hsa-miR-23a~27a~24-2 cluster members inhibit aggressiveness of breast cancer cells by commonly targeting NCOA1, NLK and RAP1B.

Targeted therapy is receiving considerable attention from the researchers around the globe owing to the increased drug-resistance and incidences of cancer recurrences. MicroRNAs (miRNAs) exhibits tremendous potential as a candidate for molecular targeted therapy in cancer. Unfortunately, majority of research related to microRNAs are focussed on either a particular miRNA or a set of unrelated miRNAs. There is lack of holistic knowledge on differential co-expression of miRNA clusters in regulating the gene expression under physiological conditions. Previously, we reported the cooperative effect of hsa-miR-23a~27a~24-2 cluster in inducing ER (Endoplasmic Reticulum) stress-mediated apoptotic cell death of HEK cells. In the present study, we have investigated the common anti-cancer effects of individual members of this cluster. Our in silico analysis identified twelve common target genes distributed across three independent clusters. Furthermore, we found NCOA1, NLK, and RAP1B to fall in a single cluster with NCOA1 as a central hub molecule. Prognostic analysis showed profound involvement of these three genes in the breast cancer progression and metastasis. We further demonstrated that alteration in the levels of individual members of miR-23a~27a~24-2 cluster commonly regulates the invasive migration of breast cancer cells by modulating EMT and cytoskeletal pathway proteins. Our results reveal a new insight into the therapeutic potential of individual members of the pro-apoptotic hsa-miR-23a~27a~24-2 cluster family against metastatic breast cancer.

Inhibition of microRNA-128-3p attenuates hypercholesterolemia in mouse model.

AIMS: Hypercholesterolemia remains a critical risk factor for cardiovascular diseases and there is an urgent need to develop effective alternative therapeutics. Herein, we investigated the effects of miR-128-3p inhibition on serum cholesterol levels using a hypercholesterolemic mouse model. MATERIALS AND METHODS: Five injections of anti-miR-128-3p (AM-128) treatment were given, and the cholesterol profile in serum and liver was quantified. We validated the underlying gene network using qRT-PCR, western blotting, ELISA, and dual luciferase assays. KEY FINDINGS: AM-128 treatment inhibits cholesterol biosynthesis by upregulating INSIG1 and downregulating HMGCR (3-hydroxy-3-methylglutaryl-CoA reductase) expression. The serum cholesterol clearance by SR-B1 (scavenger receptor class B member 1) and LDLR (low density lipoprotein receptors) was also increased. Furthermore, the catabolism of cholesterol by CYP7A1 (cytochrome P450 family 7 subfamily A member 1) was increased. SIGNIFICANCE: Our results confirmed a critical role of miR-128-3p inhibition in lowering serum cholesterol and suggest its potential therapeutic implications in reversing hypercholesterolemia.

Mitochondrial microRNA (MitomiRs) in cancer and complex mitochondrial diseases: current status and future perspectives.

Mitochondria are not only important for cellular bioenergetics but also lie at the heart of critical metabolic pathways. They can rapidly adjust themselves in response to changing conditions and the metabolic needs of the cell. Mitochondrial involvement as well as its dysfunction has been found to be associated with variety of pathological processes and diseases. mitomiRs are class of miRNA(s) that regulate mitochondrial gene expression and function. This review sheds light on the role of mitomiRs in regulating different biological processes-mitochondrial dynamics, oxidative stress, cell metabolism, chemoresistance, apoptosis,and their relevance in metabolic diseases, neurodegenerative disorders, and cancer. Insilico analysis of predicted targets of mitomiRs targeting energy metabolism identified several significantly altered pathways (needs in vivo validations) that may provide a new therapeutic approach for the treatment of human diseases. Last part of the review discusses about the clinical aspects of miRNA(s) and mitomiRs in Medicine.

Susceptibility to high-altitude pulmonary edema is associated with circulating miRNA levels under hypobaric hypoxia conditions.

Hypobaric hypoxia poses stress to sojourners traveling to high-altitude. A cascade of physiological changes occurs to cope with or adapt to hypobaric hypoxia. However, an insufficient physiological response to the hypoxic condition resulting from imbalanced vascular homeostasis pathways results in high-altitude pulmonary edema (HAPE). The present study aims to identify the implication of miRNAs associating with HAPE and adaptation. We analyzed the expression of 1,113 miRNAs in HAPE-patients (HAPE-p), HAPE-free controls (HAPE-f), and highland natives (HLs). Based on miRNA profiling and in silico analyses, miR-124-3p emerged relevantly. We observed a significant overexpression of miR-124-3p in HAPE-p. In silico analyses revealed a direct interaction of miR-124-3p with vascular homeostasis and hypoxia-associated genes NOS3 (endothelial nitric oxide synthase), Apelin, and ETS1 (V-Ets avian erythroblastosis virus E2 oncogene homolog 1). Moreover, the transcript and biolevel expression of these genes were significantly decreased in HAPE-p when compared with HAPE-f or HLs. Our in vitro analysis in human umbilical vein endothelial cells demonstrated a significant knockdown of these genes both at transcript and protein levels following miR-124-3p overexpression. Conclusively, our results showed that miR-124-3p might play a plausible role in HAPE pathophysiology by inhibiting the expression of NOS3, Apelin, and ETS1.

Differential expression of p52 and RelB proteins in the metastatic and non-metastatic groups of uveal melanoma with patient outcome.

PURPOSE: Non-canonical NFκB (NC-NFκB) pathway plays an influential role in metastasis, which promotes cancer proliferation and progression. The aim of the study was to examine the expression of NC-NFκB proteins and their correlation with clinicopathological factors associated with metastatic cases of uveal melanoma (UM) and with the patient outcome. METHOD: Expression of NC-NFκB proteins (p52, RelB, and co-expression of p52/RelB) was evaluated in 75 formalin-fixed cases of uveal melanoma by immunohistochemistry. Validation of nuclear immunoreactivity was done by western blotting. Transcriptional status of NC-NFκB genes was assessed in 60 fresh tumor tissues by quantitative real-time PCR. Co-immunoprecipitation was performed to determine the presence of native p52/RelB heterodimer in UM. Prognostic relevance was determined using Cox proportional hazard and Kaplan-Meier methods. RESULTS: Immunohistochemical expression of p52, RelB, and their co-expression was observed in 81%, 68.7%, 56.2% of metastatic cases, respectively, while their expression was seen only in 38%, 33% and 30% of non-metastatic cases. Loss of BAP-1 was correlated with expression of p52 and RelB proteins. Co-immunoprecipitation assay confirmed the putative interaction of p52 with RelB protein in metastatic cases of uveal melanoma. Co-expression of p52/RelB and expression of p52 protein was significantly correlated with decreased metastasis-free survival (MFS) (p = 0.004; p = 0.002) and overall survival (OS) (p = 0.004; p = 0.032), while the RelB expression only correlated with reduced MFS (p = 0.003). CONCLUSION: Our data showed that non-canonical NFκB proteins were significantly higher in metastatic cases and associated with poor outcome of the patients. Furthermore, the p52 protein could be used as a potential therapeutic biomarker for metastatic cases in uveal melanoma.

Identification of canonical NFκB (C-NFκB) pathway in uveal melanoma and their relation with patient outcome.

Inflammation in uveal melanoma (UM) is linked to a bad prognosis. It is rare type of cancer, of which the metastases are usually fatal within a year. Infiltration with an inflammatory infiltrate increases with disease progression but does not seem to inhibit metastasis. The Canonical NFκB (C-NFκB) pathway is known to play a crucial role in tumor inflammation. We therefore, studied the expression of canonical NFκB proteins and their prognostic relevance in UM. Our study evaluated the expression of C-NFκB proteins (p65, p50, and c-Rel) by using immunohistochemistry on sections from 75 formalin-fixed UM. Activation of the NFκB subunit was determined on fresh tumor specimens by measuring the DNA-binding activity in nuclei using an NFκB ELISA assay. Real-time PCR was performed on frozen material on 58 tumors. The presence of native C-NFκB heterodimers (p65/p50 and c-Rel/p50) was confirmed by co-immunoprecipitation followed by Western blotting. We observed a high nuclear immunoreactivity of p65, p50, and c-Rel proteins in 54, 60 and 41% UM cases, respectively. Expression of C-NFκB proteins significantly correlated with parameters which are related to the inflammatory environment of UM. Nuclear immunoreactivity of p65 and p50 was associated with lower patient survival (p = 0.041; p = 0.048) while c-Rel was not. Our finding reveals that C-NFκB proteins expressed are more often in UM with inflammation than those without inflammation. Activation of the canonical NFκB pathway is more frequent in high risk UM patients. These observations might help to understand the behaviour of high risk tumors, with upregulation of C-NFκB proteins contributing to tumor aggressiveness.

MiR-195 regulates mitochondrial function by targeting mitofusin-2 in breast cancer cells.

Mitochondrial dynamics is a highly dysregulated process in cancer. Apoptosis and mitochondrial fission are two concurrent events wherein increased mitochondrial fragmentation serves as a hallmark of apoptosis. We have shown earlier that miR-195 exerts pro-apoptotic effects in breast cancer cells. Herein, we have demonstrated miR-195 as a modulator of mitochondrial dynamics and function. Imaging experiments upon miR-195 treatment have shown that mitochondria undergo extensive fission. We validated mitofusin2 as a potential target of miR-195. This may provide a molecular explanation for the respiratory defects induced by miR-195 over-expression in breast cancer cells. Active, but not total, mitochondrial mass, was reduced with increasing levels of miR-195. We have further shown that miR-195 enhances mitochondrial SOD-2 expression but does not affect PINK1 levels in breast cancer cells. Collectively, we have revealed that miR-195 is a modulator of mitochondrial dynamics by targeting MFN2 thereby impairing mitochondrial function. Concomitantly, it enhances the scavenger of reactive oxygen species (SOD-2) to maintain moderate levels of oxidative stress. Our findings suggest a therapeutic potential of miR-195 in both ER-positive as well as ER-negative breast cancer cells.

Mutational Analysis of the Mitochondrial DNA Displacement-Loop Region in Human Retinoblastoma with Patient Outcome.

Alteration in mitochondrial DNA plays an important role in the development and progression of cancer. The Displacement Loop (D-loop) region of mitochondrial DNA (mtDNA) is the regulatory region for its replication and transcription. Therefore, we aimed to characterize mutations in the D-loop region of mitochondrial DNA along with the morphological changes and analyzed their impact on survival in retinoblastoma patients. mtDNA D-loop region was amplified by Nested-Polymerase Chain Reaction (Nested-PCR) and mutations were analyzed in 60 tumor samples from retinoblastoma patients by DNA sequencing. Transmission electron microscopy was performed on 5 retinoblastoma specimens. Mutations were correlated with clinical, histopathological parameters and patient survival. D-loop mutations were found in total of 52/60 (86.6%) patients. The most common mutations were T to C and C to T followed by A to G. There were 5.81% mutations which were not previously reported in the MITOMAP database. A73G (83.33%) were the most frequent mutations found in our cases and it was statistically significant with poor tumor differentiation and age. In addition, this study was further analyzed for morphological changes in retinoblastoma that had disorganized, swollen and less numbers of mitochondria on electron microscopy. This is the first study showing high frequency of mtDNA mutation which might be due to abnormal morphology of mitochondria in retinoblastoma. Our results indicate that pathogenic mtDNA D-loop mutations may be involved in tumorigenesis of retinoblastoma tumor.

PI3K/AKT/mTOR activation and autophagy inhibition plays a key role in increased cholesterol during IL-17A mediated inflammatory response in psoriasis.

Psoriasis is an immune-mediated inflammatory disease of the skin. Previous studies including ours have shown that IL-17A plays a major role in its pathogenesis; however, its precise molecular mechanism of action is not well understood. Cytokines like TNF α and IL-23 are also important in mediating the disease and some studies have also reported autophagy as a novel mechanism by which cytokines controls the immune response. Herein, we investigated the effect of IL-17A on autophagy and reveal crosstalk between autophagy and cholesterol signaling in keratinocytes. Our results suggest that IL-17A stimulated keratinocytes activated PI3K/AKT/mTOR signaling and inhibited autophagy by simultaneously inhibiting autophagosome formation and enhancing autophagic flux. Western blotting was utilized to detect the expression of autophagic markers (LC3 and p62), PI3K, mTOR and AKT. Induction of autophagy by mTOR inhibitor rapamycin and/or starvation also inhibited the levels of IL-17A secreted IL-8, CCL20 and S100A7 in keratinocytes. Herein, we also observed that inhibition of autophagy by IL-17A was accompanied by enhanced cellular cholesterol levels which in turn regulated the autophagic flux. To investigate crosstalk between autophagy and cellular cholesterol, we used methyl-ß-cyclodextrin (MßCD), which disrupts detergent-insoluble microdomains (DIMs) by depleting cells of cholesterol and checked autophagy. Decreased expression of LC3-II in psoriatic lesional skin compared to non-lesional skin and induction of autophagy by anti-psoriatic drug methotrexate in keratinocytes further confirms the role of autophagy in psoriasis. Our findings suggest that modulators of autophagy and/or cholesterol levels may be developed, and also may lead to new therapeutic agents for psoriasis treatment.

miR-107 functions as a tumor suppressor in human esophageal squamous cell carcinoma and targets Cdc42.

Previously, we reported significantly decreased expression of tissue and circulating miR-107 in esophageal cancer (EC). However, its role in esophageal tumorigenesis still remains elusive. Therefore, the aim of the present study was to analyze the role of miR-107 in esophageal squamous cell carcinoma (ESCC). The role of miR-107 in ESCC was evaluated using MTT assay, cell cycle analysis by flow cytometry, annexin assay, colony formation assay and scratch assay. Overexpression of miR-107 in KYSE-410 cells suppressed cell proliferation at 72 h post-transfection (p=0.0001). Moreover, a significant increase in the G0/G1 population (p<0.001) and a significant decrease in the G2/M (p=0.032) population was also observed in the miR-107-treated cells as compared to the negative control (NC). Notably, miR-107 overexpression attenuated the colony formation potential of ESCC cells by 41.83% as compared to the NC (p=0.007). miR-107 mimic inhibited ESCC cell migration in a time-dependent manner, reducing the wound closure to only 50.41±7.23% at 72 h post-transfection (p=0.041). Further analysis by Matrigel invasion assay revealed a significant decrease in the migratory and invasive abilities of the KYSE-410 cells at 72 h post miR-107 transfection. qRT-PCR analysis showed decreased expression of one of the newly identified targets of miR-107, Cdc42, at the mRNA level. Further validation by western blotting confirmed a significant reduction in the identified target at the protein level. In addition, the relative luciferase activity of the reporter containing Cdc42 3'UTR was significantly decreased upon miR-107 co-transfection, indicating it to be a direct target of miR-107. Our results herein document that miR-107 functions as a tumor suppressor and inhibits the proliferation, migration and invasion of ESCC cells. Moreover, this is the first report showing Cdc42 as a downstream target of miR-107.

MicroRNA: a connecting road between apoptosis and cholesterol metabolism.

Resistance to apoptosis leads to tumorigenesis and failure of anti-cancer therapy. Recent studies also highlight abrogated lipid/cholesterol metabolism as one of the root causes of cancer that can lead to metastatic transformations. Cancer cells are dependent on tremendous supply of cellular cholesterol for the formation of new membranes and continuation of cell signaling. Cholesterol homeostasis network tightly regulates this metabolic need of cancer cells on cholesterol and other lipids. Genetic landscape is also shared between apoptosis and cholesterol metabolism. MicroRNAs (miRNAs) are the new fine tuners of signaling pathways and cellular processes and are known for their ability to post-transcriptionally repress gene expression in a targeted manner. This review summarizes the current knowledge about the cross talk between apoptosis and cholesterol metabolism via miRNAs. In addition, we also emphasize herein recent therapeutic modulations of specific miRNAs and their promising potential for the treatment of deadly diseases including cancer and cholesterol related pathologies. Understanding of the impact of miRNA-based regulation of apoptosis and metabolic processes is still at its dawn and needs further research for the development of future miRNA-based therapies. As both these physiological processes affect cellular homeostasis, we believe that this comprehensive summary of miRNAs modulating both apoptosis and cholesterol metabolism will open uncharted territory for scientific exploration and will provide the foundation for discovering novel drug targets for cancer and metabolic diseases.

Prognostic significance of NADPH oxidase-4 as an indicator of reactive oxygen species stress in human retinoblastoma.

BACKGROUND: Reactive oxygen species (ROS) have been shown to enhance the proliferation of cancer cells. NADPH oxidases (NOX4) are a major intracellular source of ROS and are found to be associated with cancer and tumor cell invasion. Therefore, the purpose of this study is to evaluate the expression of NOX4 protein in human retinoblastoma. METHODS: Immunohistochemical expression of NOX4 protein was analyzed in 109 specimens from prospective cases of retinoblastoma and then correlated with clinicopathological parameters and patient survival. Western blotting confirmed and validated the immunoreactivity of NOX4 protein. RESULTS: In our study we found a male preponderance (55.9 %), and 25/109 (22.9 %) were bilateral. Massive choroidal invasion was the histopathological high-risk factor (HRF) most frequently observed, in 42.2 % of the cases. NOX4 protein was expressed in 67.88 % (74/109) of primary retinoblastoma cases and was confirmed by Western blotting. NOX4 was statistically significant with massive choroidal invasion and pathological TNM staging. There was a statistically significant difference in overall survival in patients with NOX4 expression (p = 0.0461). CONCLUSION: This is the first study to show the expression of NOX4 protein in retinoblastoma tumors. Hence, a retinoblastoma tumor may exhibit greater ROS stress. This protein may prove to be useful as a future therapeutic target for improving the management of retinoblastoma.

Gene expression profiling reveals the role of RIG1 like receptor signaling in p53 dependent apoptosis induced by PUVA in keratinocytes.

Photochemotherapy using 8-methoxypsoralen in combination with UVA radiation (PUVA) is an effective treatment for various skin dermatosis including psoriasis however its molecular mechanism is not clear. Previously we demonstrated that PUVA differentially regulates miRNA expression profile with a significant up-regulation of hsa-miR-4516. To study in detail the molecular mechanism of PUVA in keratinocytes, we investigated the genome wide transcriptomic changes using Illumina whole genome gene expression beadchip. Microarray analysis revealed 1932 differentially expressed gene and their Insilico analysis revealed Retinoic Acid Inducible Gene-I (RIG-1) signaling, apoptosis and p53 pathway to be associated with PUVA induced effects. We demonstrate that miR-4516 mediated down-regulation of UBE2N promotes p53 nuclear translocation and pro-apoptotic activity of PUVA is independent of IRF3 but is mediated by the RIG-I in a p53 and NFκB dependent manner. Additionally, PUVA inactivated the AKT/mTOR pathway in concert with inhibition of autophagy and suppressed cell migration. Taken together this study broadens our understanding about the mechanism of action of PUVA providing possible new strategy targeting proapoptotic function of RIG-1, a regulator of innate immune response or p53 for psoriasis therapy.

MicroRNA-195 inhibits proliferation, invasion and metastasis in breast cancer cells by targeting FASN, HMGCR, ACACA and CYP27B1.

De novo lipogenesis, a hallmark for cancers is required for cellular transformation. Further it is believed that resistance to apoptosis and epithelial-to-mesenchymal-transition(EMT) facilitates metastasis via over-expression of anti-apoptotic Bcl-2. Previously we demonstrated that hsa-miR-195 targets BCL2, induces apoptosis and augmented the effect of etoposide in breast cancer cells. However, the mechanism behind its function remains elusive. Herein gene expression profiling was done in presence/absence of hsa-miR-195 in Breast cancer cells. IPA revealed mitochondrial dysfunction, fatty acid metabolism and xenobiotic metabolism signalling among the top processes being affected. For the first time we herein identified ACACA, FASN (the key enzymes of de novo fatty acid synthesis), HMGCR (the key enzyme of de novo cholesterol synthesis) and CYP27B1 as direct targets of hsa-miR-195. We further showed that ectopic expression of hsa-miR-195 in MCF-7 and MDA-MB-231 cells not only altered cellular cholesterol and triglyceride levels significantly but also resulted in reduced proliferation, invasion and migration. We further demonstrated that over expression of hsa-miR-195 decreased the Mesenchymal markers expression and enhanced Epithelial markers. In conclusion we say that hsa-miR-195 targets the genes of de novo lipogenesis, inhibits cell proliferation, migration, and invasion which potentially opens new avenues for the treatment of breast cancer.

Moxifloxacin and ciprofloxacin induces S-phase arrest and augments apoptotic effects of cisplatin in human pancreatic cancer cells via ERK activation.

BACKGROUND: Pancreatic cancer, one of the most dreadful gastrointestinal tract malignancies, with the current chemotherapeutic drugs has posed a major impediment owing to poor prognosis and chemo-resistance thereby suggesting critical need for additional drugs as therapeutics in combating the situation. Fluoroquinolones have shown promising and significant anti-tumor effects on several carcinoma cell lines. METHODS: Previously, we reported growth inhibitory effects of fourth generation fluoroquinolone Gatifloxacin, while in the current study we have investigated the anti-proliferative and apoptosis-inducing mechanism of older generation fluoroquinolones Moxifloxacin and Ciprofloxacin on the pancreatic cancer cell-lines MIA PaCa-2 and Panc-1. Cytotoxicity was measured by MTT assay. Apoptosis induction was evaluated using annexin assay, cell cycle assay and activation of caspase-3, 8, 9 were measured by western blotting and enzyme activity assay. RESULTS: Herein, we found that both the fluoroquinolones suppressed the proliferation of pancreatic cancer cells by causing S-phase arrest and apoptosis. Blockade in S-phase of cell cycle was associated with decrease in the levels of p27, p21, CDK2, cyclin-A and cyclin-E. Herein we also observed triggering of extrinsic as well as intrinsic mitochondrial apoptotic pathway as suggested by the activation of caspase-8, 9, 3, and Bid respectively. All this was accompanied by downregulation of antiapoptotic protein Bcl-xL and upregulation of proapoptotic protein Bak. Our results strongly suggest the role of extracellular-signal-regulated kinases (ERK1/2), but not p53, p38 and c-JUN N-terminal kinase (JNK) in fluoroquinolone induced growth inhibitory effects in both the cell lines. Additionally, we also found both the fluoroquinolones to augment the apoptotic effects of broad spectrum anticancer drug Cisplatin via ERK. CONCLUSION: The fact that these fluoroquinolones synergize the effect of cisplatin opens new insight into therapeutic index in treatment of pancreatic cancer.

Prognostic significance of mitochondrial oxidative phosphorylation complexes: Therapeutic target in the treatment of retinoblastoma.

PURPOSE: Altered energy metabolism plays an important role in the development and progression of cancer. The objective of this study was to elucidate the role of mitochondrial oxidative phosphorylation complexes and their prognostic significance in retinoblastoma (Rb). METHODS: Immunohistochemistry was performed on 109 primary enucleated retinoblastoma tissues for mitochondrial OXPHOS complexes and their expression was confirmed by western blotting. RESULTS: Histopathological high risk factors (HRFs) were identified in 42.2% cases. Mitochondrial OXPHOS complexes III, IV and V were expressed in more than 50% of primary retinoblastoma cases each whereas mitochondrial complex I was expressed in only 29/109 (26.60%) cases by immunohistochemistry. Loss of mitochondrial complex I correlated well with poor tumor differentiation and tumor invasion (p < 0.05) whereas expression of mitochondrial complexes III, IV and V was associated with better survival (Kaplan-Meier method). CONCLUSIONS: This was the first study predicting a relevant role of mitochondrial OXPHOS complexes and highlights the prognostic significance with patient outcome in retinoblastoma. Loss of mitochondrial complex I immunoexpression could prove to be a useful independent prognostic biomarker to identify high risk retinoblastoma patients. Differential expression of these mitochondrial complexes is a novel finding and may be used as an attractive future anticancer target in primary retinoblastoma tumors. FINANCIAL DISCLOSURE: The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Expression of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins in human retinoblastoma.

BACKGROUND: Regulation of apoptosis is a complex process that involves a number of genes, including Bcl-2, Bcl-x, Bax and other Bcl-2 family members. The aim of the present study is to assess the expression of Bcl- 2 and Bax in retinoblastoma, and correlate them with clinical and histopathological parameters. METHODS: The expression of Bcl-2 and Bax proteins were examined using immunohistochemistry, Western blotting and reverse transcriptase-polymerase chain reaction in a series of 60 prospective cases of primary retinoblastoma tissues. RESULTS: Immunohistochemistry showed expression of Bcl-2 in 40/60 (66.6%), whereas Bax expression was found only in 18/60 (30%) cases, and these correlated with mRNA expression. The Western blotting results also correlated well with the immunohistochemical expression of Bcl-2 (25 kDa) and Bax (21 kDa) proteins. Bcl-2 was expressed in 96% (24/25) of invasive tumours and in 45.7% (16/35) of non-invasive tumours. Expression of Bcl-2 significantly correlated with tumour invasiveness (P = 0.0274) and poor differentiation (P = 0.0163), whereas loss of Bax correlated with massive choroidal invasion and Pathological Tumor-Node-Metastasis (pTNM) (P = 0.0341). However, no correlation was found between Bax and Bcl-2 expression. CONCLUSIONS: Our findings suggest that these apoptotic regulatory proteins may serve as poor prognostic markers and can be used as a therapeutic target for the treatment of invasive retinoblastoma. Further functional studies are required to explore the role of Bax and Bcl-2 in retinoblastoma.

STAT6 silencing up-regulates cholesterol synthesis via miR-197/FOXJ2 axis and induces ER stress-mediated apoptosis in lung cancer cells.

MiRNAs and transcription factors have emerged as important regulators for gene expression and are known to regulate various biological processes, including cell proliferation, differentiation and apoptosis. Previously, using genome-wide expression profiling studies, we have shown an inverse relationship of STAT6 and cholesterol biosynthesis and also identified FOXJ2 binding sites in the upstream region of 3 key genes (HMGCR, HMGCS1 and IDI1) of the cholesterol synthesis pathway. Our previous study also provided clues toward the anti-apoptotic role played by STAT6. For better understanding of the cellular response and underlying signaling pathways activated by STAT6 silencing, we examined the changes in miRNome profile after the siRNA-mediated silencing of STAT6 gene in NCI-H460 cells using LNA-based miRNA microarray. Our analysis showed significant downregulation of miRNAs, let-7b and miR-197, out of which miR-197 was predicted to target FOXJ2. We here show that miR-197 not only negatively regulates FOXJ2 expression through direct binding to its respective binding site in its 3'UTR but also alters total cholesterol levels by regulating genes associated with cholesterol biosynthesis pathway. We further demonstrated that STAT6 silencing elicited ER stress-mediated apoptosis in NCI-H460 cells through C/EBP homologous protein (CHOP) induction, alteration of BH3 only proteins expression and ROS production. The apoptosis induced by STAT6 downregulation was partially reversed by NAC, the ROS scavenger. Based on the above findings, we suggest that ER stress plays a major role in STAT6-induced apoptosis.

Activation of AKT/GSK3ß pathway by TDZD-8 attenuates kainic acid induced neurodegeneration but not seizures in mice.

Activation of glycogen synthase kinase3ß (GSK3ß), an enzyme that regulates a multitude of cellular signaling pathways, is implicated in neurodegenerative processes observed in an array of CNS diseases. We examined the hypothesis that the pathological changes in an acute kainic acid (KA) induced excitotoxicity model, relevant to human temporal lobe epilepsy (TLE), could be sensitive to inhibition of GSK3ß by 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) treatment in Swiss albino mice. Immediate seizure responses due to KA were recorded. Neurodegenerative and morphogenic changes were examined by western blot analysis and light microscopy, respectively, 48 h after KA administration. Although tonic-clonic seizure episodes evoked by KA were unaffected, TDZD-8 pretreatment decreased KA mediated elevation in caspase-3 cleavage as well as increased Bcl2 and phospho-GSK3ß (Ser9; pGSK3ß(Ser9)) expression. Likewise, microscopic examination also revealed that pretreatment with TDZD-8 attenuated cell damage elicited by KA in the CA1, CA3 and DG regions. In all the above parameters, the combined effect of a sub-effective dose of sodium valproate (SVP) with TDZD-8 was higher than that of solitary TDZD-8 treatment. The findings suggest that activated GSK3ß orchestrated neurodegenerative alterations following KA treatment and its inhibition by TDZD-8 affords a distinct neuroprotective profile by activating Akt/GSK3ß pathway which might act upstream of Bax/Bcl2 and caspase-3 pathways. Compounds targeting GSK3ß activity might represent a novel therapeutic option for exploration as an adjunct to conventional anti-epileptic drugs in preventing neurodegenerative processes in TLE.