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Abstrak :
Latar Belakang: Breast Cancer Stem Cells (BCSC) merupakan populasi sel kanker payudara yang mempunyai sifat sel punca. BCSC menjaga stabilitas tumor dengan menginisiasi pembentukan populasi sel kanker baru serta memberikan kekebalan terhadap terapi. BCSC dapat berinteraksi dengan lingkungan mikro tumor yang melepaskan berbagai sitokin dan growth factor, termasuk TGF-β1. Melalui mekanisme autoinduksi, TGF-β1 dapat meningkatkan produksi TGF-β1 endogen dan pensinyalan autokrinnya. Pensinyalan autokrin TGF-β1 dapat meningkatkan pengaruh tumor promotor TGF-β1 dalam perkembangan kanker melalui penguatan karakter kepuncaan dan sifat tumorigenik. Penelitian ini bertujuan untuk enganalisis efek pemberian TGF-β1 rekombinan manusia pada sel punca kanker payudara (aldehyde dehydrogenase positive, ALDH+) terhadap ekspresi marker kepuncaan melalui pensinyalan autokrin TGF-β1. Sebagai pembanding digunakan kanker payudara subtipe triple negative (TNBC). Metode: BCSCs manusia (ALDH+) dan TNBC (MDA-MB-231) dikultur dalam Dulbecco's Modified Eagle Medium/Nutrient Mixture F12/HG (DMEM F12/HG) dengan suplemen 0,1 ng/ml protein rekombinan TGF-β1 manusia (rhTGF-β1) selama periode 1, 2 dan 4 jam. Medium kultur kemudian diganti dengan DMEM F12/HG tanpa serum selama 24 jam. Tingkat ekspresi mRNA reseptor TGF-β tipe 1 (TβR1), TGF-β1, faktor transkripsi pengikat oktamer 4 (OCT4), dan anggota A1 keluarga aldehida dehidrogenase 1 (ALDH1A1) dianalisis menggunakan real time reverse transcriptase polymerase chain reactions (RT-qPCR). Kadar protein TGF- β1 dalam media kultur ditentukan dengan menggunakan enzyme-linked immunosorbent assay (ELISA). Sifat tumorigenik sel diuji dengan uji mammosphere forming unit (MFU assay). Hasil: Tingkat ekspresi mRNA dan protein TGF-β1 BCSC setelah perlakuan tampak meningkat namun tidak pada TNBC. mRNA TβR1 BCSCs meningkat pada periode perlakuan 1 dan 2 jam, sedangkan pada TNBC hanya pada periode 1 jam. Penanda kepuncaan ALDH1A1 dan OCT4 tampak meningkat pada BCSC namun tidak pada TNBC. Uji MFU menunjukkan sifat tumorigenik kedua kelompok sel terutama pada periode perlakuan 2 jam tampak meningkat. Kesimpulan: Perlakuan TGF-β1 dalam konsentrasi rendah dan dalam waktu singkat memicu autoinduksi pada BCSCs yang menyebabkan peningkatan ekspresi gen kepuncaan melalui pensinyalan autokrin. Sedangkan pada TNBC, peningkatan ekspresi marker kepuncaan tidak terjadi. Namun demikian, sifat tumorigenik BCSC dan TNBC tetap meningkat. ......Background: Breast Cancer Stem Cells (BCSC) is a population of breast cancer cells that have stem cell characteristics. BCSCs maintain tumor stability by initiating the formation of new cancer cell populations and providing resistance to therapy. BCSCs can interact with the tumor microenvironment which releasing various cytokines and growth factors, including TGF-β1. Through the autoinduction mechanism, TGF-β1 can increase endogenous TGF-β1 production and autocrine signaling. TGF-β1 autocrine signaling can increase the tumor promoter role of TGF-β1 in cancer development by enhancing the stemness and tumorigenic properties. This study aims to analyze the effect of Human TGF-β1 recombinant protein treatment to breast cancer stem cells (aldehyde dehydrogenase positive, ALDH+) on the expression of stemness marker through TGF-β1 autocrine signaling. Triple negative breast cancer (TNBC) was used as a comparison. Methods: Human BCSCs (ALDH+) and TNBC (MDA-MB-231) were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F12/HG (DMEM F12/HG) with 0.1 ng / ml recombinant protein of human TGF-β1 supplementation (rhTGF- β1) over 1, 2 and 4 hour periods. The culture medium was then replaced with DMEM F12/HG serum-free for 24 hours. The expression levels of the TGF-β receptor type 1 (TβR1), TGF-β1, octamer-binding transcription factor 4 (OCT4), and members of the A1 family of aldehyde dehydrogenase 1 (ALDH1A1) mRNA were analyzed using real time reverse transcriptase polymerase chain reactions (RT-qPCR). TGF-β1 protein levels in conditioned medium were determined using an enzyme-linked immunosorbent assay (ELISA). The tumorigenic properties of cells were tested by the mammosphere forming unit (MFU) assay. Results: The expression level of mRNA and TGF-β1 BCSC protein after treatment appeared to be increased but not in TNBC. mRNA TβR1 BCSCs increased in the treatment period of 1 and 2 hours, whereas in TNBC only in the 1 hour period. The markers of ALDH1A1 and OCT4 expression appeared to be increased in BCSC but not in TNBC. The MFU test showed that the tumorigenic properties of both cell groups, especially in the 2 hour treatment period, appeared to be increasing. Conclusion: Treatment of TGF-β1 in low concentrations and in a short time triggered autoinduction of BCSCs and leads to the increased expression of stemness genes marker via autocrine signaling. Whereas in TNBC, this increase in the expression of the stemness markers did not occur. However, the tumorigenic nature of BCSC and TNBC continues to increase.

Abstrak :
Latar belakang: Data dari World Health Organization, Centers for Disease Control and Prevention dan Kementerian Kesehatan RI menunjukan bahwa kanker payudara merupakan kanker yang paling umum pada wanita. Penyembuhan kanker melalui berbagai cara telah banyak dilakukan, namun pertumbuhan kembali dari kanker telah banyak dilaporkan. Sel punca kanker diyakini berperan dalam pertumbuhan kanker maupun rekurensi setelah pengobatan. Berdasarkan beberapa riset, CD44+/CD24- sel punca kanker memiliki potensial yang tinggi untuk menimbulkan kanker. Beberapa gen memiliki peran sebagai faktor transkripsi yang berkontribusi dalam pertumbuhan kanker dan beberapa berperan juga dalam mempertahankan tingkat pluripotensi kanker. c-Myc merupakan salah satu gen yang mempertahankan iPS (induced pluripotent stem cells) bersama dengan KLF4, Oct4, SOX2 dan Nanog. Namun demikian, selama pertumbuhan kanker, lingkungan mikro dari kanker menjadi hipoksia. Berhubungan dengan ini, pengaruh hipoksia terhadap ekspresi gen yang berfungsi dalam pluripotensi masih belum jelas. Oleh karena itu, eksperimen ini menyelidiki ekspresi gen c-Myc dalam sel punca kanker yang diinduksi hipoksia. Metode: Sel punca kanker payudara CD44+/CD24- diinduksi oleh beberapa durasi hipoksia (0 jam, 0.5 jam, 4 jam, 6 jam dan 24 jam). Total RNA sel kemudian diekstraksi dan mRNA gen c-Myc diamplifikasi melalui one-step qRT-PCR. Ekspresi relative dari gen c-Myc dilakukan dengan formula Livak berdasarkan nilai Ct yang diperoleh dengan gen 18S. Sebagai kontrol, konfirmasi ekspresi gen c-Myc dikonfirmasi melalui elektroforesis. Hasil: Ekspresi c-Myc pada sampel sel punca kanker payudara CD44+/CD24- yang diinduksi hipoksia selama 0.5 jam sedikit mengalami peningkatan dibandingkan dengan sampel 0 jam walaupun tidak signifikan. Ekspresi c-Myc pada sampel yang diinduksi hipoksia selama 4, 6 dan 24 jam menurun dibandingkan sampel yang tidak diinduksi hipoksia. Kesimpulan: Ekspresi c-Myc pada sel punca kanker CD44+/CD24- yang digunakan dalam eksperimen ini cenderung menurun pada 3 durasi hipoksia yang berbeda (4 jam, 6 jam dan 24 jam) pada kondisi in vitro. ......Background: Data from World Health Organization, Centers for Disease Control and Prevention and Kementerian Kesehatan RI show that breast cancer is the most common cancer among women. Eradicating cancer through several treatments have been done but there are cases in which cancer relapse is reported. Cancer stem cells have been found to develop the cancer as well as play important role in cancer regrowth. According to some researches, CD44+/CD24- breast cancer stem cells potential to cancer development is high. Several genes which have role as transcription factors may contribute to cancer growth and some act to maintain the cancer stemness and pluripotency level. c-Myc is one gene which maintains iPS (induced pluripotent stem cells) along with KLF4, Oct4, SOX2 and Nanog. However, during the cancer growth the cancer microenvironment becomes hypoxic. In accordance to this, the effect of hypoxia towards the gene expression acting in cancer pluripotency was not yet clear. Therefore c-Myc expression in hypoxia-induced breast cancer stem cells was assessed in this research. Method: The CD44+/CD24- breast cancer stem cells (BCSCs) are induced by several hypoxia durations (0 hour, 0.5 hour, 4 hours, 6 hours and 24 hours) in hypoxia chamber. The mRNA of BCSCs is extracted through RNA isolation procedure. Following this, qRT-PCR procedure is done to amplify the mRNA. The Ct (cycle threshold) obtained from qRT-PCR are calculated using Livak formula to get the c-Myc relative expression from the samples. Ct of 18S is used to normalize the c-Myc Ct. Electrophoresis is done next to confirm the c-Myc expression. Results: c-Myc expression in 0.5 hour hypoxia induced CD44+/CD24- breast cancer stem cells sample is slightly high than in 0 hour hypoxia induced sample, even though the increase is not significant. Meanwhile, c-Myc expression in 4, 6 and 24 hours hypoxia induced samples are lower than 0 hour hypoxia induced sample. Conclusion: c-Myc expression from the breast cancer stem cell CD44+/CD24- samples used in this experiment tend to have gradual decrease during 3 different periods (0 hour, 0.5 hour, 4 hours, 6 hours and 24 hours) of hypoxia in vitro.

Abstrak :
Latar Belakang: Beberapa bukti menunjukkan bahwa quiescent cancer stem cell (CSC) terlibat dalam resistans terhadap gefitinib pada adenokarsinoma paru sebagai mekanisme nonmutasi. Kami sebelumnya telah mempublikasikan bahwa gefitinib- resistant persister (GRP) mengekspresikan stemness factor dengan level yang tinggi dan memiliki ciri khas fenotip CSC. Studi terbaru menunjukkan bahwa FBXW7, merupakan jenis protein F-box, memainkan peran penting dalam pemeliharaan quiescent CSC dengan memediasi degradasi protein c-MYC melalui proses ubiquination. Tujuan dari penelitian ini adalah untuk mengetahui peran FBXW7 dalam resistans terhadap gefitinib pada adenokarsinoma paru dengan mutasi EGFR. Metode: Cell line dari sel adenokarsinoma paru, PC9, yang mengandung mutasi sensitif EGFR dipajankan pada gefitinib dengan konsentrasi tinggi untuk mengembangkan GRP. Kami mencoba melakukan abrogasi ekspresi gen FBXW7, dan mengevaluasi sensitivitasnya terhadap gefitinib dan populasi CD133-positive stem cell di GRP. Kami juga memasukkan plasmid FUCCI melalui proses infeksi lentiviral ke dalam sel dan kemudian menyelidiki siklus sel dan sel pada fase G0 dalam GRP. Selanjutnya, kami telah mengembangkan model gefitinib-resistant tumor (GRT) dengan menyuntikkan sel PC9 ke dalam mencit NOG diikuti dengan pemberian gefitinib setelah pertumbuhan tumor, dan mengevaluasi ekspresi mRNA dan ekspresi protein dari penanda terkait quiescence, FBXW7 in vivo. Hasil: GRP menunjukkan ekspresi yang tinggi dari penanda cancer stem cell, CD133 dan penanda terkait quiescence, FBXW7 dan ekspresi c-MYC yang rendah pada tingkat protein secara in vitro. Analisis siklus sel menunjukkan bahwa mayoritas GRP berada pada fase G0/G1. TIndakan abrogasi gen FBXW7 menurunkan populasi sel CD133-positive di GRPs. Abrogasi FBXW7 juga meningkatkan kerentanan sel terhadap gefitinib, membalikkan populasi sel fase G0/G1 menjadi sel S/G2/M, dan menurunkan jumlah sel GRP. Secara in vivo, pada GRT setelah pengobatan gefitinib menunjukkan ekspresi FBXW7 yang tinggi dan ekspresi c-MYC yang rendah. Kami juga menemukan bahwa ekspresi FBXW7 dalam sel CD133-positive meningkat dan ekspresi c-MYC menurun pada mencit dan pada 9 dari 14 spesimen tumor dari pasien adenokarsinoma paru dengan mutasi EGFR resistan terhadap gefitinib. Kesimpulan: Temuan ini menunjukkan bahwa FBXW7 dapat memainkan peran penting dalam pemeliharaan quiescence pada gefitinib-resistant lung CSC pada adenokarsinoma paru dengan mutasi positif EGFR ......Background: Accumulating evidence indicates that quiescent cancer stem cells (CSCs) are involved in the resistance to gefitinib in non-small cell lung cancer (NSCLC) as non-mutational mechanism. We have previously reported that gefitinib-resistant persisters (GRPs) highly expressed stemness factors and had characteristic features of the CSCs phenotype. Recent studies demonstrate that FBXW7, a type of F-box protein, plays an important role in the maintenance of quiescent CSC by mediating the degradation of c-MYC protein by ubiquination. The aim of this study is to figure out the role of FBXW7 in the resistance to gefitinib in lung adenocarcinoma with EGFR mutation. Methods: lung adenocarcnoma cell lines, PC9, harboring sensitive-EGFR mutation were exposed to high concentration of gefitinib in order to develop GRPs. We tried to knockdown FBXW7 gene expression, and evaluated their sensitivity to gefitinib and CD133-positive stem cell population in GRPs. We also introduced FUCCI plasmid via lentiviral infection in the cells and then investigated the cell cycle and G0-phase cells in GRPs. Furthermore, we established gefitinib-resistant tumor (GRT) model by injecting PC9 cells into NOG-mice followed by gefitinib administration after tumor growth, and evaluated mRNA and protein expression of quiescence-related markers including FBXW7 in vivo. Results: In vitro, GRPs showed high expression of stem cell marker CD133 and quiescence-related markers including FBXW7 and low expression of c-MYC at protein level. Cell cycle analysis revealed that majority of GRPs existed in G0/G1 phase. Silencing of FBXW7 gene reduced CD133-positive cell population in GRPs. Knockdown of FBXW7 also increased susceptibility of cells to gefitinib, reversed population of G0/G1 cells to G2/S/M cells, and decreased cell number of GRPs. In vivo, GRTs after gefitinib treatment revealed high expression of FBXW7 and low expression of c-MYC. We also found that FBXW7 expression in CD133-positive cells was increased and c-MYC expression was decreased in mice and in 9 out of 14 tumor specimens from EGFR-mutant lung adenocarcinoma patients with acquired resistance to gefitinib. Conclusion: These findings suggest that FBXW7 plays a pivotal role in the maintenance of quiescence in gefitinib-resistant lung CSCs in EGFR mutation- positive lung adenocarcinoma.

Abstrak :
It is pointed out that cancer stem cell is a cell type within a tumor that possesses the capacity of cell-renewal and can give rise to the heterogeneous lineages of cancer cells that comprise the tumor. It is emphasized that a cancer stem cell is a tumor initiating cell. That conventional chemotherapy kills most cells in a tumor, but cancer stem cells remain intact is discussed. Vast applications of stem cells, cancer stem cells, mesenchymal stem cells, and human pluripotent stem cells are discussed. Because human embryonic stem cells possess the potential of producing unlimited quantities of any human cell type, considerable focus is placed on their therapeutic potential in this volume. Because of the pluripotency of embryonic stem cells, this volume discusses various applications such as tissue engineering, regenerative medicine, pharmacological and toxicological uses. The role of these cells in cell differentiation is also included. The role of cancer stem cells of breast, colon, and melanoma tumors in response to antitumor therapy is detailed. The role of cancer stem cells, specifically in the deadliest brain cancer, glioblastoma multiforme, is explained. Transplantation of bone marrow-derived stem cells for myocardial infarcation and use of mesenchymal stem cells in orthopedics are described.

Abstrak :
The difference among pluripotent stem cells, multipotent stem cells, and unipotent stem cells is pointed out. Vast therapeutic applications of the following specific stem cells in disease and tissue injury are discussed: human embryonic stem cells, human mesenchymal stem cells, germ cell-derived pluripotent stem cells, induced pluripotent stem cells, human umbilical cord blood-derived stem cells, breast tumor stem cells,and hematopoietic stem cells. Because of the potential of human embryonic stem cells to produce unlimited quantities of any human cell type, considerable focus is placed on their therapeutic potential. Because of their pluripotency, these cells have been used in various applications such as tissue engineering, regenerative medicine, pharmacological and toxicological studies, and fundamental studies of cell differentiation. The formation of embryoid bodies, which are three-dimensional aggregates of embryonic stem cells, is explained as this is the first step in cell differentiation. Such embryoid body culture has been widely used as a trigger for the in vitro differentiation of embryonic stem cells. The basic capacity of self-renewal of human embryogenic stem cells is explained. The role of TGF-beta in the propagation of human embryonic stem cells is discussed. The differentiation of human embryonic stem cells into neurons, hepatocytes, cardiomyocytes, and retinal cells is fully explained. Donor policies for hematopoietic stem cells are also explained.

Abstrak :
Breast cancer stem cells (BCSCs) dengan petanda Aldehida dehidrogenase 1-positif (ALDH1+) merupakan populasi minor dari sel-sel tumor dengan kemampuan tumorigenik yang tinggi dan bertahan terhadap stres oksidatif. Manganese superoksida dismutase (MnSOD) merupakan pertahanan utama terhadap superoksida yang diekspresikan spesifik di mitokondria, yang merupakan salah satu sumber utama stres oksidatif di dalam sel.  Sejauh ini, belum diketahui peranan MnSOD terhadap ketahanan hidup dan kepuncaan BCSC. Transfeksi in vitro pada BCSC (ALDH1+) dilakukan dengan menggunakan siRNA MnSOD spesifik dalam kondisi kultur standar. Total RNA dan protein diekstraksi dengan menggunakan TriPure® Isolation Reagent dan RIPA® lysis buffer. Viabilitas sel diukur dengan menggunakan trypan exclusion assay. Ekspresi relatif mRNA MnSOD dan OCT4 dianalisis dengan menggunakan one-step qRT-PCR. Aktivitas MnSOD diukur dengan menggunakan uji inhibisi xantin oksidase (RanSOD® kit). Kadar superoksida sel diukur dengan menggunakan uji dihidroetidium dan tumorigenik diukur dengan menggunakan mammosphere-forming unit. Setelah diinkubasi selama 48 jam dengan menggunakan siRNA dengan menggunakan dosis 80 pmol. Ekspresi relatif mRNA MnSOD mengalami penekanan sejumlah 0,17-kali (p<0,01), penurunan aktivitas spesifik MnSOD sebesar 70,4 %, peningkatan kadar superoksida sel menjadi 1,13-kali, penurunan ekspresi OCT4 menjadi 1,08-kali (p<0,05) dan penurunan mamosphere forming unit efficiency menjadi 36,5 % (p<0,05) dibandingkan dengan kontrol negatif. Viabilitas BCSC (ALDH1+) menurun sebanyak 75 %(p<-0,05) dibandingkan kontrol negatif. Hasil penelitian ini menunjukkan bahwa penekanan ekspresi MnSOD dapat menjadi target yang menjanjikan untuk menurunkan kepuncaan dan tumorigenitas BCSC (ALDH1+). ......Aldehyde dehydrogenase 1-positive (ALDH1+) breast cancer stem cells (BCSCs) are a small population of tumor cells with high capacity of tumorigenicity and oxidative stress. Manganese superoxide dismutase (MnSOD) is specifically expressed in mitochondria as the primary defense against superoxides, which are one of the causes of oxidative stress in cells. The aim of this study was to determine the impact of suppressing MnSOD expression using small interfering RNA (siRNA) on the stemness, tumorigenicity, and viability of BCSCs. In vitro transfection of ALDH1+ BCSCs was performed using 33 and 66 µM specific MnSOD siRNA under standard culture conditions. Total RNA and protein were extracted from the transfected cells using TriPure® Isolation Reagent and RIPA® lysis buffer. Cell viability was measured using a trypan blue exclusion assay. The relative expression of MnSOD and OCT4 mRNAs was analyzed by one step qRT-PCR. MnSOD activity was determined by xanthine oxidase inhibition assay (RanSOD® kit). Cellular superoxides were measured using a dihydroethidium assay and tumorigenicity was observed with mammosphere-forming unit.  After siRNA incubation for 48 hours, MnSOD was suppressed by 0.176-fold (p<0.01), MnSOD enzyme specific activity was reduced 70.4%, cellular superoxide levels increased by 1.13-fold, OCT4 expression was suppressed by 1.98-fold (p<0.05), and mammosphere-forming unit decreased by 36.5% (p<0.05) compared with the corresponding negative controls. The viability of the ALDH1+ BCSCs was reduced 75% (p< 0.05). Our results suggest that suppression of MnSOD expression may be a promising target to reduce stemness and tumorigenicity of ALDH1+ BCSCs.

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 :
ABSTRAK
Lingkungan mikro tumor berperan penting dalam meregulasi sifat kepuncaan, proliferasi, ketahanan terhadap apoptosis, dan metabolisme sel punca kanker. Penelitian ini bertujuan untuk menganalisis efek modulasi lingkungan ekstraseluler melalui kondisi hipoksia dan alkalinisasi pada metabolisme glukosa dan ketahanan hidup sel punca kanker CSC payudara manusia CD24-/CD44 . Pada penelitian in vitro eksperimental ini, CSC payudara manusia dikultur pada kondisi hipoksia dan kondisi alkali. Kultur sel diinkubasi selama 30 menit, 4, 6, 24, dan 48 jam pada suhu 37 C kemudian dilakukan analisis status metabolisme glukosa, regulasi pH, ketahanan hidup, dan penanda kepuncaan serta pluripotensi CSC payudara menggunakan berbagai teknik yaitu qRT-PCR, kolorimetri, fluorometri, dan aktivitas enzimatik. Kondisi hipoksia menyebabkan peningkatan ekspresi mRNA dan konsentrasi HIF1? sehingga mengaktivasi gen ndash;gen yang berada di bawah regulasinya. Hipoksia juga menyebabkan penekanan proliferasi namun meningkatkan ketahanan terhadap apoptosis. Alkalinisasi menyebabkan peningkatan pH ekstraseluler pHe yang menstimulasi peningkatan aktivitas dan ekspresi mRNA gen regulator pH seluler. Status metabolisme menunjukkan peningkatan aktivitas glikolisis anaerobik disertai peningkatan ekspresi transporter GLUT1. Alkalinisasi menyebabkan penekanan proliferasi CSC payudara bahkan kematian sel. Sebagai kesimpulan, modulasi lingkungan ekstraseluler baik melalui hipoksia maupun alkalinisasi dapat meningkatkan aktivitas glikolisis yang selanjutnya mempengaruhi ketahanan hidup dan kepuncaan CSC payudara CD24-/CD44 .Kata kunci: lingkungan mikro, hipoksia, alkalinisasi, glikolisis, sel punca kanker payudara.

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ABSTRACT
This study was aimed to analyze the effect of extracellular pH and O2 level modulation on glucose metabolism and survival of the human CD24 CD44 breast cancer stem cells BCSCs . The primary BCSCs CD24 CD44 cells were cultured under hypoxia 1 O2 or under supplementation of sodium bicarbonate 100 mM for various periods. After each incubation periods, the pH regulation, glucose metabolism, survival, stemness and pluripotency markers were analyzed using various techniques including qRT PCR, colorimetry, fluorometry, dan enzymatic reactions. This study demonstrated that hypoxia caused an increase of HIF1 mRNA expression and protein level, and shifted metabolic states to be more glycolytic. Hypoxia also promoted the suppression of cell proliferation and induced the apoptosis evasion. Alkalinization caused a high pHe then stimulated an increase of mRNA and activity of cellular pH regulator that lead to upregulation of anaerobic glycolysis. Alkalinization inhibited BCSCs proliferation and promoted apoptosis. To conclude, modulation of the extracellular environment of human BCSCs through hypoxic condition and alkalinization could shift the metabolic state toward the anaerobic glycolysis which in turn affected the proliferation and survival.Keywords Breast cancer stem cells, tumor hypoxia, extracellular pH, cell viability, stemness

Abstrak :
ABSTRAK
Latar Belakang: Trombositosis pada pasien kanker payudara KPD diduga berkontribusi pada penyebaran dan sifat invasi sel punca kanker payudara. Modifikasi lingkungan mikro tumor dapat dilakukan untuk meningkatkan efektivitas terapi anti kanker. Belum diketahui apakah platelet derived growth factor PDGF -AB dalam lisat trombosit LT juga berperan terhadap cancer stem cell CSC payudara CD24-/CD44 .Tujuan: Penelitian ini bertujuan untuk menganalisis efek LT dan PDGF-AB didalamnya sebagai lingkungan mikro tumor pada proliferasi dan sifat invasi sel punca kanker payudara CD24-/CD44 yang ditandai dengan kadar matrix metalloproteinase-9 MMP-9 dan epithelial-cadherin E-cadherin . Metode: Penelitian ini merupakan studi eksperimental pada kultur sel punca KPD yang diberi LT dari pasien KPD dan donor sehat. Darah semua donor dilakukan pemeriksaan hematologi dan diproses untuk mendapatkan platelet rich plasma PRP . Jumlah trombosit per ?L PRP setiap donor dihitung. PRP diproses untuk mendapatkan LT. Kadar PDGF-AB LT diukur. LT 0,01 ditambahkan ke dalam medium dulbecco rsquo;s modified eagle rsquo;s medium DMEM -F12 untuk kultur sel punca KPD. Setelah inkubasi 48 jam, total jumlah sel, population doubling time PDT dan viabilitas sel dihitung dan dinormalisasikan terhadap nilai kontrolnya. Ekspresi MMP-9 dan E-cadherin dipilih sebagai penanda biologi sifat invasi dan diukur dengan metode enzyme-linked immunosorbent assay ELISA . Jumlah total sel, PDT, viabilitas sel, kadar MMP-9 dan E-cadherin dibandingkan antara pasien KPD dan donor sehat lalu dianalisis korelasinya dengan jumlah trombosit dan kadar PDGF-AB dalam lisat trombosit. Hasil: Jumlah trombosit dan kadar PDGF-AB dalam LT pasien KPD lebih tinggi dibandingkan LT donor sehat, keduanya dengan nilai p=0,02. LT pasien KPD memicu proliferasi sel punca KPD lebih baik dibandingkan LT donor sehat p

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ABSTRACT
Background Thrombocytosis in breast cancer BC patient is supposed to play a role in the invasiveness of breast cancer stem cells. Modification of tumor microenvironment was proposed to increase the efficacy of anticancer therapy. Aim This study aimed to analyze the effect of platelet lysate PL as well as its platelet derived growth factor PDGF AB content as a tumor microenvironment on the CD24 CD44 breast cancer stem cell BCSC proliferation and invasiveness. Methods This experimental study treated BCSC culture with PL from BC patients or healthy donors. Venous blood from all donors were subjected to hematology test and processed to obtain PRP. Platelet counts in PRP were determined. PRP was processed to obtain PL. PDGF AB contents in PL were measured. PL 0.01 was supplemented into dulbecco rsquo s modified eagle rsquo s medium DMEM F12 medium and used for culturing the CD24 CD44 BCSCs . After 48 hours, total cell count, population doubling time PDT , and cell viability were calculated and normalized to its control. Matrix metalloproteinase 9 MMP 9 and E cadherin was used as biological marker for CSC invasiveness and measured by enzyme linked immunosorbent assay ELISA method. Total cell count, PDT, cells viability as well as MMP 9 and E cadherin levels between BCSC, healthy donor platelet lysate and control group were compared and their correlation with platelet count in PRP and PDGF AB levels in platelet lysates were analyzed. Results Platelet counts and PDGF AB levels were higher in BC patient PL compared to healthy donor group, both with a p value of 0.02. BC patient PL could stimulate the proliferation of BCSCs higher than healthy donor PL p