Investigation of the apoptotic effect of the 147-DENA monoclonal antibody in breast cancer treatment
Abstract
Aim: Breast cancer is the most frequently diagnosed cancer type in women worldwide and displays extensive molecular heterogeneity. This study aimed to evaluate the effects of 147-DENA, a monoclonal antibody developed against the oncodevelopmental antigen diamino neuraminic acid, on two distinct breast cancer cell lines regarding cytotoxicity, apoptosis, morphological changes, and gene expression modulation.
Materials and Methods: Breast cancer cells MCF-7 and MDA-MB-231, along with the normal breast epithelial cell line MCF-10A, were cultured under standard conditions. Various concentrations of the 147-DENA monoclonal antibody were applied to the cells, and the IC50 value was determined using the WST-1 assay. Subsequently, apoptosis analysis was confirmed by examining the levels of pro-apoptotic and anti-apoptotic proteins using the immunofluorescence staining method. Cell morphology was evaluated using scanning electron microscopy to identify apoptosis-specific structural changes. Changes in gene expression related to cell metabolism and apoptosis were analyzed using RT-PCR.
Results: The monoclonal antibody 147-DENA induced cytotoxic effects in MCF-7 and MDA-MB-231 breast cancer cells while sparing the non-tumorigenic epithelial MCF-10A cell line. Further analysis revealed that 147-DENA treatment led to an increased expression of apoptotic proteins and decreased levels of anti-apoptotic proteins, indicating a clear pro-apoptotic shift. Morphologically, the cells exhibited characteristic features of apoptosis, including cellular shrinkage and membrane blebbing. Additionally, altered expression patterns in genes associated with apoptosis and cell metabolism highlighted the therapeutic potential of 147-DENA.
Conclusion: 147-DENA selectively targets breast cancer cells by inducing apoptosis without damaging normal epithelial cells. Its capability to modulate crucial apoptotic pathways and gene expression highlights its potential as a targeted therapeutic strategy for breast cancer, especially in highly heterogeneous tumor profiles.
Project Number
TYL-2019-20885
References
- Jokhadze N, Das A and Dizon DS: Global cancer statistics: A healthy population relies on population health. CA Cancer J Clin 74: 224–226, 2024.
- Bray Bsc F, Laversanne | Mathieu, Hyuna |, et al.: Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74: 229–263, 2024.
- Baldo BA: Monoclonal Antibodies Approved for Cancer Therapy. In: Safety of Biologics Therapy. Springer International Publishing, Cham, pp57–140, 2016.
- Lu R-M, Hwang Y-C, Liu I-J, Lee C-C, Tsai H-Z, Li H-J and Wu H-C: Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27: 1, 2020.
- KÖHLER G and MILSTEIN C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495–497, 1975.
- Quinteros DA, Bermúdez JM, Ravetti S, Cid A, Allemandi DA and Palma SD: Therapeutic use of monoclonal antibodies: general aspects and challenges for drug delivery. In: Nanostructures for Drug Delivery. Elsevier, pp807–833, 2017.
- Qu B, Ziak M, Zuber C and Roth J: Poly (alpha 2,8-deaminoneuraminic acid) is expressed in lung on a single 150-kDa glycoprotein and is an oncodevelopmental antigen. Proceedings of the National Academy of Sciences 93: 8995–8998, 1996.
- Inoue S, Kitajima K and Inoue Y: Identification of 2-Keto-3-deoxy-D-glycero-D-galactonononic acid (KDN, Deaminoneuraminic Acid) Residues in Mammalian Tissues and Human Lung Carcinoma Cells. Journal of Biological Chemistry 271: 24341–24344, 1996.
- Harbeck N and Gnant M: Breast cancer. The Lancet 389: 1134–1150, 2017.
- Baudino T: Targeted Cancer Therapy: The Next Generation of Cancer Treatment. Curr Drug Discov Technol 12: 3–20, 2015.
- Pérez-Herrero E and Fernández-Medarde A: Advanced targeted therapies in cancer: Drug nanocarriers, the future of chemotherapy. European Journal of Pharmaceutics and Biopharmaceutics 93: 52–79, 2015.
- Weiss J, Glode A, Messersmith WA and Diamond J: Sacituzumab govitecan: breakthrough targeted therapy for triple-negative breast cancer. Expert Rev Anticancer Ther 19: 673–679, 2019.
- Gül N and van Egmond M: Antibody-Dependent Phagocytosis of Tumor Cells by Macrophages: A Potent Effector Mechanism of Monoclonal Antibody Therapy of Cancer. Cancer Res 75: 5008–5013, 2015.
- Lee YT, Tan YJ and Oon CE: Molecular targeted therapy: Treating cancer with specificity. Eur J Pharmacol 834: 188–196, 2018.
- Inoue S, Kitajima K, Sato C and Go S: Human KDN (Deaminated Neuraminic Acid) and Its Elevated Expression in Cancer Cells: Mechanism and Significance. pp669–678, 2011.
- Pistritto G, Trisciuoglio D, Ceci C, Garufi A and D’Orazi G: Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies. Aging 8: 603–619, 2016.
- D’Arcy MS: Cell death: a review of the major forms of apoptosis, necrosis and autophagy. Cell Biol Int 43: 582–592, 2019.
- Li J, Yang Z, Li Y, et al.: Cell apoptosis, autophagy and necroptosis in osteosarcoma treatment. Oncotarget 7: 44763–44778, 2016.
- Ramirez MLG and Salvesen GS: A primer on caspase mechanisms. Semin Cell Dev Biol 82: 79–85, 2018.
- Sharma SS and Pledger WJ: The non-canonical functions of p27 Kip1 in normal and tumor biology. Cell Cycle 15: 1189–1201, 2016.
- Bencivenga D, Stampone E, Azhar J, et al.: p27Kip1 and Tumors: Characterization of CDKN1B Variants Identified in MEN4 and Breast Cancer. Cells 14: 188, 2025.
- Liu J, Xiao Q, Xiao J, et al.: Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduct Target Ther 7: 3, 2022.
- Hinz N, Baranowsky A, Horn M, et al.: Knockdown of AKT3 Activates HER2 and DDR Kinases in Bone-Seeking Breast Cancer Cells, Promotes Metastasis In Vivo and Attenuates the TGFβ/CTGF Axis. Cells 10: 430, 2021.
- Hinz N and Jücker M: Distinct functions of AKT isoforms in breast cancer: a comprehensive review. Cell Communication and Signaling 17: 154, 2019.
- Ghalehbandi S, Yuzugulen J, Pranjol MZI and Pourgholami MH: The role of VEGF in cancer-induced angiogenesis and research progress of drugs targeting VEGF. Eur J Pharmacol 949: 175586, 2023.
Abstract
Amaç: Meme kanseri, dünya genelinde kadınlarda en sık teşhis edilen kanser türü olup geniş bir moleküler heterojenite sergilemektedir. Bu çalışma, onkogelişimsel antijen olan deaminonöraminik aside karşı geliştirilmiş bir monoklonal antikorun iki farklı meme kanseri hücre hattı üzerindeki sitotoksisite, apoptoz, morfolojik değişiklikler ve gen ekspresyonu modülasyonu yönünden etkilerini değerlendirmeyi amaçlamıştır.
Gereç ve Yöntem: Meme kanseri hücreleri MCF-7 ve MDA-MB-231 ile normal meme epitel hücre hattı olan MCF-10A, standart koşullar altında kültüre edilmiştir. Hücrelere 147-DENA monoklonal antikorunun çeşitli konsantrasyonları uygulanarak WST-1 testi ile IC50 değeri belirlenmiştir. Ardından, apoptoz analizi, pro-apoptotik ve anti-apoptotik protein seviyelerinin immün floresan boyama yöntemiyle incelenmesi sonucu doğrulanmıştır. Hücre morfolojisi, apoptoza özgü yapısal değişikliklerin belirlenmesi için taramalı elektron mikroskobu kullanılarak değerlendirilmiştir. Hücre metabolizması ve apoptozla ilişkili gen ekspresyon değişiklikleri ise RT-PCR analizi ile incelenmiştir.
Bulgular: Monoklonal antikor 147-DENA, MCF-7 ve MDA-MB-231 meme kanseri hücrelerinde sitotoksik etki gösterirken, non-tümorijenik epitel hücre hattı olan MCF-10A üzerinde herhangi bir sitotoksik etkiye neden olmamıştır. Daha ileri analizler, 147-DENA uygulamasının pro-apoptotik proteinlerin ekspresyonunu artırırken anti-apoptotik protein seviyelerini azalttığını ve bu durumun belirgin bir pro-apoptotik eğilime işaret ettiğini ortaya koymuştur. Morfolojik olarak hücrelerde, hücre büzülmesi ve membran kabarcıklanması gibi apoptozun karakteristik özellikleri gözlenmiştir. Buna ek olarak, apoptoz ve hücre metabolizması ile ilişkili genlerdeki ekspresyon değişiklikleri, 147-DENA’nın terapötik potansiyelini vurgulamaktadır.
Sonuç: 147-DENA, apoptozu indükleyerek meme kanseri hücrelerini seçici olarak hedef alırken, normal epitel hücrelere zarar vermemiştir. Kritik apoptotik yolakları ve gen ekspresyonunu modüle edebilme yeteneği, 147-DENA’nın özellikle yüksek derecede heterojen tümör profillerinde meme kanseri için hedefe yönelik bir tedavi stratejisi olarak potansiyelini vurgulamaktadır.
Ethical Statement
Bu çalışma, Ege Üniversitesi Hayvan Deneyleri Yerel Etik Kurulu tarafından onaylanmış olup, 2015-023 onay numarası ile yürütülmüştür.
Supporting Institution
Ege Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi
Project Number
TYL-2019-20885
Thanks
Çalışma, Ege Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi TYL-2019-20885 numaralı proje kapsamında desteklenmiştir.
References
- Jokhadze N, Das A and Dizon DS: Global cancer statistics: A healthy population relies on population health. CA Cancer J Clin 74: 224–226, 2024.
- Bray Bsc F, Laversanne | Mathieu, Hyuna |, et al.: Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 74: 229–263, 2024.
- Baldo BA: Monoclonal Antibodies Approved for Cancer Therapy. In: Safety of Biologics Therapy. Springer International Publishing, Cham, pp57–140, 2016.
- Lu R-M, Hwang Y-C, Liu I-J, Lee C-C, Tsai H-Z, Li H-J and Wu H-C: Development of therapeutic antibodies for the treatment of diseases. J Biomed Sci 27: 1, 2020.
- KÖHLER G and MILSTEIN C: Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256: 495–497, 1975.
- Quinteros DA, Bermúdez JM, Ravetti S, Cid A, Allemandi DA and Palma SD: Therapeutic use of monoclonal antibodies: general aspects and challenges for drug delivery. In: Nanostructures for Drug Delivery. Elsevier, pp807–833, 2017.
- Qu B, Ziak M, Zuber C and Roth J: Poly (alpha 2,8-deaminoneuraminic acid) is expressed in lung on a single 150-kDa glycoprotein and is an oncodevelopmental antigen. Proceedings of the National Academy of Sciences 93: 8995–8998, 1996.
- Inoue S, Kitajima K and Inoue Y: Identification of 2-Keto-3-deoxy-D-glycero-D-galactonononic acid (KDN, Deaminoneuraminic Acid) Residues in Mammalian Tissues and Human Lung Carcinoma Cells. Journal of Biological Chemistry 271: 24341–24344, 1996.
- Harbeck N and Gnant M: Breast cancer. The Lancet 389: 1134–1150, 2017.
- Baudino T: Targeted Cancer Therapy: The Next Generation of Cancer Treatment. Curr Drug Discov Technol 12: 3–20, 2015.
- Pérez-Herrero E and Fernández-Medarde A: Advanced targeted therapies in cancer: Drug nanocarriers, the future of chemotherapy. European Journal of Pharmaceutics and Biopharmaceutics 93: 52–79, 2015.
- Weiss J, Glode A, Messersmith WA and Diamond J: Sacituzumab govitecan: breakthrough targeted therapy for triple-negative breast cancer. Expert Rev Anticancer Ther 19: 673–679, 2019.
- Gül N and van Egmond M: Antibody-Dependent Phagocytosis of Tumor Cells by Macrophages: A Potent Effector Mechanism of Monoclonal Antibody Therapy of Cancer. Cancer Res 75: 5008–5013, 2015.
- Lee YT, Tan YJ and Oon CE: Molecular targeted therapy: Treating cancer with specificity. Eur J Pharmacol 834: 188–196, 2018.
- Inoue S, Kitajima K, Sato C and Go S: Human KDN (Deaminated Neuraminic Acid) and Its Elevated Expression in Cancer Cells: Mechanism and Significance. pp669–678, 2011.
- Pistritto G, Trisciuoglio D, Ceci C, Garufi A and D’Orazi G: Apoptosis as anticancer mechanism: function and dysfunction of its modulators and targeted therapeutic strategies. Aging 8: 603–619, 2016.
- D’Arcy MS: Cell death: a review of the major forms of apoptosis, necrosis and autophagy. Cell Biol Int 43: 582–592, 2019.
- Li J, Yang Z, Li Y, et al.: Cell apoptosis, autophagy and necroptosis in osteosarcoma treatment. Oncotarget 7: 44763–44778, 2016.
- Ramirez MLG and Salvesen GS: A primer on caspase mechanisms. Semin Cell Dev Biol 82: 79–85, 2018.
- Sharma SS and Pledger WJ: The non-canonical functions of p27 Kip1 in normal and tumor biology. Cell Cycle 15: 1189–1201, 2016.
- Bencivenga D, Stampone E, Azhar J, et al.: p27Kip1 and Tumors: Characterization of CDKN1B Variants Identified in MEN4 and Breast Cancer. Cells 14: 188, 2025.
- Liu J, Xiao Q, Xiao J, et al.: Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduct Target Ther 7: 3, 2022.
- Hinz N, Baranowsky A, Horn M, et al.: Knockdown of AKT3 Activates HER2 and DDR Kinases in Bone-Seeking Breast Cancer Cells, Promotes Metastasis In Vivo and Attenuates the TGFβ/CTGF Axis. Cells 10: 430, 2021.
- Hinz N and Jücker M: Distinct functions of AKT isoforms in breast cancer: a comprehensive review. Cell Communication and Signaling 17: 154, 2019.
- Ghalehbandi S, Yuzugulen J, Pranjol MZI and Pourgholami MH: The role of VEGF in cancer-induced angiogenesis and research progress of drugs targeting VEGF. Eur J Pharmacol 949: 175586, 2023.
Details
| Primary Language | Turkish |
|---|---|
| Subjects | Cancer Therapy (Excl. Chemotherapy and Radiation Therapy) |
| Journal Section | Research Article |
| Authors | |
| Project Number | TYL-2019-20885 |
| Publication Date | December 8, 2025 |
| Submission Date | April 21, 2025 |
| Acceptance Date | August 6, 2025 |
| Published in Issue | Year 2025 Volume: 64 Issue: 4 |
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