Kemoterapi yanıtının yeniden düzenlenmesi: venetoklaks, artmış DNA hasarı ve ferroptotik yolaklar aracılığıyla insan akciğer kanseri hücrelerinde etopozide yanıtı artırmaktadır

Abstract

Amaçlar: Venetoklaks, seçici bir Bcl-2 inhibitörü küçük molekül olup, FDA tarafından akut myeloid lösemi ve kronik lenfositik lösemi tedavisi için onaylanmıştır. Hematolojik malignitelerde umut verici sonuçlar göstermesine rağmen, akciğer kanserindeki terapötik potansiyeli halen belirsizliğini korumaktadır. Bu çalışmada, klasik kemoterapötikler olan etopozid ve sisplatinin venetoklaks ile kombinasyonunun insan akciğer kanseri hücrelerindeki etkisi ve altta yatan mekanizmalar araştırılmıştır. Yöntemler: Sisplatin ve etopozid tek başına veya venetoklaks (8 µM) ile kombinasyon halinde insan küçük hücreli dışı akciğer kanseri hücrelerine (A549) uygulanmış, hücre canlılığı MTT testi ile belirlenmiştir. Elde edilen sitotoksisite sonuçları L929 normal hücreleri ile karşılaştırılmıştır. Reaktif oksijen türleri (ROS) üretimi, antioksidanlar (N-asetilsistein, mannitol) ve ferroptoz inhibitörleri (deferoksamin, ferrostatin-1) varlığında veya yokluğunda konfokal mikroskopi ile görüntülenmiştir. Ayrıca, serbest demir havuzu ve lizozomal lipid birikimi Hoechst/Calcein-AM/Neutral Red üçlü boyama yöntemiyle saptanmıştır. Lipid peroksidasyonu malondialdehit tayini ile ölçülmüştür. DNA hasarı belirteci H2AX ve ferroptoz belirteci transferrinin mRNA düzeyleri RT-qPCR ile değerlendirilmiştir. Bulgular: Bulgular, venetoklaksın (8 µM) A549 hücrelerini etopozide duyarlılaştırdığını, ancak sisplatin aktivitesi üzerinde anlamlı bir etkisi olmadığını göstermiştir. İleri analizler, etopozid ve venetoklaks birlikte uygulandığında ROS üretiminin belirgin şekilde arttığını ve bu etkinin ferroptoz inhibitörleri tarafından baskılandığını ortaya koymuştur. Görüntüleme analizleri ayrıca, etopozid–venetoklaks kombinasyonunu takiben serbest demir havuzunun ve lizozomal lipidlerin arttığını göstermiştir. Gen ekspresyon analizleri ise H2AX ve transferrin düzeylerinin kombinasyonun uygulandığı grupta anlamlı biçimde yükseldiğini ortaya koymuştur. Sonuç: Bu çalışma, venetoklaksın akciğer kanseri hücrelerini ferroptotik yolaklar ve DNA hasarı aracılığıyla etopozide duyarlılaştırdığını ilk kez göstermektedir.

Keywords

• Sisplatin
• etopozid
• ferroptoz
• akciğer kanseri
• venetoklaks

Rewiring chemotherapy response: venetoclax sensitizes human lung cancer cells to etoposide via enhanced DNA damage and ferroptotic pathways

Abstract

Aims: Venetoclax is a selective Bcl-2 inhibitor small molecule, approved by FDA for acute myeloid and chronic lymphocytic leukemia. Despite the promising results in hematological malignancies, its therapeutic potential in lung cancer remains unclear. In the present study, the effect of conventional chemotherapeutics, etoposide and cisplatin, in combination with venetoclax was investigated along with the underlying mechanisms in lung cancer cells. Methods: Human non-small cell lung cancer cells (A549) were administered with cisplatin and etoposide either as single agents or in combination with venetoclax (8 µM), and the cell viability was determined with MTT assay. The acquired cytotoxicity was compared with L929 normal cells. ROS generation was visualized by confocal microscopy in the presence or absence of antioxidants (N-acetylcysteine, mannitol) and ferroptosis inhibitors (deferoxamine, ferrostatin-1). Furthermore, labile iron pool and lysosomal lipid were detected with Hoechst/Calcein-AM/Neutral red triple staining. Lipid peroxidation was determined with malondialdehyde assay. The mRNA expressions of DNA damage marker H2AX and ferroptotic marker transferrin were evaluated with RT-qPCR. Results: Results indicated that venetoclax (8 µM) sensitizes A549 cells to etoposide (p<0.001) while having no significant impact on cisplatin activity (p>0.05). Further analyses demonstrated that co-treatment of etoposide and venetoclax significantly increased ROS generation, which was inhibited by ferroptosis inhibitors (p<0.001). Additionally, imaging analyses revealed that both labile iron pool and lysosomal lipid increase following etoposide-venetoclax co-treatment. Gene expression analyses indicated that H2AX and transferrin were both significantly upregulated in co-treated groups (p<0.05). Conclusion: This study demonstrated for the first time that venetoclax sensitizes lung cancer cells to etoposide via ferroptotic pathways and DNA damage.

Keywords

• Cisplatin
• etoposide
• ferroptosis
• lung cancer
• venetoclax

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