Targeted inhibition of colorectal cancer proliferation: The dual-modulatory role of 2,4-DTBP on anti-apoptotic Bcl-2 and Survivin proteins.

The anti-apoptotic proteins, Bcl-2 and Survivin, are consistently overexpressed in numerous human malignancies, notably in colorectal cancer. 2,4-Di-tert-butylphenol (2,4-DTBP) is a naturally occurring phenolic compound known for its diverse biological activities, including anti-cancer properties. The mechanism behind 2,4-DTBP-induced inhibition of cell proliferation and apoptosis in human colorectal cancer cells, specifically regarding Bcl-2 and Survivin, remains to be elucidated. In this study, we employed both in silico and in vitro methodologies to underpin this interaction at the molecular level. Molecular docking demonstrated a substantial binding affinity of 2,4-DTBP towards Bcl-2 (ΔG = -9.8 kcal/mol) and Survivin (ΔG = -5.6 kcal/mol), suggesting a potential inhibitory effect. Further, molecular dynamic simulations complemented by MM-GBSA calculations confirmed the significant binding of 2,4-DTBP with Bcl-2 (dGbind = -54.85 ± 6.79 kcal/mol) and Survivin (dGbind = -32.36 ± 1.29 kcal/mol). In vitro assays using HCT116 colorectal cancer cells revealed that 2,4-DTBP inhibited proliferation and promoted apoptosis in both a dose- and time-dependent manner. Fluorescence imaging and scanning electron microscopy illustrated the classical features associated with apoptosis upon 2,4-DTBP exposure. Cell cycle analysis through flow cytometry highlighted a G1 phase arrest and apoptosis assay demonstrated increased apoptotic cell population. Notably, western blotting results indicated a decreased expression of Bcl-2 and Survivin post-treatment. Considering the cytoprotective roles of Bcl-2 and Survivin through the inhibition of mitochondrial dysfunction, our findings of disrupted mitochondrial bioenergetics, characterized by reduced ATP production and oxygen consumption, further accentuate the functional impairment of these proteins. Overall, the integration of in silico and in vitro data suggests that 2,4-DTBP holds promise as a therapeutic agent targeting Bcl-2 and Survivin in colorectal cancer.

Structural evidence of the conversion of nitric oxide (NO) to nitrite ion (NO2-) by lactoperoxidase (LPO): Structure of the complex of LPO with NO2- at 1.89 Å resolution.

Lactoperoxidase (LPO) is a heme containing mammalian enzyme which uses hydrogen peroxide (H2O2) to catalyze the conversion of substrates into oxidized products. LPO is found in body fluids and tissues such as milk, saliva, tears, mucosa and other body secretions. The previous structural studies have shown that LPO converts substrates, thiocyanate (SCN-) and iodide (I-) ions into oxidized products, hypothiocyanite (OSCN-) and hypoiodite (IO-) ions respectively. We report here a new structure of the complex of LPO with an oxidized product, nitrite (NO2-). This product was generated from NO using the two step reaction of LPO by adding hydrogen peroxide (H2O2) in the solution of LPO in 0.1 M phosphate buffer at pH 6.8 as the first step. In the second step, NO gas was added to the above mixture. This was crystallized using 20% (w/v) PEG-3350 and 0.2 M ammonium iodide at pH 6.8. The structure determination showed the presence of NO2- ion in the distal heme cavity of the substrate binding site of LPO. The structure also showed that the propionate group which is linked to pyrrole ring D of the heme moiety was disordered. Similarly, the side chain of Asp108, which is covalently linked to heme moiety, was also split into two components. As a result of these changes, the conformation of the side chain of Arg255 was altered allowing it to form new interactions with the disordered carboxylic group of propionate moiety. These structural changes are indicative of an intermediate state in the catalytic reaction pathway of LPO.

A Peptide Bond from the Inter-lobe Segment in the Bilobal Lactoferrin Acts as a Preferred Site for Cleavage for Serine Proteases to Generate the Perfect C-lobe: Structure of the Pepsin Hydrolyzed Lactoferrin C-lobe at 2.28 Å Resolution.

C-lobe represents the C-terminal half of lactoferrin which is a bilobal 80 kDa iron binding glycoprotein. The two lobes are designated as N-lobe (Ser1-Glu333) and C-lobe (Arg344-Arg689). The N- and C-lobes are connected by a 10-residue long α-helical peptide (Thr334-Thr343). Both lobes adopt similar conformations and have identical iron binding sites. The bilobal lactoferrin was hydrolyzed in a limited proteolysis using pepsin at pH 2.0. It produced a 40 kDa and fully functional C-lobe which was purified and crystallized at pH 8.0. The structure determination revealed that the structure contained residues from Tyr342 to Arg689 representing a fully functional monoferric C-lobe. It showed that pepsin cleaved lactoferrin at the peptide bond Arg341-Tyr342 which is part of the inter-lobe decapeptide. Interestingly, the two previously determined structures of the enzymatically produced C-lobe using trypsin and proteinase K also cleaved lactoferrin at the same peptide bond Arg341-Tyr342. This was a striking result as the three enzymes, pepsin, trypsin and proteinase K have different specificity requirements and yet they cleaved the bilobal lactoferrin at the same peptide bond and generated an identical and fully functional C-lobe. This shows that the observed cleavage site in lactoferrin adopts a highly favourable conformation for proteolysis. It is noteworthy that the three enzymes with different specificities cut the protein at the same peptide bond which may be of physiological significance because the antibacterial action of lactoferrin is extended further through the C-lobe.

Heavy metal pollution and risk assessment by the battery of toxicity tests.

The current study was carried out on dominant fish Oreochromis niloticus and water collected from the polluted Yamuna River, Agra, India. The heavy metals in water, recorded as follows: Fe > Mn > Zn > Cu > Ni > Cr > Cd and all were found to be above the prescribed limits. According to metal pollution index, exposed muscle (49.86), kidney (47.68) and liver (45.26) have been recorded to have higher bioaccumulation. The blood biochemical analysis of exposed O. niloticus indicated significant increase in activities of aspartate aminotransferase (+ 343.5%), alkaline phosphatase (+ 673.6%), alanine aminotransferase (+ 309.1%), and creatinine (+ 494.3%) over the reference. However, a significant decrease in albumin (A): globulins (G) ratio (- 87.86%) was observed. Similarly, the exposed fish also showed significant increase in total leucocyte count (+ 121%), differential leucocyte count, respiratory burst (+ 1175%), and nitric oxide synthase (+ 420%). The histological examination of liver and kidney showed tissue injury. Moreover, micronuclei (0.95%), kidney shaped nuclei (1.2%), and lobed nuclei (0.6%) along with DNA damage in the form of mean tail length in the liver (20.7 µm) and kidney (16.5 µm) was observed in the exposed O. niloticus. Potential health risk assessments based on estimated daily intake, target hazard quotient, hazard index, and target cancer risk indicated health risks associated with the consumption of these contaminated fishes. In conclusion, the present study showed that exposure to heavy metals contaminated water can alter immunological response; induce histopathological alterations and DNA damage in the studied fish. The consumption of this contaminated water or fish could have serious impact on human health.

D-ribose and pathogenesis of Alzheimer's disease.

It is estimated that the global prevalence of dementia will rise as high as 24 million and predicted to be double in every 20 years which is attributed to the fact that the ageing population is increasing and so more individuals are at risk of developing neurodegenerative diseases like Alzheimer's. Many scientists favored glycation of proteins such as tau, amyloid beta (Aß) etc. as one of the important risk factor in Alzheimer's disease (AD). Since, D-ribose shows highest glycation ability among other sugars hence, produces advanced glycation end products (AGEs) rapidly. However, there are several other mechanisms suggested by researchers through which D-ribose may cause cognitive impairments. There is a concern related to diabetic patients since they also suffer from D-ribose metabolism, may be more prone to AD risk. Thus, it is imperative that the pathogenesis and the pathways involved in AD progression are explored in the light of ribosylation and AGEs formation for identifying suitable diagnostics marker for early diagnosis or finding promising therapeutic outcomes.

Studies on conformational changes induced by binding of pendimethalin with human serum albumin.

Pendimethalin (PND) is a widely used herbicide in modern means of agricultural practices. So, its toxic residues exist extensively in the environment and can enter human body. Therefore, the in vitro interaction of PND with human serum albumin (HSA) has been explored by employing various biophysical, molecular docking and dynamics simulation studies as well as enzyme kinetics to unravel its binding mechanism. The binding constant of the PND-HSA complex was about 104 M-1 using Fluorescence quenching spectra. The negative value of Gibbs free energy change (ΔG0 = -32.0 kJ mol-1) indicates this interaction is a spontaneous process. A large negative ΔH0 and positive ΔS0 suggests that hydrophobic interactions and H-bonding are involved in the binding process of PND with HSA. The binding of PND can cause conformational and micro-environmental changes in HSA molecule, as shown by various biophysical and molecular dynamics simulation studies. The site marker competition and molecular docking and simulation experiments affirmed that the binding of PND to HSA occurs at or near site I. Esterase-like activity of HSA exhibited decline in the presence of PND revealed the direct involvement of Lys199 of subdomain IIA (Sudlow's site I) in the binding process.

Pendimethalin-induced oxidative stress, DNA damage and activation of anti-inflammatory and apoptotic markers in male rats.

Male Wistar rats were exposed to herbicide, pendimethalin (PND) at varying oral doses of 62.5, 125 and 250 mg/kg b.w. for 14 days. Toxiological effects were assessed in terms of oxidative stress, DNA damage, histopathological alterations and induction of anti-inflammatory and apoptotic responses linked Bax, Bcl-2, IFN-γ, TNF-α and caspase-3 gene expression. In comparison with respective untreated controls, all exposure groups of PND exhibited significant changes in the oxidative stress markers (protein carbonylation and lipid peroxidation) and antioxidant defenses (GSH, SOD, CAT and GST) in liver and kidney tissues. The histopathological changes including leucocyte infiltration, pyknotic nuclei, necrosis, large bowman's space, shrinked renal cortex, were observed in the liver and kidney tissues of PND exposed rats. Significant DNA damage was recorded through comet assay in liver and kidney cells of treated animals as compared to control. Alteration in anti-inflammatory and apoptotic genes expression determined by RT-PCR, revealed the activation of intrinsic apoptotic pathway(s) under the PND induced cellular stress. A pronounced increase in Bax expression, caspase-3 activities and decreased Bcl-2 expressions were also associated with PND-induced apoptosis. Data from this study suggests that PND induces cellular toxicity and genetic perturbations which can alter the normal cellular and physiological functioning in rats.

Publisher Correction: Multiple biomarker responses (serum biochemistry, oxidative stress, genotoxicity and histopathology) in Channa punctatus exposed to heavy metal loaded waste water.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Multiple biomarker responses (serum biochemistry, oxidative stress, genotoxicity and histopathology) in Channa punctatus exposed to heavy metal loaded waste water.

Experiments were conducted to investigate the health of fish Channa punctatus inhabiting heavy metal-loaded waste water. Heavy metals in the order of Fe > Mn > Zn > Co > Ni > Cu = Cr were present in the waste water. Gills had high metal load followed by liver and then kidney. Albumin, albumin to globulin (A:G) ratio, triglyceride, high density lipoprotein (HDL) and very low density lipoprotein (VLDL) were found to be lower but phospholipid, low density lipoprotein (LDL), total protein, lipid and cholesterol were higher as compared to the reference. Oxidative stress markers such as superoxide dismutase (SOD), catalase (CAT), glutathione S transferase (GST) and lipid peroxidation (LPO) were significantly higher in all tissues, whereas reduced glutathione (GSH) levels were comparatively low. Damage to DNA was observed with significantly higher mean tail length of comets in the exposed fish gill cells (30.9 µm) followed by liver (24.3 µm) and kidney (20.6 µm) as compared to reference fish (5.2, 4.8 and 5.9 µm respectively). Histopathology in gill, liver and kidney also showed marked damage. Integrated biochemical, oxidative stress, genotoxicity and histopathological findings are valuable biomarkers for native fish adaptive patterns, and monitoring of water quality/pollution of freshwater ecosystems.

Computational study involving identification of endocrine disrupting potential of herbicides: Its implication in TDS and cancer progression in CRPC patients.

Several environmental pollutants, including herbicides, act as endocrine disrupting chemicals (EDCs). They can cause cancer, diabetes, obesity, metabolic diseases and developmental problems. Present study was conducted to screen 608 herbicides for evaluating their endocrine disrupting potential. The screening was carried out with the help of endocrine disruptome docking program, http://endocrinedisruptome.ki.si (Kolsek et al., 2013). This program screens the binding affinity of test ligands to 12 major nuclear receptors. As high as 252 compounds were capable of binding to at least three receptors wherein 10 of them showed affinity with at-least six receptors based on this approach. The latter were ranked as potent EDCs. Majority of the screened herbicides were acting as antagonists of human androgen receptor (hAR). A homology modeling approach was used to construct the three dimensional structure of hAR to understand their binding mechanism. Docking results reveal that the most potent antiandrogenic herbicides would bind to hydrophobic cavity of modeled hAR and may lead to testicular dysgenesis syndrome (TDS) on fetal exposure. However, on binding to T877 mutant AR they seem to act as agonists in castration-resistant prostate cancer (CRPC) patients.