Disertasi

Efek modulasi lingkungan ekstraseluler melalui hipoksia dan alkalinisasi pada metabolisme glukosa dan ketahanan hidup sel punca kanker payudara manusia. = The effects of extracellular modulation through hypoxia and alkalinization on the human breast cancer stem cell’s glucose metabolism and survival.

Latar belakang: Sel punca kanker (CSC) merupakan subpopulasi minor di dalam tumor padat, termasuk kanker payudara, yang bersifat tumorigenik dan bertanggungjawab pada progresivitas tumor, metastasis, rekurensi, dan resistensi terapi. 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 CSC payudara manusia CD24-/CD44+. Metode: Penelitian eksperimental ini merupakan penelitian in vitro menggunakan teknik kultur sel sebagai metode dasar penelitian. Kondisi hipoksia dibuat dengan cara menurunkan konsentrasi O2 melalui peningkatan konsentrasi gas N2 sehingga komposisi gas di dalam inkubator menjadi O2 1%; CO2 5%; dan N2 94%. Alkalinisasi dilakukan dengan cara menambahkan agen pengalkali sodium bikarbonat (NaHCO3) pada medium kultur. Sel kemudian diinkubasi selama 30 menit, 4 , 6, 24, dan 48 jam dengan suhu 37°C pada masing-masing kondisi ekstraseluler tersebut di atas. Khusus untuk alkalinisasi, dilakukan alkalinisasi ulangan setelah 24 jam alkalinisasi awal kemudian inkubasi dilanjutkan dengan durasi waktu yang sama. Setelah masing- masing waktu inkubasi tercapai dilakukan analisis status metabolisme glukosa, regulasi pH, ketahanan hidup, dan penanda kepuncaan serta pluripotensi CSC payudara CD24-/CD44+ menggunakan berbagai teknik berupa qRT-PCR, kolorimetri, fluorometri, dan reaksi enzimatik. Hasil: Kondisi hipoksia menyebabkan peningkatan ekspresi mRNA dan konsentrasi HIF1α sehingga mengaktivasi gen–gen yang berada di bawah regulasinya yaitu CA9 dan GLUT1. Konsekuensi peningkatan regulasi HIF1α berupa peningkatan status metabolisme glikolisis anaerobik. Hipoksia juga menyebabkan penekanan proliferasi namun meningkatkan ketahanan terhadap apoptosis. Alkalinisasi menyebabkan peningkatan pH ekstraseluler (pHe) yang menstimulasi peningkatan ekspresi mRNA gen regulator pH seluler, CA9 dan aktivitas anhidrase karbonat ekstraseluler (CAe). Status metabolisme menunjukkan peningkatan aktivitas glikolisis yang ditandai dengan peningkatan konsumsi glukosa disertai peningkatan ekspresi transporter GLUT1, peningkatan produksi laktat, dan aktivitas laktat dehidrogenase serta penurunan konsumsi oksigen. Regulasi ini tampaknya melibatkan peran HIF1α. Alkalinisasi terlihat menyebabkan penekanan proliferasi CSC payudara bahkan kematian sel. Gen penanda kepuncaan dan pluripotensi CSC payudara memiliki pola ekspresi mRNA yang berbeda–beda setelah alkalinisasi namun cenderung menurun jika lingkungan ekstraseluler dipertahankan pada kondisi alkali. 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.


Background: Cancer stem cells (CSCs) are considered as a minor subpopulation of cells within a solid tumor, including breast cancer, that they have been proved to be tumorigenic and responsible for tumor progression, metastasis, recurrence, and therapy resistance. The microenvironment is believed to play a key role in regulating stemness, proliferation, survival, and metabolism of CSCs. The aim of this study is to analyze the effects of extracellular modulation through hypoxic condition and alkalinization toward glucose metabolism and survival of human CD24-/CD44+ breast cancer stem cells (BCSCs). Methods: In this in vitro experimental study, the hypoxic condition was achieved by setting the gas composition of the incubator to 1% O2; 5% CO2; and 94% N2. While alkalinization was created through sodium bicarbonate (NaHCO3) supplementation into the BCSCs culture medium. The cells then were incubated 30 minutes; 4, 6, 24, and 48 hours within this two conditions. For alkalinization, re-alkalinization was conducted after 24 hours from the first alkalinization then incubated further for the next 24 hours. The purpose of re-alkalinization was to maintain a high pHe of the culture. 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. Results: Hypoxia caused an increase of HIF1α mRNA expression and protein level. Under the regulation of this hypoxic transcription factor, BCSCs metabolic states shifted to be more glycolytic and created a low pH of the extracellular environment. Hypoxia also promoted the suppression of cell proliferation and induced the evasion to apoptosis. Alkalinization caused a high pHe then stimulated an increase of mRNA and the activity of cellular pH regulator, CA9. Alkalinization seemed to inhibit BCSCs proliferation and promoted cell death when the high pHe was maintained through re-alkalinization. Metabolic states were upregulated to anaerobic glycolysis that was demonstrated by low oxygen consumption, high glucose consumption along with high GLUT1 mRNA expression, high lactate production, and high lactate dehydrogenase (LDH) activity. Stemness and pluripotency markers had a different pattern at mRNA expression level in response to the alkalinization but seemed to decrease when the alkaline condition was maintained. Conclusion: Modulation of extracellular environment of human CD24-/CD44+ BCSCs through hypoxic condition and alkalinization could shift the metabolic state toward the anaerobic glycolysis which in turn affected the proliferation and survival.
Keywords: tumor microenvironment, hypoxia, alkalinization, metabolic states, breast cancer stem cells.