The stimulation of apoptosis via mitochondrial (intrinsic) pathway in HeLa cells by some anthraquinone derivates

Abstract

Objective: The aim of this study was to investigate the effects of Ant-3, -4, and -6 anthraquinone derivatives on the mitochondrial (intrinsic) apoptotic pathway in HeLa cells. Methods: Mitochondrial membrane potential changes, Caspase-9 activity, apoptotic activity, and protein expression levels involved in the intrinsic pathway were measured. The TUNEL method was used to determine the apoptotic index. Results: Maximum Caspase-9 activation was observed after 6 hours of incubation with Ant-6 (3 μM) and Ant-4 (5 μM).The highest mitochondrial membrane potential change occurred after 36 hours of treatment with Ant-4 (5 μM).The apoptotic index, determined by the TUNEL method, peaked in the Ant-4 (5 μM) group after 48 hours. Increased expression of AIF and EndoG proteins was observed after 48 hours in HeLa cells treated with Ant-4 and mitoxantrone. Conclusion: These findings show promising results in in vitro experiments. However, further clinical studies are needed to validate the therapeutic potential of these newly synthesized anthraquinone derivatives in the treatment of cervical cancer.

Keywords

• Anthraquinone
• apoptosis
• caspase-9
• HeLa
• intrinsic pathway
• Western Blot

Project Number

This work has been supported by Anadolu University Scientific Research Projects Coordination Unit under grant number 1501S007.

Bazı Antrakinon Türevleri ile HeLa Hücrelerinde Mitokondriyal (İntrinsik) Yolak Üzerinden Apoptozun Uyarılması

Abstract

Amaç: Bu çalışmanın amacı, Ant-3, -4 ve -6 antrakinon türevlerinin HeLa hücrelerinde mitokondriyal (intrinsik) apoptotik yolak üzerindeki etkilerini araştırmaktır. Yöntemler: Mitokondriyal membran potansiyeli değişiklikleri, Kaspaz-9 aktivitesi, apoptotik aktivite ve intrinsik yolağa dahil olan proteinlerin ekspresyon seviyeleri ölçülmüştür. Apoptotik indeksin belirlenmesinde TUNEL yöntemi kullanılmıştır. Bulgular: Ant-6 (3 μM) ve Ant-4 (5 μM) ile 6 saatlik inkübasyon sonrası maksimum Kaspaz-9 aktivasyonu gözlenmiştir. Mitokondriyal membran potansiyelindeki en yüksek değişiklik, Ant-4 (5 μM) ile 36 saatlik uygulama sonrası meydana gelmiştir. TUNEL yöntemiyle belirlenen apoptotik indeks, Ant-4 (5 μM) grubunda 48 saat sonunda en yüksek seviyeye ulaşmıştır. Ant-4 ve mitoksantron ile tedavi edilen HeLa hücrelerinde 48 saat sonunda AIF ve EndoG proteinlerinin ekspresyonunda artış gözlenmiştir. Sonuç: Bu bulgular, in vitro deneylerde umut verici sonuçlar ortaya koymaktadır. Bununla birlikte, bu yeni sentezlenen antrakinon türevlerinin rahim ağzı kanseri tedavisindeki terapötik potansiyelini doğrulamak için ileri klinik çalışmalara ihtiyaç vardır.

Keywords

• Antrakinon
• apoptoz
• kaspaz-9
• HeLa
• intrinsik yolak
• Western Blot

Project Number

This work has been supported by Anadolu University Scientific Research Projects Coordination Unit under grant number 1501S007.

Ethical Statement

Funding This work has been supported by Anadolu University Scientific Research Projects Coordination Unit under grant number 1501S007.

References

1. [1] Sung, H., Ferlay, J., Siegel, R. L., Laversanne, M., Soerjomataram, I., Jemal, A., & Bray, F. (2021). Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 71(3), 209–249. https://doi.org/10.3322/caac.21660
2. [2] Ma, C., Gu, Y., Liu, C., Tang, X., Yu, J., Li, D., & Liu, J. (2023). Anti-cervical cancer effects of Compound Yangshe granule through the PI3K/AKT pathway based on network pharmacology. J. Ethnopharmacol., 301, 115820. https://doi.org/10.1016/j.jep.2022.115820
3. [3] Bray, F., Laversanne, M., Sung, H., Ferlay, J., Siegel, R. L., Soerjomataram, I., & Jemal, A. (2024). Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin, 74(3), 229–263. https://doi.org/10.3322/caac.21834
4. [4] Wang, L., Zhao, Y., Wang, Y., & Wu, X. (2018). The role of galectins in cervical cancer biology and progression. Biomed. Res. Int., 2018, 2175927. https://doi.org/10.1155/2018/2175927
5. [5] Jin, Y. M., Liu, S. S., Chen, J., Chen, Y. N., & Ren, C. C. (2018). Robotic radical hysterectomy is superior to laparoscopic and open radical hysterectomy in the treatment of cervical cancer. PLoS One, 13(3), e0193033. https://doi.org/10.1371/journal.pone.0193033
6. [6] Malik, E. M., & Müller, C. E. (2016). Anthraquinones as pharmacological tools and drugs. Med. Res. Rev., 36(4), 705–748. https://doi.org/10.1002/med.21391
7. [7] Damiani, R. M., Moura, D. J., Viau, C. M., Caceres, R. A., Henriques, J. A. P., & Saffi, J. (2016). Pathways of cardiac toxicity: Comparison between chemotherapeutic drugs doxorubicin and mitoxantrone. Arch. Toxicol., 90, 2063–2076. https://doi.org/10.1007/s00204-016-1759-y
8. [8] Ly, J. D., Grubb, D. R., & Lawen, A. (2003). The mitochondrial membrane potential (Δψm) in apoptosis: An update. Apoptosis, 8, 115–128. https://doi.org/10.1023/A:1022945107762

9. [9] Huang, Q., Lu, G., Shen, H. M., Chung, M. C., & Ong, C. N. (2007). Anti-cancer properties of anthraquinones from rhubarb. Med. Res. Rev., 27(5), 609–630. https://doi.org/10.1002/med.20094
10. [10] Khan, K., Karodi, R., Siddiqui, A., Thube, S., & Rub, R. (2011). Development of anti-acne gel formulation of anthraquinone-rich fraction from Rubia cordifolia (Rubiaceae). Int. J. Appl. Res. Nat. Prod., 4(4), 28–36.
11. [11] Davis, R. H., Agnew, P. S., & Shapiro, E. (1986). Antiarthritic activity of anthraquinones found in Aloe vera for podiatric medicine. J. Am. Podiatr. Med. Assoc., 76(2), 61–68. https://doi.org/10.7547/87507315-76-2-61
12. [12] Wuthi-udomlert, M., Kupittayanant, P., & Gritsanapan, W. (2010). In vitro evaluation of antifungal activity of anthraquinone derivatives of Senna alata. J. Health Res., 24(3), 117–122.
13. [13] Jackson, T., Verrier, J., & Kochanek, P. (2013). Anthraquinone-2-sulfonic acid (AQ2S) is a novel neurotherapeutic agent. Cell Death Dis., 4(1), e451. https://doi.org/10.1038/cddis.2012.187
14. [14] Demarque, D. P., Silva, R. M. P., Santos, L. F., Leopoldino, A. M., Espreafico, E. M., & Lopes, N. P. (2018). Cytotoxicity of structurally diverse anthranoids and correlation with mechanism of action and side effects. J. Pharm. Pharm. Sci., 21(1), 347–353. https://doi.org/10.18433/jpps30077
15. [15] Preobrazhenskaya, M. N., Shchekotikhin, A. E., Shtil, A. A., & Huang, H. S. (2006). Antitumor anthraquinone analogues for multidrug-resistant tumor cells. J. Med. Sci., 26(1), 1–4.
16. [16] Onder, N. I., Incesu, Z., & Özkay, Y. (2015). Synthesis and evaluation of new dithiocarbamic acid 6,11-dioxo-6,11-dihydro-1H-anthra[1,2-d]-imidazol-2-ylmethyl esters. Arch. Pharm., 348(7), 508–517. https://doi.org/10.1002/ardp.201500063
17. [17] Erdogan, M. K., Agca, C. A., & Askin, H. (2019). Enhanced antiproliferative and apoptotic effects of 5-fluorouracil combined with Pistacia eurycarpa extracts on human colorectal cancer cells. Biol. Divers. Conserv., 12(1), 27–38. https://doi.org/10.5505/biodicon.2019.57441
18. [18] Li, P., Zhou, L., Zhao, T., Liu, X., Zhang, P., Liu, Y., Zheng, X., & Li, Q. (2017). Caspase-9: Structure, mechanisms, and clinical application. Oncotarget, 8(14), 23996–24008. https://doi.org/10.18632/oncotarget.15098
19. [19] Singh, M., Malhotra, L., Haque, M. A., Kumar, M., Tikhomirov, A., Litvinova, V., Korolev, A. M., Ethayathulla, A., Das, U., & Shchekotikhin, A. E. (2021). Heteroarene-fused anthraquinone derivatives as potential modulators for human aurora kinase B. Biochimie, 182, 152–165. https://doi.org/10.1016/j.biochi.2020.12.024
20. [20] Okon, E., Gaweł-Bęben, K., Jarzab, A., Koch, W., Kukula-Koch, W., & Wawruszak, A. (2023). Therapeutic potential of 1,8-dihydroanthraquinone derivatives for breast cancer. Int. J. Mol. Sci., 24(21), 15789. https://doi.org/10.3390/ijms242115789
21. [21] Luo, S. K., Li, J., Hong, W. D., Zhao, Y., & Tong, X. Z. (2005). Preliminary report of fludarabine, mitoxantrone and dexamethasone in treating refractory or relapsed multiple myeloma. Zhongguo Ai Zheng Za Zhi, 24(12), 1518–1521.
22. [22] Yaoxian, W., Hui, Y., Yunyan, Z., Yanqin, L., Xin, G., & Xiaoke, W. (2013). Emodin induces apoptosis of human cervical cancer HeLa cells via intrinsic mitochondrial and extrinsic death receptor pathways. Cancer Cell Int., 13, 71. https://doi.org/10.1186/1475-2867-13-71
23. [23] Wijesekara, I., Zhang, C., Van Ta, Q., Vo, T. S., Li, Y. X., & Kim, S. K. (2014). Physcion from marine-derived fungus Microsporum sp. induces apoptosis in human cervical carcinoma HeLa cells. Microbiol. Res., 169(4), 255–261. https://doi.org/10.1016/j.micres.2013.09.001
24. [24] Radogna, F., Diederich, M., & Ghibelli, L. (2008). Melatonin antagonizes the intrinsic pathway of apoptosis via mitochondrial targeting of Bcl-2. J. Pineal Res., 44(3), 316–325. https://doi.org/10.1111/j.1600-079X.2007.00532.x
25. [25] Yang, J., Liu, X., Bhalla, K., Kim, C. N., Ibrado, A. M., Cai, J., Peng, T. I., Jones, D. P., & Wang, X. (1997). Prevention of apoptosis by Bcl-2: Release of cytochrome c from mitochondria blocked. Science, 275(5303), 1129–1132. https://doi.org/10.1126/science.275.5303.1129
26. [26] Chiang, J. H., Yang, J. S., Ma, C. Y., Yang, M. D., Huang, H. Y., Hsia, T. C., Kuo, H. M., Wu, P. P., Lee, T. H., & Chung, J. G. (2011). Danthron, an anthraquinone derivative, induces DNA damage and caspase cascade-mediated apoptosis in SNU-1 human gastric cancer cells through mitochondrial permeability transition pores and Bax-triggered pathways. Chem. Res. Toxicol., 24(1), 20–29. https://doi.org/10.1021/tx100248s