BRAF ve CDKN2A mutant insan A375 melanom hücre hattında Vemurafenib ve Palbosiklib kombinasyonunun sitotoksisite ve apoptozis üzerine etkisi
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Dosyalar
Tarih
2016
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Yayıncı
Ege Üniversitesi, Tıp Fakültesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Son yıllarda hedefe yönelik tedavi stratejilerinin gelişmesi sağkalımda önemli iyileşme sağlamış olsa da melanom küratif olmayan ve fatal seyreden bir kanserdir. BRAF mutant melanom hastalarında tedavide kullanılan BRAF inhibitörleri anlamlı klinik fayda sağlamakta ancak ilaçlara erken dönemde direnç gelişimi ilaçların etkilerini sınırlandırmaktadır. Etkin tedavi sağlamak amacıyla oluşturulan kombinasyon rejimlerinin progresyonsuz ve genel sağkalıma etkisi yeterli değildir. Melanomda CDK 4/6-siklin D-Rb yolağı hücre siklusunu kontrol eder. Bu yolakta mutasyon varlığının gösterilmesi yolağı tedavideki hedeflerden biri haline getirmiştir. Biz bu çalışmamızda, CDK 4/6 inhibitörü Palbosiklib ve BRAF inhibitörü Vemurafenib'in tek ajan ve kombine tedavide sitotoksisite ve apoptozis üzerine etkilerini inceledik. Homozigot BRAF ve CDKN2A mutant A375 hücre hattına 48 saat Palbosiklib uygulamasıyla ilacın sitotoksik etkisi sırasıyla 5 ϻM konsantrasyonda %7, 10 ϻM'da %15, 25 ϻM'da %40, 50 ϻM'da %77, 75 ϻM'da %88, 100 ϻM'da %93 saptanırken, Vemurafenib ile; 5 ϻM konsantrasyonda %34.5, 10 ϻM'da %50, 25 ϻM'da %63.8, 50 ϻM'da %67, 75 ϻM'da %73, 100 ϻM'da %82.5 oranında saptandı. İlaçların çeşitli konsantrasyonlarındaki kombinasyonlarında sitotoksisitenin sinerjistik etkiyle arttığı gözlendi. Palbosiklib'in IC50 değeri 25.2 ϻM, Vemurafenib'in IC50 değeri 12.23 ϻM'dı. IC50 değerine yaklaşan konstantrasyonlarda sitotoksik etkinin apoptozis yoluyla olduğu ve kombinasyon ile apoptozisin arttığı gösterildi. Düşük konsantrasyonlarda apoptozis gözlenmedi. Bu çalışma ile Palbosiklib'in tek başına BRAF mutant melanom hücre hattında sitotoksik etkili olduğu, Vemurafenib ile kombinasyonunda ise sinerjizma ile sitotoksisitenin arttığı ve sitotoksisitede apoptozis'in rol oynayabileceği ilk kez gösterilmiştir. Sonuç olarak, bu çalışma Palbosiklib'in gelecekte melanom tedavisinde tek başına ve kombine olarak etkin bir terapötik ajan olabileceğini desteklemektedir.
In the recent years, despite of improvements in outcome and survival at melanoma patients mainly due to new systemic treatment strategies with targeted therapy, melanoma is still a non-curative and fatal cancer. Although BRAF inhibitors, such as Vemurafenib, are effective on BRAF mutant melanomas, developing resistance limits the efficency of drug in long-term period. Combination strategies did not improve progression free and overall survival, sufficiently. CDK 4/6 (cyclin dependent kinase)-cyclin D-Rb (retinoblastoma) pathway controls cell cycle progression and the presence of mutations on this pathway in melanoma results to become therapeutic target. In this study, we observed cytotoxic and apoptotic effects of Palbosiklib and Vemurafenib as a single agent and combination. After treating A375 cell line, homozygous BRAF and CDKN2A mutant, with Palbocyclib and Vemurafenib during 48 hours, cytotoxic effects of drug were tested. The cytotoxic effects of Palbosiklib are, respectively, %7 in 5 ϻM concentration, %15 in 10 ϻM, %40 in 25 ϻM, %77 in 50 ϻM, %88 in 75 ϻM and %93 in 100 ϻM. The cytotoxic effects of Vemurafenib are, respectively, %34.5 in 5 ϻM concentration, %50 in 10 ϻM, %63.8 in 25 ϻM, %67 in 50 ϻM, %73 in 75 ϻM, %82.5 in 100 ϻM. It was shown that concurrent applications of drugs are more cytotoxic than single agents and also this effect is sinergistic. IC50 value for Palbosiklib is 25.2 ϻM, for Vemurafenib is 12.23 ϻM. In high drug concentrations which are close to IC50 value, apoptosis is triggered with both single agent treatment and concurrent treatment. It is not seem any apoptotic effect with low concentrations of drugs. In this study, for the first time, it was shown that Palbocyclib is a cytotoxic agent on BRAF mutant melanoma cell lines as a single agent or combination therapy with Vemurafenib and synergistic effect might play a role in combination therapy, moreover this cytotoxic effect may be related with apoptosis. In conclusion, these results support single agent Palbocyclib and Palbocyclib/Vemurafenib combination may be novel therapeutic treatment strategies for BRAF mutant melanoma patients in future.
In the recent years, despite of improvements in outcome and survival at melanoma patients mainly due to new systemic treatment strategies with targeted therapy, melanoma is still a non-curative and fatal cancer. Although BRAF inhibitors, such as Vemurafenib, are effective on BRAF mutant melanomas, developing resistance limits the efficency of drug in long-term period. Combination strategies did not improve progression free and overall survival, sufficiently. CDK 4/6 (cyclin dependent kinase)-cyclin D-Rb (retinoblastoma) pathway controls cell cycle progression and the presence of mutations on this pathway in melanoma results to become therapeutic target. In this study, we observed cytotoxic and apoptotic effects of Palbosiklib and Vemurafenib as a single agent and combination. After treating A375 cell line, homozygous BRAF and CDKN2A mutant, with Palbocyclib and Vemurafenib during 48 hours, cytotoxic effects of drug were tested. The cytotoxic effects of Palbosiklib are, respectively, %7 in 5 ϻM concentration, %15 in 10 ϻM, %40 in 25 ϻM, %77 in 50 ϻM, %88 in 75 ϻM and %93 in 100 ϻM. The cytotoxic effects of Vemurafenib are, respectively, %34.5 in 5 ϻM concentration, %50 in 10 ϻM, %63.8 in 25 ϻM, %67 in 50 ϻM, %73 in 75 ϻM, %82.5 in 100 ϻM. It was shown that concurrent applications of drugs are more cytotoxic than single agents and also this effect is sinergistic. IC50 value for Palbosiklib is 25.2 ϻM, for Vemurafenib is 12.23 ϻM. In high drug concentrations which are close to IC50 value, apoptosis is triggered with both single agent treatment and concurrent treatment. It is not seem any apoptotic effect with low concentrations of drugs. In this study, for the first time, it was shown that Palbocyclib is a cytotoxic agent on BRAF mutant melanoma cell lines as a single agent or combination therapy with Vemurafenib and synergistic effect might play a role in combination therapy, moreover this cytotoxic effect may be related with apoptosis. In conclusion, these results support single agent Palbocyclib and Palbocyclib/Vemurafenib combination may be novel therapeutic treatment strategies for BRAF mutant melanoma patients in future.
Açıklama
Anahtar Kelimeler
A375 Hücre Hattı, CDK 4/6, CDKN2A, BRAF, MAPK, Vemurafenib, Palbosiklib, Sitotoksisite, Melanom, A375 Cell Line, Cytotoxicity, Malignant, Melanoma
