Kapsaisinin H2452 Mezotelyoma Hücre Hattından Seçilen Klon larda Sitotoksisitesinin ve Kaspaz-3 Üzerine Etkilerinin Belirlenmesi

Gizem Ceylan; Sabahattin Cömertpay

doi:10.18016/ksutarimdoga.vi.673591

EN TR

Determination of the Cytotoxicity of Capsaicin and its Effects on Caspase-3 in the Clones Selected from H2452 Mesothelioma Cell Line

Abstract

Mesothelioma is a type of cancer that develops from the mesothelium tissue that covers the surface of the internal organs such as the heart, lungs and abdomen, and its tumors are thought to have polyclonal origin. Apoptotic effects of capsaicin on cancer cells are known. In this study; we tried to determine the cytotoxicity of capsaicin and its effects on caspase-3 expression and activation on four (clone-I, -II, -III and -IV) clones selected from the H2452 mesothelioma cell line. For this purpose; firstly, capsaicin cytotoxicity, i.e. IC50 values of clones were determined by the method of reduction of MTS [3- (4,5) -dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium] salt to formazan. It has been demonstrated that the type of the clones was affecting the IC50’s of capsaicin statistically significantly (p <0.01). Subsequently, capsaicin was applied to selected clones at an average concentration of IC50 (251.6 µM) and cell growth curves were plotted. Accordingly; after 72 hours of capsaicin treatment, clone-I was observed to contain a higher number of viable cells (p <0.001) than other clones. By qRT-PCR and Western blotting performed following the application, mRNA and protein levels of caspase-3 molecule were determined in capsaicin-treated (KAP), ethanol-treated (EtOH) and untreated control cells. As a result, the capsaicin cytotoxicity was found to differ significantly between the selected clones, and the amount of inactive and active forms of the caspase-3 protein and the relative caspase-3 mRNA levels showed different results for each clone. When all the findings were evaluated together, the idea that the cell populations forming mesothelioma tumors may have a polyclonal origin appears to be supported.

Keywords

Mesothelioma,Capsaicin,CAP,Tumor heterogeneity,Apoptosis

Kapsaisinin H2452 Mezotelyoma Hücre Hattından Seçilen Klon larda Sitotoksisitesinin ve Kaspaz-3 Üzerine Etkilerinin Belirlenmesi

Öz

Mezotelyoma; kalp, akciğer ve karın gibi iç organların yüzeyini kaplayan mezotelyum dokusundan gelişen bir kanser türüdür ve tümörlerinin poliKlon al kökene sahip olduğu düşünülmektedir. Kapsaisinin kanser hücreleri üzerindeki apoptotik etkileri bilinmektedir. Bu çalışmada; H2452 mezoteliyomahücre hattından seçilmiş dört (I, II, III ve IV numaralı) Klon üzerinde kapsaisinin sitotoksisitesi ve kaspaz-3 ifadesine ve aktivasyonuna etkileri tespit edilmeye çalışılmıştır. Bu amaçla; öncelikle, Klon ların IC50 değerleri MTS [3-(4,5)-dimetiltiazol-2-il)-5-(3-karboksimetoksifenil)-2-(4-sülfofenil)-2H-tetrazolyum] tuzunun formazana indirgenmesi metoduyla belirlenmiş ve kapsaisin sitotoksisitesinin bazı Klon lar arasında istatistiksel olarak anlamlı bir fark gösterdiği (p<0.01) ortaya konulmuştur. Daha sonra, seçilen Klon lara ortalama IC50 derişiminde (251.6 µM) kapsaisin uygulanmış ve hücre büyüme eğrileri çizilmiştir. Buna göre; 72 saat sonunda, Klon I’in diğer Klon lardan daha yüksek sayıda (p<0.001) canlı hücre içerdiği gözlenmiştir. Bunu takiben gerçekleştirilen qRT-PCR ve Western Blotlama ile; kapsaisin uygulanmış (KAP), etanol uygulanmış (EtOH) ve herhangi bir uygulama görmemiş, kontrol grubu hücrelerinde kaspaz-3 molekülünün sırasıyla mRNA ve protein seviyesindeki değişimler tespit edilmiştir. Sonuç olarak; kapsaisin sitotoksistesinin seçilen Klon ile anlamlı bir değişiklik gösterdiği tespit edilmiş, ve kaspaz-3 proteinin inaktif ve aktif biçimlerinin miktarı ile bağıl kaspaz-3 mRNA seviyesi her bir Klon için diğerlerinden farklı sonuçlar ortaya koymuştur. Tüm bulgular birlikte değerlendirildiğinde, mezotelyoma tümörlerini oluşturan hücre popülasyonlarının poliKlon al bir kökene sahip olabileceği fikri destekleniyor görünmektedir.

Anahtar Kelimeler

Mezoteliyoma,Kapsaisin,KAP,Tümör heterojenitesi,Apoptoz

Supporting Institution

Kahramanmaraş Sütçü İmam Üniversitesi Bilimsel Araştırma Projeleri Birimi

Project Number

2017/2-11 YLS

Thanks

Bu çalışma Kahramanmaraş Sütçü İmam Üniversitesi Bilimsel Araştırma Projeleri Birimi 2017/2-11 YLS Nolu proje kapsamında gerçekleştirilmiştir.

References

Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A 2018. Global Cancer Statistics 2018:GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin 68(6): 394-424.
Ceylan G 2019. Kanserli ve Ölümsüzleştirilmiş Mezotelyum Hücrelerinden Seçilen Klonlar Üzerine Kapsaisin Etkisinin İncelenmesi. Kahramanmaraş Sütçü İmam Üniversitesi Fen Bilimleri Enstitüsü Tarımsal Biyoteknoloji Ana Bilim Dalı, Yüksek Lisans Tezi, 62 sy.
Comertpay S, Pastorino S, Mika T, Mezzapelle R, Strianese O, Napolitano A, Baumann F, Weigel T, Friedberg J, Sugarbaker P, Krausz T, Wang E, Powers A, Gaudino G, Shreya K, Pass HI, Parsons LB, Yang H, Carbone M 2014. Evaluation of Clonal Origin of Malignant Mesothelioma. Journal of Translational Medicine 12(301).
Comertpay S, Ceylan G 2019. Öncül Afidikolin ve Nokodazol Muamelesinin Kanserli ve Ölümsüzleştirilmiş Mezotelyum Hücrelerinde Kapsaisin Sitotoksitesine Etkisi. KSÜ Tarım ve Doğa Dergisi 22(3): 456-465..
Elmore S 2007. Apoptosis:A Review of Programmed Cell Death. Toxicol Pathol 35(4): 495-516.
Helvacı N, Comertpay S 2018. In Vitro Evaluation of the Effects of Capsaicin on Normal and Cancerous Cells of Human Cartilage. Turkısh Journal of Biology 42: 422-434.
Harada J, Sugimoto M 1999. Activation of Caspase-3 in b-amyloid-induced Apoptosis of Cultured Rat Cortical Neurons. Brain Research 842, 311–323.
Hiddinga BI, Rolfo C, van Meerbeeck JP 2015. Mesothelioma Treatment: Are We on Target? A review. Journal of Advanced Reserch 6(3): 319-330.

Huang SP, Chen JC, Wu CC, Chen CT, Tang NY, Ho YT, Lo C, Lin JP, Chung JG, Lin JG 2009. Capsaicin-Induced Apoptosis in Human Hepatoma HepG2 Cells. Anticancer Research 29: 165-174.
Huh HC, Lee SY, Lee SK, Park NH, Han IS 2011. Capsaicin Induces Apoptosis of Cisplatin-Resistant Stomach Cancer Cells by Causing Degradation of Cisplatin-Inducible Aurora-A Protein. Nutrition and Cancer 63(7): 1095-1103.
Ito K, Nakazato T, Yamato K, Miyakawa Y, Yamada T, Hozumi N, Segawa K, Ikeda Y, Kizaki M 2004. Induction of Apoptosis in Leukemic Cells by Homovanillic Acid Derivative, Capsaicin, through Oxidative Stress: Implication of Phosphorylation of p53 at Ser-15 Residue by Reactive Oxygen Species. Cancer Research 64: 1071–1078.
Jin T, Wu H, Wang Y, Peng H 2016. Capsaicin Induces Immunogenic Cell Death in Human Osteosarcoma Cells. Experimental and Therapeutic Medicine 12(2): 765-770.
Kim SH, Liu CY, Fan PW, Hsieh CH, Lin HY, Lee MC, Fang K 2016. The Aqueous Extract of Brucea Javanica Suppresses Cell Growth and Alleviates Tumorigenesis of Human Lung Cancer Cells by Targeting Mutated Epidermal Growth Factor Receptor. Drug Des Devel Ther 10, 3599-3609.
Li H, Krstin S, Wang S, Wink M 2018. Capsaicin and Piperine Can Overcome Multidrug Resistance in Cancer Cells to Doxorubicin. Molecules 23 (3).
Martinvalet D, Zhu P ve Lieberman J 2005. Granzyme A induces caspase-independent mitochondrial damage, a required first step for apoptosis. Immunity 22(3): 355-370.
Metintas S, Batirel HF, Bayram H, Yilmaz U, Karadag M, Ak G, Metintas M 2017. Turkey National Mesothelioma Surveillance and Environmental Asbestos Exposure Control Program. International Journal of Environmental Research and Public Health 14(11).
Mori A, Lehmann S, O’Kelly J, Kumagai T, Desmond JC, Pervan M, McBride WH, Kizaki M, Koeffler HP 2006. Capsaicin, a Component of Red Peppers, Inhibits the Growth of Androgen-Independent, p53 Mutant Prostate Cancer Cells. Cancer Research 66(6).
Riedl SJ, Shi Y 2004. Molecular Mechanisms of Caspase Regulation During Apoptosis. Nature Reviews Molocular Cell Biology 5(11): 897-907.
Robinson BWS, Musk AW, Lake RA 2005. Malignant mesothelioma. The Lancet 366(9483), 397-408.
Scherpereel A, Wallyn F, Albelda SM, Munck C 2018. Novel Therapies for Malignant Pleural Mesothelioma. The Lancet Oncology 19(3): e161-e172.
Schmittgen TD, Livak KJ 2008. Analyzing Real-Time PCR Data by the Comparative CT Method. Nature Protocols 3(6), 1101-1108.
Segerman A, Niklasson M, Haglund C, Bergström T, Jarvius M, Xie Y, Westermark A, Sönmez D, Hermansson A, Kastemar M, Naimaie-Ali Z, Nyberg F, Berglund M, Sundström M,
Hesselager G, Uhrbom L, Gustafsson M, Larsson R, Westermark B 2016. Clonal Variation in Drug and Radiation Response among Glioma-Initiating Cells Is Linked to Proneural- Mesenchymal Transition. Cell Reports 17, 2994–3009.
Song Z, Chang H, Han N, Liu Z, Liu Y, Wang H, Shao J, Wang Z, Gao H, Yin J 2017. He-Wei Granules (HWKL) Combat Cisplatin-Induced Nephrotoxicity and Myelosuppression in Rats by Inhibiting Oxidative Stress, Inflammatory Cytokines and Apoptosis. RSC Advances 7(32), 19794-19807.
Zhang R, Humphreys I, Sahu PR, Shi Y, Srivastava SK 2008. In Vitro and In Vivo Induction of Apoptosis by Capsaicin in Pancreatic Cancer Cells is Mediated Through ROS Generation and Mitochondrial Death Pathway. Apoptosis 13(12):1465-78.

Details

Primary Language

Turkish

Subjects

Structural Biology

Journal Section

Research Article

Authors

Gizem Ceylan ^*
0000-0002-9627-5492
Türkiye

Sabahattin Cömertpay
0000-0002-2364-8171
Türkiye

Publication Date

October 31, 2020

Submission Date

January 15, 2020

Acceptance Date

March 13, 2020

Published in Issue

Year 2020 Volume: 23 Number: 5

DOI

https://doi.org/10.18016/ksutarimdoga.vi.673591

IZ

https://izlik.org/JA33HX29YC

Cite

RIS / Bibtex

APA

Ceylan, G., & Cömertpay, S. (2020). Kapsaisinin H2452 Mezotelyoma Hücre Hattından Seçilen Klon larda Sitotoksisitesinin ve Kaspaz-3 Üzerine Etkilerinin Belirlenmesi. Kahramanmaraş Sütçü İmam Üniversitesi Tarım Ve Doğa Dergisi, 23(5), 1125-1134. https://doi.org/10.18016/ksutarimdoga.vi.673591