METHODS: In this study, CD24 population from the MCF-7 spheroid was sorted and subjected to spheroid formation test, stem cell markers immunofluorescence, invasion and migration test as well as microRNA expression profiling.
RESULTS: Sorted MCF-7 CD24 cells from primary spheroids were able to reform its 3D spheroid shape after 7 days in non-adherent culture conditions. In contrast to the primary spheroids, the expression of SOX-2, CD44, CD49f and Nanog were dim in MCF-7 CD24+ cells. Remarkably, MCF-7 CD24 cells were found to show high expression of ALDH1 protein which may have resulted in these cells exhibiting higher resistance against doxorubicin and cisplatin when compared to that of the parental cells. Moreover, microRNA profiling has shown that the absence of cancer stem cell properties were consistent with the downregulation of major cancer stem cells related pathways including Hedgehog, Wnt and MAPK signalling pathways. However, the upregulated pathways such as adherans junctions, focal adhesion and tight junction suggest that CD24+ cells were probably at an epithelial-like state of cell transition.
CONCLUSION: In conclusion, neglected CD24+ cells in MCF-7 spheroid did not exhibit typical breast CSCs properties. The presence of miRNAs and their analysed pathways suggested that these cells could be a distinct intermediate cell state in breast CSCs.
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-β1, TβR1, 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-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.
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.
METHOD: B. frutescens leaves extracts were prepared using Soxhlet apparatus with solvents of different polarity. The selective cytotoxicity of these extracts at various concentrations (20 to 160 μg/ml) were tested using cell viability assay after 24, 48 and 72 h of treatment. The IC50 value in human breast cancer (MCF-7 and MDA-MB-231) and mammary breast (MCF10A) cell lines were determined. Apoptotic study using AO/PI double staining was performed using fluorescent microscope. The glucose uptake was measured using 2-NBDG, a fluorescent glucose analogue. The phytochemical screening was performed for alkaloids, flavonoids, tannins, triterpenoids, and phenols.
RESULTS: B. frutescens leaves extracts showed IC50 value ranging from 10 -127μg/ml in MCF-7 cells after 72 h of treatment. Hexane extract had the lowest IC50 value (10μg/ml), indicating its potent selective cytotoxic activity. Morphology of MCF-7 cells after treatment with B. frutescens extracts exhibited evidence of apoptosis that included membrane blebbing and chromatin condensation. In the glucose uptake assay, B. frutescens extracts suppressed glucose uptake in cancer cells as early as 24 h upon treatment. The inhibition was significantly lower compared to the positive control WZB117 at their respective IC50 value after 72 h incubation. It was also shown that the glucose inhibition is selective towards cancer cells compared to normal cells. The phytochemical analysis of the extract using hexane as the solvent in particular gave similar quantities of tannin, triterpenoids, flavonoid and phenols. Presumably, these metabolites have a synergistic effect in the in vitro testing, producing the potent IC50 value and subsequently cell death.
CONCLUSION: This study reports the potent selective cytotoxic effect of B. frutescens leaves hexane extract against MCF-7 cancer cells. B. frutescens extracts selectively suppressed cancer cells glucose uptake and subsequently induced cancer cell death. These findings suggest a new role of B. frutescens in cancer cell metabolism.
METHODS: Postbiotic metabolites (PM) produced by six strains of L. plantarum were determined for their antiproliferative and cytotoxic effects on normal human primary cells, breast, colorectal, cervical, liver and leukemia cancer cell lines via MTT assay, trypan blue exclusion method and BrdU assay. The toxicity of PM was determined for human and various animal red blood cells via haemolytic assay. The cytotoxicity mode was subsequently determined for selected UL4 PM on MCF-7 cells due to its pronounced cytotoxic effect by fluorescent microscopic observation using AO/PI dye reagents and flow cytometric analyses.
RESULTS: UL4 PM exhibited the lowest IC50 value on MCF-7, RG14 PM on HT29 and RG11 and RI11 PM on HL60 cell lines, respectively from MTT assay. Moreover, all tested PM did not cause haemolysis of human, dog, rabbit and chicken red blood cells and demonstrated no cytotoxicity on normal breast MCF-10A cells and primary cultured cells including human peripheral blood mononuclear cells, mice splenocytes and thymocytes. Antiproliferation of MCF-7 and HT-29 cells was potently induced by UL4 and RG 14 PM respectively after 72 h of incubation at the concentration of 30% (v/v). Fluorescent microscopic observation and flow cytometric analyses showed that the pronounced cytotoxic effect of UL4 PM on MCF-7 cells was mediated through apoptosis.
CONCLUSION: In conclusion, PM produced by the six strains of L. plantarum exhibited selective cytotoxic via antiproliferative effect and induction of apoptosis against malignant cancer cells in a strain-specific and cancer cell type-specific manner whilst sparing the normal cells. This reveals the vast potentials of PM from L. plantarum as functional supplement and as an adjunctive treatment for cancer.
AIM OF THE STUDY: This study aimed to investigate the in vitro antiproliferative effects and apoptotic events of the ionic liquid extract of Graviola fruit (IL-GFE) on MCF-7 breast cancer cells and their cytokinetics behaviour to observe their potential as a therapeutic alternative in cancer treatment.
MATERIALS AND METHODS: The cell viability assay of the extract was measured using tetrazolium bromide (MTT assay) to observe the effects of Graviola fruit extract. Then the cytokinetics behaviour of MCF-7 cells treated with IL-GFE is observed by plotting the growth curve of the cells. Additionally, the cell cycle distribution and apoptosis mechanism of IL-GFE action on MCF-7 cancer cells were observed by flow cytometry.
RESULTS: IL-GFE exhibited anti-proliferative activity on MCF-7 with the IC50 value of 4.75 μg/mL, compared to Taxol with an IC50 value of 0.99 μg/mL. IL- GFE also reduced the number of cell generations from 3.71 to 1.67 generations compared to 2.18 generations when treated with Taxol. Furthermore, the anti-proliferative activities were verified when the growth rate was decreased dynamically from 0.0077 h to 1 to 0.0035 h-1. Observation of the IL-GFE-treated MCF-7 under microscope demonstrated detachment of cells and loss of density. The growth inhibition of the cells by extracts was associated with cell cycle arrest at the G0/G1 phase, and phosphatidylserine externalisation confirms the anti-proliferation through apoptosis.
CONCLUSIONS: ionic liquid Graviola fruit extract affect the cytokinetics behaviour of MCF-7 cells by reducing cell viability, induce apoptosis and cell cycle arrest at the G0/G1 phase.
AIM OF THE STUDY: To develop a natural biodegradable macromolecule i.e. Chitosan (CS)-coated-DAUN-PLGA-poly(lactic-co-glycolic acid)-Nanoparticles (NPs) with an aim to improve oral-DAUN bioavailability and to develop as well as validate UHPLC-MS/MS (ESI/Q-TOF) method for plasma quantification and pharmacokinetic analysis (PK) of DAUN.
RESULTS: A particle size (198.3 ± 9.21 nm), drug content (47.06 ± 1.16 mg/mg) and zeta potential (11.3 ± 0.98 mV), consisting of smooth and spherical shape was observed for developed formulation. Cytotoxicity studies for CS-DAUN-PLGA-NPs revealed; a comparative superiority over free DAUN-S (i.v.) in human breast adenocarcinoma cell lines (MCF-7) and a higher permeability i.e. 3.89 folds across rat ileum, as compared to DAUN-PLGA-NPs (p
MATERIALS AND METHODS: Cytotoxicity screening of chalepin against MCF7 cells was conducted using SRB assay. Apoptosis induction was examined by established morphological and biochemical assays including phase contrast and Hoechst/PI staining fluorescence microscope. Similarly, Annexin-V/FITC and TUNEL assays were conducted using flow cytometry whereas caspase-3 activity was evaluated using microplate reader.
KEY FINDINGS: The result indicates remarkable cytotoxic activity against MCF7 cells, whereas it shows moderate cytotoxic activity against MDA-MB231 cells. Interestingly, chalepin did not present any toxicity against MRC5 normal cell line. Morphological examination using both phase contrast and fluorescence microscope displays typical apoptotic features such as membrane blebbing, DNA fragmentation, chromatin condensation and apoptotic bodies' formation following chalepin treatment against MCF7 cells at different concentration for 48 h. Apoptosis induction is significantly associated with externalisation of phosphatidylserine, and DNA fragmentation in MCF7 cells chalepin treated cells when compared with control. The protein expressions of caspase-8, 9 and cleaved PARP1 were upregulated which correlated well with increased caspase-3 activity.
SIGNIFICANCE: From our recent findings, chalepin was able to induced apoptosis in MCF7 cells and therefore, could be evaluated further as a potential source of anticancer agent for cancer treatment such as breast cancer.