Research Spotlight

Pleiotropic Hes-1 Concomitant with its Differential Activation Mediates Neural Stem Cell Maintenance and Radial Glial Propensity in Developing Neocortex.

Sivadasan Bindu Dhanesh1, Chandramohan Subashini1, Paul Ann Riya1, Vazhanthodi Abdul Rasheed1 and Jackson James 1

Cereb Cortex. 2016 Jul 11. [Epub ahead of print] DOI: 10.1093/cercor/bhw207

Abstract

Notch signaling pathway and its downstream effector Hes-1 are well known for their role in cortical neurogenesis. Despite the canonical activation of Hes-1 in developing neocortex, recent advances have laid considerable emphasis on Notch/CBF1-independent Hes-1 (NIHes-1) expression with poor understanding of its existence and functional significance. Here, using reporter systems and in utero electroporation, we could qualitatively unravel the existence of NIHes-1 expressing neural stem cells from the cohort of dependent progenitors throughout the mouse neocortical development. Though Hes-1 expression is maintained in neural progenitor territory at all times, a simple shift from Notch-independent to -dependent state makes it pleiotropic as the former maintains the neural stem cells in a non-dividing/slow-dividing state, whereas the latter is very much required for maintenance and proliferation of radial glial cells. Therefore, our results provide an additional complexity in neural progenitor heterogeneity regarding differential Hes-1 expression in the germinal zone during neo-cortical development.

In Vitro Evaluation of the Antioxidant, 3,5-Dihydroxy-4-ethyl-trans-stilbene (DETS) Isolated from Bacillus cereus as a Potent Candidate against Malignant Melanoma.

Nath LR1,Kumar SN2,Das AA3,Nambisan B4,Shabna A1, Mohandas C4and Anto RJ1

Front Microbiol. 2016 Apr 14;7:452. doi: 10.3389/fmicb.2016.00452. eCollection 2016.

Abstract

3,5-dihydroxy Q1 -4-ethyl-trans-stilbene (DETS) is a natural stilbene, which was first identified as bioactive bacterial secondary metabolite isolated from Bacillus cereus associated with a rhabditid entomopathogenic nematode. The present study was intended to investigate the antioxidant and anticancer activity of this compound in vitro. Antioxidant activity was investigated by assaying DPPH free radical scavenging, superoxide radical-(O2..) scavenging, hydroxyl radical scavenging and metal chelating activity, which proved that the compound is a powerful antioxidant. The metal chelating activity of DETS was higher than butylated hydroxyanisol (BHA) and gallic acid, two well-known antioxidants. As the molecule exhibited strong antioxidant potential, it was further evaluated for cytotoxic activity toward five cancer cells of various origins. Since the compound has a strong structural similarity with resveratrol (trans- 3,4,5-trihydroxystilbene), a well-studied chemopreventive polyphenolic antioxidant, its anticancer activity was compared with that of resveratrol. Among the five cancer cells studied, the compound showed maximum cytotoxicity toward the human melanoma cell line, [A375, IC50: 24.01 μM] followed by cervical [HeLa-46.17 μM], colon [SW480- 47.28 μM], liver [HepG2- 69.56 μM] and breast [MCF-7- 84.31 μM] cancer cells. A375 was much more sensitive to DETS compared to the non-melanoma cell line, A431, in which the IC50 of the compound was more than double (49.60 μM). In the present study, the anticancer activity of DETS against melanoma was confirmed by various apoptosis assays. We also observed that DETS, like resveratrol, down-regulates the expression status of major molecules contributing to melanoma progression, such as BRAF, β-catenin and Brn-2, all of which converge in MITF-M, the master regulator of melanoma signaling. The regulatory role of MITF-M in DETS-induced cytotoxicity in melanoma cells was confirmed by comparing the cytotoxicity of DETS in A375 cells (IC50-24.01 μM), with that in SK-MEL-2 (IC50-67.6 μM), another melanoma cells which highly over-express MITF-M. The compound arrests the cells at S-G2 transition state of the cell cycle, as resveratrol. Our results indicate that DETS is a powerful antioxidant, having anticancer efficacy comparable with that of resveratrol, and is a potential candidate to be explored by in vivo studies and in-depth mechanistic evaluation. To our knowledge, this is the first report on the antioxidant and anticancer properties of DETS.

Transfer RNA Derived Small RNAs Targeting Defense Responsive Genes Are Induced during Phytophthora capsici Infection in Black Pepper (Piper nigrum L.).

Asha S1, Soniya EV1

Front Plant Sci. 2016 Jun 1;7:767. doi: 10.3389/fpls.2016.00767. eCollection 2016.

Abstract

Small RNAs derived from transfer RNAs were recently assigned as potential gene regulatory candidates for various stress responses in eukaryotes. In this study, we report on the cloning and identification of tRNA derived small RNAs from black pepper plants in response to the infection of the quick wilt pathogen, Phytophthora capsici. 5'tRFs cloned from black pepper were validated as highly expressed during P. capsici infection. A high-throughput systematic analysis of the small RNAome (sRNAome) revealed the predominance of 5'tRFs in the infected leaf and root. The abundance of 5'tRFs in the sRNAome and the defense responsive genes as their potential targets indicated their regulatory role during stress response in black pepper. The 5'Ala(CGC) tRF mediated cleavage was experimentally mapped at the tRF binding sites on the mRNA targets of Non-expresser of pathogenesis related protein (NPR1), which was down-regulated during pathogen infection. Comparative sRNAome further demonstrated sequence conservation of 5'Ala tRFs across the angiosperm plant groups, and many important genes in the defense response were identified in silico as their potential targets. Our findings uncovered the diversity, differential expression and stress responsive functional role of tRNA-derived small RNAs during Phytophthora infection in black pepper.

Hypothetical protein Rv3423.1 of Mycobacterium tuberculosis is a histone acetyltransferase.

Jose L1,Ramachandran R1,Bhagavat R2, Gomez RL1,Chandran A1, Raghunandanan S1, Omkumar RV3, Chandra N2, Mundayoor S1, Kumar RA1

FEBS J. 2016 Jan;283(2):265-81. doi: 10.1111/febs.13566. Epub 2015 Nov 26.

Abstract

We isolated an 8 kDa mycobacterial hypothetical protein, Rv3423.1, from the chromatin of human macrophages infected with Mycobacterium tuberculosis H37Rv. Bioinformatics predictions followed by in vitro biochemical assays with purified recombinant protein showed that Rv3423.1 is a novel histone acetyltransferase that acetylates histone H3 at the K9/K14 positions. Transient transfection of macrophages containing GFP-tagged histone H1 with RFP-tagged Rv3423.1 revealed that the protein co-localizes with the chromatin in the nucleus. Co-immunoprecipitation assays confirmed that the Rv3423.1-histone interaction is specific. Rv3423.1 protein was detected in the culture filtrate of virulent but not avirulent M. tuberculosis. Infection of macrophages with recombinant Mycobacterium smegmatis constitutively expressing Rv3423.1 resulted in a significant increase in the number of intracellular bacteria. However, the protein did not seem to offer any growth advantage to free-living recombinant M. smegmatis. It is highly likely that, by binding to the host chromatin, this histone acetyltransferase from M. tuberculosis may manipulate the expression of host genes involved in anti-inflammatory responses to evade clearance and to survive in the intracellular environment..

DEPTOR promotes survival of cervical squamous cell carcinoma cells and its silencing induces apoptosis through downregulating PI3K/AKT and by up-regulating p38 MAP kinase.

Srinivas KP1, Viji R1, Dan VM1, Sajitha IS1, Prakash R1, Rahul PV1, Santhoshkumar TR1, Lakshmi S2, Pillai MR1

Oncotarget. 2016 Mar 16. doi: 10.18632/oncotarget.8131. [Epub ahead of print]

Abstract

DEPTOR is an endogenous inhibitor of mTOR complexes, de-regulated in cancers. The present study reveals a vital role for DEPTOR in survival of cervical squamous cell carcinoma (SCC). DEPTOR was found to be overexpressed in both cervical SCC cells and tissues and it's silencing in cervical SCC cells induced apoptosis, mainly by up-regulation of p38 MAPK and by inhibiting PI3K/AKT pathway via a feed-back inhibition from mTORC1-S6K. DEPTOR silencing resulted in reduced expression of the nitric oxide synthases iNOS and eNOS, as well as increased activation of ERK1/2 and p38 MAP kinases. Activation of AKT signaling by overexpression of constitutively active-AKT (CA-AKT) failed to overcome the apoptosis caused by DEPTOR silencing. Similarly pharmacological inhibition of ERK also failed to control apoptosis. However pharmacological inhibition of p38 MAPK rescued the cells from apoptosis, indicating the major role of p38 MAPK in cell death induced by DEPTOR silencing. DEPTOR was also found to regulate ERK1/2 in an AKT dependent manner. DEPTOR knockdown induced cell death in SiHa cells overexpressing the anti-apoptotic Bcl-2 and Bcl-xL, indicating strong survival role of DEPTOR in these cells. DEPTOR overexpression activated PI3K/AKT by relieving the negative feed-back inhibition from mTORC1-S6K. DEPTOR regulation was also observed to be independent of HPV E6/E7 oncoproteins, but it might be a molecular co-factor contributing to cervical carcinogenesis. In summary, DEPTOR is found to promote survival of cervical SCC cells and its reduction induced apoptosis via differential effects on PI3K/AKT and p38 MAPK and can be a potential target in cervical SCC.

Downregulation of vimentin in macrophages infected with live Mycobacterium tuberculosis is mediated by Reactive Oxygen Species

P. P. Mahesh, R. J. Retnakumar ∧ Sathish Mundayoor

Scientific Reports 6, Article number: 21526 (2016) doi:10.1038/srep21526

Abstract

Mycobacterium tuberculosis persists primarily in macrophages after infection and manipulates the host defence pathways in its favour. 2D gel electrophoresis results showed that vimentin, an intermediate filament protein, is downregulated in macrophages infected with live Mycobacterium tuberculosis H37Rv when compared to macrophages infected with heat- killed H37Rv. The downregulation was confirmed by Western blot and quantitative RT-PCR. Besides, the expression of vimentin in avirulent strain, Mycobacterium tuberculosis H37Ra- infected macrophages was similar to the expression in heatkilled H37Rv- infected macrophages. Increased expression of vimentin in H2O2- treated live H37Rvinfected macrophages and decreased expression of vimentin both in NAC and DPI- treated heat-killed H37Rv-infected macrophages showed that vimentin expression is positively regulated by ROS. Ectopic expression of ESAT-6 in macrophages decreased both the level of ROS and the expression of vimentin which implies that Mycobacterium tuberculosis-mediated downregulation of vimentin is at least in part due to the downregulation of ROS by the pathogen. Interestingly, the incubation of macrophages with anti-vimentin antibody increased the ROS production and decreased the survival of H37Rv. In addition, we also showed that the pattern of phosphorylation of vimentin in macrophages by PKA/ PKC is different from monocytes, emphasizing a role for vimentin phosphorylation in macrophage differentiation.

ERK mediated upregulation of death receptor 5 overcomes the lack of p53 functionality in the diaminothiazole DAT1 induced apoptosis in colon cancer models: efficiency of DAT1 in Ras-Raf mutated cells.

Thamkachy R1, Kumar R1, Rajasekharan KN2, Sengupta S3

Mol Cancer. 2016 Mar 8;15:22. doi: 10.1186/s12943-016-0505-7

Abstract

BACKGROUND: p53 is a tumour suppressor protein that plays a key role in many steps of apoptosis, and malfunctioning of this transcription factor leads to tumorigenesis. Prognosis of many tumours also depends upon the p53 status. Most of the clinically used anticancer compounds activate p53 dependent pathway of apoptosis and hence require p53 for their mechanism of action. Further, Ras/Raf/MEK/ERK axis is an important signaling pathway activated in many cancers. Dependence of diaminothiazoles, compounds that have gained importance recently due to their anticancer and anti angiogenic activities, were tested in cancer models with varying p53 or Ras/Raf mutational status.
METHODS: In this study we have used p53 mutated and knock out colon cancer cells and xenograft tumours to study the role of p53 in apoptosis mediated by diaminothiazoles. Colon cancer cell lines with varying mutational status for Ras or Raf were also used. We have also examined the toxicity and in vivo efficacy of a lead diaminothiazole 4-Amino-5-benzoyl-2-(4-methoxy phenylamino)thiazole (DAT1) in colon cancer xenografts.
RESULTS: We have found that DAT1 is active in both in vitro and in vivo models with nonfunctional p53. Earlier studies have shown that extrinsic pathway plays major role in DAT1 mediated apoptosis. In this study, we have found that DAT1 is causing p53 independent upregulation of the death receptor 5 by activating the Ras/Raf/MEK/ERK signaling pathway both in wild type and p53 suppressed colon cancer cells. These findings are also confirmed by the in vivo results. Further, DAT1 is more efficient to induce apoptosis in colon cancer cells with mutated Ras or Raf.
CONCLUSIONS: Minimal toxicity in both acute and subacute studies along with the in vitro and in vivo efficacy of DAT1 in cancers with both wild type and nonfunctional p53 place it as a highly beneficial candidate for cancer chemotherapy. Besides, efficiency in cancer cells with mutations in the Ras oncoprotein or its downstream kinase Raf raise interest in diaminothiazole class of compounds for further follow-up.

Plumbagin, a naphthaquinone derivative induces apoptosis in BRCA 1/2 defective castrate resistant prostate cancer cells as well as prostate cancer stem-like cells.

R S R1, K H S1, Somasundaram V2, S SK1, Nadhan R1, Nair RS3, Srinivas P4

Pharmacol Res. 2016 Mar;105:134-45. doi: 10.1016/j.phrs.2016.01.012. Epub 2016 Jan 22

Abstract

Eventhough the role of BRCA1/2 in hereditary prostatic cancer is being unleashed at a rapid rate; their optimal clinical management remains undefined. Cancer stem cells are thought to be responsible for cancer chemoresistance and relapse, thus they represent a significant concern for cancer prognosis and therapy. In this study, we have analyzed the effect of Plumbagin (PB) and structurally related naphthaquinones on BRCA1/2 silenced prostate cancer cells and the ability of PB to target stem cells. Our cell proliferation studies showed that both PC-3 and DU145 cells were more sensitive to PB, though all the compounds induced mitochondrial potential loss, DNA fragmentation and morphological changes which are indicative of apoptosis. Both BRCA1/2 siRNA transfected PC-3 and DU145 cells exhibited increased sensitivity to PB. Gene expression profiling post PB treatment in BRCA1/2 silenced cells revealed that PB has a putative role in tumor suppression in BRCA defective cancers. Using flow cytometric analysis we have proved that PB has the putative ability to directly target CSCs. Overall studies suggest that PB's antitumour mechanisms holds promise for novel therapeutic approaches against BRCA mutated cancers as well as CSCs.