Study of molecular mechanisms of anti-tumor effect of hemorphin-7 in vivo

Keywords:  hemorphin, calcineurin, calmodulin, sarcoma-45, doxorubicin, µ-opioid receptors (MOR) 

Hemorphin-7 (H7, YPWTQRF) is a member of hemorphins family, an endogenous nonclassical opioid peptide derived from haemoglobin (Hb), demonstrating a wide spectrum of biological activities [For review see Ref. 26]. H7 exhibits anti-inflammatory and immunoregulatory properties [30, 17]. It was shown the small, but significant passage of hemorphin-7 across the blood-brain-barrier [26], however a- and β- globin mRNAs were identified in mouse brain, implying the synthesis of globin in the central nervous system (CNS) [27].  Brain catepsin B was shown to participate in the generation of several hemorphins, including hemorphin-7 from LVV-hemorphin-7 (LVV-H7) in vitro [2]. It should be noted that LVV-H7 contains the amino acid sequence of all known hemorphins. By using in vivo microdialysis in combination with electrospray mass spectrometry in vivo processing of hemorphin-7 from LVV-hemorphin-7 in rat brain and blood was shown [31]. It has been shown that H7, like other hemorphins, in vitro modulates Ca²⁺/calmodulin (CaM)-dependent protein phosphatase calcineurin activity by binding to CaM, exhibiting a concentration-dependent biphasic response on enzyme activity [5, 7, 10, 17]. Calcineurin is known as a key enzyme in the signal transduction cascade leading to T cell activation. This enzyme controls gene expression of several cytokines (interleukin (IL)-2, tumor necrosis factor a, (TNFa), IL-4, IL-5  etc.) and other regulatory proteins via dephosphorylation and nuclear translocation of  NFATc (nuclear factor of activated T cell) family members [33]. Importantly, H7 was shown to regulate DNA-binding activity of NFAT, AP-1 and NF-kB transcription factors in stimulated human Jurkat T cells [6]. Moreover, it has been demonstrated that H7 in nM concentrations negatively regulates IL-2 promoter transcriptional activity [17]. It has been revealed that H-7 in µM concentrations significantly (2,5 fold)  inhibits in vitro DNA methylation in pathophysiology of tumor (sarcoma-45) [18]. It is well known that methylation of DNA often correlates with the lack of transcriptional activity, and inhibition of DNA methylation is a specific characteristic of anti-tumor drugs [15, 21]. It has been reported that some of the members of hemorphins family (e.g. LVVYPW (MP-2), VVYPWTQ (valorphin)) exert anti-tumor properties [8, 11, 25].  It has also been shown that µ-opioid receptors (MOR) agonists exert anti-tumor properties [22-23]. Because H7 is the most potent among the hemorphins in MOR binding [37], and, moreover, H7 has a capacity to induce the release of -endorphin from the pituitary tissue into circulation [26], it is suggested that H-7 may exhibit anti-tumor properties as well. This view is supported by several reports from different laboratories indicating that Ca2+/CaM/calci-neurin/NFAT pathway is involved in pathophysiology of cancer [24, 28].

It is to be noted that hemorphins change the sensitivity of CaM to its antagonists (trifluoperazine (TFP), chlorpromazine, W-7, vinblastine and vincristine) [3]. All of these compounds are drugs that are implicated in the treatment of different diseases, including cancer [20, 35]. Therefore in 1992, we predicted that hemorphins had good prospects for applied medicine as drugs without side effect [3].

Taking into consideration all of the above mentioned the aims of the  present study were: 

1. to examine the effects of intraperitoneal (ip) injections of synthetic LVV-H7 and H7 alone  and H7 in combination with anti-tumor agent doxorubicin (DOX) on tumor growth in rats bearing S-45; 
2. to study the effect of LVV-H7 and H7 on plasma and brain calcineurin activity in sarcoma-45 bearing rats; 
3. to determine the in vivo effect of LVV-H7 and H7on thermodynamic parameters of DNAs, isolated from S-45 and S-45 treated with  hemorphin (LVV-H7 and H7)  and H7+DOX; 
4. to examine if LVV-H7 and H7 can directly bind to DNA and form DNA-hemorphin complex.

Materials and Methods

In the experiments the synthetic hemorphins (H7 and LVV-H7) and anticancer drug DOX (anthracycline antibiotic, trade name Adriamycin) were used. The experiments were carried out on Wistar line rats of both sexes weighing 100-120 g at the time of experiment. Rats provided by Animal House of the Institute of Biochemistry NAS RA, were caged in groups of 5 with food and water given ad libitum. Rats were randomly divided into 5 groups: control group (healthy rats, n=6) and 4 experimental groups inoculated with sarcoma-45 (S-45) by the method of Chernov W., 1971 [14]. The first experimental group (n=10) received daily single ip injection of LVV-H7 (1mg/kg) dissolved in saline (0.9% NaCl) (0.5 ml), during 8 days. The second experimental group (n=10) received daily single ip injection of H7 (1mg/kg) dissolved in saline, during 8 days. The third experimental group (n=10) received daily mutual ip injection of H7 (1mg/kg) + DOX (2mg/kg) during 8 days. The fourth experimental group (n=10) received daily 0.5 ml of saline injection. The rats in control group were injected by saline (0.5 ml). On the 14th day of the experiment animals were decapitated. The brains and tumors were rapidly removed, frozen and stored at -70^o C until use. The trunk blood was immediately collected into sodium citrate (3.2%)-coated vacutainer tubes. Then, the blood samples were centrifugated at 1500 rpm for 10 min, separated into aliquots and stored at -70^o C. 

Calcineurin Activity 

Calcineurin activity was measured by spectrofluorimetric assay using 4-methylumbelliferyl phosphate (4-MUP) as a substrate  [1]. A typical enzyme assay was performed in 1 ml of incubation mixture, containing 50 mM Tris-HCl, pH 7.5, 0.5 mM DTT, 1 mg/ml bovine serum albumin (BSA), 1 mM MgCl2, 0.3 mM CaCl2, 1 µM 4-MUP, and necessary amount of enzyme (100 000 x g soluble brain protein fraction or plasma). One unit of enzyme activity is defined as amount of enzyme that caused the formation of 0.1 nM of  4-methylumbelliferon (4-MU) at 32^o C for 1 h. The quantity of 4-MU was determined fluorimetrically using a Perkin-Elmer MPF-44A spectrofluorimeter. The fluorescence was measured at 445 nm (Exitation at 365 nm). As control the substrate and enzyme were incubated separately. 

Thermodynamic investigation of DNA

Excess heat capacity (ΔCp) versus temperature (ToC) profiles for the thermally induced transitions of DNAs isolated from S-45 and S-45 treated with hemorphins (LVV-H7 and H7) alone or with H7 in combination with DOX were measured by using a differential scanning microcalorimetry (DSM). Measurements were conducted on a DASM-4 microcalorimeter (Russia). In these experiments, the heating rate was 1oC/min at temperature ranges 10-100^oC. Enthalpies (ΔH) were calculated from the areas under the experimental ΔCp versus ToC curves using Scal Dos software. The DNAs were dissolved in 0.1 M sodium chloride–sodium citrate (SSC) buffer (pH 6.9) containing 15 mM NaCl, 1.5 mM sodium citrate. The concentration of solution of DNAs isolated from S-45 was 288 μg/ml and concentrations of DNAs isolated from S-45 treated with H7, LVV-H7 and H7+DOX were in the range of 300-304 μg/ml respectively. 

Detection of DNA-peptide complex

The formation of DNA-H7 and DNA-LVV-H7 complexes in vitro was detected by differential absorbance measurements, which were conducted on a SPECORD UV/VIS spectrophotometer (Germany). Absorbance was measured at 200-350 nm. DNA (at concentration of 28 μg/ml), isolated from healthy rats’ liver, was reacted with LVV-H7 or H7 in 1 mM NaCl (pH 6.9). Registration of DNA-H7 and DNA-LVV-H7 complexes was detected in µM concentration ranges of peptides.

The results were analyzed using Origin version 6.1 software. Statistical significance – p‹0.05. All data were expressed as means ± SEM.

Results and Disscussion

The effects of LVV-H7 and H7 alone, as well as the effect of H7 in combination with DOX have been studied on tumor growth after 8 days of treatment of rats bearing S-45. As one can see (fig.1), treatment of rats with H7 or H7+DOX induced statistically significant inhibition of tumor growth, by 44, 9 % and 57, 3% respectively in comparison with S-45 bearing rats, which received daily single injection of 0,9% NaCl during 8 days. It is suggested that H7 and DOX act synergistically. It should be noted that DOX alone induces the inhibition of tumor growth on 52% (data not shown). We did not observe statistically significant inhibition of tumor growth by treatment of rats with LVV-H7. The mechanism of DOX action has been linked to DNA damage, topoisomerase II inhibition, and irons sequestration with subsequent free radical generation, rendering cells more vulnerable to apoptosis [19, 32, 36]. In addition, it has been reported that DOX pretreatment sensitize prostate cancer cell lines to TRAIL (TNF-related apoptosis inducing ligand)-induced apoptosis, which correlates with the loss of anti-apoptotic protein cFLIP (cellular FLICE inhibitory protein) expression. Moreover, DOX generates a pro-apoptotic phenotype by phosphorylation of EF-2 (elongation factor-2), which in turn also degrades cFLIPs [36,19].  Importantly, CaM antagonist TFP modulates cytotoxicity of DOX in vitro and anti-tumor effect of DOX in vivo and this effect is dependent on the level of DOX resistance [16]. Another CaM antagonist vincristine attenuates DOX cardiotoxicity. Treatment with DOX+vincristine activated pro-survival signal Akt (protein kinase B) and diminished cytochrome C release. It is suggested that these findings may have clinical implication [13]. As one can see (Fig.1) H7 potentiates the anti-tumor effect of DOX. Because hemorphins are suggested to be allosteric regulators of CaM, and have a capacity to change the sensitivity of CaM to its antagonists (e.g. TFP, vinctistine and etc.), it is suggested, that H7 may make the action of DOX less toxic, acting by  binding  to  CaM.   Using  the  ICPL  (isotope  coded 

 Fig.1. Effect of hemorphin (LVV-H7 or H7) alone and H7 in combination with DOX on tumor growth in rats bearing S-45, after 8 days of in vivo treatment. * p‹0.05.

protein label) technology with MALDI-MS-MS mass spectrometric analysis, ferritin H chain is identified as a potential target for hemorphin in mouse brain [4]. This protein has already been reported to be involved in the pathophysiology of cancer [29]. In addition, melanoma-derived ferritin H chain isoform is able to stimulate up regulation of p53 andmediate apoptosis involving Fas (CD95) [12]. It is thought that H7 by up regulation of ferritin expression may mediate the process of apoptosis in cancer cells. It should be noted that β-endorphin has been recorded to increase apoptosis in lung cancer cells [23]. H7 may affect apoptotic processes by inducing the release of β-endorphin as well. Thus, synergistic action of H7 and DOX in inhibition of tumor growth may be based on different mechanisms (e.g. increase in apoptosis, release of β-endorphin, regulation of ferritin expression etc.). 

In the present work we have studied the influence of ip administration of hemorphins (H7 and LVV-H7 ) on calcineurin activity in pathophysiology of S-45. We have observed reduction of calcineurin activity in brain by 58,16% and in plasma by 63, 44% respectively, in comparison with calcineurin activity in brain and plasma of healthy rats, treated with saline (Fig.2 A and B). These data correlate with literature data, indicating to down-regulation of calcineurin activity in pathophysiology of cancer 28. It has been demonstrated for the first time that both H7 and LVV-H7 are able to recover in vivo calcineurin activity in the brain and plasma of S-45 bearing rats. Both H7 and LVV-H7 after 8 days of treatment of S-45 rats, induce increase in brain (by 20,1% and 36,83% respectively)  and  plasma  (by  47,44%  and  44,14% respectively)  calcineurin 

Fig. 2. The effect of ip administration of LVV-H7 and H7 on calcineurin activity in the brain (A) and plasma(B).  

** p‹ 0.01; *** p‹0.001.

activity in comparison with brain and plasma calcineurin activity of S-45 untreated rats. The data obtained confirm our previous findings that calcineurin is involved in the molecular mechanisms of hemorphins action in physiology and pathophysiology 5, 8-10. It is well known that calcineurin participates in gene expression and production of IL-2 via calcineurin/NFAT pathway, so now it is clear why anti-cancer effect of LVVYPW (MP-2) is bound with restoration of IL-2 synthesis suppressed in tumor bearing organism [16]. LVVYPW induce the increase of calcineurin activity and calcineurin, in turn, stimulates IL-2 synthesis on gene transcription level. The data obtained give as reason to suggest that anti-tumor effect of hemorphins is associated with the regulation of calcineurin activity by hemorphins. However in the present study we have shown, that though LVV-H7 demonstrates the inhibition of tumor growth (by 23,8%), the data obtained are not statistically significant. At the same time it has been shown that LVV-H7, likewise H7, in vivo recovered calcineurin activity. To clear up these results, there is need for further investigations.

In order to reveal if  H7 and LVV-H7, likewise LVVYPW [8], may in vivo directly affect DNA structure, we have studied the thermodynamic characteristics of DNAs, isolated from S-45 bearing rats and S-45 bearing rats treated with H7, LVV-H7 and H7 + DOX.

Using differential scanning microcalorimetry (DSM) we revealed the changes in heat capacity (ΔCp) of  DNAs, isolated from S-45 and S-45 treated with LVV-H7, H7 and H7+ DOX (Fig.3). Using automated data processing software Scal Dos the values of enthalpies (ΔH) of studied DNAs was determined. Data obtained give us reason to suggest that as a result of in vivo treatment of S-45 with LVV-H7, H7 and H7+DOX, the interaction of DNA with peptides takes place. The values of enthalpy for DNAs, isolated S-45, S-45 treated with H7, H7 +DOX and LVV-H7, were 9,  23 kcal/mol,  10,17 kcal/mol, 

Fig. 3.DSM thermograms for DNAs isolated from S-45 and S-45 treated with LVV-H7, H7, H7+DOX. Enthalpies (ΔH) were calculated from the areas under the experimental 

ΔC_p versus T^oC curves.

10,1 kcal/mol and 12,8 kcal/mol respectively. It should be noted that the value of enthalpy for DNA isolated from S-45 treated by LVVYPW (LVV-H3) was found out to be 16,2 kcal/mol [8]. The data obtained give us reason to suggest that as a result of in vivo treatment of S-45 with LVV-H7  the hydrophobic interaction of DNA with peptide, due to N-terminal  LVV amino acid sequence, takes place, which induces the stabilization of DNA by increasing value of enthalpy (12,88 kcal/mol) in comparison with enthalpy of S-45 DNA (9,23 kcal/mol). The values of enthalpy for DNAs, isolated from S-45, treated with H7 (10,17 kcal/mol) or H7+DOX (10,1 kcal/mol), have small differences in comparison with that for DNA, isolated from  untreated S-45 (9, 23 kcal/mol). It is suggested that H-7 induces weeker interaction with DNA than LVV-H7 or LVV-H3. It seems very likely that anti-tumor action of H-7 is bound with the ability of this peptide to induce the cancer cells apoptosis. This view is supported by finding that the values of enthalpy for DNAs isolated from S-45 treated with H7 or H7+DOX are almost equal (10,17 kcal/mol and 10,1 kcal/mol, respectively). One can also speculate that the differences in values of enthalpy between DNAs isolated from S-45, treated with H7 and treated with LVV-H7 may, possibly, explain lack of anti-tumor effect for LVV-H7.

We used the spectrophotometric approach to confirm the formation of DNA-H7 and DNA-LVV-H7 complexes in vitro by measuring differential absorbance spectra (Fig.4). Registration of complexes with changed confor¬mation was detected in µM concentration ranges of peptides. 

Fig. 4. Differential absorbance spectra for DNA-LVV-H7 and DNA-H7 complexes in comparison with absorbance spectrum for DNA from healthy rats liver.

Thus, the present study demonstrates that Ca²⁺/CaM/calcineurin/NFAT pathway is involved in the molecular mechanisms of anti-tumor effect of H7 in vivo. One can speculate that H7 via modulation of Ca²⁺/CaM/calcineurin/ NFAT pathways may affect different genes expression, including the expression of tumor suppressing genes. This view is supported by our recent findings indicating that H7 is able to regulate DNA-binding activity of NFAT, AP-1 and NFkB transcription factors in stimulated Jurkat T cells [6]. The ability of H7 to induce the inhibition of DNA methylation and to make changes in thermodynamic parameters of DNA may also affect transcriptional activity. 

Taking into account all above mentioned, we propose that anti-tumor effect of  H7 is based on implication of different mechanisms directed to recover the homeostatic disturbance in the organism in pathophysiology of  tumor.

References

1. Anthony F.A., Merat D.L., and Cheung W.Y. A spectrofluorimetric assay of calmodulin-dependent protein phosphatase using 4-methylumbelliferyl phosphate. Anal. Biochem., 1986, 155, p. 103-107.
2. Barkhudaryan N. Hemorphins: Processing and possible mechanism of action in the brain and immune system. Proc. Int. Conf. Biochemical and Molecular-Biological Aspects of the Brain Immune System (A. Galoyan Ed.), Yerevan, 2001, p.132-139.
3. Barkhudaryan N., Barsegian K., Avetisian N., Zakaryan T., Galoyan A. The changes of the sensitivity of calmodulin against its antagonists under the influence of hypothalamic coronaro-constrictory peptide factors. Neurokhimiya (RAS & NAS RA), 1992, 11, p.228-234.
4. Barkhudaryan N., Dosch D., Gabrielyan A., Kellermann J., Lottspeich F. Study of  the effect of hemorphins on mouse brain proteome by using different proteome analysis approaches. Proc. Int. Conf.” Biotechnology and Health” & DAAD Alumni seminar,  Yerevan, 2008, pp.36-41. 
5. Barkhudaryan N., Gambarov S., Gyulbayazyan T., Nahapetyan K. LVV-hemorphin-4 modulates Ca²⁺/calmodulin-dependent pathways in the immune system by the same mechanism as in the brain. J. Mol. Neurosci., 2002, 18:203-210. 
6. Barkhudaryan N. H.,  Hunanyan O.V., Gabrielyan A.V., Serfling E. Hemorphin-7 regulates DNA-binding activity of NF-AT, AP-1 and NF-κB transcription factors in stimulated  Jurkat T cells. Blood  (Ministry Health RA), 2010, 10, p. 70-74.
7. Barkhudaryan N., Orosz F., Liliom K., Barsegyan K., Ovadi J., Galoyan A. New data on binding of hypothalamic coronaro-constrictory peptide factors with calmodulin. Neurokhimiya (RAS & NAS RA), 1991, 10, p.155-166.
8. Barkhudaryan N.H., Sarukhanyan F.P., Garybyan D.V., Stepanyan H.M., Grigoryan I.E., Dalyan Y.B. LVVYPW demonstrates the ability to affect DNA structure. Proc. Int. Conf. “Biotechnology and Health” & DAAD Alumni seminar, Yerevan, 2009, p. 21-29.
9. Barkhudaryan N., Zakaryan H., Sarukhanyan F., Gabrielyan A., Dosch D., Kellermann J., Lottspeich F. Hemorphins act as homeostatic agents in response to endotoxin-induced stress. Neurochem. Res., 2010, 33, p. 925-933.
10. Barsegyan K., Barkhudaryan N.,  Galoyan A. The investigation of the effect of native and synthetic coronaro-constrictory peptide factors on Ca2+, calmodulin-dependent phosphoprotein phosphatase activity. Neurokhimiya (RAS & NAS RA), 1992, 11, p.141-149.
11. Blishchenko E.Yu.,  Sazonova O.V.,  Kalinina O.A. et al.  Anti-tumor effect of valorphin in vitro and in vivo. Combined action with cytostatic drugs, Cancer Biology & Therapy, 2005, 4, p. 118-124.
12. Bresgen N., Ohlenschläger I., Fiedler B., Wacht N., Zach S., Dunkelmann B., Arosio P., Kuffer E., Lottspeich F., Ecke P.M. Ferritin – a  mediator of apoptosis? J. Cell Physiol., 2007, 212, p.157-164.
13. Chatterjee K., Zhang J., Tao R., Honbo N., Karliner J.S. Vincristine attenuates doxorubicine cardiotoxicity. Biochem.Biophys. Res-Commun., 2008, 373, p.555-560.
14. Chernov W.A. In: Methods of experimental chemotherapy. Moscow, 1971, p.357-403.
15. Dong W., Tu S., Xie J., Sun P., Wu Y., Wang L. Frequent promoter hypermethylation and transcriptional downregulation of BTG4 gene in gastric cancer. Biochem. Biophys. Res. Comun., 2009, 387, p.132-138.
16. Ganapathi R., Schmidt H., Grabowski D., Melia M., Ratliff N. Modulation in vitro and in vivo of cytotoxicity but not cellular levels of doxorubicin by calmodulin inhibitor trifluoperazine is dependent on the level of resistance. Br. J. Cancer, 1988, 58, p.335-400.
17. Hunanyan O.V. Hemorphin-7 regulates interleukin-2 promoter activity by Ca2+/calmodulin/calcineurin/NFAT signalling pathway. Medical Sci.Arm. NAS RA, 2011, LI (N3): 43-50.
18. Hunanyan O., Garibyan D., Nersesyan L., Stepanyan H., Sarukhanyan F., Zakaryan H., Barkhudaryan N. Hemorphin-7 may affect transcriptionally activity in pathophysiology by inhibiting of DNA methylation. In: Biotechnology and Health, & DAAD Alumni seminar, Yerevan,  2008, p.148-149.
19. Kelly M.M., Hoel B.D., Voelkel-Johnson C. Doxorubicin pretreatment sensitizes prostate cancer cell lines to trail induced apoptosis, which correlates with the loss of c-FLIP expression. Cancer Biol.Ther., 2002, 1, p.520-527.
20. Lazo J.S., and Larner J.M. Individual antineoplastic drugs. In: Human Pharmacology, Molecular to Clinical, 3rd ed. (Brody T.M., Larner J., and Minneman K.P., eds.), Mosby, St. Louis, MO, 1998, p, 599-613.
21. Lin J., Lai M.,  Huang Q., Ruan W., Ma Y., Cui J. Reactivation of IGFBP7 by DNA demethylation inhibits human colon cancer cell growth in vitro. Cancer Biol. Ther., 2008, 7, p. 1896-1900.
22. Maneckjee R., Biswas R., Vonderhaar B.K., Binding of opioids to human MCF-7 breast cancer cells and their effects on growth. Cancer. Res., 1990, 50, p. 2234-2238.
23. Maneckjee R., Minna J.D. Opioids induce while nicotine suppresses apoptosis in human colon cancer. Cell Growth Differ., 1994, p. 1033-1040.
24. Medyouf H., Alcalde H., Berthier C., Guillemin M.C., dos Santos N.R., Janin A., Decaudin D., de The H., Ghysdael J. Targeting calcineurin activation as a therapeutic strategy for T-cell acute lymphoblastic leukemia.Nature Medicine, 2007, 13, p.736-741.
25. Mikhailova A., Belevskaya R.G., Kalyzhnaya M., Fonina L.A., Liashenko V.A., Petrov R.V. Mielopeptide-2 recovers Intwerleukin-2 synthesis and interleukin-2 receptor expression in human T lymphocytes by tumor products or measels virus. J. Immunother., 2006, 29, p.306-312.
26. Nyberg F., Sanderson K., Glamsta E.-L. The hemorphins: a new class of opioid peptides derived from the blood protein hemoglobin. Biopolymers, 1997, 43:147-156.
27. Ohyagi Y., Yamada T., and Goto I. Hemoglobin as a novel protein developmentally regulated in neurons. Brain Res., 1994, 635 p.323-327.
28. Padma S., Sowjanya A.P., Poli U.R., Jain M., Rao B.N., Ramakrishna G. Downregulation of calcineurin activity in cervical carcinoma. Cancer Cell Int., 2005, 5, p. 7-12. 
29. Raje D. Muchtar H., Oshowo A., Clark I. What proportion of patients referred to secondary care with iron deficiency anemia have colon cancer? Chir., M.S., Dis. Colon  Rectum, 2007, 50, p. 1211-1214.
30. Sanderson K., Nyberg F., Khalil Z. Modulation of pheripheral inflammation by locally administered hemorphin-7. Inflamm. Res., 1998, 47:49-55.
31. Sanderson Nydahl, K., Pierson, J., Nyberg, F., Caprioli, R.M., Andren P.E. In vivo processing of LVV-hemorphin-7 in rat brain and blood utilizing microdialysis combined with electrospray mass spectrometry. Rapid Commun. Mass Spectrom., 2003, 17, p. 838-844.
32. Seiter K.Toxicity of the topoisomerase II inhibitors. Expert Opin.Drug.Saf., 2005, 4, p.219-234.
33. Serfling E., Berberich-Siebelt F., Chuvpilo S., Jankevics E., Klein-Hessling S., Twardzik T., Avots A. The role of NF-AT transcription factors in Tcell activation and differentiation. Biochim.Biophys. Acta, 1498, 2000, p. 1-18.
34. Viola J.P.B., Carvalho L.D.S., Fonseca B.P.F., Teixeira L.K. NFAT transcription factors: from cell cycle to tumor development. Braz. J.Med.Biol.Res., 2005, 38, p.335-344.
35. Weiss B., Sellinger-Barnette M., Winkler J.D., Schechter L.E., Prozialek W.C. Calmodulin antagonists: structure-activity relationships. In Calmodulin Antagonists and Cellular Physiology (Hidaka H., and Hartshorne D.J., eds), Academic Press, Inc., Orlando, FL, 1985, p.45-62.
36. White S.J., Kasman L.M., Kelly M.M., Lu P., Spruill L., McDermott P.J., Voelkel Johnson C. Doxorubicin generates a pro-apoptotic phenotype by phosphorylation of EF-2. Free Radic.Biol. Med., 2007, 43, p. 1313-1321.
37. Zhao Q., Garreau I., Sannier F., Piot J.M. Opioid peptides derived from hemoglobin: Hemorphins Biopoly. Peptide Sci., 1997, 43, p.75-98.

Вопросы, ответы, комментарии