The effects of lithium, metformin and everolimus substances on cell growth in 2D and 3D Ishikawa endometrial carcinoma cell culture

Abstract

Purpose: Our aim is to study the effects of the single and combined treatments of Everolimus, Metformin, and Lithium Chloride in two-dimensional (2D, monolayer) and three-dimensional (3D, spheroid) cell cultures of Ishikawa cells, which comprise the endometrial cancer cell line. Materials and methods: As part of the study, the effects of single and combined forms of Everolimus, Metformin, and Lithium Chloride were determined on cell viability, invasion, colony formation and apoptosis, and PI3K/AKT/mTOR pathway. Cell viability was assessed using XTT assay. CASP3, CASP8, CASP9, FASL, FADD, TNF, TRADD, BAX, P53, PI3KCA, PI3KCB, PTEN, MTOR, AKT1 genes were evaluated with RT-PCR, apoptosis was evaluated by flow cytometry and 3D spheroid results were evaluated with invert microscope analysis. Results: Everolimus, metformin, and lithium's IC50 levels were found at 48 hours to be 37.46 nM, 48.59 mM, and 100 μM, respectively. It was determined that the invasive capacities of Ishikawa cells in treatment groups, as well as cell colony formation were significantly reduced. In addition, Ishikawa spheroid cells were significantly suppressed compared with the control groups. RT-PCR results revealed that substances and their combinations affect genes associated with PI3K/AKT/mTOR pathway and apoptosis. Flow cytometry results showed notably increased apoptosis by single and combined treatments. Conclusion: As a result, the single and combination forms of everolimus, metformin, and lithium have reduced cell proliferation, induced apoptosis, and decreased mTOR activation through various mechanisms in Ishikawa cells. However, our study has shown that Eve alone and triple combination therapy (Eve+Met+Lit) are more effective than other therapies in the treatment of endometrial cancer.

Keywords

• Endometrial cancer
• PI3K/AKT/mTOR pathway
• everolimus
• metformin
• lithium

Lityum, metformin ve everolimus maddelerinin 2D ve 3D Ishikawa endometrial karsinom hücre kültüründe hücre büyümesi üzerine etkileri

Abstract

Amaç: Amacımız endometrium kanser hücre hattı olan Ishikawa hücrelerinin iki boyutlu (2D, monolayer) ve üç boyutlu (3D, spheroid) hücre kültürlerinde Everolimus, Metformin ve Lityum Klorür'ün tekli ve kombine tedavilerinin etkilerini incelemektir. Gereç ve yöntem: Çalışma kapsamında, Everolimus, Metformin ve Lityum Klorürün tekli ve kombine formlarının hücre canlılığı, invazyon, koloni oluşumu ve apoptoz ve PI3K/AKT/mTOR yolağı üzerindeki etkileri belirlendi. Hücre canlılığı XTT testi kullanılarak değerlendirilmiştir. CASP3, CASP8, CASP9, FASL, FADD, TNF, TRADD, BAX, P53, PI3KCA, PI3KCB, PTEN, MTOR, AKT1 genleri RT-PCR ile, apoptoz Flow sitometri ile ve 3D sferoid sonuçları invert mikroskop analizi ile değerlendirildi. Bulgular: Everolimus, metformin ve lityumun IC50 seviyeleri 48 saatte sırasıyla 37.46 nM, 48.59 mM ve 100 μM olarak bulundu. Tedavi gruplarındaki Ishikawa hücrelerinin invazyon kapasitelerinin yanı sıra hücre koloni oluşumunun da önemli ölçüde azaldığı tespit edilmiştir. Ayrıca, Ishikawa sferoid hücreleri kontrol gruplarına kıyasla önemli ölçüde baskılanmıştır. RT-PCR sonuçları, maddelerin ve kombinasyonlarının PI3K/AKT/mTOR yolağı ve apoptoz ile ilişkili genleri etkilediğini ortaya koymuştur. Flow sitometri sonuçları tekli ve kombine tedavilerin apoptozu belirgin şekilde arttırdığını göstermiştir. Sonuç: Sonuç olarak, everolimus, metformin ve lityumun tekli ve kombinasyon formları, Ishikawa hücrelerinde çeşitli mekanizmalar yoluyla hücre çoğalmasını azaltmış, apoptozu indüklemiş ve mTOR aktivasyonunu azaltmıştır. Bununla birlikte, çalışmamız tek başına Eve ve üçlü kombinasyon tedavisinin (Eve+Met+Lit) endometriyal kanser tedavisinde diğer tedavilerden daha etkili olduğunu göstermiştir.

Keywords

• Endometriyal kanser
• PI3K/AKT/mTOR yolağı
• everolimus
• metformin
• lityum

References

1. 1. Ferlay J, Soerjomataram I, Dikshit R, et al. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 2015;136:359-386. https://doi.org/10.1002/ijc.29210
2. 2. Colombo N, Creutzberg C, Amant F, et al. 2 ESMO-ESGO-ESTRO consensus conference on endometrial cancer: diagnosis, treatment and follow-up. Ann Oncol 2016;27:16-41. https://doi.org/10.1093/annonc/mdv484
3. 3. Nyen TV, Moiola CP, Colas E, Annibali D, Amant F. Modeling endometrial cancer: past, present, and future. Int J Mol Sci 2018;19:2348. https://doi.org/10.3390/ijms19082348
4. 4. Bokhman JV. Two pathogenetic types of endometrial carcinoma. Gynecol Oncol 1983;15:10-17. https://doi.org/10.1016/0090-8258(83)90111-7
5. 5. Piulats JM, Guerra E, Gil Martín M, et al. Molecular approaches for classifying endometrial carcinoma. Gynecol Oncol 2017;145:200-207. https://doi.org/10.1016/j.ygyno.2016.12.015
6. 6. Korch C, Spillman MA, Jackson TA, et al. DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination. Gynecol Oncol 2012;127:241-248. https://doi.org/10.1016/j.ygyno.2012.06.017
7. 7. Mendivil A, Schuler KM, Gehrig PA. Non-endometrioid adenocarcinoma of the uterine corpus: A review of selected histological subtypes. Cancer Control 2009;16:46-52. https://doi.org/10.1177/107327480901600107
8. 8. Alzahrani AS. PI3K/Akt/mTOR inhibitors in cancer: at the bench and bedside Semin Cancer Biol 2019;59:125-132. https://doi.org/10.1016/j.semcancer.2019.07.009

9. 9. Peng Y, Wang Y, Zhou C, Mei W, Zenget C. PI3K/Akt/mTOR pathway and its role in cancer therapeutics: are we making headway? Front Oncol 2022;12:818128(e-1-17). https://doi.org/10.3389/fonc.2022.819128
10. 10. Rubinstein M, Shen S, Monk BJ, et al. Looking beyond carboplatin and paclitaxel for the treatment of advanced/recurrent endometrial cancer. Gynecol Oncol 2022;167:540-546. https://doi.org/10.1016/j.ygyno.2022.10.012
11. 11. Soliman PT, Westin SN, Iglesias DA, et al. Everolimus, letrozole, and metformin in women with advanced or recurrent endometrioid endometrial cancer: a multi-center, single Arm, Phase II Study. Clin Cancer Res 2020;26:581-587. https://doi.org/10.1158/1078-0432.CCR-19-0471
12. 12. Oza AM, Elit L, Tsao MS, et al. Phase II study of temsirolimus in women with recurrent or metastatic endometrial cancer: a trial of the NCIC clinical trials group. J Clin Oncol 2011;29:3278-3285. https://doi.org/10.1200/JCO.2010.34.1578
13. 13. Hasskarl J. Everolimus. Recent Results Cancer Res 2018;211:101-123 https://doi.org/10.1007/978-3-319-91442-8_8
14. 14. Sarbassov DD, Guertin DA, Sabatini DM, et al. Phosphorylation and regulation of Akt/PKB by the rictormTOR complex. Science 2005;307:1098-1101. https://doi.org/10.1126/science.1106148
15. 15. O’Reilly KE, Rojo F, She QB, et al. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt Cancer Res 2006;66:1500-1508. https://doi.org/10.1158/0008-5472.CAN-05-2925
16. 16. Wang X, Yue P, Chan CB, et al. Inhibition of mammalian target of rapamycin induces phosphatidylinositol 3-kinase-dependent and Mnk-mediated eukaryotic translation initiation factor 4E phosphorylation. Mol Cell Biol 2007;27:7405-7413. https://doi.org/10.1128/MCB.00760-07
17. 17. Vidal F, Araujo WM, Cruz AL, et al. Lithium reduces tumorigenic potential in response to EGF signaling in human colorectal cancer cells. Int J Oncol 2011;38:1365-1373. https://doi.org/10.3892/ijo.2011.955
18. 18. Cho YJ, Kim JH, Yoon J, et al. Constitutive activation of glycogen synthase kinase-3beta correlates with better prognosis and cyclin-dependent kinase inhibitors in human gastric cancer. BMC Gastroenterol 2010;10:91. https://doi.org/10.1186/1471-230X-10-91
19. 19. Bilir A, Ergüven M, Yazihan N, Aktas E, Gulperi Oktem G, Akin Sabanci A. Enhancement of vinorelbine-induced cytotoxicity and apoptosis by clomipramine and lithium chloride in human neuroblastoma cancer cell line SH-SY5Y. J Neurooncol 2010;100:385-395. https://doi.org/10.1007/s11060-010-0209-6
20. 20. Bilir A, Erguven M, Ermis E, Sencan M, Yazihan N. Combination of imatinib mesylate with lithium chloride and medroxyprogesterone acetate is highly active in Ishikawa endometrial carcinoma in vitro. J Gynecol Oncol 2011;22:225-232. https://doi.org/10.3802/jgo.2011.22.4.225
21. 21. Liu H, Scholz C, Zang C, et al. Metformin and the mTOR inhibitor everolimus (RAD001) sensitize breast cancer cells to the cytotoxic effect of chemotherapeutic drugs in vitro. Anticancer Res 2012;32:1627-1637.
22. 22. Chen YH, Wu JX, Yang SF, et al. Metformin potentiates the anticancer effect of everolimus on cervical cancer in vitro and in vivo. Cancers 2021;13:4612. https://doi.org/10.3390/cancers13184612
23. 23. Wang Y, Wei J, Li L, et al. Combined use of metformin and everolimus is synergistic in the treatment of breast cancer cells. Oncol Res 2015;22:193-201. https://doi.org/10.3727/096504015X14348950540999
24. 24. Wong C, Vosburgh E, Levine AJ, Cong L, Xu EY. Human neuroendocrine tumor cell lines as a three-dimensional model for the study of human neuroendocrine tumor therapy. J Vis Exp 2012:e4218. https://doi.org/10.3791/4218
25. 25. Friedrich J, Seidel C, Ebner R, Kunz Schughart LA. Spheroid-based drug screen: considerations and practical approach. Nat Protoc 2009;4:309-324.
26. 26. Zhu H, Han B, Pan X, Qi H, Xu L. Thiazolidenediones induce tumour-cell apoptosis through the Akt-GSK3β pathway. J Clin Pharm Ther 2012;37:65-70. https://doi.org/10.1111/j.1365-2710.2011.01251.x
27. 27. Slomovitz BM, Coleman RL. The PI3K/AKT/mTOR pathway as a therapeutic target in endometrial cancer. Clin Cancer Res 2012;18:856-864. https://doi.org/10.1158/1078-0432.CCR-12-0662
28. 28. Barra F, Evangelisti G, Desideri LF, et al. Investigational PI3K/AKT/mTOR inhibitors in development for endometrial cancer. Expert Opin Investig Drugs 2019;28:131-142. https://doi.org/10.1080/13543784.2018.1558202
29. 29. Stringer EM, Fleming GF. Hormone therapy plus mTOR inhibitors in the treatment of endometrial carcinoma. Eur Endocrinol 2013;9:18-21. https://doi.org/10.17925/EE.2013.09.01.18
30. 30. Leslie R, Downes CP. PTEN function: how normal cells control it and tumour cells lose it. Biochem J 2004;382:1-11. https://doi.org/10.1042/BJ20040825
31. 31. De Melo AC, Paulino E, Garces ÁHI. A review of mTOR pathway Inhibitors in gynecologic cancer. Oxid Med Cell Longev 2017;2017:4809751. https://doi.org/10.1155/2017/4809751
32. 32. Bjornsti MA, Houghton PJ. The TOR pathway: a target for cancer therapy. Nat Rev Cancer 2004;4:335-348. https://doi.org/10.1038/nrc1362
33. 33. Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev 2000;18:1926-1945. https://doi.org/10.1101/gad.1212704
34. 34. Faivre S, Kroemer G, Raymond E. Current development of mTOR inhibitors as anticancer agents. Nat Rev Drug Discov 2006;5:671-688. https://doi.org/10.1038/nrd2062
35. 35. Hollander MC, Blumenthal GM, Dennis PA. PTEN loss in the continuum of common cancers, rare syndromes and mouse models. Nat Rev Cancer 2011;11:289-301. https://doi.org/10.1038/nrc3037
36. 36. Mutter GL. PTEN, a protean tumor suppressor. Am J Pathol 2001;158:1895-1898. https://doi.org/10.1016/S0002-9440(10)64656-1
37. 37. Pabona JMP, Burnett AF, Brown DM, et al. Metformin promotes anti-tumor biomarkers in human endometrial cancer cells. Reprod Sci 2020;27:267-277. https://doi.org/10.1007/s43032-019-00019-2
38. 38. Roncolato F, Lindemann K, Willson ML, et al. PI3K/AKT/mTOR inhibitors for advanced or recurrent endometrial cancer. Cochrane Database Syst Rev 2019;10,CD012160. https://doi.org/10.1002/14651858.CD012160.pub2
39. 39. Shoji K, Oda K, Kashiyama T, et al. Genotype-dependent efficacy of a dual PI3K/mTOR inhibitor, NVP-BEZ235, and an mTOR inhibitor, RAD001, in endometrial carcinomas. PLoS One 2012;7:e37431. https://doi.org/10.1371/journal.pone.0037431
40. 40. Wu H, Goel V, Haluska FG, et al. PTEN signaling pathways in melanoma. Oncogene 2003;22:3113-3122. https://doi.org/10.1038/sj.onc.1206451
41. 41. Janku F, Wheler JJ, Westin SN, et al. PI3K/AKT/mTOR inhibitors in patients with breast and gynecologic malignancies harboring PIK3CA mutations. J Clin Oncol 2012;30,777-782. https://doi.org/10.1200/JCO.2011.36.1196
42. 42. Sun H, Enomoto T, Fujita M, et al. Mutational analysis of the PTEN gene in endometrial carcinoma and hyperplasia. Am J Clin Pathol 2001;115:32-38. https://doi.org/10.1309/7JX6-B9U9-3P0R-EQNY
43. 43. Kong D, Suzuki A, Zou TT, et al. PTEN1 is frequently mutated in primary endometrial carcinomas. Nat Genet 1997;17:143-144. https://doi.org/10.1038/ng1097-143
44. 44. Hayes MP, Douglas W, Ellenson LH. Molecular alterations of EGFR and PIK3CA in uterine serous carcinoma. Gynecol Oncol 2009;113:370-373. https://doi.org/10.1016/j.ygyno.2008.12.021