Combined Application of Honokiol and 2-Deoxyglucose against MCF7 Breast Cancer Cells Under Hypoxia

Breast cancer is the most common cancer among women. Epidemiologists estimate that over 2.3 million new cases of breast cancer are diagnosed worldwide each year. Natural compounds represent promising molecules for the development of antitumor drugs; among them, lignans show significant antiproliferative effects against breast cancer cells. The goal of the study was to analyse the antiproliferative effects of lignan honokiol on MCF7 breast cancer cells, find synergistic combinations of honokiol with 2-deoxyglucose, and evaluate the effects of the combinations found on cells in hypoxia. The antiproliferative effects of the compounds were evaluated by the MTT test, and protein expression analysis was performed by immunoblotting. Honokiol inhibited MCF7 cell growth with an IC50 value of 19.7 μM. Synergistic combinations of honokiol with the glycolysis inhibitor 2-deoxyglucose were detected; the compounds at low doses caused significant suppression of MCF7 cell growth. The established combinations of compounds inhibited HIF-1α expression and were effective in hypoxia, considered as the leading factor of chemotherapeutic resistance. Oestrogen receptor alpha (ERα) is the main growth driver of hormone-dependent breast tumours. Honokiol combined with 2-deoxyglucose reduced ERα expression in MCF7 cells, and expression of the hormone-dependent protein GREB1 was also downregulated. Honokiol at a concentration of 15 μM in combination with 6 mM 2-deoxyglucose induced the cleavage of PARP (a marker of apoptosis) in MCF7 cells after 48 h of incubation. The cells treated with the combination of honokiol and 2-deoxyglucose demonstrated a decrease in the expression of cyclin D1. Thus, honokiol represents a promising basis for the development of antitumor agents; the combination of this natural compound with glycolysis inhibitors can be used to reduce the applied doses

Targeting AKT Kinase in Hydroxytamoxifen-Resistant Breast Cancer Cells

More than 650,000 people die each year from breast cancer, making it a particularly significant disease worldwide. The development of about 70% of breast tumors depends on steroid hormones, namely, estrogens. Estrogens trigger signaling pathways that support tumor growth and progression. Hydroxytamoxifen (HT) halting estrogen-induced tumor growth is among the most effective drugs in current anticancer therapy. The purpose of this work was to investigate approaches to overcome breast cancer cell resistance to hydroxytamoxifen. Cells with resistance to the antiestrogen hydroxytamoxifen were obtained by long-term incubation of parental MCF7 cells with this drug. Estrogen receptor α (ERα) expression and progesterone receptor (PR) expression were analyzed by immunoblotting. The resistant MCF7/HT cells were found not to lose ERα expression. These cells were found to have slightly reduced ERα activity when compared to parental MCF7 cells. The expression of PR, one of the ERα targets, was downregulated in hydroxytamoxifen-resistant cells. AKT kinase belongs to the PI3K/AKT/mTOR signaling pathway, and its activity is associated with resistance. Three types of AKT inhibitors were evaluated, including AKT inhibitor IV (6-(2-benzothiazolyl)-1-ethyl-2-[2-(methylphenylamino)ethenyl]-3-phenyl-1H-benzimidazolium, monoiodide), 10-DEBC (2-chloro-N,N-diethyl-10H-phenoxazine-10-butanamine, monohydrochloride), and luminespib (HSP90 inhibitor, 5-[2,4-dihydroxy-5-(1-methylethyl)phenyl]-N-ethyl-4-[4-(4-morpholinylmethyl)phenyl]-3-isoxazolecarboxamide). All three compounds showed high antiproliferative activity against hydroxytamoxifen-resistant cells. The IC50 value of 10-DEBC was 4.2 µM, and this was when the AKT inhibitor IV was more active with IC50 value of 390 nM. The HSP90 inhibitor luminespib, which reduces AKT expression, showed the highest activity against parental and hydroxytamoxifen-resistant breast cancer cells, with IC50 values of 14 and 18 nM, respectively. Thus, the hydroxytamoxifen-resistant cells were found to partially retain hormone signaling and to be sensitive to selective AKT inhibitors. The best effects were discovered for HSP90-AKT blocker luminespib, with an IC50 value of about 20 nM

Inhibitory Effects of 5-Fluorouracil on the Growth of 4-Hydroxytamoxifen-Resistant and Sensitive Breast Cancer Cells

Cancer is one of the leading causes of death worldwide, accounting for about 10 million deaths a year, or nearly one in six deaths. The most common types of cancer are breast, colorectal, lung, and prostate cancers. Prolonged application of hormone drugs leads to the development of resistance. The development of agents with high activity against resistant cells is a challenge. It is important to create novel targeted compounds and search for active molecules among those previously developed. The study aimed to evaluate the sensitivity of 4-hydroxytamoxifen-resistant cells to 5-fluorouracil (5-FU) and analyse the signalling pathways that are regulated by 5-FU in breast cancer cells. Antiproliferative activity of compounds was assessed by the MTT assay, and immunoblotting was used to evaluate the expression of proteins in breast cancer cells. Activity of 5-FU was evaluated on parental MCF7 cells and a cell subline with resistance to 4-hydroxytamoxifen (HT), named MCF7/HT. The MCF7/HT cells showed high sensitivity to 5-FU. Expression of oestrogen receptor α (ERα, a key driver of breast cancer growth) in MCF7 and MCF7/HT cells was not sensitive to 5-FU treatment. In both parental and resistant cells, 5-FU induced changes in the activity of several signalling proteins. 5-FU activated AKT, extracellular signal-regulated kinase 1/2 (ERK 1/2) and upregulated cyclin D1 expression. The data suggest that 5-FU should be further investigated as a chemotherapeutic for hormone-resistant cancers; the combination of 5-FU with novel apoptosis inducer LCTA-3344 is considered effective to inhibit the growth of breast cancer cells, including those that are hormone-resistant

Targeting hormone-resistant breast cancer cells with docetaxel: a look inside the resistance

Aim: The study aims to analyze the effect of long-term incubation of ERα-positive MCF7 breast cancer cells with 4-hydroxytamoxifen (HT) on their sensitivity to tubulin polymerization inhibitor docetaxel.Methods: The analysis of cell viability was performed by the MTT method. The expression of signaling proteins was analyzed by immunoblotting and flow cytometry. ERα activity was evaluated by gene reporter assay. To establish hormone-resistant subline MCF7, breast cancer cells were treated with 4-hydroxytamoxifen for 12 months.Results: The developed MCF7/HT subline has lost sensitivity to 4-hydroxytamoxifen, and the resistance index was 2. Increased Akt activity (2.2-fold) and decreased ERα expression (1.5-fold) were revealed in MCF7/HT cells. The activity of the estrogen receptor α was reduced (1.5-fold) in MCF7/HT. Evaluation of class III β-tubulin expression (TUBB3), a marker associated with metastasis, revealed the following trends: higher expression of TUBB3 was detected in triple-negative breast cancer MDA-MB-231 cells compared to hormone-responsive MCF7 cells (P < 0.05). The lowest expression of TUBB3 was found in hormone-resistant MCF7/HT cells (MCF7/HT < MCF7 < MDA-MB-231, approximately 1:2:4). High TUBB3 expression strongly correlated with docetaxel resistance: IC50 value of docetaxel for MDA-MB-231 cells was greater than that for MCF7 cells, whereas resistant MCF7/HT cells were the most sensitive to the drug. The accumulation of cleaved PARP (a 1.6-fold increase) and Bcl-2 downregulation (1.8-fold) were more pronounced in docetaxel-treated resistant cells (P < 0.05). The expression of cyclin D1 decreased (2.8-fold) only in resistant cells after 4 nM docetaxel treatment, while this marker was unchanged in parental MCF7 breast cancer cells.Conclusion: Further development of taxane-based chemotherapy for hormone-resistant cancer looks highly promising, especially for cancers with low TUBB3 expression