Cell Fate following Irradiation of MDA-MB-231 and MCF-7 Breast Cancer Cells Pre-Exposed to the Tetrahydroisoquinoline Sulfamate Microtubule Disruptor STX3451.

A tetrahydroisoquinoline (THIQ) core is able to mimic the A and B rings of 2-methoxyestradiol (2ME2), an endogenous estrogen metabolite that demonstrates promising anticancer properties primarily by disrupting microtubule dynamic instability parameters, but has very poor pharmaceutical properties that can be improved by sulfamoylation. The non-steroidal THIQ-based microtubule disruptor 2-(3-bromo-4,5-dimethoxybenzyl)-7-methoxy-6-sulfamoyloxy-1,2,3,4-tetrahydroisoquinoline (STX3451), with enhanced pharmacokinetic and pharmacodynamic profiles, was explored for the first time in radiation biology. We investigated whether 24 h pre-treatment with STX3451 could pre-sensitize MCF-7 and MDA-MB-231 breast cancer cells to radiation. This regimen showed a clear increase in cytotoxicity compared to the individual modalities, results that were contiguous in spectrophotometric analysis, flow cytometric quantification of apoptosis induction, clonogenic studies and microscopy techniques. Drug pre-treatment increased radiation-induced DNA damage, with statistically more double-strand (ds) DNA breaks demonstrated. The latter could be due to the induction of a radiation-sensitive metaphase block or the increased levels of reactive oxygen species, both evident after compound exposure. STX3451 pre-exposure may also delay DNA repair mechanisms, as the DNA damage response element ataxia telangiectasia mutated (ATM) was depressed. These in vitro findings may translate into in vivo models, with the ultimate aim of reducing both radiation and drug doses for maximal clinical effect with minimal adverse effects.

Cholecalciferol Inhibits Cell Growth and Induces Apoptosis in the CaSki Cell Line.

Vitamin D has displayed anti-cancer actions in numerous in vitro studies. Here, we investigated the anti-cancer actions of cholecalciferol, a vitamin D precursor, on a metastatic cervical cancer cell line, namely, CaSki. Experimental cultures were incubated for 72 h and treated with cholecalciferol (10-1000 ng/mL). In the present study, cell count, viability, proliferation and cell cycle were analyzed by a crystal violet assay, trypan blue assay, Ki67 proliferation, and a cell cycle assay, respectively. Biomarkers of apoptosis, necrosis, and autophagic cell death were measured by the Caspase 3/7 and Annexin V/7-AAD Muse™ assays, a LC3-II assay, and a lactate dehydrogenase release assay, respectively. The ultrastructural features of cell death were assessed by transmission electron microscopy. A statistical analysis was performed using a one-way ANOVA and Bonferroni's post-hoc analysis test, and p < 0.05 is considered statistically significant here. The results identify statistical decreases in cell count and viability at high-dose treatments (100 and 1000 ng/mL). In addition, significant increases in apoptotic biochemical markers and apoptotic ultrastructure are shown to be present at high-dose treatments. In conclusion, high-dose cholecalciferol treatments inhibit cell count and viability, which are both mediated by apoptotic induction in the CaSki cell line.

Rooibos tea extracts inhibit osteoclast formation and activity through the attenuation of NF-κB activity in RAW264.7 murine macrophages.

Rooibos tea is a naturally sweet and aromatic tea that is native to the Western Cape province of South Africa. Rooibos is usually fermented to produce the traditional reddish brown colour and has been found to have numerous health benefits. These include beneficial effects on osteoblasts; however, its effects on osteoclast formation and activity are unknown. Osteoclasts are large, multinucleated cells responsible for bone resorption. Binding of RANKL to its receptor on osteoclast precursors triggers the NF-κB signalling pathway leading to the formation of osteoclasts. Certain bone destructive diseases, such as osteoporosis, are characterised by overactive osteoclasts. The inhibition of osteoclasts may offer a potential mode to prevent these diseases. The polyphenol contents of both fermented and unfermented tea extracts were similar although the radical scavenging activity of fermented rooibos tea was lower. Both tea extracts were not cytotoxic and inhibited osteoclast formation. Fermented rooibos tea extract caused a greater reduction in osteoclast resorption and the associated gene expression when compared with unfermented rooibos tea. Both tea extracts were shown to attenuate NF-κB activity. Fermented rooibos was found to have a more potent inhibitory effect on osteoclasts than unfermented rooibos extract and therefore may have a beneficial effect on bone health.

Commercial Honeybush (Cyclopia spp.) Tea Extract Inhibits Osteoclast Formation and Bone Resorption in RAW264.7 Murine Macrophages-An in vitro Study.

Honeybush tea, a sweet tasting caffeine-free tea that is indigenous to South Africa, is rich in bioactive compounds that may have beneficial health effects. Bone remodeling is a physiological process that involves the synthesis of bone matrix by osteoblasts and resorption of bone by osteoclasts. When resorption exceeds formation, bone remodeling can be disrupted resulting in bone diseases such as osteoporosis. Osteoclasts are multinucleated cells derived from hematopoietic precursors of monocytic lineage. These precursors fuse and differentiate into mature osteoclasts in the presence of receptor activator of NF-kB ligand (RANKL), produced by osteoblasts. In this study, the in vitro effects of an aqueous extract of fermented honeybush tea were examined on osteoclast formation and bone resorption in RAW264.7 murine macrophages. We found that commercial honeybush tea extract inhibited osteoclast formation and TRAP activity which was accompanied by reduced bone resorption and disruption of characteristic cytoskeletal elements of mature osteoclasts without cytotoxicity. Furthermore, honeybush tea extract decreased expression of key osteoclast specific genes, matrix metalloproteinase-9 (MMP-9), tartrate resistant acid phosphatase (TRAP) and cathepsin K. This study demonstrates for the first time that honeybush tea may have potential anti-osteoclastogenic effects and therefore should be further explored for its beneficial effects on bone.

Novel in silico-designed estradiol analogues are cytotoxic to a multidrug-resistant cell line at nanomolar concentrations.

PURPOSE: 2-Methoxyestradiol (2ME) is a promising anti-cancer agent that disrupts the integrity and dynamics of the spindle network. In order to overcome the pharmacokinetic constraints of this compound, a panel of sulphamoylated estradiol analogues were in silico-designed by our laboratory. In this study, we analysed the potential of each analogue to induce cell death on a panel of cancer cell lines. Moreover, the mechanism of action of the most effective compounds was determined. METHODS: Cytotoxicity screening of the compounds and intermediates was performed on five different cancer cell lines to determine IG50 values. An in vitro tubulin polymerization assay was done to determine the effect of the drugs on tubulin polymerization while their intracellular effects on the microtubule network were assessed by immunofluorescence microscopy. RESULTS: IG50 calculations showed that the sulphamoylated analogues induce cytotoxicity at nanomolar concentrations in all cell lines, including the P-glycoprotein pump overexpressing multidrug-resistant uterine sarcoma cell line. The non-sulphamoylated compounds were only cytotoxic at micromolar ranges, if at all. The sulphamoylated compounds inhibited pure tubulin polymerization in a dose-dependent manner and induced microtubule destruction in cells after 24-h exposure. CONCLUSION: Results revealed that the novel sulphamoylated 2ME derivatives have potential as anti-cancer drugs, possibly even against chemoresistant cancer cells. These compounds disrupt the intracellular microtubule integrity which leads to mitotic block of the cells.

Influence of estradiol analogue on cell growth, morphology and death in esophageal carcinoma cells

2-Methoxyestradiol-bis-sulphamate is a bis-sulphamoylated derivative of the naturally occurring 17-beta-estradiol metabolite namely 2-methoxyestradiol. 2-Methoxyestradiol-bis-sulphamate is regarded as a potential anticancer drug with increased antiproliferative activity when compared to 2-methoxyestradiol. The aim of this pilot in vitro study was to determine the influence of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and possible induction of certain types of cell death in the SNO esophageal carcinoma cell line. A dose-dependent study (0.2-1.0 microM) was conducted with an exposure time of 24 hours. Data revealed that 2-methoxyestradiol-bis-sulphamate reduced cell numbers statistically significantly to 74% after exposure to 0.4 microM of the drug. Morphological studies including light microscopy demonstrated hallmarks of apoptosis, while fluorescent microscopy revealed both the presence of apoptosis and autophagy as types of cell death being induced in SNO cells after 24 hours of exposure to 0.4 microM 2-methoxyestradiol-bis-sulphamate.

Influence of estradiol analogue on cell growth, morphology and death in esophageal carcinoma cells

2-Methoxyestradiol-bis-sulphamate is a bis-sulphamoylated derivative of the naturally occurring 17-beta-estradiol metabolite namely 2-methoxyestradiol. 2-Methoxyestradiol-bis-sulphamate is regarded as a potential anticancer drug with increased antiproliferative activity when compared to 2-methoxyestradiol. The aim of this pilot in vitro study was to determine the influence of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and possible induction of certain types of cell death in the SNO esophageal carcinoma cell line. A dose-dependent study (0.2-1.0 microM) was conducted with an exposure time of 24 hours. Data revealed that 2-methoxyestradiol-bis-sulphamate reduced cell numbers statistically significantly to 74% after exposure to 0.4 microM of the drug. Morphological studies including light microscopy demonstrated hallmarks of apoptosis, while fluorescent microscopy revealed both the presence of apoptosis and autophagy as types of cell death being induced in SNO cells after 24 hours of exposure to 0.4 microM 2-methoxyestradiol-bis-sulphamate.(AU)

Influence of estradiol analogue on cell growth, morphology and death in esophageal carcinoma cells.

2-Methoxyestradiol-bis-sulphamate is a bis-sulphamoylated derivative of the naturally occurring 17-beta-estradiol metabolite namely 2-methoxyestradiol. 2-Methoxyestradiol-bis-sulphamate is regarded as a potential anticancer drug with increased antiproliferative activity when compared to 2-methoxyestradiol. The aim of this pilot in vitro study was to determine the influence of 2-methoxyestradiol-bis-sulphamate on cell growth, morphology and possible induction of certain types of cell death in the SNO esophageal carcinoma cell line. A dose-dependent study (0.2-1.0 microM) was conducted with an exposure time of 24 hours. Data revealed that 2-methoxyestradiol-bis-sulphamate reduced cell numbers statistically significantly to 74% after exposure to 0.4 microM of the drug. Morphological studies including light microscopy demonstrated hallmarks of apoptosis, while fluorescent microscopy revealed both the presence of apoptosis and autophagy as types of cell death being induced in SNO cells after 24 hours of exposure to 0.4 microM 2-methoxyestradiol-bis-sulphamate.

In vitro effects of Sutherlandia frutescens water extracts on cell numbers, morphology, cell cycle progression and cell death in a tumorigenic and a non-tumorigenic epithelial breast cell line.

Sutherlandia frutescens is a South African herb traditionally used for internal cancers, diabetes, a variety of inflammatory conditions and recently to improve the overall health in cancer and HIV/AIDS patients. The in vitro effects of S. frutescens extracts were evaluated on cell numbers, morphology, cell cycle progression and cell death. Dose-dependent studies (2-10 mg/ml) revealed a decrease in malignant cell numbers when compared to their controls. S. frutescens extracts (10 mg/ml) decreased cell growth in a statistically significantly manner to 26% and 49% (P<0.001) in human breast adenocarcinoma (MCF-7) and human non-tumorigenic epithelial mammary gland cells (MCF-12A) respectively after 72 h of exposure. Cell density was significantly compromised and hypercondensed chromatin, cytoplasmic shrinking, membrane blebbing and apoptotic bodies were more pronounced in the MCF-7 cell line. Both S. frutescens-treated cell lines exhibited and increased tendency for acridine orange staining, suggesting increased lysosomal and/or autophagy activity. Flow cytometry showed an increase in the sub G(1) apoptotic fraction and an S phase arrest in both the 5 mg/ml and 10 mg/ml S. frutescens-treated cells. S. frutescens induced an increase in apoptosis in both cell lines as detected by Annexin V and propidium iodide flow cytometric measurement. At 10 mg/ml, late stages of apoptosis were more prominent in MCF-7 S. frutescens-treated cells when compared to the MCF-12A cells. Transmission electron microscopy revealed hallmarks of increased vacuolarization and hypercondensed chromatin, suggesting autophagic and apoptotic processes. The preliminary study demonstrates that S. frutescens water extracts exert a differential action mechanism in non-tumorigenic MCF-12A cells when compared to tumorigenic MCF-7 cells, warranting future studies on this multi-purpose medicinal plant in southern Africa.

Influence of 2-methoxyestradiol on MCF-7 cells: an improved differential interference contrasting technique and Bcl-2 and Bax protein expression levels.

Proteins of the B-cell lymphoma 2 family are crucial for the regulation of apoptosis. B-cell lymphoma 2-associated X is a pro-apoptotic protein, while B-cell lymphoma 2 protein opposes apoptosis. The influence of 1 microM 2-methoxyestradiol was investigated on the expression levels of these two proteins in MCF-7 cells. 2-Methoxyestradiol exposure did not influence B-cell lymphoma 2 protein expression levels after 24 h of exposure. In contrast, B-cell lymphoma 2-associated X protein levels were significantly reduced. An improved differential interference contrasting technique revealed compromised cell density and the presence of a mitotic block in exposed cells. The study proposes that the influence of 2-methoxyestradiol on the expression of these proteins may be time- and cell type dependent and thus not evident during the mitotic block observed. Investigation of the regulation of the B-cell lymphoma 2 family will allow researchers to consider signaling pathways for diseases where apoptosis can potentially be controlled.

Influence of 2-methoxyestradiol on MCF-7 cells: an improved differential interference contrasting technique and Bcl-2 and Bax protein expression levels

Proteins of the B-cell lymphoma 2 family are crucial for the regulation of apoptosis. B-cell lymphoma 2-associated X is a pro-apoptotic protein, while B-cell lymphoma 2 protein opposes apoptosis. The influence of 1 microM 2-methoxyestradiol was investigated on the expression levels of these two proteins in MCF-7 cells. 2-Methoxyestradiol exposure did not influence B-cell lymphoma 2 protein expression levels after 24 h of exposure. In contrast, B-cell lymphoma 2-associated X protein levels were significantly reduced. An improved differential interference contrasting technique revealed compromised cell density and the presence of a mitotic block in exposed cells. The study proposes that the influence of 2-methoxyestradiol on the expression of these proteins may be time- and cell type dependent and thus not evident d uring the mitotic block observed. Investigation of the regulation of the B-cell lymphoma 2 family will allow researchers to consider signaling pathways for diseases where apoptosis can potentially be controlled.

Influence of 2-methoxyestradiol on MCF-7 cells: an improved differential interference contrasting technique and Bcl-2 and Bax protein expression levels

Proteins of the B-cell lymphoma 2 family are crucial for the regulation of apoptosis. B-cell lymphoma 2-associated X is a pro-apoptotic protein, while B-cell lymphoma 2 protein opposes apoptosis. The influence of 1 microM 2-methoxyestradiol was investigated on the expression levels of these two proteins in MCF-7 cells. 2-Methoxyestradiol exposure did not influence B-cell lymphoma 2 protein expression levels after 24 h of exposure. In contrast, B-cell lymphoma 2-associated X protein levels were significantly reduced. An improved differential interference contrasting technique revealed compromised cell density and the presence of a mitotic block in exposed cells. The study proposes that the influence of 2-methoxyestradiol on the expression of these proteins may be time- and cell type dependent and thus not evident d uring the mitotic block observed. Investigation of the regulation of the B-cell lymphoma 2 family will allow researchers to consider signaling pathways for diseases where apoptosis can potentially be controlled.(AU)

In vitro effects of 2-methoxyestradiol on cell morphology and Cdc2 kinase activity in SNO oesophageal carcinoma cells.

The effects of 1 x 10(-6) M exogenous 2-methoxyestradiol (2 ME) were determined on cell morphology and cell division cycle (Cdc) 2 kinase activity in SNO oesophageal carcinoma cells. Mitotic indices revealed an increase in metaphase cells (11.2%) when compared to the 0.5% vehicle-treated cells after 18 h of exposure to 2 ME. Vehicle-treated control cells did not show any hallmarks of apoptosis after 18 h of exposure to dimethyl sulphoxide. Only 0.5% of 2 ME-treated cells showed characteristics of apoptosis. Conversely, increased morphological hallmarks of apoptosis were observed in SNO-treated cells after 21.5 h of 2 ME exposure. When compared to the 0.5% in vehicle-treated cells, 4.7% of cells were in apoptosis. Furthermore, 34.1% of cells were blocked in metaphase after 21.5 h of 2 ME exposure compared to 0.6% of vehicle-control cells. In addition, Cdc2 kinase activity was statistically significantly increased (1.3-fold) (p<0.005) in 2 ME-treated cells when compared to vehicle-treated controls. The present preliminary study suggests that the accumulation observed in metaphase cells and the increase in Cdc2 kinase activity caused by 2 ME are consistent with morphological hallmarks of mitotic arrest and disrupted mitotic spindle formation, thus leading to induction of apoptosis in SNO cells.

Influence of 2-methoxyestradiol on cell morphology and cdc2 kinase activity in WHCO3 esophageal carcinoma cells.

The influence of 1 x 10(-6) M exogenous 2-methoxyestradiol (2ME) was investigated on nuclear and cytoplasmic morphology, as well as Cdc (cell division cycle) 2 kinase activity in WHCO3 esophageal carcinoma cells. Mitotic indices after 18 h of 2ME exposure revealed an increase in metaphase cells (9.0%) when compared to the vehicle-treated cells (0.9%). 2ME-treated cells showed apoptotic cells at 5.6% after 18 h of exposure to dimethyl sulphoxide, compared to 0.9% in vehicle-treated cells. Increased morphological characteristics of apoptosis were observed in 2ME-treated cells after 21.5 h of exposure. Twelve percent of cells were in apoptosis when compared to the 1.6% of vehicle-treated cells. Furthermore, 42.4% of cells were arrested in metaphase after 21.5 h of 2ME exposure compared to 2.9% of vehicle-control cells present in metaphase. Cdc2 kinase activity was statistically significantly increased (1.7-fold) (P < 0.005) after 18 h of 2ME exposure when compared to vehicle-treated controls. Although the mechanism of 2ME's action on esophageal carcinoma cells is not yet elucidated, the present study revealed that 2ME caused metaphase arrest, as well as an increase in Cdc2 kinase activity that culminated in the induction of apoptosis in these cells.