Pankreas Kanserinde Toll-Like Reseptör 4 (TLR4) Yolağının İmmün Check-Point VISTA Üzerindeki Etkilerinin Araştırılması
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Tarih
2021
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Necmettin Erbakan Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Pankreas duktal adenokarsinom (PDAC), pankreasın maling tümörlerinden olup en yaygın
görülen tipidir ve Dünya çapında kansere bağlı ölümlerin yedincisi olarak bilinmektedir. Pankreas kanseri
erken evrelerde herhangi bir semptom göstermediğinden dolayı hızlıca yayılım göstermektedir ve bundan
dolayı teşhisi oldukça zordur. Cerrahi, kemoterapi ve radyoterapi dahil olmak üzere pankreas kanseri için
tedavi yöntemlerinin başarı oranı düşük ve nüksü kaçınılmazdır. İmmunoterapötik yaklaşımlar ilerlemiş
malignitelerin tedavisi için ortaya çıkmıştır. İmmun check-point inhibitörleri, kanser tedavisinde tümörün
büyümesini engellerken aynı zamanda kemoterapinin etkinliğini arttırdığı için oldukça umut vericidir.
İmmunoterapide son zamanlarda hedef haline gelmiş T hücre aktivasyonunun V-domain immunoglobin
baskılayıcısı (VISTA) esas olarak hematopoietik hücrelerde ve kanser hücrelerinde yüksek seviyede
eksprese olan transmembran proteinidir. Prostat kanseri, küçük hücreli olmayan akciğer kanseri (NSCLC)
ve kolorektal karsinom gibi birçok insan kanserinde bağışıklıktan kaçınma ve hayatta kalmak için T
hücresi ile ilişkili yanıtı baskılayan negatif immun check-point olarak bilinmektedir. Kanserle ilişkili
VISTA’nın, pankreas kanserinde M2 makrofajlarından olan CD68+ makrofajlarında yüksek seviyede
eksprese olduğu tespit edilmiştir. Ancak pankreas kanserinde VISTA’nın bağlantılı olduğu sinyal
yolakları ve klinik önemi hakkında çok az bilgi bulunmaktadır. Toll-like reseptörler (TLR’ler) mikrobik
hücre duvarının spesifik bileşenlerini tanıyan hem doğuştan gelen hem de adaptif immun hücreleri aktive
eden, evrimsel olarak korunmuş patern tanıma reseptörü (PRR) olarak bilinmektedir. Kanserde
immunosupresif faktörlerin salgılanmasına ek olarak apoptoz direncini de arttırmaktadır. Pankreas kanser
hücrelerinde TLR4 yüksek seviyede eksprese olmaktadır ve kötü prognozla ilişkilidir. İmmun check-point
VISTA ile TLR4 ekspresyonu arasında potansiyel bağlantılar henüz keşfedilmemiştir. Pankreas
kanserinde VISTA ve TLR4’ün downstream sinyal yolaklarındaki bağlantıların hedeflenmesi, immun
check-point inhibitörleri ve onkogenik yolak inhibitörleri üzerine yapılacak klinik çalışmalar için fayda
sağlayacaktır.
Bu çalışmada pankreas kanserinin gelişimi ve proliferasyonunda TLR4 ve down stream
yolağında yer alan IRAK4 moleküllerinin VISTA ile arasındaki bağlantılarının araştırılması
hedeflenmiştir. Bu amaçla pankreas kanseri hücre hattı olan PANC-1 hücrelerine TLR4 antagonisti olan
Naloxone ve VISTA-siRNA uygulaması yapılmıştır. Öncelikle TLR4 geninin PANC-1 hücreleri üzerinde
proliferasyona olan etkisinin incelenmesi ve Naloxone ilacının IC50 dozunu bulmak için MTT analizi
yapılmıştır. IC50 dozu tespit edildikten sonra TLR4,IRAK4 ve VISTA’nın gen ekspresyonu seviyesinde
baskılanmasına qRT-PCR ile bakılmıştır. Sonraki aşamada VISTA blokasyonunun TLR4 gen
ekspresyonundaki etkisine bakmak için VISTA-siRNA uygulanmıştır. MTT analizi ile hücre proliferasyonundaki azalma, qRT-PCR ile hedef genlerin ekspresyonlarındaki azalma anlamlı bir şekilde
tespit edilmiştir. Bu sonuçlardan yola çıkarak Naloxone ve VISTA-siRNA’nın PANC-1 hücrelerine
beraber transfekte edildiğindeki sinerjik etkilerini görebilmek için Naloxone’un etken dozu ile VISTAsiRNA kombinasyon halinde hücrelere uygulanmıştır. qRT-PCR ile kontrol grubuna göre uygulama
yapılan grup kıyaslandığında VISTA ile TLR4 ve onun downstream yolağında yer alan IRAK4
molekülünün ekspresyon seviyesinde anlamlı bir azalma gözlemlenmiştir.
Sonuç olarak PANC-1 hücre hattında TLR4 sinyal yolağı ile VISTA gen ekspresyonu arasında
anlamlı bir ilişki ve pankreas kanseri proliferasyonunda ve ilerlemesinde bu hedef genlerin tehlikeli bir
potansiyele sahip olduğu görülmüştür. Çalışmamızdan elde edilen bulgular, ileri analizler ile
geliştirilmelidir. Verilerimize göre pankreas kanseri ilerlemesinde TLR4 sinyal yolağının immun checkpoint VISTA’ya aracılık ettiği düşünüldüğünden ilerki çalışmalarda gerekli inhibitörler kullanılarak diğer
sinyal yolakları ile potansiyel bağlantıların tespit edilmesi gerekmektedir. VISTA ve TLR4 sinyal yolağı
arasında bulunan ilişkinin ve bağlantılı olduğu sinyal yolaklarının tespit edilmesi durumunda kanser
tedavisinde immun check-point inhibitörlerinin cevap belirlemede önemli bir belirteç olabileceğini
düşünmekteyiz.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignant tumors of the pancreas, and is known worldwide as the seventh in cancer-related deaths. Since pancreatic cancer does not show any symptoms in the early stages, it spreads rapidly and is therefore difficult to diagnose. Treatment methods for pancreatic cancer, including surgery, chemotherapy, and radiotherapy, have a low success rate and inevitable recurrence. Immunotherapeutic approaches have emerged for the treatment of advanced malignancies. Immune check-point inhibitors are very promising as they prevent the growth of the tumor in cancer treatment and at the same time increase the effectiveness of chemotherapy. The Vdomain immunoglobin suppressor of T cell activation (VISTA), which has recently become a target in immunotherapy, is a highly expressed transmembrane protein primarily in hematopoietic cells and cancer cells. Prostate cancer is known as a negative immune check-point that suppresses T cell-related response for immune avoidance and survival in many human cancers such as non-small cell lung cancer (NSCLC) and colorectal carcinoma. Cancer-associated VISTA has also been found to be highly expressed in CD68 + macrophages, which are M2 macrophages in pancreatic cancer. However, there is less information about the signal pathways associated with VISTA in pancreatic cancer and its clinical significance. Tolllike receptors (TLRs) are known as evolutionarily conserved pattern recognition receptors (PRRs), which activate both innate and adaptive immune cells that recognize specific components of the microbial cell wall. In addition to the secretion of immunosuppressive factors in cancer, it also increases apoptosis resistance TLR4 is highly expressed in pancreatic cancer cells and is associated with a poor prognosis. Potential links between VISTA and TLR4 expression have not yet been discovered. Targeting the linkages in the downstream signaling pathways of VISTA and TLR4 in pancreatic cancer would be useful for clinical studies on immune checkpoint inhibitors and oncogenic pathway inhibitors. In this study, it was aimed to investigate the connections between TLR4 and IRAK4 molecules in the down stream pathway with VISTA in the development and proliferation of pancreatic cancer. For this purpose, TLR4 antagonist Naloxone and VISTA-siRNA were transfected into PANC-1 cells, a pancreatic cancer cell line. First of all, MTT analysis was performed to examine the effect of TLR4 gene on proliferation on PANC-1 cells and to find the IC50 dose of the Naloxone drug. After the IC50 dose was determined, suppression of the level of TLR4, IRAK4 and VISTA gene expression was shown in qRT-PCR. In the next step, VISTA-siRNA was applied to determine the effect of VISTA blocking on TLR4 gene expression. The decrease in cell proliferation was detected by MTT analysis, and the decrease in target genes expressions was determined significantly by qRT-PCR. Based on these results, in order to see the synergistic effect of Naloxone and VISTA-siRNA when transfected into PANC-1 cells together, the active dose of Naloxone and VISTA-siRNA were applied to the cells in combination. When the treated group was compared with the control group by qRT-PCR, a significant decrease was observed in the expression level of VISTA and TLR4 molecule in its downstream pathway with IRAK4. As a result, a significant relationship between TLR4 signaling pathway and VISTA gene expression in PANC-1 cell line and these target genes have a dangerous potential in the proliferation and progression of pancreatic cancer. Findings obtained from our study should be developed with further analysis. According to our data, since it is thought that the TLR4 signaling pathway mediates the immune check-point VISTA in the progression of pancreatic cancer, potential connections with other signaling pathways should be determined by using the necessary inhibitors in future studies. If the findings of the study are supported by further analysis, it is thought that the relationship between VISTA and TLR4 signaling pathways and their associated signal pathways may be an important marker in determining the response of immune check-point inhibitors in cancer treatment.
Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignant tumors of the pancreas, and is known worldwide as the seventh in cancer-related deaths. Since pancreatic cancer does not show any symptoms in the early stages, it spreads rapidly and is therefore difficult to diagnose. Treatment methods for pancreatic cancer, including surgery, chemotherapy, and radiotherapy, have a low success rate and inevitable recurrence. Immunotherapeutic approaches have emerged for the treatment of advanced malignancies. Immune check-point inhibitors are very promising as they prevent the growth of the tumor in cancer treatment and at the same time increase the effectiveness of chemotherapy. The Vdomain immunoglobin suppressor of T cell activation (VISTA), which has recently become a target in immunotherapy, is a highly expressed transmembrane protein primarily in hematopoietic cells and cancer cells. Prostate cancer is known as a negative immune check-point that suppresses T cell-related response for immune avoidance and survival in many human cancers such as non-small cell lung cancer (NSCLC) and colorectal carcinoma. Cancer-associated VISTA has also been found to be highly expressed in CD68 + macrophages, which are M2 macrophages in pancreatic cancer. However, there is less information about the signal pathways associated with VISTA in pancreatic cancer and its clinical significance. Tolllike receptors (TLRs) are known as evolutionarily conserved pattern recognition receptors (PRRs), which activate both innate and adaptive immune cells that recognize specific components of the microbial cell wall. In addition to the secretion of immunosuppressive factors in cancer, it also increases apoptosis resistance TLR4 is highly expressed in pancreatic cancer cells and is associated with a poor prognosis. Potential links between VISTA and TLR4 expression have not yet been discovered. Targeting the linkages in the downstream signaling pathways of VISTA and TLR4 in pancreatic cancer would be useful for clinical studies on immune checkpoint inhibitors and oncogenic pathway inhibitors. In this study, it was aimed to investigate the connections between TLR4 and IRAK4 molecules in the down stream pathway with VISTA in the development and proliferation of pancreatic cancer. For this purpose, TLR4 antagonist Naloxone and VISTA-siRNA were transfected into PANC-1 cells, a pancreatic cancer cell line. First of all, MTT analysis was performed to examine the effect of TLR4 gene on proliferation on PANC-1 cells and to find the IC50 dose of the Naloxone drug. After the IC50 dose was determined, suppression of the level of TLR4, IRAK4 and VISTA gene expression was shown in qRT-PCR. In the next step, VISTA-siRNA was applied to determine the effect of VISTA blocking on TLR4 gene expression. The decrease in cell proliferation was detected by MTT analysis, and the decrease in target genes expressions was determined significantly by qRT-PCR. Based on these results, in order to see the synergistic effect of Naloxone and VISTA-siRNA when transfected into PANC-1 cells together, the active dose of Naloxone and VISTA-siRNA were applied to the cells in combination. When the treated group was compared with the control group by qRT-PCR, a significant decrease was observed in the expression level of VISTA and TLR4 molecule in its downstream pathway with IRAK4. As a result, a significant relationship between TLR4 signaling pathway and VISTA gene expression in PANC-1 cell line and these target genes have a dangerous potential in the proliferation and progression of pancreatic cancer. Findings obtained from our study should be developed with further analysis. According to our data, since it is thought that the TLR4 signaling pathway mediates the immune check-point VISTA in the progression of pancreatic cancer, potential connections with other signaling pathways should be determined by using the necessary inhibitors in future studies. If the findings of the study are supported by further analysis, it is thought that the relationship between VISTA and TLR4 signaling pathways and their associated signal pathways may be an important marker in determining the response of immune check-point inhibitors in cancer treatment.
Açıklama
Yüksek Lisans Tezi
Anahtar Kelimeler
Pankreas duktal adenokarsinom, siRNA, TLR4, VISTA, İmmünoterapi, İmmun-check point, Pancreatic ductal adenocarcinoma, Immunotherapy, Immune-check point
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Baş Topcu, K. S. (2021). Pankreas kanserinde Toll-like Reseptör 4 (TLR4) yolağının immün check-point VISTA üzerindeki etkilerinin araştırılması. (Yayınlanmamış Yüksek Lisans Tezi). Necmettin Erbakan Üniversitesi, Sağlık Bilimleri Enstitüsü Hemşirelik Anabilim Dalı, Konya.
