Exposure to BPA and BPS during pregnancy disrupts the bone mineralization in the offspring.

Exposure to plastic-derived estrogen-mimicking endocrine-disrupting bisphenols can have a long-lasting effect on bone health. However, gestational exposure to bisphenol A (BPA) and its analogue, bisphenol S (BPS), on offspring's bone mineralization is unclear. The effects of in-utero bisphenol exposure were examined on the offspring's bone parameters. BPA and BPS (0.0, 0.4 µg/kg bw) were administered to pregnant Wistar rats via oral gavage from gestational day 4-21. Maternal exposure to BPA and BPS increased bone mineral content and density in the offspring aged 30 and 90 days (P < 0.05). Plasma analysis revealed that alkaline phosphatase, and Gla-type osteocalcin were significantly elevated in the BPS-exposed offspring (P < 0.05). The expression of BMP1, BMP4, and their signaling mediators SMAD1 mRNAs were decreased in BPS-exposed osteoblast SaOS-2 cells (P < 0.05). The expression of extracellular matrix proteins such as ALPL, COL1A1, DMP1, and FN1 were downregulated (P < 0.05). Bisphenol co-incubation with noggin decreased TGF-ß1 expression, indicating its involvement in bone mineralization. Altered mineralization could be due to dysregulated expression of bone morphogenetic proteins and signalling mediators in the osteoblast cells. Thus, bisphenol exposure during gestation altered growth and bone mineralization in the offspring, possibly by modulating the expression of Smad-dependent BMP/TGF-ß1 signalling mediators.

M1 polarization induction by lead and amyloid peptides in microglial cells: Implications for neurodegeneration process.

Neurodegeneration in conditions like Alzheimer's and Parkinson's disease is influenced by genetic and environmental factors. This study explores the potential neurodegenerative effects of lead (Pb) toxicity and amyloid beta peptides (Aßp 1-40 and Aßp 25-35) by promoting M1 polarization in microglial cells. To this end, we investigated and observed that IC50 concentrations of Pb (22.8 µM) and Aßp 25-35(29.6 µM). Our results demonstrated significant Pb uptake (31.13% at 25 µM Pb) and increased intracellular ROS levels (77.1%) upon treatment with Pb in combination of both Aßp 1-40 and Aßp 25-35. Protein carbonylation significantly increased (73.12 nmol/mL) upon treatment with Pb in combination of both Aßp 1-40 and Aßp 25-35, indicating oxidative damage and compromised cellular defenses against oxidative stress along with elevated DNA oxidative damage (164.9 pg/mL of 8-OH-dG) upon treatment with Pb in combination with both Aßp 1-40 and Aßp 25-35. Microglial polarization showed elevated M1 markers (inducible nitric oxide synthase and cyclooxygenase 2) and reduced M2 markers (arginase-1 and cluster of differentiation 206), suggesting Pb's role in inducing neurodegenerative microglial polarization. These findings provide insights into the complex molecular events contributing to Pb-induced neurotoxicity and neurodegenerative diseases.

Unveiling Potential Neurotoxic Mechansisms: Pb-Induced Activation of CDK5-p25 Signaling Axis in Alzheimer's Disease Development, Emphasizing CDK5 Inhibition and Formation of Toxic p25 Species.

Alzheimer's disease (AD) is a complex neurodegenerative disorder with an etiology influenced by various genetic and environmental factors. Heavy metals, such as lead (Pb), have been implicated in AD pathogenesis, but the underlying mechanisms remain poorly understood. This study investigates the potential neurodegenerative role of Pb and amyloid ß peptides (1-40 and 25-35) via their interaction with cyclin-dependent kinase 5 (CDK5) and its activator, p25, in an attempt to unravel the molecular basis of Pb-induced neurotoxicity in neuronal cells. To this end, a CDK5 inhibitor was utilized to selectively inhibit CDK5 activity and investigate its impact on neurodegeneration. The results revealed that Pb exposure led to elevated Pb uptake (56.7% at 15 µM Pb) and disturbances in intracellular calcium (19.6% increase upon Pb treatment). The results revealed a significant decrease in total antioxidant capacity (by 88.6% upon Pb treatment) and also elevation in protein carbonylation (by 26.2% upon Pb and Aßp's combination treatment), indicative of oxidative damage, suggesting an impaired cellular defence against oxidative stress and elevated DNA oxidative damage (178 pg/ml and 182 pg/ml of 8-OH-dG upon Pb and All treatment). Additionally, dysregulations in levels of calpain, p25-35 and CDK5 are observed and markers associated with antioxidant metabolism (phospho-Peroxiredoxin 1), DNA damage responses (phospho-ATM and phospho-p53), and nuclear membrane disruption (phospho-lamin A/C) were observed, supporting the role of Pb-induced CDK5-p25 signaling in AD pathogenesis. These findings shed light on the intricate molecular events underlying Pb-induced neurotoxicity and provide valuable insights into the mechanisms that contribute to AD development.

Deleterious effects of combination of lead and ß-amyloid peptides in inducing apoptosis and altering cell cycle in human neuroblastoma cells.

Lead (Pb) is a toxic pollutant known to cause several abnormalities related to the brain, including cognitive dysfunction, and it is ubiquitous in nature. ß-amyloid peptides (AP) are crucially involved in Alzheimer's disease (AD). It has been reported that there is a connection between lead and amyloid peptides in exerting similar kinds of altered functions in the brain and long-term exposure to lead leads ultimately to increased beta amyloid formation in the brain, lethal to human brain cells. There is still a lack of information on the mechanism by which Pb affects AP formation, exerting combined toxicity in AD patients. To fill the gap, we have systematically analyzed the toxicity individually and in combination of Pb and AP in human brain cells. We found that the combination of Pb and AP exerted a higher toxicity than individual exposures in human neuroblastoma cells. The lower inhibitory concentration values were determined by both time and concentration dependent manner on using MTT assay. The data resulted in the development of enhanced toxicity on exposure to Pb with both the combinations of AP(1-40) or (25-35) and with all combinations in human brain cells compared to individual exposures to Pb (1-40) or AP(25-35). The severe apoptotic effect and alteration in cell cycle by arresting at the S-phase evidenced the increased toxicity of combinational exposure to Pb and AP on human neuroblastoma cells. Furthermore, the quantitative determination of LDH and caspase-3 activity indicated the induction of severe toxicity. We conclude that both are synergistically associated with effects such as arresting the cell cycle and triggering apoptosis during the progression of Alzheimer's disease.

Cytotoxicity and Proteasome Inhibition by Alkaloid Extract from Murraya koenigii Leaves in Breast Cancer Cells-Molecular Docking Studies.

Murraya koenigii (curry tree) leaves are rich in bioactive compounds such as flavonoids, alkaloids, and coumarins. Alkaloids from M. koenigii leaves have antianalgesic, antiulcerogenic, antiobesity, and antitumor activities. In this study, we tested the cytotoxic and proteasome-inhibitory potential of a total alkaloid extract (TAE) from M. koenigii leaves in the breast cancer cell line MDA-MB-231. The TAE decreased cell viability with an IC50 of 14.4 µg/mL and altered growth kinetics of breast cancer cells. TAE (32 µg/mL) arrested cells (35%) in the "S" phase of the cell cycle and induced apoptosis. The 26S proteasome, a multicatalytic protease complex, promotes tumor cell proliferation and protects tumor cells from apoptosis. The TAE and mahanine, a carbazole alkaloid present in M. koenigii leaves, preferentially inhibited the trypsin-like, but not the chymotrypsin-like proteolytic activity of the proteasome with an IC50 of 162 µg/mL and 287 µM, respectively. In silico analysis of 26 compounds from M. koenigii leaves revealed significant docking scores for mahanine and two other carbazole alkaloids with the ß2 and ß5 subunits of the catalytic 20S proteasome. Taken together, this study demonstrates that inhibition of the proteasome is an important biological activity of M. koenigii alkaloids, which may lead to cancer cell death.

Novel approach to target cancer stem cells for therapy.

Even though lots of efforts have been made to find different strategies for cancer treatments, currently available therapeutic approaches are chemotherapy, radiation and surgery or combination of these. These treatments prolonged the survival of patients but did not assure complete cure of the disease. Recent scientific evidences suggest that cancer stem cells (CSC) are responsible for recurrence, resistance and existence of this disease even after various therapeutic treatments. Therefore, we hypothesize that the best approach is to target CSCs along with cancer cells for complete remission of the disease. Before targeting these cells, studying their morphological, proliferation, behavioral aspects, physico-chemical interaction and characterizations are very important. For therapeutic approach the differentiation capacity of these cells to cancer cells with or without drugs is critical. To study basic parameters; the best approach would be aseptic sorting of CSCs from cancer cells based on specific cell surface markers by flowcytometer or magnetic cell sorter. The sorted cells have to be grown in culture conditions and treat with optimum concentrations of drugs to target CSC and cancer cell to find appropriate potential combination.

Brain delivery of transferrin coupled indinavir submicron lipid emulsions--pharmacokinetics and tissue distribution.

Indinavir, an antiretroviral protease inhibitor used in treatment of HIV infection has limited penetration into brain due to efflux of P-glycoprotein. The aim of this work was to develop transferrin coupled submicron lipid emulsions (SLEs) containing indinavir for delivery to brain. Stearylamine containing SLEs were prepared, characterized, tested for stability and optimized formulation (SLE-4) was developed. Transferrin was coupled to get SLE-6 by water soluble EDC method and purified by gel filtration. The coupled transferrin was quantified by modified Bradford dye assay method. The fluorescent dye (DiD oil) incorporated SLEs were used to check the brain specific delivery of SLEs. The in vivo pharmacokinetic and tissue distribution were conducted in mice. During pharmacokinetic studies, there was no significant difference in the serum levels of indinavir from SLE-1, SLE-4 and SLE-6 formulations at all time points. In tissue distribution studies the therapeutic availability (TA) of indinavir in brain from SLE-6 was 4.69, 3.1 and 1.7 times higher than drug solution, SLE-1 and SLE-4 respectively whereas, the TA of indinavir from SLE-4 was 2.76 and 1.82 times the drug solution and SLE-1. The brain to serum ratios with SLE-6 were above one indicates the brain specific delivery. The brain delivery of indinavir was improved with transferrin ligand attachment to SLEs by receptor mediated transcytosis.

The guanine nucleotide exchange factor, C3G regulates differentiation and survival of human neuroblastoma cells.

Neuronal differentiation involving neurite growth is dependent on environmental cues which are relayed by signalling pathways to actin cytoskeletal remodelling. C3G, the exchange factor for Rap1, functions in pathways leading to actin reorganization and filopodia formation, processes required during neurite growth. In the present study, we have analyzed the function of C3G, in regulating neuronal cell survival and plasticity. Human neuroblastoma cells, IMR-32 induced to differentiate by serum starvation or by treatment with nerve growth factor (NGF) or forskolin showed enhanced C3G protein levels. Transient over-expression of C3G stimulated neurite growth and also increased responsiveness to NGF and serum deprivation induced differentiation. C3G-induced neurite growth was dependent on both its catalytic and N-terminal regulatory domains, and on the functions of Cdc42 and Rap1. Knockdown of C3G using small hairpin RNA inhibited forskolin and NGF-induced morphological differentiation of IMR-32 cells. Forskolin-induced differentiation was dependent on catalytic activity of C3G. Forskolin and NGF treatment resulted in phosphorylation of C3G at Tyr504 predominantly in the Golgi. C3G expression induced the cell cycle inhibitor p21 and C3G knockdown enhanced cell death in response to serum starvation. These findings demonstrate a novel function for C3G in regulating survival and differentiation of human neuroblastoma cells.

C3G is required for c-Abl-induced filopodia and its overexpression promotes filopodia formation.

The Rap1 guanine nucleotide exchange factor, C3G (also known as Rap1GEF-1) is involved in signaling from growth factors, cytokines and integrins and plays a role in cell adhesion and migration, but the mechanism by which C3G regulates various cellular functions is poorly understood. We, therefore, investigated the ability of C3G to affect actin cytoskeleton-dependent morphological changes in cells. Using RNA interference, we provide evidence that C3G is required for c-Abl-induced filopodia during cell spreading on fibronectin. C3G expression induces actin cytoskeletal reorganization and promotes filopodia formation independent of its catalytic activity. It showed enrichment at filopodia tips characteristic of molecules involved in filopodia dynamics. C3G-induced filopodia were not inhibited by dominant negative mutants of Rho, Rac and Cdc42, but required Abl catalytic activity. Coexpression of N-Wasp-Crib inhibited C3G induced as well as c-Abl-induced filopodia and wiskostatin, a pharmacological inhibitor of N-Wasp attenuates C3G-induced filopodia. Cellular C3G interacts with c-Abl and C3G expression results in enhanced localization of endogenous c-Abl in the cytoplasm. We suggest that C3G and c-Abl function in an interdependent manner, in linking external signals to remodeling the cytoskeleton to induce filopodia.

Phosphorylated guanine nucleotide exchange factor C3G, induced by pervanadate and Src family kinases localizes to the Golgi and subcortical actin cytoskeleton.

BACKGROUND: The guanine nucleotide exchange factor C3G (RapGEF1) along with its effector proteins participates in signaling pathways that regulate eukaryotic cell proliferation, adhesion, apoptosis and embryonic development. It activates Rap1, Rap2 and R-Ras members of the Ras family of GTPases. C3G is activated upon phosphorylation at tyrosine 504 and therefore, determining the localization of phosphorylated C3G would provide an insight into its site of action in the cellular context. RESULTS: C3G is phosphorylated in vivo on Y504 upon coexpression with Src or Hck, two members of the Src family tyrosine kinases. Here we have determined the subcellular localization of this protein using antibodies specific to C3G and Tyr 504 phosphorylated C3G (pY504 C3G). While exogenously expressed C3G was present mostly in the cytosol, pY504 C3G formed upon Hck or Src coexpression localized predominantly at the cell membrane and the Golgi complex. Tyrosine 504-phosphorylated C3G showed colocalization with Hck and Src. Treatment of Hck and C3G transfected cells with pervanadate showed an increase in the cytosolic staining of pY504 C3G suggesting that tyrosine phosphatases may be involved in dephosphorylating cytosolic phospho-C3G. Expression of Src family kinases or treatment of cells with pervanadate resulted in an increase in endogenous pY504 C3G, which was localized predominantly at the Golgi and the cell periphery. Endogenous pY504 C3G at the cell periphery colocalized with F-actin suggesting its presence at the subcortical actin cytoskeleton. Disruption of actin cytoskeleton by cytochalasin D abolished phospho-C3G staining at the periphery of the cell without affecting its Golgi localization. CONCLUSIONS: These findings show that tyrosine kinases involved in phosphorylation of C3G are responsible for regulation of its localization in a cellular context. We have demonstrated the localization of endogenous C3G modified by tyrosine phosphorylation to defined subcellular domains where it may be responsible for restricted activation of signaling pathways.