Dasatinib ile muamele edilen k562 hücre serisinde, dasatinib'in, pp2a katalitik ve regulatuvar alt birimlerinde, enzim aktivitesine ve protein ekspresyonu üzerine etkisinin değerlendirilmesi
Küçük Resim Yok
Tarih
2015
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Kronik miyeloid lösemi (KML), kemik iliğinde beyaz kan hücrelerinde başlayan bir kanser tipi olup, hastaların neredeyse tamamında saptanan BCR/ABL translokasyonuna bağlı artmış tirozin kinaz aktivitesiyle karakterizedir. Translokasyon sonucu yeni kimerik gen oluşumuna bağlı aktivasyona neden olduğu bilinen ilk yeniden düzenlenme Philadelphia (Ph) kromozomudur. 9 ve 22 numaralı kromozomlar arasındaki translokasyon sonrasında meydana gelir. 9q34 bölgesine yerleşmiş olan ABL1 geninin 5' kısmına yakın bir bölgeden kırılması sonrasında 3' tarafında kalan parçanın, 22q11 BCR geninin 3' kısmına yakın bir bölgeden kırılması ve kırılan bu bölgeye füzyonu sonrasında ortaya çıkar. Füzyon sonrasında ortaya çıkan yeni kimerik gen, artmış tirozin kinaz aktivitesine ve anormal hücre lokalizasyonuna sahip bir ABL1 proteininin üretilmesine neden olur. Bu yeniden düzenlemeyle oluşan BCR-ABL1 kinaz aktivitesini inhibe etmek için hastalar Tirozin Kinaz İnhibitörleri (TKI) ile tedavi edilmektedir. Bu amaçla, Dasatinib BCR-ABL1'i inhibe etmek için uygulanan ikinci-nesil bir tirozin kinaz inhibitörüdür. KML'de İmatinib dirençli ya da tolere olmayan hastaların tedavisinde kullanılan güvenli ve etkili bir inhibitördür. Dasatinib, lösemide hücrelerin büyüme ve bölünmesiyle ilgili sinyalleri inhibe eder ve sonuç olarak sinyallerin bloklanması hücrenin ölümüyle sonuçlanır. Dasatinib, birinci-nesil TKI olan imatinibden 325 kat daha etkili olsa da KML hastalarının %20'si genel olarak ilaç tedavisine yanıt vermemektedir. Bu durum, tedavide etkin olabilecek alternatif ilaçların gündeme gelmesini ve olası direnç mekanizmalarının aydınlatılmasını gerektirmektedir. BCR-ABL1-indüklü KML'de genomik ya da fonksiyonel değişime uğramış birçok hücresel olayda fonksiyon gören protein fosfotaz 2A (PP2A) gibi yapıların incelenmesi bu sorulara yanıtlardan biri olabilir. Protein fosfotazlar, hücrede fosforilasyon-defosforilasyon dengesinin sağlanması açısından son derece önemlidir. Yapılan çalışmalarda KML'de (PP2A) aktivitesinin değiştiği saptanmıştır. PP2A, hücresel homeostazı sağlayan en önemli serin-treonin fosfotazdır. PP2A, 65kDa yapısal A alt birim ve 36 kDa katalitik C altbirim ve düzenleyici B alt birimden oluşur. Yapılan birçok çalışma sonucu, hematolojik malignansilerde PP2A ekspresyonunun ve/veya fonksiyonunun inhibe olduğu ortaya koyulmuştur. Bu çalışmada amacımız, dasatinib ile muamele edilen K562 kronik myleoid lösemi hücre serisinde, PP2A enzim aktivitesi ve alt birimlerinin protein düzeyinde ekspresyon değişimini saptamaktır. Bu amaç ile dasatinibin KML'ye model oluşturan K562 hücre serisindeki sitotoksik etkisi WST-1 testi ile, apoptotik etkisi Annexin V ve Apo Direct Tunnel metodu ve sitostatik etkisi ise Hücre Döngüsü Analizi ile değerlendirilmiştir. PP2A'nın apoptoz ve hücresel sağkalım üzerindeki etkisini belirlemek için PP2A inhibitörü olan Okadaik asit (OA) ile çalışılmıştır. Dasatinib ile muamele edilen K562 hücre serisindeki değişen PP2A'nın enzim aktivitesi serin/treonin fosfataz analiz kiti, PP2A'nın alt birimlerindeki protein ifade düzeyleri ise Western Blot analizi ile değerlendirilmiştir. Dasatinibin, K562 hücre serisindeki sitotoksik etksi 4,6 Nm olarak belirlenmiştir. OA'nın PP2A'yı baskılanmasından dolayı apoptozda artış belirlenmiştir. İnhibitörler nedeniyle baskılanan PP2A'dan dolayı hücre döngüsü tutuklanmasında da değişiklikler kaydedilmiştir. Dasatinib ile muamele edilen hücrelerin PP2A enzim aktivitesi 72.saatte kontrole kıyasla azalmıştır. Western Blot analizi ile dasatinibin PP2A C alt birimin protein seviyesinin kontrole kıyasla azaldığı bulunmuştur. Sonuç olarak, dasatinibin hücre homeostazında rol oynayan PP2A'nın enzim seviyesini ve protein seviyesinde değişime neden olduğu belirlenmiştir. Böylece PP2A inhibitörlerinin KML tedavisinde terapötik hedef olarak katkı sağlayabileceğini düşünmekteyiz
Chronic myeloid leukemia (CML), a group of cancer that usually begins in white blood cells and observed in almost all patients belonging this group, is characterized by the increased tyrosine kinase activity depends on BCR/ABL translocation. Resulting in translocation, that is known to cause activation depending on new chimeric gene formation is anew rearrengment Philadelphia (Ph) chromosome. This anew rearrengment occurs after translocation which is between chromosome 9 and 22. This chromosome has small acrocentric structure that is observed in 90% of chronic myeloid leukemia (CML) patients. It occurs after breaking from nearby the 5' region of the ABL1 gene in 9q34 region, by the break of the remaining part on the side 3' from nearby 3' region of 22q11 BCR gene and their fusion into this region. New chimeric gene, emerged after fusion, causes increased tyrosie kinase activity and production of ABL1 protein having abnormal cell localization. The patients have being generally treated using tyrosine kinase inhibitors (TKIs) to inhibit this BCR-ABL1 kinnase activity, occured by rearrangement. For this purpose, dasatinib is the second-generation tyrosine kinase inhibitör that is used for inhibition of Bcr-Abl1. In CML, it has been using to treat patients with Imatinib-resistant or intolerant CML-AP and it is so effective and safety. Dasatinib shows its effect in leukemia by inhibiting signals related to cell growth and splitting and eventually cell die as a result of blocking signals. Although dasatinib is 325 fold more effective than that of imatinib that is first-generation tyrosine kinase inhibitor, 20% of CMLpatients generally dont respond to treatment with certain drugs. Therefore, alternative drugs should be used and the resistant mechanism should be enlightened. This problem may occur in CML that was induced by BCR-ABL1. This case, in BCR-ABL1-induced KML, one of responses to these questions can be provided by the investigations of structures such as protein phosphatase 2A (PP2A) changed genetically or functionally that functions as tumor suppressor in some cancers patients with imatinib-resistant or -intolerant CML-AP Protein phosphatases have major role in maintenance phosphorylation-dephosphorylation balance in cell. Studies related to KML have shown that the activity of PP2A is changed. PP2A is the most important serine/threonine phosphatase ensuring cellular homeostasis. PP2A consist of 65 kDa A, 36 kDa C subunit and regulatory B subunit. Most studies have shown that PP2A expression/or function in hematological malignancies is inhibited. In our study, we aimed to evaluate PP2A enzyme activity in dasatinib treated K562 chronic myleoid leukemia cell line and the expression changes in protein levels. The cytotoxic effects of dasatinib on K562 cell line, were evaluated by WST-1 analysis. Apoptotic situation was determined by using Annexin V and Apo Direct Tunnel assays and cycletest plus DNA Reagent Kit was used to determine cytoctatic effcets of PP2A. We also used okadaic acidas PP2A inhibitor to assesed major role of PP2A in apoptosis. To analyse the changes of PP2A enyzme and protein levels we used serine/theroninephosphatase assay and western blot analysis, respectively. For this aim, the effect of dasatinib on KML and the cytotoxic effect of dasatinib on K562 cell line, forming the model, were evaluated by WST-1 test, apoptotic effect by Annexin V and Apo Direct Tunnel method, and stochastic effect by cell cycle analysis. Okadaic acid, an inhibitor of PP2A, was studied to be able to determine the effect of PP2A on apoptosis and cellular survival. Enzyme activity of the PP2A, changing in K562 cell line, treated by dasatinib and protein levels in subunits of the PP2A were evaluated using serine/threonine phosphatase analysis kit and Western Blot analysis, respectively. The cytotoxic effect of dasatinib in K562 cell line was found to be 4,6 nM at 48th hour. The increase in apoptosis owing to the repression of PP2A by OA was detected. The changes in cell cycle-arresting owing to the repressed PP2A by inhibitors were also detected. The PP2A enzyme activity of cells, treated by dasatinib, increased compared to that of the control, depending on the time. It was found that protein level belonging to the PP2A C catalytic subunit of dasatinib decreased compared to that of the control. Consequently, treatment with okadaic acid revealed the increase in apoptosis and cell cycle-arresting at G2/M transition owing to the PP2A suppressing. Phosphatase activity of PP2A decreased at 72nd hours. When treated with dasatinib. Protein level of catalytic subunit of PP2A slightly decreased. It is also found that the PP2A having a role in cell homeostasis of dasatinibin gave rise to the change in enzyme and protein levels. Thus, we could say that targeting PP2A inhibitors may be promosing therapeutic aim to treat CML.
Chronic myeloid leukemia (CML), a group of cancer that usually begins in white blood cells and observed in almost all patients belonging this group, is characterized by the increased tyrosine kinase activity depends on BCR/ABL translocation. Resulting in translocation, that is known to cause activation depending on new chimeric gene formation is anew rearrengment Philadelphia (Ph) chromosome. This anew rearrengment occurs after translocation which is between chromosome 9 and 22. This chromosome has small acrocentric structure that is observed in 90% of chronic myeloid leukemia (CML) patients. It occurs after breaking from nearby the 5' region of the ABL1 gene in 9q34 region, by the break of the remaining part on the side 3' from nearby 3' region of 22q11 BCR gene and their fusion into this region. New chimeric gene, emerged after fusion, causes increased tyrosie kinase activity and production of ABL1 protein having abnormal cell localization. The patients have being generally treated using tyrosine kinase inhibitors (TKIs) to inhibit this BCR-ABL1 kinnase activity, occured by rearrangement. For this purpose, dasatinib is the second-generation tyrosine kinase inhibitör that is used for inhibition of Bcr-Abl1. In CML, it has been using to treat patients with Imatinib-resistant or intolerant CML-AP and it is so effective and safety. Dasatinib shows its effect in leukemia by inhibiting signals related to cell growth and splitting and eventually cell die as a result of blocking signals. Although dasatinib is 325 fold more effective than that of imatinib that is first-generation tyrosine kinase inhibitor, 20% of CMLpatients generally dont respond to treatment with certain drugs. Therefore, alternative drugs should be used and the resistant mechanism should be enlightened. This problem may occur in CML that was induced by BCR-ABL1. This case, in BCR-ABL1-induced KML, one of responses to these questions can be provided by the investigations of structures such as protein phosphatase 2A (PP2A) changed genetically or functionally that functions as tumor suppressor in some cancers patients with imatinib-resistant or -intolerant CML-AP Protein phosphatases have major role in maintenance phosphorylation-dephosphorylation balance in cell. Studies related to KML have shown that the activity of PP2A is changed. PP2A is the most important serine/threonine phosphatase ensuring cellular homeostasis. PP2A consist of 65 kDa A, 36 kDa C subunit and regulatory B subunit. Most studies have shown that PP2A expression/or function in hematological malignancies is inhibited. In our study, we aimed to evaluate PP2A enzyme activity in dasatinib treated K562 chronic myleoid leukemia cell line and the expression changes in protein levels. The cytotoxic effects of dasatinib on K562 cell line, were evaluated by WST-1 analysis. Apoptotic situation was determined by using Annexin V and Apo Direct Tunnel assays and cycletest plus DNA Reagent Kit was used to determine cytoctatic effcets of PP2A. We also used okadaic acidas PP2A inhibitor to assesed major role of PP2A in apoptosis. To analyse the changes of PP2A enyzme and protein levels we used serine/theroninephosphatase assay and western blot analysis, respectively. For this aim, the effect of dasatinib on KML and the cytotoxic effect of dasatinib on K562 cell line, forming the model, were evaluated by WST-1 test, apoptotic effect by Annexin V and Apo Direct Tunnel method, and stochastic effect by cell cycle analysis. Okadaic acid, an inhibitor of PP2A, was studied to be able to determine the effect of PP2A on apoptosis and cellular survival. Enzyme activity of the PP2A, changing in K562 cell line, treated by dasatinib and protein levels in subunits of the PP2A were evaluated using serine/threonine phosphatase analysis kit and Western Blot analysis, respectively. The cytotoxic effect of dasatinib in K562 cell line was found to be 4,6 nM at 48th hour. The increase in apoptosis owing to the repression of PP2A by OA was detected. The changes in cell cycle-arresting owing to the repressed PP2A by inhibitors were also detected. The PP2A enzyme activity of cells, treated by dasatinib, increased compared to that of the control, depending on the time. It was found that protein level belonging to the PP2A C catalytic subunit of dasatinib decreased compared to that of the control. Consequently, treatment with okadaic acid revealed the increase in apoptosis and cell cycle-arresting at G2/M transition owing to the PP2A suppressing. Phosphatase activity of PP2A decreased at 72nd hours. When treated with dasatinib. Protein level of catalytic subunit of PP2A slightly decreased. It is also found that the PP2A having a role in cell homeostasis of dasatinibin gave rise to the change in enzyme and protein levels. Thus, we could say that targeting PP2A inhibitors may be promosing therapeutic aim to treat CML.
Açıklama
Anahtar Kelimeler
Tıbbi Biyoloji, Medical Biology
