Sekar Arumsari1, Dimas Ramadhian Noor2, Syarifah Dewi3, Resda Akhra Syahrani1, Septelia Inawati Wanandi4
1 Molecular Biology and Proteomics Core Facilities, Indonesian Medical Education and Research Institute, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia.
2Molecular Biology and Proteomics Core Facilities, Indonesian Medical Education and Research Institute, Faculty of Medicine, Universitas Indonesia; Departments of Master Program in Biomedical Sciences, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia.
3Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia.
4Molecular Biology and Proteomics Core Facilities, Indonesian Medical Education and Research Institute; Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia.
DOI: 10.4103/jnsbm.JNSBM_113_19
ABSTRACT
Introduction: 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. Materials and Methods: 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 using one-step quantitative reverse transcription polymerase chain reaction. 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. Results: After siRNA incubation for 48 h, 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). Conclusion: Our results suggest that suppression of MnSOD expression may be a promising target to reduce stemness and tumorigenicity of ALDH1+ BCSCs.
Keywords: Breast cancer stem cells, enzyme activity, manganese superoxide dismutase, OCT4, superoxides.
