Gene Expression In MCF-7 Cells Research Articles

Overcoming Breast Cancer Drug Resistance: A Novel Approach Using siRNA-Mediated P-glycoprotein Downregulation to Enhance Vinorelbine Efficacy

Purpose: Cancer, the second leading cause of mortality worldwide, represents a global health challenge, primarily due to drug resistance. Vinorelbine (Vino) is a chemotherapeutic agent that disrupts cancer cell growth by targeting microtubules and inducing apoptosis. However, drug resistance remains a formidable obstacle. This resistance is caused by various factors including genetic mutations, drug efflux mechanisms, and DNA repair systems. Resolution of this challenge requires an innovative approach. This study investigated the potential of small interfering RNA (siRNA) to target and downregulate a vinorelbine-resistant MCF-7/ADR breast cancer cell line. Methods: Cells were cultured in Dulbecco’s modified Eagle’s medium 10% fetal bovine serum/penicillin/streptomycin. An siRNA targeting ABCB1 was designed and synthesized, and the cells were transfected with siRNA at final concentrations of 10, 20, and 30 nM. The3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to assess cell viability. ABCB1 mRNA expression levels were determined by real-time PCR. Results: MCF-7 cells exhibited a higher sensitivity to vinorelbine than MCF-7/ADR cells. MCF-7/ADR cells exhibited resistance to vinorelbine at concentrations, 12.50 and 25.00 μM. Treatment with siRNA significantly reduced ABCB1 expression by 2.93-fold (p = 0.0001). Similarly, co-treatment with siRNA and vinorelbine produced a substantial 2.89-fold decrease in ABCB1 gene expression in MCF-7 cells compared to that in MCF-7/ADR cells (P =0.0001). Conclusion: The results of the present study indicate that the concurrent use of siRNA and vinorelbine holds substantial promise as a therapeutic approach to overcome ABCB1-mediated multidrug resistance (MDR) in breast cancer. It is necessary to conduct comprehensive clinical trials to determine the true effectiveness of this combination therapy.

Baicalein prevents capecitabine-induced heart damage in female Wistar rats and enhances its anticancer potential in MCF-7 breast cancer cells

AimWe investigated the ability of baicalein (BAI) to enhance the anticancer potential of capecitabine (CAP) in the MCF-7 cell line and its protective effect on CAP-induced cardiotoxicity in female Wistar rats. Methods and key findingsIn vitro study involved evaluating the effect of BAI and/or CAP on cell viability, cell cycle progression, and BAX and Bcl2 gene expression in MCF-7 cells. Co-treatment of BAI with CAP significantly reduced the viability of MCF-7 cells, improved their cytotoxic effect, markedly elevated the percentage of the sub-G1 population, drastically reduced the G2/M population, and significantly altered the mRNA expression of BAX and Bcl2 genes compared with each treatment alone. In vivo study revealed that the oral administration of CAP (140 mg/kg BW) to adult female rats significantly elevated the levels of serum creatine kinase-myocardial band (CK-MB), lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β and cardiac TNF-α, IL-1β malondialdehyde (MDA) concentration, whereas it reduced the serum and cardiac total antioxidant capacity (TAC), level of cardiac glutathione (GSH) and activity of glutathione peroxidase (GPx) with a vast array of circulatory, inflammatory, degenerative, and necrotic alterations in the cardiac tissue. Furthermore, CAP administration significantly upregulated the mRNA expression of NF-κB, TLR4, MyD88, ATF6, CHOP, and JNK genes. Concurrent administration of BAI (200 mg/kg BW) and CAP significantly improved the biochemical alterations and cardiac oxidant/antioxidant status and architecture. In addition, it modulated the TLR4/MyD88/NF-κB pathway and endoplasmic reticulum stress. SignificanceAltogether, BAI can augment the anticancer potential of CAP and alleviate its cardiotoxic effects during cancer treatment.

Aryl Hydrocarbon Receptor Directly Regulates VTCN1 Gene Expression in MCF-7 Cells.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates the toxicity of dioxins and polycyclic aromatic hydrocarbons. Recent studies have suggested that AhR is involved in cancer immunity. In the present study, we examined whether AhR regulates the expression of immune checkpoint genes in breast cancer cells. We discovered that the mRNA expression of V-set domain containing T cell activation inhibitor 1 (VTCN1) that negatively regulates T cell immunity was upregulated by AhR agonists in breast cancer cell lines, MCF-7 and T47D. Furthermore, AhR knockout or knockdown experiments clearly demonstrated that upregulation of VTCN1 gene expression by 3-methylcholanthrene was AhR dependent. Luciferase reporter and chromatin immunoprecipitation assays revealed that this upregulation of VTCN1 gene expression was induced by the recruitment of AhR to the AhR responsive element in the VTCN1 gene promoter in MCF-7 cells. Taken together, AhR directly regulates VTCN1 gene expression in MCF-7 cells.

Rational design of small-molecules to recognize G-quadruplexes of c-MYC promoter and telomere and the evaluation of their in vivo antitumor activity against breast cancer.

DNA G4-structures from human c-MYC promoter and telomere are considered as important drug targets; however, the developing of small-molecule-based fluorescent binding ligands that are highly selective in targeting these G4-structures over other types of nucleic acids is challenging. We herein report a new approach of designing small molecules based on a non-selective thiazole orange scaffold to provide two-directional and multi-site interactions with flanking residues and loops of the G4-motif for better selectivity. The ligands are designed to establish multi-site interactions in the G4-binding pocket. This structural feature may render the molecules higher selectivity toward c-MYC G4s than other structures. The ligand–G4 interaction studied with 1H NMR may suggest a stacking interaction with the terminal G-tetrad. Moreover, the intracellular co-localization study with BG4 and cellular competition experiments with BRACO-19 may suggest that the binding targets of the ligands in cells are most probably G4-structures. Furthermore, the ligands that either preferentially bind to c-MYC promoter or telomeric G4s are able to downregulate markedly the c-MYC and hTERT gene expression in MCF-7 cells, and induce senescence and DNA damage to cancer cells. The in vivo antitumor activity of the ligands in MCF-7 tumor-bearing mice is also demonstrated.

Anti-Tumor Activity and Underlying Mechanism of Phomoxanthone B in MCF7 Cells.

Xanthones are a class of heterocyclic natural products, which are promising sources of anti-cancer leads. Phomoxanthone B (PXB) and Phomoxanthone A(PXA)are xanthone dimers. PXA is wellstudied as an anti-cancer agent, but PXB is not. In our study, PXB was isolated from the endophytic fungus Phomopsis sp. By254. The purpose of this study was to identify the underlying anti-tumor mechanisms of PXB in breast cancer MCF7 cell line. Apoptosis, cell cycle, proliferation, invasion, and migration assays were used to assess the anti-tumor activity of PXB. RNA sequencing was used to analyze the effect of PXB treatment on gene expression in MCF7 cells. PXB showed cytotoxicity towards a variety of tumor cells, especially MCF7 cells. PXB inhibited the migration and invasion, arrested cell cycle at G2/M phase, and induced apoptosis associated with caspase-3 activation in MCF7 cells. The detailed transcriptome analysis revealed that PXB affected several pathways related to tumorigenesis, metabolisms, and oxidative phosphorylation in MCF7 cells. KEGG transcriptome analysis revealed that PXB upregulated pro-survival signal pathways, such as MAPK, PI3K-AKT, and STAT3 pathways. We found that PXB also significantly upregulated the expression of IL24, DDIT3, and XAF1, which may contribute to PXB-induced apoptosis. We further found that PXB may downregulate oxidative phosphorylation by decreasing the expression of electron transport chain genes, especially MT-ND1, which is a potential unfavorable prognostic marker for ER-positive breast cancer. PXB exerts strong cytotoxicity against human tumor cells and has a potential for ER-positive breast cancer treatment.

The effects of the esterified Quercetin with omega3 and omega6 fatty acids on viability, nanomechanical properties, and BAX/BCL-2 gene expression in MCF-7 cells.

Quercetin is one of the major flavonoids and it appears to have cytotoxic effects on various cancer cells through regulating the apoptosis pathway genes such as BAX and BCL2. Combination of Quercetin (Q) with other compounds can increase its effectiveness. In the present study, the effects of the Quercetin and its esterified derivatives on viability, nanomechanical properties of cells, and BAX/BCL-2 gene expression were investigated. Using the MTT and flow cytometry assays, the cytotoxic potential, apoptosis, and necrosis were investigated. The AFM assay was performed to find the nanomechanical properties of cells as the elastic modulus value and cellular adhesion forces. The BAX/BCL2 gene expression was investigated through the Real-Time PCR. The results showed that the esterification of Quercetin with linoleic acid (Q-LA) and α-linolenic acid (Q-ALA) increased the cytotoxic potential of Q. The elastic modulus value and cellular adhesion forces were increased using the esterified derivatives and the highest ratio of BAX/BCL2 gene expression was observed in Q-LA. Esterified Quercetin derivatives have a higher cytotoxic effect than the un-esterified form in a dose-dependent manner. Esterified derivatives caused the nanomechanical changes and pores formation on the cytoplasmic membrane. One of the internal apoptosis pathway regulation mechanisms of these compounds is increasing the BAX/BCL2 gene expression ratio.

TET2 Inhibits PD-L1 Gene Expression in Breast Cancer Cells through Histone Deacetylation.

Simple SummaryProgrammed cell death ligand 1 (PD-L1) is an essential immune checkpoint molecule that helps tumor cells to escape the immune surveillance. The aim of the current study was to investigate the epigenetic mechanisms underlying the aberrant expression of PD-L1 in breast cancer cells. Here, we identified TET2 as a negative regulator of PD-L1 gene transcription in breast cancer cells. Mechanistically, TET2 recruits HDAC1/2 to the PD-L1 promoter and facilitates the deacetylation of H3K27ac, resulting to the suppression of PD-L1 gene transcription. Our work reveals an unanticipated role of TET2-HDAC1/2 complex in the regulation of PD-L1 gene expression, providing new insights into the epigenetic mechanisms that drive immune evasion during breast cancer pathogenesis.Activation of PD-1/PD-L1 checkpoint is a critical step for the immune evasion of malignant tumors including breast cancer. However, the epigenetic mechanism underlying the aberrant expression of PD-L1 in breast cancer cells remains poorly understood. To investigate the role of TET2 in the regulation of PD-L1 gene expression, quantitative reverse transcription PCR (RT-qPCR), Western blotting, chromatin immunoprecipitation (ChIP) assay and MeDIP/hMeDIP-qPCR were performed on MCF7 and MDA-MB-231 human breast cancer cells. Here, we reported that TET2 depletion upregulated PD-L1 gene expression in MCF7 cells. Conversely, ectopic expression of TET2 inhibited PD-L1 gene expression in MDA-MB-231 cells. Mechanistically, TET2 protein recruits histone deacetylases (HDACs) to PD-L1 gene promoter and orchestrates a repressive chromatin structure to suppress PD-L1 gene transcription, which is likely independent of DNA demethylation. Consistently, treatment with HDAC inhibitors upregulated PD-L1 gene expression in wild-type (WT) but not TET2 KO MCF7 cells. Furthermore, analysis of the CCLE and TCGA data showed a negative correlation between TET2 and PD-L1 expression in breast cancer. Taken together, our results identify a new epigenetic regulatory mechanism of PD-L1 gene transcription, linking the catalytic activity-independent role of TET2 to the anti-tumor immunity in breast cancer.

The Impacts of Simulated Microgravity on The Cell Viability and Claudin-1 and Claudin-3 Expression of MCF-7 Breast Cancer Cells

It has been believed that microgravity directly can alter the structure, morphology, and function of biosystems and numerous research have been performed to recognize these alterations. Claudin proteins are the tight junctions’ main components. Additionally, they are crucial for the protection of the differentiated state of epithelial cells as well as for cell-cell interaction. This study aimed to explore the probable correlation between the claudin-1 and claudin-3 expression and microgravity condition. Additionally, examined the impacts of microgravity condition on cell morphology and viability. The gene expression in MCF-7 cells were assessed by real-time quantitative RT PCR. Afterward, the morphology and cellular viability of the cells were evaluated by an inverted microscope, MTT assay, and flow cytometry analysis. After 72 h of simulated microgravity, the claudin-1 and claudin-3 expression increased significantly (P<0.05). Also, MCF-7 cells after 72 h exposure to microgravity simulation comprised rounded cells, which were grouped and linked to each other making multicellular spheroids. However, microgravity simulation after 24 or 72 h did not have a remarkable effect on the viability of cells. The consequence of this research lied in the fact that simulated microgravity could not be a direct cure for breast cancer treatment. However, microgravity research can offer a unique in vitro tool to explore biomechanical effects in the biology of cancer. The findings obtained from this investigation can open fascinating research lines in astrobiology, biophysics, and cancer biology and can be utilized to improve survivability and life quality for malignancy patients.

Bisphenolic compounds alter gene expression in MCF-7 cells through interaction with estrogen receptor α

Plasticizers released from microplastic are increasingly viewed with concern. While adverse health effects induced by bisphenol A and its analogues on marine animals are well documented in the literature, the endocrine potential of bisphenolic compounds on human health remains elusive. We applied next generation sequencing (NGS) with the estrogen receptor α (ERα) positive human breast cancer cell line MCF-7 treated with 17-β-estradiol (E2), bisphenol A (BPA), bisphenol B (BPB), bisphenol Z (BPZ) and tetramethyl bisphenol A (4MeBPA). We used molecular docking, microscale thermophoresis, ERα activation assay, and cell cycle experiments on MCF-7 and ERα overexpressing HEK293 cells to verify the impact of the compounds on ERα. 14 genes were found upregulated (ADORA1, DDIT4, CELSR2, FOSL2, JUN, HSPA13, IER3, IGF1R, PGR, RUNX2, SLC7A11, SLC7A2, SLC7A5, STC2) and 3 genes were downregulated (BCAS3, PHF19, PRKCD) in almost all samples. These genes are associated with cell growth, invasion, migration, apoptosis and cancer development. We further confirmed the binding, activation and proliferative effect of BPA, BPB, BPZ, and 4MeBPA on ERα. We provide evidence for the endocrine potential of bisphenolic compounds and give insights into their molecular effects in MCF-7 cells.

Triaryl dicationic DNA minor-groove binders with antioxidant activity display cytotoxicity and induce apoptosis in breast cancer

Despite advances in cancer treatment modalities, DNA still stands as one of the targets for anticancer agents. DNA minor groove binders (MGBs) represent an important investigational chemotherapeutic class with promising cytotoxic capacity. Herein this study reports the potent cytotoxic effect of a series of repurposed flexible bis-imidamides 1–4, triaryl bis-guanidine 5 and bis-N-substituted guanidines 6,7 having a 1,4-diphenoxybenzene scaffold backbone on MCF-7 and MDA-MB-231 breast cancer cell lines. Of these compounds, imidamide 4 was chosen for further in-vitro, in-vivo and molecular dynamics (MD) studies owing to its promising anti-tumor activity, with IC50 values on MCF-7 and MDA-MB-231 breast cancer cell lines of 1.9 and 2.08 μM, respectively. Annexin V/propidium iodide apoptosis assay revealed apoptosis induction on imidamide 4 treated MCF-7 cells. RT-PCR assay results demonstrated the proapoptotic effect of compound 4 through increase of mRNA levels of the pro-apoptotic genes; p53, PUMA, and Bax, and inhibiting the anti-apoptotic Bcl-2 gene expression in MCF-7 cells. Moreover, compound 4 induced a G0/G1 cell-cycle arrest in MCF-7 in a dose-dependent manner. Corroborating in-vivo experiments on Ehrlich ascites carcinoma (EAC)-bearing mice, reflected the anticancer strength of derivative 4. For further target validation, molecular dynamics (MD) studies demonstrated an energetically favorable binding of imidamide 4 with the DNA minor groove AT rich site. In effect, imidamide 4 can be viewed as a promising hit dicationic compound with good cytotoxic and apoptotic inducing activity against breast cancer that can be adopted for future optimization.

The Role of Harmine in Up-regulating p53 Gene Expression and Inducing Apoptosis in MCF-7 Cell Line

Background: As a major tumor suppressor gene, P53 plays a principal role in the apoptosis of cancer cells. Harmine is a harmal-derived alkaloid with antioxidant and anticancer properties. This study was designed to assess the ability of harmine to express P53 gene and stimulate apoptosis in breast cancer cell line. Methods: MCF-7 cell line was cultured and treated with harmine. Half maximal inhibitory concentration (IC50) assay was carried out through MMT method following 24 h of treatment. Flow cytometry technique was employed to measure apoptotic cells and real-time PCR was performed to estimate P53 gene expression in tumor cells. Results: The IC50 for the harmine extract in MCF-7 cells was 30 μM. Harmine administration in all treated groups resulted in a significant increase in apoptosis and P53 gene expression in MCF-7 cells (P < 0.00001). Conclusions: It seems that harmine administration is able to induce apoptosis in MCF-7 cells through the up-regulation of P53 expression.

The Cytotoxic Effects of Thymol as the Major Component of Trachyspermum ammi on Breast Cancer (MCF-7) Cells

Natural phenolic compounds have inhibition effects on various malignancies. Thymol is one of these compounds present in several plant sources such as ajowan (Trachyspermum ammi) fruits. In this study, thymol was evaluated for its potential cytotoxic activity as well as its effect on apoptotic gene expression in breast cancer cell line. We used the GC-MS technique to identify the essential oil constituents of ajowan. MCF-7 cells were treated by various concentrations of thymol and half maximum inhibitory concentration (IC50) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, morphological alterations and changes of P21, P53 and Mcl-1 gene expression in MCF-7 cells were investigated by microscopic examination and real-time RT-PCR assay. Data from MTT assay indicated that the IC50 values of thymol on MCF-7 cells for 48 h and 72 h were 54 and 62 μg/mL, respectively. Furthermore, this compound significantly affected gene expressions of P53 and P21, but not Mcl-1. Thymol can induce the apoptosis process in MCF-7, and hence it can be considered an anticancer agent in the future.

Fluid Shear Stress Induces Drug Resistance to Doxorubicin and Paclitaxel in the Breast Cancer Cell Line MCF7

AbstractCirculating tumor cells (CTCs) are known to have cancer stem cell (CSC) properties and survive physiological conditions of fluid shear stress (FSS). However, current chemotherapy screening techniques do not adequately recapitulate this FSS environment and are not predictive of a drug response. In this study, MCF7 and MDA‐MB‐231 cells under FSS are used as an in vitro model of CTCs. The effects of doxorubicin (DOX) and paclitaxel on sheared cells using WST8 assay and stemness (CD44+/CD24−) and apoptosis (Annexin V+/7‐AAD+) using flow cytometry are tested. Quantitative polymerase chain reaction is used to test gene expression. It is shown that suspension‐cultured and FSS treated MCF7 cells increase in drug resistance, especially with DOX. There is a synergistic increase in the CD44+/CD24− CSC‐like population and an increase in drug resistance‐related gene expression in MCF7 cells co‐treated with FSS and drugs. There is also a correlated increase in STAT3 and NANOG expression under FSS. To the best of the authors' knowledge, this is the first report to suggest that the increase in CSC‐like cells from FSS contributes to drug resistance via the STAT3/NANOG pathway. This increase in CTC drug resistance also highlights the importance of implementing FSS, which is unavailable in current drug screening techniques.

Pyroptosis of MCF7 Cells Induced by the Secreted Factors of hUCMSCs.

Human umbilical cord mesenchymal stem cells (hUCMSCs) are superior to other sources of mesenchymal stem/stromal cells (MSCs), and they are used as a novel tool for cell-based cancer therapy. However, the mechanism underlying hUCMSC-induced cancer cell death is not clear. In the present study, we aimed to evaluate the effect of secreted factors of hUCMSCs on the breast cancer cell line MCF7 by exposing them to the conditioned medium (CM) of hUCMSCs. We evaluated the morphological changes, cell viability, cell cycle, apoptosis, DNA fragmentation, and interleukin-1β (IL-1β) secretion of CM-exposed MCF7 cells. The results showed that the secreted factors of hUCMSCs could cause MCF7 cell death by inducing pyroptosis. We also sequenced the total RNA, and the differentially expressed genes (DEGs) were subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. A total of 2597 (1822 upregulated and 775 downregulated) genes were identified and 14 pathways were significantly enriched. The results showed that the expression of the pyroptosis-related genes NLRP1 and CASP4 and the inflammation-related pathways changed significantly in MCF7 cells exposed to the CM. To the best of our knowledge, this study is the first to report that the secreted factors of hUCMSCs can cause MCF7 cell pyroptosis. Furthermore, it is the first to examine the global gene expression in MCF7 cells exposed to CM. These results will provide valuable information for further studies on the mechanism of MCF7 cell pyroptosis induced by the secreted factors of hUCMSCs. It will also help understand the effect of hUCMSCs on cell-based breast cancer therapy.

Flightless-I mediates the repression of estrogen receptor α target gene expression by the glucocorticoid receptor in MCF-7 cells.

The human homologue of flightless-I (FLII) belong to the gelsolin protein family and contain a gelsolin-like domain at the C-terminus and a leucine-rich repeat (LRR) domain at the N-terminus. FLII regulates estrogen receptor alpha (ERα) and glucocorticoid receptor (GR)-mediated transcription by direct interaction through different domains, suggestive of its potential role in the crosstalk between the ERα and GR signaling pathway. Here, we demonstrate that FLII plays a critical role in GR-mediated repression of ERα target gene expression. In FLII-depleted cells, the reduction in 17-β-estradiol (E2)-induced ERα occupancy following treatment with dexamethasone (Dex) at the estrogen responsive element (ERE) site of the ERα target gene was significantly inhibited. The ERE binding of GR by the cotreatment with E2 and Dex was significantly inhibited by FLII depletion, indicating that FLII is required for the recruitment of GR at the ERE sites of ERα target genes. In addition, the recruitment of ERα-induced FLII to ERE sites was significantly reduced by Dex treatment. In protein binding assays, GR inhibited the E2-induced interaction between ERα and FLII, suggesting that GR interferes with the binding of ERα and FLII at the ERα target genes, resulting in the release of ERα and FLII from EREs. Taken together, our data reveal an unknown mechanism by which the transcription coactivator FLII regulates the GR-mediated repression of ERα target gene expression in MCF-7 cells.

Glaucarubulone glucoside from Castela macrophylla suppresses MCF-7 breast cancer cell growth and attenuates benzo[a]pyrene-mediated CYP1A gene induction.

Quassinoids often exhibit antioxidant and antiproliferative activity. Emerging evidence suggests that these natural metabolites also display chemopreventive actions. In this study, we investigated the potential for the quassinoid glaucarubulone glucoside (Gg), isolated from the endemic Jamaican plant Castela macrophylla (Simaroubaceae), to display potent cytotoxicity and inhibit human cytochrome P450s (CYPs), particularly CYP1A enzymes, known to convert polyaromatic hydrocarbons into carcinogenic metabolites. Gg reduced the viability of MCF-7 breast adenocarcinoma cells (IC50 = 121 nm) to a greater extent than standard of care anticancer agents 5-fluorouracil, tamoxifen (IC50 >10 μm) and the tamoxifen metabolite 4-hydroxytamoxifen (IC50 = 2.6 μm), yet was not cytotoxic to non-tumorigenic MCF-10A breast epithelial cells. Additionally, Gg induced MCF-7 breast cancer cell death. Gg blocked increases in reactive oxygen species in MCF-10A cells mediated by the polyaromatic hydrocarbon benzo[a]pyrene (B[a]P) metabolite B[a]P 1,6-quinone, yet downregulated the expression of genes that promote antioxidant activity in MCF-7 cells. This implies that Gg exhibits antioxidant and cytoprotective actions in non-tumorigenic breast epithelial cells and pro-oxidant, cytotoxic actions in breast cancer cells. Furthermore, Gg inhibited the activities of human CYP1A according to non-competitive kinetics and attenuated the ability of B[a]P to induce CYP1A gene expression in MCF-7 cells. These data indicate that Gg selectively suppresses MCF-7 breast cancer cell growth without impacting non-tumorigenic breast epithelial cells and blocks B[a]P-mediated CYP1A induction. Taken together, our data provide a rationale for further investigations of Gg and similar plant isolates as potential agents to treat and prevent breast cancer. Copyright © 2017 John Wiley & Sons, Ltd.

CLDN6-induced apoptosis via regulating ASK1-p38/JNK signaling in breast cancer MCF-7 cells.

Claudin 6 (CLDN6), a member of tight junction protein claudin (CLDN) family, inhibits proliferation and induces apoptosis of MCF-7 breast cancer cells. However, these molecular mechanisms of CLDN6-induced apoptosis remain largely elusive. We previously found that restoration of human CLDN6 gene expression was correlated with the expression level of apoptosis signal-regulating kinase1 (ASK1) using cDNA array and bioinformatics analysis. ASK1, a mitogen-activated protein kinase kinase kinase, is involved in environmental stress-activation of the c-jun N-terminal kinase (JNK) and p38 pathways, which contribute to apoptosis-associated tumor cell death. In the present study, we show that the restoration of CLDN6 gene expression in MCF-7 cells marhedly decreased ASK1 phosphorylation at Ser967. Activated ASK1ser967 further induced the activation of downstream targets, JNK and p38 kinase. MCF-7/CLDN6 stable transfection cell clone treated with TRX1, an ASK1 inhibitor, showed suppressed JNK and p38 activation, and showed substantially increased survival and colony formation and reduced percent of apoptotic cells using TUNEL staining and DNA ladder. Furthermore, TRX1 treatment increased Bcl-2/Bax ratio and reduced caspase-3 cleavage in MCF-7/CLDN6 stable transfection cell clone. Therefore, these data show that CLDN6 mediates ASK1-p38/JNK apoptotic signaling in MCF-7 cells, and it is correlated with constitutive deregulation of the balance of Bcl-2 family proteins and activation of caspase-3.

Alternative Viewpoint Against Breast Cancer Based on Selective Serotonin Receptors 5HTR3A and 5HTR2A Antagonists that can Mediate Apoptosis in MCF-7 Cell Line.

Neurotransmitters had progressive effects on various cancers via their different type of receptors. This study was conducted to determine the pattern of serotonin receptors, respectively, 5HTR2A and 5HTR3A gene expression in MCF-7 cells and evaluate their selective antagonist effects on them. RT-PCR was performed to determine the pattern of serotonin receptor gene expression in human breast cancer cell line (MCF-7). MCF-7 cells were cultured and treated via different doses of tropisetron (5HTR3A antagonist) and ketanserin (5HTR2A antagonist) for 48 hours. Oxidative and reductive enzyme activity was carried out by MTT assay. Subsequently, nuclear morphology of cells was observed by mixed dye florescent staining. To validate cell proliferation inhibition, Real time PCR was carried out for determining the descending rate of proliferating cell nuclear antigen (PCNA) gene expression in treating MCF-7 cells. Assessment of quantification of apoptosis and its discrimination with necrosis at single cell level using Flowcytometry technique was performed. Results showed that 5HTR2A and 5HTR3A have expression in MCF-7 cells. Based on our finding, tropisetron and ketanserin had suppression effects on MCF-7 cells proliferation. (93.35% in tropisetron 50 µmoll(-1) and 72.36% in Ketanserin 25µmoll(-1) concentration). Therefore, the use of tropisetron and ketanserin as an antagonist of serotonin receptor may be as new approaches are recommended for the treatment of breast cancer cells.

CTCF May Not Directly Regulate ERα mRNA Expression in the ER+ MCF7 Breast Cancer Cell Line

Introduction: CTCF is an evolutionally conserved 11-zinc finger protein factor involved in an array of processes whose deregulation could lead to cellular transformation. Through interactions with ERα binding regions and ERregulated genes, CTCF was shown to compartmentalize the cellular genome into domains. It also colocalized with ERα in MCF7 cells and had interactions with ERα during histone deacetylase recruitment and fork-head activity. A fast-running isoform was previously shown to be expressed in breast cancer tissue but not in normal breast tissue. It is not clear whether there is a regulatory relationship between CTCF and ERα in breast cancer. Aim: To determine whether CTCF expression regulated ERα expression in the ER+ MCF7 breast cancer cell line. Methods: MCF7 breast cancer cells were transfected with either CTCF expression vectors or siRNA against CTCF. Following CTCF over-expression and knock-down, changes in endogenous expression of ERα gene and protein expression were monitored by quantitative polymerase chain reaction (using MIQE guidelines) and western blot analysis respectively. Results: CTCF plasmid overexpression and siRNA knockdown was associated with cell rounding but with 96.4% and 95.7% cell viability respectively. Increase in CTCF mRNA on over-expression was associated with a rise in CTCF protein expression. siRNA knockdown of CTCF mRNA was accompanied by a corresponding decrease in CTCF protein expression. CTCF over-expression and knockdown appeared to inhibit the ability to detect ERα protein expression by western blotting. Neither the over-expression nor knockdown of CTCF altered ERα mRNA expression as detected by QPCR. Conclusion: Alterations in CTCF mRNA expression did not affect ERα gene expression in MCF7 cells suggesting that CTCF interactions with the estrogen receptor in breast cancer may not be mediated via direct regulation of ERα mRNA expression.

Cinnamomum cassia Suppresses Caspase-9 through Stimulation of AKT1 in MCF-7 Cells but Not in MDA-MB-231 Cells.

Background Cinnamomum cassia bark is a popular culinary spice used for flavoring and in traditional medicine. C. cassia extract (CE) induces apoptosis in many cell lines. In the present study, particular differences in the mechanism of the anti-proliferative property of C. cassia on two breast cancer cell lines, MCF-7 and MDA-MB-231, were elucidated.Methodology/Principal FindingsThe hexane extract of C. cassia demonstrated high anti-proliferative activity against MCF-7 and MDA-MB-231 cells (IC50, 34±3.52 and 32.42 ±0.37 μg/ml, respectively). Oxidative stress due to disruption of antioxidant enzyme (SOD, GPx and CAT) activity is suggested as the probable cause for apoptosis initiation. Though the main apoptosis pathway in both cell lines was found to be through caspase-8 activation, caspase-9 was also activated in MDA-MB-231 cells but suppressed in MCF-7 cells. Gene expression studies revealed that AKT1, the caspase-9 suppressor, was up-regulated in MCF-7 cells while down-regulated in MDA-MB-231 cells. Although, AKT1 protein expression in both cell lines was down-regulated, a steady increase in MCF-7 cells was observed after a sharp decrease of suppression of AKT1. Trans-cinnamaldehyde and coumarin were isolated and identified and found to be mainly responsible for the observed anti-proliferative activity of CE (Cinnamomum cassia).ConclusionActivation of caspase-8 is reported for the first time to be involved as the main apoptosis pathway in breast cancer cell lines upon treatment with C. cassia. The double effects of C. cassia on AKT1 gene expression in MCF-7 cells is reported for the first time in this study.