Cytostatic/Cytotoxic Effects of 5‐Fluorouridine Nucleolipids on Colon,Hepatocellular, and Renal Carcinoma Cells: in vitro Identification of a Potential Cytotoxic Multi‐Anticancer Drug

The insufficient penetration through the cell membranes is one of the major drawbacks of chemotherapeutics such as 5‐fluorouracil (5‐FU; 1 ). To improve the penetration, a useful strategy is the attachment of lipophilic moieties. Thus, we have synthesized a series of nucleolipid derivatives of 5‐fluorouridine (5‐FUrd; 2a ), carrying lipophilic moieties at N(3) and/or at the 2′,3′‐O position, i.e., 3a, 3b, 4 – 7 , and tested their cytostatic/cytotoxic activities towards three carcinoma cell lines (colon (HT‐29), hepatocellular (HepG2), and renal (RENCA)) in comparison with 5‐FU ( 1 ) and 5‐FUrd ( 2a ). After 48 h of incubation, four derivatives, 3a, 3b, 5 , and 7 , showed inhibitory effects on the survival of HT‐29, HepG2, and RENCA cells. Additionally, to differentiate between anticancer and side‐effects, we tested the cytotoxicity of the derivatives in human macrophages. Interestingly, the derivatives 4, 5 , and 6 did not exhibit any effects on survival of THP‐1 macrophages. Furthermore, we investigated the apoptosis induction of compound 1 and 2a , and the above‐mentioned derivatives in HT‐29 cells. Derivative 5 showed the highest significant (p<0.05; p<0.01) increase of the apoptosis at 80 μM after 2‐h or 4‐h treatment, as well as after 6‐h incubation at 40 μM (p<0.05). Real‐time PCR revealed that 40‐μM derivative 5 showed a 1.8‐fold increase of the pro‐apoptotic caspase‐3 gene and a twofold significant increase (p<0.01 and p<0.05 vs. control and 1 , resp.) of the tumor suppressor TP53 gene, whereas the other compounds did not show any effect. We demonstrated that some 5‐FUrd derivatives such as compound 5 are more effective than 5‐FU or 5‐FUrd concerning a cytotoxic (vs. cytostatic (5‐FU, 5‐FUrd)) effect on different cancer cell lines, but without cytotoxic side‐effects on differentiated macrophages. Thus, compound 5 is suggested as a novel potent cytotoxic multi‐anti‐cancer drug.     

Synthesis of 5‐Fluorouridine Nucleolipid Derivatives and Their Cytostatic/Cytotoxic Activities on Human HT‐29 Colon Carcinoma Cells

One of the major drawbacks of chemotherapeutics is their insufficient penetration through cell membranes due to a high hydrophobicity. Thus, we have synthesized a series of selected nucleolipid derivatives of 5‐fluorouridine (5‐FUrd; 2a ), carrying lipophilic moieties at N(3) and/or in the 2′,3′‐O‐position (i.e., 3a – 7a and 3c ), and tested their cytostatic/cytotoxic activities using HT‐29 human colon carcinoma cells, in comparison with, e.g., 5‐FU ( 1 ) and 5‐FUrd ( 2a ). Incorporation and intracellular localization of the substances under test were performed after conjugation with the fluorochrome Atto 425. We showed that all 5′‐O‐labelled Atto 425 derivatives were incorporated by the human HT‐29 cells and accumulated in their cytoplasm. Moreover, after 24‐h treatment of HT‐29 human colon carcinoma cells, 1 or 2a (10, 20, 40, or 80 μM ) revealed a significant (14–23 or 33–45%, resp.) decrease of the viability in comparison with the (negative) control. Interestingly, derivatives 3a and 3c (40 and 80 μM ) led to a significant (77–95 or 89–96%, resp.) inhibition of survival of human HT29 cells, i.e., these two substances were ca. 63–72% or ca. 75%, respectively more effective than 5‐FU ( 1 ; positive control). Furthermore, derivative 5a showed a significant, i.e., 30 and 86%, inhibition of the survival at 40 and 80 μM , respectively in comparison with the (negative) control. Some synthesized 5‐FUrd derivatives turned out to be more effective than 5‐FU ( 1 ) or 5‐FUrd ( 2a ).     

Synthesis and Characterization of Novel Thiazolo[3,2‐a]pyrimidine Derivatives and Evaluation of Antioxidant and Cytotoxic Activities

A series of novel thiazolo[3,2‐a]pyrimidines were synthesized and characterized by FT‐IR, ¹H, ¹³C‐NMR and mass techniques. Their antioxidant activities were investigated by 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging assay and the results showed that all the synthesized compounds exhibit good antioxidant activity. In addition, it was found that any substituent on the aromatic ring of the products plays an important role in their antioxidant activity. In vitro cytotoxicity of compounds 4a – 4j was investigated using MTT cell viability assay. Among these compounds, 6‐ethyl 2,3‐dimethyl 5‐(4‐chlorophenyl)‐7‐methyl‐2,3‐dihydro‐5H‐[1,3]thiazolo[3,2‐a]pyrimidine‐2,3,6‐tricarboxylate ( 4e ) bearing a chlorine substituent displayed the highest cytotoxic effect (IC₅₀=6.26±0.6 μm ) in comparison with doxorubicin (IC₅₀=0.68±0.1 μm ) as a standard after 72 h. Therefore, it is assumed that these compounds could be used as effective antioxidant and cytotoxic agents.     

Guanosine Nucleolipids: Synthesis,Characterization, Aggregation and X‐Ray Crystallographic Identification of Electricity‐Conducting G‐Ribbons

The lipophilization of β‐d ‐riboguanosine ( 1 ) with various symmetric as well as asymmetric ketones is described (→ 3a – 3f ). The formation of the corresponding O‐2′,3′‐ketals is accompanied by the appearance of various fluorescent by‐products which were isolated chromatographically as mixtures and tentatively analyzed by ESI‐MS spectrometry. The mainly formed guanosine nucleolipids were isolated and characterized by elemental analyses, ¹H‐, ¹³C‐NMR and UV spectroscopy. For a drug profiling, static topological polar surface areas as well as ¹⁰logP_OW values were calculated by an increment‐based method as well as experimentally for the systems 1‐octanol‐H₂O and cyclohexane‐H₂O. The guanosine‐O‐2′,3′‐ketal derivatives 3b and 3a could be crystallized in (D₆)DMSO – the latter after one year of standing at ambient temperature. X‐ray analysis revealed the formation of self‐assembled ribbons consisting of two structurally similar 3b nucleolipid conformers as well as integrated (D₆)DMSO molecules. In the case of 3a ? DMSO, the ribbon is formed by a single type of guanosine nucleolipid molecules. The crystalline material 3b ? DMSO was further analyzed by differential scanning calorimetry (DSC) and temperature‐dependent polarization microscopy. Crystallization was also performed on interdigitated electrodes (Au, distance, 5 μm) and visualized by scanning electron microscopy. Resistance and amperage measurements clearly demonstrate that the electrode‐bridging 3b crystals are electrically conducting. All O‐2′,3′‐guanosine ketals were tested on their cytostatic/cytotoxic activity towards phorbol 12‐myristate 13‐acetate (PMA)‐differentiated human THP‐1 macrophages as well as against human astrocytoma/oligodendroglioma GOS‐3 cells and against rat malignant neuroectodermal BT4Ca cells.     

4‐Cyano‐α‐methyl‐l‐phenylalanine as a Spectroscopic Marker for the Investigation of PeptaibioticMembrane Interactions

Two analogs of the ten‐amino acid residue, membrane‐active lipopeptaibiotic trichogin GA IV, mono‐labeled with 4‐cyano‐α‐methyl‐L ‐phenylalanine, a potentially useful fluorescence and IR absorption probe of the local microenvironment, were synthesized by the solid‐phase methodology and conformationally characterized. The single modification was incorporated either at the N‐terminus (position 1) or near the C‐terminus (position 8) of the peptide main chain. In both cases, the replaced amino acid was the equally helicogenic α‐aminoisobutyric acid (Aib) residue. We performed a solution conformational analysis by use of FT‐IR absorption, CD, and 2D‐NMR spectroscopies. The results indicate that both labeled analogs essentially maintain the overall helical propensity of the naturally occurring lipopeptaibiotic. Peptide? membrane interactions were assessed by fluorescence and ATR‐IR absorption techniques. Analogies and differences between the two peptides were highlighted. Taken together, our data confirm literature results that some of the spectroscopic parameters of the 4‐cyanobenzyl chromophore are sensitive markers of the local microenvironment.     

Nucleolipids of the Cancerostatic 5‐Fluorouridine: Synthesis,Adherence to Oligonucleotides,and Incorporation in Artificial Lipid Bilayers

5‐Fluorouridine ( 1a ) was converted to its N(3)‐farnesylated nucleoterpene derivative 8 by direct alkylation with farnesyl bromide ( 4 ). Reaction of the cancerostatic 1a with either acetone, heptan‐4‐one, nonadecan‐10‐one, or hentriacontan‐16‐one afforded the 2′,3′‐O‐ketals 2a – 2d . Compound 2b was then first farnesylated (→ 5 ) and subsequently phosphitylated to give the phosphoramidite 6 . The ketal 2c was directly 5′‐phosphitylated without farnesylation of the base to give the phosphoramidite 7 . Moreover, the recently prepared cyclic 2′,3′‐O‐ketal 11 was 5′‐phosphitylated to yield the phosphoramidite 12 . The 2′,3′‐O‐isopropylidene derivative 2a proved to be too labile to be converted to a phosphoramidite. All novel derivatives of 1a were unequivocally characterized by NMR and UV spectroscopy and ESI mass spectrometry, as well as by elemental analyses. The lipophilicity of the phosphoramidite precursors were characterized by both their retention times in RP‐18 HPLC and by calculated log P values. The phosphoramidites 6, 7 , and 12 were exemplarily used for the preparation of four terminally lipophilized oligodeoxynucleotides carrying a cyanine‐3 or a cyanine‐5 residue at the 5′‐(n–1) position (i.e., 14 – 17 ). Their incorporation in an artificial lipid bilayer was studied by single‐molecule fluorescence spectroscopy and fluorescence microscopy.     

Synthesis of β‐Ketoamide Curcumin Analogs for Anti‐Diabetic and AGEs Inhibitory Activities

Two different series of novel β‐ketoamide curcumin analogs enriched in biological activities have been synthesized. The synthesized compounds were screened for their in vitro anti‐diabetic and AGEs inhibitory activities and exhibited potent to good anti‐diabetic and AGEs inhibitory activities. The molecular docking study was also performed with the α‐amylase enzyme.     

Synthesis and Antiproliferative Activities of Novel Substituted 5‐Anilino‐α‐Glucofuranose Derivatives

In order to find novel antitumor candidate agents with high efficiency and low toxicity, 14 novel substituted 5‐anilino‐α‐glucofuranose derivatives have been designed, synthesized and evaluated for antiproliferative activities in vitro. Their structures were characterized by NMR (¹H and ¹³C) and HR‐MS, and configuration (R/S) at C(5) was identified by two‐dimensional ¹H,¹H‐NOESY‐NMR spectrum. Their antiproliferative activities against human tumor cells were investigated by MTT assay. The results demonstrated that most of the synthesized compounds had antiproliferative effects comparable to the reference drugs gefitinib and lapatinib. In particular, (5R)‐5‐O‐(3‐chloro‐4‐{[5‐(4‐fluorophenyl)thiophen‐2‐yl]methyl}anilino)‐5‐deoxy‐1,2‐O‐(1‐methylethylidene)‐α‐glucofuranose ( 9da ) showed the most potent antiproliferative effects against SW480, A431 and A549 cells, with IC₅₀ values of 8.57, 5.15 and 15.24 μm , respectively. This work suggested 5‐anilino‐α‐glucofuranose as an antitumor core structure that may open a new way to develop more potent anti‐cancer agents.     

5α‐Androst‐16‐en‐3α‐ol β‐D‐Glucuronide,Precursor of 5α‐Androst‐16‐en‐3α‐ol in Human Sweat

5α‐Androst‐16‐en‐3α‐ol (α‐androstenol) is an important contributor to human axilla sweat odor. It is assumed that α‐andostenol is excreted from the apocrine glands via a H₂O‐soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H₂O‐soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α‐androstenol, β‐androstenol sulfates, 5α‐androsta‐5,16‐dien‐3β‐ol (β‐androstadienol) sulfate, α‐androstenol β‐glucuronide, α‐androstenol α‐glucuronide, β‐androstadienol β‐glucuronide, and α‐androstenol β‐glucuronide furanose. The occurrence of α‐androstenol β‐glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative‐ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α‐androstenol was observed after incubation of the sterile human sweat or α‐androstenol β‐glucuronide with a commercial glucuronidase enzyme, the urine‐isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have β‐glucuronidase activities. We demonstrated that if α‐ and β‐androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H₂O‐soluble precursor of α‐androstenol in apocrine secretion should be a β‐glucuronide.     

Synthesis of 3‐Methylidene‐1‐tosyl‐2,3‐dihydroquinolin‐4(1H)‐ones as Potent Cytotoxic Agents

An efficient synthetic strategy to 3‐methylidene‐2,3‐dihydroquinolin‐4(1H)‐ones variously substituted in position 2 has been developed. The title compounds were synthesized in the reaction sequence involving reaction of diethyl methylphosphonate with methyl 2‐(tosylamino)benzoate, condensation of thus formed diethyl 2‐oxo‐2‐(2‐N‐tosylphenyl)ethylphosphonate with various aldehydes followed by successful application of the obtained 3‐(diethoxyphosphoryl)‐1,2‐dihydroquinolin‐4‐ols as Horner–Wadsworth–Emmons reagents for the olefination of formaldehyde. Also, enantioselective approach to the target compounds has been evaluated using 3‐dimenthoxyphosphoryl group as a chiral auxiliary. Single X‐ray crystal analysis of (2S)‐3‐(dimenthoxyphosphoryl)‐2‐phenyl‐1‐tosyldihydroquinolin‐4‐ol revealed the presence of strong resonance‐assisted hydrogen bond (RAHB). The obtained 3‐methylidene‐2,3‐dihydroquinolin‐4(1H)‐ones were then tested for their cytotoxic activity against two leukemia cell lines NALM‐6 and HL‐60 and a breast cancer MCF‐7 cell line. All compounds showed very high cytotoxic activity with the IC₅₀ values mostly below 1 μm in all three cancer cell lines. The selected analogs were also tested on human umbilical vein endothelial cells (HUVEC) and on human mammary gland/breast cells (MCF‐10A) to evaluate their influence on normal cells. Since one of the most serious problems in cancer chemotherapy is the development of drug resistance, the mRNA levels and activity of ABCB1 transporter considered to be the most important factor engaged in drug resistance, were evaluated in MCF‐7 cells treated with two selected analogs. Both compounds were strong ABCB1 transporter inhibitors that could prevent efflux of anticancer drugs from cancer cells.     

Novel grafted agar disks for the covalent immobilization of β‐D‐galactosidase

Novel grafted agar disks were prepared for the covalent immobilization of β‐D‐galactosidase (β‐gal). The agar disks were activated through reacting with ethylenediamine or different molecular weights of Polyethyleneimine (PEI), followed by glutaraldehyde (GA). The modification of the agar gel and the binding of the enzyme were verified by Fourier Transform Infrared (FTIR) and elemental analysis. Moreover, the agar's activation process was optimized, and the amount of immobilized enzyme increased 3.44 folds, from 38.1 to 131.2 U/g gel, during the course of the optimization process. The immobilization of β‐gal onto the activated agar disks caused its optimum temperature to increase from 45°C to 45–55°C. The optimum pH of the enzyme was also shifted towards the acidic side (3.6–4.6) after its immobilization. Additionally, the Michaelis‐Menten constant (K_m) increased for the immobilized β‐gal as compared to its free counterpart whereas the maximum reaction rate (V_max) decreased. The immobilized enzyme was also shown to retain 92.99% of its initial activity after being used for 15 consecutive times. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 675–684, 2015.     

Fed‐batch synthesis of galacto‐oligosaccharides with Aspergillus oryzae β‐galactosidase using optimal control strategy

Fed‐batch synthesis of galacto‐oligosaccharides (GOS) from lactose with β‐galactosidase from Aspergillus oryzae was evaluated experimentally and reaction yield was maximized via optimal control technique. The optimal lactose and enzyme feed flow rate profiles were determined using a model for GOS synthesis previously reported by the authors. Experimentally it was found that fed‐batch synthesis allowed an increase on the maximum total GOS concentration from 115 (batch synthesis) to 218 g L^?1 as consequence of the increase in total sugars concentration from 40 to 58% w/w. Such high concentration of total sugars was not attainable in batch operation because of the low solubility of lactose at the reaction temperature (40°C). Simulations predicted a GOS yield of 32.5 g g^?1 in fed‐batch synthesis under optimal conditions, while experimentally the same yield as in batch synthesis was obtained (28 g g^?1). Besides, an enrichment of total oligosaccharides in GOS with a high polymerization degree (GOS‐5 and GOS‐6) was observed in the fed‐batch synthesis. Experimental profiles for all sugars were similar to the ones predicted by simulation, which supports the use of this methodology for the optimization of GOS synthesis. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:59–67, 2014     

Characterization of an Importin α/β‐recognized nuclear localization signal in β‐dystroglycan

β‐dystroglycan (β‐DG) is a widely expressed transmembrane protein that plays important roles in connecting the extracellular matrix to the cytoskeleton, and thereby contributing to plasma membrane integrity and signal transduction. We previously observed nuclear localization of β‐DG in cultured cell lines, implying the existence of a nuclear targeting mechanism that directs it to the nucleus instead of the plasma membrane. In this study, we delineate the nuclear import pathway of β‐DG, characterizing a functional nuclear localization signal (NLS) in the β‐DG cytoplasmic domain, within amino acids 776–782. The NLS either alone or in the context of the whole β‐DG protein was able to target the heterologous GFP protein to the nucleus, with site‐directed mutagenesis indicating that amino acids R⁷⁷⁹ and K⁷⁸⁰ are critical for NLS functionality. The nuclear transport molecules Importin (Imp)α and Impβ bound with high affinity to the NLS of β‐DG and were found to be essential for NLS‐dependent nuclear import in an in vitro reconstituted nuclear transport assay; cotransfection experiments confirmed the dependence on Ran for nuclear accumulation. Intriguingly, experiments suggested that tyrosine phosphorylation of β‐DG may result in cytoplasmic retention, with Y⁸⁹² playing a key role. β‐DG thus follows a conventional Impα/β‐dependent nuclear import pathway, with important implications for its potential function in the nucleus. J. Cell. Biochem. 110: 706–717, 2010. © 2010 Wiley‐Liss, Inc.     

Isolation and 2‐D‐DIGE proteomic analysis of intracellular and extracellular forms of Listeria monocytogenes

The pathogenicity of Listeria monocytogenes is related to its ability of invading and multiplying in eukaryotic cells. Its main virulence factors are now well characterized, but limited proteomic data is available concerning its adaptation to the intracellular environment. In this study, L. monocytogenes EGD (serotype 1/2a) grown in human THP‐1 monocytes (24 h) were successfully separated from host organelles and cytosolic proteins by differential and isopycnic centrifugation. For control, we used cell homogenates spiked with bacteria grown in broth. Proteomes from both forms of bacteria were compared using a 2‐D‐DIGE approach followed by MALDI‐TOF analysis to identify proteins. From 1684 distinct spots, 448 were identified corresponding to 245 distinct proteins with no apparent contamination of host proteins. Amongst them, 61 show underexpression (stress defense; transport systems, carbon metabolism, pyrimidines synthesis, D ‐Ala‐D ‐Ala ligase) and 22 an overexpression (enzymes involved in the synthesis of cell envelope lipids, glyceraldehyde‐3‐phosphate, pyruvate and fatty acids). Our proteomic analysis of intracellular L. monocytogenes (i) suggests that bacteria thrive in a more favorable environment than extracellularly, (ii) supports the concept of metabolic adaptation of bacteria to intracellular environment and (iii) may be at the basis of improved anti‐Listeria therapy.     

Mir‐29b promotes human aortic valve interstitial cell calcification via inhibiting TGF‐β3 through activation of wnt3/β‐catenin/Smad3 signaling

Herein, we hypothesized that pro‐osteogenic MicroRNAs (miRs) could play functional roles in the calcification of the aortic valve and aimed to explore the functional role of miR‐29b in the osteoblastic differentiation of human aortic valve interstitial cells (hAVICs) and the underlying molecular mechanism. Osteoblastic differentiation of hAVICs isolated from human calcific aortic valve leaflets obtained intraoperatively was induced with an osteogenic medium. Alizarin red S staining was used to evaluate calcium deposition. The protein levels of osteogenic markers and other proteins were evaluated using western blotting and/or immunofluorescence while qRT‐PCR was applied for miR and mRNA determination. Bioinformatics and luciferase reporter assay were used to identify the possible interaction between miR‐29b and TGF‐β3. Calcium deposition and the number of calcification nodules were pointedly and progressively increased in hAVICs during osteogenic differentiation. The levels of osteogenic and calcification markers were equally increased, thus confirming the mineralization of hAVICs. The expression of miR‐29b was significantly increased during osteoblastic differentiation. Furthermore, the osteoblastic differentiation of hAVICs was significantly inhibited by the miR‐29b inhibition. TGF‐β3 was markedly downregulated while Smad3, Runx2, wnt3, and β‐catenin were significantly upregulated during osteogenic induction at both the mRNA and protein levels. These effects were systematically induced by miR‐29b overexpression while the inhibition of miR‐29b showed the inverse trends. Moreover, TGF‐β3 was a direct target of miR‐29b. Inhibition of miR‐29b hinders valvular calcification through the upregulation of the TGF‐β3 via inhibition of wnt/β‐catenin and RUNX2/Smad3 signaling pathways.     

Asymmetric Allylation of α‐Ketoester‐Derived N‐Benzoylhydrazones Promoted by Chiral Sulfoxides/N‐Oxides Lewis Bases: Highly Enantioselective Synthesis of Quaternary α‐Substituted α‐Allyl‐α‐Amino Acids

Chiral sulfoxides/N‐oxides (R)‐ 1 and (R,R)‐ 2 are effective chiral promoters in the enantioselective allylation of α‐keto ester N‐benzoylhydrazone derivatives 3a , 3b , 3c , 3d , 3e , 3f , 3g to generate the corresponding N‐benzoylhydrazine derivatives 4a , 4b , 4c , 4d , 4e , 4f , 4g , with enantiomeric excesses as high as 98%. Representative hydrazine derivatives 4a , 4b were subsequently treated with SmI₂, and the resulting amino esters 5a , 5b with LiOH to obtain quaternary α‐substituted α‐allyl α‐amino acids 6a , 6b , whose absolute configuration was assigned as (S), with fundament on chemical correlation and electronic circular dichroism (ECD) data. Chirality 25:529–540, 2013. © 2013 Wiley Periodicals, Inc.     

Activation of Wnt/β‐catenin signalling is required for TGF‐β/Smad2/3 signalling during myofibroblast proliferation

Fibrosis in animal models and human diseases is associated with aberrant activation of the Wnt/β‐catenin pathway. Despite extensive research efforts, effective therapies are still not available. Myofibroblasts are major effectors, responsible for extracellular matrix deposition. Inhibiting the proliferation of the myofibroblast is crucial for treatment of fibrosis. Proliferation of myofibroblasts can have many triggering effects that result in fibrosis. In recent years, the Wnt pathway has been studied as an underlying factor as a primary contributor to fibrotic diseases. These efforts notwithstanding, the specific mechanisms by which Wnt‐mediated promotes fibrosis reaction remain obscure. The central role of the transforming growth factor‐β (TGF‐β) and myofibroblast activity in the pathogenesis of fibrosis has become generally accepted. The details of interaction between these two processes are not obvious. The present investigation was conducted to evaluate the level of sustained expression of fibrosis iconic proteins (vimentin, α‐SMA and collagen I) and the TGF‐β signalling pathway that include smad2/3 and its phosphorylated form p‐smad2/3. Detailed analysis of the possible molecular mechanisms mediated by β‐catenin revealed epithelial–mesenchymal transition and additionally demonstrated transitions of fibroblasts to myofibroblast cell forms, along with increased activity of β‐catenin in regulation of the signalling network, which acts to counteract autocrine TGF‐β/smad2/3 signalling. A major outcome of this study is improved insight into the mechanisms by which epithelial and mesenchymal cells activated by TGFβ1‐smad2/3 signalling through Wnt/β‐catenin contribute to lung fibrosis.