Molecular insights into epoxyazadiradione induced death in triple-negative breast cancer cells: A system biology approach.

Epoxyazadiradione is an important limonoid with immense pharmacological potential. We have reported previously that epoxyazadiradione (EAD) induces apoptosis in triple negative breast cancer cells (MDA-MB 231) by modulating diverse cellular targets. Here, we identify the key genes/pathways responsible for this effect through next-generation sequencing of the transcriptome from EAD treated cells and integrated molecular data analysis using bioinformatics. In silico analysis indicated that EAD displayed favourable drug-like properties and could target multiple macromolecules relevant to TNBC. RNA sequencing revealed that EAD treatment results in the differential expression of 1838 genes in MDA-MB 231 cells, with 752 downregulated and 1086 upregulated. Gene set enrichment analysis of these genes suggested that EAD disrupts protein folding in the endoplasmic reticulum, triggering the unfolded protein response (UPR) and potentially leading to cell death. EAD also induced oxidative stress and DNA damage, downregulated pathways linked to metabolism, cell cycle progression, pro-survival signalling, cell adhesion, motility and inflammatory response. The identification of protein cluster and hub genes were also done. The validation of the identified hub genes gave an inverse correlation between their expression in EAD treated cells and TNBC patient samples. Thus, the identified hub genes could be explored as therapeutic or diagnostic markers for TNBC. Hence, EAD appears to be a promising therapeutic candidate for TNBC by targeting various hallmarks of cancer, including cell death resistance, uncontrolled proliferation and metastasis. To conclude, the identified pathways and validated targets for EAD will provide a roadmap for further in vivo studies and preclinical/clinical validation required for potential drug development.

Synergistic effects of epoxyazadiradione (EAD) and paclitaxel against triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancer (TNBC) is the most aggressive and chemo-resistant form of breast cancer subtype, and chemotherapy is a vital treatment option for that. Paclitaxel is an effective chemo drug for TNBC. However, in clinical settings, paclitaxel has adverse side effects. The synergistic combination is the most promising method for overcoming undesirable toxicity and achieving a beneficial therapeutic outcome. Previous reports, including our study, showed certain anticancer potential of epoxyazadiradione (EAD), the neem limonoid, in different types of cancer cells, including TNBC. OBJECTIVE: This study was designed to investigate the possible synergistic effects of EAD and paclitaxel against TNBC cells. METHODS: We examined the effects of EAD and paclitaxel alone and in combination in MDA-MB 231 cells, and the percentage cytotoxicity was used to calculate synergism. Characteristic apoptotic changes were observed by visualizing cellular morphology, nuclear fragmentation and membrane integrity. We further estimated anti-migratory potential of experimental compounds by wound healing assay. The reduction in inflammation during combinatorial treatment was evaluated by observing NF-κB translocation. RESULTS: The combined treatment with EAD (5 µM) and paclitaxel (5 nM), which were used at doses lower than their individual IC50 concentrations, showed a synergistic effect in MDA-MB-231 cells. This combination effectively induced apoptosis and antimigration and reduced the inflammatory reactions induced by the higher dose of paclitaxel. CONCLUSION: To conclude, EAD could be the drug of choice for combined treatment with paclitaxel in a chemotherapy regimen.

Ginger exosome-like nanoparticles (GELNs) induced apoptosis, cell cycle arrest, and anti-metastatic effects in triple-negative breast cancer MDA-MB-231 cells.

Ginger exosome-like nanoparticles (GELNs) have been extensively implicated in alleviating inflammation, maintaining intestinal microbiome and are considered competent drug delivery vehicles. Despite this, the current knowledge of the GELN interaction with cancer cells is limited. Triple-negative breast cancer (TNBC), an aggressive variant lacking efficient therapeutics, necessitates novel natural counterparts with minimal side effects. This study investigates the action of GELNs isolated from ginger rhizomes against TNBC cells. GELNs were isolated by ultracentrifugation and characterized physicochemically. The interaction of GELNs with TNBC cells (MDA-MB-231) was studied in detail. The GELNs induced a concentration-dependent decrease in cell viability in MDA-MB-231 cells without affecting the normal cell lines tested. GELNs induced apoptosis as indicated by morphological changes, nuclear fragmentation, membrane damage, phosphatidyl serine translocation, ROS generation, drop in mitochondrial membrane potential, expression of apoptotic specific proteins, and increased caspase activity. GELNs also instigated cell cycle arrest, retarded cell migration and colony formation in TNBC cells. These findings report a novel action of GELNs against TNBC cells and a closer look at the underlying molecular mechanism of this interspecies communication. This opens newer prospects for using dietary ELNs to target therapeutically challenging cancers.

Studies on the mode of action of synthetic diindolylmethane derivatives against triple negative breast cancer cells.

Diindolylmethane (DIM) is a metabolic product of indole-3-carbinol (I3C), the major phytochemicals present in cruciferous vegetables, which can modulate multiple signalling pathways in cancer. The present study deals with the mechanism of action of two synthetic biaryl conjugates of DIM in triple negative breast cancer cells. Out of 12 DIM derivatives tested, two compounds, DIM-1 and DIM-4, exhibit cytotoxicity with GI50 values of 9.83 ± 0.2195 µM and 8.726 ± 0.5234 µM, respectively, in 2D culture. In 3D culture, DIM-1 and DIM-4 show GI50 values of 24.000 ± 0.7240 µM and 19.230 ± 0.3754 µM, respectively. The non-toxic nature of the compounds was also established by the toxicity studies using the zebrafish model system. The two compounds induced apoptosis and anoikis in the cancer cells, which was confirmed by morphological analysis, nuclear fragmentation, membrane integrity assay, caspase activity measurements and modulation of pro/anti-apoptotic proteins. The compounds inhibited cell migration and MMP-2 and MMP-9 activities indicating their anti-metastatic property. They also reduced the expression of active Ras, phosphorylated forms of PI3K, Akt and mTOR. Immunofluorescence studies revealed the reduced expression of EGFR and pEGFR in treated cells. To conclude, DIM-1 and DIM-4 induced anti-breast cancer effects by blocking EGF receptor and subsequently inhibiting Ras-mediated PI3K-Akt-mTOR signalling pathway.

Dietary Exosome-Like Nanoparticles: An Updated Review on Their Pharmacological and Drug Delivery Applications.

Exosomes are lipid bilayer membrane-bound extracellular vesicular structures (30-150 nm) mainly released by eukaryotic cells of animal origin. Exosome-like nanoparticles (ELNs) are the vesicular structures originating from plant sources with features similar to eukaryotic animal cell derived exosomes. ELNs derived from dietary sources (dietary ELNs) have exceptional pharmacological potential in alleviating many diseases and are good in maintaining intestinal health through the manipulation of the gut microbiome. The dietary ELNs being highly biocompatible find their application in targeted therapy as well. They are being established as promising drug delivery agents and can also be developed into dietary supplements. This review highlights the ELNs derived from various dietary sources, their diversity in molecular compositions, potential health benefits, and drug delivery applications. Few clinical trials are attempted with dietary ELNs which are also described in the review along with their properties that can be exploited for the food and pharma industries in the future.

Semi-synthetic diversification of coronarin D, a labdane diterpene, under Ugi reaction conditions.

The prevalence of 5-hydroxydihydrofuran-2(3H)-one moiety in natural products is exploited for the first time using coronarin D, a labdane diterpene, to afford Ugi reaction product 1a and interrupted Ugi product 2a. The potential of the Ugi reaction was further extended to l-phenylalanine, 2-aminopyridine, and d-glucosamine, which afforded Ugi reaction products 3a-f, 4, and 5a-d, respectively. Cytotoxicity studies in RAW cells reveal that compounds 3e and 5b were non-toxic up to 50 µM, and these compounds were able to reduce the LPS stimulated NO production in RAW cells in par with the standard anti-inflammatory drug dexamethasone.

Epoxyazadiradione induced apoptosis/anoikis in triple-negative breast cancer cells, MDA-MB-231, by modulating diverse cellular effects.

Triple-negative breast cancer (TNBC) is one of the most aggressive forms of its kind, which accounts for 15-20% of all breast cancers. As this cancer form lacks hormone receptors, targeted chemotherapy remains the best treatment option. Apoptosis and anoikis (detachment-induced cell death) induction by small molecules can prevent TNBC metastasis to a greater extent. Epoxyazadiradione (EAD) is a limonoid from the neem plant with an anticancer property. Here, we demonstrate that EAD induced mitochondria-mediated apoptosis and anoikis in TNBC cells (MDA-MB-231). Apart from this, it promotes antimigration, inhibition of colony formation, downregulation of MMP-9 and fibronectin, induction of G2/M phase arrest with downregulation of cyclin A2/cdk2, interference in cellular metabolism, and inhibition of nuclear factor kappa-B (NF-kB) nuclear translocation. Moreover, a significant reduction is observed in the expression of EGFR on the plasma membrane and nucleus upon treatment with EAD. Among the diverse cellular effects, anoikis induction, metabolic interference, and downregulation of membrane/nuclear EGFR expression by EAD are reported here for the first time. To summarize, EAD targets multiple cellular events to induce growth arrest in TNBC, and hence can be developed into the best antineoplastic agent in the future.

Exosomes and exosomal RNAs in breast cancer: A status update.

Improved treatment of breast cancer, the world's second most common cancer, requires identification of new sensitive prognostic and diagnostic biomarkers. Exosomes are lipid-bilayer extracellular vesicles of size 30-150 nm, released by all cell types, including breast cancer cells. Cellular communication is the primary function attributed to them. This review discusses the potential utility of exosomes and exosomal RNAs (microRNAs [miRNAs]/long non-coding RNAs [LncRNAs]) in breast cancer biology and treatment. The existing literature shows that exosomes play a significant role in breast tumorigenesis and progression through transfer miRNAs and LncRNAs. These miRNAs and LncRNAs function by post-transcriptionally regulating their target mRNAs, eventually leading to modulation of expression/repression. Over the past two decades, numerous publications point towards diagnostic and therapeutic applications of exosomal miRNAs/LncRNAs. Until now, we do not have clinically approved exosome-based therapeutics. Therefore, it is high time that clinicians and cancer researchers utilise exosome's benefits through randomised clinical trials for better management of breast cancer.

Therapeutic Perspectives of Food Bioactive Peptides: A Mini Review.

Bioactive peptides are short chain of amino acids (usually 2-20) that are linked by amide bond in a specific sequence which have some biological effects in animals or humans. These can be of diverse origin like plant, animal, fish, microbe, marine organism or even synthetic. They are successfully used in the management of many diseases. In recent years increased attention has been raised for its effects and mechanism of action in various disease conditions like cancer, immunity, cardiovascular disease, hypertension, inflammation, diabetes, microbial infections etc. Bioactive peptides are more bioavailable and less allergenic when compared to total proteins. Food derived bioactive peptides have health benefits and its demand has increased tremendously over the past decade. This review gives a view on last two years research on potential bioactive peptides derived from food which have significant therapeutic effects.

Morus alba Leaf Lectin (MLL) Sensitizes MCF-7 Cells to Anoikis by Inhibiting Fibronectin Mediated Integrin-FAK Signaling through Ras and Activation of P38 MAPK.

Lectins are a unique class of carbohydrate binding proteins/glycoproteins, and many of them possess anticancer properties. They can induce cell cycle arrest and apoptosis, inhibit protein synthesis, telomerase activity and angiogenesis in cancer cells. In the present study, we have demonstrated the effect of Morus alba leaf lectin (MLL) on anoikis induction in MCF-7 cells. Anoikis induction in cancer cells has a significant role in preventing early stage metastasis. MLL treatment in monolayers of MCF-7 cells caused significant detachment of cells in a time and concentration dependent manner. The detached cells failed to re-adhere and grew even to culture plates coated with different matrix proteins. DNA fragmentation, membrane integrity studies, annexin V staining, caspase 9 activation and upregulation of Bax/Bad confirmed that the detached cells underwent apoptosis. Upregulation of matrix metalloproteinase 9 (MMP-9) caused a decrease in fibronectin (FN) production which facilitated the cells to detach by blocking the FN mediated downstream signaling. On treatment with MLL, we have observed downregulation of integrin expression, decreased phosphorylation of focal adhesion kinase (FAK), loss in FAK-integrin interaction and active Ras. MLL treatment downregulated the levels of phosphorylated Akt and PI3K. Also, we have studied the effect of MLL on two stress activated protein kinases p38 MAPK and JNK. p38 MAPK activation was found to be elevated, but there was no change in the level of JNK. Thus our study substantiated the possible antimetastatic effect of MLL by inducing anoikis in MCF-7 cells by activation of caspase 9 and proapoptotic Bax/Bad by blockage of FN mediated integrin/FAK signaling and partly by activation of p38 MAPK.

Antifungal, Anticancer and Aminopeptidase Inhibitory Potential of a Phenazine Compound Produced by Lactococcus BSN307.

A bioactive compound was purified from the culture medium of a new strain of Lactococcus BSN307 by solvent extraction followed by chromatographic techniques. This bioactive compound was identified to belong to phenazine class of compounds by MS, NMR and FTIR. The phenazine compound showed antifungal activity against Aspergillus niger, Penicillium chrysogenum as well as Fusarium oxysporum by disc diffusion assay in addition to antioxidant potential as demonstrated by DPPH scavenging assay. The compound demonstrated selective cytotoxicity against cancer cell lines HeLa and MCF-7 where IC50 was achieved with 20 and 24 µg/mL respectively. At the same time no cytotoxicity was occurred in normal H9c2 cells. The bioactive found to be inhibitory to both leucine and proline aminopeptidases and thus revealed its potential as metalloenzyme inhibitor. This study, for the first time reports the production of phenazine class of compounds by lactic acid bacteria.

Anticancer activity of synthetic bis(indolyl)methane-ortho-biaryls against human cervical cancer (HeLa) cells.

Bis(indolyl)methane appended biaryls were designed, synthesized and evaluated in human cervical cancer cell lines (HeLa) for their anticancer activities and compared against normal rat cardiac myoblasts (H9C2) cells. Compounds 1-12 were synthesized, with variations in one of the phenyl unit, in a single step by condensation of biaryl-2-carbaldehydes with indole in the presence of para-toluenesulfonic acid. Compound 1 exhibited a GI50 value of 11.00 ± 0.707 µM and the derivatives, compounds 4 and 11 showed a GI50 value of 8.33 ± 0.416 µM and 9.13 ± 0.177 µM respectively in HeLa cells and was found to be non-toxic to H9C2 cells up to 20 µM. Furthermore, compounds 1, 4 and 11 induced caspase dependent cellular apoptosis in a concentration-dependent manner, reduced mitochondrial membrane potential, inhibited the cell migration and downregulated the production of MMP-2 and MMP-9 in HeLa cells.

Enhancement in intramolecular interactions and in vitro biological activity of a tripodal tetradentate system upon complexation.

Novel biomimetic mononuclear complexes, [Fe()Cl2](+) () and [Cu()(H2O)](2+) () based on naphthalimide appended tripodal tetradentate ligand ( = 2,2',2''-(3,3',3''-(2,2',2''-nitrilotris(methylene)tris(1H-benzo[d]imidazole-2,1-diyl))tris(propane-3,1-diyl))tris(1H-benzo-[de]isoquinoline-1,3(2H)-dione)) have been synthesized and characterized by various analytical and spectral techniques. In addition, the structures of the ligand () and complex were established unambiguously through X-ray crystal structure analysis. Uniquely, the coordination with a metal ion modified the ligand scaffold to interact efficiently with ct-DNA (groove binding) as well as protein (hydrophobic and/or electrostatic interactions). We have determined the affinity of these complexes for DNA/protein and the values are found to be in the range, KDNA = 0.34-1.01 × 10(4) M(-1) and KBSA = 4.1-5.0 × 10(5) M(-1). Furthermore, the fluorescence quenching of BSA with complexes and occurs through a static mechanism and affects the conformation of BSA around the tryptophan residues. The in vitro biological studies of these systems employing HeLa cell lines indicated that both these complexes exhibited enhanced cytotoxicity (IC50 = 32 ± 0.19 and 10 ± 0.21 µM for complexes and , respectively), when compared to the ligand () (IC50 = 150 µM). Interestingly, both the complexes ( and ) were found to be non-toxic to normal H9C2 cell lines. The mechanism of in vitro biological activity of these complexes has been evaluated through a variety of techniques: acridine orange/ethidium bromide, DAPI staining studies, annexin V-FITC/PI and poly(ADP-ribose)-polymerase (PARP) cleavage, which confirmed the apoptotic mediated cell death. Our results demonstrate the importance of complexation of the naphthalimide ligand () as well as the potential of these biomimetic metal complexes as cytotoxic and anticancer agents.

2,4-Di-tert-butyl phenol as the antifungal, antioxidant bioactive purified from a newly isolated Lactococcus sp.

The volatile organic compound 2,4-di-tert-butyl phenol (2,4 DTBP) was purified from the cell free supernatant of a newly isolated Lactococcus sp. by solvent extraction and chromatographic techniques. Molecular characterization of the compound by ESI-MS, (1)H NMR and FTIR analysis revealed the structure, C14H22O. Fungicidal activity was demonstrated against Aspergillus niger, Fusarium oxysporum and Penicillium chrysogenum by disc diffusion assay. Among the cell lines tested for cytotoxicity of this compound (normal cell line H9c2 and cancer cell lines HeLa and MCF-7), a remarkable cytotoxicity against HeLa cells with an IC50 value of 10 µg/mL was shown. A biocontrol experiment with 2,4 DTBP supplemented fraction prevented growth of the abovementioned fungi on wheat grains. The study further strengthens the case for development of biopreservatives and dietary antioxidants from lactic acid bacteria for food applications.

Control of spoilage fungi by protective lactic acid bacteria displaying probiotic properties.

Thirty-six lactic acid bacteria belong to Lactococcus, Lactobacillus, Enterococcus, and Pediococcus were isolated, and the spectrum of antifungal activity was verified against Fusarium oxysporum (KACC 42109), Aspergillus niger (KACC 42589), Fusarium moniliforme (KACC 08141), Penicillium chrysogenum (NII 08137), and the yeast Candida albicans (MTCC 3017). Three isolates, identified as Pediococcus pentosaceus (TG2), Lactobacillus casei (DY2), and Lactococcus (BSN) were selected further, and their antifungal compounds were identified by ESI-MS and HPLC analysis as a range of carboxylic acids along with some unidentified, higher molecular weight compounds. An attempt to check out the shelf life extension of wheat bread without fungal spoilage was performed by fermenting the dough with the Lactococcus isolate. Apart from growth in low pH and tolerance to bile salts, probiotic potential of these three isolates was further substantiated by in vitro screening methods that include transit tolerance to the conditions in the upper human gastrointestinal tract and bacterial adhesion capacity to human intestinal cell lines.

Antioxidant rich Morus alba leaf extract induces apoptosis in human colon and breast cancer cells by the downregulation of nitric oxide produced by inducible nitric oxide synthase.

Morus species had been used widely in the traditional medicines for various diseases. In this study we report the in vitro antiproliferative activity of the methanol extract of Morus alba. The extract is capable of inducing cytotoxicity in human colon cancer (HCT-15) cells (IC(50) = 13.8 µg/ml) and breast cancer (MCF-7) cells (IC(50) = 9.2 µg/ml), resulted in significant morphological changes of the cells, fragmentation of DNA, and caspase-3 activation- characteristics of apoptosis. It downregulated the amount of nitric oxide (NO) produced as a result of inducible nitric oxide (iNOS) activation. The HPLC analysis of the extract showed epicatechin (20%), myricetin (10%), quercetin hydrate (12%), luteolin (12%), and kaempferol (6%) as the major active components and ascorbic acid, gallic acid, pelargonidine, and p-coumaric acid as the minor components. To the best of our knowledge, this is the first report showing the downregulation of iNOS and induction of apoptosis by M. alba extract.

Purification of a lectin from M. rubra leaves using immobilized metal ion affinity chromatography and its characterization.

Lectins represent a heterogeneous group of proteins/glycoproteins with unique carbohydrate specificity, with wide range of biomedical applications. The multi-step purification protocols generally used for purification of lectin result in a significant reduction in the final yield and activity. In the present study, Morus rubra lectin (MRL) was purified to homogeneity from the leaves using a single-step immobilized metal ion affinity chromatography (IMAC) procedure. The approximate molecular weight of purified MRL resolved as a single band on SDS-PAGE was 52 kDa. Final percentage yield of purified lectin by IMAC was calculated as 74.7 %. Purified MRL was specific to three sugars, galactose, D-galactosamine and N-acetyl-D-galactosamine, and rendered haemagglutination (HA) activity towards different human blood group RBCs. MRL showed stability over a wide range of temperature (up to 80 °C) and pH (4-11). Chelation of the lectin with EDTA did not alter HA which indicates that metal ion is not required for activity. In the presence of Fe(2+), Ca(2+), Zn(2+), Ni(2+), Mn(2+), Na(+) and K(+), HA activity was reduced to 50 %, whereas the presence of trivalent metal ions (Fe3(+) and Al(3+)) and Cu(2+) did not affect the activity. In the presence of Mg(2+) and Hg(2+), only 25 % of HA activity remained.

Purified mulberry leaf lectin (MLL) induces apoptosis and cell cycle arrest in human breast cancer and colon cancer cells.

Medicinal values of mulberry are known to humans from ancient ages. The white mulberry, Morus alba L. is a rich source of many bioactive phytochemicals. Earlier investigations in our laboratory lead to the purification and characterization of an anti-proliferative lectin (MLL) from the leaves of this plant. Further to that, here we have investigated the mechanism of cell death induction by MLL on human breast cancer (MCF-7) and colon cancer (HCT-15) cells. Cells were treated with GI(50) concentration (concentration of lectin required for 50% inhibition of cell growth) of MLL (8.5 µg/ml for MCF-7 and 16 µg/ml for HCT-15) for 24 h to induce cell death. The induction of apoptosis was studied by morphological analysis, DNA fragmentation, apoptotic cell staining and caspase 3 activity assay. Apoptotic cells in sub G0-G1 phase were monitored using flow cytometry. MLL induced significant morphological changes and DNA fragmentation associated with apoptosis in MCF-7 and HCT-15 cells. Positive annexin V and acridine orange/ethidium bromide stained cells indicated apoptosis induction by MLL. Up-regulation of caspase 3 activity was also found in cells treated with MLL. Flow cytometry analysis showed an increase in the percentage of cells in sub G0-G1 phase confirming the MLL induced apoptosis. In conclusion, MLL induced apoptosis in MCF-7 and HCT-15 cells in a caspase dependent manner.

Purification and characterization of a novel anti-proliferative lectin from Morus alba L. leaves.

A novel anti-proliferative lectin was purified from Morus alba L. (Mulberry) leaves by a two step chromatographic procedure namely, immobilized metal ion affinity chromatography (IMAC) and convective interaction media (CIM) based anion exchange chromatography. The purified mulberry leaf lectin (MLL) was specific to galactose, galactosamine and N-acetyl galactosamine (GalNAc). MLL was homogenous with a molecular weight of ~56kDa in silver stained SDS-PAGE. The lectin showed RBC agglutination activity up to 40°C and was independent of pH above pH 6. Haemagglutination activity of purified MLL was not dependent on any metal ions. However, with high concentration of trivalent metal ions, Fe3+ and Al3+ and the divalent metal ion Fe2+, a three fold increase in agglutination activity was observed. The purified MLL showed an anti-proliferative activity towards human breast cancer cells (MCF-7) and colon cancer cells (HCT-15) with a higher potency towards MCF-7 cells. This is the first report on the anti-proliferative activity of a GalNAc specific lectin from M. alba.

Cell survival, activation and apoptosis of hepatic stellate cells: modulation by extracellular matrix proteins.

AIM: Cytokines and growth factors released by various hepatic cells exert both paracrine and autocrine effects on hepatic stellate cell (HSC) activation during liver injury. The aim of the present study was to examine whether the surrounding extracellular matrix (ECM) influences the activation, transdifferentiation and survival of HSCs. METHODS: An in vitro model system of isolated HSCs maintained in culture on different matrix protein substrata was employed. RESULTS: The rate of loss of HSC-specific retinol uptake activity and gain of myofibroblast-like activity such as (35)[S] proteoglycan synthesis varied in cells maintained on different matrix proteins and was in the order collagen I > collagen IV >/= laminin. (3)[H]-thymidine incorporation by HSCs maintained on different matrix proteins varied and was in the order collagen I > collagen IV > laminin. MTT assay revealed that the growth inhibition in response to curcumin was significantly low in cells maintained on collagen I. Apoptotic marker activities such as DNA fragmentation, 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) staining, annexin staining and caspase-3 activities showed that cells maintained on collagen I showed minimal apoptosis than those maintained on collagen IV, laminin and polylysine, showing the influence of ECM on HSC apoptosis. Experiments using blocking antibodies showed that the collagen I effect was mediated through alpha(2)beta(1) integrin. CONCLUSIONS: These results indicate that ECM influences activation, transdifferentiation and survival of HSCs, and suggest that apart from diffusible factors, the surrounding ECM also influences HSC behavior critical in both the progression of the fibrosis and the restitution of the liver during recovery after hepatic injury.