Clinacanthus Nutans Induces Antiproliferative and Apoptosis in Human Breast Cancer Cells Through Targeted Apoptosis Pathway

Clinacanthus nutans dichloromethane fraction (CN-Dcm) extract has previously been proven to suppress breast cancer (MCF7) cell proliferation. Despite this, the molecular mechanisms involved in C. nutans extract-treated MCF7 cells are unknown. Hence, the molecular mechanism of apoptosis in treated MCF7 was investigated in this current study. This study was intended to subfractionate CN-Dcm extract using column chromatography and analysed the treated MCF7 cells using the CellTiter 96® AQueous One Solution Cell Proliferation (MTS) assay, Annexin V/propidium iodide (PI) assay, western blot and reverse transcription-qualitative polymerase chain reaction (RT-qPCR). Out of nine subfraction extracts (SF1 to SF9), SF2 extract strongly inhibited MCF7 cells with the lowest IC50 value (23.51 ± 0.99 µg/mL) and substantially induced apoptosis in the MCF7 cells. SF2 extract significantly downregulated BCL-2 expression and upregulated P53, BAX, BID, BCL-2, caspase-8, caspase-9 and caspase-3 expressions in treated MCF7 cells. Therefore, SF2 extract was analysed using liquid chromatography coupled to quadrupole time–of–flight mass spectrometry (LC-QTOF-MS), which confirmed the presence of bioactive chemical compounds. Thus, it can be concluded that the compounds found in SF2 extract may potentially cause apoptosis in MCF7 cells through intrinsic and extrinsic pathways.

Non-homogeneous dispersion of graphene in polyacrylonitrile substrates induces a migrastatic response and epithelial-like differentiation in MCF7 breast cancer cells

Background Recent advances from studies of graphene and graphene-based derivatives have highlighted the great potential of these nanomaterials as migrastatic agents with the ability to modulate tumor microenvironments. Nevertheless, the administration of graphene nanomaterials in suspensions in vivo is controversial. As an alternative approach, herein, we report the immobilization of high concentrations of graphene nanoplatelets in polyacrylonitrile film substrates (named PAN/G10) and evaluate their potential use as migrastatic agents on cancer cells. Results Breast cancer MCF7 cells cultured on PAN/G10 substrates presented features resembling mesenchymal-to-epithelial transition, e.g., (i) inhibition of migratory activity; (ii) activation of the expression of E-cadherin, cytokeratin 18, ZO-1 and EpCAM, four key molecular markers of epithelial differentiation; (iii) formation of adherens junctions with clustering and adhesion of cancer cells in aggregates or islets, and (iv) reorganization of the actin cytoskeleton resulting in a polygonal cell shape. Remarkably, assessment with Raman spectroscopy revealed that the above-mentioned events were produced when MCF7 cells were preferentially located on top of graphene-rich regions of the PAN/G10 substrates. Conclusions The present data demonstrate the capacity of these composite substrates to induce an epithelial-like differentiation in MCF7 breast cancer cells, resulting in a migrastatic effect without any chemical agent-mediated signaling. Future works will aim to thoroughly evaluate the mechanisms of how PAN/G10 substrates trigger these responses in cancer cells and their potential use as antimetastatics for the treatment of solid cancers. Graphical Abstract

Co-Inhibition of P-gp and Hsp90 by an Isatin-Derived Compound Contributes to the Increase of the Chemosensitivity of MCF7/ADR-Resistant Cells to Doxorubicin

Breast cancer is a complex and multi-drug resistant (MDR) disease, which could result in the failure of many chemotherapeutic clinical agents. Discovering effective molecules from natural products or by derivatization from known compounds is the interest of many research studies. The first objective of the present study is to investigate the cytotoxic combinatorial, chemosensitizing, and apoptotic effects of an isatin derived compound (5,5-diphenylimidazolidine-2,4-dione conjugated with 5-substituted isatin, named HAA2021 in the present study) against breast cancer cells (MCF7) and breast cancer cells resistant to doxorubicin (MCF7/ADR) when combined with doxorubicin. The second objective is to investigate the binding mode of HAA2021 withP-glycoprotein (P-gp) and heat shock protein 90 (Hsp90), and to determine whether their co-inhibition by HAA2021 contribute to the increase of the chemosensitization of MCF7/ADR cells to doxorubicin. The combination of HAA2021, at non-toxic doses, with doxorubicin synergistically inhibited the proliferation while inducing significant apoptosis in MCF7 cells. Moreover, HAA2021 increased the chemosensitization of MCF7/ADR cells to doxorubicin, resulting in increased cytotoxicity/selectivity and apoptosis-inducing efficiency compared with the effect of doxorubicin or HAA2021 alone against MCF7/ADR cells. Molecular modeling showed that two molecules of HAA2021 bind to P-gp at the same time, causing P-gp inhibitory effect of the MDR efflux pump, and accumulation of Rhodamine-123 (Rho123) in MCF7/ADR cells. Furthermore, HAA2021 stably interacted with Hsp90α more efficiently compared with 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), which was confirmed with the surface plasmon resonance (SPR) and molecular modeling studies. Additionally, HAA2021 showed multi-target effects via the inhibition of Hsp90 and nuclear factor kappa B (NF-𝜅B) proteins in MCF7 and MCF7/ADR cells. Results of real time-PCR also confirmed the synergistic co-inhibition of P-gp/Hsp90α genes in MCF7/ADR cells. Further pharmacokinetic and in vivo studies are warranted for HAA2021 to confirm its anticancer capabilities.

Extracellular Vesicles Induce an Aggressive Phenotype in Luminal Breast Cancer Cells Via PKM2 Phosphorylation

BackgroundAerobic glycolysis is a hallmark of glucose metabolism in cancer. Previous studies have suggested that cancer cell–derived extracellular vesicles (EVs) can modulate glucose metabolism in adjacent cells and promote disease progression. We hypothesized that EVs originating from cancer cells can modulate glucose metabolism in recipient cancer cells to induce cell proliferation and an aggressive cancer phenotype.MethodsTwo breast cancer cell lines with different levels of glycolytic activity, MDA-MB-231 cells of the claudin-low subtype and MCF7 cells of the luminal type, were selected and cocultured as the originating and recipient cells, respectively, using an indirect coculture system, such as a Transwell system or a microfluidic system. The [18F]fluorodeoxyglucose (FDG) uptake by the recipient MCF7 cells was assessed before and after coculture with MDA-MB-231 cells. Proteomic and transcriptomic analyses were performed to investigate the changes in gene expression patterns in the recipient MCF7 cells and MDA-MB-231 cell-derived EVs.ResultsFDG uptake by the recipient MCF7 cells significantly increased after coculture with MDA-MB-231 cells. In addition, phosphorylation of PKM2 at tyrosine-105 and serine-37, which is necessary for tumorigenesis and aerobic glycolysis, was highly activated in cocultured MCF7 cells. Proteomic profiling revealed the proliferation and dedifferentiation of MCF7 cells following coculture with MDA-MB-231 cells. Transcriptomic analysis demonstrated an increase in glycolysis in cocultured MCF7 cells, and the component analysis of glycolysis-related genes revealed that the second most abundant component after the cytoplasm was extracellular exosomes. In addition, proteomic analysis of EVs showed that the key proteins capable of phosphorylating PKM2 were present as cargo inside MDA-MB-231 cell-derived EVs.ConclusionsThe phenomena observed in this study suggest that cancer cells can induce a phenotype transition of other subtypes to an aggressive phenotype to consequently activate glucose metabolism via EVs. Therefore, this study could serve as a cornerstone for further research on interactions between cancer cells.

Diospyros Kaki Leaves Decoction Phytochemical Characterization and Bioactivities Evaluation: LC-MS-Qtof Identification, Antioxidant Activity, Enzyme Inhibition and Cytotoxicity Toward HepG2 and Mcf-7 Cell Lines

Diospyros kaki known as Persimmon has played an important role in Chinese medicine, and its leaves are used in pharmaceuticals, cosmetics, and beverages due to their biological properties. The aim of this study was to evaluate chemical composition, antioxidant activity, enzyme inhibition. The main compounds of Diospyros kaki leaves decoctions were identified using high-resolution liquid chromatography – high-resolution tandem mass spectrometry (LC-HRMS-MS) interfaced with a Quadrupole Time-of-Flight (Q-tof). The obtained results showed that the aqueous extract had a high content of total phenolsand flavonoids and showed strong antioxidant activity (EC50 between 3.79±0.19 and 12.05±0.11µg/mL). In addition, these extracts were able to inhibit3-hydroxy-3-methylglutaril-CoA (HMG-CoA) reductase and acetylcholinesterase (IC50 ranging from 58.35±0.69 to 210.21±28.9 µg/mL). The cytotoxicity against human liver cells HepG2 and breast MCF7 cell lines was also evaluated. No cytotoxicity to HepG2 and MCF7 cells was detected. These results indicate that Diospyros kaki leaves could be considered as attractive source of health-promoting ingredients and a promising plant for the development of new functional foods and pharmaceutical products.

Investigation of the effect of Prunus Amygdalus Amara on the expression of some genes of apoptosis and immortality in breast cancer cells (MCF-7)

Background: Anti-cancer effects of almond nuts or oil have been approved, but there are a few pieces of research that have evaluated, in detail, almond and other seeds' effects on cancer. Therefore, in the present project, the aim was to explore the regulatory effect of the bitter almond extract (Prunus amygdalus Batsch) on the apoptotic and anti-cancer potency of MCF-7 cells. Objectives: In the current experimental research, the Almond effect on MCF7 cells was evaluated by investigating the expression and the balance between Bcl-2, Bax genes to unmark the potential molecular mechanism. Methods: For 24 and 48h, the MCF7 cells were treated with the bitter almond extract (187.5-3000 µg/mL). MTT assay was used to assess the viability, and Real-time-PCR was applied to determine the expression of Bax and Bcl-2, facing β-actin. Results: Our results revealed a significant difference between different extract concentrations on the viability of MCF7 cell lines in 24 and 48 h; cell viability decreased time-dependently (P < 0.05). After 24 and 48h of extract facing MCF7 cells, the evaluated IC50 value was 3000 and 1500 µg/mL, respectively. Based on Real Time-PCR analysis, after 24 and 48 h, the mRNA levels of BCL-2 decreased by the extract, whereas BAX was in the MCF-7 cell line. Conclusion: From the results, it can be concluded that bitter almond extract has anti-cancer properties that may influence the apoptotic pathways by regulating relative gene expression.

EGFR Transgene Stimulates Spontaneous Formation of MCF7 Breast Cancer Cells Spheroids with Partly Loss of HER3 Receptor

Multicellular spheroids with 3D cell–cell interactions are a useful model to simulate the growth conditions of cancer. There is evidence that in tumor spheroids, the expression of various essential molecules is changed compared to the adherent form of cell cultures. These changes include growth factor receptors and ABC transporters and result in the enhanced invasiveness of the cells and drug resistance. It is known that breast adenocarcinoma MCF7 cells can spontaneously form 3D spheroids and such spheroids are characterized by high expression of EGFR/HER2, while the natural phenotype of MCF7 cells is EGFRlow/HER2low. Therefore, it was interesting to reveal if high epidermal growth factor receptor (EGFR) expression is sufficient for the conversion of adherent MCF7 to spheroids. In this study, an MCF7 cell line with high expression of EGFR was engineered using the retroviral transduction method. These MCF7-EGFR cells assembled in spheroids very quickly and grew predominantly as a 3D suspension culture with no special plates, scaffolds, growth supplements, or exogenous matrixes. These spheroids were characterized by a rounded shape with a well-defined external border and 100 µM median diameter. The sphere-forming ability of MCF7-EGFR cells was up to 5 times stronger than in MCF7wt cells. Thus, high EGFR expression was the initiation factor of conversion of adherent MCF7wt cells to spheroids. MCF7-EGFR spheroids were enriched by the cells with a cancer stem cell (CSC) phenotype CD24−/low/CD44− in comparison with parental MCF7wt cells and MCF7-EGFR adhesive cells. We suppose that these properties of MCF7-EGFR spheroids originate from the typical features of parental MCF7 cells. We showed the decreasing of HER3 receptors in MCF7-EGFR spheroids compared to that in MCFwt and in adherent MCF7-EGFR cells, and the same decrease was observed in the MCF7wt spheroids growing under the growth factors stimulation. To summarize, the expression of EGFR transgene in MCF7 cells stimulates rapid spheroids formation; these spheroids are enriched by CSC-like CD24−/CD44− cells, they partly lose HER3 receptors, and are characterized by a lower potency in drug resistance pomp activation compared to MCF7wt. These MCF7-EGFR spheroids are a useful cancer model for the development of anticancer drugs, including EGFR-targeted therapeutics.

In silico analysis and in planta production of recombinant ccl21/IL1β protein and characterization of its in vitro anti-tumor and immunogenic activity

CCL21 has an essential role in anti-tumor immune activity. Epitopes of IL1β have adjuvant activity without causing inflammatory responses. CCR7 and its ligands play a vital role in the immune balance; specifically, in transport of T lymphocytes and antigen-presenting cells such as dendritic cells to the lymph nodes. This study aimed to produce epitopes of CCL21 and IL1β as a recombinant protein and characterize it’s in vitro anti-tumor and immunogenic activity. A codon-optimized ccl21/IL1β gene was designed and synthesized from human genes. Stability and binding affinity of CCL21/IL1β protein and CCR7 receptor were examined through in silico analyses. The construct was introduced into N. tabacum to produce this recombinant protein and the structure and function of CCL21/IL1β were examined. Purified protein from transgenic leaves generated a strong signal in SDS PAGE and western blotting assays. FTIR measurement and MALDI-TOF/TOF mass spectrography showed that ccl21/IL-1β was correctly expressed in tobacco plants. Potential activity of purified CCL21/IL1β in stimulating the proliferation and migration of MCF7CCR7+ cancer cell line was investigated using the wound healing method. The results demonstrated a decrease in survival rate and metastasization of cancer cells in the presence of CCL21/IL1β, and IC 50 of CCL21 on MCF7 cells was less than that of non-recombinant protein. Agarose assay on PBMCsCCR7+ showed that CCL21/IL1β has biological activity and there is a distinguishable difference between chemokinetic (CCL21) and chemotactic (FBS) movements. Overall, the results suggest that CCL21/IL1β could be considered an effective adjuvant in future in vivo and clinical tests.