THE ANTICANCER ACTIVITY OF JUGLONE VIA INDUCING PROGRAMMED CELL DEATH IN PANCREATIC CANCER CELLS

Abstract

Aim: Pancreatic cancer (PC), has a poor prognosis with its silent course until the advanced level without any symptoms. Additionally, currently limited treatment options makes developing alternative treatment strategies and agents mandatory. In researches conducted to investigate new therapeutic agents for cancer natural products are main sources. Among all, Juglone, a natural naphthoquinone, stands out with its cytotoxic effect in cancer cells, induction of apoptosis and autophagy, inhibition of antiangiogenesis and cancer cell migration. Thus, in our study, we aimed to investigate the anticancer activity and the apoptotic effects of juglone on BxPC-3 Pancreatic cancer cells. Method: To evaluate the apoptotic effects; proapoptotic Bax, antiapoptotic Bcl-2 and an important inhibitor of apoptosis Birc5 (Survivin) gene expressions were determined by qPCR analysis. We also confirmed the results for apoptosis by performing immunofluorescence analysis using Annexin V-FITC. Findings: According to qPCR analysis, juglone induced statistically significant 2.24, 1.57, 2.43 fold increases of Bax gene the expressions at 10, 15 and 20µM doses, respectively. We detected low fold increases after juglone treatments for Bcl-2 gene expression at other treatments except 20µM dose. This changes were not statistically significant. Juglone decreased survivin gene expression at all treatment doses while 2.39 fold decrease at 20 μM doses of juglone was statistically significant. Also, we confirmed clearly by the immunofluorescence analysis that juglone increased apoptosis dose-dependently in BxPC-3 cells. Results: Taken together all the results of gene expression and immunfloresence analysis our study suggests that juglone shows anticancer activity by inducing apoptosis possibly enhancing intrinsic apoptotic pathway in Pancreatic cancer cells. Keywords: juglone, pancreatic cancer, Bax gene, Bcl-2 gene, survivin gene, apoptosis,

Keywords

• juglone
• pancreatic cancer
• Bax gene
• Bcl-2 gene
• survivin gene
• apoptosis

JUGLONUN PANKREAS KANSERİ HÜCRELERİNDE PROGRAMLI HÜCRE ÖLÜMÜNÜ İNDÜKLEYEREK GÖSTERDİĞİ ANTİKANSER ETKİSİ

Abstract

Amaç: Pankreas kanseri (PK), belirti vermeden ileri düzeye kadar sessiz seyri ile kötü prognoza sahiptir. Ek olarak, halihazırda sınırlı olan tedavi seçenekleri, alternatif tedavi stratejileri ve teröopatik ajanların geliştirilmesini zorunlu kılmaktadır. Kanser için yeni terapötik ajanları araştırmak amacıyla yapılan araştırmalarda doğal ürünler temel kaynaklardır. Bunların arasında doğal bir naftokinon olan Juglon, kanser hücrelerinde sitotoksik etkisi, apoptoz ve otofajiyi indüklemesi, antianjiyogenez ve kanser hücresi göçünü engellemesi ile öne çıkmaktadır. Bu nedenle çalışmamızda juglonun BxPC-3 Pankreas kanseri hücreleri üzerindeki antikanser aktivitesini ve apoptotik etkilerini araştırmayı amaçladık. Yöntem: Apoptotik etkileri değerlendirmek için; proapoptotik Bax, antiapoptotik Bcl-2 ve önemli apoptoz inhibitörü Birc5 (Survivin) gen ekspresyonları qPCR analizi ile belirlendi. Annexin V-FITC ile immünofloresan analizi yaparak apoptoz sonuçlarını da doğruladık. Bulgular: qPCR analizine göre juglon, 10, 15 ve 20µM dozlarda Bax gen ekspresyonlarında sırasıyla 2,24, 1,57, 2,43 kat düzeyinde istatistiksel olarak anlamlı artışlara neden oldu. Bcl-2 gen ekspresyonu için, juglon tedavilerinden sonra 20µM doz dışındaki diğer uygulamalarda düşük kat artışları saptadık. Bu değişiklikler istatistiksel olarak anlamlı değildi. Juglon, tüm tedavi dozlarında survivin gen ekspresyonunu azaltırken, 20 μM juglon uygulmasındaki 2.39 kat azalma istatistiksel olarak anlamlıydı. qPCR analizine ek olarak, juglonun BxPC-3 hücrelerinde doza bağlı olarak apoptozu arttırdığını immünofloresan analizi ile doğruladık. Sonuç: Gen ekspresyonu ve immünfloresans analizinin tüm sonuçları birlikte ele alındığında, çalışmamız juglonun pankreas kanseri hücrelerinde muhtemelen intrinsik apoptotik yolu indükleyerek antikanser aktivite gösterdiğini ortaya koymuştur.

Keywords

• JUGLON
• PANKREAS KANSERİ
• APOPTOZ
• BAX GENİ
• BCL-2 GENİ
• SURVIVIN GENİ

References

1. Altieri, D.C. (2003). Survivin, versatile modulation of cell division and apoptosis in cancer. Oncogene 22:8581–9. https:// doi: 10.1038/sj.onc.1207113.
2. Andric, M., Nikolic, N., Boskovic, M., Milicic, B., Skodric, S., Basta Jovanovic, G., et al. (2012). Survivin gene promoter polymorphism-31G/C as a risk factor for keratocystic odontogenic tumor development. European journal of oral sciences, 120(1): 9-13. https://doi.org/10.1111/j.1600-0722.2011.00919.x
3. Avci. E., Arikoglu, H., Erkoç Kaya, . (2016). Investigation of juglone effects on metastasis and angiogenesis in pancreatic cancer cells. Gene 588:74–78. http://dx.doi.org/10.1016/j.gene.2016.05.001
4. Azmi, A.S., Wang, Z., Philip, P.A., et al (2011). Emerging Bcl-2 inhibitors for the treatment of cancer. Expert Opin Emerg Drugs 16:59-70 https:// doi: 10.1517/14728214.2010.515210.
5. Bengtsson, A., Andersson, R., Ansari, D. (2020). The actual 5-year survivors of pancreatic ductal adenocarcinoma based on real-world data. Science Reports,10:16425. https://doi: 10.1038/s41598-020-73525-y.
6. Calcabrini C., Catanzaro E., Bishayee A., Turrini E., Fimognari C. Marine Sponge Natural Products with Anticancer Potential: An Updated Review. Marine Drugs. 2017;15:310. https://doi: 10.3390/md15100310.
7. Catanzaro, E., Greco, G., Potenza, L., Calcabrini, C., Fimognari, C. (2018). Natural Products to Fight Cancer: A Focus on Juglans regia. Toxins (Basel). 14;10(11):469. doi: 10.3390/toxins10110469.
8. Ji, Y.B., Qu, Z.Y., Zou, X. (2009). Juglone-induced apoptosis in human gastric cancer SGC-7901 cells via the mitochondrial pathway. Exp Toxicol Pathol, 63(1-2):69-78. https:// doi: 10.1016/j.etp.2009.09.010.

9. Jin. Z., El-Deiry WS. (2005). Overview of cell death signaling pathways. Cancer Biology & Therapy, 4(2):139–63. https://doi: 10.4161/cbt.4.2.1508.
10. Khan, M. (1999). γ-Sitosterol, a cytotoxic sterol from Markhamia zanzibarica and Kigelia africana. Fitoterapia, 70:96–97. https://doi: 10.1016/S0367-326X(99)00005-2.
11. Kim, R. (2005). Recent advances in understanding the cell death pathways activated by anticancer therapy. Cancer, 103(8):1551–60. https://doi: 10.1002/cncr.20947.
12. Kitagawa, R.R., Vilegas, W., Carlos, I.Z., et al (2011). Antitumor and immunomodulatory effects of the naphthoquinone 5-methoxy-3, 4-dehydroxanthomegnin. Revista Brasileira de Farmacognosia 21(6):1084-1088. https://doi.org/10.1590/S0102-695X2011005000136
13. Kumar M.R.S., Aithal K., Rao B.N., Udupa N., Rao B.S.S. Cytotoxic, genotoxic and oxidative stress induced by 1,4-naphthoquinone in B16F1 melanoma tumor cells. Toxicology. In Vitro. 2009;23:242–250. https:// doi: 10.1016/j.tiv.2008.12.004.
14. Los, M., Van de Craen, M., Penning, L.C., Schenk, H., Westendorp, M., Baeuerle, P.A., et al. (1995). Requirement of an ICE/CED-3 protease for Fas/APO-1-mediated apoptosis. Nature, 375(6526):81–3. https://doi: 10.1038/375081a0.
15. McGuigan, A., Kelly, P., Turkington, R.C., Jones, C., Coleman, H.G., McCain, RS. (2018) Pancreatic cancer: a review of clinical diagnosis, epidemiology, treatment and outcomes. World Journal of Gastroenterol 24(43):4846–4861. https:// 10.3748/wjg.v24.i43.4846
16. Naidu, M.U.R., Ramana, G.V., Rani, P.U., Mohan, I.K., Suman, A., Roy, P. (2004). Chemotherapy-induced and/or radiation therapy-induced oral mucositis-complicating the treatment of cancer. Neoplasia 6(5), 423–431. https://doi: 10.1593/neo.04169
17. Navya, P.N., Kaphle, A., Srinivas, S.P., Bhargava SK, Rotello VM, Daima HK et al. (2019) Current trends and challenges in cancer management and therapy using designer nanomaterials. Nano Convergence 6, 23. https://doi.org/10.1186/s40580-019-0193-2
18. Oltvai Z.N., Milliman, C.L. and Korsmeyer, S.J. (1993) Bcl–2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell, 74, 609–619. https:// doi: 10.1016/0092-8674(93)90509-o.
19. Ouyang, L., Shi, Z., Zhao, S., Wang F.-T., Zhou§, T.-T., Liu, B., and Bao, J.-K. (2012). Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis Cell Proliferation, 45:487–498 https:// doi: 10.1111/j.1365-2184.2012.00845.x.
20. Pavan V., Ribaudo G., Zorzan M., Redaelli M., Pezzani R., Mucignat-Caretta C., Zagotto G. (2017). Antiproliferative activity of Juglone derivatives on rat glioma. Natural Product Research. 2017;31:632–638. https://doi: 10.1080/14786419.2016.1214830.
21. Pereira, N.P., Corrêa, J.R. (2018). Pancreatic cancer: treatment approaches and trends. Journal of Cancer Metastasis Treatment 4:30. https://10.20517/2394-4722.2018.13
22. Ravelo, Á.G., Estévez-Braun, A., and Pérez-Sacau, E. (2003) The chemistry and biology of lapachol and related natural products α and β-lapachones. Studies in Natural Products Chemistry 29, 719−760. https:// 10.1016/S1572-5995(03)80017-0
23. Sah, N.K., Khan, Z., Khan, G.J., et al (2006). Structural, functional and therapeutic biology of survivin. Cancer Letters 244:164–71. https:// doi: 10.1016/j.canlet.2006.03.007.
24. Tang, Y.T., Li, Y., Chu, P., Dong Ma, X., Tang,Z.Y., Sun, Z.L. (2022). Molecular biological mechanism of action in cancer therapies: Juglone and its derivatives, the future of development. Biomedicine & Pharmacotherapy, 148;112785, https://doi.org/10.1016/j.biopha.2022.112785.
25. Verma, R.P. (2006). Anti-cancer activities of 1,4-naphthoquinones: A QSAR study. Anti-Cancer Agents in Medicinal Chemistry, 6:489–499. https://doi: 10.2174/187152006778226512.
26. Wang, P., Gao, C., Wang, W., Yao, L.P., Zhang, J., Zhang, S.D., Li, J., Fang, S.H., Fu, Y.J. (2018) Juglone induces apoptosis and autophagy via modulation of mitogen-activated protein kinase pathways in human hepatocellular carcinoma cells. Food Chem Toxicol, 116(Pt B):40-50. https:// doi: 10.1016/j.fct.2018.04.004.
27. Xu, H.L., Yu, X.F., Qu, S.C., et al. (2012). Juglone, from Juglans mandshruica Maxim, inhibits growth and induces apoptosis in human leukemia cell HL-60 through a reactive oxygen species-dependent mechanism. Food and Chemical Toxicology 50:590–596. https:// doi: 10.1016/j.fct.2012.01.002. Epub 2012 Jan 14.
28. Yazdani, N., Sayahpour, F.A., Haghpanah, V., et al. (2012). Survivin gene polymorphism association with papillary thyroid carcinoma, Pathology-Researh and Practice, 15:208(2):100-3. https:// doi: 10.1016/j.prp.2011.12.009. Epub 2012 Jan 30.
29. You, W., Henneberg, M,. (2017). Cancer incidence increasing globally: the role of relaxed natural selection. Evolutinary Applications, 11(2), 140–152. https://doi.org/10.1111/eva.12523
30. Yuan, S., Akey, C.W. (2013). Apoptosome structure, assembly, and procaspase activation. Structure, 21(4):501–15. doi: 10.1016/j.str.2013.02.024.