Glioblastomada Artemisia absinthium ve Temozolomidin Kombine Uygulamasının Apoptoz, Otofaji ve Nitrik Oksit Sentaz Üzerine Etkileri

Abstract

Artemisia absinthium (pelin otu; WO), antikanser potansiyeli ile bilinen bir bitkidir; bu çalışma, WO’nun tek başına ve Temozolomid (TMZ) ile birlikte uygulanmasının T98-G glioblastoma hücrelerinde sitotoksisite, apoptoz, otofaji, mitokondriyal membran potansiyeli, hücre göçü ve nitrik oksit sentaz (NOS) yanıtları üzerindeki etkilerini kapsamlı bir şekilde değerlendirmektedir. Çalışma esnasında sitotoksisite, apoptoz, otofaji, mitokondriyal membran potansiyeli ve hücre göçü sırasıyla MTT, Annexin V-FITC/PI, JC-1, monodansilkadaverin (MDC) boyaması ve yara iyileşme testi kullanılarak analiz edilmiştir. eNOS, iNOS ve LC3-II protein düzeyleri immünfloresan ve western blot yöntemleri kullanılarak incelenmiştir. WO, T98-G hücre canlılığını doz- ve zamana bağlı olarak anlamlı şekilde azaltmış, özellikle 48 ve 72 saatlerde belirgin sitotoksisite gözlenmiştir. Kombine tedavi erken (%24.6) ve geç (%40.5) apoptozu artırmış ve hücre canlılığını %31.9’a düşürmüştür. Kombinasyon tedavisi, tek ajanlarla karşılaştırıldığında çekirdek çevresi ve sitoplazmik birikimi artırarak otofajik vakuolleri belirgin şekilde yükseltmiştir. WO ve TMZ kombinasyonu, mitokondriyal membran potansiyelini sinerjistik biçimde bozarak apoptozun mitokondriyal yolak üzerinden gerçekleştiğini düşündürmüştür. WO, T98-G hücre göçünü güçlü biçimde inhibe ederken, TMZ ve kombinasyon tedavisinin baskılayıcı etkileri orta düzeyde kalmıştır. T98-G hücrelerinde WO IC₅₀, eNOS (p<0.0001), iNOS (p<0.0001) ve LC3 (p<0.01) düzeylerini anlamlı şekilde artırırken; TMZ IC₅₀ iNOS düzeyini orta derecede yükseltmiştir (p<0.05). Kombine tedavi, eNOS üzerinde ek bir etki göstermeksizin iNOS ve LC3 düzeylerini maksimum düzeye çıkarmıştır (p<0.0001). Ön değerlendirme niteliğindeki Western blot analizleri, immünfloresans bulgularını niteliksel olarak destekleyerek eNOS, iNOS ve LC3 ifadelerinin tedaviye bağlı tutarlı değişim gösterdiğini ortaya koymuştur. Bu bulgular, WO’nun TMZ etkinliğini artırma potansiyelini vurgulamakta ve glioblastoma tedavisinde ileri araştırmalar için temel oluşturmaktadır.

Keywords

• T98-G hücre hattı
• programlanmış hücre ölümü
• mitokondriyal disfonksiyon
• hücre göçü

Supporting Institution

Manisa Celal Bayar Üniversitesi. Bilimsel Araştırma Projeleri Koordinasyon Birimi.

Project Number

2022-025

Ethical Statement

Bu çalışma için etik kurul onayı gerekmemektedir.

Thanks

Yazarlar, bu çalışmayı destekleyen Manisa Celal Bayar Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi'ne teşekkür ederler (Proje No. 2022-025).

Effects of Combined Artemisia absinthium and Temozolomide Treatment on Apoptosis, Autophagy, and Nitric Oxide Synthase in Glioblastoma

Abstract

Artemisia absinthium (wormwood; WO) is a plant known for its anticancer potential and this study comprehensively evaluates the effects of WO, alone and in combination with Temozolomide (TMZ), on cytotoxicity, apoptosis, autophagy, mitochondrial membrane potential, cell migration, and nitric oxide synthase (NOS) responses in T98-G glioblastoma cells. Cytotoxicity, apoptosis, autophagy, mitochondrial membrane potential, and cell migration were assessed using MTT, Annexin V-FITC/PI, JC-1, monodansylcadaverine (MDC) staining, and wound healing assay, respectively. Protein levels of eNOS, iNOS, and LC3-II were examined using immunofluorescence and western blotting. WO significantly reduced T98-G cell viability in a dose- and time-dependent manner with pronounced cytotoxicity observed at 48 and 72 hours. The combined treatment increased early (24.6%) and late (40.5%) apoptosis, reducing cell viability to 31.9%. Combined treatment markedly increased autophagic vacuoles, enhancing perinuclear and cytoplasmic accumulation compared with single agents. Combined WO and TMZ treatment disrupted mitochondrial membrane potential more prominently than either agent alone, suggesting that apoptosis is mediated via the mitochondrial pathway. WO strongly inhibited T98-G cell migration, whereas TMZ and the combination had moderate suppressive effects. In T98-G cells, WO IC₅₀ significantly increased eNOS (p<0.0001), iNOS (p<0.0001), and LC3 (p<0.01), while TMZ IC₅₀ moderately increased iNOS (p<0.05) and combined treatment maximally elevated iNOS and LC3 (p<0.0001) without further affecting eNOS. Preliminary Western blot analyses qualitatively corroborated the immunofluorescence data, indicating consistent treatment-dependent trends in eNOS, iNOS, and LC3-II expression. These findings highlight the potential of WO in enhancing TMZ efficacy and provide a basis for further investigation in glioblastoma therapy.

Keywords

• T98-G cell line
• programmed cell death
• mitochondrial dysfunction
• cell migration

Supporting Institution

Manisa Celal Bayar University. Scientific Research Projects Committee.

Project Number

2022-025

Ethical Statement

Ethics committee approval is not required for this study.

Thanks

The authors thank the Scientific Research Projects Committee of Manisa Celal Bayar University for supporting this work (Project No. 2022-025).

References

1. Batash, R., Asna, N., Schaffer, P., Francis, N., & Schaffer, M. (2017). Glioblastoma multiforme, diagnosis and treatment; recent literature review. Current Medicinal Chemistry, 24(27), 3002–3009. https://doi.org/10.2174/0929867324666170516123206
2. Batiha, G.E., Olatunde, A., El-Mleeh, A., Hetta, H.F., Al-Rejaie, S., Alghamdi, S., Zahoor, M., Magdy Beshbishy, A., Murata, T., Zaragoza-Bastida, A., & Rivero-Perez, N. (2020). Bioactive compounds, pharmacological actions, and pharmacokinetics of wormwood (Artemisia absinthium). Antibiotics (Basel, Switzerland), 9(6), 353. https://doi.org/10.3390/antibiotics9060353
3. Bora, K.S., & Sharma, A. (2010). Neuroprotective effect of Artemisia absinthium L. on focal ischemia and reperfusion-induced cerebral injury. Journal of Ethnopharmacology, 129(3), 403–409. https://doi.org/10.1016/j.jep.2010.04.030
4. Caner, A., Döşkaya, M., Değirmenci, A., Can, H., Baykan, S., Uner, A., Başdemir, G., Zeybek, U., & Gürüz, Y. (2008). Comparison of the effects of Artemisia vulgaris and Artemisia absinthium growing in western Anatolia against trichinellosis (Trichinella spiralis) in rats. Experimental Parasitology, 119(1), 173–179. https://doi.org/10.1016/j.exppara.2008.01.012
5. Dehelean, C.A., Marcovici, I., Soica, C., Mioc, M., Coricovac, D., Iurciuc, S., Cretu, O.M., & Pinzaru, I. (2021). Plant-derived anticancer compounds as new perspectives in drug discovery and alternative therapy. Molecules (Basel, Switzerland), 26(4), 1109. https://doi.org/10.3390/molecules26041109
6. He, M., Yasin, K., Yu, S., Li, J., & Xia, L. (2023). Total Flavonoids in Artemisia absinthium L. and evaluation of ıts anticancer activity. International Journal of Molecular Sciences, 24(22), 16348. https://doi.org/10.3390/ijms242216348
7. Kashyap, D., Tuli, H.S., Yerer, M.B., Sharma, A., Sak, K., Srivastava, S., Pandey, A., Garg, V.K., Sethi, G., & Bishayee, A. (2021). Natural product-based nanoformulations for cancer therapy: Opportunities and challenges. Seminars in Cancer Biology, 69, 5–23. https://doi.org/10.1016/j.semcancer.2019.08.014
8. Koyuncu I. (2018). Evaluation of anticancer, antioxidant activity and phenolic compounds of Artemisia absinthium L. Extract. Cellular and Molecular Biology (Noisy-le-Grand, France), 64(3), 25–34. https://doi.org/10.14715/cmb/2018.64.3.5

9. Mahmoudi, M., Ebrahimzadeh, M.A., Ansaroudi, F., Nabavi, S.F., & Nabavi, S.M. (2009). Antidepressant and antioxidant activities of Artemisia absinthium L. at flowering stage. African Journal of Biotechnology, 8(24), 7170–7175. https://doi.org/10.5897/AJB09.753
10. Mughees, M., Wajid, S., & Samim, M. (2020). Cytotoxic potential of Artemisia absinthium extract loaded polymeric nanoparticles against breast cancer cells: Insight into the protein targets. International Journal of Pharmaceutics, 586, 119583. https://doi.org/10.1016/j.ijpharm.2020.119583
11. Nazeri, M., Mirzaie-Asl, A., Saidijam, M., & Moradi, M. (2020). Methanolic extract of Artemisia absinthium prompts apoptosis, enhancing expression of Bax/Bcl-2 ratio, cell cycle arrest, caspase-3 activation and mitochondrial membrane potential destruction in human colorectal cancer HCT-116 cells. Molecular Biology Reports, 47(11), 8831–8840. https://doi.org/10.1007/s11033-020-05933-2
12. Park, J.H., & Lee, H.K. (2022). Current understanding of hypoxia in glioblastoma multiforme and its response to immunotherapy. Cancers, 14(5), 1176. https://doi.org/10.3390/cancers14051176
13. Parpura, V., Heneka, M.T., Montana, V., Oliet, S.H., Schousboe, A., Haydon, P.G., Stout, R.F., Jr, Spray, D.C., Reichenbach, A., Pannicke, T., Pekny, M., Pekna, M., Zorec, R., & Verkhratsky, A. (2012). Glial cells in (patho) physiology. Journal of Neurochemistry, 121(1), 4–27. https://doi.org/10.1111/j.1471-4159.2012.07664.x
14. Pfeffer, C.M., & Singh, A.T.K. (2018). Apoptosis: A target for anticancer therapy. International Journal of Molecular Sciences, 19(2), 448. https://doi.org/10.3390/ijms19020448
15. Ramazani, A., Sardari, S., Zakeri, S., & Vaziri, B. (2010). In vitro antiplasmodial and phytochemical study of five Artemisia species from Iran and in vivo activity of two species. Parasitology Research, 107(3), 593–599. https://doi.org/10.1007/s00436-010-1900-4
16. Singh, N.P., & Lai, H.C. (2004). Artemisinin induces apoptosis in human cancer cells. Anticancer Research, 24(4), 2277–2280.
17. Tsamesidis, I., Papadimitriou-Tsantarliotou, A., Christodoulou, A., Amanatidou, D., Avgeros, C., Stalika, E., Bousnaki, M., Michailidou, G., Beketova, A., Eleftheriou, P., Bikiaris, D.N., Vizirianakis, I.S., & Kontonasaki, E. (2024). Investigating the cytotoxic effects of Artemisia absinthium extract on oral carcinoma cell line. Biomedicines, 12(12), 2674. https://doi.org/10.3390/biomedicines12122674
18. Wang, H., Liu, Y., Wang, Y., Xu, T., Xia, G., & Huang, X. (2023). Umbelliprenin induces autophagy and apoptosis while inhibits cancer cell stemness in pancreatic cancer cells. Cancer Medicine, 12(14), 15277–15288. https://doi.org/10.1002/cam4.6170
19. Wei, X., Xia, L., Ziyayiding, D., Chen, Q., Liu, R., Xu, X., & Li, J. (2019). The extracts of Artemisia absinthium L. suppress the growth of hepatocellular carcinoma cells through induction of apoptosis via endoplasmic reticulum stress and mitochondrial-dependent pathway. Molecules, 24(5), 913. https://doi.org/10.3390/molecules24050913