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Abstrak :
Latar Belakang: Sel punca kanker merupakan populasi sel minor yang memiliki kemampuan self-renewal dan proliferasi tak terbatas sehingga bersifat tumorigenik dan diduga berperan dalam penurunan sensitivitas terhadap berbagai terapi kanker. Tamoksifen merupakan terapi lini pertama pada kanker payudara ER positif namun penggunaan jangka panjangnya menimbulkan masalah resistensi. Beberapa faktor yang diduga berperan dalam penurunan sensitivitas sel terhadap Tamoksifen yakni modulasi pensinyalan estrogen melalui ER?66; dan ER?36 (yang diketahui memperantarai pensinyalan non-genomik), serta ekspresi transporter effluks seperti MRP2 yang berperan dalam penurunan kadar Tamoksifen intraseluler. Penelitian ini bertujuan untuk menganalisis efek pemaparan Tamoksifen berulang pada sel punca kanker payudara CD24-/CD44+, dalam kaitannya mengenai sensitivitas terapi melalui perubahan ekspresi estrogen reseptor alfa dan transporter efluks MRP2. Metode: Selpunca kanker payudara CD24-/CD44+ dipaparkan Tamoksifen 1 ?M selama 21 hari dengan DMSO sebagai kontrol negatif. Viabilitas sel setelah pemaparan Tamoksifen diuji dengan metode trypan blue exclusion. Sifat tumorigenik sel setelah pemaparan (CD24-/CD44+(T)) diuji dengan mammossphere formation assay dan dibandingkan dengan sel CD24-/CD44+(0) yang belum dipaparkan Tamoksifen. Ekspresi mRNA Oct4, c-Myc, ER?66, ER?36 dan MRP2 dianalisis dengan one step quantitative RT-PCR. Hasil: Terjadi penurunan sensitivitas sel punca kanker payudara CD24-/CD44+(T) yang dipaparkan Tamoksifen selama 21 hari yang ditunjukkan dengan kenaikan viabilitas sel hingga 125,2%. Tamoksifen tidak dapat menekan sifat tumorigenik sel CD24-/CD44+(T) yang dibuktikan melalui jumlah mammosfer yang tidak berbeda bermakna dibandingkan dengan CD24-/CD44+(0). Penurunan sensitivitas sel CD24-/CD44+(T) juga dibuktikan melalui peningkatan ekspresi Oct4 dan c-Myc; keduanya merupakan petanda pluripotensi dan c-Myc juga dikenal sebagai petanda keganasan. Parameter penurunan sensitivitas seperti ER?66, ER?36 dan MRP2 juga menunjukkan peningkatan ekspresi pada hari ke-15 namun menurun kembali pada hari ke-21 yang menunjukkan adanya mekanisme regulasi lain yang mungkin terlibat dalam penurunan sensitivitas sel punca kanker payudara terhadap Tamoksifen. Kesimpulan: Pemaparan Tamoksifen berulang dapat menurunkan sensitivitas sel punca kanker payudara CD24-/CD44+ melalui perubahan ekspresi estrogen reseptor alfa dan transporter efluks MRP2. ......Background: Cancer stem cells are minor population of cells possessing self-renewal and unlimited proliferation abilities which support their tumorigenicity and role in decreased sensitivity to many cancer therapies. Tamoxifen is a first line therapy for breast cancer patients with positive ER status. Nonetheless, after 5 years of its long term use eventually leads to recurrence and resistance in 50% of patients receiving tamoxifen therapy. Among some factors that might play role in decreased sensitivity to tamoxifen are modulation of estrogen signaling through ER?66 and ER?36 (the latter known for its non-genomic estrogen signaling), and expression of efflux transporter such as MRP2 responsible for decreased intracellular tamoxifen level. The objective of this study is to analyze the effects of long term tamoxifen exposure toward decreased sensitivity of the breast cancer stem cells CD24-/CD44+ through changes in expression of estrogen receptor alpha and efflux transporter MRP2. Methods: Breast cancer stem cells CD24-/CD44+ were exposed to 1 ?M tamoxifen for 21 days with DMSO as negative control. After exposure with 1 ?M tamoxifen, the cell viability were tested by the trypan blue exclusion method. Cell tumorigenicity of tamoxifen-exposed CD24-/CD44+(T) and CD24-/CD44+(0) (before treatment) were tested by the mammosphere formation assay. The expression of Oct4, c-Myc, ER?66, ER?36 andMRP2 mRNAs were analyzed by one step quantiative RT-PCR. Results: A decreased sensitivity of the breast cancer stem cells CD24-/CD44+ exposed with 1 ?M tamoxifen for 21 days was observed as indicated by an increased cell viability up to 125.2%. In the presence of tamoxifen, breast cancer stem cells CD24-/CD44+(T) exhibited tumorigenic properties as indicated in no significant difference in the formation of mammosphere unit compared to those of CD24-/CD44+(0). After exposure with 1 ?M tamoxifen for 21 days, an elevated level of Oct4 and c-Myc expressions were observed; both are known as pluripotency markers and the latter also known as marker of aggresiveness. Parameters for a decreased sensitivity such as ER?66, ER?36 and MRP2 also exhibited an elevated expression after 15 days of exposure, but the decreased expression after 21 days of exposure suggests that there might be another mechanism involved in decreased sensitivity of the breast cancer stem cells toward tamoxifen. Conclusion: Long term tamoxifen exposure may decrease the sensitivity of the breast cancer stem cells CD24-/CD44+ through changes in expression of estrogen receptor alpha and efflux transporter MRP2.

Abstrak :
Latar Belakang: Sel punca kanker merupakan populasi sel minor yang memiliki kemampuan self-renewal dan proliferasi tak terbatas sehingga bersifat tumorigenik dan diduga berperan dalam penurunan sensitivitas terhadap berbagai terapi kanker. Tamoksifen merupakan terapi lini pertama pada kanker payudara ER positif namun penggunaan jangka panjangnya menimbulkan masalah resistensi. Beberapa faktor yang diduga berperan dalam penurunan sensitivitas sel terhadap Tamoksifen yakni modulasi pensinyalan estrogen melalui ERα66; dan ERα36 (yang diketahui memperantarai pensinyalan non-genomik), serta ekspresi transporter effluks seperti MRP2 yang berperan dalam penurunan kadar Tamoksifen intraseluler. Penelitian ini bertujuan untuk menganalisis efek pemaparan Tamoksifen berulang pada sel punca kanker payudara CD24-/CD44+, dalam kaitannya mengenai sensitivitas terapi melalui perubahan ekspresi estrogen reseptor alfa dan transporter efluks MRP2. Metode: Selpunca kanker payudara CD24-/CD44+ dipaparkan Tamoksifen 1 μM selama 21 hari dengan DMSO sebagai kontrol negatif. Viabilitas sel setelah pemaparan Tamoksifen diuji dengan metode trypan blue exclusion. Sifat tumorigenik sel setelah pemaparan (CD24-/CD44+(T)) diuji dengan mammossphere formation assay dan dibandingkan dengan sel CD24-/CD44+(0) yang belum dipaparkan Tamoksifen. Ekspresi mRNA Oct4, c-Myc, ERα66, ERα36 dan MRP2 dianalisis dengan one step quantitative RT-PCR. Hasil: Terjadi penurunan sensitivitas sel punca kanker payudara CD24-/CD44+(T) yang dipaparkan Tamoksifen selama 21 hari yang ditunjukkan dengan kenaikan viabilitas sel hingga 125,2%. Tamoksifen tidak dapat menekan sifat tumorigenik sel CD24-/CD44+(T) yang dibuktikan melalui jumlah mammosfer yang tidak berbeda bermakna dibandingkan dengan CD24-/CD44+(0). Penurunan sensitivitas sel CD24-/CD44+(T) juga dibuktikan melalui peningkatan ekspresi Oct4 dan c-Myc; keduanya merupakan petanda pluripotensi dan c-Myc juga dikenal sebagai petanda keganasan. Parameter penurunan sensitivitas seperti ERα66, ERα36 dan MRP2 juga menunjukkan peningkatan ekspresi pada hari ke-15 namun menurun kembali pada hari ke-21 yang menunjukkan adanya mekanisme regulasi lain yang mungkin terlibat dalam penurunan sensitivitas sel punca kanker payudara terhadap Tamoksifen. Kesimpulan: Pemaparan Tamoksifen berulang dapat menurunkan sensitivitas sel punca kanker payudara CD24-/CD44+ melalui perubahan ekspresi estrogen reseptor alfa dan transporter efluks MRP2. ......Background: Cancer stem cells are minor population of cells possessing self-renewal and unlimited proliferation abilities which support their tumorigenicity and role in decreased sensitivity to many cancer therapies. Tamoxifen is a first line therapy for breast cancer patients with positive ER status. Nonetheless, after 5 years of its long term use eventually leads to recurrence and resistance in 50% of patients receiving tamoxifen therapy. Among some factors that might play role in decreased sensitivity to tamoxifen are modulation of estrogen signaling through ERα66 and ERα36 (the latter known for its non-genomic estrogen signaling), and expression of efflux transporter such as MRP2 responsible for decreased intracellular tamoxifen level. The objective of this study is to analyze the effects of repeated tamoxifen exposure toward decreased sensitivity of the breast cancer stem cells CD24-/CD44+ through changes in expression of estrogen receptor alpha and efflux transporter MRP2. Methods: Breast cancer stem cells CD24-/CD44+ were exposed to 1 μM tamoxifen for 21 days with DMSO as negative control. After exposure with 1 μM tamoxifen, the cell viability were tested by the trypan blue exclusion method. Cell tumorigenicity of tamoxifen-exposed CD24-/CD44+(T) and CD24-/CD44+(0) (before treatment) were tested by the mammosphere formation assay. The expression of Oct4, c-Myc, ERα66, ERα36 andMRP2 mRNAs were analyzed by one step quantiative RT-PCR. Results: A decreased sensitivity of the breast cancer stem cells CD24-/CD44+ exposed with 1 μM tamoxifen for 21 days was observed as indicated by an increased cell viability up to 125.2%. In the presence of tamoxifen, breast cancer stem cells CD24-/CD44+(T) exhibited tumorigenic properties as indicated in no significant difference in the formation of mammosphere unit compared to those of CD24-/CD44+(0). After exposure with 1 μM tamoxifen for 21 days, an elevated level of Oct4 and c-Myc expressions were observed; both are known as pluripotency markers and the latter also known as marker of aggresiveness. Parameters for a decreased sensitivity such as ERα66, ERα36 and MRP2 also exhibited an elevated expression after 15 days of exposure, but the decreased expression after 21 days of exposure suggests that there might be another mechanism involved in decreased sensitivity of the breast cancer stem cells toward tamoxifen. Conclusion: Repeated tamoxifen exposure may decrease the sensitivity of the breast cancer stem cells CD24-/CD44+ through changes in expression of estrogen receptor alpha and efflux transporter MRP2.

Abstrak :
Kurkumin diketahui dapat menghambat drug efflux transporter, khususnya pada penelitian ini overekspresi multidrug resistance protein 1 MRP1 dan MRP2, yang berdasarkan penelitian terdahulu, diduga menyebabkan resistensi MCF-7 pada pemberian endoksifen dan estradiol berulang. Penelitian ini bertujuan untuk membuktikan apakah kurkumin dapat menghambat ekspresi MRP1 dan MRP2 pada MCF-7 yang diberikan endoksifen dan estradiol berulang. Metode: Penelitian eksperimental ini menggunakan cDNA hasil sintesis isolasi RNA, dimana kelompok perlakuan dipaparkan pada MCF-7 3x per minggu, hingga 8 minggu. Perlakuan dibagi menjadi DMSO kontrol negatif , Endoksifen 1,000 nM/L ?-Estradiol 1 nM/L EB, kontrol positif , Endoksifen 1,000 nM/L ?-Estradiol 1 nM/L Kurkumin 8.5?M EBK8.5, dan Endoksifen 1,000 nM/L ?-Estradiol 1 nM/L Kurkumin 17?M EBK17 . Tingkat ekspresi mRNA relatif MRP1 dan MRP2 diukur dengan qRT-PCR dan dihitung dengan metode livak. Hasil: Terdapat peningkatan ekspresi mRNA pada perlakuan EB pada MRP1 dan MRP2, relatif terhadap DMSO. Peningkatan ekspresi mRNA meningkat pada EBK8.5, dan menurun pada EBK17 pada MRP1. Terjadi sebaliknya pada MRP2. Kesimpulan: Kurkumin dapat bekerja sebagai terapi pendamping pada terapi endoksifen dan estradiol untuk menurunkan resistensi dari MRP1 dan MRP2. ......Background: Curcumin is known to inhibit drug efflux transporter overexpression, such as multidrug resistance protein 1 MRP1 dan MRP2, which in previous experiment, suspected as a causal of MCF 7 cell resistancy given endoxifen and estradiol repeatedly. This study aims to prove whether curcumin can inhibit expression of MRP1 and MRP2 in MCF 7 given endoxifen and estradiol repeatedly. Method: This experimental design uses cDNA from RNA isolation synthesis, which treatment group given repeatedly on MCF 7 3x per week until 8th week. The treatment groups are DMSO negative control, Endoxifen 1,000 nM L Estradiol 1 nM L EB, positive control, Endoxifen 1,000 nM L Estradiol 1 nM L Curcumin 8.5 M EBK8.5, and Endoxifen 1,000 nM L Estradiol 1 nM L Curcumin 17 M EBK17. Relative mRNA expression in MRP1 and MRP2 is measured with qRT PCR and quantified with Livak method. Result: There is an increased mRNA expression in treatment of EB in MRP1 and MRP2, relative to DMSO. Increased mRNA expression is higher on EBK8.5, and lower on EBK17 in MRP1, and conversely in MRP2. Conclusion: Curcumin could work as adjuvant for Endoxifen and Estradiol therapy to decrease resistancy caused by mRNA expression of MRP1and MRP2.