Introduction
At present time the study of the properties of peptides [Dudgeon W. D. et al, 2016.] has a great interest. Peptides have the same structure as proteins, but the size of these molecules is smaller. It is also important to note that short peptides, being a natural metabolic product present in the body, cannot be detected in blood or urine. In this regard, the study of the properties has an interest on individual structures on cell cultures.
Peptide IPH PRO contains a complex of low molecular weight peptides and has a normalizing effect on prostate function.
Experimental studies have shown that the IPH PRO peptide regulates metabolic processes in the prostate cells, increase their spare capacity, suggesting that the efficacy of peptide IPH PRO to normalize the functions of the prostate gland in men of different age in violation having a different genesis.
Prostate diseases currently occupy a significant place among urological diseases, tend to increase morbidity and are becoming increasingly important social. A considerable share is occupied by malignant tumors.
In this regard, the aim of the present study was to examination of the cytoprotoctive and oncoprotective and other properties of the peptide.
Study design
1 phase.
To assess the cytostatic and oncoprotective properties of the peptide IPH PRO in relation to the urine-reproductive system, we have chosen embryonic stem cells (ESCs), which belong to the pluripotent type, which means that they can be differentiated into all three primary germ sheets: ectoderm, endoderm and mesoderm, from which organs and glands of the urogenital and other systems are formed in the future. The human embryo reaches the blastocyst stage, from which stem cells are obtained on 5-6 days after fertilization.
In normal gender differentiation, the final structure of all sex structures corresponds to a set of gender chromosomes in cells (XX or XY). Ovarian formation requires a set of chromosomes 46,XX, as well as genetic factors such as DAX-1 and WNT-4 signal molecule. The formation of the male phenotype requires the presence of the Y chromosome, in particular the SRY gene (Sex-determining Region Y), which in combination with the genes SOX9, SF1, WT1 and some others determines the development of testicles from undifferentiated gonads.
The first testicular hormone, the Mullerian Inhibiting Factor (MIF), begins to be produced at 6-7 weeks of fetal development. Activation of this gene requires the functioning of a gene encoding the steroidogenic factor SF1. SF1 belongs to a large family of nuclear receptors, which are DNA-binding transcription factors. It is a protein consisting of 461 amino acids, which has a DNA-binding domain (DBD), a ligand-binding domain (LBD) and two activating domains — AF-1 (function activator 1) and AF-2 (function activator 2).
Approximately by the week 8 of fetal life, Leydig cells in the fetal testicles begin to produce testosterone. In the second half of fetal life, low testosterone levels are maintained by LG secreted by the fetus’ own pituitary gland. Under the action of testosterone wolffian ducts begin to differentiate into testicular appendages, deferent duct and seminal vesicles. The formation of the external genitals also requires the presence of an active metabolite of testosterone — dihydrotestosterone. The effects of testosterone and dihydrotestosterone implemented through the genes of the androgen receptor.
In the presence of genotype XX and the normal structure of long and short shoulders of X chromosomes, bipotent gonads are transformed into ovaries on approximately weeks 10-11 of fetal life. This only occurs in the absence of a factor in the development of the testes (encoded by the gene SRY), testosterone and Mullerian Inhibiting Factor (MIF), and requires the involvement of DAX-1 (a gene which is located in the locus DSS) and molecule WNT-4. The female phenotype is formed independently of the female sex hormones, but the fetal testicles with their hormones affect the initial female potential of undifferentiated gonads.
Thus, the main genes involved in the formation of organs and glands of the urogenital system can be divided into:
- Genes involved in the differentiation of the gonads and the ontogeny of the genital organs:
— male: SRY, SOX9, WT1, gene encoding steroidogenic factor SF1,
— female: DAX-1 gene and WNT-4 molecule gene.
- Genes responsible for protein synthesis of sex -hormones and receptors to these hormones: hormones and receptors for hormones: FSH (gene follicle stimulating hormone), LH (luteinizing hormone gene), AMN (gene antimullerian hormone), AMHR (gene receptor AMHR), TAR (the gene for the androgen receptor), GnRH (gene gonadotropin-releasing hormone), GnRH-R (GnRH receptor gene), LH-R (receptor gene and LH — luteinizing hormone), FSH-R (the receptor gene FSH — follicle stimulating hormone).
2 phase.
The second phase of the experimental study was the evaluation of marker biological active molecules by immunofluorescence method using primary antibodies to SSEA-4 (1:150, Abcam) and p53 protein (1:50, Abcam).
We have selected the following marker biologically active molecules:
1) Stage-specific embryonic antigen-4 (SSEA-4),
expressed on stem cells, is a glycosphingolipid, which is over expressed in some types of cancer and is associated with the progression of the disease.
Recent studies show excessive expression of these antigens in prostate cancer. In addition, increased expression of SSEA-4 on the cell surface is associated with loss of cell-cell interactions and increased migration phenotype, indicating the important role of SSEA-4 in cancer invasion.
In connection with these data, we set a goal to study the effect of peptide regulator of biological processes IPH PRO on the suppression of SSEA-4. For this experiment, we selected cells of sc5 line capable for expressing SSEA-4.
2) Aging of polypotent cells in culture is associated with increased activity of the p53 gene. In mice with mutation in the p53 gene, there was increased resistance to the development of tumors, combined with a reduction in life expectancy. P53 protein plays a key role in endogenous antitumor mechanisms [Donekover et al., 2002]. P53 protein controls the course of cell cycle processes, as well as the absence of damage in the genome that could lead to further development of pathology. Protein p53 is a transcriptional factor that serves as a suppressor of malignant tumors by activating apoptosis in the tissues of the body. The p53 protein is activated when DNA is damaged, as well as when stimuli that can lead to such damage or are a signal of cell aging and a violation of its functional activity [Arshad H. et al., 2010]. P53-dependent apoptosis avoids the accumulation of mutations, and, when they have already occurred, p53-dependent apoptosis allows to eliminate such potentially dangerous cells [Burtis C., Ashwood E., Bruns D., 2006].
The research
To study the properties of the peptide IPH PRO we used the following cell cultures of the Russian collection of vertebrate cell cultures (RKKK P):
SC5
Origin: human, embryonic stem cells (ESCs), blastocyst (5-6 days of development), obtained as a result of IVF
Science. 1998. 282: 1145 — 1147;
Ontogenesis. 2011. 42 (4): 249 — 263;
Cytology. 2012. 54 (1): 5 — 16.
Morphology: colonies of round cells with high nuclear/ cytoplasmic attitude.
Method of cultivation: monolayer; colonies attached to mitotically inactivated (mitomycin-C) feeder layer of mesenchymal cells of the bone marrow of the human embryo.
Cultivation conditions: medium — Knockout Dulbecco’s modified Eagles medium serum — Knockout Serum Replacement 20% other ingredients — NEAA 1%, L-glutamine 2mM, 2 — mercaptoethanol 0.1 mM, bFGF — 8 ng/ml.
The procedure of replanting — mechanical replanting of ESC culture was carried out under the control of a microscope by dividing the colony into fragments using a disposable scalpel and transferring them to a new layer of the feeder; daily change of environment, replanting every 5-6 days.
Cryopreservation — growth medium, 10% DMSO, 5×105 cells/ml in ampoule.
Viability after cryopreservation: 60 % (trypan blue on the zero passage)
Contamination control: bacteria, fungi and Mycoplasma are not detected.
Control of the identity of the species: karyological analysis.
Karyology: 2n= 46, modal chromosome number 46 (98.0+0.9 %), normal human karyotype (46, XX), number of polyploids (0.2 + 0.2%).
DNA profile (STR): Amelogenin: X, X
CSF1PO: 12, 13
D13S317: 8, 11
D16S539: 9, 12
D5S818: 9, 11
D7S820: 8,10,12
THO1: 6, 9.3
TPOX: 10, 11
vWA: 17,17
Other characteristic:
The average time of one cell population doubling is 28.2 hours.
Line immortalized, hosted more than 120 doubling the cell population.
Expression of surface antigens typical for human ESCs: SSEA-4, TRA-1-60 and transcription factors OS-4, Nanog.
The ability to spontaneous differentiation into derivatives of the 3 germinal leaves.
Ability to form in vivo teratomas containing derivatives of 3 germinal leaves.
Field of application: cell biology, embryology.
Collections: Institute of Cytology of the Russian Academy of Sciences (figure 1) (Federal state budgetary institution of science «Institute of Cytology of the Russian Academy of Sciences»)
Method of research
Group for the study:
Group 1 — the measurement of the expression of molecules before the study,
Group 2 — control (addition of nutrient medium, serum albumin incubation),
Group 3 — addition of a control peptide of Glu-TRP dipeptide at a concentration of 100 micrograms (µg);
Group 4-addition of peptide IPH PRO 100 micrograms (mcg).
For the study, IPH PRO and Glu-Trp peptides in the form of a lyophilized powder were used, which were dissolved with sterile water for injection in a volume of 10 ml to a final peptide concentration of 100 µg.
For most dissociated cell cultures, as previously shown, the most effective is the concentration of peptides 100 µg/ml on long-term experience of peptides [Linkova N. S. et al., 2016; Khavinson V. et al. 2017,].
As a control, selected immune protective peptide Glu-Trp with the known and well described in the literature properties [Morozov V. G., Khavinson V. Kh., Malinin V. V., 2000, Khavinson V. Kh., Morozov V. G., 2001, Khavinson V. Kh., Kuznik B. I., Linkova N. With., Pronyaeva V. E., 2013].
PCR-method with the use of own primers and reagents of the Novocasta company and sets of monoclonal antibodies produced by Biosource (Belgium) were used to measure the level of gene expression.
Cell swabs were treated with appropriate primary antibodies according to the standard Protocol:
- Triple washing with phosphate-salt buffer – FSB (pH=7,2) for 5 minutes;
- Permeabilize cells with 0.1% Triton X-100, dissolved in the FSB, for 15 minutes;
- Rinsing in three changes of the FSB (for 5 minutes);
- Incubation in 1% bovine serum albumin (diluted FSB, pH 7.5) for 30 minutes to block nonspecific binding;
- Incubation with primary antibodies, 60 minutes;
- Rinsing in three changes of PBS (5 minutes);
- Incubation with secondary antibodies conjugated with fluorochrome Alexa Fluor 488 (1:1000, Abcam) 30 minutes at room temperature in the dark;
- Rinsing in three changes of the FSB in 5 min;
- paining cell nuclei dye Hoechst 33258 (Sigma, USA) (1:100 from stock solution in dH2O) for 1 minute (a dye used as a fluorescent marker DNA, when you link to which its fluorescence increases).
- Flushing in FSB 5 minutes;
- Conclusion of finished products under the cover glass in the mounting medium Dako Fluorescent Mounting Medium (Dako).
The study of drugs was carried out in a confocal microscope Olympus FluoView FV1000 at an increase of 200, 400, 600. By blue color the expression of the studied marker fluoresces. Conducted measurement of the relative areas of expression in %. The relative area of expression was calculated as the ratio of the area occupied by immune positive cells to the total area of cells in the field of view and expressed as a percentage. The dynamics of gene expression is measured in conventional units, the base level is taken as 10.
Statistical processing of results
Statistical processing of experimental data included calculation of arithmetic mean, standard deviation and confidence interval for each sample and was carried out in Statistica 11.0. If the data were normally distributed, the differences in means were determined by the Student criteria (t).
The results of the study and their discussion of the Effect of the peptide IPH PRO on the expression of genes responsible for the human reproductive system
Thus, in figure 2 and figure 3, has been presented that the peptide IPH PRO significantly increases the expression of genes responsible for the normal formation of both male genital organs and factors that form the hormonal system.
*p<0.05 compared to baseline data;
**p<0.05 compared to control;
***p<0.05 between the indicators of the level of expression in the application of Glu-Trp and IPH PRO.
Figure 2. Total expression of genes involved in the differentiation of the gonads and the ontogeny of male genital organs: SRY, SOX9, WT1, a gene that encodes steroidogenic factor SF1.
*p<0.05 compared to baseline data;
**p<0.05 compared to control;
***p<0.05 between the indicators of the level of expression in the application of Glu-Trp and IPH PRO.
Figure 3. Total expression of genes responsible for protein synthesis of sex -hormones and receptors to these hormones: hormones and receptors for hormones: FSH (gene follicle stimulating hormone), LH (luteinizing hormone gene), AMN (gene antimullerian hormone), AMHR (gene receptor AMHR), TAR (the gene for the androgen receptor), GnRH (gene gonadotropin-releasing hormone), GnRH-R (GnRH receptor gene), LH-R (receptor gene and LH — luteinizing hormone), FSH-R (the receptor gene FSH- follicle stimulating hormone.)
Thus, from the above data it can be seen that under the influence of the peptide IPH PRO in human cell culture there is a statistically significant increase in the expression of genes responsible for the ontogenesis of male genital organs and hormonal background. These data show that the peptide IPH PRO significantly increases the «cascade» of signal molecules in human cell culture, which is necessary for the activation of proliferation and differentiation of stem cells into male genital cells, in particular, prostate cells.
The effect of the peptide IPH PRO on the expression of SSEA-4 in cultures of human cells
Figure 4 shows that the use of the peptide IPH PRO reduces the expression of SSEA-4 on 4 times from the initial level, which is detected in large numbers in prostate cancer and other tumors in the human body.
*p<0.05 compared to baseline data;
**p<0.05 compared to control;
***p<0.05 between the indicators of the level of expression in the application of Glu-Trp and IPH PRO.
Figure 4. The effect of the peptide IPH PRO on the expression of SSEA-4 in cultures of human cells.
Thus, the use of the peptide IPH PRO is expressed oncoprotector character, in particular, in malignant tumors of the prostate according to the level of expression of the marker SSEA-4 on human cell culture.
Effect of IPH PRO peptide on p53 protein expression in human cell cultures
Figure 4 has presented that the use of the peptide IPH PRO increases the production of protein p53, which is a transcriptional factor that serves as a suppressor of malignant tumors by activating apoptosis in the tissues of the body, which leads to a conclusion about the antitumor properties of the peptide under study.
*p<0.05 compared to baseline data;
**p<0.05 compared to control;
***p<0.05 between the indicators of the level of expression in the application of Glu-Trp and IPH PRO.
Figure 5. The effect of the peptide IPH PRO on the expression of p53 in the culture of human cells.
P53-dependent apoptosis also avoids the accumulation of mutations, and, in the case where they have already arisen, p53-dependent apoptosis allows to eliminate such potentially dangerous cells for the body, which allows us to conclude about the cytoprotective effect of the peptide under study.
The data indicate a high oncoprotective activity of the peptide IPH PRO in relation to the cells of the reproductive system of men according to the expression of biological molecules in cell culture.
Conclusion
The studies confirm the high biological activity of the peptide IPH PRO in relation to the control of the normal formation of the reproductive system in humans at the genetic level according to the expression of genes responsible for the formation of male genitals and hormones on cell culture.
The data indicate a high oncoprotective activity of the peptide IPH PRO in respect of prostate cells according to the expression of biological molecules in cell culture.
Literature
Linkova N.S., Drobantseva A. O., Orlova O. A., Kuznetsova E. P., Polyakova O.V., Kvetnoy I.M., Khinson V.Kh. Peptide regulation of functions of skin fibroblasts upon aging in vitro // Cell technologies in biology and medicine. — 2016. — №1. — P. 40-44.
Mutovin G. R. Fundamentals of clinical genetics (genomics and proteomics of hereditary pathology). Textbook for universities in 2 volumes. Issue. 3. Moscow: GEOTAR-media, 2008.
Khavinson V. Kh. Peptide regulation of aging. SPb.: Science, 2009. – p. 50
Arshad H., Ahmad Z., Hasan S.H. Gliomas: correlation of histologic grade, Ki67 and p53 expression with patient survival // Asian Pac J Cancer Prev. — 2010. — Vol.
— N 6. — P 1637-1640;
Lin L, Achermann JC. Steroidogenic factor-1 (SF-1, Ad4BP, NR5A1) and disorders of testis development// Sex Dev. -2008;2(4-5):200-209.
Sivasubramaniyan K, Harichandan A, Schilbach K.Expression of stage-specific embryonic antigen-4 (SSEA-4) defines spontaneous loss of epithelial phenotype in human solid tumor cells//Glycobiology-2015 Aug;25(8):902-17.