, advent of the precursors (buds) of the hind legs, changes in the shape of mouth and other second-order morphological changes typical to mature animals. 

The results were averaged within each group and diffe­rences between diffe­rent groups were evaluated on Wilkoxon-Mann-Whitney’s U-criterion.

Results

Dynamics of SMAP level on embryonic stages and metamorphosis in Xenopus laevis

In the 1st series of studies the results revealed the dynamics of the level of SMAP in the organism of the animals throughout the early stages of ontogenesis (embryo­genesis and metamorphosis) of Xenopus laevis. It was noticed that after slight downregulation of SMAP immediately after fertilization of the roe, on the forth­coming stages till finalization of the neurula its level remained unchanged, on the same values. Thereafter continuous and significant (p < 0.01 and p < 0.001) downregulation of the SMAP level, inter­rupted once in the form of its slight upregulation in the interval between middle of the late tailbud stage (37th stage) and onset of the pre-metamorphosis stage (39th stage; Figure 1), was observed.

Figure 1. Changes of SMAP level in early stages of ontogenesis in Xenopus laevis. **- p < 0.01; *** - p < 0.001 relatively to preceding point.

Role of SMAP in regulation of embryonic development and metamorphosis in Xenopus laevis

In the series of studies incubation of the embryos of Xenopus laevis on the blastula and gastrula stages in fresh water containing  SMAP at a dose of 50 and 100 μg/ml led to delay of their development and, finally, to death of all the em­bryos of this group within 4 days of observation. At the same time the embryos of the intact and control groups left alive and developed normally, without lethality of any specimens.

In the of studies blocking of SMAP activity with the anti-SMAP poly­clonal antibodies in the experimental group realized significant effect on the rate of animal passing through embryo­genesis. Particularly, it was shown that single addition of the anti-SMAP antibodies at a dose of into the incu­bation milieu of the embryos of Xenopus laevis, being on the of deve­lopment, after 14 days resulted in their passing ahead (two stages earlier) of the meta­morphosis stage (50th stage) relatively to the rate of passing of this stage by the animals of the intact (and control groups (p = 0.01). Along with it, animals of the and experimental groups under effects of SMAP of both doses were on the 49th stage of deve­lop­ment passing ahead metamorphosis by one stage earlier relatively to the animals of intact and control groups (p = 0.01).

So, up to the end of the neurula stage of embryogenesis of Xenopus laevis the level of SMAP in the organism remains unchanged, while its continuous down­regulation on the next stages is observed. Addition of SMAP to the embryos at the stages of blastula and gastrula leads to delay of embryonic development and, finally, to death of all the embryos. Blocking of SMAP activity by anti-SMAP anti­bodies on the stage of pre-metamorphosis of Xenopus laevis leads to noticeable passing ahead of the metamorphosis stage by the embryos.

Discussion

The results of the studies indicate to close involvement of SMAP, realizing serotonin functions on sub-cellular level, in the processes of cell proliferation and differentiation. As the initial stages of embryogenesis are characterized with high indexes of cell proliferation, remaining of the SMAP level stable, in the form of smooth horizontal line during theses stages up to the end of neurula stage shows that such unchanged amount of SMAP is required for molecular support of inten­sive cell proliferation.

As it is known from the literature, on the next after neurula stages of emb­ryogenesis and initial stages of development including metamorphosis, decline of cell proliferation and, in opposite, step-by-step strengthening of cell dif­ferentiation are observed [6]. Gradual downregulation of SMAP on the said stages indicates that its level is synchronized negatively to realization and/or regulation of differen­ti­ation processes, thus giving grounds to making a conclusion that SMAP itself is engaged in negative regulation of cell differentiation, probably, through switching on differentiation-launching genes on the background of SMAP downregulation.

The above stated conclusions, issuing from the results of the of the stu­dies, were confirmed later by the results of the undertaken . Parti­cu­larly, artificial upregulation of the SMAP level in the embryos through its addi­tion to the incuba­tion milieu of embryos, staying on the blastula and gastrula stages, under the both applied doses brought to cessation of the embryo deve­lop­ment and total death of the embryos. So, SMAP upregulation on the initial stages of embryo­gene­sis rea­lizes cytotoxic effects on the embryonic cells.

The observed cytotoxic effects of SMAP on the onset of embryogenesis of Xenopus laevis may be related to induction by SMAP of conformational changes of chromatin trans­ducting it into the condensed inactive form. This idea is con­firmed by our ealier studies in which SMAP administration to the sturgeon juve­niles led to significant downregulation (by over 50%) of the level of mutagenic changes in the somatic cells induced by soil sediments from Baku Bay containing high levels of heavy metals and polyaromatic hydro­carbons relatively to the control group kept under the similar polluted conditions. In the control animals this contamination induced 5-times elevation of mutations relatively to the intact animals kept in fresh water [7]. These earlier obtained data give grounds to make a conclusion that SMAP brings chromatin to the condensed, folded state, this way providing its protection from the effects of adverse factors.

Along with it, antibodies-mediated blo­cking of SMAP activity on the 37th stage of development significantly fomented passing ahead me­ta­­mor­phosis (by two stages earlier) by the embryos of the 3rd experimental group rela­tive­ly to the intact and control animals, this way sup­por­ting the above proposed idea of existing signi­ficant negative regulation of the differentiation pro­cesses of embryonic cells by SMAP, issuing from its downregulation on post-neurula stages of development. Besides, such passing ahead of metamor­phosis, though in somehow fainter degree (by one stage earlier), was noticed for the effects of SMAP at both studied doses. However, these effects of SMAP itself are, probably, due to the pheno­me­non of down-regu­lation of SMAP-accepting receptors resulting from their inter­nalization into the embryo­nic cells under the effect of applied doses of SMAP. So, these results give grounds in the future for appli­ca­tion of anti-SMAP antibodies for the purpose of forced differen­tiation of imma­ture embryonic-resem­bling cells, par­ticularly cancer stem cells, having many common features with the embryonic cells. The idea of possible impact on cancer stem cells through modulating micro­environmental stimuli, leading to their differentiation, has been put forward by different researchers [3,8,9].

References

  1. Fulawka1 L, Donizy P, Halon A (2014) Cancer stem cells – the current status of an old concept: literature review and clinical approaches. Biological Research 47: 66-75.
  2. Pan Q, Li Q, Liu S, Ning N (2015) Concise Review: Targeting Cancer Stem Cells Using Immunologic Approaches. Stem Cells 33: 2085-2092. [Crossref]
  3. 2021 Copyright OAT. All rights reserv
  4. Cojoc M, Mäbert K, Muders MH, Dubrovska A (2015) A role for cancer stem cells in therapy resistance: cellular and molecular mechanisms. Semin Cancer Biol 31: 16-27. [Crossref]
  5. Mekhtiev AA (2000) Revealing in the rat brain protein, possessing with anticonsolidation properties. Bulletin of Exper Biol Med 129:147-150.
  6. Catty D (1991) Antibodies. A Practical Approach, Oxford University Press, Oxford, New York, Tokyo, p. 384.
  7. Sive HL, Grainger RM, Harland RM (2010) Early Development of Xenopus laevis. A Laboratory Manual Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York p. 340.
  8. Mekhtiev AA, Movsum-zadeh SK (2008) Antimutagenic activity of serotonergic system and underlying mechanisms in sturgeon juveniles (Acipenser guelden¬staedti persicus) and goldfish (Carassius auratus). J Evol Biochem Physiol 44: 476-481.
  9. Radosevich J, Aqil M, Bassiony M (2015) Rethinking the tumor stem cell theory. Tumor Biology 36: Suppl. 1: S24.
  10. Sell S (2004) Stem cell origin of cancer and differentiation therapy. Crit Rev Oncol Hematol 51: 1-28. [Crossref]

Editorial Information

Editor-in-Chief

Ciro Rinaldi
University Federico II Naples

Article Type

Research Article

Publication history

Received date: May 25, 2017
Accepted date: June 14, 2017
Published date: June 19, 2017

Copyright

© 2017 Aminov AV. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Citation

Aminov AV and Mekhtiev AA (2017) Application of embryonic models for elaboration of anticarcinogenic preparations of desired action. Hematol Med Oncol 2: DOI: 10.15761/HMO.1000133

Corresponding author

Mekhtiev AA

Department of Molecular Basis of Integrative Activity, A.I. Karayev Institute of Physiology, Azerbaijan NAS 78 Sharif-zadeh St. Baku, AZ1100 Azerbaijan, Baku.

E-mail : bhuvaneswari.bibleraaj@uhsm.nhs.uk

Figure 1. Changes of SMAP level in early stages of ontogenesis in Xenopus laevis. **- p < 0.01; *** - p < 0.001 relatively to preceding point.