Исследование биологической активности липосомного сангвинарина на культурах опухолевых клеток и простейших

Статья в Elpub

Вестник Томского государственного университета. Биология. 2018; : 99-117

Исследование биологической активности липосомного сангвинарина на культурах опухолевых клеток и простейших

Луценко С. В., Черемных Е. Г., Седякина Н. Е., Молдогазиева Н. Т., Громовых Т. И., Фельдман Н. Б.

Аннотация

Получена липосомная форма сангвинарина на основе лецитина и холестерина, характеризующаяся средним размером частиц 108,5±2,2 нм, дзета-потенциалом -34,7±1,4 мВ и способностью к пролонгированному высвобождению действующего вещества. Проведено исследование активности липосомного сангвинарина на культурах опухолевых клеток и клеток простейших. Липосомная форма сангвинарина обладала цитотоксической активностью, близкой к активности свободного вещества, в отношении опухолевых клеток карциномы молочной железы человека линии MCF-7 (IC₅₀ 14,5 и 9,4 мкМ соответственно). Минимальная подавляющая концентрация липосомного сангвинарина в отношении инфузорий P. caudatum составляла 0,49 мкМ. Препарат оказывал выраженное стимулирующее действие на процесс тромбообразования, а также функциональную активность системы комплемента в отношении инфузорий T. pyriformis, в 2 раза сокращая время их полужизни в сыворотке крови. Полученные результаты позволяют рассматривать липосомный сангвинарин в качестве перспективного противоопухолевого и антипротозойного средства.

Список литературы

1. Kosina P., Walterova D., Ulrichova J., Lichnovsky V., Stiborova M., Rydlova H., Vicar J., Krecman V., Brabec M.J., Simanek V. Sanguinarine and chelerythrine: assessment of safety on pigs in ninety days feeding experiment // Food and Chemical Toxicology. 2004. Vol. 42. PP. 85-91. doi: 10.1016/j.fct.2003.08.007

2. Zdarilova A., Vrublova E., Vostalova J., Klejdus B., Stejskal D., Proskova J., Kosina P., Svobodova A., Vecera R., Hrbac J., Cernochova D., Vicar J., Ulrichova J., Simanek V. Natural feed additive of Macleaya cordata: safety assessment in rats a 90-day feeding experiment // Food and Chemical Toxicology. 2008. Vol. 46. PP. 3721-3726. doi: 10.1016/j. fct.2008.09.054

3. Miao F., Yang X.J., Zhou L., Hu H.J., Zheng F., Ding X.D., Sun D.M., Zhou C.D., Sun W. Structural modification of sanguinarine and chelerythrine and their antibacterial activity // Natural Product Research. 2011. Vol. 25. PP. 863-875. doi: 10.1080/14786419.2010.482055

4. Yang X.J., Miao F., Yao Y., Cao F.J., Yang R., Ma Y.N., Qin B.F., Zhou L. In vitro antifungal activity of sanguinarine and chelerythrine derivatives against phytopathogenic fungi // Molecules. 2012. Vol. 17. PP. 13026-13035. doi: 10.3390/molecules171113026

5. Tan G.T., Pezzuto J.M., Kinghorn A.D., Hughes S.H. Evaluation of natural products as inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase // Journal of Natural Products. 1991. Vol. 54. PP. 143-154. doi: 10.1021/np50073a012

6. Miao F., Yang X.J., Ma Y.N., Zheng F., Song X.P., Zhou L. Structural modification of sanguinarine and chelerythrine and their in vitro acaricidal activity against Psoroptes cuniculi // Chemical and Pharmaceutical Bulletin (Tokyo) 2012. Vol. 60. PP. 1508-1513. doi: 10.1248/cpb.c12-00618

7. Yao J.-Y., Li X.-L., Shen J.-Y., Pan X.-Y., Hao G.-J., Xu Y., Ying W.-L., Ru H.-S., Liu X.-L. Isolation of bioactive components from Chelidonium majus L. with activity against Trichodina sp. // Aquaculture. 2011. Vol. 318. PP. 235-238. doi: 10.1016/j. aquaculture.2011.04.035

8. Wang G.X., Zhou Z., Jiang D.X., Han J., Wang J.F., Zhao L.W., Li J. In vivo anthelmintic activity of five alkaloids from Macleaya microcarpa (Maxim) Fedde against Dactylogyrus intermedius in Carassius auratus // Veterinary Parasitology. 2010. Vol. 171. PP. 305-313. doi: 10.1016/j.vetpar.2010.03.032

9. Lenfeld J., Kroutil M., Marsalek E,. Slavik J., Preininger V., Simanek V. Antiinflammatory activity of quaternary benzophenanthridine alkaloids from Chelidonium majus // Planta Medica. 1981. Vol. 43. PP. 161-165. doi: 10.1055/s-2007-971493

10. Tsai I.L., Wun M.F., Teng C.M., Ishikawa T., Chen I.S. Anti-platelet aggregation constituents from Formosan Toddalia asiatica // Phytochemistry. 1998. Vol. 48. PP. 1377-1382. doi: 10.1016/S0031-9422(97)00678-X

11. Xu J.Y., Meng Q.H., Chong Y., Jiao Y., Zhao L., Rosen E.M., Fan S. Sanguinarine is a novel VEGF inhibitor involved in the suppression of angiogenesis and cell migration // Molecular and Clinical Oncology. 2013. Vol. 1, № 2. PP. 331-336. doi: 10.3892/mco.2012.41

12. Gaziano R., Moroni G., Bue C., Miele M.T., Sinibaldi-Vallebona P., Pica F. Antitumor effects of the benzophenanthridine alkaloid sanguinarine: Evidence and perspectives // World Journal of Gastrointestinal Oncology. 2016. Vol. 8. PP. 30-39. doi: 10.4251/wjgo.v8.i1.30

13. Maseri A., Cianflone D. Inflammation in acute coronary syndromes // European Heart Journal Supplements. 2002. Vol. 4 (Suppl. B). PP. B8-B13. doi: 10.1016/S1520-765X(02)90009-X

14. Baigent C., Blackwell L., Collins R., Emberson J., Godwin J., Peto R., Buring J., Hennekens C., Kearney P., Meade T., Patrono C., Roncaglioni M.C., Zanchetti A. Aspirin in the primary and secondary prevention of vascular disease: collaborative meta-analysis of individual participant data from randomised trials // Lancet. 2009. Vol. 373. PP. 1849-1860. doi: 10.1016/S0140-6736(09)60503-1

15. Patrono C., Garda Rodriguez L.A., Landolfi R., Baigent C. Low-dose aspirin for the prevention of atherothrombosis // The New England Journal of Medicine. 2005. Vol. 353. PP. 2373-2383. doi: 10.1056/NEJMra052717

16. Simmons D.L., Botting R.M., Hla T. Cyclooxygenase isozymes: the biology ofprostaglandin synthesis and inhibition // Pharmacological Reviews. 2004. Vol. 56. PP. 387-437. doi: 10.1124/pr.56.3.3

17. Jeng J.H., Wu H.L., Lin B.R., Lan W.H., Chang H.H., Ho Y.S., Lee P.H., Wang Y.J., Wang J.S., Chen Y.J., Chang M.C. Antiplatelet effect of sanguinarine is correlated to calcium mobilization, thromboxane and cAMP production // Atherosclerosis. 2007. Vol. 191. PP. 250-258. doi: 10.1016/j.atherosclerosis.2006.05.023

18. Lavalle G.E., Bertagnolli A.C., Tavares W.L., Cassali G.D. Cox-2 expression in canine mammary carcinomas: correlation with angiogenesis and overall survival // Veterinary Pathology. 2009. Vol. 46. PP. 1275-1280. doi: 10.1354/vp.08-VP-0226-C-FL

19. Han M.H., Yoo Y.H., Choi Y.H. Sanguinarine-induced apoptosis in human leukemia U937 cells via Bcl-2 downregulation and caspase-3 activation // Chemotherapy. 2008. Vol. 54. PP. 157-165. doi: 10.1159/000140359

20. Adhami V.M., Aziz M.H., Reagan-Shaw S.R., Nihal M., Mukhtar H., Ahmad N. Sanguinarine causes cell cycle blockade and apoptosis of human prostate carcinoma cells via modulation of cyclin kinase inhibitor-cyclin-cyclin-dependent kinase machinery // Molecular Cancer Therapeutics. 2004. Vol. 3. PP. 933-940.

21. Park S.Y., Jin M.L., Kim Y.H., Lee S.J., Park G. Sanguinarine inhibits invasiveness and the MMP-9 and COX-2 expression in TPA-induced breast cancer cells by inducing HO-1 expression // Oncology Reports. 2014. Vol. 31. PP. 497-504. doi: 10.3892/or.2013.2843

22. Zhang X., Lu S., Han J., Sun S., Wang L., Li Y. Preparation, characterization and in vivo distribution of solid lipid nanoparticles loaded with syringopicroside // Pharmazie. 2011. Vol. 66. PP. 404-407. doi: 10.1691/ph.2011.0350

23. Riss T.L., Moravec R.A., Niles A.L., Duellman S., Benink H.A., Worzella T.J., Minor L. Cell Viability Assays. In: Sittampalam G.S., Coussens N.P., Brimacombe K., et al., eds. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-. Avaliable at: http://www.ncbi.nlm.nih. gov/books/NBK144065/

24. Черемных Е.Г., Кулешин А.В., Кулешина О.Н. Биотестирование пищевых добавок на инфузориях // Вестник Российского университета дружбы народов. Серия: Экология и безопасность жизнедеятельности. 2011. № 3. С. 5-12. Available at: https://elibrary.ru/ item.asp?id=16757361

25. Kuleshina O.N., Kozlov L.V., Cheremnykh E.G. A universal method for measuring functional activity of complement in humans, laboratory, domestic, and agricultural animals, amphibians, and birds // Bulletin of Experimental Biology and Medicine. 2014. Vol. 157, № 2. РР. 285-287. doi: 10.1007/s10517-014-2546-5

26. Ivanov P.A., Faktor M.I., Karpova N.S., Cheremnykh E.G., Brusov O.S. Complement-mediated death of ciliate Tetrahymenapyriformis caused by human blood serum // Bulletin of Experimental Biology and Medicine. 2016. Vol. 160, №6. PP. 775-778. doi: 10.1007/ s10517-016-3307-4

27. Riddick T.M., Zeta-Meter, Inc. Control of colloid stability through zeta potential: with a closing chapter on its relationship to cardiovascular disease. Livingston Pub. Co., 1968. 372 p.

28. Feldman N.B., Kuryakov V.N., Sedyakina N.E., Gromovykh T.I., Lutsenko S.V. Preparation of liposomes containing benzophenanthridine alkaloid sanguinarine and evaluation of its cytotoxic activity // International Journal of Nanotechnology. 2018. Vol. 15, № 4/5. PP. 280-287. doi: 10.1504/IJNT.2018.094785

29. Луценко С.В., Громовых Т.И., Каширин В.В., Курьяков В.Н., Баранова А.А., Садыкова В.С., Фельдман Н.Б. Исследование in vitro противоопухолевой и антимикробной активности препарата пэгилированных липосом с сангвинарином // Антибиотики и химиотерапия. 2018. Т. 63, № 3-4. С. 3-7.

30. Stanley S.L. Jr. Amoebiasis // Lancet. 2003. Vol. 361, №9362. РР. 1025-1034. doi: 10.1016/ S0140-6736(03)12830-9

31. Desjeux P. Leishmaniasis: current situation and new perspectives // Comparative immunology, microbiology and infectious diseases. 2004. Vol. 27, №5. РР. 305-318. doi: 10.1016/j.cimid.2004.03.004

32. Nimir A.R., SaliemA., Ibrahim I.A. Ophthalmic parasitosis: a review article // Interdisciplinary Perspectives on Infectious Diseases. 2012. Article 587402. doi: 10.1155/2012/587402

Tomsk State University Journal of Biology. 2018; : 99-117

Study of the biological activity of liposomal sanguinarine on cultures of tumor cells and protozoa

Lutsenko S. V., Cheremnykh E. G., Sedyakina N. E., Moldogazieva N. T., Gromovykh T. I., Feldman N. B.

Abstract

Sanguinarine is a benzophenanthridine alkaloid with antimicrobial, antiviral, antiparasitic, anti-inflammatory, antiplatelet, antiangiogenic, and antitumor activity. One of the important properties of sanguinarine is a pronounced ability to suppress thrombogenesis, tumor growth and metastasis. However, the low solubility of sanguinarine in biological fluids limits its medical use. The present research was devoted to the development of the liposomal form of sanguinarine and the study of its biological activity. We obtained liposomes with sanguinarine on the basis of lecithin and cholesterol by the method of hydration of a thin film with buffer, followed by sonication and extrusion through a polycarbonate membrane with a pore size of 100 nm. Purification of liposomal dispersion from a drug that was not included in the vesicles was performed by gel filtration chromatography. We studied the morphology of the obtained liposomal particles by scanning electron microscopy; particle size and zeta potential were determined by dynamic light scattering. The study of the dynamics of sanguinarine release was conducted using the method of dialysis; quantitative analysis of the released sanguinarine from liposomes was performed using reverse-phase HPLC. The cytotoxic activity (CTA) of liposomal preparation against tumor cells of human breast carcinoma MCF-7 line was determined by the MTT assay. The toxicity and biological effects of liposomal sanguinarine on the cultures of Paramecium caudatum Ehrenberg and Tetrahymena pyriformis WH1, as well as the study of the effect of the drug on the complement system, were evaluated using the automated video registration system "BioLaT" (Russia). According to electron microscopy data, the obtained liposomes were spherical nanosized particles (See Fig. 1). The mean size of the obtained liposomal particles with sanguinarine included in their composition, determined using the method of the dynamic light scattering, was 108.5±2.2 nm, and the zeta potential was -34.7±1.4 mV. The effectiveness of sanguinarine inclusion in liposomes was quite high and amounted to 72.8±4.8%. The study of the dynamics of sanguinarine release from liposomes in conditions close to physiological (pH 7.4; 37°C) showed that this process occurs at the highest rate in the first 2 h of incubation. Then, the process is prolonged (release of about 50% sanguinarine after 6 h of incubation, and about 93% after 70 h) (See Fig. 2). Liposomal sanguinarine showed dose-dependent cytotoxic activity against tumor cells of human carcinoma MCF-7 in the micromolar concentration range ^е Fig. 3). The CTA of liposomal sanguinarine (IC₅₀ 14.5 |M) was slightly lower than the activity of free sanguinarine (IC₅₀ 9.4 |M), which can be explained by the prolonged release of sanguinarine from liposomes into the cell medium, as well as by the specificity of compartmentalization and intracellular release of the drug when it is absorbed by tumor cells by endocytosis. The prolonged release and the property of preferential accumulation of liposomes in tumor tissue can have a positive effect on therapeutic efficacy in the application of liposomal sanguinarine in vivo. The effect of liposomal sanguinarine on the survival of P. caudatum ciliates was dose-dependent (See Fig. 4). The minimum inhibitory concentration of liposomal sanguinarine was 0.49 |M. At concentrations from 0.245 |mM and below, the drug did not cause cell death for 2 h; over the next 24 h, the death of the ciliates was neither observed. Thus, liposomal sanguinarine has a pronounced cytotoxic effect on P. caudatum, a representative of the protozoa, which can serve as the basis for the development of antiprotozoal drugs. To identify pathogenic Protozoa species spectrum vulnerable to the action of liposomal sanguinarine, additional research is required. We also assessed the influence of liposomal sanguinarine on the protective blood systems - coagulation and the complement system. The effect of liposomal sanguinarine on thrombus formation in vitro was evaluated in citrate plasma after its recalcification according to the time of the onset of thrombus formation and the resulting clot density (See Fig. 5). The clot size in plasma solutions with the addition of the drug was significantly smaller compared with the control. At the same time, liposomal sanguinarine induces the formation of a clot after 7 min of incubation, whereas in the control the formation of a clot begins only after 14 min of incubation. Thus, under the conditions of this experiment, liposomal sanguinarine had a pronounced stimulating effect on thrombus formation. Stimulation of thrombosis by liposomal sanguinarine can be caused both by direct activation of coagulation enzymes and by the induction of enzymatic reactions of the coagulation system, which can efficiently proceed on the surface of liposomal nanoparticles. The study of the effect of liposomal sanguinarine in a non-toxic concentration of 60 ng/ml on the functional activity of the complement system against T. pyriformis ciliates showed that the half-life of the ciliates as a target of the complement system in the medium containing serum and liposomal sanguinarine (T₅₀ 21.7 min) reduced approximately twice compared with the control (T₅₀ 41.6 min) (See Fig. 6). In the absence of serum in the samples, liposomal sanguinarine at a concentration of 60 ng/ml, on the contrary, had a stimulating effect on T. pyriformis growth - the value of the proliferation coefficient for native cells was 2.1±0.2, and for the treated cells it was 6.4±0.8. The obtained data may indicate the activating effect of liposomal sanguinarine with respect to the assembly of the membrane attack complex of the complement system on the surface of T. pyriformis cells, causing their death. This effect allows to envisage the prospect of using liposomal sanguinarine as an immunostimulating agent. Thus, the pronounced cytotoxic antitumor and antiprotozoal activity, demonstrated in experiments in vitro, makes it possible to consider liposomal sanguinarine as a promising antitumor and antiprotozoal agent. The detected effect of thrombosis stimulation by liposomal sanguinarine seems to be important when selecting the dose of the drug introduced into the bloodstream. The paper contains 6 Figures and 32 References.

References

13. Maseri A., Cianflone D. Inflammation in acute coronary syndromes // European Heart Journal Supplements. 2002. Vol. 4 (Suppl. B). PP. B8-B13. doi: 10.1016/S1520-765X(02)90009-X

16. Simmons D.L., Botting R.M., Hla T. Cyclooxygenase isozymes: the biology ofprostaglandin synthesis and inhibition // Pharmacological Reviews. 2004. Vol. 56. PP. 387-437. doi: 10.1124/pr.56.3.3

24. Cheremnykh E.G., Kuleshin A.V., Kuleshina O.N. Biotestirovanie pishchevykh dobavok na infuzoriyakh // Vestnik Rossiiskogo universiteta druzhby narodov. Seriya: Ekologiya i bezopasnost' zhiznedeyatel'nosti. 2011. № 3. S. 5-12. Available at: https://elibrary.ru/ item.asp?id=16757361

27. Riddick T.M., Zeta-Meter, Inc. Control of colloid stability through zeta potential: with a closing chapter on its relationship to cardiovascular disease. Livingston Pub. Co., 1968. 372 p.

29. Lutsenko S.V., Gromovykh T.I., Kashirin V.V., Kur'yakov V.N., Baranova A.A., Sadykova V.S., Fel'dman N.B. Issledovanie in vitro protivoopukholevoi i antimikrobnoi aktivnosti preparata pegilirovannykh liposom s sangvinarinom // Antibiotiki i khimioterapiya. 2018. T. 63, № 3-4. S. 3-7.

30. Stanley S.L. Jr. Amoebiasis // Lancet. 2003. Vol. 361, №9362. RR. 1025-1034. doi: 10.1016/ S0140-6736(03)12830-9

31. Desjeux P. Leishmaniasis: current situation and new perspectives // Comparative immunology, microbiology and infectious diseases. 2004. Vol. 27, №5. RR. 305-318. doi: 10.1016/j.cimid.2004.03.004

32. Nimir A.R., SaliemA., Ibrahim I.A. Ophthalmic parasitosis: a review article // Interdisciplinary Perspectives on Infectious Diseases. 2012. Article 587402. doi: 10.1155/2012/587402