Hasil Pencarian  ::  Simpan CSV :: Kembali

Abstrak :
Latar Belakang : Kanker payudara merupakan salah satu kanker tersering pada wanita. Saat ini keberhasilan pengobatan terhadap kanker payudara masih rendah selain karena progesifitas tumor, tumor juga kadang bersifat resisten terhadap pengobatan, adanya metastasis dan meningkatnya agresivitas. Penelitian menunjukkan adanya peranan dari subset populasi sel punca kanker payudara (breast cancer stem cells, BCSC) yang menyokong pada sifat keganasan kanker ini. Selain Oct4 sebagai penanda kepuncaan sel, salah satu penanda yang dipakai dalam menyortir BCSC adalah aldehyde dehydrogenase (ALDH), dan dari 19 subfamili ALDH diteliti isoform ALDH1A1 dan ALDH1A3 yang dominan berperan pada BCSC. Selain itu adanya kondisi hipoksia turut mendukung menyebabkan kegasanan kanker payudara meningkat. Penelitian ini bertujuan untuk melihat pengaruh hipoksia pada ekspresi mRNA ALDH1A1 dan ALDH1A3 dan hubungannya dengan kepuncaan dan ketahanan hidup sel punca kanker payudara ALDH+. Metode: Sampel menggunakan kultur sel BCSC ALDH dan sel MCF-7. Dilakukan inkubasi hipoksia (O2 1) dan normoksia (O2 20) selama 6, 24 dan 48 jam kemudian dilakukan analisis ekspresi HIF1, ALDH1A1, ALDH1A3 dan Oct4. Selanjutnya dilakukan analisis viabilitas dan pengukuran mammosphere forming unit (MFU). Hasil : Studi menunjukkan kondisi hipoksia menyebabkan peningkatan ekspresi ALDH1A3 pada sel BCSC ALDH sedangkan ALDH1A1 mengalami penurunan ekspresi dibandingkan dengan sel normoksia, sementara pada sel MCF-7 terjadi hal sebaliknya. Ekspresi Oct4 mengalami peningkatan pada awal hipoksia (6 jam) kemudian menurun. Terjadi penurunan MFU, sedangkan ketahanan hidup sel tetap stabil. Kesimpulan : Kondisi hipoksia bisa menyebabkan penurunan sifat kepuncaan pada sel BCSC ALDH ditinjau dari penanda self-renewal, pluripotensi dan tumorigenitas, tetapi ketahanan hidup sel tetap stabil. ......Background: Breast cancer is one of frequent cancer-related disease among women. Complete successful therapy to breast cancer was still a big challenge considering the tumour progression, therapy resistancy, metastatic ability and increasing aggressivity. Studies shown that breast cancer stem cells (BCSC) subset were involved to maintain the malignancy. Besides the well-known cell stemness marker Oct4, researchers also suggested aldehyde dehydrogenase (ALDH) as a marker of BCSC, in which ALDH1A1 and ALDH1A3 believed to play dominant role. In addition, tumour hypoxia might also support increasing malignancy. This study aimed to analyze the expressions of ALDH1A1 and ALDH1A3 mRNA and their correlation with stemness properties and cell survival of hypoxic BCSC ALDH+. Method(s): Samples were obtained using BCSC ALDH and MCF-7 cells. The cells then being treated with hypoxia (O2 1) and normoxia (O2 20) conditions for 6, 24 and 48 hours respectively. We also measured the cells survival and mammosphere forming unit (MFU) capability to analyze cell proliferation. Result(s): Our study showed that ALDH1A3 in BCSC ALDH hypoxia cells was expressed at significantly higher level but ALDH1A1 was at lower level compared to their respective normoxia cells, while we found the opposite results in MCF-7 cells. Oct4 expression was increased at early hypoxia (6 hours) then declined shortly. MFU was reduced but cells survival remained stable. Conclusion(s): Hypoxic condition decreased the stemness properties of BCSC ALDH considering the self renewal, pluripotency, and tumorigenicity markers. However, cells survival remained stable.

Abstrak :
Tingginya rekurensi pada kanker kolorektal (KKR) disebabkan karena terapi saat ini belum mempertimbangkan keberadaan lingkungan mikrotumor dan kepuncaan. Kepuncaan adalah sifat dari sel punca kanker yang memiliki kemampuan self-renewal, pluripotensi, dan tumorigenic. Penelitian kami sebelumnya menunjukan bahwa sekretom fibroblas dari adenokarsinoma kolorektal meningkatkan ekspresi CD133 dan CD44 dalam sel Lestari HT-29. Namun peran sekretom fibroblas tersebut terhadap sifat kepuncaan masih perlu diteliti lebih lanjut. Penelitian ini bertujuan untuk mengetahui pengaruh sekretom fibroblas adenokarsinoma kolorektal area tumor dibandingkan area nontumor dan fibroblas normal terhadap sifat kepuncaan, MnSOD, dan STAT3 pada sel lestari HT-29. Pemberian Conditioned Medium (CM) yang mengandung sekretom fibroblas adenokarsinoma kolorektal area tumor (CM-T) dan nontumor pasangannya (CM-NT) pada kultur sel lestari HT-29 sudah dilakukan oleh peneliti sebelumnya. Fibroblas normal (NF) diisolasi dari jaringan preputium. Semua fibroblas ditanam dalam media kultur bebas serum selama 24 jam untuk mengumpulkan CM. Kemudian, CM ditambahkan ke kultur sel lestari HT-29 selama 72 jam. Ekspresi mRNA Oct4 dan ALDH1A1 dianalisis dengan qRT-PCR. Ekspresi protein STAT3, pSTAT3, SOD, dan Oct4 dianalisis dengan western blot. Pemberian CM-T meningkatkan ekspresi mRNA OCT4 dan ALDH1A1 signifikan dibandingkan dengan kontrol, CM-NT, dan CM-NF. Kami menyimpulkan bahwa pemberian sekretom fibroblas KKR meningkatkan ekspresi OCT4 dan ALDH1A1 secara signifikan dibandingkan dengan fibroblas non tumor pasangannya dan fibroblas normal

---

The high recurrence in colorectal cancer (CRC) is because current therapy has not considered the presence of a tumor microenvironment and stemness. Stemness is a characteristic of cancer stem cells that have properties like self-renewal, pluripotent and tumorigenic abilities. Our previous study has demonstrated that the secretomes of fibroblasts isolated from colorectal carcinoma (CRC) patients could upregulated the expression of CD133 and CD44 in the HT29 CRC cell line. However, the role of CRC fibroblasts secretomes in CRC stemness is needed to be further investigated. Therefore, the present study aimed to investigate the effect of fibroblast secretomes from CRC patients in comparison with the secretomes from normal fibroblasts on the expression of stemness markers, MnSOD, and STAT3 on HT-29 cells. The supplementation of Conditioned Medium (CM) from adenocarcinoma colorectal fibroblast (CM-T) and its nontumor partner (CM-NT) in HT-29 cell cultures has been done by our previous study. Normal fibroblasts (NF) were isolated from prepuce tissue. All fibroblasts were grown in free-serum culture medium for 24 hours to collect conditioned medium (CM). Then, CM was supplemented to HT-29 CRC cells for 72 hours. The effects of CM-T on the mRNA expression of OCT4 and ALDH1A1 were analysed using qRT-PCR. Supplementation of CM-T significantly increased OCT4 and ALDH1A1 mRNA expressions compared to that of CM-NT, CM-NF, and control. We conclude that secretomes from CRC patients upregulate the expression of CRC stemness.

Abstrak :
Kanker payudara adalah kanker dengan insidensi dan tingkat mortalitas tertinggi untuk wanita di Indonesia berdasarkan Cancer Country Profile oleh WHO pada 2014. Salah satu hipotesis terbaru dalam dunia onkologi adalah keberadaan sel punca kanker yang mendorong tumorigenesis, metastasis, therapy resistance dan remission pada kasus kanker ganas. Sel punca kanker dianggap hidup dalam kondisi hipoksia in vivo, sejalan dengan keadaan sel punca pada umumnya. Kondisi inilah yang mendorong sel punca kanker untuk memiliki karakteristik stemness yang menganugerahkan mereka kapasitas self-renewal dan pluripotency hingga akhirnya memperoleh ciri malignansi. Sebagai salah satu cara untuk mendalami sifat unik ini, kultur sel punca kanker payudara yang telah difraksinasi menggunakan marker CD44 /CD24-, diekspos terhadap hipoksia dengan interval berbeda. Perubahan ekspresi dari Oct4 sebagai core regulator dan ALDH1 sebagai modulator dari stemness akan diukur dan dibandingkan dalam kondisi hipoksia dan normoksia. ......Breast cancer has the highest incidence and mortality rate in Indonesian women based on WHO Cancer Country Profiles 2014. One of the emerging hypothesis in the oncology world is on cancer stem cells, which are responsible for the tumorigenesis, metastasis, therapy resistance and remission in many cancer types and cases. Similar to stem cells, cancer stem cells live around hypoxic surrounding in vivo and this condition granted the cancer stem cells pluripotency and self renewal capability, thus the characteristic of stemness and malignancy. Breast cancer has been shown to also contain cancer stem cells and so to study the unique trait under hypoxic condition, the cells, which have been fractionated by markers CD44 and CD24 , are subjected to hypoxia. The expression of Oct4 and ALDH1 as the core regulator and modulator of stemness respectively are assessed and further compared between hypoxic and normoxic groups.

Abstrak :
ABSTRAK
Saat ini terdapat banyak sekali studi yang sedang berjalan tentang sel punca pluripoten dan potensinya untuk menjadi terapi regeneratif pada masa yang akan datang. Sampai sekarang, sel punca embrionik adalah satu-satunya sumber untuk sel punca pluripoten yang telah dipelajari dengan baik. Akan tetapi, banyak sekali tantangan untuk memakai sel punca embrionik ini seperti masalah etik dan penolakan sistem imun tubuh. Belakangan ini, banyak sekali studi yang sedang berjalan dalam menginvestigasi cara baru dalam pemograman ulang reprogramming sel manusia untuk menjadi induced pluripotent stem cell iPSC . OCT4 Octamer-binding faktor transkripsi 4 adalah salah satu protein krusial yang bertanggung jawab atas pembaharuan self-renewal sel punca embrionik. Maka dari itu, studi ini bertujuan untuk menginvestigasi kemampuan OCT4, dengan bantuan VP22 viral protein , untuk dilokalisasi ke dalam sel manusia dewasa HepG2 , yang akhirnya dapat membuat pemograman ulang sel HepG2 menjadi iPSC. Pada penelitian ini, sel HepG2 diinkubasi dengan protein fusi rekombinan VP22-OCT4 selama 1 jam dan 6 jam. Kemudian, proses indirect immunofluorescence staining dilaksanakan yang mencakup inkubasi dengan mouse monoklonal IgG antibodi primer terhadap OCT4 Ab1 dan dilanjutkan dengan goat anti-mouse IgG antibodi sekunder, FITC konjugat Ab2 . Pengamatan dengan mikroskop confocal dilaksanakan untuk melihat adanya imunofluoresen. Pada hasil yang diperoleh, terdapat perbedaan yang bermakna antara kelompok kontrol dan kelompok eksperimen untuk kedua periode inkubasi p=0.005 untuk 1 jam dan p=0,000 untuk 6 jam , serta perbedaan bermakna juga terjadi antara kelompok sel HepG2 dengan VP22-OCT4 diinkubasi selama 1 jam dan 6 jam p=0,044 . Untuk menyimpulkan hasil tersebut, protein rekombinan VP22-OCT4 dapat dilokalisasi ke dalam sel HepG2 dalam waktu inkubasi selama 1 jam dan 6 jam.

---

ABSTRACT
There are many studies ongoing about the remarkable potential of pluripotent stem cell as the future regenerative therapy. Embryonic stem cell is the only source that has been studied well for it. However, many constraints arise regarding this matter like ethical problems and risk of cell transplantation rejection. Recently, there are many research undergone in exploring new approach through reprogramming somatic cell to become induced pluripotent stem cell iPSC . OCT4 Octamer binding transcription factor 4 is one of the proteins that is responsible for the self renewal of embryonic stem cell. Therefore, this study aimed to investigate the ability of OCT4 transcription factor, with the help of VP22 viral protein , to be localized into adult somatic cells HepG2 , which in turn could reprogram it into iPSC. In this study, HepG2 cells are isolated with VP22 OCT4 recombinant fusion protein for 1 hour and 6 hours, which then followed by isolating with mouse monoclonal IgG primary antibody against OCT4 Ab1 and goat anti mouse IgG secondary antibody, FITC conjugate Ab2 respectively for the indirect immunofluorescence staining function. Confocal microscope is utilized to observe the presence of immunofluorescence. The result of the experiment showed a significant difference between the control and the experimental groups for both incubation period p 0.005 for 1 hour and p 0.000 for 6 hours . Moreover, there is also a significance difference between the group of HepG2 cells with VP22 OCT4 incubated for 1 hour and 6 hours p 0.044 . In conclusion, VP22 OCT4 recombinant protein can be translocated inside the HepG2 cells under 1 hour and 6 hours of incubation periods.

Abstrak :
LatarBelakang: Keberadaan sel Oct4 pada suatu struktur merupakan indikasi adanya sel dengan kapasitas pluripoten, disebut juga sel punca pluripoten yang sedang gencar diteliti karena manfaatnya. Dalam proses pengembangan sel punca pluripoten, masih ditemukan hambatan yaitu pengisolasian sumber sel dari embryo yang dianggap kontroversial. Dengan karakteristik kulit dan pandangan sebagai material yang terbuang paska sirkumsisi, kulit prepusium diduga memiliki sel punca pada lingkungan histologinya dan dirasa bisa menjadi sumber baru yang tidak kontroversial untuk sel punca pluripoten. Metode: Sampel diambil dari sirkumsisi massal yang kemudian diolah dengan proses histoteknik dan dilakukan proses staining oleh Hematoxylin-Eosin staining dan Immunohistochemistry Oct4 staining di Laboratorium Histologi Fakultas Kedokteran Universitas Indonesia. Slide sampel yang diperoleh kemudian dilakukan mikrofoto menggunakan optilab dan dianalisa. Hasil: Sel Oct4 ditemukan pada kulit prepusium. Namun sel tersebut hanya ditemukan di beberapa lokasi, yaitu kelenjar sebasea, folikel rambut, pembuluh darah, dan lapisan hipodermis dari kulit. Kesimpulan: 80 dari sampel kulit prepusium yang diambil memiliki sel Oct4 dan sel hanya di temukan pada 4 lokasi spesifik kelenjar sebasea, hair follicle, pembuluh darah dan lapisan hipodermis.

---

Background The presence of Oct4 cell in a structure indicates the presence of pluripotent cell, which popular nowadays being on researched due to its benefit. In the development of the cell, an obstacle is found such as the isolation process of the cell rsquo s source, embryonic stem cell, is considered as controversial. With its skin characteristic and views as discarded material, preputial skin expected to possessed stem cell in its histological environment and has the potential to be new source of pluripotent stem cell that is not controversial. Method Sample was taken from mass circumcision event, which then undergo histotechnique process involved the staining with Hematoxylin Eosin Staining and Immunohistochemistry Oct4 staining in Histology Laboratory, Fakultas Kedokteran Universitas Indonesia. Sample slide that obtain then being analyzed by microphoto of the slide under the microscope. Results Oct4 cells are found in the preputial skin. However, these cell only found limited in several locations such as sebaceous gland, hair follicle, blood vessel, and hypodermis layer of the skin. Conclusion 80 of the preputial skin sample possessed the Oct4 cells and these cells are only found in 4 specific locations sebaceous gland, hair follicle, blood vessel, and hypodermis layer.

Abstrak :
[ABSTRAK
Glioma adalah tumor otak primer yang sampai saat ini sering timbul resistensi terapi. Sel punca glioma diduga berperan penting dalam resistensi dan rekurensi sel tumor. Sel punca glioma memiliki penanda permukaan CD133 dan mampu berpluripotensi dengan mengekspresikan Oct4. Kondisi hipoksia tumor juga berperan dalam self renewal sel punca glioma. Tujuan dari penelitian ini adalah untuk mengetahui hubungan keberadaan sel punca glioma dengan keganasan, pluripotensi dan kondisi hipoksia. Cross sectional digunakan sebagai desain penelitian dengan jumlah sampel sebanyak 35 jaringan, terdiri atas 15 glioma derajat keganasan tinggi dan 20 glioma derajat keganasan rendah. Pengukuran ekspresi relatif mRNA CD133, Oct4 dan HIF-1α menggunakan metode qRTPCR. Protein HIF-1α dilihat ekspresinya melalui teknik imunohistokimia. Ekspresi relatif mRNA CD133 dan Oct4 lebih tinggi bermakna (p < 0.05, Mann- Whitney) pada glioma derajat keganasan tinggi dibanding glioma derajat keganasan rendah. Protein HIF-1α lebih tinggi bermakna (p < 0,01, Mann- Whitney) pada glioma derajat keganasan tinggi dibanding glioma derajat keganasan rendah. Terdapat hubungan ekspresi sel punca glioma CD133 dengan pluripotensi serta kondisi hipoksia (r = 0,518, r = 0,339; Spearman?s rho) serta pluripotensi dengan kondisi hipoksia pada derajat keganasan tinggi (r = 0,749; Spearman?s rho). Ekspresi relatif mRNA CD133, Oct4 dan HIF-1α meningkat seiring dengan peningkatan derajat keganasan. Terdapat hubungan yang bermakna antara keberadaan penanda sel punca glioma CD133 dengan pluripotensi dan kondisi hipoksia pada glioma derajat keganasan tinggi.

---

ABSTRACT
Glioma is primary brain tumor with frequent therapeutic resistance. Glioma cancer stem cells were considered to play a role in resistance and recurrence of tumor cells. Glioma cancer stem cells expressed CD133 on their surface and capable of pluripotency as expressed by Oct4 positive. Tumor hypoxic condition also play a role in glioma cancer stem cells self renewal. Aim of this study is to investigate correlation between glioma cancer stem cells, degree of malignancy, pluripotency and hypoxia. Design of this study is cross sectional with 35 glioma samples comprises of 20 low grade malignant glioma and 15 high grade malignant glioma. Expression of mRNA CD133, Oct4 and HIF-1α were measured using qRT-PCR. HIF-1α protein expression was detected by immunohistochemistry from glioma sample. mRNA CD133 and Oct4 expression significantly higher (p < 0.05, Mann-Whitney) in high grade malignant glioma compared to low grade malignant glioma. HIF-1α tissue expression significantly higher (p < 0,01, Mann- Whitney) in high grade malignant glioma compared to low grade malignant glioma. There was correlation between expression of CD133 glioma cancer stem cells marker with pluripotency and hypoxia (r = 0,518, r = 0,543; Spearman?s rho) and pluripotency with hypoxia in high grade malignant glioma (r = 0,749; Spearman?s rho). mRNA CD133, Oct4 and HIF-1α expression increased with high grade malignant glioma. There was significant correlation between CD133 glioma cancer stem cell marker with pluripotency and hypoxia in high grade malignant glioma, Glioma is primary brain tumor with frequent therapeutic resistance. Glioma cancer stem cells were considered to play a role in resistance and recurrence of tumor cells. Glioma cancer stem cells expressed CD133 on their surface and capable of pluripotency as expressed by Oct4 positive. Tumor hypoxic condition also play a role in glioma cancer stem cells self renewal. Aim of this study is to investigate correlation between glioma cancer stem cells, degree of malignancy, pluripotency and hypoxia. Design of this study is cross sectional with 35 glioma samples comprises of 20 low grade malignant glioma and 15 high grade malignant glioma. Expression of mRNA CD133, Oct4 and HIF-1α were measured using qRT-PCR. HIF-1α protein expression was detected by immunohistochemistry from glioma sample. mRNA CD133 and Oct4 expression significantly higher (p < 0.05, Mann-Whitney) in high grade malignant glioma compared to low grade malignant glioma. HIF-1α tissue expression significantly higher (p < 0,01, Mann- Whitney) in high grade malignant glioma compared to low grade malignant glioma. There was correlation between expression of CD133 glioma cancer stem cells marker with pluripotency and hypoxia (r = 0,518, r = 0,543; Spearman’s rho) and pluripotency with hypoxia in high grade malignant glioma (r = 0,749; Spearman’s rho). mRNA CD133, Oct4 and HIF-1α expression increased with high grade malignant glioma. There was significant correlation between CD133 glioma cancer stem cell marker with pluripotency and hypoxia in high grade malignant glioma]

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: Rekayasa jaringan tulang memerlukan tiga komponen utama, yaitu sel punca, scaffold, dan faktor pertumbuhan. IGF-1 merupakan salah satu faktor pertumbuhan yang berperan dalam proliferasi dan diferensiasi sel osteoblast. IGF-1 akan berikatan dengan reseptornya, yaitu IGF-1R untuk mengaktivasi jalur hilir. Dalam sirkulasi tubuh manusia, IGF berikatan dengan IGFBP-3 yang dapat memperpanjang waktu paruh serta menghambat IGF-1 berikatan dengan IGF-1R. Pada penelitian sebelumnya, tercatat bahwa tidak ada perbedaan kemampuan proliferasi dan diferensiasi antara DPSC subjek normal dan subjek CLP, namun ada perbedaan signifikan dalam jumlah ekspresi IGF-1. OCT-4, SOX-2 dan NANOG merupakan faktor transkripsi utama pluripotensi yang telah diteliti dapat mengatur pluripotensi, pembaruan diri, proliferasi, serta diferensiasi DPSC. Penelitian terbaru mencatat peningkatan ekspresi ketiga gen tersebut pasca dilakukan penghambatan jalur GSK-3 dan m-TOR yang merupakan jalur hilir dari aksi IGF-1 pada sel DPSC. Namun, belum diketahui secara pasti ekspresi ketiga gen tersebut pada DPSC subjek normal dan CLP setelah dilakukannya penghambatan IGF-1 menggunakan anti IGF-1R dan IGFBP-3. Tujuan: Menganalisis pengaruh anti IGF-1 dan IGFBP-3 terhadap ekspresi gen OCT4, SOX2, dan NANOG pada DPSC subjek normal dan CLP. Metode: Sampel RNA DPSC subjek normal (n=4) dan DPSC subjek CLP (n=3), sebelum dan setelah diberikan perlakuan anti IGF-1R atau IGFBP-3, diperoleh dari bahan biologis tersimpan di Laboratorium Oral Biologi Fakultas Kedokteran Gigi Universitas Indonesia. Selanjutnya, ekspresi gen OCT4, SOX2, NANOG, dan housekeeping gene GAPDH diuji dengan two step Real-Time PCR (RT-PCR). Hasil: Tidak terdapat perbedaan ekspresi gen OCT4, SOX2, dan NANOG, baik antara DPSC subjek normal dan CLP sebelum dan setelah diberikan perlakuan anti IGF-1R dan IGFBP-3 (p³0,05). Kesimpulan: Perlakuan anti IGF-1R dan IGFBP-3 tidak memengaruhi tingkat ekspresi gen OCT4, SOX2, dan NANOG sel punca pulpa gigi permanen subjek normal dan subjek celah bibir dan palatum ......Background: Bone tissue engineering requires three main components, namely stem cells, scaffold, and growth factors. IGF-1 is a growth factor that plays role in osteoblast proliferation and differentiation. IGF-1 will bind to its receptor, namely IGF-1R, to activate the downstream pathway. In the human body circulation, IGF binds to IGFBP-3 which can inhibit IGF-1 from binding to IGF-1R. Previous studies noted that there were no differences in the ability to proliferate and differentiate between DPSC from normal subjects and CLP subjects, yet there were significant differences in the level of IGF-1 expression. OCT-4, SOX-2 and NANOG are core pluripotency factors which regulate pluripotency, self-renewal, proliferation and differentiation of DPSC. Recent study has noted an increase in the expression of these three genes after inhibition of GSK-3 and m-TOR pathways, which are the downstream pathways of IGF-1 on DPSC cells. However, the expression of these three genes in DPSC from normal and CLP subjects after inhibition of IGF-1 using anti IGF-1R and IGFBP-3 is still unknown. Objective: To analyze the effect of anti IGF-1 and IGFBP-3 on OCT4, SOX2, and NANOG gene expression in DPSC of normal and CLP subjects. Methods: RNA samples of DPSC from normal and CLP subjects, before and after being treated with anti-IGF-1R or IGFBP-3, were obtained from Laboratory of Oral Biology, Faculty of Dentistry, Universitas Indonesia. Furthermore, the expression of OCT4, SOX2, NANOG, and housekeeping gene GAPDH were tested using two step Real-Time PCR (RT-PCR). Results: There was no difference between the expression of the OCT4, SOX2, and NANOG in DPSC from normal and CLP subjects before and after anti IGF-1R and IGFBP-3 treatment (p≥0.05). Conclusion: Anti-IGF-1R and IGFBP-3 did not affect the expression level of OCT4, SOX2, and NANOG in dental pulp stem cells of normal subjects and cleft lip and palate subjects.

Abstrak :
Pendahuluan: Kanker payudara triple-negatif merupakan subset keganasan yang menantang terkait dengan prognosis buruk akibat rendahnya ekspresi HER2 dan reseptor hormon, yang mengakibatkan kurangnya modalitas terapi yang ditargetkan secara spesifik. Selain itu, bagian dari keganasan ini mempunyai tingkat kekambuhan dini dan metastasis yang tinggi. Manganese superoxide dismutase (MnSOD), suatu enzim yang sangat penting untuk keseimbangan redoks, terlibat dalam tumorigenesis karena peran gandanya. Awalnya, MnSOD bertindak sebagai penekan tumor selama tumorigenesis awal, namun perannya bergeser seiring perkembangan penyakit. Kanker payudara triple-negatif diperkaya dengan subpopulasi sel dengan potensi tumorigenik tinggi dan stres oksidatif yang dikenal sebagai sel punca kanker. Sel-sel ini mengekspresikan faktor transkripsi Oct4 dan Sox2, yang mengatur pembaruan diri dan kepuncaan. Studi ini menyelidiki efek penekanan ekspresi MnSOD melalui KO gen CRISPR/Cas9 pada sel punca kanker BCSC triple-negatif dengan mengukur ekspresi mRNA OCT4 dan SOX2. Metode: Penelitian ini menggunakan kelompok kontrol BCSC tripel-negatif BT-549 tipe wild-type dan dua kelompok BT-549 KO MnSOD yang diberi perlakuan. Untuk setiap kelompok, tiga sampel ulangan digunakan. Ekspresi mRNA OCT4 dan SOX2 di setiap kelompok kontrol dan kelompok perlakuan dideteksi oleh RT-qPCR. Hasil masing-masing dihitung dengan Metode Livak untuk mengekstraksi rasio ekspresi. Uji T dua sampel dilakukan untuk menghitung signifikansinya. Hasil: Temuan ini menunjukkan tren penurunan ekspresi SOX2 dan ekspresi mRNA OCT4 yang tidak konsisten. Kesimpulan: Penekanan MnSOD dapat menjadi target potensial untuk mengubah sifat pluripotensi BCSC triple-negatif dengan memberikan efek tidak langsung pada ekspresi mRNA OCT4 dan SOX2. ......Introduction: Triple-negative breast cancer represents a challenging subset of malignancy associated with poor prognosis due to low expression of HER2 and hormone receptors, resulting in the lack of specific targeted therapeutic modalities. Additionally, this subset of malignancy acquires a high rate of early recurrence and metastasis. Manganese superoxide dismutase (MnSOD), an enzyme paramount for redox balance, is implicated in tumorigenesis by its dual role. Initially, MnSOD acts as a tumor suppressor during early tumorigenesis, but its role shifts as the disease advances. Triple-negative breast cancer is enriched with a subpopulation of cells with high tumorigenic potential and oxidative stress known as cancer stem cells (CSC). These cells expressed Oct4 and Sox2 transcription factors, which regulate self-renewal and stemness. This study investigates the effect of suppressing MnSOD expression through CRISPR/Cas9 gene knockout on the stemness of triple-negative BCSCs by measuring OCT4 and SOX2 mRNA expression. Methods: This study utilized a control group of wild-type BT-549 triple-negative BCSCs and two treated groups of MnSOD-knockout BT549 BCSCs. For each group, three replicate samples were used. The mRNA expression of OCT4 and SOX2 in each control and treated group was detected by RT-qPCR. Their respective results were calculated by the Livak Method to extract the expression ratio. A two-sample T-test was performed to calculate the significance. Results: The findings demonstrate a decreasing trend of SOX2 expression and an inconsistent OCT4 mRNA expression. Conclusion: MnSOD suppression may present as a potential target for altering the pluripotency properties of triple-negative BCSCs by exerting an indirect effect on OCT4 and SOX2 mRNA expression.

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.