Potent growth suppressive activity of curcumin in human breast cancer cells: Modulation of Wnt/β-catenin signaling

Abnormal activation of the Wnt/β-catenin signaling pathway and subsequent upregulation of β-catenin driven downstream targets—c-Myc and cyclin D1 is associated with development of breast cancer. The objective of our study was to determine if curcumin could modulate the key elements of Wnt pathway in breast cancer cells; an effect that might underscore its usefulness for chemoprevention/treatment of this malignancy. Curcumin showed a cytotoxic effect on MCF-7 cells with 50% inhibitory concentration (IC₅₀) of 35 μM; while IC₅₀ for MDA-MB-231 cells was 30 μM. Treatment with low cytostatic dose of 20 μM curcumin showed G₂/M arrest in both breast cancer cells. The effect of curcumin (20 μM) treatment on expression of Wnt/β-catenin pathway components in breast cancer cells (MCF-7 and MDA-MB-231) was analyzed by immunofluorescence and Western blotting. Curcumin was found to effectively inhibit the expression of several Wnt/β-catenin pathway components—disheveled, β-catenin, cyclin D1 and slug in both MCF-7 and MDA-MB-231. Immunofluorescence analysis showed that curcumin markedly reduced the nuclear expression of disheveled and β-catenin proteins. Further, the protein levels of the positively regulated β-catenin targets—cyclin D1 and slug, were downregulated by curcumin treatment. The expression levels of two integral proteins of Wnt signaling, GSK3β and E-cadherin were also altered by curcumin treatment. In conclusion, our data demonstrated that the efficacy of curcumin in inhibition of cell proliferation and induction of apoptosis might occur through modulation of β-catenin pathway in human breast cancer cells.     

Development of polymorphic EST-SSR markers and their applicability in genetic diversity evaluation in Rhododendron arboreum

Molecular Biology Reports - The genus Rhododendron, known for large impressive flowers is widely distributed throughout the world. Rhododendrons have limited genetic information, despite of...     

A study of oxidative stress in cervical cancer- an institutional study

Cervical cancer is the second most common cause of cancer-related death among women worldwide, especially in developing countries. Oxidative stress has been associated with cervical cancer. Many studies demonstrated that the low level of antioxidants induces the production of free radicals that cause lipid peroxidation, DNA, and protein damage leading to mutations that favors malignant transformation. This is a case-control institutional study conducted to evaluate the level of oxidative stress in cervical cancer patients and the age-matched healthy controls. We measured level of TBARS expressed as MDA, activity of SOD and GSH level by the spectrophotometric method, and level of 8-OHdG was estimated using a competitive sandwich ELISA assay. Our results showed a significant increase in the level of lipid peroxidation in group IV when compared to the control, group II and group III (p < 0.001). The activity of SOD was also significantly higher in group IV when compared to the control group (p < 0.001), group II (p < 0.001), and group III (p < 0.001). The level of GSH was also significantly lower in group IV when compared to the control group (p < 0.01), group II (p < 0.01), and group III (p < 0.01). The level of 8-OHdG was significantly higher in group IV than in the other groups (p < 0.01). The results suggest that oxidative stress is involved in the pathogenesis of cervical cancer, which is demonstrated by an increased level of lipid peroxidation and higher levels of 8-OHdG and an altered antioxidant defense system.     

Extraction of total phenolic content from Azadirachta indica or (neem) leaves: Kinetics study

The objective of this work is to put forth the optimization and kinetics of total phenolic compounds extraction from Azadirachta indica leaves in a stirred batch extraction. Various experiential extraction parameters have been studied for maximum extraction of the total phenolic compounds. The maximum yield of total phenolic compounds was found to be 10.80?mg?g^?1 of dried neem powder under the optimized conditions. The extraction kinetics behavior followed first-order kinetics with diffusion coefficient ranged from 1.8?×?10^?12 to 3.2?×?10^?12?m² s^?1 for all sets. Activation energy (E_a) value for the extraction of the total phenolic compounds was found to be 22.87?kJ?mol^?1. The kinetic expression model developed by Spiro and Siddique showed a good agreement with the experimental outcomes. The obtained results can be used to scale up the operations for industrial purposes.     

Identification and expression analysis of group 3 LEA family genes in sorghum [Sorghum bicolor (L.) Moench]

Sorghum with its remarkable adaptability to drought and high temperature provides a model system for grass genomics and resource for gene discovery especially for abiotic stress tolerance. Group 3 LEA genes from barley and rice have been shown to play crucial role in abiotic stress tolerance. Here, we present a genome-wide analysis of LEA3 genes in sorghum. We identified four genes encoding LEA3 proteins in the sorghum genome and further classified them into LEA3A and LEA3B subgroups based on the conservation of LEA3 specific motifs. Further, expression pattern of these genes were analyzed in seeds during development and vegetative tissues under abiotic stresses. SbLEA3A group genes showed expression at early stage of seed development and increased significantly at maturity, while SbLEA3B group genes expressed only in matured seeds. Expression of SbLEA3 genes in response to abiotic stresses such as soil moisture deficit (drought), osmotic, salt, and temperature stresses, and exogenous ABA treatments was also studied in the leaves of 2-weeks-old seedlings. ABA and drought induced the expression of all LEA3 genes, while cold and heat stress induced none of them. Promoter analysis revealed the presence of multiple ABRE core cis-elements and a few low temperature response (LTRE)/drought responsive (DRE) cis-elements. This study suggests non-redundant function of LEA3 genes in seed development and stress tolerance in sorghum.     

Unfolding during urea denaturation of a low molecular weight phytocystatin (thiol protease inhibitor) purified from Phaseolus mungo (Urd)

In the present study, two phytocystatins were purified to homogeneity as peaks I and II with molecular weights of 19 kDa and 17 kDa, respectively, as determined by SDS-PAGE and mass spectrometry. Both PMCs I and II were purified with a greater than 1000-fold purification and overall yield of about 16-18%. The effect of urea on PMC I and II was analysed by fluorescence and Circular Dichroism (CD) spectroscopy. Fluorescence studies suggest a red shift of the maximum emission at higher urea concentrations. PMC I and II are extremely stable protein inhibitors with regards to temperature and pH stability. FTIR studies show predominant alpha-helical structure in both the cystatins. CD analysis results show change in urea concentration-dependent loss in ellipticity, as well as in the shape of the CD spectrum compared to the intact phytocystatin.     

Impact of deglycosylation and thermal stress on conformational stability of a full length murine igG2a monoclonal antibody: Observations from molecular dynamics simulations

With the rise of antibody based therapeutics as successful medicines, there is an emerging need to understand the fundamental antibody conformational dynamics and its implications towards stability of these medicines. Both deglycosylation and thermal stress have been shown to cause conformational destabilization and aggregation in monoclonal antibodies. Here, we study instabilities caused by deglycosylation and by elevated temperature (400 K) by performing molecular dynamic simulations on a full length murine IgG2a mAb whose crystal structure is available in the Protein Data bank. C^α‐atom root mean square deviation and backbone root mean square fluctuation calculations show that deglycosylation perturbs quaternary and tertiary structures in the C_H2 domains. In contrast, thermal stress pervades throughout the antibody structure and both Fabs and Fc regions are destabilized. The thermal stress applied in this study was not sufficient to cause large scale unfolding within the simulation time and most amino acid residues showed similar average solvent accessible surface area and secondary structural conformations in all trajectories. C_H3 domains were the most successful at resisting the conformational destabilization. The simulations helped identify aggregation prone regions, which may initiate cross‐β motif formation upon deglycosylation and upon applying thermal stress. Deglycosylation leads to increased backbone fluctuations and solvent exposure of a highly conserved APR located in the edge β‐strand A of the C_H2 domains. Aggregation upon thermal stress is most likely initiated by two APRs that overlap with the complementarity determining regions. This study has important implications for rational design of antibody based therapeutics that are resistant towards aggregation. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Gadd45alpha does not modulate the carboplatin or 5-fluorouracil-induced apoptosis in human papillomavirus-positive cells

Gadd45alpha is shown to be induced by a wide spectrum of DNA-damaging agents and implicated in negative regulation of cell growth by causing G2-M arrest or induction of apoptosis. In the present study, we explored the involvement of p53 in the promoter activation of Gadd45alpha as well as the role of Gadd45alpha in carboplatin (Carb) or 5-fluorouracil (5-FU)-induced apoptosis in human papillomavirus virus (HPV)-positive HEp-2 and HeLa cells. We report that Carb or 5-FU upregulate Gadd45alpha and p53 in both these cells. Transient transfection of chloramphenicol acetyl transferase (CAT)-reporter construct driven by Gadd45alpha promoter clearly indicated that Gadd45alpha upregulation was mediated through activation of its promoter. Inhibition of p53 function by dominant-negative-p53 expression partially suppressed the activation of Gadd45alpha promoter. Further, the induction of apoptosis was assessed by detection of poly (ADP-ribose) polymerase (PARP) cleavage by Western blot analysis. Inhibition of upregulated Gadd45alpha expression by antisense expression vector did not modulate the Carb or 5-FU-induced apoptosis. Overall, we conclude that Gadd45alpha promoter activation partially depends on p53 function in HPV-positive cells. Moreover, Gadd45alpha protein does not modulate Carb or 5-FU-induced apoptosis in these cells.