Fucoidan'ın androjene-duyarlı ve-duyarsız prostat kanseri hücre dizileri üzerindeki terapötik etkisi

Androgen blockers like Bicalutamide are being used in the treatment of androgen-sensitive prostate cancer, while chemotherapeutic agents like Docetaxel are being used in the treatment of androgen-insensitive prostate cancer. Nevertheless, by time patients are gaining resistance to these treatments and die. Therefore, new agents which do not affect healthy cells and only kill the targeted cancer cells are being investigated to increase the patients' life quality. One of these agents is Fucoidan, a sulphated polysaccharide isolated from brown seaweed. Because of Fucoidan's anti-tumoral specifity, we decided to investigate Fucoidan's effects on prostate cancer. In our study, we used the normal prostate epithelial cell line WPMY1, the androgen-sensitive prostate cancer cell line LNCaP and the androgen-insensitive prostate cell line DU145. Fucoidan's effects on cytotoxicity, apoptosis and cell cycle gene expressions in these cell lines were investigated and statistically analyzed. Also, synergistic effects of Fucoidan with Bicalutamide and Docetaxel which are used in the treatment of prostate cancer were investigated. The IC50 value of Bicalutamide in the LNCaP cell line was determined as 13 µM; while, the IC50 value of Docetaxel in the DU145 cell line was determined as 1.75 nM at 72 hours. Fucoidan's effective dose was designated as 1 mg/ml. When Fucoidan and Docetaxel were used together in the DU145 cell line they showed a synergistic effect; however, when Fucoidan and Bicalutamide were used together in the LNCaP cell line they did not show a synergistic effect. Exposure to Fucoidan increased the apoptotic cell number by 7.4-fold in the LNCaP cell line and 2-fold in the DU145 cell line after 72 hours. No apoptotic effects on the WPMY1 cell line could be observed after 72 hours of exposure to Fucoidan. Fucoidan has upregulated 12 genes that function in cell cycle control by 2-4 folds in the LNCaP cell line. But, these gene expression changes could not be seen in the DU145 and WPMY1 cell lines. In conclusion, Fucoidan is effective on both the androgen-sensitive and androgen-insensitive phases of prostate cancer and does not show any detrimental effects on healthy prostate epithelial cells. We propose that Fucoidan is more effective on the androgen receptor pathway and by fully understanding this mechanism, Fucoidan can be used as a complementary treatment of prostate cancer in the future.Androjene-duyarlı prostat kanserinin tedavisinde Bicalutamide gibi androjen blokerleri kullanılırken, androjene-duyarsız prostat kanserinde Docetaxel gibi kematerapötik ajanlar kullanılmaktadır. Ancak, hastalar bu tedavilere belli bir süre sonra direnç kazanıp ölürler. Bu nedenle hastaların yaşam kalitesini arttırmak amacıyla sağlıklı hücrelere zarar vermeyen, ancak kanser hücrelerini hedefli bir şekilde öldürebilecek özellikteki yeni doğal ajanlar araştırılmaktadır. Bunlardan biri kahverengi deniz yosunundan elde edilen ve sülfatlanmış bir polisakkarit olan Fucoidan' dır. Fucoidan anti-tümöral özellik gösterdiğinden, çalışmamızda Fucoidan' ın prostat kanseri üzerindeki etkilerini araştırmayı planladık. Çalışmamızda normal prostat epiteli hücre dizisi olan WPMY1, androjene-duyarlı prostat kanseri hücre dizisi olan LNCaP ve androjene-duyarsız prostat kanseri hücre dizisi olan DU145 kullanıldı. Fucoidan' ın bu hücreler üzerindeki sitotoksik, apoptotik etkileri ile hücre döngüsünde görev alan genlerin ekspresyonunda meydana getirdiği değişiklikler araştırılıp istatistiksel olarak değerlendirildi. Ayrıca, Fucoidan' ın prostat kanserinin tedavisinde kullanılan Bicalutamide ve Docetaxel ile birlikte kullanıldığında sinerjistik etkisinin olup olmadığı araştırıldı. Bicalutamide' nin LNCaP hücre dizisindeki IC50 değeri 13 µM; Docetaxel' in DU145 hücre dizisindeki IC50 değeri ise 1.75 nM olarak belirlendi. Fucoidan için etken doz olarak 1 mg/ml seçildi. Fucoidan, Docetaxel ile birlikte verildiğinde DU145 hücre dizisinde sinerjik bir etki gösterdi; ancak, Bicalutamide birlikte verildiğinde LNCaP hücre dizisinde sinerjik bir etki oluşturmadı. Fucoidan, LNCaP hücre dizisinde 72 saat sonunda apoptotik hücre sayısında 7.4-katlık bir artış sağladı, DU145 hücre dizisinde ise 72 saat sonunda bu artış 2-kattı. WPMY1 hücre dizisinde 72 saat sonunda apoptotik bir artış görülmedi. Fucoidan, LNCaP hücre dizisinde hücre döngüsü ve kontrolünde etkili genlerin 12 tanesinin 2-4 kat artış göstermesine neden oldu. DU145 ve WPMY1 hücre dizilerinde ise bu gen ekspresyonu değişiklikleri gözlenmedi. Sonuç olarak, Fucoidan prostat kanserinin androjene-duyarlı ve androjene-duyarsız iki evresinde etkilidir ve sağlıklı prostat hücreleri üzerinde herhangi zararlı bir etkisi bulunmamaktadır. Fucoidan' ın androjen reseptör yolağı üzerinden daha etkili olduğu düşünülmektedir ve bu mekanizmanın tam anlaşılmasıyla, Fucoidan' ın gelecekte prostat kanserinin tamamlayıcı tedavisinde kullanılabileceği düşünülmektedi

Genetically modified antigen specific NK cells

cell receptor (TCR) gene therapy can be used to redirect the activity of cytotoxic T cells towards tumor antigens. However, due to the heterodimeric nature of the TCR molecule, TCRα and TCRβ chains introduced by gene delivery have a risk of pairing with the endogenously expressed β or α chains in the T cell. This phenomenon, called mispairing, gives rise to problematic mixed TCR dimers with unpredictable specificity. Our group has previously shown the proof-of-concept for NK cells genetically modified to express functional TCRs (TCR-NK cells) to overcome the mispairing problem. TCR-NK cells expressing CD3δ, CD3γ, CD3ε and TCRα/ß on their cell surface can detect an antigenic peptide presented by MHC-I and selectively show antigen-specific cytotoxic activity. Since NK cells already express CD3ζ, its overexpression in TCR-NK cells does not increase antigen-specific triggering. On the contrary, increased background activity against non-specific targets is observed in TCR-NK cells with CD3ζ overexpression. The underlying mechanism of this effect is yet unknown. IV In this study, we aimed to analyze in detail the gene expression profiles of TCR-NK cells to understand the transcriptomic changes associated with genetic modification. Our results demonstrate that the use of CD3ζ overexpression dramatically effects gene expression profile and might lead to aberrant behavior of TCR-NK cells. We conclude that the inclusion of CD3ζ in vector design may be deleterious to the antigen-specificity of TCR-NK cells. Further development of TCR-NK cells should rely on expression of only CD3δγε components of the CD3 comple

Engineering antigen‐specific NK cell lines against the melanoma‐associated antigen tyrosinase via TCR gene transfer

Introduction of Chimeric Antigen Receptors to NK cells has so far been the main practical method for targeting NK cells to specific surface antigens. In contrast, T cell receptor (TCR) gene delivery can supply large populations of cytotoxic T‐lymphocytes (CTL) targeted against intracellular antigens. However, a major barrier in the development of safe CTL‐TCR therapies exists, wherein the mispairing of endogenous and genetically transferred TCR subunits leads to formation of TCRs with off‐target specificity. To overcome this and enable specific intracellular antigen targeting, we have tested the use of NK cells for TCR gene transfer to human cells. Our results show that ectopic expression of TCR α/β chains, along with CD3 subunits, enables the functional expression of an antigen‐specific TCR complex on NK cell lines NK‐92 and YTS, demonstrated by using a TCR against the HLA‐A2‐restricted tyrosinase‐derived melanoma epitope, Tyr368‐377. Most importantly, the introduction of a TCR complex to NK cell lines enables MHC‐restricted, antigen‐specific killing of tumor cells both in vitro and in vivo. Targeting of NK cells via TCR gene delivery stands out as a novel tool in the field of adoptive immunotherapy which can also overcome the major hurdle of “mispairing” in TCR gene therapy