ELF3 plays an important role in defining TRAIL sensitivity in breast cancer by modulating the expression of decoy receptor 2 (DCR2).

BACKGROUND: TRAIL protein on binding to its cognate death receptors (DR) can induce apoptosis specifically in breast tumor cells sparing normal cells. However, TRAIL also binds to decoy receptors (DCR) thereby inhibiting the apoptotic pathways thus causing TRAIL resistance. Also, one of the barriers due to which TRAIL-based therapy could not become FDA-approved might be because of resistance to therapy. Therefore, in the current study we wanted to explore the role of transcription factors in TRAIL resistance with respect to breast cancer. METHODS: Microarray data from TRAIL-sensitive (TS) and TRAIL-resistant (TR) MDA-MB-231 cells were reanalyzed followed by validation of the candidate genes using quantitative PCR (qPCR), immunoblotting and immunofluorescence technique. Overexpression of the candidate gene was performed in MDA-MB-231 and MCF7 cells followed by cell viability assay and immunoblotting for cleaved caspase-3. Additionally, immunoblotting for DCR2 was carried out. TCGA breast cancer patient survival was used for Kaplan-Meier (KM) plot. RESULTS: Validation of the candidate gene i.e. ELF3 using qPCR and immunoblotting revealed it to be downregulated in TR cells compared to TS cells. ELF3 overexpression in MDA-MB-231 and MCF7 cells caused reversal of TRAIL resistance as observed using cell viability assay and cleaved caspase-3 immunoblotting. ELF3 overexpression also resulted in DCR2 downregulation in the MDA-MB-231 and MCF7 cells. Furthermore, KM analysis found high ELF3 and low DCR2 expression to show better patient survival in the presence of TRAIL. CONCLUSION: Our study shows ELF3 to be an important factor that can influence TRAIL-mediated apoptosis in breast cancer. Also, ELF3 and DCR2 expression status should be taken into consideration while designing strategies for successful TRAIL-based therapy.

CDH1 overexpression sensitizes TRAIL resistant breast cancer cells towards rhTRAIL induced apoptosis.

PURPOSE: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is well known for its unique ability to induce apoptosis in cancer cells but not normal cells. However, a subpopulation of cancer cells exist that does not respond to toxic doses of TRAIL. In this study, we aimed to identify key factors regulating TRAIL resistance in breast cancer. METHODS: rhTRAIL (recombinant human TRAIL) resistant cells (TR) isolated from TRAIL sensitive MDA-MB-231 parental cells (TS) were confirmed using trypan blue assay, cell viability assay and AO/EtBr (acridine orange/ethidium bromide) staining. Microarray was performed followed by analysis using DAVID and Cytoscape bioinformatics software to identify the candidate hub gene. Gene expression of the candidate gene was confirmed using real-time PCR and western blot. Candidate gene was overexpressed via transient transfection to identify its significance in the context of rhTRAIL. Breast cancer patient data was obtained from The Cancer Genome Atlas (TCGA) database. RESULTS: Whole transcriptome analysis identified 4907 differentially expressed genes (DEGs) between TS and TR cells. CDH1 was identified as the candidate hub gene, with 18-degree centrality. We further observed CDH1 protein to be downregulated, overexpression of which increased apoptosis in TR cells after rhTRAIL treatment. TCGA patient data analysis also showed CDH1 mRNA to be low in TRAIL resistant patient group compared to TRAIL sensitive group. CONCLUSION: CDH1 overexpression sensitizes TR cells towards rhTRAIL induced apoptosis. Therefore, we can hypothesize that CDH1 expression should be taken into account while performing TRAIL therapy in breast cancer.

Reactive oxygen species-dependent upregulation of death receptor, tumor necrosis factor receptor 1, is responsible for theophylline-mediated cytotoxicity in MDA-MB-231 breast cancer cells.

Theophylline, a methylxanthine drug, has been used as a therapy for respiratory diseases. Recently, it has also been shown to have a potential in treating different cancers. Also, it has shown promising results in clinical trials for AML in combination therapy. Subsequently, studies have shown theophylline to kill breast cancer cells but not normal breast cells. Therefore, in this study, we have explored the molecular mechanism underlying the cytotoxic effect of theophylline on breast cancer cells. Theophylline-treated cancer cells were analyzed for the transcript and protein expression of candidate apoptotic genes such as TNFR1, caspase-8, -9, -3 using qPCR and immunoblotting, respectively. Cell viability and apoptosis was measured in the presence or absence of TNFR1 inhibitor, R7050, using AO/EtBr staining and MTT assay, respectively. Similarly, oxidative stress was studied by analyzing ROS in the presence or absence of ROS inhibitor, NAC, using DCFDA assay. Theophylline caused reduced cell viability in cancer but not normal cells. Theophylline-treated breast cancer cells showed increased expression of death receptor, TNFR1, along with elevated levels of active caspase-8, -9 and -3. Inhibition of TNFR1 reduced caspase-dependent apoptosis even in the presence of theophylline. Theophylline further caused increased ROS generation, inhibition of which resulted in reduced TNFR1-mediated apoptosis. Theophylline also increased cathepsin activity, which was reduced on exposure of cells to TNFR1 inhibitor, R7050. We conclude that ROS-mediated activation of TNFR1 is responsible for caspase-3 and cathepsin-dependent cell death in breast cancer cells on exposure to theophylline.

Enhanced expression of death receptor 5 is responsible for increased cytotoxicity of theophylline in combination with recombinant human TRAIL in MDA-MB-231 breast cancer cells.

Background: Theophylline has been reported to induce cytotoxicity and cell cycle arrest in cancer cells. On the other hand, TRAIL, a secretory ligand, is known for its unique ability to induce cell death only in tumor cells. In the present study, we elucidated the mechanism behind the cytotoxic effect of theophylline in combination with recombinant human TRAIL (rhTRAIL) on cancer cell line MDA-MB-231. Materials and Methods: Cytotoxicity of theophylline in combination with TRAIL was measured via trypan blue assay and MTT assay. Protein levels were assessed using Western hybridization. Reactive oxygen species (ROS) levels were measured using 2',7'-dichlorofluorescin diacetate and mitochondrial membrane potential (MMP) assay was conducted using tetramethylrhodamine, ethyl ester. Results: We observed theophylline in combination with rhTRAIL to be significantly cytotoxic to the cancer cells in comparison to theophylline and rhTRAIL alone. Next, western hybridization showed combination treatment to upregulate cleaved form of caspase-8, 9 and 3, in comparison to the cells treated with rhTRAIL and theophylline alone. Theophylline in combination also increased the levels of ROS and reduced MMP. Interestingly, combination treatment increased the protein level of death receptor 5 (DR5), sensitizing the cells towards TRAIL-induced apoptosis. Conclusion: Theophylline in combination with TRAIL significantly increases cytotoxicity in the MDA-MB-231 breast cancer cell line when compared to theophylline and rhTRAIL alone via upregulation of DR5 levels.

Exploring the Molecular Mechanism of Cinnamic Acid-Mediated Cytotoxicity in Triple Negative MDA-MB-231 Breast Cancer Cells.

BACKGROUND: Cinnamic Acid (CA), also known as 3-phenyl-2-propenoic acid, is a naturally occurring aromatic fatty acid found commonly in cinnamon, grapes, tea, cocoa, spinach and celery. Various studies have identified CA to have anti-proliferative action on glioblastoma, melanoma, prostate and lung carcinoma cells. OBJECTIVE: Our objective was to investigate the molecular mechanism underlying the cytotoxic effect of CA in killing MDA-MB-231 triple negative breast cancer cells. METHODS: We performed MTT assay and trypan blue assay to determine cell viability and cell death, respectively. Comet analysis was carried out to investigate DNA damage of individual cells. Furthermore, AO/EtBr assay and sub-G1 analysis using flow cytometry were used to study apoptosis. Protein isolation followed by immunoblotting was used to observe protein abundance in treated and untreated cancer cells. RESULTS: Using MTT assay, we have determined CA to reduce cell viability in MDA-MB-231 breast cancer cells and tumorigenic HEK 293 cells but not in normal NIH3T3 fibroblast cells. Subsequently, trypan blue assay and comet assay showed CA to cause cell death and DNA damage, respectively, in the MDA-MB-231 cells. Using AO/EtBr staining and sub-G1 analysis, we further established CA to increase apoptosis. Additionally, immunoblotting showed the abundance of TNFA, TNF Receptor 1 (TNFR1) and cleaved caspase-8/-3 proapoptotic proteins to increase with CA treatment. Subsequently, blocking of TNFA-TNFR1 signalling by small molecule inhibitor, R-7050, reduced the expression of cleaved caspase-8 and caspase-3 at the protein level. CONCLUSION: Thus, from the above observations, we can conclude that CA is an effective anticancer agent that can induce apoptosis in breast cancer cells via TNFA-TNFR1 mediated extrinsic apoptotic pathway.