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.

Role of TGF-ß1 in human breast cancer stem cells.

OBJECTIVE: To investigate the auto-induction of transforming growth factor-b1 (TGF-ß1) in breast cancer stem cells (BCSCs) and its effect on cell viability and stemness. METHODS: Human BCSCs (aldehyde dehydrogenase positive; ALDH+) were grown in serum-free Dulbecco's Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12) and treated for periods of 1, 2 and 4 hours with 0.1 ng/ml recombinant human TGF-ß1 protein (rhTGF-ß1). The medium was then replaced with serum-free DMEM/F12 without rhTGF-ß1 for 24 hours. Cell viability was determined using a trypan blue exclusion assay. Type 1 TGF-ß receptor (TßR1), TGF-ß1, octamer-binding transcription factor 4 (OCT4) and aldehyde dehydrogenase 1 family member A1 (ALDH1A1) messenger RNA (mRNA) expression levels were analysed using quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR). The TGF-ß protein level in the culture medium was determined using an enzyme-linked immunosorbent assay (ELISA). RESULTS: The expression levels of rhTGF-ß1, TGF-ß1 and TßR1 mRNA significantly increased in BCSCs compared to control after treatment for 1 and 2 hours but decreased after 4 hours. This is in line with alteration of stemness gene, OCT4 and ALDH1A1 mRNA expressions. However, the secretion of newly synthesised TGF-ß1 significantly increased after 2 hours. In contrast, viable BCSCs decreased after 1 hour and then gradually increased 2.7 times compared to control after 4 hours. CONCLUSIONS: TGF-ß1 treatment in low concentration and for short period of time triggers its auto-induction in BCSCs, leading to increased cell viability and stemness gene expression via autocrine signalling.

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.

Mechanisms of Cytotoxicity of Chemical Agents to Giant Cell Tumors: An In Vitro Study.

BACKGROUND: Various chemical agents have been used as an adjuvant treatment for giant cell tumor (GCT). However, the comparative effect of these chemicals remains unclear. METHODS: Multinucleated and spindle cells from cultured GCT patients, characterized by Nanog and Oct4 expression with RT-PCR, were directly administered, in vitro, with concentrations of 1%, 3%, and 5% of H2O2 and 75%, 85%, and 95% of ethanol for 10 minutes and concentrations of 0.003%, 0.005%, 0.01%, 0.03%, 0.1%, and 0.3% of H2O2 for 5 minutes and were incubated for 24 hours. Cell morphology, cell viability, and flow cytometry after various concentrations of H2O2 and ethanol exposure were assessed. RESULTS: H2O2 in all concentrations caused loss of cell viability. The number of viable cells after H2O2 exposure was related to the concentration-dependent effect. The initial viable spindle-shaped cell, multinucleated giant cell, and round-epithelioid cell had morphological changes into fragmented nonviable cells after exposure to H2O2. Flow cytometry using Annexin V showed cell death due to necrosis, with the highest concentration amounting to 0.3%. CONCLUSION: Administering local chemical adjuvants of H2O2 in vitro caused loss of viable GCT cells. The number of viable cells after H2O2 exposure was related to the concentration-dependent effect, whereas reducing concentration of H2O2 may cause loss of viability and morphology of cultured GCT cells with the apoptotic mechanism.

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.