Progesteronun HepG2 Karaciğer Kanseri Hücre Canlılığı ve Karaciğer Fonksiyon Testleri Üzerindeki Etkilerine Karşı D Vitamininin Etkisi

Yıl 2024, , 108 - 116, 30.06.2024

Melek Naz Akkuş , Hale Bayram , Mustafa Sıtar , Belgin Selam , Mehmet Cıncık , Yaprak Dönmez Çakıl

https://doi.org/10.52827/hititmedj.1439617

Öz

Amaç: Progesteron, adrenal bezler ve yumurtalıklar tarafından sentezlenen, yapısal olarak birçok farklı hormonun da öncüsü olan bir sinyal molekülüdür. D vitamini ise diğer vitaminlerden farklı olarak ekzojen alımın yanında endojen olarak da sentezlenebilen ancak eksiklik durumu güncel tıp dünyasında büyük tartışmalara neden olan steroid yapıda bir hormondur. Bu çalışmada amaç, progesteronun HepG2 hücre proliferasyonu ve karaciğer enzim aktivitelerine etkisini belirlemek, ayrıca D vitamininin progesteronun oluşturduğu sitotoksik etkileri engellemedeki rolünü incelemektir.
Gereç ve Yöntem: HepG2 hepatoselüler kanser hücre kültürü ortamına uygulanacak progesteron ve D vitamini dozlarının belirlenmesi için öncelikle her iki hormon için ayrı sitotoksisite çalışmaları yapılmıştır. Ardından progesteron ve D vitamini, deney ve kontrol gruplarına tek başlarına veya birlikte belirli dozlarda uygulanmıştır. HepG2 hücre canlılığı, morfolojik özellikleri ve karaciğer enzim aktiviteleri gruplar arasında karşılaştırmalı olarak değerlendirilmiştir.
Bulgular: Hücrelere uygulanan 1 mM ve 2 mM progesteron dozlarında kontrol grubuna kıyasla hücre canlılığında azalma olduğu saptandı. Ek olarak, 1 mM ve 2 mM progesteron uygulananlarda AST ve LDH aktivite değerlerinde de anlamlı olarak düşüklük bulundu. D vitamininin 0,008 μM ve 166,667 μM dozları aralığında HepG2 hücrelerinde sitotoksik bir etkiye sahip olmadığı belirlendi ve 2,5 μM dozda uygulandı. Yalnızca D vitamini uygulanan hücrelerde ALT, AST ve LDH enzim aktivite değerlerinde anlamlı bir farklılık görülmedi. Yalnızca progesteron uygulanan hücrelerle, progesteron+D vitamininin birlikte uygulandığı hücreler arasında hücre canlılığı ve karaciğer enzim düzeyleri benzerlik gösterdi.
Sonuç: Kullanılan doz ve inkübasyon sürelerinde D vitamininin progesteronun sebep olduğu sitotoksik etkileri engellemede etkili olmadığı düşünülmektedir.

Anahtar Kelimeler

D vitamini, hepatosellüler karsinom, HepG2, karaciğer fonksiyon testleri, progesteron

Etik Beyan

Hücre kültürü çalışması olduğu için etik kurul onayına gerek bulunmamaktadır.

Destekleyen Kurum

Bu çalışma Melek Naz Akkuş’un yüksek lisans projesi kapsamında Maltepe Üniversitesi Bilimsel Araştırma Projeleri tarafından desteklenmiştir.

Proje Numarası

Bu çalışma Melek Naz Akkuş’un yüksek lisans projesi kapsamında Maltepe Üniversitesi Bilimsel Araştırma Projeleri tarafından desteklenmiştir.

Teşekkür

Atlas Üniversitesi Tıbbi Farmakoloji Anabilim Dalı Öğretim Üyesi Doç. Dr. Zeynep Güneş Özünal’a kıymetli destekleri için teşekkür ederiz.

Effect of Vitamin D Against Progesterone-induced Effects on HepG2 Liver Cancer Cell Viability and Liver Function Tests

Öz

Objective: Progesterone is a signaling molecule synthesized by the adrenal glands and ovaries and
is structurally the precursor of many different hormones. Vitamin D, unlike other vitamins, is a steroid
hormone that can be synthesized endogenously as well as supplied exogenously. Vitamin D deficiency
is a matter of considerable controversy in the world of medicine. The objective of this study was to
investigate the impact of progesterone on the proliferation and liver enzyme activities of HepG2 cells, as
well as to assess the potential of vitamin D in mitigating the cytotoxic effects induced by progesterone.
Material and Method: Cytotoxicity studies were conducted on HepG2 hepatocellular cancer cells to
determine the appropriate doses of progesterone and vitamin D when applied alone or in combination.
The hormones were administered to the experiment and control groups, either alone or in combination
at specific doses. Subsequently, HepG2 cell viability, morphology and liver enzyme activities were
measured comparatively between the groups.
Results: The results indicated a decrease in cell viability in the cells treated with 1 mM and 2 mM
progesterone when compared to the control group. In addition, AST and LDH activity values were
significantly lower with 1 mM and 2mM progesterone. Vitamin D was found not to have a cytotoxic
effect on HepG2 cells between doses of 0.008 μM and 166.667 μM. Therefore, a dose of 2.5 μM was
selected for further applications. No significant difference in ALT, AST, and LDH enzyme activity values
was observed when only vitamin D was administered. Similar cell viability and enzyme activities were
demonstrated when progesterone was administered alone or in combination with vitamin D.
Conclusion: At the doses and incubation periods used in the current study, vitamin D was found to be
ineffective in preventing the cytotoxic effects caused by progesterone.

Anahtar Kelimeler

Hepatocellular carcinoma, HepG2, liver function tests, progesterone, Vitamin D

Proje Numarası

Bu çalışma Melek Naz Akkuş’un yüksek lisans projesi kapsamında Maltepe Üniversitesi Bilimsel Araştırma Projeleri tarafından desteklenmiştir.

Kaynakça

• Sundström-Poromaa I, Comasco E, Sumner R, Luders E. Progesterone – Friend or foe? Frontiers in Neuroendocrinology 2020;59:100856.
• Taraborrelli S. Physiology, production and action of progesterone. Acta Obstet Gynecol Scand 2015;94(Suppl 161):8–16.
• Manocha A, Kankra M, Singla P, Sharma A, Ahirwar AK, Bhargava S. Clinical significance of reproductive hormones. Current Medicine Research and Practice 2018;8(3):100–108.
• Xu L, Yuan Y, Che Z, et al. The Hepatoprotective and Hepatotoxic Roles of Sex and Sex-Related Hormones. Front Immunol 2022;13:939631.
• Van Der Linden M, Buckingham K, Farquhar C, Kremer JA, Metwally M. Luteal phase support for assisted reproduction cycles. Cochrane Database Syst Rev 2015;7:CD009154.
• Tetruashvili N, Domar A, Bashiri A. Prevention of Pregnancy Loss: Combining Progestogen Treatment and Psychological Support JCM. 2023;12(5):1827.
• Tsur A, Leonard SA, Kan P, et al. Vaginal Progesterone Is Associated with Intrahepatic Cholestasis of Pregnancy. Am J Perinatol 2023;40(11):1158–1162.
• Zipori Y, Bachar G, Farago N, et al. Vaginal progesterone treatment for the prevention of preterm birth and intrahepatic cholestasis of pregnancy: A case-control study. European Journal of Obstetrics & Gynecology and Reproductive Biology 2020;253:117–120.
• Kinci MF, Şehirli Kinci Ö, Karakaş Paskal E. Gebeliğin İntrahepatik Kolestazı. Muğla Sıtkı Koçman Üniversitesi Tıp Dergisi 2021 Aug 31;8(2):158–162.
• Yılmaz S, Üstün Y, Hızlı D, Deveer R. Gebeliğin İntrahepatik Kolestazı. Gazi Med J. 2012;(23):138–144.
• Choudhary NS, Bodh V, Chaudhari S, Saraf N, Saigal S. Norethisterone Related Drug Induced Liver Injury: A Series of 3 Cases. Journal of Clinical and Experimental Hepatology 2017;7(3):266–268.
• Giustina A, Lazaretti-Castro M, Martineau AR, Mason RS, Rosen CJ, Schoenmakers I. A view on vitamin D: a pleiotropic factor? Nat Rev Endocrinol 2024. doi: 10.1038/s41574-023- 00942-0. Epub ahead of print. PMID: 38253860.
• Saponaro F, Saba A, Zucchi R. An Update on Vitamin D Metabolism. IJMS 2020;21(18):6573.
• Kitson MT, Roberts SK. D-livering the message: The importance of vitamin D status in chronic liver disease. Journal of Hepatology 2012;57(4):897–909.
• Celik S, Golbasi H, Gulucu S, et al. Role of Vitamin B12 and Vitamin D levels in intrahepatic cholestasis of pregnancy and correlation with total bile acid. Journal of Obstetrics and Gynaecology 2022;42(6):1847–1852.
• Gençosmanoğlu Türkmen G, Vural Yilmaz Z, Dağlar K, et al. Low serum vitamin D level is associated with intrahepatic cholestasis of pregnancy. J of Obstet and Gynaecol 2018;44(9):1712–1718.
• Wikström Shemer E, Marschall H. Decreased 1,25‐dihydroxy vitamin D levels in women with intrahepatic cholestasis of pregnancy. Acta Obstet Gynecol Scand 2010;89(11):1420–1423.
• Seraphin G, Rieger S, Hewison M, Capobianco E, Lisse TS. The impact of vitamin D on cancer: A mini review. The Journal of Steroid Biochemistry and Molecular Biology 2023;231:106308.
• Young MRI, Xiong Y. Influence of vitamin D on cancer risk and treatment: Why the variability? Trends Cancer Res 2018;13:43–53.
• Van Den Hof WFPM, Coonen MLJ, Van Herwijnen M, et al. Validation of gene expression profiles from cholestatic hepatotoxicants in vitro against human in vivo cholestasis. Toxicology in Vitro 2017;44:322–329.
• Strober W. Trypan Blue Exclusion Test of Cell Viability. CP in Immunology [Internet]. 1997 Mar [cited 2024;21(1). Available from: https://currentprotocols.onlinelibrary.wiley. com/doi/10.1002/0471142735.ima03bs21
• Ozgur E, Guler G, Kismali G, Seyhan N. Mobile Phone Radiation Alters Proliferation of Hepatocarcinoma Cells. Cell Biochem Biophys 2014;70(2):983–991.
• Estiú MC, Monte MJ, Rivas L, et al. Effect of ursodeoxycholic acid treatment on the altered progesterone and bile acid homeostasis in the mother placenta foetus trio during cholestasis of pregnancy. Brit J Clinical Pharma 2015;79(2):316–329.
• Toyoda Y, Endo S, Tsuneyama K, et al. Mechanism of Exacerbative Effect of Progesterone on Drug-Induced Liver Injury. Toxicological Sciences 2012;126(1):16–27.
• Du T, Xiang L, Zhang J, et al. Vitamin D improves hepatic steatosis in NAFLD via regulation of fatty acid uptake and β-oxidation. Front Endocrinol 2023;14:1138078.
• Bozic M, Guzmán C, Benet M, et al. Hepatocyte vitamin D receptor regulates lipid metabolism and mediates experimental diet-induced steatosis. Journal of Hepatology 2016;65(4):748–757.
• Eitah HE, Attia HN, Soliman AAF, et al. Vitamin D ameliorates diethylnitrosamine-induced liver preneoplasia: A pivotal role of CYP3A4/CYP2E1 via DPP-4 enzyme inhibition. Toxicology and Applied Pharmacology 2023;458:116324.
• Goto RL, Tablas MB, Prata GB, et al. Vitamin D3 supplementation alleviates chemically-induced cirrhosis-associated hepatocarcinogenesis. The Journal of Steroid Biochemistry and Molecular Biology 2022;215:106022. Gholamalizadeh M, Jarrahi AM, Akbari ME, et al. Association between FTO gene polymorphisms and breast cancer: the role of estrogen. Expert Review of Endocrinology & Metabolism 2020;15(2):115–121.
• Fragni M, Fiorentini C, Rossini E, et al. In vitro antitumor activity of progesterone in human adrenocortical carcinoma. Endocrine 2019;63(3):592–601.
• Pedernera E, Gómora MJ, Morales-Vásquez F, Pérez-Montiel D, Mendez C. Progesterone reduces cell survival in primary cultures of endometrioid ovarian cancer. J Ovarian Res 2019;12(1):15.
• Chang WT, Lin HL, Chang KL, et al. Progesterone increases epirubicin’s apoptotic effects in HepG2 cells by S phase cell cycle arrest. Hepatogastroenterology 2010;57(97):107–113.
• Lima MA, Silva SV, Jaeger RG, Freitas VM. Progesterone decreases ovarian cancer cells migration and invasion. Steroids 2020;161:108680.
• Dressing GE, Alyea R, Pang Y, Thomas P. Membrane Progesterone Receptors (mPRs) Mediate Progestin Induced Antimorbidity in Breast Cancer Cells and Are Expressed in Human Breast Tumors. HORM CANC 2012;3(3):101–112.
• Goncharov AI, Maslakova AA, Polikarpova AV, et al. Progesterone inhibits proliferation and modulates expression of proliferation— R elated genes in classical progesterone receptor-negative human BxPC3 pancreatic adenocarcinoma cells. The Journal of Steroid Biochemistry and Molecular Biology 2017 Jan;165:293–304.
• Alese MO, Bamisi OD, Alese OO. Progesterone modulates cadmium-induced oxidative stress and inflammation in hepatic tissues of Wistar rats. Int J Clin Exp Pathol 2021;14(10):1048–1055.
• Gocek E, Studzinski GP. Vitamin D and differentiation in cancer. Critical Reviews in Clinical Laboratory Sciences 2009;46(4):190–209.
• Marques LA, Semprebon SC, Biazi BI, et al. Vitamin D3 and Salinomycin synergy in MCF-7 cells cause cell death via endoplasmic reticulum stress in monolayer and 3D cell culture. Toxicology and Applied Pharmacology 2022;452:116178.
• Zhang X, Li P, Bao J, et al. Suppression of Death Receptor-mediated Apoptosis by 1,25-Dihydroxyvitamin D3 Revealed by Microarray Analysis. Journal of Biological Chemistry 2005;280(42):35458–35468.
• Zheng W, Peng W, Qian F, et al. Vitamin D suppresses CD133+/CD44 + cancer stem cell stemness by inhibiting NF-κB signaling and reducing NLRP3 expression in triple-negative breast cancer. Cancer Chemother Pharmacol [Internet]. 2024 Mar 8 [cited 2024 May 7]; Available from: https://link.springer. com/10.1007/s00280-024-04660-w
• Veeresh PKM, Basavaraju CG, Dallavalasa S, et al. Vitamin D3 Inhibits the Viability of Breast Cancer Cells In Vitro and Ehrlich Ascites Carcinomas in Mice by Promoting Apoptosis and Cell Cycle Arrest and by Impeding Tumor Angiogenesis. Cancers 2023;15(19):4833.
• Özerkan D, Özsoy N, Yılmaz E. Vitamin D and melatonin protect the cell’s viability and ameliorate the CCl4 induced cytotoxicity in HepG2 and Hep3B hepatoma cell lines. Cytotechnology 2015;67(6):995–1002.
• Carlberg C, Velleuer E. Vitamin D and the risk for cancer: A molecular analysis. Biochemical Pharmacology 2022;196:114735.
• Öner Ç, İsan H, Aktaş RG, Çolak E. Vitamin D’nin Hepatoselüler Karsinom Üzerindeki Etkisi. Osmangazi Journal of Medicine 2020;42(3):301-310.