The Effects of Althaea officinalis L. Extract on Cell Viability, Invasion, and Apoptosis in Prostate Cancer

Abstract

This study aims to evaluate the cytotoxic effects of methanol (AOME) and ethyl acetate (AOEE) extracts obtained from the flowers of Althaea officinalis L., which is known for its high antioxidant capacity, on prostate cancer cell lines (LNCaP and PC3), as well as their inhibitory effects on cell migration and invasion, and their influence on pro-apoptotic gene expression. Cell viability was assessed using the MTT assay, while cell migration and invasion were analyzed using the scratch assay. Changes in pro-apoptotic gene expression were evaluated by real-time PCR (RT-qPCR), as determined at the IC₅₀ concentrations obtained from cytotoxicity tests. The results showed that AOME and AOEE induced dose-dependent cytotoxicity in both cell lines. The IC₅₀ values were found to be 1.1×10⁻³ μg/mL (AOME) and 11×10⁻⁵ μg/mL (AOEE) for PC3 cells, and 0.21×10⁻³ μg/mL (AOME) and 0.45×10⁻³ μg/mL (AOEE) for LNCaP cells. Scratch assay analyses demonstrated that both extracts significantly inhibited cell migration and invasion compared to the control group. Additionally, RT-qPCR analyses revealed that pro-apoptotic gene expression was significantly increased, and anti-apoptotic gene expression was significantly decreased in treated cells (p<0.05). These findings suggest that A. officinalis extracts possess properties that reduce cell viability, inhibit migration and invasion, and promote apoptosis in prostate cancer cells, indicating that the extract may have therapeutic potential against prostate cancer and warrant further validation through comprehensive molecular investigations.

Keywords

• Althaea officinalis
• apoptosis
• prostate cancer
• wound healing

Supporting Institution

This study was funded by Balıkesir University Coordinator of Scientific Research Projects (Project no: 2021/014). The authors thank to BAUN BAP Unit for their supports.

Project Number

2021/014

Thanks

We sincerely thank Dr. Muhammed Enes Bulut for his valuable support to this study.

Althaea officinalis L. Ekstraktının Prostat Kanserinde Hücre Canlılığı, İnvazyon ve Apoptoz Üzerindeki Etkileri

Abstract

Bu çalışmanın amacı, yüksek antioksidan kapasitesi ile bilinen Althaea officinalis L. çiçeklerinden elde edilen metanol (AOME) ve etil asetat (AOEE) ekstrelerinin prostat kanseri hücre hatları (LNCaP ve PC3) üzerindeki sitotoksik etkilerini, hücre göçü ve invazyonu üzerindeki inhibe edici etkilerini ve pro-apoptotik gen ekspresyonuna etkilerini değerlendirmektir. Hücre canlılığı MTT testi ile değerlendirilmiş, hücre göçü ve invazyonu scratch testi ile analiz edilmiştir. Pro-apoptotik gen ekspresyonundaki değişiklikler, sitotoksisite testlerinden elde edilen IC₅₀ konsantrasyonlarında değerlendirilmiştir. Sonuçlar, AOME ve AOEE’nin her iki hücre hattında doz bağımlı sitotoksisite indüklediğini göstermiştir. IC₅₀ değerleri PC3 hücreleri için 1,1×10⁻³ μg/mL (AOME) ve 11×10⁻⁵ μg/mL (AOEE), LNCaP hücreleri için ise 0,21×10⁻³ μg/mL (AOME) ve 0,45×10⁻³ μg/mL (AOEE) olarak bulunmuştur. Scratch testi analizleri, her iki ekstrenin de hücre göçü ve invazyonunu kontrol grubuna göre anlamlı şekilde engellediğini göstermiştir. Ayrıca, RT-qPCR analizleri, tedavi edilen hücrelerde pro-apoptotik gen ekspresyonunun anlamlı şekilde arttığını ve anti-apoptotik gen ekspresyonunun anlamlı şekilde azaldığını ortaya koymuştur (p<0,05). Bu bulgular, A. officinalis ekstrelerinin hücre canlılığını azalttığını, göç ve invazyonu engellediğini ve apoptosis’i teşvik ettiğini göstermekte olup, ekstrenin prostat kanseri üzerinde terapötik potansiyele sahip olabileceğini ve bu durumun kapsamlı moleküler çalışmalarla doğrulanması gerektiğini düşündürmektedir.

Keywords

• Althaea officinalis
• apoptoz
• prostat kanseri
• yara iyileşmesi

Supporting Institution

Bu çalışma, Balıkesir Üniversitesi Bilimsel Araştırma Projeleri Koordinatörlüğü (Proje No: 2021/014) tarafından desteklenmiştir. Desteklerinden dolayı BAUN BAP Birimi’ne teşekkür ederiz.

Project Number

2021/014

Thanks

Bu çalışmaya değerli katkılarından dolayı Dr. Muhammed Enes Bulut’a içtenlikle teşekkür ederiz.

References

1. 1. Kosova F, Arı Z. Prostat kanseri ve apoptozis ilişkisi. J Clin Exp Invest. 2011;2(1):124–131.
2. 2. Peng X, Zhuang DD, Guo QS. Induction of S phase arrest and apoptosis by ethyl acetate extract from Tetrastigma hemsleyanum in human hepatoma HepG2 cells. Tumour Biol. 2015;36(4):2541–2550.
3. 3. Rawla P. Epidemiology of prostate cancer. World J Oncol. 2019;10(2):63–89.
4. 4. Kılınç K, Demir S, Turan İ, Menteşe A, Örem A, Sönmez M, Aliyazıcıoğlu Y. Rosa canina extract has antiproliferative and proapoptotic effects on human lung and prostate cancer cells. Nutr Cancer. 2020;72(2):273–282.
5. 5. Berkovich L, Earon G, Ron I, Rimmon A, Vexler A, Lev-Ari S. Moringa oleifera aqueous leaf extract down-regulates nuclear factor-kappaB and increases cytotoxic effect of chemotherapy in pancreatic cancer cells. BMC Complement Altern Med. 2013;13:212.
6. 6. Hage-Sleiman R, Mroueh M, Daher CF. Pharmacological evaluation of aqueous extract of Althaea officinalis flower grown in Lebanon. Pharm Biol. 2011;49(3):327–333.
7. 7. Al-Snafi AE. The pharmaceutical importance of Althaea officinalis and Althaea rosea: a review. Int J PharmTech Res. 2013;5(3):1378–1385.
8. 8.Zhang Y, Kong F, Zhang L, Li C. Modulatory effect of Althaea officinalis L. root extract on cisplatin-induced cytotoxicity and cell proliferation in A549 human lung cancer cell line. Trop JPharm Res. 2017;15(12):2647–2652.

9. 9.Livak KJ, Schmittgen TD. Analysis of relative gene expressiondata using real-time quantitative PCR and the 2(-Delta DeltaC(T)) method. Methods. 2001;25(4):402–408.
10. 10.Elmastaş M, Öztürk L, Erenler R, Gökçe İ. Determination of antioxidant activity of marshmallow flower (Althaea officinalis L.). Anal Lett. 2004;37(9):1859–1869.
11. 11.Elmastaş M, Erenler R, Demirtaş İ, Öztürk L. Superoxide anion scavenging activity of marshmallow flower (Althaea officinalis L.). 7th Int Electron Conf Synth Org Chem (ECSOC-7). 2003;1–30.
12. 12.Kobayashi T, Nakata T, Kuzumaki T. Effect of flavonoids on cell cycle progression in prostate cancer cells. Cancer Lett. 2002;176(1):17–23.
13. 13.Shah SMA, Naveed A, Akram M, Shah PA. Pharmacological activity of Althaea officinalis L. J Med Plants Res.2011;5(24):5662–5666.
14. 14.Sadighara P, Gharibi S, Moghadam Jafari A, Jahed Khaniki G,Salari S. The antioxidant and flavonoids contents of Althaea officinalis L. flowers based on their color. Avicenna JPhytomed. 2012;2(3):113–117.
15. 15.Ciobanu M, Pirvu L, Paun G, et al. Development of a new(bio)hybrid matrix based on Althaea officinalis and Betonica officinalis extracts loaded into mesoporous silica nanoparticles for bioactive compounds with therapeutic applications. J Drug Deliv Sci Technol. 2019;51:605–613.
16. 16.Kadhum HH, Abd AH, Al-Shammari AM. Anti-proliferative activity of Althaea officinalis extracts on Iraqi breast cancer cell line AMJ13. Iraqi J Med Sci. 2021;19(2):--.
17. 17.Farhat C, Younes H, Alyamani OA, Mrad M, Hourani N, et al. Chemical characterization and in vitro biological evaluation of aqueous extract of Althaea officinalis L. flower grown inLebanon. J Herbal Med. 2022;34:100575.
18. 18.Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26(4):239–257.
19. 19.Li X, Marani M, Yu J, Nan B, Roth JA, Kagawa S, Fang B, Denner L, Marcelli M. Adenovirus-mediated Bax overexpression for the induction of therapeutic apoptosis in prostate cancer. Cancer Res. 2001;61(1):186–191.
20. 20.Chaabane W, User SD, El-Gazzah M, Jaksik R, Sajjadi E, Rzeszowska-Wolny J, Los MJ. Autophagy, apoptosis, mitoptosis and necrosis: interdependence between those pathways and effects on cancer. Arch Immunol Ther Exp(Warsz). 2013;61(1):43–58.
21. 21.Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35(4):495–516.
22. 22.Koff JL, Ramachandiran S, Bernal-Mizrachi L. A time to kill:targeting apoptosis in cancer. Int J Mol Sci. 2015;16(2):2942–2955.
23. 23.Peña-Blanco A, García-Sáez AJ. Bax, Bak and beyond – mitochondrial performance in apoptosis. FEBS J. 2018;285(3):416–431.
24. 24.Dozmorov MG, Hurst RE, Culkin DJ, Kropp BP, Frank MB,Osban J, Penning TM, Lin HK. Unique patterns of molecularprofiling between human prostate cancer LNCaP and PC3 cells.Prostate. 2009;69(10):1077–1090.
25. 25.Ravenna L, Principessa L, Verdina A, Salvatori L, Russo MA,Petrangeli E. Distinct phenotypes of human prostate cancercells associate with different adaptation to hypoxia and pro-inflammatory gene expression. PLoS One. 2014;9(5):e96250.
26. 26.Tai S, Sun Y, Squires JM, Zhang H, Oh WK, Liang CZ, Huang J.PC3 is a cell line characteristic of prostatic small cell carcinoma. Prostate. 2011;71(15):1668–1679.
27. 27.Calabrese EJ, Mattson MP. How does hormesis impact biology,toxicology, and medicine? NPJ Aging Mech Dis. 2017;3:13.
28. 28.Trachootham D, Alexandre J, Huang P. Targeting cancer cellsby ROS-mediated mechanisms: a radical therapeutic approach. Nat Rev Drug Discov. 2009;8(7):579–591.
29. 29.Son Y, Cheong YK, Kim NH, Chung HT, Kang DG, Pae HO.Mitogen-activated protein kinases and reactive oxygen species: How can ROS activate MAPK pathways? J Signal Transduct. 2011;2011:792639.
30. 30.Rawlings JS, Rosler KM, Harrison DA. The JAK/STATsignaling pathway. J Cell Sci. 2004;117(Pt 8):1281–1283.
31. 31.Hanahan D. Hallmarks of cancer: a 2012 perspective. AnnOncol. 2012;23(9):ix23.
32. 32.Weng CJ, Yen GC. Flavonoids, a ubiquitous dietary phenolic subclass, exert extensive in vitro anti-invasive and in vivo anti-metastatic activities. Cancer Metastasis Rev. 2012;31(1–2):323–351.
33. 33.Eroglu C, Avci E, Secme M, Dodurga Y, Vural H, Kurar E.The combination effect of ferulic acid and gemcitabine on expression of genes related apoptosis and metastasis in PC3prostate cancer cells. Eur J Biol. 2018;77(1):32–37.
34. 34.Subhawa S, Naiki-Ito A, Kato H, Naiki T, Komura M, Nagano-Matsuo A, Yeewa R, Inaguma S, Chewonarin T, Banjerdpongchai R, Takahashi S. Suppressive Effect and Molecular Mechanism of Houttuynia cordata Thunb. Extract against Prostate Carcinogenesis and Castration-Resistant Prostate Cancer. Cancers (Basel). 2021;13(14):3403.
35. 35.Satari A, Amini SA, Raeisi E, Lemoigne Y, Heidarian E. Synergetic Impact of Combined 5-Fluorouracil and Rutin onApoptosis in PC3 Cancer Cells through the Modulation of P53Gene Expression. Adv Pharm Bull. 2019;9(3):462-469.
36. 36.Han R, Yang H, Li Y, Ling C, Lu L. Valeric acid acts as a novel HDAC3 inhibitor against prostate cancer. Med Oncol.2022;39(12):213.
37. 37.Tseng JC, Wang BJ, Wang YP, Kuo YY, Chen JK, Hour TC,Kuo LK, Hsiao PJ, Yeh CC, Kao CL, Shih LJ, Chuu CP.Caffeic acid phenethyl ester suppresses EGFR/FAK/Akt signaling, migration, and tumor growth of prostate cancer cells.Phytomedicine. 2023;116:154860.
38. 38.Senthilkumar K, Arunkumar R, Elumalai P, Sharmila G,Gunadharini DN, et al. Quercetin inhibits invasion, migration and signalling molecules involved in cell survival and proliferation of prostate cancer cell line (PC3). Cell Biochem Funct. 2011;29(2):87–95.