Non-tumorigenic Epithelial Cells Research Articles

Giant obscurin regulates migration and metastasis via RhoA-dependent cytoskeletal remodeling in pancreatic cancer.

Obscurins, encoded by the OBSCN gene, are giant cytoskeletal proteins with structural and regulatory roles. Large scale omics analyses reveal that OBSCN is highly mutated across different types of cancer, exhibiting a 5-8% mutation frequency in pancreatic cancer. Yet, the functional role of OBSCN in pancreatic cancer progression and metastasis has to be delineated. We herein show that giant obscurins are highly expressed in normal pancreatic tissues, but their levels are markedly reduced in pancreatic ductal adenocarcinomas. Silencing of giant obscurins in non-tumorigenic Human Pancreatic Ductal Epithelial (HPDE) cells and obscurin-expressing Panc5.04 pancreatic cancer cells induces an elongated, spindle-like morphology and faster cell migration via cytoskeletal remodeling. Specifically, depletion of giant obscurins downregulates RhoA activity, which in turn results in reduced focal adhesion density, increased microtubule growth rate and faster actin dynamics. Although OBSCN knockdown is not sufficient to induce de novo tumorigenesis, it potentiates tumor growth in a subcutaneous implantation model and exacerbates metastasis in a hemispleen murine model of pancreatic cancer metastasis, thereby shortening survival. Collectively, these findings reveal a critical role of giant obscurins as tumor suppressors in normal pancreatic epithelium whose loss of function induces RhoA-dependent cytoskeletal remodeling, and promotes cell migration, tumor growth and metastasis.

Extracellular vesicles released by non-small cell lung cancer cells drive invasion and permeability in non-tumorigenic lung epithelial cells

Extracellular vesicles (EVs) released from non-small cell lung cancer (NSCLC) cells are known to promote cancer progression. However, it remains unclear how EVs from various NSCLC cells differ in their secretion profile and their ability to promote phenotypic changes in non-tumorigenic cells. Here, we performed a comparative analysis of EV release from non-tumorigenic cells (HBEC/BEAS-2B) and several NSCLC cell lines (A549, H460, H358, SKMES, and Calu6) and evaluated the potential impact of NSCLC EVs, including EV-encapsulated RNA (EV-RNA), in driving invasion and epithelial barrier impairment in HBEC/BEAS-2B cells. Secretion analysis revealed that cancer cells vary in their secretion level, with some cell lines having relatively low secretion rates. Differential uptake of NSCLC EVs was also observed, with uptake of A549 and SKMES EVs being the highest. Phenotypically, EVs derived from Calu6 and H358 cells significantly enhanced invasion, disrupted an epithelial barrier, and increased barrier permeability through downregulation of E-cadherin and ZO-1. EV-RNA was a key contributing factor in mediating these phenotypes. More nuanced analysis suggests a potential correlation between the aggressiveness of NSCLC subtypes and the ability of their respective EVs to induce cancerous phenotypes.

Protein S-mediated signal transduction pathway regulates lung cancer cell proliferation, migration and angiogenesis

Objective/BackgroundProtein S (PS; encoded by the PROS1 gene), a key vitamin K-dependent anticoagulant protein, is emerging as a key structural and functional protein that is overexpressd in various malignancies, but how PS signals to promote lung cancer progression is unclear. MethodsWe used immortalized, nontumorigenic human lung epithelial cell line NL-20, A549 cells as experimental cellular models for lung cancer, and human microvascular endothelial cells (HMEC-1) as a model system for angiogenesis. A loss- and gain-of-function approach was then used to analyze the role of tumor-derived PS and their natural TAM receptors Tyro3 and MerTK in regulating cell proliferation, migration, anchorage-independent growth, and capillary-like tube formation, all prominent attributes of the metastatic phenotype of tumor cells. ResultsEvidence is now provided that regulation of PROS1 gene expression using either stable cell lines expressing lentiviral-short hairpin RNA (shRNAs) or a replication-incompetent adenovirus alters the phosphorylation of several major signaling pathways, including Erk, PKB/Akt, p38, and focal adhesion kinase (FAK), and modulates PS-dependent Tyro3- and MerTK-mediated cell migration, proliferation, and anchorage-independent growth of lung cancer cells, and endothelial cell capillary-like tube formation. ConclusionThese finding suggest that the PS-Tyro3 and -MerTK axis mediates important signaling pathways to promote lung cancer progression. Genetic inhibition of endogenous PS may serve as a promising target for anticancer drug development.

Anti-proliferative activity of yerba mate (Ilex paraguariensis) aqueous extracts on human colorectal cancer cell lines

Background: Yerba mate, a popular, tea-like beverage prepared from the dried leaves of Ilex paraguariensis, is widely consumed, and has several reported health benefits. Compared with other herbal teas, the effect of yerba mate on human cells in the context of cancer has not been extensively studied. The method of extraction of bioactive compounds from the yerba mate leaves plays an important role in its effect on cancer cells. Methods: In this study we assessed the viability, anti-proliferative, and apoptotic effect of the aqueous yerba mate extract, prepared using the same conditions employed for consumption, on different human colorectal cancer cell lines (Caco-2, HT-29, and HCT116) and on the non-tumorigenic human colon epithelial cell line (NCM460).Results: Cytotoxicity of aqueous yerba mate extract was studied and a dose-dependent decrease in viability was observed in all the tested cell lines. At 24 hrs., viability decreased to 19.7% with Caco-2 cells, 2.7% with HCT116, and 8.4% with HT-29 cells at a concentration of 4.8 mg/mL of yerba mate extract. The effect was less prominent on the NCM460 cell line where the viability of cells at the same concentration was 65.2%. Yerba mate extract also showed concentration-dependent anti-proliferative effects as determined by the WST-1 proliferation kit. IC50 values ranged between 0.22-0.69 mg/mL at 24 hr for cell lines tested. To study whether cell death was due to apoptosis, Caco-2 cells were stained with Annexin V-FITC assay and an increase in the percentage of late apoptotic Caco-2 cells was observed with yerba mate extract at 0.6-4.8 mg/mL. Cell cycle analysis using DNA content by flow cytometry showed an increase in the percentage of Caco-2 cells in the subG0/G1 phase and the G0/G1 phase after treatment with 2.4 mg/mL extract. Collectively, our data suggest that yerba mate aqueous extract exhibits an anti-proliferative effect on tested cell lines by inducing apoptosis. Conclusions: Yerba mate aqueous extract exhibits a strong anti-proliferative activity against adenocarcinoma cell lines studied and constitutes a promising functional food adjuvant to anti-cancer therapy. Further work is needed to identify active components and mechanisms of action. Keywords: Aqueous extract, yerba mate, anti-proliferative activity, adenocarcinoma cell lines

Effective Targeting of Raf-1 and Its Associated Autophagy by Novel Extracted Peptide for Treating Breast Cancer Cells.

Breast cancer is one of the most common causes of death in women worldwide and has harmful influence on their psychological state during therapy. Multikinase inhibitors have become effective drugs for treating a variety of cancer diseases such as breast cancer. A purified short peptide (H-P) was isolated from the natural honey and tested for its potential regulatory role in breast cancer cells compared with the effectiveness of the anticancer drug, Sorafenib (SOR), using MCF-7, EFM-19, and MCF-10A cell lines. Furthermore, we investigated the direct connection between Raf-1 activation and cellular autophagy as potential targets of SOR and H-P extract using RNA interference. Interestingly, the treatment with H-P showed competitive regulation of phosphorylated Raf-1, MEK1/2, and matched autophagy-related LC3B without any detectable toxic effects in the non-tumorigenic epithelial cells. Unlike SOR, the regulation of Raf-1 protein and autophagic machinery by the novel H-P extract showed neglected levels of the released proinflammatory cytokine. This regulation of cytokine secretion by H-P resulted in decreasing the expression level of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) in treated cells. Moreover, the transfection of MCF-7 cells with small interference RNA (siRNA) antagonist Raf-1 expression markedly reduced the expression of LC3B, while it increased the expression of NF-kB1 and NF-kB2, indicating the potential cross-link between Raf-1, autophagy, and NF-kB effector. Collectively, these findings suggest that H-P-mediated Raf-1, MEK1/2, LC3B, and NF-kB provide a novel and efficacious multikinase inhibitor for treating breast cancer without detectable cytotoxic effects.

Extracellular vesicles from MDA-MB-231 breast cancer cells stimulated with insulin-like growth factor 1 mediate an epithelial-mesenchymal transition process in MCF10A mammary epithelial cells.

Insulin-like growth factor-1 (IGF-1) plays an important role in function and development of the mammary gland. However, high levels of IGF-1 has been associated with an increased risk of breast cancer development. Epithelial-mesenchymal transition (EMT) is a process where epithelial cells lose their epithelial characteristics and acquire a mesenchymal phenotype, which is considered one of the most important mechanisms in cancer initiation and promotion of metastasis. Extracellular vesicles (EVs) are released into the extracellular space by different cell types, which mediate intercellular communication and play an important role in different physiological and pathological processes, such as cancer. In this study, we demonstrate that EVs from MDA-MB-231 breast cancer cells stimulated with IGF-1 (IGF-1 EVs) decrease the levels of E-cadherin, increase the expression of vimentin and N-cadherin and stimulate the secretion of metalloproteinase-9 in mammary non-tumorigenic epithelial cells MCF10A. IGF-1 EVs also induce the expression of Snail1, Twist1 and Sip1, which are transcription factors involved in EMT. Moreover, IGF-1 EVs induce activation of ERK1/2, Akt1 and Akt2, migration and invasion. In summary, we demonstrate, for the first time, that IGF-1 EVs induce an EMT process in mammary non-tumorigenic epithelial cells MCF10A.

Circulating extracellular vesicles from individuals at high-risk of lung cancer induce pro-tumorigenic conversion of stromal cells through transfer of miR-126 and miR-320

BackgroundExtracellular vesicles (EVs) containing specific subsets of functional biomolecules are released by all cell types and analysis of circulating EVs can provide diagnostic and prognostic information. To date, little is known regarding the role of EVs both as biomarkers and potential key players in human lung cancer.MethodsPlasma EVs were isolated from 40 cancer-free heavy-smokers classified according to a validated 24-microRNA signature classifier (MSC) at high (MSCpos-EVs) or low (MSCneg-EVs) risk to develop lung cancer. EVs origin and functional properties were investigated using in vitro 3D cultures and in vivo models. The prognostic value of miRNAs inside EVs was assessed in training and in validation cohorts of 54 and 48 lung cancer patients, respectively.ResultsDifferent membrane composition, biological cargo and pro-tumorigenic activity were observed in MSCpos vs MSCneg-EVs. Mechanistically, in vitro and in vivo results showed that miR-126 and miR-320 from MSCpos-EVs increased pro-angiogenic phenotype of endothelial cells and M2 polarization of macrophage, respectively. MSCpos-EVs prompted 3D proliferation of non-tumorigenic epithelial cells through c-Myc transfer. Moreover, hypoxia was shown to stimulate the secretion of EVs containing c-Myc from fibroblasts, miR-126-EVs from endothelial cells and miR-320-EVs from granulocytes. Lung cancer patients with higher levels of mir-320 into EVs displayed a significantly shorter overall survival in training [HR2.96] and validation sets [HR2.68].ConclusionOverall our data provide a new perspective on the pro-tumorigenic role of circulating EVs in high risk smokers and highlight the significance of miR-320-EVs as a new prognostic biomarker in lung cancer patients.

Abstract 2364: MicroRNA-1205 directly targets ONECUT2 in neuroendocrine prostate cancer cells

Abstract One of six deaths globally is due to cancer. In 2018, prostate cancer (PCa) represented 1.28 million cases worldwide, the second most frequent cancer diagnosis and fifth leading cause of death in men. Currently, the mortality rate of PCa has experienced a steady increase due to the progression of metastatic castration-resistant prostate cancer (mCRPC). As an androgen driven disease, androgen deprivation therapies (ADTs) have been employed for treatment. However, despite ADTs, mCRPC develops by maintaining AR signaling, which can eventually lead to progression of AR-independent neuroendocrine PCa (NEPC). NEPC is observable in 1 in 5 men with mCRPC and associated with poor outcome. Therefore, further understanding the mechanisms of progression to mCRPC and NEPC is required. The 8q24 human chromosomal region associated with Increased PCa incidence and aggressiveness contains the PVT1 (Plasmocytoma Variant Translocation 1) locus. The PVT1 locus encodes six microRNAs, including microRNA-1205 (miR-1205) in PCa. Our laboratory previously confirmed that miR-1205 is underexpressed in PCa tissue in comparison to normal prostatic tissue and in CRPC cells in comparison to non-CRPC cells. To understand the molecular mechanism of miR-1205, we performed RNA pull down and RNA sequencing analysis to identify miR-1205 molecular targets in non-tumorigenic RWPE-1 prostate epithelial cells and PCa cells (MDA PCa 2b (metastatic PCa), PC-3 (NEPC), and C4-2B (CRPC)). We identified one cut homeobox 2 (ONECUT2 or OC2) as a direct molecular target of miR-1205. ONECUT2 is a master regulator of transcriptional factors that drives NEPC by through regulating hypoxia signaling or inducing neuroendocrine markers. ONECUT2 enrichment was approximately 3-fold higher in RWPE-1 (P=0.26), 2-fold in PC-3 (P=0.06), 1-fold in C4-2B (P=0.05) and MDA PCa 2b (P=0.02) cells. The significant binding of miR-1205 to ONECUT-2 in the PC-3 NEPC cell line suggests that miR-1205 regulation of ONECUT2 may be an important molecular mechanism in NEPC. Consequently, further study of miR-1205 and ONECUT2 in NEPC is required. Citation Format: Bachelard Dieujuste, Michelle Naidoo, Olorunseun Ogunwobi. MicroRNA-1205 directly targets ONECUT2 in neuroendocrine prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2364.

4-Propargyl-substituted 1H-pyrroles induce apoptosis and autophagy via extracellular signal-regulated signaling pathway in breast cancer.

Novel pyrrole derivatives (PDs) with propargyl units (1-7) were investigated for their anticancer activity on breast cancer cells. The MTT assay was used to assess the cell viability. Morphological changes in human breast cancer cells were visualized under a phase-contrast microscope. Apoptosis and autophagy were detected using the DNA fragmentation assay and staining by autophagic vacuoles, respectively. The levels of apoptosis- and autophagy-related proteins such as cytochrome c, Bcl-2, LC3-I/II were investigated by Western blot analysis. The effect of PDs on the ERK1/2 signaling pathway was investigated using specific inhibitors. All the tested PDs were found to be active in the range of 36.7 ± 0.2 to 459.7 ± 4.2 µM. Compounds 3 and 4 showed cytotoxic activity in breast cancer cells, but were found to be safer with lower cytotoxicity on human nontumorigenic epithelial breast cells. Compound 4 induced apoptosis, whereas compound 3 induced autophagy. Both compounds inhibited the ERK signaling pathway in breast cancer cells. The present study revealed that both synthesized PDs induced different programmed cell death types by inhibiting the ERK signaling pathway in two genotypically different breast cancer cells. Therefore, novel PDs might be promising anticancer agents for breast cancer therapy and further structural modifications of PDs may yield promising anticancer agents.

IGF2BP2 knockdown inhibits LPS‑induced pyroptosis in BEAS‑2B cells by targeting caspase 4, a crucial molecule of the non‑canonical pyroptosis pathway

Insulin-like growth factor 2 (IGF2) mRNA-binding protein 2 (IGF2BP2) is a secreted protein that can bind to IGF2 and has been reported to promote inflammation. The data from the ENCORI database have predicted that IGF2BP2 can bind caspase 4, which mediates pyroptosis and promotes airway inflammation and lipopolysaccharide (LPS)-induced lung injury. The present study investigated whether IGF2BP2 can regulate LPS-induced lung cell inflammation by targeting caspase 4. Therefore, the non-tumorigenic lung epithelial cell line Beas-2B was transfected with short hairpin RNA (shRNA)-IGF2BP2 and stimulated with LPS. A number of parameters, including cell viability, production of interleukin (IL)-1β and IL-18, activation of gasdermin D (GSDMD) and the expression levels of IGF2BP2, caspase 4 and cleaved-caspase 1, were subsequently assessed using CCK-8, ELISA kits, western blotting and immunofluorescence staining, respectively. RNA pull-down assay was used to probe the possible interaction between IGF2BP2 and caspase 4 RNA. LPS treatment was found to inhibit cell viability, trigger IL-1β and IL-18 production and increase IGF2BP2 expression in a concentration-dependent manner. Compared with cells transfected with shRNA-negative control, cells that were transfected with shRNA-IGF2BP2 exhibited enhanced cell viability, reduced IL-1β and IL-18 concentrations, decreased GSDMD activation in addition to reduced expression levels of caspase 4 and cleaved-caspase 1 following stimulation with 1 µg/ml LPS. Concomitantly, the effects of IGF2BP2 silencing on caspase 4 expression were higher compared with those noted on caspase 1. In addition, binding of IGF2BP2 to caspase 4 RNA was also observed. To conclude, data from the present study suggest that IGF2BP2 knockdown inhibited LPS-induced Beas-2B cell inflammation by targeting caspase 4, thereby inhibiting the non-canonical pyroptosis pathway.

MicroRNA-145 transcriptionally regulates Semaphorin 3A expression in prostate cancer cells.

Prostate cancer (PCa) is one of the most prevalent cancer types among males. Differential expression of microRNAs is associated with various cancers including PCa. Although mature microRNAs are preferentially located in the cytoplasm, several studies identified mature human microRNAs in purified nuclei and miR-145 has been found to be predominantly expressed in the nuclei of benign tissues compared to tumor lesions. However, the nuclear functions of miR-145 are yet limited. Here, we aimed at investigating the inductive role of miR-145 on the expression of Semaphorin 3A(SEMA3A) in PCa cell lines. To study the regulatory potential of miR-145 in the transcriptional level in PCa, we overexpressed miR-145 in PC3 and DU145 cells, and confirmed its upregulation by quantitative-real-time-PCR. Then we investigated the tumor suppressor potential of miR-145 upon inducing SEMA3A expression using cell viability assay, western blot analysis, Chromatin Immunoprecipitation assay and luciferase reporter assay. Our results revealed that p53, miR-145, and SEMA3A expressions are significantly downregulated in PC3 and DU145 cells compared to nontumorigenic prostate epithelial PNT1a cells. miR-145 overexpression in PCa cells induced the expression of SEMA3A at both messenger RNA and protein levels. Furthermore, increased miR-145 expression enriched RNA Pol-II antibody on the promoter of SEMA3A and induced luciferase activity controlled by SEMA3A promoter. In this study, we showed that the functions of miR-145 are not limited to gene silencing, and found that it may lead to changes in gene expression in the transcriptional level.

Piperine and Celecoxib synergistically inhibit colon cancer cell proliferation via modulating Wnt/β-catenin signaling pathway

BackgroundCelecoxib (CXB), a selective COX-2 inhibitor NSAID, has exhibited prominent anti-proliferative potential against numerous cancers. However, its low bioavailability and long term exposure related cardiovascular side effects, limit its clinical application. In order to overcome these limitations, natural bioactive compounds with lower toxicity profile are used in combination with therapeutic drugs. Therfore, in this study Piperine (PIP), a natural chemo-preventive agent possessing drug bioavailability enhancing properties, was considered to be used in combination with low doses of CXB. PurposeWe hypothesized that the combination of PIP with CXB will have a synergistic anti-proliferative effect on colon cancer cells. Study designThe potency of PIP and CXB alone and in combination was evaluated in HT-29 human colon adenocarcinoma cells and mechanism of growth inhibition was investigated by analyzing the players in apoptotic and Wnt/β-catenin signaling pathways. MethodsThe effect of PIP on the oral bioavailability of CXB in mice was investigated using HPLC analysis. The study investigated the synergistic anti-proliferative effect of CXB and PIP on HT-29 cells and IEC-6 non-tumorigenic rat intestinal epithelial cells by SRB cell viability assay. Further, the cellular and molecular mechanism(s) involved in the anti-proliferative combinatorial effect was extensively explored in HT-29 cells by flow cytometry and western blotting. The in vivo efficacy of this combination was studied in CT26.WT tumor syngeneic Balb/c mice model. ResultsPIP as a bioenhancer increased the oral bioavailability of CXB (129%). The IC50 of CXB and PIP were evaluated to select doses for combination treatment of HT-29 cells. The drug combinations having combination index (CI) less than 1 were screened using CompuSyn software. These combinations were significantly cytotoxic to HT-29 cells but IEC-6 were least effected. Further, the mechanism behind CXB and PIP mediated cell death was explored. The co-treatment led to reactive oxygen species generation, mitochondrial dysfunction, caspase activation and enhanced apoptosis in HT-29 cells. Additionally, the combination treatment synergistically modulated Wnt/β-catenin pathway, downregulated the stemness markers and boosted therapeutic response in CT26 syngeneic Balb/c mice. ConclusionThe outcomes of the study suggests that combining CXB and PIP offers a novel approach for the treatment of colon cancer.

Elevated levels of expression of cytochrome P450 3A4 in a human liver epithelial cell line in differentiation-inducing conditions.

Cytochrome P450 (CYP) enzymes, especially CYP3A4 play a major role in the metabolism of xenobiotics in human liver. CYP3A4-expressing human liver or hepatoma cell lines may be good cell substitutes of human hepatocytes for drug metabolism studies. However, there are only a few cell lines expressing high levels of CYP3A4. The aim of this study is to investigate the expression of CYP3A4 and its mechanism in an immortalized non-tumorigenic human liver epithelial cell line, THLE-5b in differentiation-inducing conditions. When THLE-5b cells were cultivated in culture medium supplemented with hepatocytic differentiation-inducing factors, they showed hepatocytic morphology. In addition, elevated levels of expression not only of α1-antitrypsin (AAT) and albumin (ALB) mRNAs, but also of CYP3A4 mRNA, which are functional hepatocyte markers, were observed compared with the control. Among hepatocytic differentiation-inducing factors, dexamethasone (DEX) and insulin-transferrin-sodium selenite (ITS) seemed to be involved in elevation of expression of CYP3A4 mRNA. The mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126 or the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 reduced CYP3A4 mRNA levels of THLE-5b cells. Furthermore, the CpG site of the CYP3A4 promoter region in THLE-5b cells was found to be unmethylated, although in low CYP3A4-expressing HepG2 cells, the site was methylated. In conclusion, THLE-5b cells, which are unmethylated at the CpG site of the CYP3A4 promoter region, express CYP3A4 mRNA through the MEK/ERK1/2 and PI3K/Akt signaling pathways and acquire hepatocytic functions in differentiation-inducing conditions. Thus, THLE-5b cells could be a useful cell system for the study of drug metabolism.

S100A8/A9 mediate the reprograming of normal mammary epithelial cells induced by dynamic cell\u2013cell interactions with adjacent breast cancer cells

To understand the potential effects of cancer cells on surrounding normal mammary epithelial cells, we performed direct co-culture of non-tumorigenic mammary epithelial MCF10A cells and various breast cancer cells. Firstly, we observed dynamic cell–cell interactions between the MCF10A cells and breast cancer cells including lamellipodia or nanotube-like contacts and transfer of extracellular vesicles. Co-cultured MCF10A cells exhibited features of epithelial-mesenchymal transition, and showed increased capacity of cell proliferation, migration, colony formation, and 3-dimensional sphere formation. Direct co-culture showed most distinct phenotype changes in MCF10A cells followed by conditioned media treatment and indirect co-culture. Transcriptome analysis and phosphor-protein array suggested that several cancer-related pathways are significantly dysregulated in MCF10A cells after the direct co-culture with breast cancer cells. S100A8 and S100A9 showed distinct up-regulation in the co-cultured MCF10A cells and their microenvironmental upregulation was also observed in the orthotropic xenograft of syngeneic mouse mammary tumors. When S100A8/A9 overexpression was induced in MCF10A cells, the cells showed phenotypic features of directly co-cultured MCF10A cells in terms of in vitro cell behaviors and signaling activities suggesting a S100A8/A9-mediated transition program in non-tumorigenic epithelial cells. This study suggests the possibility of dynamic cell–cell interactions between non-tumorigenic mammary epithelial cells and breast cancer cells that could lead to a substantial transition in molecular and functional characteristics of mammary epithelial cells.

Proteome of breast cancer derived microvesicles

The aim of the research. To examine the proteomic profi le of breast cancer exosomes. Material and methods. Cell lines used for this study were MDA-MB-231 female epithelial breast cancer cells (ATCC HTB-26) and MCF10A non-tumorigenic epithelial breast tissue cells. MVs were isolated using diff erential ultracentrifugation. Samples were lysed, reduced, alkylated, digested, and analyzed by an Orbitrap Fusion mass spectrometer. MS raw fi les were analyzed using MaxQuant version 1.6.12.0. Peptides were searched against the human UniProt FASTA database using the Andromeda search engine, integrated into MaxQuant. Results. MVs derived from MCF10A and MDA-MB-231 cell lines were analyzed, and 1427 and 547 proteins were identifi ed in the MDA-MB-231 and MCF10A-derived MVs, respectively. In total, 455 proteins were common to both MDA-MB-231 and MCF10A MVs. MVs derived from MCF10A and MDAMB-231 cell lines were analyzed, and 1427 and 547 proteins were identifi ed in the MDA-MB-231 and MCF10A-derived MVs, respectively. In total, 455 proteins were common to both MDA-MB-231 and MCF10A MVs. Th e unique MDA-MB-231 MV proteins were searched against the DisGeNET human diseases database. Out of 972 MDA-MB-231 MV proteins, 112 were cancer-related while 32 were specifi cally associated with BC. In the MDA-MB-231 MV proteome, 23 Wnt signaling pathway proteins were identifi ed based on their GO biological process. Proteomic analysis identifi ed enzymes OAT, TALDO1, and BLMH were only in MVs from metastatic MDA-MB-231 cell line. The specific activity of OAT and TALDO1 was higher in MV fractions of MDA-MB-231 in comparison to the non-cancerous MCF10A cell line-derived MVs. Th ese fi ndings might suggest that these enzymes might play a role in BC. In our present study, we found that some enzymes identifi ed from MV fractions were already proposed to play a role in cancer therapy as therapeutic targets (OAT, TALDO1) and resistance against chemotherapy agents (BLMH).

Exploring the Anti-Cancer Mechanism of Novel 3,4'-Substituted Diaryl Guanidinium Derivatives.

We previously identified a guanidinium-based lead compound that inhibited BRAF through a hypothetic type-III allosteric mechanism. Considering the pharmacophore identified in this lead compound (i.e., “lipophilic group”, “di-substituted guanidine”, “phenylguanidine polar end”), several modifications were investigated to improve its cytotoxicity in different cancer cell lines. Thus, several lipophilic groups were explored, the di-substituted guanidine was replaced by a secondary amine and the phenyl ring in the polar end was substituted by a pyridine. In a structure-based design approach, four representative derivatives were docked into an in-house model of an active triphosphate-containing BRAF protein, and the interactions established were analysed. Based on these computational studies, a variety of derivatives was synthesized, and their predicted drug-like properties calculated. Next, the effect on cell viability of these compounds was assessed in cell line models of promyelocytic leukaemia and breast, cervical and colorectal carcinomas. The potential of a selection of these compounds as apoptotic agents was assessed by screening in the promyelocytic leukaemia cell line HL-60. The toxicity against non-tumorigenic epithelial MCF10A cells was also investigated. These studies allowed for several structure-activity relationships to be derived. Investigations on the mechanism of action of representative compounds suggest a divergent effect on inhibition of the MAPK/ERK signalling pathway.

Synthesis, in vitro biological activity, hydrolytic stability and docking of new analogs of BIM-23052 containing halogenated amino acids.

One of the potent somatostatin analogs, BIM-23052 (DC-23-99) D-Phe-Phe-Phe-D-Trp-Lys-Thr-Phe-Thr-NH2, has established in vitro growth hormone inhibitory activity in nM concentrations. It is also characterized by high affinity to some somatostatin receptors which are largely distributed in the cell membranes of many tumor cells. Herein, we report the synthesis of a series of analogs of BIM-23052 containing halogenated Phe residues using standard solid-phase peptide method Fmoc/OtBu-strategy. The cytotoxic effects of the compounds were tested in vitro against two human tumor cell lines-breast cancer cell line and hepatocellular cancer cell line, as well as on human non-tumorigenic epithelial cell line. Analogs containing fluoro-phenylalanines are cytotoxic in μM range, as the analog containing Phe (2-F) showed better selectivity against human hepatocellular cancer cell line. The presented study also reveals that accumulation of halogenated Phe residues does not increase the cytotoxicity according to tested cell lines. The calculated selective index reveals different mechanisms of antitumor activity of the parent compound BIM-23052 and target halogenated analogs for examined breast tumor cell lines. All peptides tested have high antitumor activity against the HepG2 cell line (IC50 ≈ 100μM and SI > 5) compared to breast cells. This is probably due to the high permeability of the cell membrane and the higher metabolic activity of hepatocytes. In silico docking studies confirmed that all obtained analogs bind well with the somatostatin receptors with preference to ssrt3 and ssrt5. All target compounds showed high hydrolytic stability at acid and neutral pH, which mimic physiological condition in stomach and human plasma.

Thidiazuron decreases epithelial-mesenchymal transition activity through the NF-kB and PI3K/AKT signalling pathways in breast cancer.

Breast cancer is the major type among the women population globally. The treatment of cancer metastasis has made modest progress due to multiple factors. Thidiazuron (TDZ) is a novel plant growth regulator that has been shown to have anticancer effects. Therefore, we explored the anti‐metastatic potentials of TDZ in cell lines by assessing its potential to suppress the epithelial‐mesenchymal transition (EMT). We pretreated the BEAS‐2B and breast cancer (MDA‐MB‐231) cells with TDZ and deliberated alteration in a cell viability, mammosphere, migration, NF‐кB signalling, PI3K/AKT signalling and matrix metalloproteinase (MMP) expression and analysed the EMT induction by TGF‐β/TNF‐α‐stimulated BEAS‐2B cells. Treatment with TDZ (5‐50 μmol) diminished the migration and invasion of the extremely metastatic MDA‐MB‐231 cells. Additionally, TDZ treatment led to down‐regulation of uPAR, uPA, VEGF and MMP‐2/‐9 expression and up‐regulation of TIMP‐1/2 expression in these cells. Furthermore, TDZ treatment blocked invasion and EMT in non‐tumorigenic BEAS‐2B epithelial cells stimulated with TGF‐β/TNF‐α.TDZ prevents EMT and may thus block metastasis of breast cancer cells.

S-allylcysteine induces cytotoxic effects in two human lung cancer cell lines via induction of oxidative damage, downregulation of Nrf2 and NF-κB, and apoptosis.

In this study, we investigated the putative cytotoxic effect elicited by the garlic-derived compound S-allylcysteine (SAC) in two human cancer cell lines (HCC827 and NCI-H1975) in order to develop an experimental approach to the therapeutic potential of this molecule for lung cancer. Cells were incubated for 24, 48 and 72 h in the presence of SAC (10 or 20 mM), which resulted in a concentration- and time-dependent decrease in cell viability and culture confluence in both cell lines. These effects were contrasted with - and validated through - those observed in an immortalized but nontumorigenic epithelial cell line from human bronchial epithelium (BEAS-2B, negative control) and an adenocarcinoma human alveolar basal epithelial cell line (A549, positive control). SAC (20 mM at 72 h) also increased the oxidative damage to lipids, augmented apoptosis, and decreased the expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) and the nuclear factor kappa B (NF-κB) proteins in HCC827 and NCI-H1975 cells. Our results establish the efficacy of SAC in reducing malignant growth and proliferation of lung tumor cells. This effect is mediated by the induction of oxidative damage associated with the downregulation of Nrf2 and NF-κB and their corresponding signaling pathways.

Function and mechanism exploration of zinc finger protein 64 in lung adenocarcinoma cell growth and metastasis

This paper aims to discover the effect of Zinc Finger Protein 64 (ZFP64) and Notch pathway on lung adenocarcinoma cell. ZFP64 expression in cancer tissue and overall survival analysis was identified by TCGA-LUAD. ZFP64 expressions in tumor tissue (n = 30) and adjacent tissue (n = 30), and in human nontumorigenic bronchial epithelial cell line BEAS-2B and human lung adenocarcinoma cell lines (H23, H1975, H2228, and H2085) were measured via quantitative real-time polymerase chain reaction (qRT-PCR). H1975 cell viability, cell cycle progression, and migration after transfection or under Notch inhibitor MK-0752 treatment were detected through MTT assay, flow cytometer, and wound healing assay, respectively. Expressions of notch intracellular domain (NICD) and hairy and enhancer of split 1 (Hes-1) in H1975 cell were determined by western blot. Epithelial-mesenchymal transition (EMT)-related proteins (E-Cadherin and Vimentin) expressions were identified through qRT-PCR and western blot. ZFP64 expression in lung adenocarcinoma tissue and lung adenocarcinoma cell lines was higher and related to poor prognosis. After transfection, H1975 cell viability, migration, and expressions of Vimentin, NICD and Hes-1 were upregulated yet cell percentage in G0/G1 phase, E-cadherin expression was downregulated by overexpressed ZFP64. However, Notch inhibitor MK-0752 inhibited the effects of overexpressed ZFP64 on H1975 cell viability, cell cycle, migration, EMT progress, and Notch pathway activation. Overexpressed ZFP64 promoted the development of lung adenocarcinoma cells by activating Notch pathway.