Phytosynthesis of silver nanoparticles using aqueous sandalwood (Santalum album L.) leaf extract: Divergent effects of SW-AgNPs on proliferating plant and cancer cells.

The biogenic approach for the synthesis of metal nanoparticles provides an efficient eco-friendly alternative to chemical synthesis. This study presents a novel route for the biosynthesis of silver nanoparticles using aqueous sandalwood (SW) leaf extract as a source of reducing and capping agents under mild, room temperature synthesis conditions. The bioreduction of Ag+ to Ago nanoparticles (SW-AgNPs) was accompanied by the appearance of brown color, with surface plasmon resonance peak at 340-360 nm. SEM, TEM and AFM imaging confirm SW-AgNP's spherical shape with size range of 10-32 nm. DLS indicates a hydrodynamic size of 49.53 nm with predominant negative Zeta potential, which can contribute to the stability of the nanoparticles. FTIR analysis indicates involvement of sandalwood leaf derived polyphenols, proteins and lipids in the reduction and capping of SW-AgNPs. XRD determines the face-centered-cubic crystalline structure of SW-AgNPs, which is a key factor affecting biological functions of nanoparticles. This study is novel in using cell culture methodologies to evaluate effects of SW-AgNPs on proliferating cells originating from plants and human cancer. Exposure of groundnut calli cells to SW-AgNPs, resulted in enhanced proliferation leading to over 70% higher calli biomass over control, enhanced defense enzyme activities, and secretion of metabolites implicated in biotic stress resistance (Crotonyl isothiocyanate, Butyrolactone, 2-Hydroxy-gamma-butyrolactone, Maltol) and plant cell proliferation (dl-Threitol). MTT and NRU were performed to determine the cytotoxicity of nanoparticles on human cervical cancer cells. SW-AgNPs specifically inhibited cervical cell lines SiHa (IC50-2.65 ppm) and CaSki (IC50-9.49 ppm), indicating potential use in cancer treatment. The opposing effect of SW-AgNPs on cell proliferation of plant calli (enhanced cell proliferation) and human cancer cell lines (inhibition) are both beneficial and point to potential safe application of SW-AgNPs in plant cell culture, agriculture and in cancer treatment.

Impact of Serotonin Pathway Gene Polymorphisms and Serotonin Levels in Suicidal Behavior.

OBJECTIVE: Suicide is a significant public health issue and a major cause of death in all ages worldwide. Previous studies have shown the involvement of genetics in suicidal behaviour. This study aimed to assess the role of the genetic variants of the serotonin transporter genes (5HTTLPR, SLC6A4 intron 2) and receptor gene (5HTR2AT102C) in individuals who died of suicide. The study compares the serum levels of serotonin between the cases and controls. METHODS: We conducted a case control study with 120 cases and 126 controls. Socio-economic details of the subjects were collected using a semi-structured proforma and psychological autopsy was used to collect details of medical and other clinical conditions. Blood was drawn after taking informed consent and serum levels of serotonin were estimated by ELISA. Genotyping was performed using appropriate primers followed by polymerase chain reaction (PCR) and a restriction fragment length polymorphism (RFLP). RESULTS: Mean age was 32.59 ± 12.58 for cases and 33.64 ± 9.78 for controls. The risk associated LL genotype of 5HTTLPR was higher among cases. The heterozygous 12/10 genotype of SLC6A4 intron 2 polymorphism was increased among controls. Serum levels of serotonin were lower among cases. Variant genotypes of all the 3 polymorphisms showed significant interaction (OR= 39.26) indicating that this model may increase suicidal tendency. CONCLUSION: The findings of this study suggest that low serum levels of serotonin and two variants of the serotonin gene may influence suicide behaviour in a south Indian population.

Inflammation-induced PELP1 expression promotes tumorigenesis by activating GM-CSF paracrine secretion in the tumor microenvironment.

The inflammatory tumor microenvironment has been implicated as a major player fueling tumor progression and an enabling characteristic of cancer, proline, glutamic acid, and leucine-rich protein 1 (PELP1) is a novel nuclear receptor coregulator that signals across diverse signaling networks, and its expression is altered in several cancers. However, investigations to find the role of PELP1 in inflammation-driven oncogenesis are limited. Molecular studies here, utilizing macrophage cell lines and animal models upon stimulation with lipopolysaccharide (LPS) or necrotic cells, showed that PELP1 is an inflammation-inducible gene. Studies on the PELP1 promoter and its mutant identified potential binding of c-Rel, an NF-κB transcription factor subunit, to PELP1 promoter upon LPS stimulation in macrophages. Recruitment of c-Rel onto the PELP1 promoter was validated by chromatin immunoprecipitation, further confirming LPS mediated PELP1 expression through c-Rel-specific transcriptional regulation. Macrophages that overexpress PELP1 induces granulocyte-macrophage colony-stimulating factor secretion, which mediates cancer progression in a paracrine manner. Results from preclinical studies with normal-inflammatory-tumor progression models demonstrated a progressive increase in the PELP1 expression, supporting this link between inflammation and cancer. In addition, animal studies demonstrated the connection of PELP1 in inflammation-directed cancer progression. Taken together, our findings provide the first report on c-Rel-specific transcriptional regulation of PELP1 in inflammation and possible granulocyte-macrophage colony-stimulating factor-mediated transformation potential of activated macrophages on epithelial cells in the inflammatory tumor microenvironment, reiterating the link between PELP1 and inflammation-induced oncogenesis. Understanding the regulatory mechanisms of PELP1 may help in designing better therapeutics to cure various inflammation-associated malignancies.

Cloning and functional characterization of human Pak1 promoter by steroid hormones.

P21-activated kinase 1 (Pak1) is known to be involved in a plethora of functions including cell growth, survival and can lead to cell transformation and tumor progression especially in breast tissue. Multiple studies have shown Pak1 dysregulation as a change in DNA copy number as well as gene expression levels, suggesting many regulatory mechanisms at transcriptional and translational level. However, very little is known about the transcriptional regulation of the human Pak1 promoter. Here, we focus on Pak1 promoter regulation by steroid hormones along with their respective receptors that are also crucial players in breast tissue function and tumorigenesis. Our results show high Pak1 expression in breast cancer cell lines and in breast tumor tissue. It also suggests that Pak1 is hormone responsive, whose expression can be modulated by steroid hormones namely, estrogen in the form of 17ß-estradiol (E2) and progesterone (P4). Sequence analysis of a 3.2kb Pak1 proximal promoter region shows the presence of PRE (progesterone response element) and ERE (estrogen response element) half sites, that were further cloned and characterized. Results from promoter analysis showed that Pak1 promoter activity is mediated by PR via its binding to PRE present on the Pak1 promoter that was further reaffirmed in vitro by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP). Our results together suggest that it is the PR isoform B regulates Pak1 promoter. To our knowledge, this is the first study to report the detailed characterization and transcriptional regulation of the human Pak1 promoter by steroid hormones.

Small-molecule WNK inhibition regulates cardiovascular and renal function.

The With-No-Lysine (K) (WNK) kinases play a critical role in blood pressure regulation and body fluid and electrolyte homeostasis. Herein, we introduce the first orally bioavailable pan-WNK-kinase inhibitor, WNK463, that exploits unique structural features of the WNK kinases for both affinity and kinase selectivity. In rodent models of hypertension, WNK463 affects blood pressure and body fluid and electro-lyte homeostasis, consistent with WNK-kinase-associated physiology and pathophysiology.

Molecular mechanism of anti-cancer activity of phycocyanin in triple-negative breast cancer cells.

BACKGROUND: Triple-negative breast cancers represent an important clinical challenge, as these cancers do not respond to conventional endocrine therapies or other available targeted agents. Phycocyanin (PC), a natural, water soluble and non-toxic molecule is shown to have potent anti-cancer property. METHODS: In this study, we determined the efficacy of PC as an anti-neoplastic agent in vitro on a series of breast cancer cell lines. We studied effects of PC in inducing DNA damage and apoptosis through western blot and qPCR. Also, anti-metastatic and anti-angiogenic properties were studied by classic wound healing and vasculogenic mimicry assays. RESULTS: We found that triple negative MDA-MB-231 cells were most sensitive to PC (IC50 : 5.98 ± 0.95 µM) as compared to other cells. They also showed decreased cell proliferation and reduced colony formation ability upon treatment with PC. Profile of Cell cycle analysis showed that PC caused G1 arrest which could be attributed to decreased mRNA levels of Cyclin E and CDK-2 and increased p21 levels. Mechanistic studies revealed that PC induced apoptosis as evident by increase in percentage of annexin positive cells, increase in γ-H2AX levels, and by changing the Bcl-2/Bax ratio followed by release of cytochrome C and increased Caspase 9 levels. MDA MB 231 cells treated with PC resulted in decreased cell migration and increased cell adhesive property and also showed anti-angiogenic effects. We also observed that PC suppressed cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) production. All these biological effects of phycocyanin on MDA MB 231 cells could be attributed to decreased MAPK signaling pathway. We also observed that PC is non-toxic to non-malignant cells, platelets and RBC's. CONCLUSION: Taken together, these findings demonstrate, for the first time, that PC may be a promising anti-neoplastic agent for treatment of triple negative breast cancers.

Breast tumors with elevated expression of 1q candidate genes confer poor clinical outcome and sensitivity to Ras/PI3K inhibition.

Genomic aberrations are common in cancers and the long arm of chromosome 1 is known for its frequent amplifications in breast cancer. However, the key candidate genes of 1q, and their contribution in breast cancer pathogenesis remain unexplored. We have analyzed the gene expression profiles of 1635 breast tumor samples using meta-analysis based approach and identified clinically significant candidates from chromosome 1q. Seven candidate genes including exonuclease 1 (EXO1) are consistently over expressed in breast tumors, specifically in high grade and aggressive breast tumors with poor clinical outcome. We derived a EXO1 co-expression module from the mRNA profiles of breast tumors which comprises 1q candidate genes and their co-expressed genes. By integrative functional genomics investigation, we identified the involvement of EGFR, RAS, PI3K / AKT, MYC, E2F signaling in the regulation of these selected 1q genes in breast tumors and breast cancer cell lines. Expression of EXO1 module was found as indicative of elevated cell proliferation, genomic instability, activated RAS/AKT/MYC/E2F1 signaling pathways and loss of p53 activity in breast tumors. mRNA-drug connectivity analysis indicates inhibition of RAS/PI3K as a possible targeted therapeutic approach for the patients with activated EXO1 module in breast tumors. Thus, we identified seven 1q candidate genes strongly associated with the poor survival of breast cancer patients and identified the possibility of targeting them with EGFR/RAS/PI3K inhibitors.

Crowd sourcing a new paradigm for interactome driven drug target identification in Mycobacterium tuberculosis.

A decade since the availability of Mycobacterium tuberculosis (Mtb) genome sequence, no promising drug has seen the light of the day. This not only indicates the challenges in discovering new drugs but also suggests a gap in our current understanding of Mtb biology. We attempt to bridge this gap by carrying out extensive re-annotation and constructing a systems level protein interaction map of Mtb with an objective of finding novel drug target candidates. Towards this, we synergized crowd sourcing and social networking methods through an initiative 'Connect to Decode' (C2D) to generate the first and largest manually curated interactome of Mtb termed 'interactome pathway' (IPW), encompassing a total of 1434 proteins connected through 2575 functional relationships. Interactions leading to gene regulation, signal transduction, metabolism, structural complex formation have been catalogued. In the process, we have functionally annotated 87% of the Mtb genome in context of gene products. We further combine IPW with STRING based network to report central proteins, which may be assessed as potential drug targets for development of drugs with least possible side effects. The fact that five of the 17 predicted drug targets are already experimentally validated either genetically or biochemically lends credence to our unique approach.