Stiffness assisted cell-matrix remodeling trigger 3D mechanotransduction regulatory programs.

Engineered matrices provide a valuable platform to understand the impact of biophysical factors on cellular behavior such as migration, proliferation, differentiation, and tissue remodeling, through mechanotransduction. While recent studies have identified some mechanisms of 3D mechanotransduction, there is still a critical knowledge gap in comprehending the interplay between 3D confinement, ECM properties, and cellular behavior. Specifically, the role of matrix stiffness in directing cellular fate in 3D microenvironment, independent of viscoelasticity, microstructure, and ligand density remains poorly understood. To address this gap, we designed a nanoparticle crosslinker to reinforce collagen-based hydrogels without altering their chemical composition, microstructure, viscoelasticity, and density of cell-adhesion ligand and utilized it to understand cellular dynamics. This crosslinking mechanism utilizes nanoparticles as crosslink epicenter, resulting in 10-fold increase in mechanical stiffness, without other changes. Human mesenchymal stem cells (hMSCs) encapsulated in 3D responded to mechanical stiffness by displaying circular morphology on soft hydrogels (5 kPa) and elongated morphology on stiff hydrogels (30 kPa). Stiff hydrogels facilitated the production and remodeling of nascent extracellular matrix (ECM) and activated mechanotransduction cascade. These changes were driven through intracellular PI3AKT signaling, regulation of epigenetic modifiers and activation of YAP/TAZ signaling. Overall, our study introduces a unique biomaterials platform to understand cell-ECM mechanotransduction in 3D for regenerative medicine as well as disease modelling.

Curcumin stably interacts with DNA hairpin through minor groove binding and demonstrates enhanced cytotoxicity in combination with FdU nucleotides.

We report, based on biophysical studies and molecular mechanical calculations that curcumin binds DNA hairpin in the minor groove adjacent to the loop region forming a stable complex. UV-Vis and fluorescence spectroscopy indicated interaction of curcumin with DNA hairpin. In this novel binding motif, two É£ H of curcumin heptadiene chain are closely positioned to the A16-H8 and A17-H8, while G12-H8 is located in the close proximity of curcumin α H. Molecular dynamics (MD) simulations suggest, the complex is stabilized by noncovalent forces including; π-π stacking, H-bonding and hydrophobic interactions. Nuclear magnetic resonance (NMR) spectroscopy in combination with molecular dynamics simulations indicated curcumin is bound in the minor groove, while circular dichroism (CD) spectra suggested minute enhancement in base stacking and a little change in DNA helicity, without significant conformational change of DNA hairpin structure. The DNA:curcumin complex formed with FdU nucleotides rather than Thymidine, demonstrated enhanced cytotoxicity towards oral cancer cells relative to the only FdU substituted hairpin. Fluorescence co-localization demonstrated stability of the complex in biologically relevant conditions, including its cellular uptake. Acridine orange/EtBr staining further confirmed the enhanced cytotoxic effects of the complex, suggesting apoptosis as mode of cell death. Thus, curcumin can be noncovalently complexed to small DNA hairpin for cellular delivery and the complex showed increased cytotoxicity in combination with FdU nucleotides, demonstrating its potential for advanced cancer therapy.

Robust design of some selective matrix metalloproteinase-2 inhibitors over matrix metalloproteinase-9 through in silico/fragment-based lead identification and de novo lead modification: Syntheses and biological assays.

Broad range of selectivity possesses serious limitation for the development of matrix metalloproteinase-2 (MMP-2) inhibitors for clinical purposes. To develop potent and selective MMP-2 inhibitors, initially multiple molecular modeling techniques were adopted for robust design. Predictive and validated regression models (2D and 3D QSAR and ligand-based pharmacophore mapping studies) were utilized for estimating the potency whereas classification models (Bayesian and recursive partitioning analyses) were used for determining the selectivity of MMP-2 inhibitors over MMP-9. Bayesian model fingerprints were used to design selective lead molecule which was modified using structure-based de novo technique. A series of designed molecules were prepared and screened initially for inhibitions of MMP-2 and MMP-9, respectively, as these are designed followed by other MMPs to observe the broader selectivity. The best active MMP-2 inhibitor had IC50 value of 24nM whereas the best selective inhibitor (IC50=51nM) showed at least 4 times selectivity to MMP-2 against all tested MMPs. Active derivatives were non-cytotoxic against human lung carcinoma cell line-A549. At non-cytotoxic concentrations, these inhibitors reduced intracellular MMP-2 expression up to 78% and also exhibited satisfactory anti-migration and anti-invasive properties against A549 cells. Some of these active compounds may be used as adjuvant therapeutic agents in lung cancer after detailed study.

Bio-assay Guided Isolation of Anti-cancer Compounds from Anthocephalus cadamba Bark.

Anthocephalus cadamba, an important plant in the traditional system of medicine in India, is reported to possess anticancer activity. Guided by bio-assay tests using human colorectal (HCT116) and hepatocellular carcinoma (HepG2) cell lines, it has been shown to contain three active constituents, the triterpenoid saponins 3-O-[α-L-rhamnopyranosyl]-quinovic acid (1) and 3-O-[α-L-rhamnopyranosyl]-quinovic acid 28-O-[ß-D-glucopyranosyl] ester (2), and the alkaloid cadambine (3). The structures of the isolated compounds were established using spectroscopic techniques. The isolated compounds demonstrated concentration dependent inhibition of both the cell lines, where compound 3 proved to be the most potent inhibitor of cell line HCT116 (IC50 45 +/- 4 µg/mL) and compound 2 demonstrated maximum inhibitory activity against HepG2 cell line with an IC50 value of 89 +/- 7 µg/mL.

Effect of corchorusin-D, a saikosaponin like compound, on B16F10 melanoma cells (in vitro and in vivo).

Corchorusin-D (COR-D), isolated from Corchorus acutangulus, was reported to induce apoptosis in leukemic cells. However, no studies concerning its activity on melanoma cells have been reported. We have evaluated its in vitro anti-cancer activity on melanoma cells (B16F10, SK-MEL-28, and A375). The results demonstrate that COR-D showed maximum inhibition of B16F10 cells in vitro. COR-D induced mitochondrial dysfunction and altered the Bax/Bcl-2 ratio with down regulation of pro-caspases 9 and activation of caspase 3 in B16F10 cells, triggering intrinsic pathway of apoptosis. Moreover, it inhibited the in vivo B16F10 tumor growth and increased the survival rate of mice. Greater number of Annexin V-FITC and propidium iodide (PI)-positive tumor cells signified that COR-D induced apoptosis in vivo also. The reduction in tumor growth is well correlated with decreased microvascular density of the tumor cells in treated mice. In conclusion, this study reveals that COR-D-induced mitochondrial dysfunction is responsible for the induction of apoptotic cell death.

Corchorusin-D directed apoptosis of K562 cells occurs through activation of mitochondrial and death receptor pathways and suppression of AKT/PKB pathway.

Saponins, plant glycosides, have been reported to possess anti-cancer properties. Therefore the effect of corchorusin-D (COR-D), a compound isolated from Corchorus acutangulus, was studied in the chronic myelogenous leukemic cell line K562, using MTT assay, phase contrast and confocal microscopy, annexin V binding, cell cycle analysis and western blotting. COR-D inhibited cell growth in K562 cells and showed increased number of Annexin V FITC binding cells. Characteristic apoptotic changes, seen under phase contrast and confocal microscopes with accumulation of cells in the sub-G0 phase. The apoptosis involved drop in Bcl-2/Bax ratio, loss of mitochondrial membrane potential, release of cytochrome c in cytosol followed by activation of caspases 9 and 3, and cleavage of PARP. Down-regulation of pro-caspase 10 was observed along with formation of death-inducing signaling complex between TNF-R1 and TRADD. COR-D suppressed PDK1 and AKT with activation of MAP kinase family members ERK1/2, JNK1/2 and p38. Thus it induced apoptosis by activating mitochondrial and death receptor pathways and suppressing AKT/PKB rather than MAP kinase pathway. Significant enhancement of apoptosis, noted using specific inhibitors of ERK1/2, p38 and JNK1/2, suggests that COR-D can enhance apoptosis in K562 cells in combination with MAP kinase inhibitors.

Effect of nanoclay and SEBS-g-MA on the morphology and properties of immiscible poly(methyl methacrylate)/polystyrene blend.

In recent years, nanoclays are being used as compatibilizer for various immiscible polymer blends. However, little work has been done on the morphology of immiscible polymer blends in presence of both the nanoclay and a reactive compatibilizer. Here, we report the synergistic effect of nanoclay and SEBS-g-MA on the morphology and properties of (70/30 w/w) PMMA/PS blend. Scanning electron microscopy study of the blend with various amount of nanoclay and SEBS-g-MA indicated a reduction in the average domain sizes (D) of dispersed PS phase in PMMA matrix compared to that in the pure blend. Addition of both SEBS-g-MA and nanoclay significantly lowered the D of PS in the blend compared to that with only SEBS-g-MA or clay. X-ray diffraction study and transmission electron microscopy revealed the presence of intercalated clay platelets in PMMA matrix, as well as, at the interface of the (70/30 w/w) PMMA/PS blend-clay nanocomposites. Addition of SEBS-g-MA in the blend-clay nanocomposites promoted the exfoliation of clays in PMMA matrix. Thus, exfoliated clay platelets in PMMA matrix effectively restricted the coalescence of dispersed PS domains while SEBS-g-MA improved the adhesion between the phases at the interface. At certain loading (phr), storage modulus, elongation at break and thermal stability of the blend were greatly improved when both the nanoclay and SEBS-g-MA were present in the blend. The use of reactive compatibilizer and nanoclay in polymer blends may lead to a high performance material which combines the advantages of compatibilized polymer blends and the merits of polymer nanocomposites.

Anti-leukemic activity of Dillenia indica L. fruit extract and quantification of betulinic acid by HPLC.

The methanolic extract of Dillenia indica L. fruits showed significant anti-leukemic activity in human leukemic cell lines U937, HL60 and K562. This finding led to fractionation of the methanolic extract, on the basis of polarity, in which the ethyl acetate fraction showed the highest anti-leukemic activity. A major compound, betulinic acid, was isolated from the ethyl acetate fraction by silica gel column chromatography and was identified and characterized. Betulinic acid could explain the anti-leukemic activity of the methanolic extract and the ethyl acetate fraction. Hence the quantitative estimation of betulinic acid was approached in methanolic extract and fractions using HPLC.

Corchorusin-D, a saikosaponin-like compound isolated from Corchorus acutangulus Lam., targets mitochondrial apoptotic pathways in leukemic cell lines (HL-60 and U937).

PURPOSE: The presence of triterpene saponins in Corchorus acutangulus Lam. has been reported. However, no studies concerning biological activity of the plant extracts have been done so far. In the present study, the anti-leukemic activity of the methanol extract of aerial parts (ME) of C. acutangulus has been investigated, and efforts have been made to identify the active ingredient responsible for this activity. METHODS: The anti-leukemic activity of ME, its fractions and corchorusin-D (COR-D), the active ingredient, was investigated in leukemic cell lines U937 and HL-60 using cell viability and MTT assays. The molecular pathways leading to the activity of COR-D were examined by confocal microscopy, flow-cytometry, caspase and Western blot assays. RESULTS: ME, its n-butanolic fraction and COR-D inhibited cell growth and produced significant cytotoxicity in leukemic cell lines U937 and HL-60. COR-D produced apoptotic cell death via mitochondrial disfunction and was found to pursue the intrinsic pathway by inciting the release of apoptosis-inducing factors (AIFs) from mitochondria. COR-D-induced translocation of Bax from cytosol to mitochondria facilitating caspase-9 activation and up regulation of downstream pathways leading to caspase-3 activation and PARP cleavage, which resulted in the subsequent accumulation of cells in the sub-G0 phase followed by DNA fragmentation. CONCLUSIONS: COR-D possesses significant anti-leukemic activity in U937 and HL-60 cell lines by acting on the mitochondrial apoptotic pathways. Since the necrotic body formation is low after COR-D treatment, the occurrence of inflammation in in vivo systems could be reduced, which represents a positive indication in view of therapeutic application.

Synergistic effect of nanoclay and EPR-g-MA on the properties of nylon6/EPR blends.

The effect of EPR-g-MA, nanoclay and a combination of the two on phase morphology and properties of (70/30 w/w) nylon6/EPR blends prepared by melt processing technique has been studied. We found that the number average domain diameter (D(n)) of the dispersed EPR phase in the blend decreased in presence of EPR-g-MA, and clay. This observation indicated that nanoclay could be used as an effective compatibilizer in nylon6/EPR blend. X-ray diffraction study and TEM analysis of the blend/clay nanocomposites revealed the delaminated clay morphology and preferential location of the exfoliated clay platelets in nylon6 phase. Thus, selective dispersion of the clay platelets with delaminated morphology in the matrix phase acted as the barrier and thereby prevented the coalescence of dispersed EPR domains during melt processing. However, addition of nanoclay in the blend did not improve the impact property of the blend. Interestingly, the impact strength of the nylon6/EPR/EPR-g-MA/clay blend was much higher than the nylon6/EPR/EPR-g-MA and nylon6/EPR/clay blends at certain compositions with comparable D(n) values. This could be explained by assuming the presence of delaminated clay platelets at the interface, which were stiffer than the polymers. Hence, the bending energy of the interfaces was high.