online ISSN 2415-3176
print ISSN 1609-6371
logoExperimental and Clinical Physiology and Biochemistry
  • 9 of 12
Up
ECPB 2018, 82(2): 64–70
https://doi.org/10.25040/ecpb2018.02.064
Research articles

Peculiarities of Changes in Ca2 +-activated, Mg2+-dependent ATP-hydrolase Activity of the Endoplasmic Reticulum of Blood Lymphocytes in the Patients with Ovarian Cancer

R. BARYLYAK, D. VOROBETS, O. ONUFROVYCH, Z. VOROBETS
Abstract

The activity and kinetic parameters of endoplasmic reticulum Ca2+,Mg2+-ATPase (SERCA) of blood lymphocytes in patients with ovarian cancer (OC) have been examined. It is known that cancer cells usually have the disturbance of calcium homeostasis. Although the changes of Ca2+-signaling can not be considered as a prerequisite for the initiation of cancer, the consequences of the changes of the structure and functioning of Ca2+-channels or other Ca2+-transporting systems in cancer cells can be significant in the tumor progression. SERCA is an intracellular Ca2+ -store and a multifunctional organelle that performs a variety of Ca2+-dependent functions involved in the homeostatic and signaling mechanisms. Plasma membrane Ca2+, Mg2+-ATPase and endoplasmic reticulum Ca2+,Mg2+-ATPase are two Ca2+-transporting structures which regulate Ca2+-concentration in cytosol. It is assumed that the endoplasmic reticulum Ca2+,Mg2+-ATPase in the lymphocytes plays a leading role in the process of reducing the Ca2+ level in cytosol. It has been found that SERCA activity of blood lymphocytes in the almost healthy individuals has been (2.25 ± 0.17) μmol Рі / min per 1 mg of protein. According to the stats, the SERCA activity of blood lymphocytes in the patients with OC has been significantly different from the control group. Its value has been decreased by 1.5 times (p < 0.001), compared to the almost healthy subjects. The decrease of the plasma membrane Ca2+,Mg2+-ATPase activity that has been demonstrated earlier and in SERCA activity of blood lymphocytes in the patients with OC, indicates the increase of [Ca2+]i in cytosol of lymphocytes.

Both Ca2+,Mg2+-ATPase and Na+,K+-ATPase use the energy of ATP hydrolysis to carry ions against their electrochemical gradient. Therefore, the changed concentration of ATP in the incubation medium may affect the rate of ATP-hydrolysis reaction. The dependence of Ca2+,Mg2+-ATPase activity on the substrate concentration (ATP) in the incubation medium has been determined by the value of the affinity constant of the substrate (KATP). It has been calculated by determining the Ca2+,Mg2+-ATPase activity in the incubation medium containing the substrate in the concentration ranging from 0.1 to 5.0 mM (with a constant concentration of Ca2+ - 0.05 mM and Mg2+- 5 mM).

It has been found that the increase of ATP concentration in the incubation medium in the concentration ranging from 0.1 to 4.0 mM has led to the gradual monotonic increase of SERCA activity of lymphocytes in the almost healthy subjects. The maximum ATP-hydrolase activities of SERCA of blood lymphocytes in the healthy donors and patients with OC have been noticed when ATP concentration in the incubation medium has been 4 mM. Researching of the concentration dependence of Ca2+, Mg2+-ATPase activity on ATP concentration shows that throughout the range of the substrate concentrations, the SERCA activity in the patients with OC has been decreased compared to the control group.

In order to determine the main kinetic parameters of ATP hydrolysis using SERCA and to elucidate a possible mechanism of the change of the enzyme activity of blood lymphocytes in the patients with OC, the curves of concentration dependencies have been depicted on the Lineweaver-Burk plot.

It has been found that the maximum rate of ATP-hydrolysis of SERCA in the healthy subjects has been (2.2 ± 0.2) μmol Рі/min per 1 mg of protein. The maximum rate of ATPase reaction catalyzed by SERCA in the patients with OC has decreased in 1.3 times (p < 0.05) compared to the control group. According the the stats, the affinity constants of SERCA for ATP in both groups have been also significantly different, what indicates their different affinity for the substrate. Specifically, SERCA affinity to ATP in the healthy donors has been (0.13 ± 0.04) mM and its value in the patients with OC has been in 2.15 times higher.

The obtained data indicate that inhibition of SERCA activity in lymphocytes occurs caused by both reduction of the maximum rate of ATP hydrolysis and reduction of SERCA affinity for the substrate (the affinity constant for ATP has been increased).

Article recieved: 08.05.2018

Keywords: ovarian cancer, lymphocytes, endoplasmic reticulum, Ca2+, Mg2+-ATPase

Full text: PDF (Ukr) 398K

References
  1. 1. Ait Ghezali L, Arbabian A, Roudot H, Brouland JP, Baran-Marszak F, Salvaris E et al. Induction of endoplasmic reticulum calcium pump expression during early leukemic B cell differentiation. J Exp Clin Cancer Res. 2017;36(1):87. doi.org/10.1186/s13046-017-0556-7
  2. 2. Arbabian A, Brouland JP, Gelebart P, Kovàcs T, Bobe R, Enouf J et all. Endoplasmic reticulum calcium pump and cancer. Biofactors. 2011;37(3):139-49. doi.org/10.1002/biof.142
  3. 3. Barylyak RV, Iefremova UP, Onufrovych OK, Melnyk OV, Vorobets DZ, Vorobets ZD. Characterization of Са2+,Мg2+-ATPase of blood lymphocytes in women with ovar- ian cancer. Regulatory Mechanisms in Biosystems. 2018;9(1):85-9. doi.org/10.15421/021812
  4. 4. Bergner A, Huber RM. Regulation of the endoplasmic reticulum Ca2+-store in cancer. Anti-Cancer Agents in Medicinal Chemistry. 2008;8 (7):705-09. doi.org/10.2174/187152008785914734
  5. 5. Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Scandinavian Journal of Clinical and Laboratory Investigation. 1968;21(97):77- 89.
  6. 6. Chemaly ER, Troncone L, Lebeche D. SERCA control of cell death and survival. Cell Calcium. 2018; 69:46-61. doi.org/10.1016/j.ceca.2017.07.001
  7. 7. Dang D, Rao R. Calcium-ATPases: Gene disorders and dysregula- tion in cancer. Biochimica et Biophysica Acta. 2016;1863(6):1344-50. doi.org/10.1016/j.bbamcr.2015.11.016
  8. 8. Fafula RV, Yefremova UP, Lychkovska NE, Melnyk OV, Vorobets ZD, Kulachkovskyi OR. Methodological approach to the study of the enzymatic spectrum of lymphocytes at pathological conditions using the detergent saponin (ultrastructural study). Bulletin of Biology and Medicine. 2012;1(96):163-6 (in Ukrainian).
  9. 9. Fafula RV, Lychkovska NE, Yefremova UP, Vorobets ZD. Enzymatic activity of Ca2+-transporting Mg2+-dependent ATPase of peripheral blood lymphocytes in patients with rheumatoid arthritis. Medical chemistry. 2011; 13 (4): 69-72 (in Ukrainian).
  10. 10. Lapovets L, Lutsyk B. Laboratorian immunology. K.: Areal; 2004. 173. (in Ukrainian).
  11. 11. Lipskaia L, Hulot JS, Lompre AM. Role of sarco/endoplasmic reticulum calcium content and calcium ATPase activity in the control of cell growth and proliferation. Pflugers Arch. 2009;457(3):673-85. doi.org/10.1007/s00424-007-0428-7
  12. 12. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenolreagent. The Journal of Biological Chemistry. 1951;193:265-75.
  13. 13. Mishell BB. Selected methods in cellular immunology. San Francisko; W. H. Freeman and Company. 1980; 486-91.
  14. 14. Monteith GR, Davis FM, Roberts-Thomson SJ. Calcium channels and pumps: Changes and concequences. The Journal of Biological Chemistry. 2012;287(38):31666-73. doi.org/10.1074/jbc.R112.343061
  15. 15. Monteith GR, Prevaskaya N, Roberts-Thomson SJ. The calcium-cancer signalling nexus. Nature Reviews Cancer. 2017; 17:367–8016. doi.org/10.1038/nrc.2017.18
  16. 16. Padanyi R, Paszty K, Hegedus L, Varga K, Papp B, Penniston JT et al. Multifaceted plasma membrane Ca2+ pumps: From structure to intracellular Ca2+ han- dling and cancer. Biochimica et Biophysica Acta. 2016;1863(6):1351-63. doi.org/10.1016/j.bbamcr.2015.12.011
  17. 17. Papp B, Brouland J-P, Arbabian A, Gélébart P, Kovács T, Bobe R et al. Endoplasmic reticulum calcium pumps and cancer cell differentiation. Biomolecules. 2012;2 (1):165-86. doi.org/10.3390/biom2010165
  18. 18. Paryzhak SYa, Yakubets OI, Vorobets ZD. Markers and regulatory mechanisms in ovarian carcinoma. The Ukrainian Biochemical Journal. 2014;86(4):36-50 (in Ukrainian). doi.org/10.15407/ubj86.04.036
  19. 19. Peters AA, Milevskiy MJ, Lee WC, Curry MC, Smart CE, Saunus JM et al. The calcium pump plasma membrane Ca2+-ATPase 2 (PMCA2) regulates breast cancer cellproliferation and sensitivity to doxorubicin. Scientific Reports. 2016;6: 25505-17. doi.org/10.1038/srep25505
  20. 20. Pinto MC, Kihara AH, Goulart VA, Tonelli FM, Gomes KN, Ulrich H et al. Calcium signaling and cell proliferation. Cellular Signaling. 2015;27(11):2139-49. doi.org/10.1016/j.cellsig.2015.08.006
  21. 21. Rathbun W, Betlach V. Estimation of enzymically produced orthophosphate in the presence of cysteine and adenosine triphosphate. Anal. Biochem. 1969; 28: 436-47. doi.org/10.1016/0003-2697(69)90198-5
  22. 22. Seo JA, Kim B, Dhanasekaran DN, Tsang BK, Song YS. Curcumin induces apoptosis by inhibiting saeco/ando- plasmic reticulum Ca2+-ATPase activity in ovarian cancer cells. Cancer Lett. 2016;371(1):30-7. doi.org/10.1016/j.canlet.2015.11.021
  23. 23. Wang H, Haas M, Liang M, Cai T, Tian J, Li S et al. Ouabain assembles signaling cascades through the caveolar Na+/K+–ATPase. J Biol Chem. 2004;279(17):17250-59. doi.org/10.1074/jbc.M313239200


Програмування - Roman.im