Honokiol inhibits the growth of hormone-resistant breast cancer cells: its promising effect in combination with metformin.

Background and purpose: Primary and metastatic breast cancers still represent an unmet clinical need for improved chemotherapy and hormone therapy. Considerable attention has been paid to natural anticancer compounds, especially lignans. The study aimed to evaluate the activity of several lignans against breast cancer cells and assess the effect of leading lignans on signaling pathways in combination with metformin. Experimental approach: Human breast cancer cell lines MCF7 (hormone-dependent), MDA-MB-231, and SKBR3 (hormone-independent) were used. A hormone-resistant MCF7/hydroxytamoxifen (HT) subline was obtained by long-term cultivation of the MCF7 line with hydroxytamoxifen. Antiproliferative activity was assessed by the MTT test; the expression of signaling pathway proteins was evaluated by immunoblotting analysis. Findings/Results: We evaluated the antiproliferative activity of lignans in breast cancer cells with different levels of hormone dependence and determined the relevant IC50 values. Honokiol was chosen as the leading compound, and its IC50 ranged from 12 to 20 µM, whereas for other tested lignans, the IC50 exceeded 50 µM. The accumulation of cleaved PARP and a decrease in the expression of Bcl-2 and ERα in MCF7/HT were induced following the combination of honokiol with metformin. Conclusions and implications: Honokiol demonstrated significant antiproliferative activity against both hormone-dependent breast cancer cells and lines with primary and acquired hormone resistance. The combination of honokiol with metformin is considered an effective approach to induce death in hormone-resistant cells. Honokiol is of interest as a natural compound with antiproliferative activity against breast cancers, including resistant tumors.

Exosomes are involved in the intercellular transfer of rapamycin resistance in the breast cancer cells.

Introduction: Resistance to chemotherapy and/or irradiation remains one of the key features of malignant tumors, which largely limits the efficiency of antitumor therapy. In this work, we studied the progression mechanism of breast cancer cell resistance to target drugs, including mTOR blockers, and in particular, we studied the exosome function in intercellular resistance transfer. Methods: The cell viability was assessed by the MTT assay, exosomes were purified by successive centrifugations, immunoblotting was used to evaluate protein expression, AP-1 activity was analyzed using reporter assay. Results: In experiments on the MCF-7 cell line (breast cancer) and the MCF-7/Rap subline that is resistant to rapamycin, the capability of resistant cell exosomes to trigger a similar rapamycin resistance in the parent MCF-7 cells was demonstrated. Exosome-induced resistance reproduces the changes revealed in MCF-7/Rap resistant cells, including the activation of ERK/AP-1 signaling, and it remains for a long time, for at least several months, after exosome withdrawal. We have shown that both the MCF-7 subline resistant to rapamycin and cells having exosome-triggered resistance demonstrate a stable decrease in the expression of DNMT3A, the key enzyme responsible for DNA methylation. Knockdown of DNMT3A in MCF-7 cells by siRNA leads to partial cell resistance to rapamycin; thus, the DNMT3A suppression is regarded as one of the necessary elements for the development of acquired rapamycin resistance. Conclusion: We propose that DNA demethylation followed by increased expression of key genes may be one of the factors responsible for the progression and maintenance of the resistant cell phenotype that includes exosome-induced resistance.

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 ß-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.

Photostability and Antiproliferative Activity of Furan Analogues of Combretastatin A-4.

Cancer is one of the most serious health problems that usually require heavy medical treatment. It is important to ensure that no additional burden is placed on patients due to the modes of administration and/or poor quality of pharmaceuticals. In this regard, understanding, quantifying, and improving the photostability (resistance to UV light or sunlight) of drugs is among the important elements that can improve the patient's quality of life. In this work, the photochemical properties of a wide range of furanone analogues of combretastatin A-4 and their antiproliferative activity against A-431 epidermoid carcinoma cells were studied in a search for compounds with improved photostability and antiproliferative activity. It was found that the incorporation of an arylidene moiety led to a significant improvement in photostability, while the antiproliferative activity strongly depends on the nature of the aryl residue in the arylidene moiety. The high photostability of arylidenes was achieved due to the delocalization of the central double bond of the 1,3,5-hexatriene system, which limited the 6π-electrocyclization. The best results in terms of antiproliferative activity were obtained for thiophene arylidene (IC50 = 0.6 µM) and 3,4-diarylfuran (IC50 = 0.047 µM). The obtained results address the lack of data available now in scientific literature on the photodegradation of combretastatin A-4 analogues and should be taken into account in studies of the side effects of pharmaceuticals based on them.

In Vitro Pharmacological Screening of Essential Oils from Baccharis parvidentata and Lippia origanoides Growing in Brazil.

In this study, the in vitro antimicrobial, antiparasitic, antiproliferative and cytotoxic activities of essential oil from Baccharis parvidentata Malag. (EO-Bp) and Lippia origanoides Kunth (EO-Lo) were explored. The relevant effects were observed against the parasitic protozoans Plasmodium falciparum, Trypanosoma cruzi, Trypanosoma brucei and Leishmania amazonensis (ranging 0.6 to 39.7 µg/mL) and malignant MCF-7, MCF-7/HT, 22Rv1, and A431 cell lines (ranging 6.1 to 31.5 µg/mL). In parallel, EO-Bp showed better selective indexes in comparison with EO-Lo against peritoneal macrophages from BALB/c mice and MRC-5 cell line. In conclusion, EO-Lo is known to show a wide range of health benefits that could be added as another potential use of this oil with the current study. In the case of EO-Bp, the wide spectrum of its activities against protozoal parasites and malignant cells, as well as its selectivity in comparison with non-malignant cells, could suggest an interesting candidate for further tests as a new therapeutic alternative.

Towards Unravelling the Role of ERα-Targeting miRNAs in the Exosome-Mediated Transferring of the Hormone Resistance.

Hormone therapy is one of the most effective breast cancer treatments, however, its application is limited by the progression of hormonal resistance, both primary or acquired. The development of hormonal resistance is caused either by an irreversible block of hormonal signalling (suppression of the activity or synthesis of hormone receptors), or by activation of oestrogen-independent signalling pathways. Recently the effect of exosome-mediated intercellular transfer of hormonal resistance was revealed, however, the molecular mechanism of this effect is still unknown. Here, the role of exosomal miRNAs (microRNAs) in the transferring of hormonal resistance in breast cancer cells has been studied. The methods used in the work include extraction, purification and RNAseq of miRNAs, transfection of miRNA mimetics, immunoblotting, reporter analysis and the MTT test. Using MCF7 breast cancer cells and MCF7/T tamoxifen-resistant sub-line, we have found that some miRNAs, suppressors of oestrogen receptor signalling, are overexpressed in the exosomes of the resistant breast cancer cells. The multiple (but not single) transfection of one of the identified miRNA, miR-181a-2, into oestrogen-dependent MCF7 cells induced the irreversible tamoxifen resistance associated with the continuous block of the oestrogen receptor signalling and the activation of PI3K/Akt pathway. We suppose that the miRNAs-ERα suppressors may act as trigger agents inducing the block of oestrogen receptor signalling and breast cancer cell transition to an aggressive oestrogen-independent state.

Secretion of Mutant DNA and mRNA by the Exosomes of Breast Cancer Cells.

Exosomes are the small vesicles that are secreted by different types of normal and tumour cells and can incorporate and transfer their cargo to the recipient cells. The main goal of the present work was to study the tumour exosomes' ability to accumulate the parent mutant DNA or RNA transcripts with their following transfer to the surrounding cells. The experiments were performed on the MCF7 breast cancer cells that are characterized by the unique coding mutation in the PIK3CA gene. Using two independent methods, Sanger sequencing and allele-specific real-time PCR, we revealed the presence of the fragments of the mutant DNA and RNA transcripts in the exosomes secreted by the MCF7 cells. Furthermore, we demonstrated the MCF7 exosomes' ability to incorporate into the heterologous MDA-MB-231 breast cancer cells supporting the possible transferring of the exosomal cargo into the recipient cells. Sanger sequencing of the DNA from MDA-MB-231 cells (originally bearing a wild type of PIK3CA) treated with MCF7 exosomes showed no detectable amount of mutant DNA or RNA; however, using allele-specific real-time PCR, we revealed a minor signal from amplification of a mutant allele, showing a slight increase of mutant DNA in the exosome-treated MDA-MB-231 cells. The results demonstrate the exosome-mediated secretion of the fragments of mutant DNA and mRNA by the cancer cells and the exosomes' ability to transfer their cargo into the heterologous cells.

Antiestrogenic and antiproliferative potency of secoisolariciresinol diglucoside derivatives on MCF-7 breast cancer cells.

Secoisolariciresinol diglucoside (SDG) is isolated from Linum usitatissimum seeds. The antiproliferative effects of SDG (1) and its derivatives secoisolariciresinol (2) and secoisolariciresinol-4', 4″-diacetate (3) have been evaluated on MCF-7 breast cancer cells and normal breast epithelial line MCF-10A. Lignan 1 has not shown cytotoxic effects on MCF-7 cells, while derivatives 2 and 3 have inhibited cell growth with IC50 values of 25 and 11 µM, respectively. Estrogen receptor alpha is a key growth driver in MCF-7 cells. Compound 1 did not affect the activity of ERα, while derivatives 2 and 3 showed significant antiestrogenic effects. Compounds 2 and 3 caused apoptosis in the MCF-7 line, determined by the cleavage of PARP. SDG derivative 3 enhanced the effect of doxorubicin. SDG derivatives can be considered as promising agents that exhibit a combined antiestrogen and proapoptotic effect in hormone-dependent breast cancer cells.

Novel d-Annulated Pentacyclic Steroids: Regioselective Synthesis and Biological Evaluation in Breast Cancer Cells.

The acid-catalyzed cyclization of benzylidenes based on 16-dehydropregnenolone acetate (16-DPA) was studied. It was found that these compounds readily undergo regioselective interrupted Nazarov cyclization with trapping chloride ion and an efficient method of the synthesis of d-annulated pentacyclic steroids based on this reaction was proposed. The structures of the synthesized pentacyclic steroids were determined by NMR and X-ray diffraction. It was found that the reaction affords a single diastereomer, but the latter can crystallize as two conformers depending on the structure. Antiproliferative activity of synthesized compounds was evaluated against two breast cancer cell lines: MCF-7 and MDA-MB-231. All tested compounds showed relatively high antiproliferative activity. The synthetic potential of the protocol developed was illustrated by the gram-scale experiment.