Murin akciğer kanseri ve fibroblast hücrelerinde otofaji ve apoptoz genlerinin deneysel profilleme ve in silico validasyonu
Öz
Amaç: Otofaji ve apoptoz, hücresel homeostazın korunmasında kritik bir rol oynayan kritik programlanmış hücre ölüm yollarıdır. Bu süreçlerin düzensizliği genellikle kanser gelişimi ve ilerlemesinde rol oynar. Amacımız, murin akciğer karsinomu hücreleri ile malign olmayan fibroblastlar arasındaki anahtar otofaji ve apoptozla ilişkili genlerin ekspresyon profillerini karşılaştırmak ve in silico analizler kullanarak düzenleyici etkileşimlerini değerlendirmektir.
Gereç ve Yöntem: KLN-205 murin akciğer karsinomu hücreleri ve fare derisi fibroblastları (MSF) standart koşullar altında kültüre edilmiştir. Atg7, SQSTM1, Beclin-1, Kaspaz-3, Kaspaz-8, Kaspaz-9, Bax ve Bcl-2'nin mRNA ekspresyon seviyeleri RT-qPCR ile ölçülmüştür. Normalize edilen veriler 2-ΔΔCT dönüşümü kullanılarak analiz edilmiş ve gruplar arasındaki istatistiksel farklılıklar eşleşmemiş t-testleri ile değerlendirilmiştir. Temel bileşen analizi (PCA) ve STRING veri tabanı kullanılarak protein-protein etkileşimi (PPI) ağlarının oluşturulması dahil olmak üzere daha geniş ekspresyon modellerini ve moleküler etkileşimleri keşfetmek için in silico analizler yapılmıştır.
Bulgular: MSF'ye kıyasla KLN-205 hücrelerinde Atg7'de anlamlı bir aşağı regülasyon gözlenmiştir (p <0.05). PCA, iki hücre tipinin farklı kümelenmesini gösterirken, volkan grafikleri ve ısı haritaları transkripsiyonel farkın varlığını desteklemiştir. STRING ağ analizi, otofaji ve apoptoz düzenleyicileri arasında, Beclin-1 ve Kaspaz-3’ün ana ağ merkezleri olarak kompakt etkileşimleri ortaya çıkarmıştır. Zenginleştirme analizleri, ‘Otofaji’, ‘Apoptoz’, ‘p53 sinyali’ ve ‘Mitofaji’ gibi aşırı temsil edilen yolakları belirlenmiştir.
Sonuç: Çalışmamız, murin akciğer karsinomu hücrelerinde otofaji ve apoptoz yolaklarının koordineli transkripsiyonel modülasyonunu vurgulamaktadır. Atg7'nin belirgin aşağı regülasyonu ve diğer anahtar genlerin değişmiş ekspresyonu, otofajik mekanizmanın fonksiyonel olarak baskılanmasını ve hücre ölümü regülasyonunun yeniden şekillenmesini önermektedir.
Anahtar Kelimeler
Akciğer kanseri Otofaji Apoptoz KLN-205 Gen ekspresyonu RT-qPCR Biyofizik
Kaynakça
- Qian S, Long Y, Tan G, Li X, Xiang B, Tao Y, vd. Programmed cell death: molecular mechanisms, biological functions, diseases, and therapeutic targets. MedComm [Internet]. 01 Aralık 2024;5(12):e70024. Available at: https://doi.org/10.1002/mco2.70024
- Gómez-Virgilio L, Silva-Lucero M-D-C, Flores-Morelos D-S, Gallardo-Nieto J, Lopez-Toledo G, Abarca-Fernandez A-M, vd. Autophagy: A Key Regulator of Homeostasis and Disease: An Overview of Molecular Mechanisms and Modulators. Cells. Temmuz 2022;11(15).
- Jalali P, Shahmoradi A, Samii A, Mazloomnejad R, Hatamnejad MR, Saeed A, vd. The role of autophagy in cancer: from molecular mechanism to therapeutic window. Front Immunol [Internet]. 2025;Volume 16. Available at: https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2025.1528230
- Saddam M, Paul SK, Habib MA, Fahim MA, Mimi A, Islam S, vd. Emerging biomarkers and potential therapeutics of the BCL-2 protein family: the apoptotic and anti-apoptotic context. Egypt J Med Hum Genet [Internet]. 2024;25(1):12. Available at: https://doi.org/10.1186/s43042-024-00485-7
- Yun CW, Jeon J, Go G, Lee JH, Lee SH. The Dual Role of Autophagy in Cancer Development and a Therapeutic Strategy for Cancer by Targeting Autophagy. Int J Mol Sci. Aralık 2020;22(1).
- Tutak I, Ozdil B, Uysal A. Voxtalisib and low intensity pulsed ultrasound combinatorial effect on glioblastoma multiforme cancer stem cells via PI3K/AKT/mTOR. Pathol Pract. 2022;239:154145.
- Dikici O, Ozdil B, Yesin TK, Dikici A, Adalı Y, Aktug H. Time-Dependent Effects of Low-Intensity Pulsed Ultrasound on Apoptosis and Autophagy in Malignant Melanoma Stem Cells. J Cell Mol Med. Haziran 2025;29(12):e70687.
- Oltulu F, Kocaturk DC, Adali Y, Ozdil B, Acikgoz E, Gurel C, vd. Autophagy and mTOR pathways in mouse embryonic stem cell, lung cancer and somatic fibroblast cell lines. J Cell Biochem. Ekim 2019;120(10):18066–76.
- Xu Y, Qian C, Wang Q, Song L, He Z, Liu W, vd. Deacetylation of ATG7 drives the induction of macroautophagy and LC3-associated microautophagy. Autophagy [Internet]. 03 Mayıs 2024;20(5):1134–46. Available at: https://doi.org/10.1080/15548627.2023.2287932
- Ostacolo K, López García de Lomana A, Larat C, Hjaltalin V, Holm KY, Hlynsdóttir SS, vd. ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism. Traffic [Internet]. 01 Nisan 2024;25(4):e12933. Available at: https://doi.org/10.1111/tra.12933
- Hasanpour SZ, Allah Bakhshian M, Mohammadi MH, Ghasemian SO, Gholami-Ahangaran M. Beclin1, Atg10 and Atg7 genes expressions as autophagy mediators in acute B-lymphoblastic leukemia. Hum Gene [Internet]. 2024;41:201311. Available at: https://www.sciencedirect.com/science/article/pii/S277304412400055X
- Devarajan E, Sahin AA, Chen JS, Krishnamurthy RR, Aggarwal N, Brun A-M, vd. Down-regulation of caspase 3 in breast cancer: a possible mechanism for chemoresistance. Oncogene. Aralık 2002;21(57):8843–51.
- Ghavami S, Hashemi M, Ande SR, Yeganeh B, Xiao W, Eshraghi M, vd. Apoptosis and cancer: mutations within caspase genes. J Med Genet [Internet]. 01 Ağustos 2009;46(8):497 LP – 510. Available at: http://jmg.bmj.com/content/46/8/497.abstract
- Park JY, Park JM, Jang JS, Choi JE, Kim KM, Cha SI, vd. Caspase 9 promoter polymorphisms and risk of primary lung cancer . Hum Mol Genet [Internet]. 15 Haziran 2006;15(12):1963–71. Available at: https://doi.org/10.1093/hmg/ddl119
- Qaed E, Liu W, Almoiliqy M, Mohamed R, Tang Z. Unleashing the potential of Genistein and its derivatives as effective therapeutic agents for breast cancer treatment. Naunyn Schmiedebergs Arch Pharmacol [Internet]. 2025;398(4):3321–43. Available at: https://doi.org/10.1007/s00210-024-03579-6
- Pu X, Storr SJ, Zhang Y, Rakha EA, Green AR, Ellis IO, vd. Caspase-3 and caspase-8 expression in breast cancer: caspase-3 is associated with survival. Apoptosis [Internet]. 2017;22(3):357–68. Available at: https://doi.org/10.1007/s10495-016-1323-5
- Boice A, Bouchier-Hayes L. Targeting apoptotic caspases in cancer. Biochim Biophys Acta - Mol Cell Res [Internet]. 2020;1867(6):118688. Available at: https://www.sciencedirect.com/science/article/pii/S016748892030046X
- Baranovsky A, Ivanov T, Granovskaya M, Papatsenko D, Pervouchine DD. Transcriptome analysis reveals high tumor heterogeneity with respect to re-activation of stemness and proliferation programs. PLoS One. 2022;17(5):e0268626.
- Kang R, Zeh HJ, Lotze MT, Tang D. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 2011;18(4):571–80.
- Marquez RT, Xu L. Bcl-2:Beclin 1 complex: multiple, mechanisms regulating autophagy/apoptosis toggle switch. Am J Cancer Res [Internet]. 2012/02/15. 2012;2(2):214–21. Available at: https://pubmed.ncbi.nlm.nih.gov/22485198
Experimental profiling and in silico validation of autophagy and apoptosis-related genes in murine lung cancer and fibroblast cells
Öz
Aim: Autophagy and apoptosis are key programmed cell death pathways that play a critical role in cellular homeostasis. Dysregulation of these pathways is frequently associated with cancer development and progression. Comparing the molecular signatures of tumor and normal cells may provide novel insights into the survival mechanisms adopted by cancer cells.
Objective: The objective of this study was to compare the expression profiles of key autophagy- and apoptosis-related genes between murine lung carcinoma cells and non-malignant fibroblasts and to assess their regulatory interactions using in silico analyses.
Materials and Methods: KLN-205 murine lung carcinoma cells and mouse skin fibroblasts (MSF) were cultured under standard conditions. The mRNA expression levels of Atg7, SQSTM1, Beclin-1, Caspase-3, Caspase-8, Caspase-9, Bax, and Bcl-2 were quantified by RT-qPCR. Normalized data were analyzed using 2-ΔΔCT method, and statistical differences between groups were evaluated using unpaired t-tests. In silico analyses were performed to explore broader expression patterns and molecular interactions, including principal component analysis (PCA) and the construction of protein–protein interaction (PPI) networks using the STRING database.
Results: A significant downregulation of Atg7 was observed in KLN-205 cells compared to MSF (p < 0.05). PCA demonstrated distinct clustering of the two cell types, while volcano plots and heatmaps supported the presence of transcriptional differences. STRING network analysis revealed compact interactions between autophagy and apoptosis regulators, with Beclin-1 and Caspase-3 as key network hubs. Enrichment analyses identified overrepresented pathways such as ‘Autophagy,’ ‘Apoptosis,’ ‘p53 signaling,’ and ‘Mitophagy.’
Conclusion: Our findings indicate that murine lung carcinoma cells exhibit coordinated transcriptional changes in autophagy and apoptosis pathways. In particular, the significant downregulation of Atg7 and altered expression of other key genes suggest a functional suppression of the autophagic machinery and a reprogramming of cell death regulation.
Anahtar Kelimeler
Lung cancer Autophagy Apoptosis KLN-205 Gene expression RT-qPCR Biophysics
Kaynakça
- Qian S, Long Y, Tan G, Li X, Xiang B, Tao Y, vd. Programmed cell death: molecular mechanisms, biological functions, diseases, and therapeutic targets. MedComm [Internet]. 01 Aralık 2024;5(12):e70024. Available at: https://doi.org/10.1002/mco2.70024
- Gómez-Virgilio L, Silva-Lucero M-D-C, Flores-Morelos D-S, Gallardo-Nieto J, Lopez-Toledo G, Abarca-Fernandez A-M, vd. Autophagy: A Key Regulator of Homeostasis and Disease: An Overview of Molecular Mechanisms and Modulators. Cells. Temmuz 2022;11(15).
- Jalali P, Shahmoradi A, Samii A, Mazloomnejad R, Hatamnejad MR, Saeed A, vd. The role of autophagy in cancer: from molecular mechanism to therapeutic window. Front Immunol [Internet]. 2025;Volume 16. Available at: https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2025.1528230
- Saddam M, Paul SK, Habib MA, Fahim MA, Mimi A, Islam S, vd. Emerging biomarkers and potential therapeutics of the BCL-2 protein family: the apoptotic and anti-apoptotic context. Egypt J Med Hum Genet [Internet]. 2024;25(1):12. Available at: https://doi.org/10.1186/s43042-024-00485-7
- Yun CW, Jeon J, Go G, Lee JH, Lee SH. The Dual Role of Autophagy in Cancer Development and a Therapeutic Strategy for Cancer by Targeting Autophagy. Int J Mol Sci. Aralık 2020;22(1).
- Tutak I, Ozdil B, Uysal A. Voxtalisib and low intensity pulsed ultrasound combinatorial effect on glioblastoma multiforme cancer stem cells via PI3K/AKT/mTOR. Pathol Pract. 2022;239:154145.
- Dikici O, Ozdil B, Yesin TK, Dikici A, Adalı Y, Aktug H. Time-Dependent Effects of Low-Intensity Pulsed Ultrasound on Apoptosis and Autophagy in Malignant Melanoma Stem Cells. J Cell Mol Med. Haziran 2025;29(12):e70687.
- Oltulu F, Kocaturk DC, Adali Y, Ozdil B, Acikgoz E, Gurel C, vd. Autophagy and mTOR pathways in mouse embryonic stem cell, lung cancer and somatic fibroblast cell lines. J Cell Biochem. Ekim 2019;120(10):18066–76.
- Xu Y, Qian C, Wang Q, Song L, He Z, Liu W, vd. Deacetylation of ATG7 drives the induction of macroautophagy and LC3-associated microautophagy. Autophagy [Internet]. 03 Mayıs 2024;20(5):1134–46. Available at: https://doi.org/10.1080/15548627.2023.2287932
- Ostacolo K, López García de Lomana A, Larat C, Hjaltalin V, Holm KY, Hlynsdóttir SS, vd. ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism. Traffic [Internet]. 01 Nisan 2024;25(4):e12933. Available at: https://doi.org/10.1111/tra.12933
- Hasanpour SZ, Allah Bakhshian M, Mohammadi MH, Ghasemian SO, Gholami-Ahangaran M. Beclin1, Atg10 and Atg7 genes expressions as autophagy mediators in acute B-lymphoblastic leukemia. Hum Gene [Internet]. 2024;41:201311. Available at: https://www.sciencedirect.com/science/article/pii/S277304412400055X
- Devarajan E, Sahin AA, Chen JS, Krishnamurthy RR, Aggarwal N, Brun A-M, vd. Down-regulation of caspase 3 in breast cancer: a possible mechanism for chemoresistance. Oncogene. Aralık 2002;21(57):8843–51.
- Ghavami S, Hashemi M, Ande SR, Yeganeh B, Xiao W, Eshraghi M, vd. Apoptosis and cancer: mutations within caspase genes. J Med Genet [Internet]. 01 Ağustos 2009;46(8):497 LP – 510. Available at: http://jmg.bmj.com/content/46/8/497.abstract
- Park JY, Park JM, Jang JS, Choi JE, Kim KM, Cha SI, vd. Caspase 9 promoter polymorphisms and risk of primary lung cancer . Hum Mol Genet [Internet]. 15 Haziran 2006;15(12):1963–71. Available at: https://doi.org/10.1093/hmg/ddl119
- Qaed E, Liu W, Almoiliqy M, Mohamed R, Tang Z. Unleashing the potential of Genistein and its derivatives as effective therapeutic agents for breast cancer treatment. Naunyn Schmiedebergs Arch Pharmacol [Internet]. 2025;398(4):3321–43. Available at: https://doi.org/10.1007/s00210-024-03579-6
- Pu X, Storr SJ, Zhang Y, Rakha EA, Green AR, Ellis IO, vd. Caspase-3 and caspase-8 expression in breast cancer: caspase-3 is associated with survival. Apoptosis [Internet]. 2017;22(3):357–68. Available at: https://doi.org/10.1007/s10495-016-1323-5
- Boice A, Bouchier-Hayes L. Targeting apoptotic caspases in cancer. Biochim Biophys Acta - Mol Cell Res [Internet]. 2020;1867(6):118688. Available at: https://www.sciencedirect.com/science/article/pii/S016748892030046X
- Baranovsky A, Ivanov T, Granovskaya M, Papatsenko D, Pervouchine DD. Transcriptome analysis reveals high tumor heterogeneity with respect to re-activation of stemness and proliferation programs. PLoS One. 2022;17(5):e0268626.
- Kang R, Zeh HJ, Lotze MT, Tang D. The Beclin 1 network regulates autophagy and apoptosis. Cell Death Differ. 2011;18(4):571–80.
- Marquez RT, Xu L. Bcl-2:Beclin 1 complex: multiple, mechanisms regulating autophagy/apoptosis toggle switch. Am J Cancer Res [Internet]. 2012/02/15. 2012;2(2):214–21. Available at: https://pubmed.ncbi.nlm.nih.gov/22485198
Ayrıntılar
| Birincil Dil | İngilizce |
|---|---|
| Konular | Kanser Hücre Biyolojisi |
| Bölüm | Araştırma Makalesi |
| Yazarlar | |
| Gönderilme Tarihi | 14 Ağustos 2025 |
| Kabul Tarihi | 28 Ekim 2025 |
| Yayımlanma Tarihi | 9 Mart 2026 |
| DOI | https://doi.org/10.19161/etd.1763314 |
| IZ | https://izlik.org/JA96LC37DR |
| Yayımlandığı Sayı | Yıl 2026 Cilt: 65 Sayı: 1 |
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