Publication: Kolon kanserinde osteonektin (SPARC) proteini ve reseptörlerinin rolü
Abstract
Amaç: Bu çalışmada ekzojen SPARC’ın HT-29 hücre hattı üzerindeki apoptotik/ sitotoksik etkileri araştırılmış ve bu hücre hattındaki etki mekanizması belirlenmeye çalışılmıştır. SPARC’ın bu etkilerine aracılık eden olası reseptörlerden stabilin-1 ve integrin αvβ3 (ilgili peptitle susturularak)’ün olası etkilerinin açığa çıkarılması amaçlanmıştır. Gereç ve yöntem: Ekzojen SPARC ve cilengitid molekülünün HT-29 hücre hattı üzerindeki toksisite etkisi Xcelligence Gerçek Zamanlı Hücre Analizörü yöntemi ile apoptotik etkisi ise kaspaz-3 ölçümü ile belirlendi. Stabilin-1 ve integrin αvβ3 reseptörlerinin (cilengitid öncesi ve sonrası) gen ekspresyonu seviyeleri GZ-PZR ile belirlenirken, reseptör sayıları akım sitometre ile ölçüldü. Bulgular: Yapılan sitotoksisite çalışmalarının sonucunda SPARC molekülünün IC50 değeri 4,57 µg/ mL, cilengitid molekülünün IC50 değeri 50 nM olarak tespit edildi. 5 µg/ mL SPARC’ın hücreyi apoptoza götürdüğü belirlenmiştir (p<0,001). SPARC ile inkübe edilen grupta integrin αvβ3’ün gen ekspresyonunda anlamlı bir artış görülürken (p<0,001) cilengitid+5µg/ mL SPARC ile inkübe edilen grupta bu gen ekspresyonunun azaldığı tespit edilmiştir (p<0,01). İntegrin αvβ3’ün reseptör sayısının kontrole kıyasla SPARC ile inkübe edilen grupta yaklaşık 2 kat arttığı, stabilin-1'in gen ekspresyonu ve reseptör sayılarında ise önemli bir değişiklik gözlenmediği belirlenmiştir (p>0,05). Sonuç: Ekzojen SPARC'ın kolon kanseri hücresinde proliferasyonu azalttığı ve apoptozu indüklediği gösterilmiştir. İntegrin αvβ3'ün kolon kanseri hücrelerinde SPARC'a aracılık eden olası reseptör olduğu düşünülmektedir. Hücre başına yüzey reseptörlerinin kantitasyonu ise antikanser tedavilerinin takibinde bir belirteç olarak düşünülebilir.
Objective: In this study, the apoptotic/ cytotoxic effects of exogenous SPARC on the HT-29 cell line were investigated and the mechanism of action in this cell line was tried to be determined. It is aimed to reveal the possible effects of stabilin-1 and integrin αvβ3 (silencing with the relevant peptide) from the possible receptors that mediate these effects of SPARC. Materials and Methods: Toxicity of exogenous SPARC and cilengitide molecule on HT-29 cell line was determined by Xcelligence Real Time Cell Analyzer method, and apoptotic effect was determined by caspase-3 measurement. The gene expression levels of stabilin-1 and integrin αvβ3 receptors (before and after cilengitide) were determined by RT-PCR, while the receptor numbers were measured with a flow cytometer. Results: As a result of the cytotoxicity studies, the IC50 value of the SPARC molecule was determined as 4,57 µg/ mL, and the IC50 value of the cilengitide molecule was determined as 50 nM. It has been determined that 5 µg/ mL SPARC lead to the cell apoptosis (p<0.001).While there was a significant increase in gene expression of integrin αvβ3 in the group incubated with SPARC (p<0.001), it was found that this gene expression decreased in the group incubated with cilengitide+5 µg/ mL SPARC (p<0.01). It was determined that the number of receptors of integrin αvβ3 increased approximately 2 times in the group incubated with SPARC compared to the control, and no significant change was observed in the gene expression and receptor numbers of stabilin-1 (p>0.05). Conclusion: It has been shown that exogenous SPARC reduces proliferation and induces apoptosis in colon cancer cell. Integrin αvβ3 is thought to be the possible receptor mediating SPARC in colon cancer cells. Quantification of surface receptors per cell can be considered as a marker in the follow-up of anticancer treatments.
Objective: In this study, the apoptotic/ cytotoxic effects of exogenous SPARC on the HT-29 cell line were investigated and the mechanism of action in this cell line was tried to be determined. It is aimed to reveal the possible effects of stabilin-1 and integrin αvβ3 (silencing with the relevant peptide) from the possible receptors that mediate these effects of SPARC. Materials and Methods: Toxicity of exogenous SPARC and cilengitide molecule on HT-29 cell line was determined by Xcelligence Real Time Cell Analyzer method, and apoptotic effect was determined by caspase-3 measurement. The gene expression levels of stabilin-1 and integrin αvβ3 receptors (before and after cilengitide) were determined by RT-PCR, while the receptor numbers were measured with a flow cytometer. Results: As a result of the cytotoxicity studies, the IC50 value of the SPARC molecule was determined as 4,57 µg/ mL, and the IC50 value of the cilengitide molecule was determined as 50 nM. It has been determined that 5 µg/ mL SPARC lead to the cell apoptosis (p<0.001).While there was a significant increase in gene expression of integrin αvβ3 in the group incubated with SPARC (p<0.001), it was found that this gene expression decreased in the group incubated with cilengitide+5 µg/ mL SPARC (p<0.01). It was determined that the number of receptors of integrin αvβ3 increased approximately 2 times in the group incubated with SPARC compared to the control, and no significant change was observed in the gene expression and receptor numbers of stabilin-1 (p>0.05). Conclusion: It has been shown that exogenous SPARC reduces proliferation and induces apoptosis in colon cancer cell. Integrin αvβ3 is thought to be the possible receptor mediating SPARC in colon cancer cells. Quantification of surface receptors per cell can be considered as a marker in the follow-up of anticancer treatments.