Growth Suppression of Mouse Pituitary Corticotroph Tumor AtT20 Cells by Curcumin: A Model for Treating Cushing's Disease

effectiveness of curcumin to suppress pituitary tumorigenesis. However the molecular mechanism that mediate this effect of curcumin are still unknown.Using the mouse corticotroph tumor cells, AtT20 cells, we report that curcumin had a robust, irreversible inhibitory effect on cell proliferation and clonogenic property. The curcumin-induced growth inhibition was accompanied by decreased NFκB activity. Further, curcumin down-regulated the pro-survival protein Bcl-xL, depolarized the mitochondrial membrane, increased PARP cleavage, which led to apoptotic cell death. Finally, curcumin had a concentration-dependent suppressive effect on ACTH secretion from AtT20 cells.The ability of curcumin to inhibit NFκB and induce apoptosis in pituitary corticotroph tumor cells leads us to propose developing it as a novel therapeutic agent for the treatment of CD

REMP software to introduce a screening REstriction site in site-directed Mutagenesis Primer

Insertion of silent mutations allowing for restriction site modification aids in the screening of successful mutants during site directed mutagenesis. Introducing a new restriction site requires the analysis of degenerate sequences within mutant primer. As the total number of degenerate codons increases, the analysis becomes increasingly laborious, time-consuming and prone to errors. Towards this, a software named as ‘REMP’ (for REstriction site in Mutant Primer) was developed and described here. From the input sequence, REMP instantaneously generates degenerate sequences having restriction sites that are 6–8 base pairs in length. The output sequences are arranged based on the number of bases changed compared to the input sequence. REMP software can be installed and run as a stand-alone program on different operating systems. Any user of REMP can edit the list of restriction sites to be considered by the software, without a need for writing a computer code or knowing a program language. © 202

Differential expression of CaMKII isoforms and overall kinase activity in rat dorsal root ganglia after injury.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) decodes neuronal activity by translating cytoplasmic Ca(2+) signals into kinase activity that regulates neuronal functions including excitability, gene expression, and synaptic transmission. Four genes lead to developmental and differential expression of CaMKII isoforms (α, β, γ, δ). We determined mRNA levels of these isoforms in the dorsal root ganglia (DRG) of adult rats with and without nerve injury in order to determine if differential expression of CaMKII isoforms may contribute to functional differences that follow injury. DRG neurons express mRNA for all four isoforms, and the relative abundance of CaMKII isoforms was γ>α>β=δ, based on the CT values. Following ligation of the 5th lumbar (L5) spinal nerve (SNL), the β isoform did not change, but mRNA levels of both the γ and α isoforms were reduced in the directly injured L5 neurons, and the α isoform was reduced in L4 neurons, compared to their contemporary controls. In contrast, expression of the δ isoform mRNA increased in L5 neurons. CaMKII protein decreased following nerve injury in both L4 and L5 populations. Total CaMKII activity measured under saturating Ca(2+)/CaM conditions was decreased in both L4 and L5 populations, while autonomous CaMKII activity determined in the absence of Ca(2+) was selectively reduced in axotomized L5 neurons 21days after injury. Thus, loss of CaMKII signaling in sensory neurons after peripheral nerve injury may contribute to neuronal dysfunction and pain

Selective Estrogen Receptor Down-Regulator and Selective Estrogen Receptor Modulators Differentially Regulate Lactotroph Proliferation

occupation, differentially modulates the biological outcome of anti-estrogens. expression and release, as well as ERE-mediated transcriptional activity. expression

Biochemical characterization of recombinant chicken riboflavin carrier protein

Chicken Riboflavin Carrier Protein (cRCP) transports riboflavin from the maternal circulation to the egg yolk for fetal development. The cRCP is a globular protein and structurally very stable due to the presence of nine intra-molecular disulphide bonds. The cRCP comprises of two domains; the larger riboflavin binding, and the smaller, oocyte receptor binding domain. With the objective to study domain folding in cRCP, these two domains of the corresponding gene were amplified, cloned, and expressed in a eukaryotic expression system to obtain soluble product. Our studies on the biochemical characterization of the recombinant proteins indicated that though the ligand binding domain assumed near-native conformation, as determined by immunological methods, it did not bind riboflavin, suggesting the interdependence of the two domains for proper organization of the riboflavin binding pocket

In Vitro Growth Suppression of Renal Carcinoma Cells by Curcumin

Background: Malignant clear cell renal carcinoma (ccRCC) is an aggressive tumor that is highly resistant to chemotherapy and radiation. Current therapeutic approaches to management of ccRCC have not significantly improved patient survival, therefore novel therapies are needed. The von Hippel-Lindau tumor suppressor gene is frequently mutated in ccRCC resulting in unregulated transcriptional activity of hypoxia-inducible factors (HIF) 1α and 2α. HIF-mediated transcription leads to increased growth factor expression and growth factor receptor (GFR)-mediated signaling. NFκB and STAT3 are phosphorylated in response to GFR activation and modulate gene expression, which promotes cell growth and invasion. Activated NFκB and STAT3 expression is associated with ccRCC pathogenesis. Purpose: The dietary polyphenol curcumin is a well-documented antitumor agent and a known inhibitor of NFκB and STAT3 activation. Given the lack of effective therapies that block ccRCC progression, our objective was to examine whether curcumin could suppress the growth and migration of ccRCC cells, and whether this suppression was mediated via inhibition of NFκB and STAT3 activity. Methods: Human ccRCC cell lines (769-p, 786-o, Caki-1, ACHN and A-498 cells) were exposed to curcumin to assess the impact of curcumin on ccRCC cell viability. To examine the mechanism by which curcumin induced cell death, we used 769-p cells, a highly aggressive human ccRCC cell line that does not express functional von Hippel-Lindau protein. The impact of curcumin on the phosphorylation status and transcriptional activity of NFκB and STAT3, in 769-p cells, was determined. Results: Our results show that in ccRCC cells curcumin decreased cell proliferation and cell viability, abolished clonogenic property, induced apoptosis and blocked cellular migration. The growth suppressive and proapoptotic effects of curcumin were accompanied by decreased phosphorylation and transcriptional activity of NFκB and STAT3. Conclusion: The ability of curcumin to induce apoptosis and inhibit migration of ccRCC cells justifies additional studies that explore the potential of developing curcumin or other NFκB and STAT3 inhibitors as novel therapeutic agents in the management of ccRCC

Curcumin suppresses constitutively activated NFκB activity, down regulates Bcl-_xl and causes mitochondrial membrane depolarization in AtT20 cells.

<p>(<i>A</i>) AtT20 cells were transiently transfected with an NFκB reporter gene, and after 24 hrs treated with either vehicle or the indicated concentrations of curcumin for 4 hrs. Cells were washed and luciferase activity in cell lysates was determined. Normalized luciferase activity was calculated, and data is presented as fold change over control. Each value is the mean ±SEM of 3 separate experiments each performed in triplicates. * indicates significant difference from control, (<i>p<0.05</i>). (<i>B</i>) AtT20 cells were treated with either vehicle or indicated concentrations of curcumin for 24 hrs. Cell lysates were harvested and equal amount of protein was subjected to western blotting with an anti-Bcl-xL Ab. The filter was stripped and reprobed with anti- β tubulin Ab to confirm equal loading. Data shown is from a single experiment, and is a representative of 2 independent experiments yielding similar results. (<i>C</i>) AtT20 cells were treated with either vehicle or indicated concentrations of curcumin for 24 hrs. Cells were washed and labeled with the dual fluorescence mitochondrial specific dye, JC-1, and analyzed by flowcytometry. The dot plots show that in vehicle and 5 µM curcumin treated cells, the % of cells emitting green fluorescence is low, and is indicative of basal apoptosis. However, treatment with 50 µM curcumin, that caused a decrease in Bcl-xL levels, significantly increased the intensity of green fluorescence. The dot plot shown is from a single experiment that is representative of 3 independent experiments. When data from the light scatter plots were quantitated (<i>D</i>) as % of gated cells, our data show that curcumin in a concentration-dependent manner, lead to increased membrane depolarization. Each value is the mean ±SEM of 3 separate experiments. * indicates significant difference from control, (<i>p<0.05</i>).</p

Curcumin induced apoptosis in AtT20 cells.

<p>(<i>A</i>) AtT20 cells were treated with the indicated concentration of curcumin for 24 hrs. Cell lysates were harvested and equal amount of protein was subjected to western blotting with an anti-total PARP Ab, to detect apoptosis. Membrane was stripped and reprobed with anti-β tubulin Ab to confirm equal loading. Data presented is from a single experiment, and is a representative of 3 separate experiments yielding similar results. (<i>B</i>) AtT20 cells were treated with the indicated concentration of curcumin for 24 hrs. Cell were washed and labelled with anexinV-FITC and propidium iodide and analyzed by flow cytometry. Data are presented as % of gated cells. Each value is the mean ±SEM of 3 separate determinations from a single experiment, and is a representative of 3 separate experiments yielding similar results. * indicates significant difference from control, (<i>p<0.05</i>).</p

Curcumin decreases secretion of ACTH.

<p>AtT20 cells were treated with the indicated concentrations of curcumin for 24 hrs, and equal amount of CM were used to detect secreted ACTH. Data were calculated as % of vehicle control and each value is the mean ±SEM of 4 separate experiments. * indicates significant difference from control, (<i>p<0.001</i>).</p