ABSTRACT The impact of the aliphatic amino-and oxy derivatives decarboxylated ornithine and Sadenosylmethionine on the rate of the L-cells growth in a tissue culture has been investigated. The known specific inhibitors of the polyamine synthesis enzymes (DFMO and MGBG) were used for control and comparison of efficacy of the substances tested. The nature of action of 1-aminooxy-3-aminopropane (APA) and S-(5-deoxyadenosile)-S-methyl--thioethylhydroxylamine (АМА) used separately or combined one with another was similar to the effect of DFMO and MGBG though less expressed. The degree of inhibition of the Lcells growth as of the 4 th day made 50-60%. By the 7 th day the tendency to not only stabilization of the growth rate but also to the recovery thereof manifested itself. This points to the reversible nature of action of the APA and AMA combinations and constitutes the difference from the effect DFMO and MGBG

E A Ryskina

I P Smirnova

M L Blagonravov

N A Shevkun

O M Kuznetsova

R I Sokuyev

S P Syatkin

T A Lobayeva

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ISSN: 0975-8585 
 
November - December 2014  RJPBCS   5(6)  Page No. 1401 
 
Research Journal of Pharmaceutical, Biological and Chemical 
Sciences 
 
Impact of the Chemical Analogues of Decarboxylated Ornithine and  
S-Adenosylmethionine on the Rate of the L-Cells Growth in the Tissue 
Culture.  
 
S P Syatkin*, I P Smirnova, O M Kuznetsova, T A Lobayeva, E A Ryskina, R I Sokuyev,  
N A Shevkun, and M L Blagonravov. 
 
Peoples’ Friendship University of Russia, 117198, Moscow, Miklouho-Maclay Street, 8, Russia. 
 
 
ABSTRACT 
 
The impact of the aliphatic amino- and oxy derivatives decarboxylated ornithine and S-
adenosylmethionine on the rate of the L-cells growth in a tissue culture has been investigated. The known 
specific inhibitors of the polyamine synthesis enzymes (DFMO and MGBG) were used for control and 
comparison of efficacy of the substances tested. The nature of action of 1-aminooxy-3-aminopropane (APA) 
and S-(5-deoxyadenosile)-S-methyl--thioethylhydroxylamine (АМА) used separately or combined one with 
another was similar to the effect of DFMO and MGBG though less expressed. The degree of inhibition of the L-
cells growth as of the 4
th
 day made 50-60%. By the 7
th
 day the tendency to not only stabilization of the growth 
rate but also to the recovery thereof manifested itself. This points to the reversible nature of action of the APA 
and AMA combinations and constitutes the difference from the effect DFMO and MGBG.  
Keywords: polyamines, putrescine, spermidine, spermine, L-cells, difluoromethylornithine (DFMO), 
methylglyoxal-bis (guanylhydrazone) (MGBG) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
*Corresponding author 
 
ISSN: 0975-8585 
 
November - December 2014  RJPBCS   5(6)  Page No. 1402 
INTRODUCTION 
 
The issue of the analysis and development of approaches to the directed impact on the mechanisms 
of regulation the cell proliferation and differentiation are currently remaining the central issue of the 
fundamental and applied research in the biology and medicine.  
 
It is well known that biogenic polyamines (PA) – spermine and spermidine as well as their precursor 
putrescine – take active participation on the process of regulation of gene activation and are the markers of 
the tumor cells proliferation [1, 2]. 
 
The use of the PA biosynthesis system as the basis for the development of the synthetic analogues of 
PA and inhibitors of the polyamine-producing enzymes resulted in the discovery of a wide range of the 
artificial chemical compounds with the potential antitumor, antiproliferative and even antiparasitic properties 
[3, 4]. 
 
The objective of this study was the analysis of impact of the aliphatic amino- and oxy derivatives 
decarboxylated ornithine and S-adenosylmethionine on the rate of the L-cells growth in a tissue culture.  
 
MATERIALS AND TECHNIQUES 
 
Quantitative measurement of the PA content 
 
The quantitative content of PA in the animal tissues was determined with the use of the fluorescent 
reagent N, N-1-dimethylaminonaphthalene-5-sulphochloride (DANS-С1) produced by "Schuchardt" (Federal 
Republic of Germany) and "Koch-Light" (England) involving the primary and secondary amine groups. A tissue 
sample for analysis and dansylation reaction was prepared according to the method [5[5]]. The PA products by 
"Schuchardt" and "Serva" (Federal Republic of Germany) were used as the tracking substances. The HPLC was 
performed with the use of the chromatographer APC/GPC-204 ("Water-milipore", USA) with the fluorescent 
detector type 420 at the excitation wavelength of 365 nm and emission 495. The chromatographic column of 
the Bondapak C18 (3.9x300 mm) with the pre-column of 3.9x20 mm was used. The chromatography was 
performed during 10 minutes in the linear methanol gradient (75-95%). The incubation mixture was 
preliminarily passed through the column C18 Sep-Pak ("Waters", USA), washed with 5 ml of methanol. The 
dansyl derivatives of amines were eluted with 2 ml of 100% methanol and used for analysis by means of HPLC 
[6]. 
Table 1: Chemical analogues of the decarboxylated ornithine and S-adenosylmethionine 
 
Name  Index Structure 
 
 
 - Difluoromethylornithine  
 
 
1- aminooxy-3-aminopropane 
S-(5- desoxyadenosile)-S-methyl 
- - thioethylhydroxylamine 
 
Methylglyoxal-bis 
(guanylhydrazone) 
 
 
DFMO 
 
 
АРА 
АМА 
 
 
 
MGBG 
 
                            CHF2     
                             |                                  
H2NCH2CH2CH2CHCOOH  
                             | 
                            NH2                                      
2HCINH2OCH2CH2CH2NH2  
 2H2SO4NH2OCH2CH2S
+
Ade  
                                      | 
                                CH3    
                        NH  
                         | | 
H3C-C=N-NH-C-NH2   
       | 
 HC=N-NH-C-NH2   
                         | | 
                             NH                                                                 
 
Fibroblasts of rats of the СЗН line  
 
The cells of the L line were grown on the Eagle nutrient medium with glutamine and 10% bovine 
blood serum. In each bottle we introduced 8000 cells per 2 ml of the nutrient medium. 105 bottles were used. 
ISSN: 0975-8585 
 
November - December 2014  RJPBCS   5(6)  Page No. 1403 
15 bottles were allocated for each of the 4 experimental groups. The first group was the control one. The 
remaining three groups were exposed to DFMO, MGBG and DFMO combined with MGBG, correspondingly. 
The finite concentration of the investigated compounds in the culture medium made 10
-4
 М. The first change 
of the culture medium and introduction of the investigated substances to the new culture medium were 
performed on the 4
th
 day after the initial inoculation. One day after that 3 bottles were taken from each group 
and the number of cells was calculated in the Goryaev chamber, the cell suspension was centrifuged at 1500 
rpm and the suspend was centrifuged for determination of Put and PA by means of HPLC. In all the remaining 
bottles the culture media was changed and the specified substances in the reference quantity were 
introduced. During the next 2 days the same actions were performed. After the 4
th
 day the cultures of the L-
cell tissue grew for another 3 days but without the change of the media and adding the chemical compounds. 
 
  The statistical processing of the findings of the study was performed with the use of the software 
package Statistica 6,0 (StatSoft, USA). The differences between the two samples were considered to be 
statistically significant at р≤0,05 [7]. 
 
Aliphatic amino- and oxy derivatives of PA. 
 
О-substituted hydroxylamines (Table 1), analogues of the products of  enzymic decarboxylation of 
ornithine and S-adenosylmethionine were synthesized at the Zelinsky Institute of Organic Chemistry of the 
Russian Academy of Sciences.   
                                                                                                      
FINDINGS AND DISCUSSION 
 
The analysis of the role of intracellular PA store in the realization of the antiproliferative effect of 
inhibitors of polyamine-producing enzymes was carried out with the use of the о-substituted derivatives of 
hydroxylamine (АМА and АРА compounds) that have demonstrated the maximum regulatory activity in the 
model test-systems [8].  
 
The schema of analysis of impact of 1-aminooxy-3-aminopropane (APA) and (5-deoxyadenosile)-5’-methyl-
-thioethylhydroxylamine (АМА) on the rate of the L-cells growth in the tissue culture and intracelullar 
content of PA was similar to the experiments with DFMO and MGBG (ref. Materials and techniques). The 
results of this series of experiments are presented in the Fig. 1 and 2.  
 
 
 
 
Figure 1: Impact of 1-aminooxy-3-aminopropane (АРА) and 5-deoxyadenosile)-5’-methyl-thioethylhydroxylamine 
(АМА) on the intracellular PA levels and growth of L-cells in the tissue culture. 
 
 Along the abscissa axis the period of cultivation of the L-cells (in days) is indicated, along the ordinate axis: at the 
left – number of L-cells (10
3
 per 1 ml of the culture medium), at the right – polycationic content (рМ per 10
6
 cells). 1 - L-
cells, 2 – Sd, 3 – Sm and 4 – putrescine. 
 
ISSN: 0975-8585 
 
November - December 2014  RJPBCS   5(6)  Page No. 1404 
The nature of action APA and АМА used separately or combined one with another was similar to the 
effect of DFMO and MGBG though less expressed. Thus, the degree of inhibition of the L-cells growth as of the 
4
th
 day made 50-60%, correspondingly (Fig. 1). By the 7
th
 day the tendency to not only stabilization of the 
growth rate but also to the recovery thereof manifested itself (Fig.2). This points to the reversible nature of 
action of the APA and AMA combinations and constitutes the difference from the effect DFMO and MGBG.  
 
 
Figure 2:   Impact of DFMO, MGBG, АРА and АМА on the rate of the L-cells growth in the tissue culture. 
 
Along the abscissa axis the period of cultivation of the L-cells (in days) is indicated, along the ordinate axis – the ratio 
of the cell quantity to the experimental and control group (%). 1 – DFMO, 2 – MGBG, 3 – DFMO+MGBG, 4 – АРА, 5 – 
АМА, 6 – АРА+АМА. 
 
The coefficient of correlation between the degree of inhibition by the investigated compounds of the 
rate of formation of polycations in the cell-free test systems from the tissues with a high mitotic index [8] and 
level of inhibition of the L-cells growth in the tissue culture by the same substances equals to 0.87. This points 
to the presence of the close positive relation between the processes of the PA synthesis and proliferation of 
the L-cells.  
 
Moreover, the changes in the PA content in the L-cells preceded and advanced by 2 days the changes 
in the rate of growth of these cells in the tissue culture. Such “independence” of the cell growth during the 
first two days against the use of the PA-synthesis inhibitors may be provided by that portion of the cell pool 
that had entered the mitosis cycle prior to the stage when the process of division becomes irreversible and 
does not depend on the changes in the PA concentration. Probably, in a single-cell culture with the 
synchronized cell division cycle the antiproliferative activity of inhibitors of the polyamine-producing enzymes 
will be higher.  
 
The results obtained also show that inhibition of the spermidine affects the cell growth rather than 
the inhibition of ODC. This is consistent with the general concept of the PA metabolism system being 
programmed to maintain the optimum level of spermidine according to the proliferative activity of a cell. The 
APA and AMA compounds demonstrated high activity as inhibitors of the PA synthesis in the cell-free test 
systems [8] and growth of the L-cells in the tissue culture. This confirms the prospectivity of the further 
investigations of this group of oxy-amino-derivatives as the new potential antitumor substances. The amino-
oxy-analogue of putrescine: 1-aminooxy-З-aminopropane (AРА) inhibits the activity of the three enzymes of 
the PA biosynthesis: ODC, AMDK and spermine-synthase [9] and like the related substances may be used as 
antitumor agents [10]. 
ISSN: 0975-8585 
 
November - December 2014  RJPBCS   5(6)  Page No. 1405 
The studies concerning the Put and PA content in the L-cells during the period of growth in the tissue 
culture affected by the inhibitors of the key enzymes for biosynthesis of these substances revealed the two 
specific features. Thus, changes in the polycationes concentration in the L-cells and the rate of growth of these 
cells in the tissue culture may be considered as two closely interrelated processes. Moreover, changes in the 
Put and PA concentration are likely to be of primary nature against the L-cells growth rate since they are ahead 
of them in terms of time as was shown by the experiments with inhibitors of the key polyamine-producing 
enzymes [6].  
 
REFERENCES 
 
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[3] Wallace HM, Fraser AV. Polyamine analogues as anticancer drugs. // Biochem Soc Trans. -2003. V-31, 
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[4] Seiler N. Thirty years of polyamine-related approaches to cancer therapy. Retrospect and prospect. 
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[7] Fadeev V. V. Editorial materials // The issues of endocrinology – 2002. – Vol. 48, № 3. – P. 47-48. 
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[9] T.O. Eloranta, A.R. Khomutov, R.M. Khomutov et al. Aminooxy Analogues of Spermidine as Inhibitors 
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[10] DL Kramer, RM Khomutov, YV Bukin et al. Cellular characterization of a new irreversible inhibitor of S-
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Impact of the Chemical Analogues of Decarboxylated Ornithine and S-Adenosylmethionine on the Rate of the L-Cells Growth in the Tissue Culture

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Impact of the Chemical Analogues of Decarboxylated Ornithine and S-Adenosylmethionine on the Rate of the L-Cells Growth in the Tissue Culture

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