Downregulation of BORIS/CTCFL leads to ROS-dependent cellular senescence and drug sensitivity in MYCN-amplified neuroblastoma.

Brother of Regulator of Imprinted Sites (BORIS) or CCCTC-binding factor like (CTCFL) is a nucleotide-binding protein, aberrantly expressed in various malignancies. Expression of BORIS has been found to be associated with the expression of oncogenes which regulate the reactive oxygen species (ROS) biogenesis, DNA double-strand break repair, regulation of stemness, and induction of cellular senescence. In the present study, we have analyzed the effects of knockdown of BORIS, a potential oncogene, on the induction of senescence and tumor suppression. Loss of BORIS downregulated the expression of critical oncogenes such as BMI1, Akt, MYCN, and STAT3, whereas overexpression increased their respective expression levels in MYCN-amplified neuroblastoma cells. BORIS knockdown exhibited high levels of ROS biogenesis, indicating an upregulated mitochondrial superoxide production and thereby induction of senescence. Our study also showed that the loss of BORIS facilitated cellular senescence through the disruption of telomere integrity via altering the expression of various proteins required for telomere capping (POT1, TRF2, and TIN1). In addition to affecting ROS production and DNA damage, BORIS knockdown sensitized the cells toward chemotherapeutic drugs and induced apoptosis. Tumor induction studies on in vivo xenograft mouse models showed that cells with loss of BORIS/CTCFL failed to induce tumors. From our study, we conclude that silencing BORIS/CTCFL influences tumor growth and proliferation by regulating key oncogenes. The results also indicated that the BORIS knockdown can cause cellular senescence and upon a combinatorial treatment with chemotherapeutic drugs can induce enhanced drug sensitivity in MYCN-amplified neuroblastoma cells.

Regulating BMI1 expression via miRNAs promote Mesenchymal to Epithelial Transition (MET) and sensitizes breast cancer cell to chemotherapeutic drug.

Polycomb group (PcG) proteinB lymphoma Mo-MLV insertion region 1 homolog (BMI1) is a transcriptional repressor that plays an important role in human carcinogenesis. MicroRNAs (miRNAs) are endogenous small non-coding RNAsthat implicate a negative regulation on gene expression. Deregulation of the expression of miRNAs has been implicated in tumorigenesis. Here, we have shown that knock-down ofBMI1increases theexpression of tumor-suppressivemiRNAs. Elevated levels of expression of miR-200a, miR-200b, miR-15a, miR-429, miR-203were observed upon knock-down of BMI1. Up-regulation of these miRNAsleads to down-regulation ofPRC1 group of proteins i.e. BMI1, RING1A, RING1B and Ub-H2A. Interestingly, overexpression of miR-200a, miR-200b and miR-15aalso produced decreased BMI1 and Ub-H2A protein expression in the CD44+ Cancer Stem Cellpopulation of MDAMB-231cells. Also,elevating the levels of BMI1 regulated miRNAspromoted Mesenchymal to Epithelial transition by regulating the expression of N-Cadherin, Vimentin, ß-Catenin, Zeb, Snail thereby resulting in decreased invasion, migration and proliferation. Here, we also report that miR-200a, miR-200b, miR-203 accretes the sensitivity of MDAMB-231 cells to the histone deacetylase inhibitor (HDACi) SAHA and miR-15a sensitized breast cancer cells to the chemotherapeutic drug cisplatin leading to apoptosis. These findings suggest that modulatingspecific miRNAs may serve as a therapeutic approach for the treatment of breast cancer.

Two way controls of apoptotic regulators consign DmArgonaute-1 a better clasp on it.

Argonaute family proteins are well conserved among all organisms. Its role in mitotic cell cycle progression and apoptotic cell elimination is poorly understood. Earlier we have established the contribution of Ago-1 in cell cycle control related to G2/M cyclin in Drosophila. Here we have extended our study in understanding the relationship of Ago-1 in regulating apoptosis during Drosophila development. Apoptosis play a critical role in controlling organ shape and size during development of multi cellular organism. Multifarious regulatory pathways control apoptosis during development among which highly conserved JNK (c-Jun N-terminal kinase) pathway play a crucial role. Here we have over expressed Ago-1 in Drosophila eye and brain by employing UAS (upstream activation sequence)-GAL4 system under the expression of eye and brain specific driver. Over expression of Ago-1 resulted in reduced number of ommatidia in the eye and produced smaller size brain in adult and larval Drosophila. A drastic reversal of the phenotype towards normal was observed upon introduction of a single copy of the dominant negative mutation of basket (bsk, Drosophila homolog of JNK) indicating an active and physical involvement of the bsk with Ago-1 in inducing developmental apoptotic process. Further study showed that Ago-1 stimulates phosphorylation of JNK through transforming growth factor-ß activated kinase 1- hemipterous (Tak1-hep) axis of JNK pathway. JNK phosphorylation results in up regulation of pro-apoptotic genes head involution defective (hid), grim & reaper (rpr) and induces activation of Drosophila caspases (cysteinyl aspartate proteinases);DRONC (Death regulator Nedd2-like caspase), ICE (alternatively Drice, Death related ICE-like caspase) and DCP1 (Death caspase-1) by inhibiting apoptotic inhibitor protein DIAP1 (Death-associated inhibitor of apoptosis 1). Further, Ago-1 also inhibits miR-14 expression to trigger apoptosis. Our findings propose that Ago-1 acts as a key regulator in controlling cell death, tumor regression and stress response in metazoan providing a constructive bridge between RNAi machinery and cell death.

Cardinal Epigenetic Role of non-coding Regulatory RNAs in Circadian Rhythm.

Circadian rhythm which governs basic physiological activities like sleeping, feeding and energy consumption is regulated by light-controlled central clock genes in the pacemaker neuron. The timekeeping machinery with unique transcriptional and post-transcriptional feedback loops is controlled by different small regulatory RNAs in the brain. Roles of the multiple neuronal genes, especially post-transcriptional regulation, splicing, polyadenylation, mature mRNA editing, and stability of translation products, are controlled by epigenetic activities orchestrated via small RNAs. Collectively, these mechanisms regulate clock and light-controlled genes for effecting pacemaker activity and entrainment. Regulatory small RNAs of the circadian circuit, timekeeping mechanism, synchronization of regular entrainment, oscillation, and rhythmicity are regulated by diversified RNA molecules. Regulatory small RNAs operate critical roles in brain activities including the neuronal clock activity. In this report, we propose the emergence of the earlier unexpected small RNAs for a historic perspective of epigenetic regulation of the brain clock system.

Erratum: miR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin.

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

miR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin.

The B-lymphoma Moloney murine leukemia virus insertion region-1 protein (BMI1) acts as an oncogene in various cancers, including breast cancer. Recent evidence suggests that BMI1 is rapidly recruited to sites of DNA double strand breaks where it facilitates histone H2A ubiquitination and DNA double strand break repair by homologous recombination. Here we show that miR-15a and miR-16 expression is decreased during the initial period after DNA damage where it would otherwise down-regulate BMI1, impairing DNA repair. Elevated miR-15a and miR-16 levels down-regulated BMI1 and other polycomb group proteins like RING1A, RING1B, EZH2 and also altered the expression of proteins associated with the BMI1 dependent ubiquitination pathway. Antagonizing the expression of miR-15a and miR-16, enhanced BMI1 protein levels and increased DNA repair. Further, overexpression of miR-15a and miR-16 sensitized breast cancer cells to DNA damage induced by the chemotherapeutic drug doxorubicin. Our results suggest that miR-15a and miR-16 mediate the down-regulation of BMI1, which impedes DNA repair while elevated levels can sensitize breast cancer cells to doxorubicin leading to apoptotic cell death. This data identifies a new target for manipulating DNA damage response that could impact the development of improved therapeutics for breast cancer.

Evolution of circadian rhythms: from bacteria to human.

The human body persists in its rhythm as per its initial time zone, and transition always occur according to solar movements around the earth over 24 h. While traveling across different latitudes and longitudes, at the pace exceeding the earth's movement, the changes in the external cues exceed the level of toleration of the body's biological clock. This poses an alteration in our physiological activities of sleep-wake pattern, mental alertness, organ movement, and eating habits, causing them to temporarily lose the track of time. This is further re-synchronized with the physiological cues of the destination over time. The mechanism of resetting of the clocks with varying time zones and cues occur in organisms from bacteria to humans. It is the result of the evolution of different pathways and molecular mechanisms over the time. There has been evolution of numerous comprehensive mechanisms using various research tools to get a deeper insight into the rapid turnover of molecular mechanisms in various species. This review reports insights into the evolution of the circadian mechanism and its evolutionary shift which is vital and plays a major role in assisting different organisms to adapt in different zones and controls their internal biological clocks with changing external cues.

Novel Triazole linked 2-phenyl benzoxazole derivatives induce apoptosis by inhibiting miR-2, miR-13 and miR-14 function in Drosophila melanogaster.

Apoptosis is an important phenomenon in multi cellular organisms for maintaining tissue homeostasis and embryonic development. Defect in apoptosis leads to a number of disorders like- autoimmune disorder, immunodeficiency and cancer. 21-22 nucleotides containing micro RNAs (miRNAs/miRs) function as a crucial regulator of apoptosis alike other cellular pathways. Recently, small molecules have been identified as a potent inducer of apoptosis. In this study, we have identified novel Triazole linked 2-phenyl benzoxazole derivatives (13j and 13h) as a negative regulator of apoptosis inhibiting micro RNAs (miR-2, miR-13 and miR-14) in a well established in vivo model Drosophila melanogaster where the process of apoptosis is very similar to human apoptosis. These compounds inhibit miR-2, miR-13 and miR-14 activity at their target sites, which induce an increased caspase activity, and in turn influence the caspase dependent apoptotic pathway. These two compounds also increase the mitochondrial reactive oxygen species (ROS) level to trigger apoptotic cell death.

Insulin/insulin-like growth factor-1 signalling (IIS) based regulation of lifespan across species.

An organism's well-being is facilitated by numerous molecular and biochemical pathways that ensure homeostasis within cells and tissues. Aging causes a gradual let-down in the maintenance of homeostasis due to various endogenous and environmental challenges, leading to amassing of damages, functional deterioration of different tissues and vulnerability to ailments. Nutrient sensing pathways that maintain glucose homeostasis in body are involved in regulation of aging. Insulin/insulin-like growth factor-1 (IGF-1) signalling (IIS) pathway was the first nutrient sensing pathway discovered to affect the aging process. This pathway is highly conserved and the most studied among different organisms. Epigenetic machineries that include DNA and histone modifying enzymes and various non-coding RNAs have been identified as important contributors to nutrition-related longevity and aging control. In this report, we present the homology and differences in IIS pathway of various organisms including worm, fly, rodent and human. We also discuss how epigenome remodelling, chromatin based strategies, small and long non-coding RNA are involved to regulate multiple steps of aging or age-related insulin homeostasis. Enhanced study of the role of IIS pathway and epigenetic mechanisms that regulate aging may facilitate progressive prevention and treatment of human age-related diseases.

Down-regulation of BORIS/CTCFL efficiently regulates cancer stemness and metastasis in MYCN amplified neuroblastoma cell line by modulating Wnt/ß-catenin signaling pathway.

BORIS/CTCFL is a vital nucleotide binding protein expressed during embryogenesis and gametogenesis. BORIS/CTCFL is the paralogue of transcriptional repressor protein CTCF, which is aberrantly expressed in various malignancies and primarily re-expressed in cancer stem cells (CSCs). The mechanism behind regulation of BORIS in various cancer conditions and tumor metastases is so far not explored in detail. The aim of the study was to understand the influence of BORIS/CTCFL on stemness and metastasis by regulating well-known oncogenes and related signaling pathways. In our study, we have identified a cross-talk between expression of BORIS/CTCFL and Wnt/ß-catenin signaling pathway, which plays a crucial role in various processes including ontogenesis, embryogenesis and maintenance of stem cell properties. Upon knockdown of BORIS/CTCFL, we observed an upregulation of Mesenchymal to Epithelial transition markers such as E-cad and downregulation of Epithelial to Mesenchymal transition markers such as N-CAD, Vimentin, SNAIL, etc. This transition was accomplished by activation of Wnt/ß-catenin signaling pathway by regulating upstream and downstream Wnt associated proteins including ß-catenin, Wnt3a/5a, CD44, MYC etc. We also identified that BMI1, an oncogene belonging to polycomb group expressed positively with levels of BORIS/CTCFL. Our study implicates the role of BORIS/CTCFL in maintenance of stemness and in transition from mesenchymal to epithelial state in MYC amplified neuroblastoma IMR-32 cells. Effectively controlling BORIS/CTCFL levels can inhibit disease establishment and hence can be considered as a potent target for cancer therapy.

Long non-coding RNA: Functional agent for disease traits.

In recent years, long non-coding RNAs (lncRNAs) have attracted the attention of researchers with their involvement in all facets of life. LncRNAs are transcripts of more than 200 nucleotides which lack defined protein coding potential. Although they do not code for proteins, a large number of them are involved in regulating gene expression and translation. The presence of numerous lncRNAs in the human genome has prompted us to investigate the contribution of these molecules to human biology and medicine. In this review, we present the potential role of lncRNAs interlinked to different human diseases and genetic disorders. We also describe their role in cellular differentiation and aging and discuss their potential importance as biomarkers and as therapeutic agents.

Prohibitin confers cytoprotection against ISO-induced hypertrophy in H9c2 cells via attenuation of oxidative stress and modulation of Akt/Gsk-3ß signaling.

Numerous hypertrophic stimuli, including ß-adrenergic agonists such as isoproterenol (ISO), result in generation of reactive oxygen species (ROS) and alteration in the mitochondrial membrane potential (Δψ) leading to oxidative stress. This process is well associated with phosphorylation of thymoma viral proto-oncogene Akt (Ser473) and glycogen synthase kinase-3ß (Gsk-3ß) (Ser9), with resultant inactivation of Gsk-3ß. In the present study, we found that the protective defensive role of prohibitin (PHB) against ISO-induced hypertrophic response in rat H9c2 cells is via attenuation of oxidative stress-dependent signaling pathways. The intracellular levels of mitochondrial membrane potential along with cellular ROS levels and mitochondrial superoxide generation were determined. In order to understand the regulation of Akt/Gsk-3ß signaling pathway, we carried out immmunoblotting for key proteins of the pathway such as PTEN, PI3K, phosphorylated, and unphosphorylated forms of Akt, Gsk-3ß, and immunofluorescence experiments of p-Gsk-3ß. Enforced expression of PHB in ISO-treated H9c2 cells suppressed cellular ROS production with mitochondrial superoxide generation and enhanced the mitochondrial membrane potential resulting in suppression of oxidative stress which likely offered potent cellular protection, led to the availability of more healthy cells, and also, significant constitutive activation of Gsk-3ß via inactivation of Akt was observed. Knockdown of PHB expression using PHB siRNA in control H9c2 cells reversed these effects. Overall, our results demonstrate that PHB confers cytoprotection against oxidative stress in ISO-induced hypertrophy and this process is associated with modulation of Akt/Gsk-3ß signaling mechanisms as evident from our PHB overexpression and knockdown experiments.

Pigmy MicroRNA: surveillance cops in Therapies kingdom.

MicroRNAs (miRNAs) are well preserved in every animal. These pigmy sized non-coding RNAs (21-23 nt), scattered in genome, are responsible for micromanaging the versatile gene regulations. Involvement of miRNAs was surveillance cops in all human diseases including cardiovascular defects, tumor formation, reproductive pathways, and neurological and autoimmune disorders. The effective functional role of miRNA can be reduced by chemical entities of antisense oligonucleotides and versatile small molecules that support the views of novel therapy of different human diseases. In this study, we have updated our current understanding for designing and synthesizing miRNA-controlling therapeutic chemicals. We have also proposed various in-vivo delivery strategies and their ongoing challenges to combat the incorporation hurdles in live cells and animals. Lastly, we have demonstrated the current progress of miRNA modulation in the treatment of different human diseases that provides an alternative approach of gene therapy.

Simultaneous delivery of Paclitaxel and Bcl-2 siRNA via pH-Sensitive liposomal nanocarrier for the synergistic treatment of melanoma.

pH-sensitive drug carriers that are sensitive to the acidic (pH = ~6.5) microenvironments of tumor tissues have been primarily used as effective drug/gene/siRNA/microRNA carriers for releasing their payloads to tumor cells/tissues. Resistance to various drugs has become a big hurdle in systemic chemotherapy in cancer. Therefore delivery of chemotherapeutic agents and siRNA's targeting anti apoptotic genes possess advantages to overcome the efflux pump mediated and anti apoptosis-related drug resistance. Here, we report the development of nanocarrier system prepared from kojic acid backbone-based cationic amphiphile containing endosomal pH-sensitive imidazole ring. This pH-sensitive liposomal nanocarrier effectively delivers anti-cancer drug (Paclitaxel; PTX) and siRNA (Bcl-2), and significantly inhibits cell proliferation and reduces tumor growth. Tumor inhibition response attributes to the synergistic effect of PTX potency and MDR reversing ability of Bcl-2 siRNA in the tumor supporting that kojic acid based liposomal pH-sensitive nanocarrier as efficient vehicle for systemic co-delivery of drugs and siRNA.

miR-15a/miR-16 induces mitochondrial dependent apoptosis in breast cancer cells by suppressing oncogene BMI1.

AIMS: miRNAs are small non-coding RNA molecules that regulate post-transcriptional gene expression. Here we have made an endeavor to search whether any miRNAs are involved in the regulation of BMI1 in breast cancer that leads to mitochondrial dependent apoptotic cell death. MAIN METHODS: Renilla luciferase reporter assay was performed to detect the ectopically expressed miRNAs that regulate the expression of 3' UTR of BMI1. MTT assay was performed to check the cytotoxicity level. Western blotting and qRT-PCR were performed to check the expression of BMI1, pro-apoptotic, anti-apoptotic proteins and mRNA expression levels respectively. JC-1 staining, Caspase-3, Caspase-6/9 assay and mitochondrial cytosolic fractionation were performed to monitor mitochondrial dependent apoptosis. Wound healing assay was performed to investigate migration. All experiments were performed upon miR-15a and miR-16 overexpression in MCF-7, MDAMB-231 breast cancer cells. KEY FINDINGS: In MCF-7, MDAMB-231 breast cancer cells luciferase reporter assay confirmed the significant reduction of reporter activity upon co-transfection of 3' UTR of BMI1 along with miR-15a and miR-16. miR-15a and miR-16 significantly down-regulated BMI1 protein and mRNA expression levels as well as anti-apoptotic protein BCL2 and up-regulated pro-apoptotic proteins. Ectopic expression of miR-15a, miR-16, increased mitochondrial ROS resulting in impaired mitochondrial membrane potential, followed by cytochrome-C release into the cytosol that activated Caspase-3 and Caspase-6/9 leading to intrinsic apoptosis. Additionally, it also inhibits migration. SIGNIFICANCE: Our results suggest that overexpression of miR-15a and miR-16 mediates down-regulation of BMI1, and leads to mitochondrial mediated apoptosis.

Novel SAHA analogues inhibit HDACs, induce apoptosis and modulate the expression of microRNAs in hepatocellular carcinoma.

In eukaryotes, transcriptional regulation occurs via chromatin remodeling, mainly through post translational modifications of histones that package DNA into structural units. Histone deacetylases (HDACs) are enzymes that play important role in various biological processes by repressing gene expression. Suberoylanilide hydroxamic acid (SAHA) is a known HDAC inhibitor that showed significant anti cancer activity by relieving gene silencing against hematologic and solid tumors. We have designed and synthesized a series of SAHA analogs C1-C4 and performed biological studies to elucidate its anti-cancer effects. It is observed that SAHA analogs significantly inhibited cell proliferation and induced apoptosis in hepatocellular carcinoma (HCC) cell lines HepG2 and SK-HEP-1. These analogs also showed non-toxic activity towards primary human hepatocytes, which describes its tumor specificity. SAHA analogs exhibited strong HDAC inhibition, which is 2-3 fold higher compared to SAHA. Moreover, these molecules induced hyper acetylation of histone H3 at various positions on the lysine residue. Further, it is observed that SAHA analogs are strong inducers of apoptosis, as they regulated the expression of various proteins involved in both extrinsic and intrinsic pathways. Interestingly, SAHA analogs induced upregulation of tumor suppressor miRNAs by activating its biogenesis pathway. Further, it is confirmed by microRNA (miRNA) prediction tools that these miRNAs are capable of targeting various anti-apoptotic genes. Based on these findings we conclude that SAHA analogs could be strong HDAC inhibitors with promising apoptosis inducing nature in HCC.

Regulation of Cell Proliferation and Migration by miR-203 via GAS41/miR-10b Axis in Human Glioblastoma Cells.

Glioma amplified sequence 41(GAS41) is a potent transcription factor that play a crucial role in cell proliferation and survival. In glioblastoma, the expression of GAS41 at both transcriptional and post transcriptional level needs to be tightly maintained in response to cellular signals. Micro RNAs (miRNA) are small non coding RNA that act as important regulators for modulating the expression of various target genes. Studies have shown that several miRNAs play role in the post-transcriptional regulation of GAS41. Here we identified GAS41 as a novel target for endogenous miR-203 and demonstrate an inverse correlation of miR-203 expression with GAS41 in glioma cell lines (HNGC2 and U87). Over expression of miR-203 negatively regulates GAS41 expression in U87 and HNGC2 cell lines. Moreover, miR-203 restrained miR-10b action by suppressing GAS41. GAS41 is essential for repressing p53 in tumor suppressor pathway during cell proliferation. Enforced expression of GAS41 produced contradictory effect on miR-203 but was able to enhance p53 tumor suppressor pathway associated protein. It was also found that miR-203 maintains the stability of p53 as knock down of p53 expression using siRNA resulted in down regulation of pri-miR and mature miR-203 expression. Conversely reconstitution of miR-203 expression induced apoptosis and inhibited migratory property of glioma cells. Taken together, we show that miR-203 is a key negative regulator of GAS41 and acts as tumor suppressor microRNA in glioma.

Drosophila maleless gene counteracts X global aneuploid effects in males.

UNLABELLED: The loss of the entire X chromosome in Drosophila males generates a genome-wide aneuploid effect. We have employed a systems biology approach (microarray) to investigate the global aneuploid effect of the maleless (mle) mutation that disrupts the binding of male specific lethal (MSL) proteins that function in dosage compensation. A large number of the genes (144) that encode a broad spectrum of cellular transport proteins and transcription factors are located mainly in the autosomes of Drosophila melanogaster. We found several such targets to be sensitive to the aneuploid effect and conserved with the X chromosome in primitive Drosophila species and Anopheles gambiae. During evolution, they shifted gradually from their X-chromosomal positions to the autosomes in the species D. melanogaster, suggesting that the counteraction of trans-acting regulatory modifiers and their targets in two separate chromosomes is evolutionarily advantageous. These findings suggest a remarkable and previously unexpected level of complexity favoring natural compensation for the aneuploid effect. We propose that the MSL complex functions in dosage compensation in two separate steps. Initially, X-linked genes including X-linked trans-acting modifiers are hyperactivated in dosage compensated males. Later, these compensated modifiers eventually mitigate inverse aneuploid effects for the equality in expression of their autosomal targets in males and females. DATABASE: Microarray data are available at the Gene Expression Omnibus (GEO) web deposit of National Centre for Biotechnology Information (NCBI) with the accession number GSE78227.

Restoring Mitochondrial Function: A Small Molecule-mediated Approach to Enhance Glucose Stimulated Insulin Secretion in Cholesterol Accumulated Pancreatic beta cells.

Dyslipidemia, particularly the elevated serum cholesterol levels, aggravate the pathophysiology of type 2 diabetes. In the present study we explored the relationship between fasting blood sugar and serum lipid parameters in human volunteers which revealed a significant linear effect of serum cholesterol on fasting blood glucose. Short term feeding of cholesterol enriched diet to rodent model resulted in elevated serum cholesterol levels, cholesterol accumulation in pancreatic islets and hyperinsulinemia with modest increase in plasma glucose level. To explore the mechanism, we treated cultured BRIN-BD11 pancreatic beta cells with soluble cholesterol. Our data shows that cholesterol treatment of cultured pancreatic beta cells enhances total cellular cholesterol. While one hour cholesterol exposure enhances insulin exocytosis, overnight cholesterol accumulation in cultured pancreatic beta cells affects cellular respiration, and inhibits Glucose stimulated insulin secretion. We further report that (E)-4-Chloro-2-(1-(2-(2,4,6-trichlorophenyl) hydrazono) ethyl) phenol (small molecule M1) prevents the cholesterol mediated blunting of cellular respiration and potentiates Glucose stimulated insulin secretion which was abolished in pancreatic beta cells on cholesterol accumulation.

A novel bisindole-PBD conjugate inhibits angiogenesis by regulating STAT3 and VEGF in breast cancer cells.

AIMS: Breast cancer is highly resistant to chemotherapeutic approach and hence, alternative strategies have been developed to fight against this heterogeneous group of disease. In particular, many studies have demonstrated about various drugs for the treatment of breast cancer. In our study, we assessed the anti-angiogenenic activities of Bisindole-PBD (5b) in MCF-7 and MDA-MB-231 cell lines. MAIN METHODS: In vitro Endothelial Cell (HUVEC) Tube Formation Assay was performed to show inhibitory role of 5b along with its role upon wound healing process in breast cancer cells in vitro. Semi-quantitative reverse transcription PCR (RT-PCR) was also done to examine the expression of VEGF in response to 5b in breast cancer cells and in HUVEC cells. siRNA transfection study explored STAT3 mediated VEGF transcription in breast cancer cells MCF-7 and MDA-MB-231. CAM assay was performed to see the role of 5b on vessel formation in chicken embryo. KEY FINDINGS: From in vitro data we have demonstrated that 5b played a role in regulation of breast cancer cell proliferation by inhibiting angiogenesis. Test drug 5b suppressed the expression VEGF at both transcriptional and post transcriptional levels. Apart from this, there was significant down regulation in STAT3 level after drug treatment, which was found to be involved in the VEGF transcription. Metastasis related MMP-2 and MMP-9 expressions were also modulated by 5b. In vivo study by Chick Chorioallantoic Membrane (CAM) Assay also showed anti-angiogenesis role of the test drug which was consistent with the in vitro data. SIGNIFICANCE: Altogether, our data demonstrated 5b as potent small molecule with anti-angiogenic activities.