Table of Contents    
ORIGINAL ARTICLE
Year : 2019  |  Volume : 10  |  Issue : 3  |  Page : 82-87  

In vitro transfection of manganese superoxide dismutase small interfering rna suppresses stemness of human breast cancer stem cells (aldehyde dehydrogenase 1-positive): Focus on OCT4 mRNA expression and mammosphere-forming capacity


1 Molecular Biology and Proteomics Core Facilities, Indonesian Medical Education and Research Institute, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
2 Molecular 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
3 Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
4 Molecular Biology and Proteomics Core Facilities, Indonesian Medical Education and Research Institute; Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

Date of Web Publication14-Jan-2020

Correspondence Address:
Septelia Inawati Wanandi
Department of Biochemistry and Molecular Biology, Faculty of Medicine, Universitas Indonesia, Jalan Salemba Raya No 6, Jakarta 10430
Indonesia
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jnsbm.JNSBM_113_19

Rights and Permissions
   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


How to cite this article:
Arumsari S, Noor DR, Dewi S, Syahrani RA, Wanandi SI. In vitro transfection of manganese superoxide dismutase small interfering rna suppresses stemness of human breast cancer stem cells (aldehyde dehydrogenase 1-positive): Focus on OCT4 mRNA expression and mammosphere-forming capacity. J Nat Sc Biol Med 2019;10, Suppl S1:82-7

How to cite this URL:
Arumsari S, Noor DR, Dewi S, Syahrani RA, Wanandi SI. In vitro transfection of manganese superoxide dismutase small interfering rna suppresses stemness of human breast cancer stem cells (aldehyde dehydrogenase 1-positive): Focus on OCT4 mRNA expression and mammosphere-forming capacity. J Nat Sc Biol Med [serial online] 2019 [cited 2020 Jan 23];10, Suppl S1:82-7. Available from: http://www.jnsbm.org/text.asp?2019/10/3/82/275574




   Introduction Top


Cancer stem cells are found within tumors and have characteristics of both cancer cells and stem cells, i.e., self-renewal, tumorigenic and pluripotent which is evident by the expression of stemness genes such as OCT4 and ALDH1.[1],[2] Cancer stem cells found in almost every type of cancer, including breast cancers, have been suggested to be responsible for anti-cancer resistance, disease recurrence, and metastasis.[3] One of the most studied breast cancer stem cells (BCSCs) markers are CD44+/CD24− and aldehyde dehydrogenase 1-positive (ALDH1+) cells. Li et al. reported that each marker exhibit distinct mechanism in the progression of breast tumorigenesis.[3] In BCSCs, CD44+/CD24− marker may be essential for cells proliferation and tumorigenicity, whereas ALDH1+ marker may involve in cell metastasis and associate with poor patients' survival rates.

Manganese superoxide dismutase (MnSOD) encodes an endogenous antioxidant enzyme that belongs to a family of superoxide dismutases (EC 1.15.1.1). MnSOD plays vital roles in the dismutation process by binding one manganese ion per subunit to convert superoxides, the most reactive oxygen species (ROS) found in the cell, into hydrogen peroxides or molecular oxygen in the mitochondrial matrix.[3] Superoxides play important roles in maintaining the redox status of the cell that controls the cell fates such as proliferation and differentiation, through the expression of redox-sensitive proteins and the activation of redox signaling pathways.[4] The imbalance between cellular antioxidant and ROS levels induces oxidative stress leading to oxidative damages of cellular macromolecules such as lipid, protein, and DNA.[5]

It is reported that MnSOD has two opposing roles in tumorigenesis.[6] In the early tumorigenesis, MnSOD antioxidant acts as a tumor suppressor by suppressing the oxidative stress, whereas, during tumor progression, MnSOD acts as an oncogene through its pro-oxidant activity to induce cell proliferation and metastasis. Our previous study had demonstrated that MnSOD expression was increased when the BSCSs were exposed to rotenone-induced oxidative stress; however, no significant change in cellular superoxides level and viability were found compared to the non-BCSCs.[7] Based on those findings, we address the research question whether MnSOD directly affects the viability and stemness of BCSCs and could further be targeted for the development of anti-cancer against BCSCs. Therefore, this study aimed to analyze the effect of MnSOD suppression using small interfering RNA (siRNA) on the stemness and viability of ALDH1+ BCSCs.


   Materials and Methods Top


Cell culture

ALDH1+ BCSCs were cultured in T25 flasks in serum-free Dulbecco's Modified Eagle Medium/Ham's F12 (DMEM/F12) (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 1% Penicillin–streptomycin (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% Amphotericin B (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cells were subcultured whenever 80% confluence was reached. The standard conditions for cell culture were 5% CO2 and 20% O2 at 37°C.[8]

Transient in vitro manganese superoxide dismutase small interfering RNA transfection

ALDH1+ BCSCs were transfected with MnSOD siRNA (Ambion, Thermo Fisher Scientific, USA), 5′-GGAACAACAGGCCUUAUUCTT-3′ (sense) and 5′-GAAUAAGGCCUGUUGUUCCTT-3′ (antisense), according to the manufacturer's protocols. Starting concentrations of 33 μM and 66 μM were used at two independent time points 48 h and 72 h at 37°C in 5% CO2.[9] Negative control siRNA number 1 (Ambion, Thermo Fisher Scientific, USA), that have a random sequence to target gene product, was used as a control. The MnSOD siRNA and Lipofectamine ® 2000 (Invitrogen, USA) were mixed in OptiMEM I (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) as the transfection medium and the assays were conducted in 12-well culture plates. The transfection medium was supplemented after 6 h and changed into the standard cell culture medium for BCSCs.

Determination of cell viability

The ALDH1+ BCSCs were harvested after 48 h treatment. Cell viability was determined using trypan blue exclusion assay (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA) as described previously.[8] Then, the cells were counted using a Luna™ automated cell counter (Logos Biosystems, South Korea).

Measurement of cellular superoxides level

The intracellular superoxide level was measured using a superoxide sensitive probe, a dihydroethidium (DHE) probe (Molecular Probes Inc., Eugene) as described previously.[9] Briefly, approximately 20,000 cells were resuspended by washing twice with phosphate buffer saline (PBS). DHE was mixed into the PBS solution to a final concentration of 20 μM and the cells were incubated for 30 min at 37°C in the darkroom. Immediately after incubation, the fluorescence intensity was measured using a fluorometer (Varioskan™ Flash Multimode Reader; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The fluorescence intensity was measured in a 96-well plate with excitation at 485 nm and emission at 530 nm. Superoxides levels were determined as a ratio of absorbance of the treatment group to that of mock cells.

Determination of relative expression of manganese superoxide dismutase and OCT4 mRNAs

Total RNA was extracted from the ALDH1+ BCSCs using a TriPure ® Isolation Reagent (Roche Diagnostic, Basal, Switzerland) and suspended in DEPC-treated water. The concentration and purity of the extracted RNA samples were determined using spectrophotometry with a wavelength of 260 nm (Varioskan™ Flash Multimode Reader; Thermo Fisher Scientific, Inc. Waltham, MA, USA). The relative expressions of the RNAs were measured using SensiFAST™ SYBR ® No-ROX One-Step Kit (Bioline, USA). The MnSOD primers were 5′-GCACTAGCAGCATGTTGAGC-3′ (forward) and 5′-ACCTTCTCCTCGGTGACGTTC-3′ (reverse),[9] and the OCT4 primers were 5′-AGGTGTTCAGCCAAACGACC-3′ (forward) and 5′-TGATCGTTTGCCCTTCTGGC-3′ (reverse).[8] Quantitative reverse transcription polymerase chain reaction was performed using Applied Biosystem ® 7500 Fast. The results were then analyzed using the Livak method's [10] and normalized to the mock cells.

Manganese superoxide dismutase activity assay

The ALDH1+ BCSCs cells were extracted using RIPA ® lysis buffer (Abcam, UK) according to the manufacturer's protocols, and the protein concentrations were determined using Bradford assay. MnSOD activity was measured using RanSOD ® (Randox Laboratories Ltd., Crumlin, UK) according to the manufacturer's protocols, as described previously.[9],[11] The protocols were modified by incubating cells in lysis buffer with 5 mM NaCN as a competitive inhibitor of Cu-Zn-SOD for 5 min.[12] The MnSOD activity is represented in units per mg of total protein.

Mammosphere-forming unit assay

ALDH1+ BCSCs after MnSOD-siRNA transfection were seeded at ultra-low attachment 96-well plates (Corning Inc., New York, USA). Each well contained 100 cells and grown in DMEM-F12 with total volume medium 100 μl/well at 37°C in an atmosphere containing 5% CO2. Formation mammospheres from the cells were determined after 3 days under an inverted microscope at ×100 (model No. IM-3, OPTIKA Srl, Ponteranica, Italy).[8] Sphere ≥700 μm in diameter were counted as a mammosphere OPTIKA Srl software version 2.7, OPTIKA ® Microscope Italy, Ponteranica, Italy (Patent registration from the General Directorate of Intellectual Property Right, Ministry of Law and Human Right, Republic of Indonesia; No. P00201607099).

Statistical analysis

All values obtained for the treated cells were compared with the values of the cells without treatment (mock cells) and negative control siRNA. Data were presented as the mean ± standard error of the mean. A statistical evaluation of the significant differences was set at P < 0.05 and P < 0.01, respectively and performed using independent t-test against negative control siRNA and mock cells.


   Results Top


The effect of manganese superoxide dismutase suppression on the enzyme activity and superoxide levels

To suppress the MnSOD expression of ALDH1+ BCSCs, we performed transient in vitro transfection using 33 μM and 66 μM MnSOD-siRNA. Following the transfection with 66 μM MnSOD siRNA for 48 h, the relative expression of MnSOD mRNA was significantly suppressed to 0.176-fold compared to the mock cells (P < 0.01) [Figure 1]. We then examined the effect of 66 μM MnSOD siRNA transfection on the MnSOD activity and also found that it was significantly suppressed (from 2.32 U/mg to 0.71 U/mg; P < 0.05) [Figure 2]. Interestingly, the suppression of MnSOD expression and activity of BCSCs could not significantly increase the cellular superoxide level compared to the negative siRNA control (~1.13-fold) [Figure 3].
Figure 1: Relative expression of manganese superoxide dismutase mRNA in aldehyde dehydrogenase 1-positive breast cancer stem cells after in vitro transfection of 33 and 66 μM Manganese superoxide dismutase small interfering RNA. The mRNA relative expression levels of Manganese superoxide dismutase were analyzed using quantitative reverse transcription polymerase chain reaction and calculated using Livak formula. Data of treated cells were normalized to the mock cells (cells without treatment) and compared to the negative control small interfering RNA. Data were presented in mean ± standard error of the mean. Statistical analysis was performed using a t-test independent assay compared to mock cells and negative control small interfering RNA. *P < 0.05 and **P < 0.01 compared to the mock cells, and ##P < 0.01 compared to negative control small interfering RNA (independent t-test)

Click here to view
Figure 2: Manganese superoxide dismutase activity in aldehyde dehydrogenase positive breast cancer stem cells after in vitro transfection of 33 and 66 μM manganese superoxide dismutase small interfering RNA. Data of treated cells were compared to the mock cells (cells without treatment) and the negative control small interfering RNA. Data were presented in mean ± standard error of the mean. Statistical analysis was performed using a t-test independent assay compared to mock cells and negative control small interfering RNA. **P < 0.01 compared to the mock cells, and #P < 0.05 compared to negative control small interfering RNA (independent t-test)

Click here to view
Figure 3: Superoxide levels of aldehyde dehydrogenase 1-positive breast cancer stem cells after in vitro transfection of 33 and 66 μM manganese superoxide dismutase – small interfering RNA. Superoxide level was presented as the ratio of fluorescence intensity of DHE probes in treated cells to mock cells (cells without treatment). Data were presented in mean ± standard error of the mean. Statistical analysis was performed using a t-test independent assay compared to mock cells and negative control small interfering RNA. **P < 0.01 compared to the mock cells

Click here to view


The effect of manganese superoxide dismutase suppression on the viability of aldehyde dehydrogenase 1-positive breast cancer stem cells

The impact of MnSOD suppression on cell viability was analyzed immediately after the siRNA treatment [Figure 4]. Our results of trypan blue exclusion assay showed that suppression of MnSOD mRNA levels by 66 μM MnSOD siRNA led to a significant decrease (0.75-fold) of ALDH1+ BCSC viability compared to the negative siRNA control (P < 0.05). Furthermore, the decrease of BCSC density after 66 μM MnSOD siRNA transfection was also observed under inverted microscope [Figure 5].
Figure 4: Viability of aldehyde dehydrogenase 1-positive breast cancer stem cells after in vitro transfection of 33 and 66 μM manganese superoxide dismutase small interfering RNA. Cell viability was measured using trypan blue exclusion assay. Data of treated cells were compared to the mock cells (cells without treatment) and the negative control small interfering RNA. Data were presented in mean ± standard error of the mean. Statistical analysis was performed using a t-test independent assay compared to mock cells and negative control small interfering RNA. #P < 0.05 compared to negative control small interfering RNA (independent t-test)

Click here to view
Figure 5: The density of aldehyde dehydrogenase 1-positive breast cancer stem cells after in vitro transfection of 33 and 66 μM manganese superoxide dismutase small interfering RNA. Cell density was visualized under an inverted microscope with ×100 and counted using an automated cell counter. (a) Cells without treatment (mock cells); (b) cells transfected with negative control small interfering RNA; (c) cells treated with 33 μM manganese superoxide dismutase small interfering RNA; and (d) cells treated with 66 μM manganese superoxide dismutase small interfering RNA

Click here to view


The effect of manganese superoxide dismutase suppression on OCT4 mRNA expression and mammosphere-forming capacity in aldehyde dehydrogenase 1-positive breast cancer stem cells

Our results showed that the MnSOD suppression by 66 μM MnSOD siRNA significantly reduced the relative expression of OCT4 mRNA (0.39-fold; P < 0.05), as shown in [Figure 6]. Then, we analyzed whether MnSOD suppression could affect the tumorigenicity of ALDH + BCSCs by measuring the MFU. After in vitro transfection of 66 μM MnSOD siRNA transfection, the number of BCSC mammospheres was significantly reduced to 36.53% (P < 0.05) compared to that of negative control siRNA cells [Figure 7]. In addition, [Figure 5]d also demonstrates that MnSOD siRNA transfected ALDH1+ BCSCs tend to lose their ability to form mammosphere.
Figure 6: Relative expression of OCT4 mRNA in Aldehyde dehydrogenase 1-positive breast cancer stem cells after in vitro transfection of 33 and 66 μM manganese superoxide dismutase small interfering RNA. The mRNA expression levels were measured using quantitative reverse transcription polymerase chain reaction and calculated using Livak formula. Data of treated cells were normalized to the mock cells (cells without treatment) and compared to the negative control small interfering RNA, respectively. Data were presented in mean ± standard error of the mean. Statistical analysis was performed using a t-test independent assay compared to mock cells and negative control small interfering RNA. *P < 0.05 and **P < 0.01 compared to the mock cells, and #P<0.05 compared to negative control small interfering RNA (independent t-test)

Click here to view
Figure 7: Mammosphere-forming capacity of aldehyde dehydrogenase 1-positive breast cancer stem cells after in vitro transfection of 33 and 66 μM manganese superoxide dismutase – Small interfering RNA. Data were presented in mean ± standard error of the mean. Statistical analysis was performed using a t-test independent assay compared to mock cells and negative control small interfering RNA. *P < 0.05 and **P < 0.01 compared to the mock cells, whereas #P < 0.05 compared to negative control small interfering RNA

Click here to view



   Discussion Top


Although the dichotomous role of MnSOD in tumorigenesis has been clearly described,[13] little is known about the role of MnSOD in maintaining the stemness of cancer stem cells. In this study, we performed in vitro transfection using MnSOD siRNA in ALDH1+ BCSCs to evaluate the effect of MnSOD on BCSC viability and stemness. The binding of complementary siRNA to MnSOD mRNA will initiate the mRNA to enter the RNA-induced-silencing (RISC) complex. Once the complex has been completely generated, mRNA will be degraded.[14] We found that in vitro transfection of 66 μM MnSOD siRNA was able to suppress not only the MnSOD mRNA synthesis but also its enzyme activity. Hardiany et al. have demonstrated that the suppression of MnSOD synthesis in human glioblastoma using specific siRNA leads to the reduction of MnSOD proteins.[9] Among other SOD isoforms, MnSOD has the highest affinity to its substrate, anion superoxides.[15] Therefore, the translational suppression of MnSOD also affects enzyme activity. Furthermore, we confirm that the suppression of MnSOD expression reduced BCSC growth as previously demonstrated in MDA-MB231 and SKBR3 cells by Kattan et al.[16] We suggest that the reduced BCSC growth after MnSOD suppression may be due to the increase of superoxide level that induces oxidative stress leading to cellular apoptosis. The previous study has shown that MnSOD suppression using siRNA increased cellular apoptosis in T98G cells,[9] while Yeung et al. found that the suppression of MnSOD increased caspase-9-dependent apoptosis and increased chemosensitization in ovarian cancer cells.[17] In this study, the superoxide levels of BCSCs transfected with 66 μM MnSOD siRNA were significantly higher compared to that of the cells without treatment. Compared to the negative siRNA control, the increase of superoxide level was not significant; however, its effect on BCSC viability was significant, suggesting that the oxidative stress occurs because of low MnSOD rather than high superoxide level. Alternatively, low MnSOD level in the cell would increase the peroxides that, in turn may induce oxidative stress and cell death.[18]

We also reveal that MnSOD suppression also reduced the stemness of BCSCs, as shown by the reduced expression of OCT4, a major pluripotent gene, in ALDH1+ BCSCs following the in vitro transfection of MnSOD siRNA. This result is also supported by the decrease of tumorigenicity of MnSOD siRNA-transfected BCSCs compared to the negative control using mammosphere-forming assay as described by Ponti et al.[19] Ji et al. have demonstrated that ROS signaling pathway decreased the expression of pluripotency markers in human embryonic stem cells along with the increase of mesodermal and endodermal expression markers.[20] Moreover, MnSOD has been shown to have an important role in maintaining the activity of OCT4 pluripotent protein by reducing its proteasomal degradation process.[21] It is reported that high expression level of MnSOD was found in estrogen-independent breast cancer cells and contributed to cell growth and invasiveness.[16] Interestingly, our parallel study demonstrated that although ER α 1 mRNA expression in ALDH1+ BCSCs was similar to that in estrogen-dependent breast cancer cells MCF-7, the MnSOD expression in ALDH1+ BCSCs was higher (6.14-fold) than that in MCF-7 cells.[22] This suggests that the high MnSOD expression in BCSCs may not be related to the estrogen signaling. To our knowledge, this is the first study emphasizing that the high MnSOD expression in BCSCs induces the high survival and stemness of BCSCs in vitro. Further,in vivo studies are necessary to elaborate on the relevance of this study on the development of anti-cancer strategy targeted to BCSCs.


   Conclusion Top


In vitro transfection of MnSOD siRNA suppresses human BCSCs (ALDH1+) as demonstrated by reduced OCT4 mRNA expression and mammosphere-forming capacity. We suggest that the suppression of MnSOD expression may be a promising target to reduce stemness and tumorigenicity of ALDH1+ BCSCs.

Acknowledgment

This research was supported by the grant of International Indexed Publication for Final Assignment of the Postgraduate Student (Hibah PITTA) 2018 from Universitas Indonesia.

Financial support and sponsorship

Publikasi Terindeks untuk Tugas Akhir Mahasiswa Universitas Indonesia (PITTA UI 2018) Grant.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Jin X, Jin X, Kim H. Cancer stem cells and differentiation therapy. Tumour Biol 2017;39:1-11.  Back to cited text no. 1
    
2.
Zhu P, Fan Z. Cancer stem cells and tumorigenesis. Biophys Rep 2018;4:178-88.  Back to cited text no. 2
    
3.
Li W, Ma H, Zhang J, Zhu L, Wang C, Yang Y. Unraveling the roles of CD44/CD24 and ALDH1 as cancer stem cell markers in tumorigenesis and metastasis. Sci Rep 2017;7:13856.  Back to cited text no. 3
    
4.
Wang R, Yin C, Li XX, Yang XZ, Yang Y, Zhang MY, et al. Reduced SOD2 expression is associated with mortality of hepatocellular carcinoma patients in a mutant p53-dependent manner. Aging (Albany NY) 2016;8:1184-200.  Back to cited text no. 4
    
5.
Nita M, Grzybowski A. The role of the reactive oxygen species and oxidative stress in the pathomechanism of the age-related ocular diseases and other pathologies of the anterior and posterior eye segments in adults. Oxid Med Cell Longev 2016;2016:1-23.  Back to cited text no. 5
    
6.
Miriyala S, Spasojevic I, Tovmasyan A, Salvemini D, Vujaskovic Z, St Clair D, et al. Manganese superoxide dismutase, MnSOD and its mimics. Biochim Biophys Acta 2012;1822:794-814.  Back to cited text no. 6
    
7.
Wanandi SI, Yustisia I, Neolaka GM, Jusman SW. Impact of extracellular alkalinization on the survival of human CD24-/CD44+breast cancer stem cells associated with cellular metabolic shifts. Braz J Med Biol Res 2017;50:e6538.  Back to cited text no. 7
    
8.
Hosea R, Hardiany NS, Ohneda O, Wanandi SI. Glucosamine decreases the stemness of human ALDH+breast cancer stem cells by inactivating STAT3. Oncol Lett 2018;16:4737-44.  Back to cited text no. 8
    
9.
Hardiany NS, Sadikin M, Siregar N, Wanandi SI. The suppression of manganese superoxide dismutase decreased the survival of human glioblastoma multiforme T98G cells. Med J Indones 2017;19:19-25.  Back to cited text no. 9
    
10.
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C (T)) method. Methods 2001;25:402-8.  Back to cited text no. 10
    
11.
Winarto H, Tan MI, Sadikin M, Wanandi SI. ARID1A expression is down-regulated by oxidative stress in endometriosis and endometriosis-associated ovarian cancer. Transl Oncogenomics 2017;9:1-7.  Back to cited text no. 11
    
12.
Rigo A, Stevanato R, Viglino P. Competitive inhibition of Cu, Zn superoxide dismutase by monovalent anions. Biochem Biophys Res Commun 1977;79:776-83.  Back to cited text no. 12
    
13.
Becuwe P, Ennen M, Klotz R, Barbieux C, Grandemange S. Manganese superoxide dismutase in breast cancer: From molecular mechanisms of gene regulation to biological and clinical significance. Free Radic Biol Med 2014;77:139-51.  Back to cited text no. 13
    
14.
Leuschner PJ, Ameres SL, Kueng S, Martinez J. Cleavage of the siRNA passenger strand during RISC assembly in human cells. EMBO Rep 2006;7:314-20.  Back to cited text no. 14
    
15.
Azadmanesh J, Borgstahl GE. A review of the catalytic mechanism of human manganese superoxide dismutase. Antioxidants (Basel) 2018;7. pii: E25.  Back to cited text no. 15
    
16.
Kattan Z, Minig V, Leroy P, Dauça M, Becuwe P. Role of manganese superoxide dismutase on growth and invasive properties of human estrogen-independent breast cancer cells. Breast Cancer Res Treat 2008;108:203-15.  Back to cited text no. 16
    
17.
Yeung BH, Wong KY, Lin MC, Wong CK, Mashima T, Tsuruo T, et al. Chemosensitisation by manganese superoxide dismutase inhibition is caspase-9 dependent and involves extracellular signal-regulated kinase 1/2. Br J Cancer 2008;99:283-93.  Back to cited text no. 17
    
18.
Buettner GR. Superoxide dismutase in redox biology: The roles of superoxide and hydrogen peroxide. Anticancer Agents Med Chem 2011;11:341-6.  Back to cited text no. 18
    
19.
Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, et al. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 2005;65:5506-11.  Back to cited text no. 19
    
20.
Ji AR, Ku SY, Cho MS, Kim YY, Kim YJ, Oh SK, et al. Reactive oxygen species enhance differentiation of human embryonic stem cells into mesendodermal lineage. Exp Mol Med 2010;42:175-86.  Back to cited text no. 20
    
21.
Sheshadri P, Ashwini A, Jahnavi S, Bhonde R, Prasanna J, Kumar A. Novel role of mitochondrial manganese superoxide dismutase in STAT3 dependent pluripotency of mouse embryonic stem cells. Sci Rep 2015;5:9516.  Back to cited text no. 21
    
22.
Wanandi SI, Syahrani RA, Arumsari S, Wideani G, Hardiany NS. Profiling of gene expression associated with stemness and aggressiveness of ALDH1A1-expressing, human breast cancer cells. Malays J Med Sci 2019;26:38-52.  Back to cited text no. 22
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]



 

Top
  
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
   Conclusion
    References
    Article Figures

 Article Access Statistics
    Viewed60    
    Printed0    
    Emailed0    
    PDF Downloaded8    
    Comments [Add]    

Recommend this journal