Breast cancer stem cells (BCSCs) express high levels of the anti-apoptotic protein, survivin. This study aimed to discover a natural active compound with anti-cancer properties that targeted survivin in human breast cancer stem cells. From the seven examined compounds, andrographolide was selected as a lead compound through in silico molecular docking with survivin, caspase-9, and caspase-3. We found that the affinity between andrographolide and survivin is higher than that with caspase-9 and caspase-3. Human CD24-/CD44+ BCSCs were treated with andrographolide in vitro for 24 hours. The cytotoxic effect of andrographolide on BCSCs was compared to that on human mesenchymal stem cells (MSCs). The expression of survivin, caspase-9, and caspase-3 mRNA was analyzed using qRT-PCR, while Thr34-phosphorylated survivin and total survivin levels were determined using ELISA and Immunoblotting assay. Annexin-V/PI flow cytometry assays were performed to evaluate the apoptotic activity of andrographolide. Our results demonstrate that the CC50 of andrographolide in BCSCs was 0.32mM, whereas there was no cytotoxic effect in MSCs. Moreover, andrographolide decreased survivin and Thr34-phosphorylated survivin, thus inhibiting survivin activation and increasing survivin mRNA in BCSCs. The apoptotic activity of andrographolide was revealed by the increase of caspase-3 mRNA and protein, as well as the increase in both the early and late phases of apoptosis. In conclusion, andrographolide can be considered an anti-cancer compound that targets BCSCs due to its molecular interactions with survivin, caspase-9, and caspase-3, which induce apoptosis. We suggest that the binding of andrographolide to survivin is a critical aspect of the effect of andrographolide.
BackgroundIt has been widely reported that breast cancer aggressiveness may be driven by breast cancer stem cells (BCSCs). BCSCs display stemness properties that include self-renewal, tumourigenicity and pluripotency. The regulation of gene expression may have important roles in BCSC stemness and aggressiveness. Thus, the aim of this study was to examine the stemness and aggressiveness gene expression profile of BCSCs compared to MCF-7 and MDA-MB-231 breast cancer cells.MethodsHuman ALDH1+ BCSCs were grown in serum-free Dulbecco’s Modified Eagle Medium (DMEM)/F12, while MCF-7 and MDA-MB-231 were cultured in DMEM supplemented with 10% foetal bovine serum under standard conditions. Total RNA was extracted using the Tripure Isolation Reagent. The relative mRNA expressions of OCT4, ALDH1A1 and CD44 associated with stemness as well as TGF-β1, TβR1, ERα1 and MnSOD associated with aggressiveness in BCSCs and MCF-7 cells were determined using the quantitative real-time PCR (qRT-PCR).ResultsThe mRNA expressions of OCT4 (5.19-fold ± 0.338; P = 0.001), ALDH1A1 (3.67-fold ± 0.523; P = 0.006), CD44 (2.65-fold ± 0.307; P = 0.006), TGF-β1 (22.89-fold ± 6.840; P = 0.015), TβR1 (3.74-fold ± 1.446; P = 0.045) and MnSOD (4.6-fold ± 1.096; P = 0.014) were higher in BCSCs than in MCF-7 but were almost similar to MDA-MB-231 cells. In contrast, the ERα1 expression of BCSCs (0.97-fold ± 0.080; P = 0.392) was similar to MCF-7 cells, indicating that BSCSs are oestrogen-dependent breast cancer cells.ConclusionThe oestrogen-dependent BCSCs express stemness and aggressiveness genes at a higher level compared to oestrogen-dependent MCF-7 but are almost similar to oestrogen-independent MDA-MB-231 cells.
<p>ABSTRAK<br />Diversitas genetik aksesi kelapa sawit Indonesia saat ini sangat<br />rendah. Dalam usaha meningkatkan keragaman genetik telah dilakukan<br />eksplorasi plasma nutfah di pusat keragaman genetik kelapa sawit di<br />Kamerun. Tujuan dari penelitian ini untuk mengetahui diversitas genetik<br />dan tingkat polimorfisme berdasarkan marka SSR aksesi-aksesi kelapa<br />sawit Kamerun. Bahan tanaman yang digunakan 49 aksesi kelapa sawit<br />Kamerun, Afrika yang ditanam di Kebun Sumber Daya Genetik (SDG)<br />Sawit Sijunjung, Sumatera Barat. DNA genomik diisolasi dari tiap<br />individu aksesi menggunakan protokol isolasi DNA untuk tanaman<br />bergetah. DNA dianalisis menggunakan 20 marka SSR. Dendrogram<br />kekerabatan dikonstruksi menggunakan metode Unweighted Pair Group<br />Method Arithmetic (UPGMA) melalui software NTSYS-pc (Numerical<br />Taxonomy and Multivariate Analysis System) versi 2.1-pc. Hasil penelitian<br />menunjukkan nilai polimorfisme information content (PIC) marka SSR<br />tinggi sebesar 0,80 (berkisar 0,63-0,91). Jumlah alel yang terdeteksi per<br />marka SSR berkisar antara 4-15 alel per lokus SSR (rata-rata 8,75).<br />Analisis filogenetik 49 aksesi menghasilkan diversitas genetik 12,5-<br />54,72% (kemiripan genetik 55,28-87,50%). Pada diversitas genetik<br />54,72%, aksesi Kamerun terbagi menjadi tujuh kelompok masing-masing<br />terdiri dari 9, 28, 4, 2, 1, 2, dan 3 aksesi. Aksesi dengan diversitas genetik<br />tinggi dan berada pada klaster berbeda, potensial digunakan sebagai calon<br />tetua dalam program pemuliaan kelapa sawit.<br />Kata kunci: Elaeis guineensis Jacq., diversitas genetik, plasma nutfah,<br />marka SSR</p><p>ABSTRACT<br />Genetic diversity of the Indonesian oil palm collection is very low.<br />To improve their genetic variability, exploration from the oil palm center<br />of origins has been done in Kamerun. The objectives of this study were to<br />determine genetic and polymorphism level of the SSR markers Cameroon-<br />originated oil palm accessions. Genetic materials used were 49 Cameroon-<br />originated oil palm accessions collected at Sijunjung Oil Palm Germplam<br />Collection Station, West Sumatera. Genomic DNA was isolated using a<br />protocol for isolating DNA from leaves rich with latex. DNA was analyzed<br />using 20 SSR markers. A dendogram was constructed using the<br />Unweighted Pair Group Method Arithmetic (UPGMA) method through the<br />Numerical Taxonomy and Multivariate Analysis System software<br />(NTSYS-pc) version 2.1-pc. Results showed that the polimorfisme<br />information content (PIC) values of the SSR markers used was high, 0.80<br />(range from 0.63-0.91). The average number of the SSR alleles detected<br />was also high, 8.75 alleles (range from 4-15 alleles per SSR locus).<br />Phylogenetic analysis of the 49 oil palm accessions resulted genetic<br />diversity of 12.5-54.72% (genetic similarity of 55.28-87.50%). At genetic<br />diversity 54.72%, the 49 accessions were divided into seven clusters, each<br />consisted of 9, 28, 4, 2, 1, 2, and 3 accesions, respectively. Accessions<br />with high genetic diversity and located at different clusters may be useful<br />as parent candidates in the future oil palm breeding programs.<br />Key words: Elaeis guineensis Jacq., genetic diversity, germplasm, SSR<br />markers</p>
A recombinant Staphylococcus equorum manganese superoxide dismutase (MnSOD) with an Asp13Arg substitution displays activity over a wide range of pH, at high temperature and in the presence of chaotropic agents, and retains 50% of its activity after irradiation with UVC for up to 45 min. Interestingly, Bacillus subtilis MnSOD does not have the same stability, despite having a closely similar primary structure and thus presumably also tertiary structure. Here, the crystal structure of S. equorum MnSOD at 1.4 Å resolution is reported that may explain these differences. The crystal belonged to space group P321, with unit-cell parameters a = 57.36, b = 57.36, c = 105.76 Å, and contained one molecule in the asymmetric unit. The symmetry operation indicates that the enzyme has a dimeric structure, as found in nature and in B. subtilis MnSOD. As expected, their overall structures are nearly identical. However, the loop connecting the helical and α/β domains of S. equorum MnSOD is shorter than that in B. subtilis MnSOD, and adopts a conformation that allows more direct water-mediated hydrogen-bond interactions between the amino-acid side chains of the first and last α-helices in the latter domain. Furthermore, S. equorum MnSOD has a slightly larger buried area compared with the dimer surface area than that in B. subtilis MnSOD, while the residues that form the interaction in the dimer-interface region are highly conserved. Thus, the stability of S. equorum MnSOD may not originate from the dimeric form alone. Furthermore, an additional water molecule was found in the active site. This allows an alternative geometry for the coordination of the Mn atom in the active site of the apo form. This is the first structure of MnSOD from the genus Staphylococcus and may provide a template for the structural study of other MnSODs from this genus.
BACKGROUND High carbon dioxide (CO2) level from indoor environments, such as classrooms and offices, might cause sick building syndrome. Excessive indoor CO2 level increases CO2 level in the blood, and over-accumulation of CO2 induces an adaptive response that requires modulation of gene expression. This study aimed to investigate the adaptive transcriptional response toward hypoxia and oxidative stress in human peripheral blood mononuclear cells (PBMCs) exposed to elevated CO2 level in vitro and its association with cell viability. METHODS PBMCs were treated in 5% CO2 and 15% CO2, representatives a high CO₂ level condition for 24 and 48 hours. Extracellular pH (pHe) was measured with a pH meter. The levels of reactive oxygen species were determined by measuring superoxide and hydrogen peroxide with dihydroethidium and dichlorofluorescin-diacetate assay. The mRNA expression levels of hypoxia-inducible factor (HIF)-1α, HIF-2α, nuclear factor (NF)-κB, and manganese superoxide dismutase (MnSOD) were analyzed using a real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). Cell survival was determined by measuring cell viability. RESULTS pHe increased in 24 hours after 15% CO₂ treatment, and then decreased in 48 hours. Superoxide and hydrogen peroxide levels increased after the 24- and 48-hour of high CO₂ level condition. The expression levels of NF-κB, MnSOD, HIF-1α, and HIF-2α decreased in 24 hours and increased in 48 hours. The increased antioxidant mRNA expression in 48 hours showed that the PBMCs were responsive under high CO2 conditions. Elevated CO2 suppressed cell viability significantly in 48 hours. CONCLUSIONS After 48 hours of high CO₂ level condition, PBMCs showed an upregulation in genes related to hypoxia and oxidative stress to overcome the effects of CO2 elevation.
BACKGROUND: Cancer stem cells (CSCs) is defined as tumor initiating cells within tumor that maintain stemness properties and tumorigenicity. Extracellular pH of CSCs in in vitro condition is important for supporting cell proliferation which may also regulate the expression of stemness markers such as OCT4. This work aimed to examine the effect of cell culture media on the proliferation and stemness of human breast cancer stem cells (BCSCs).METHODS: Human CD24-/CD44+ BCSCs were grown in Dulbecco's Modified Eagle Medium/F-12 (DMEM/F-12) with 15mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), without HEPES and adjusted to pH 7.4, or without HEPES but pH was not adjusted. BCSCs were grown under standard conditions for various days. Viable cell number was measured using trypan blue exclusion, whereas proliferation rate using MTS assay. OCT4 mRNA and protein were analyzed using quantitative real time PCR (qRT-PCR) and Western Blot assay, respectively. In vitro tumorigenic activity was determined using mammosphere formation unit (MFU) assay.RESULTS: Our results showed a higher viable cell number and proliferation of BCSCs in DMEM/F-12 HEPES (-) compared to HEPES (+) medium until 4 day incubation. OCT4 mRNA and protein level, as well as MFU of BCSCs were significantly higher in HEPES (-) compared to HEPES (+) medium on day 2.CONCLUSION: DMEM/F-12 medium without HEPES facilitates CD24-/CD44+ BCSCs to have higher proliferation and stemness on day 2 incubation compared to those with HEPES.KEYWORDS: breast cancer, cancer stem cell, OCT4, stemness, proliferation
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