Effect of Epilobium angustifolium and Serenoa repens extracts on regulation of non-genomic signaling pathway of kinases

Effect of Epilobium angustifolium and Serenoa repens extracts on regulation of non-genomic signaling pathway of kinases

Wpływ ekstraktów z Epilobium angustifolium i Serenoa repens na regulację niegenomowego szlaku sygnałowego układu kinaz

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1 Department of Pharmacology and Experimental Biology, Institute of Natural Fibers and Medicinal Plants, Poznan, Poland

2 Laboratory of Experimental Pharmacogenetics, Department of Clinical Pharmacy and Biopharmacy, Poznan University of Medical Sciences, Poznan, Poland

3 Department of Quality Control of Medicinal Products and Dietary Supplements, Institute of Natural Fibers and Medicinal Plants, Poznan, Poland

4 Chair and Department of Pharmacology, Poznan University of Medical Sciences, Poznan, Poland

5 Department of Gastroenterology and Metabolic Diseases, Medical University of Warsaw, Poland

6 Division of Gynecology and Obstetrics, Podhale Multidisciplinary Hospital in Nowy Targ

7 Department of General Pharmacology and Pharmacoeconomy, Pomeranian Medical University, Szczecin, Poland

Abstract

Objectives: Changes of kinase activity of non-genomic cellular signaling pathway may influence the effectiveness of pharmacotherapy in case of hormone-dependent tumors. Our study investigated a possible interaction at the molecular level between an aqueous herbal extract of Epilobium angustifolium as well as a lipid-sterolic fruit extract of Serenoa repens and synthetic drugs used in the treatment of hormone-dependent cancers.

Material and methods: E. angustifolium and Serenoa repens extracts were orally administered to testosterone- induced rats for 21 days. Changes of RafA/Mapk3/Mapk1 mRNA levels were analyzed by real-time quantitative PCR using target specific primers.

Results: The level of RafA mRNA slightly increased in rats receiving Epilobium angustifolium (p=0.076) and Serenoa repens (p=0.016) extracts. Administration of these extracts resulted in significantly elevated Mapk1 and Mapk3 transcripts in the investigated animals (p<0.05 for each extract). The levels of Mapk1 and Mapk3 mRNA strongly increased (p<0.05 for each extract) in animals receiving concomitantly testosterone and the extracts, while RafA transcription slightly decreased (p<0.05), as compared to controls.

Conclusions: The results of our study may indicate a potential effect of S. repens and E angustifolium extracts on the functioning of non-genomic cellular signaling kinases pathway. We investigated safety of these extracts to detect possible drug interactions between synthetic drugs used in the treatment of proliferative changes in hormone-dependent reproductive organs and herbal preparations.

Key words: menopause / rats / Serenoa repens / (pLOoELum anJustLIoOLum / / MAP kinases / non-genomic cellular signaling pathway /

Otrzymano: 30.11.2013

Zaakceptowano do druku: 10.01.2013 Corresponding author:

Radosław Kujawski

Department of Pharmacology and Experimental Biology Institute of Natural Fibers and Medicinal Plants, Wojska Polskiego 71b Street, 60-630 Poznan, Poland e-mail: kujawskiradoslaw@gmail.com

Introduction

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Streszczenie

Cel: Zmiana aktywności kinaz niegenomowego szlaku sygnalizacji komórkowej może wpływać na skuteczność farmakoterapii nowotworów hormono-zależnych. W badaniu analizowano na poziomie molekularnym wpływ wyciągu wodnego z liści ziela Epilobium angustifolium i lipidowo-sterolowego wyciągu z owoców Serenoa repens na możliwość wystąpienia interakcji z lekami syntetycznymi stosowanymi w terapii nowotworów hormono-zależnych.

Materiał i metody: Ekstrakty z Epilobium angustifolium i Serenoa repens podawano p.o. indukowanym testosteronem szczurom przez 21 dni. Zmiany poziomów mRNA kinaz RafA/MAPK3/MAPK1 analizowano metodą ilościowego PCR w czasie rzeczywistym stosując specyficzne startery.

Wyniki: U szczurów otrzymujących wyciąg z Epilobium angustifolium (p=0.016) i Serenoa repens (p=0.016) poziom transkryptu RafA uległ nieznacznemu podwyższeniu. Nastąpiła znaczna indukcja transkrypcji mRNA kinaz MAPK1 i MAPK3 u zwierząt pod wpływem badanych ekstraktów (p<0.05 dla każdego z wyciągów). U zwierząt otrzymujących jednocześnie testosteron i badane wyciągi poziomy mRNA Mapk1 i Mapk3 uległy podwyższeniu (p<0.05 dla każdego z wyciągów), podczas gdy ilość transkryptów RafA uległa nieznacznie obniżeniu (p<0.05), w porównaniu do grupy kontrolnej.

Wnioski: Wyniki badania mogą wskazywać na potencjalny wpływ wyciągów z S. repens oraz E. angustifolium na funkcjonowanie niegenomowego szlaku sygnałowego układu kinaz. W pracy odniesiono się do aspektu bezpieczeństwa stosowania badanych ekstraktów roślinnych w kontekście występowania ewentualnych interakcji pomiędzy lekami syntetycznymi stosowanymi w terapii zmian rozrostowych w hormono-zależnych narządach rozrodczych a preparatami ziołowymi.

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Conclusions

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Ta b l e I . The effect of Epilobium angustifolium extract on the transcription of RafA, Mapk1, Mapk3 mRNA.

Group RafA mRNA (%±SEM) P Mapk1 mRNA (%±SEM) P Mapk3 mRNA

(%±SEM) P

E “ 0.076 60.6“0. 0.009 6.“0.0 0.017

T 0.9“1. 0.124 104.2“.6 0.007 191.4“6.9 0.018

TE 00“1.47 <0.001 12.“4.2 <0.001 1761“2.84 <0.001

Ratios of changes in mRNA level

T/K Ĺ4. Ĺ14.04 Ĺ17.1

E/K Ĺ1.14 Ĺ.4 Ĺ4.64

TE/T Ļ0.1 Ĺ0.2 Ĺ0.2

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Ta b l e I I . The effect of Serenoa repens extract on transcription of RafA, Mapk1, Mapk3 mRNA.

Group RafA mRNA (%±SEM) P Mapk1 mRNA

(%±SEM) P Mapk3 mRNA (%±SEM) P

S 2.1“0.46 0.016 67.61“1.1 0.001 71“0.77 0.016

T 02.9“1. 0.124 104.2“.6 0.007 191.4“6.9 0.018

TS 48.“0.77 0.001 2162.“2.92 <0.001 228.9“1.76 <0.001

Ratios of changes in mRNA level

T/K Ĺ4. Ĺ14.04 Ĺ17.1

S/K Ĺ1. Ĺ.74 Ĺ6.1

TS/T Ļ0.09 Ĺ0.44 Ĺ0.

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Oświadczenie autorów

1. Radosław Kujawski – wykonanie badań laboratoryjnych, analiza wyników, zebranie literatury, przygotowanie manuskryptu.

2. Anna Bogacz – wykonanie badań laboratoryjnych, analiza statystyczna wyników.

3. Joanna Bartkowiak-Wieczorek – wykonanie badań laboratoryjnych, przygotowanie manuskryptu.

4. Monika Karasiewicz – zebranie materiału wykonanie badań laboratoryjnych.

5. Przemysław Ł. Mikołajczak – zebranie materiału, współautor tekstu pracy.

6. Beata Rakowska-Mrozikiewicz – opracowanie wyników badań, współautor tekstu pracy.

7. Hubert Wolski – przygotowanie manuskrypty, współautor tekstu pracy.

8. Bogusław Czerny – przygotowanie manuskryptu, współautor tekstu pracy.

9. Edmund Grześkowiak – współautor analizy i interpretacia wyników, współautor tekstu pracy.

10. Przemysław M. Mrozikiewicz – autor koncepcji i założeń pracy, analiza wyników, korekta i aktualizacja manuskryptu - autor zgłaszający i odpowiedzialny za manuskrypt.

Źródło finansowania: Praca była finansowana z grantu nr: N405 678240.

Konflikt interesów: Autorzy nie zgłaszają konfliktu interesów oraz nie otrzymali żadnego wynagrodzenia związanego z powstawaniem pracy.

References

1. Wulfkuhle JD, Aquino JA, Calvert VS, [et al.]. Signal pathway profiling of ovarian cancer from human tissue specimens using reverse-phase protein microarrays. Proteomics. 2003, 3 (11), 2085-2090.

2. Nicosia SV, Bai W, Cheng JQ, [et al.]. Oncogenic pathways implicated in ovarian epithelial cancer. Hematol Oncol Clin North Am. 2003, 17 (4), 927-943.

3. Wu Q, Chambliss K, Umetani M, [et al.]. Non-nuclear estrogen receptor signaling in the endothelium. J Biol Chem. 2011, 286 (17), 14737-14743.

4. Li HZ, Gao Y, Zhao XL, [et al.]. Effects of raf kinase inhibitor protein expression on metastasis and progression of human breast cancer. Mol Cancer Res. 2009, 7 (6), 832-840.

5. Chang KW, Huang YL, Wong ZR, [et al.]. Fibroblast growth factor-2 up-regulates the expression of nestin through the Ras-Raf-ERK-Sp1 signaling axis in C6 glioma cells. Biochem Biophys Res Commun. 2013, 434 (4), 854-860.

6. Smolle E, Taucher V, Pichler M, [et al.]. Targeting signaling pathways in epithelial ovarian cancer.

Int J Mol Sci. 2013, 14 (5), 9536-9555.

7. Yue W, Santen RJ, Wang J-P, [et al.]. Aromatase within the breast. Endocr Relat Cancer. 1999, 6 (2), 157-164.

8. Berstein LM, Zheng H, Yue W, [et al.]. New approaches to the understanding of tamoxifen action and resistance. Endocr Relat Cancer. 2003, 10 (2), 267-277.

9. Santen RJ, Manni A, Harvey H, Redmond C. Endocrine treatment of breast cancer in women.

Endocr Rev. 1990, 11 (2), 221-265.

10. Santen RJ, Song RX, Zhang Z, [et al.]. Long-term estradiol deprivation in breast cancer cells up-regulates growth factor signaling and enhances estrogen sensitivity. Endocr Relat Cancer.

2005, 12 (1), 61-73.

11. Borrelli F, Ernst E. Alternative and complementary therapies for the menopause. Maturitas.

2010, 66 (4), 333-343.

12. Hsieh TC, Wu JM. Mechanism of action of herbal supplement PC-SPES: elucidation of effects of individual herbs of PC-SPES on proliferation and prostate specific gene expression in androgen-dependent LNCaP cells. Int J Oncol. 2002, 20 (3), 583-588.

13. Tacklind J, Macdonald R, Rutks I, [et al.]. Serenoa repens for benign prostatic hyperplasia.

Cochrane Database Syst Rev. 2012, 12:CD001423.

14. Kujawski R, Bogacz A, Derebecka-Hołysz N, [et al.]. Medicinal plants from Epilobium genus - biological and pharmacological properties. Herba Pol. 2010, 56 (1), 66-82.

15. Stolarczyk M, Naruszewicz M, Kiss AK. Extracts from Epilobium sp. herbs induce apoptosis in human hormone-dependent prostate cancer cells by activating the mitochondrial pathway. J Pharm Pharmacol. 2013, 65 (7), 1044-1054.

16. Kosowicz J, Miskowiak B, Konwerska A, [et al.]. Pneumadin in the rat ventral prostate and its hormonal regulation. Horm Metab Res. 2004, 36 (2), 78-81.

17. Foradori CD, Weiser MJ, Handa RJ. Non-genomic actions of androgens. Front Neuroendocrinol.

2008, 29 (2), 169–181.

18. Reis-Filho JS, Tutt AN. Triple negative tumours: a critical review. Histopathology. 2008, 52(1), 108-18.

19. Riggins RB, Schrecengost RS, Guerrero MS, Bouton AH. Pathways to tamoxifen resistance.

Cancer Lett. 2007, 256 (1), 1-24.

20. Santen R, Jeng MH, Wang JP, [et al.]. Adaptive hypersensitivity to estradiol: potential mechanism for secondary hormonal responses in breast cancer patients. J Steroid Biochem Mol Biol. 2001, 79 (1-5),115–125.

21. Shin WS, Conaway M, Masamura S, [et al.]. Estradiol hypersensitivity and mitogen activated protein kinase expression in long-term estrogen deprived human breast cancer cells in vivo.

Endocrinology. 2000, 141 (1), 396-405.

22. Mrozikiewicz PM, Bogacz A, Czerny B, [et al.]. The influence of a standardized soybean extract (Glycine max) on the expression level of cytochrome P450 genes in vivo. Ginekol Pol. 2010, 81 (7), 516-250.

23. Bogacz A, Mrozikiewicz PM, Bartkowiak-Wieczorek J, [et al.]. The influence of a standardized soybean extract (Glycine max) on the expression level of CYP3A enzymes and pregnane X receptor in in vivo model. Ginekol Pol. 2010, 81 (11), 814-820.

24. Pitkin J. Alternative and complementary therapies for the menopause. Menopause Int. 2012, 18 (1), 20-27.

25. Sultan C, Terraza A, Devillier C, [et al.]. Inhibition of androgen metabolism and binding by a liposterolic extract of „Serenoa repens B” in human foreskin fibroblasts. J Steroid Biochem.

1984, 20 (1), 515-519.

26. Bedner M, Schantz MM, Sander LC, Sharplless KE. Development of liquid chromatographic methods for the determination of phytosterols in Standard Reference Materials containing saw palmetto. J Chromatogr A. 2008, 1192 (1), 74-80.

Figure 1. The influence of Serenoa repens and Epilobium angustifolium extracts on transcription of RafA, Mapk1, Mapk3 mRNA kinases.

( ± DQLPDOV WUHDWHG ZLWK Epilobium angustifolium H[WUDFW 6 ± DQLPDOV WUHDWHG ZLWK Serenoa repens H[WUDFW 7 ± WHVWRVWHURQH WUHDWHG DQLPDOV 7( ± WHVWRVWHURQH  E.

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