Mesenchymal Stem Cell-Derived Extracellular Vesicles Increase Human MCF7 Breast Cancer Cell Proliferation associated with OCT4 Expression and ALDH Activity.

OBJECTIVE: The aim of this study was to investigate the effect of mesenchymal stem cells-derived extracellular vesicles (MSC-EVs) on the human MCF7 breast cancer cell proliferation that have been considered to contain limited CSC population and its association with the expression of OCT4 and ALDH1 stemness markers. METHODS: EVs were successfully isolated from the conditioned medium of umbilical cord MSCs using size exclusion chromatography. The isolated EV fraction was verified under a transmission electron microscope (TEM). Five and ten percent (v/v) concentration of MSC-EVs were then co-cultured with MCF7 cells. To investigate MSC-EV uptake by MCF7 cells, we performed confocal microscopy analysis. Subsequently, the proliferation of co-cultured MCF7 cells was determined using trypan blue exclusion assay, while their mRNA and protein expression of OCT4 as well as ALDH activity as the marker of stemness properties were analyzed using quantitative reverse transcription polymerase chain reaction, Western Blot, and Aldefluor™ assays, respectively. RESULT: MSC-EVs were detected as round-shaped, ~100 nm sized particles under TEM. We also demonstrate that MSC-EVs can be internalized by MCF7 cells. Notably, MSC-EVs of 5% concentration increased OCT4 mRNA expression and ALDH1 activity in MCF7 cells. At 10% concentration, MSC-EVs reduced the OCT4 expression and ALDH1 activity. CONCLUSION: MSC-derived EVs modulate the stemness of MCF7 cells, either OCT4 expression or ALDH1 activity, in a concentration dependent manner along with the increase of cell proliferation.

Correction: In silico and in vitro studies on the anti-cancer activity of andrographolide targeting survivin in human breast cancer stem cells.

[This corrects the article DOI: 10.1371/journal.pone.0240020.].

In silico and in vitro studies on the anti-cancer activity of andrographolide targeting survivin in human breast cancer stem cells.

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.

Profiling of Gene Expression Associated with Stemness and Aggressiveness of ALDH1A1-Expressing Human Breast Cancer Cells.

BACKGROUND: It 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. METHODS: Human 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-ß1, TßR1, ERα1 and MnSOD associated with aggressiveness in BCSCs and MCF-7 cells were determined using the quantitative real-time PCR (qRT-PCR). RESULTS: The 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-ß1 (22.89-fold ± 6.840; P = 0.015), TßR1 (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. CONCLUSION: The 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.

Leu169Trp substitution in MnSOD from Staphylococcus equorum created an active new form of similar resistance to UVC irradiation.

Manganese superoxide dismutase from Staphylococcus equorum (MnSODSeq) maintains its activity after up to 45 minutes of UVC radiation. The enzyme occurs in a dimeric form that likely contributes to its activity and stability. Therefore, maintaining the dimeric form could be a way to improve the enzyme's stability. One of the main interactions for dimer formation occurs between Tyr168 and His31, of which the latter is also involved in the enzymatic reaction. UVC radiation may cause alterations in the electronic structure of the phenolic ring in the Tyr168 side chain: this may disrupt the Tyr168-His31 pairing and lead to enzyme instability and/or activity loss. In this report, a Leu169Trp substitution was carried out to protect the Tyr168 residue by introducing an amino acid with an aromatic side chain for better photon absorption of the UV light. Interestingly, although the substitution appeared to have a minor effect on enzyme stability and activity upon UVC irradiation, the melting temperature (TM) of the Leu169Trp mutant was different. Unlike the native protein, the TM of the mutant had not changed after UV irradiation. Thus, our effort to extend the resistance to UVC radiation was not successful, but we have discovered a biologically active new form. The present finding provides evidence that MnSODSeq maintains most of its activity and resistance to UVC irradiation as long as the dimer and its glutamate-bridge are intact, despite an alteration that destabilizes its monomeric structure. The present finding further unravels the relationship between the structure of the enzyme and its activity. Furthermore, the results may provide further insight in how to modify the enzyme to improve its characteristics for application in medicine or cosmetics.

The first crystal structure of manganese superoxide dismutase from the genus Staphylococcus.

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 Šresolution is reported that may explain these differences. The crystal belonged to space group P3221, 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/ß 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.

Structure-activity relationship of a recombinant hybrid Manganese superoxide dismutase of Staphylococcus saprophyticus/S. equorum.

Recombinant hybrid Manganese superoxide dismutase from Staphyloccus saphropyticus/S. equorum (rMnSODSeq) exhibits stability at high temperatures. The enzyme occurs as a dimer that dissociates around 52°C prior to unfolding of the monomer around 64°C, demonstrating contribution of the dimeric form to stability. Here, structure - activity relationship of rMnSODSeq was evaluated on the basis of its activity and stability in the presence of inhibitors, NaCl, denaturants, detergents, reducing agents, and at different pH values. The activity was evaluated at both 37°C and 52°C, which the latter is the temperature for dissociation of the dimer. Dimer to monomer transition coincided with significant decrease in residual activity at 52°C. However, the activity assay results at 52°C and 37°C suggest spontaneous re-association of the monomer into dimer. Intriguingly, various new species with melting temperature (TM) values other than those of the dimer or monomer were observed. These species displayed medium to comparable level of residual activities to the native at 37°C. This report suggests that dimer to monomer transition may be not the only explanation for activity loss or decrease.