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

Öz

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.

Anahtar Kelimeler

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

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

Öz

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.

Anahtar Kelimeler

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

Kaynakça

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