Curcumin Regulated of Methotrexate-Induced Cell Damage in HEK-293 Cell Line

Yıl 2021, , 38 - 43, 31.12.2020

Betül Yazğan

https://doi.org/10.34087/cbusbed.755807

Öz

Objective: Methotrexate (MTX), a conventional anti-folate, is an anti-cancer agent that is widely used for treatment of various types of cancers and it has been researched due to its side effects. Unfortunately, toxic effects of MTX is not limited just with tumour cells. It has also a toxic effect on healthy cells and organs. That is why it is an indispensability that MTX should be combined with other agents which can reduce the cellular toxicity of it at the same time not reducing its anti-cancer effectiveness. Exhaustive investigations have been performed to reveal the usage of natural antioxidants that can be reduce the unwanted side effects of anti-cancer drugs. It is postulated that Curcumin (CUR) has a protective effect on cellular toxicity in various types of cells due to its antioxidant, anti-inflammatory and anti-cancer effects. This study was designed presuming that cellular toxicity induced by MTX can be reduced by Curcumin. In this study the regulatory role of curcumin, an antioxidant agent, was investigated on MTX treated Human Embryonic Kidney Cell lines (HEK-293).
Materials and Methods: HEK-293 cells were divided into four groups as Control, CUR, MTX and MTX+CUR. Cells in the control group were incubated in cell culture medium for 48 hours without any application. After the cells in the other groups were incubated in cell culture medium for 24 hours cells in CUR group was treated with 10 μM CUR, cells in MTX group treated with 5 μM MTX and cells in the MTX+CUR group were incubated with 5 μM MTX + 10 μM CUR. After the treatments, the cells were kept incubated 24 hours in cell culture medium.
Results: MTX-induced lipid peroxidation (Lip-Px) activity (according to method of Placer et al.,), glutathione (GSH- according to Sedlak and Lindsay method) and glutathione peroxidase (GSH-Px - according to the Lawrence and Burk method) levels were measured spectrophotometrically (UV-1800) in HEK-293 cells. It has been determined that Lip-Px activity increased and GSH and GSH-Px activities decreased significantly in cells incubated with MTX. It is established that CUR treatment caused a significant decrease on the Lip-Px activity while causing a significant increment on GSH and GSH-Px activities.
Conclusion: Consequently, these findings showed that curcumin (CUR) administration, triggering the antioxidant mechanisms, can be an effective adjuvant therapy for MTX chemotherapy by regulating MTX-induced cellular stress and toxicity.

Anahtar Kelimeler

Curcumin, Glutathione peroxidase, Methotrexate, Lipid peroxidation.

Kurkumin HEK-293 Hücre Hattında Metotreksat Kaynaklı Hücresel Hasarı Düzenledi

Öz

Klasik bir antifolat olan metotreksat (MTX), çeşitli kanserlerin tedavisinde yaygın olarak kullanılan ve yan etkilerinden dolayı üzerinde çalışılan antikanser ajanlardan biridir. Ne yazık ki, MTX'in hücre üzerine toksik etkisi, sadece tümör hücreleri ile sınırlı olmayıp diğer hayati organları da etkilemektedir. Bu durum MTX’in antikanser etkinliğini azaltmadan, hücresel toksik etkilerini azaltabilecek başka ajanlarla birlikte kullanımını zorunlu kılmaktadır. Antikanser ilaçların istenmeyen yan etkilerini azaltabilecek doğal antioksidanların kullanımıyla ilgili kapsamlı araştırmalar yapılmaktadır. Yapılan çalışmalarda, kurkuminin (KUR) çeşitli dokularda meydana gelen hücresel toksisite üzerindeki koruyucu etkileri, onun antioksidan, antienflamatuar ve antikanser etkilerinin olmasına atfedilebilir. Bu çalışma, MTX’in neden olduğu hücresel toksisitenin, KUR ile azaltılabileceği varsayılarak yapıldı. MTX’e maruz bırakılan insan embriyo böbrek (HEK-293) hücre serisinde, antioksidan bir ajan olan KUR’un düzenleyici rolü araştırıldı.

HEK-293 hücreleri, Kontrol, KUR, MTX ve MTX+KUR olarak dört gruba ayrıldı. Kontrol grubundaki hücrelere herhangi bir uygulama yapılmadan, kültür ortamında 48 saat boyunca tutuldu. Diğer gruplardaki hücreler kültür ortamında 24 saat tutulduktan sonra, KUR grubundaki hücrelere 10 μM KUR, MTX grubundaki hücrelere 5 μM MTX ve MTX+KUR grubundaki hücrelere ise 5 μM MTX ve 10 μM KUR uygulandı. Uygulamaları takiben hücreler 24 saat boyunca kültür ortamında tutuldu. HEK-293 hücrelerindeki MTX kaynaklı lipit peroksidasyon (Lip-Px) aktivitesi Placer ve arkadaşlarının yöntemine göre, glutatyon (GSH) seviyeleri Sedlak ve Lindsay yöntemine göre ve glutatyon peroksidaz (GSH-Px) seviyeleri Lawrence ve Burk yöntemine göre spektrofotometrik (UV-1800) olarak ölçüldü. MTX ile inkübe edilen hücrelerde Lip-Px aktivitesinin arttığı, GSH ve GSH-Px aktivitelerinin ise önemli ölçüde azaldığı belirlenmiştir. KUR uygulamasının ise Lip-Px aktivitesini önemli ölçüde azaltırken, GSH ve GSH-Px aktivitelerini önemli ölçüde artmıştır.

Bu sonuçlar, KUR uygulamasının MTX kaynaklı hücresel stres ve toksisiteyi, antioksidan mekanizmalarla düzenleyerek, MTX kemoterapisine etkili bir yardımcı ajan olabileceğini göstermektedir.

Anahtar Kelimeler

Kurkumin, Metotreksat, Lipit peroksidasyon

Kaynakça

• 1. Widemann BC, Adamson PC, Understanding and managing methotrexate nephrotoxicity. Oncol, 2006, 6, 694-703. 2. Noha ISS, Magda MN, Azza AS, Modulatory effect of curcumin against genotoxicity and oxidative stress induced by cisplatin and methotrexate in male mice, Food and Chemical Toxicology, 2017, 370-376 . 3. Tousson E, Atteya E, El-Atrash E, Jeweely OI, Abrogation by Ginkgo Byloba leaf extract on hepatic and renal toxicity induced by methotrexate in rats, J Cancer Res Treat, 2014, 2(3), 44-51. 4. Santos MLC, de Brito BB, da Silva FAF, Botelho ACDS, de Melo FF, Nephrotoxicity in cancer treatment: An overview, World J Clin Oncol, 2020, 24, 11(4), 190-204. 5. Asvadi I, Hajipour B, Asvadi A, Asl N.A, Roshangar L, Khodadadi A, Protective effect of pentoxyfilline in renaltoxicity after methotrexate administration, European Reviewfor Medical and Pharmacological Sciences, 2011, 15, 1003–1009. 6. Farkhondeh T, Samarghandian S, Azimi-Nezhad M, Shahri AMP, Protective Effects of Curcumin Against Nephrotoxic Agents, Cardiovasc Hematol Disord Drug Targets, 2019, 19(3), 176-182. 7. Shi H, Hou C, Gu L, et al, Influence of pretreatment of piperazine ferulate on pharmacokinetic parameters of methotrexate in methotrexate-induced renal injury model rats by HPLC-MS, Asian J Pharm Sci, 2017, 12(2), 202-208. 8. El-Sheikh AA, Morsy MA, Abdalla AM, Hamouda AH, Alhaider IA, Mechanisms of thymoquinone hepatorenal protection in methotrexateinduced toxicity in rats, Mediators Inflamm, 2015, 1-12. 9. Babiak R.M, Campello A.P, Carnieri E.G, Oliveira M.B, Methotrexate: pentose cycle and oxidative stress, CellBiochemistry and Function, 1998, 16, 283–293. 10. Mora L.D.O, Antunes L.M.G, Francescato H.D.C, Bianchi M.D.L.P, The effects of oral glutamine on cisplatin-induced nephrotoxicity in rats, Pharmacol. Res, 2003, 47 (6), 517-522. 11. Daverey A, Agrawal SK, Pre and post treatment with curcumin and resveratrol protects astrocytes after oxidative stress, Brain Res, 2018, 1, 1692, 45-55. 12. Somparn P, Phisalaphong C, Nakornchai S, Unchern S, Morales N.P, Comparative antioxidant activities of curcumin and its demethoxy and hydrogenated derivatives, Biol. Pharm. Bull, 2007, 30, 74-78. 13. Ebrahimifar M, Roudsari MH, Kazemi SM, Shahmabadi HE, Kanaani L, Alavi SA, Vasfi MI, Enhancing Effects of Curcumin on Cytotoxicity of Paclitaxel, Methotrexate and Vincristine in Gastric Cancer Cells, Asian Pac J Cancer Prev, 2017, 18 (1), 65-68. 14. Aslanturk A, Uzunhisarcikli M, Protective potential of curcumin or taurine on nephrotoxicity caused by bisphenol A, Environmental Science and Pollution Research, 2020, doi: 10.1007/s11356-020-08716-1. 15. Palipoch S, Punsawad C, Chinnapun D, Suwannalert P, Amelioration of cisplatin-induced nephrotoxicity in rats by curcumin and a-tocopherol. Trop, J. Pharm. Res, 2014, 12(6), 973-979. 16. Huang C.F, Cui X.Q, Yang C.F, et al, Effects of curcumin on micronuclei formation and chromosome aberration induced by cyclophosphamide in mice, China J. Traditional Chin. Med, Pharm, 2011, 6, 42. 17. Sandur S.K, Pandey M.K, Sung B, et al, Curcumin, demethoxycurcumin, bisdemethoxycurcumin, tetrahydro-curcumin and turmerones differentially regulate anti-inflammatory and anti-proliferative responses through a ROS-independent mechanism, Carcinogenesis, 2007, 28, 1765-1773. 18. Vallianou N.G, Evangelopoulos A, Schizas N, Kazazis C, Potential Anticancer Properties and Mechanisms of Action of Curcumin, Anticancer Res, 2015, 35, 645–652. 19. Kunnumakkara A.B, Bordoloi D, Harsha C, et al, Curcumin mediates anticancer effects by modulating multiple cell signaling pathways, Clin. Sci, 2017, 131, 1781–1799. 20. Curcio M, Cirillo G, Tucci P, et al, Dextran-Curcumin Nanoparticles as a Methotrexate Delivery Vehicle: A Step Forward in Breast Cancer Combination Therapy, Pharmaceuticals (Basel) 2019, 25, 13(1):2. 21. Halliwell B, Oxidative stress and neurodegeneration: where are we now? J Neurochem, 2006, 97, 1634–1658. 22. Schieber M, Chandel NS, ROS Function in Redox Signaling and Oxidative Stress, Current Biology, 2014, 24(10), 453–462. 23. Eybl V, Kotyzova D, Koutensky J, Comparative study of natural antioxidants curcumin, resveratrol and melatonin in cadmium-induced oxidative damage in mice, Toxicology, 2006, 225, 150-156. 24. Schweizer U, Bräuer AU, Köhrle J, Nitsch R, Savaskan NE, Selenium and brain function: a poorly recognized liaison, Brain Res Brain Res Rev, 2004 45, 164–178. 25. Legallicier B, Leclere C, Monteil C, et al, The cellular toxicity of two antitumoural agents derived from platinum, cisplatinum versus oxaliplatinum, on cultures of tubular proximal cells, Drugs Exp Clin Res, 1996, 22(2), 41-50. 26. Biswas S.K, McClure D, Jimenez L.A, Megson I.L, Rahman I, Curcumin induces glutathione biosynthesis and inhibits NF-kB activation and interleukin-8 release in alveolar epithelial cells: mechanism of free radical scavenging activity, Antioxidants redox Signal, 2005, 7(1-2), 32-41. 27. Hollborn M, Chen R, Wiedemann P, Cytotoxic effects of curcumin in human retinal pigment epithelial cells, PLoS One, 2013, 8:e59603. 28. Fayi MA, Otifi H, Alshyarba M, Dera AA, Rajagopalan P, Thymoquinone and curcumin combination protects cisplatin-induced kidney injury, nephrotoxicity by attenuating NFjB, KIM-1 and ameliorating Nrf2/HO-1 signalling, J Drug Target, 2020, 5;1-10. 29. Hu A, Huang JJ, Jin XJ, et al, Curcumin suppresses invasiveness and vasculogenic mimicry of squamous cell carcinoma of the larynx through the inhibition of JAK-2/STAT-3 signaling pathway, Am J Cancer Res, 2015, 5(1), 278–288. 30. Iwona PC, Monika GG, Dorota NC, et al, Pioglitazone as a modulator of the chemoresistance of renal cell adenocarcinoma to methotrexate, Oncology Reports, 2020, 43(3), 1019-1030. 31. Placer ZA, Cushman L, Johnson BC, Estimation of products of lipid peroxidation (malonyl dialdehyde) in biological fluids, Analytical Biochem, 1966, 16, 359–364. 32. Sedlak J, Lindsay RHC, Estimation of total, protein bound and non-protein sulfhydryl groups in tissue with Ellmann’ s reagent, Analytical Biochem, 1968, 25, 192–205. 33. Lawrence RA, Burk RF, Glutathione peroxidase activity in seleniumdeficient rat liver, Biochem Biophys Res Com, 1976, 71, 952–958. 34. Weijl N.I, Elsendoorn T.J, Lentjes E.G.W.M, et al, Supplementation with antioxidant micronutrients and chemotherapy-induced toxicity in cancer patients treated with cisplatin-based chemotherapy: a randomised, double-blind, placebo-controlled study, Eur. J. Cancer, 2004, 40 (11), 1713-1723. 35. Tian F, Fan, T, Zhang Y, Jiang Y, Zhang X, Curcumin potentiates the antitumor effects of 5-FU in treatment of esophageal squamous carcinoma cells through downregulating the activation of NF-kB signaling pathway in vitro and in vivo, Acta biochimica biophysica Sinica, 2012, 44 (10), 847-855. 36. Antunes LM, Araújo MC, Darin JD, Bianchi ML, Effects of the antioxidants curcumin and vitamin C on cisplatin-induced clastogenesis in Wistar rat bone marrow cells, Mutat Res, 2000, 16, 465(1-2), 131-7. 37. Corona-Rivera A, Urbina-Cano P, Bobadilla-Morales L, Protective in vivo effect of curcumin on copper genotoxicity evaluated by comet and micronucleus assays, J. Appl. Genet, 2007, 48 (4), 389-396. 38. Liu F, Ni W, Zhang J, et al, Administration of curcumin protects kidney tubules against renal ischemia-reperfusion injury (RIRI) by modulating nitric oxide (NO) signaling pathway, Cell Physiol Biochem, 2017, 44(1), 401–411. 39. Abarikwu SO, Durojaiye M, Alabi A, Asonye B, Akiri O, Curcumin protects against gallic acid-induced oxidative stress, suppression of glutathione antioxidant defenses, hepatic and renal damage in rats, Renal Failure, 2016, 38(2), 321–329. 40. Banji OJ, Banji D, Ch K, Curcumin and hesperidin improve cognition by suppressing mitochondrial dysfunction and apoptosis induced by D-galactose in rat brain, Food Chem Toxicol, 2014, 74, 51-9. 41. Surh Y.J, Chun K.S, Cancer chemopreventive effects of curcumin. In: The Molecular Targets and Therapeutic Uses of Curcumin in Health and Disease, Springer US, 2007, 149-172. 42. Balasubramanayam M, Adaikala Koteswari A, Sampath Kumar R, Finny Monickaraj S, Uma Maheswari J, Mohan V, Kukumin-induced inhibition of cellular reactive oxygen species generation: novel therapeutic implications, J. Biosci, 2003, 28(6), 715-721. 43. Fiorillo C, Becatti M, Pensafini A, et al, Curcumin protects cardiac cells against ischemia-reperfusion injury: effects on oxidative stress, NF-kB, and JNK pathways, Free Radic. Biol. Med, 2008, 45, 839. 44. Sreejayan N, Rao M.N, Free radical scavenging activity of curcuminoids, Arzneim, 1996, 46(2), 169-171. 45. Lin X, Bai D, Wei Z, et al, Curcumin attenuates oxidative stress in RAW264.7 cells by increasing the activity of antioxidant enzymes and activating the Nrf2-Keap1 pathway, PLoS One, 2019, 21, 14(5), e0216711. doi: 10.1371/journal.pone.0216711.