Neuroprotective effects of Tacrolimus (FK-506) and Cyclosporin (CsA) in oxidative injury

The detrimental effects of hypoxic damage to central nervous system lead to energy depletion, free radical formation, lipid peroxidation (LPO), and increased calcium. We hypothesized that in vitro tacrolimus (FK-506) and cyclosporine A (CsA) could be protective against hypoxic damage in spinal cord. Dorsal columns were isolated from the spinal cord of adult rats and injured by exposure to hypoxic condition for 1 h, and treated with FK-506 (0.1 μM) and CsA (0.1 μM). After injury, reperfusion was carried out for 2 h. Tissues were collected, processed for biochemical assays, and 2,3,5-triphenyltetrazolium chloride (TTC) staining. Spinal cord hypoxia caused a significant decrease (P < 0.001) in mitochondrial ATP (30.64%) and tissue reduced glutathione (GSH) (60.14%) content. Conversely, a significant increase (P < 0.001) in tissue LPO level (57.77%) and myeloperoxidase (MPO) activity (461.24%) was observed in hypoxic group. Mitochondrial swelling was also significantly increased in hypoxic group (90.0%). Treatment with either FK-506 or CsA showed that significant neuroprotective effects (P < 0.05–0.01) were measured in various parameters in hypoxic groups. FK-506 and CsA treatment showed increase in ATP by 11.19% and 16.14% while GSH content increased by 66.46% and 77.32%, respectively. Conversely, LPO content decreased by 18.97% and 24.06% and MPO level by 42.86% and 18.66% after FK-506 and CsA treatment. Calcium uptake was also decreased in mitochondria as exhibited by the increase in absorbance by 11.19% after FK-506 treatment. TTC staining also showed increased viability after FK-506 and CsA treatment. In conclusion, present study demonstrates the neuroprotective effect of FK-506 and CsA treatment against spinal cord hypoxia induced damage is mediated via their antioxidant actions

Long Non-Coding RNA Profiling of Pediatric Medulloblastoma

BACKGROUND: Medulloblastoma (MB) is one of the most common malignant cancers in children. MB is primarily classified into four subgroups based on molecular and clinical characteristics as (1) WNT (2) Sonic-hedgehog (SHH) (3) Group 3 (4) Group 4. Molecular characteristics used for MB classification are based on genomic and mRNAs profiles. MB subgroups share genomic and mRNA profiles and require multiple molecular markers for differentiation from each other. Long non-coding RNAs (lncRNAs) are more than 200 nucleotide long RNAs and primarily involve in gene regulation at epigenetic and post-transcriptional levels. LncRNAs have been recognized as diagnostic and prognostic markers in several cancers. However, the lncRNA expression profile of MB is unknown. METHODS: We used the publicly available gene expression datasets for the profiling of lncRNA expression across MB subgroups. Functional analysis of differentially expressed lncRNAs was accomplished by Ingenuity pathway analysis (IPA). RESULTS: In the current study, we have identified and validated the lncRNA expression profile across pediatric MB subgroups and associated molecular pathways. We have also identified the prognostic significance of lncRNAs and unique lncRNAs associated with each MB subgroup. CONCLUSIONS: Identified lncRNAs can be used as single biomarkers for molecular identification of MB subgroups that warrant further investigation and functional validation

Improved Therapy for Medulloblastoma: Targeting Hedgehog and PI3K-mTOR Signaling Pathways in Combination with Chemotherapy

Aberrant activation and interactions of hedgehog (HH) and PI3K/AKT/mTOR signaling pathways are frequently associated with high-risk medulloblastoma (MB). Thus, combined targeting of the HH and PI3K/AKT/mTOR pathways could be a viable therapeutic strategy to treat high-risk patients. Therefore, we investigated the anti-MB efficacies of combined HH inhibitor Vismodegib and PI3K-mTOR dual-inhibitor BEZ235 together or combined individually with cisplatin against high-risk MB. Using non-MYC- and MYC-amplified cell lines, and a xenograft mouse model, the in vitro and in vivo efficacies of these therapies on cell growth/survival and associated molecular mechanism(s) were investigated. Results showed that combined treatment of Vismodegib and BEZ235 together, or with cisplatin, significantly decreased MB cell growth/survival in a dose-dependent-fashion. Corresponding changes in the expression of targeted molecules following therapy were observed. Results demonstrated that inhibitors not only suppressed MB cell growth/survival when combined, but also significantly enhanced cisplatin-mediated cytotoxicity. Of these combinations, BEZ235 exhibited a significantly greater efficacy in enhancing cisplatin-mediated MB cytotoxicity. Results also demonstrated that the MYC-amplified MB lines showed a higher sensitivity to combined therapies compared to non-MYC-amplified cell lines. Therefore, we tested the efficacy of combined approaches against MYC-amplified MB growing in NSG mice. In vivo results showed that combination of Vismodegib and BEZ235 or their combination with cisplatin, significantly delayed MB tumor growth and increased survival of xenografted mice by targeting HH and mTOR pathways. Thus, our studies lay a foundation for translating these combined therapeutic strategies to the clinical setting to determine their efficacies in high-risk MB patients

Exosomes Secreted Under Hypoxia Enhance Stemness in Ewing\u27s Sarcoma Through miR-210 Delivery

Intercellular communication between tumor cells within the hypoxic microenvironment promote aggressiveness and poor patient prognoses for reasons that remain unclear. Here we show that hypoxic Ewing\u27s sarcoma (EWS) cells release exosomes that promote sphere formation, a stem-like phenotype, in EWS cells by enhancing survival. Analysis of the hypoxic exosomal miRNA cargo identified a HIF-1α regulated miRNA, miR-210, as a potential mediator of sphere formation in cells exposed to hypoxic exosomes. Knockdown of HIF-1α in hypoxic EWS cells led to decreased exosomal miR-210 levels and reduced the capacity of hypoxic exosomes to form spheres. Inhibition of miR-210 in hypoxic spheres attenuated sphere formation and overexpression of miR-210 in normoxic spheres significantly enhanced the number of EWS spheres. Our results indicate that hypoxic exosomal miR-210 targets the proapoptotic protein CASP8AP2 in recipient cells. Moreover, the suppression of CASP8AP2 led to a reduction in apoptotic cells and increased sphere formation. Together, the findings in this study suggest that hypoxic exosomes promote stemness in EWS cells by delivering enriched miR-210 that is capable of down-regulating apoptotic pathways, resulting in the survival of cells with increased sphere formation. Future studies will further investigate the effects of EWS derived exosomal miRNAs on target genes and the role these interactions play in driving aggressiveness in hypoxic EWS tumors

Cardiac transcriptome profiling of diabetic Akita mice using microarray and next generation sequencing.

Although diabetes mellitus (DM) causes cardiomyopathy and exacerbates heart failure, the underlying molecular mechanisms for diabetic cardiomyopathy/heart failure are poorly understood. Insulin2 mutant (Ins2+/-) Akita is a mouse model of T1DM, which manifests cardiac dysfunction. However, molecular changes at cardiac transcriptome level that lead to cardiomyopathy remain unclear. To understand the molecular changes in the heart of diabetic Akita mice, we profiled cardiac transcriptome of Ins2+/- Akita and Ins2+/+ control mice using next generation sequencing (NGS) and microarray, and determined the implications of differentially expressed genes on various heart failure signaling pathways using Ingenuity pathway (IPA) analysis. First, we validated hyperglycemia, increased cardiac fibrosis, and cardiac dysfunction in twelve-week male diabetic Akita. Then, we analyzed the transcriptome levels in the heart. NGS analyses on Akita heart revealed 137 differentially expressed transcripts, where Bone Morphogenic Protein-10 (BMP10) was the most upregulated and hairy and enhancer of split-related (HELT) was the most downregulated gene. Moreover, twelve long non-coding RNAs (lncRNAs) were upregulated. The microarray analyses on Akita heart showed 351 differentially expressed transcripts, where vomeronasal-1 receptor-180 (Vmn1r180) was the most upregulated and WD Repeat Domain 83 Opposite Strand (WDR83OS) was the most downregulated gene. Further, miR-101c and H19 lncRNA were upregulated but Neat1 lncRNA was downregulated in Akita heart. Eleven common genes were upregulated in Akita heart in both NGS and microarray analyses. IPA analyses revealed the role of these differentially expressed genes in key signaling pathways involved in diabetic cardiomyopathy. Our results provide a platform to initiate focused future studies by targeting these genes and/or non-coding RNAs, which are differentially expressed in Akita hearts and are involved in diabetic cardiomyopathy

Differentially expressed long-non-coding RNAs (lncRNAs) in Akita heart.

<p>(A) NGS analyses showing different lncRNAs upregulated in Akita hearts. (B) Microarray analyses of lncRNAs in Akita hearts. Values are mean ± S. D. N = 3. (C) Representative Western blots and densitometric analyses of bands for Nebulin in WT and Akita hearts. Values are mean ± S. E. N = 5. (D) Representative Western blots and densitometric analyses of bands for GABA (A) receptor associated protein like-1 (GABARAPLK1) in WT and Akita hearts. Values are mean ± S. E. N = 5.</p

Transcriptome profiling of WT and Akita hearts using next generation sequencing (NGS) and microarray analyses.

<p>(A) Top-ten upregulated transcriptome in Akita by NGS analyses. (B) Top-ten downregulated transcriptome in Akita heart by NGS analyses. (C) Top-ten upregulated transcriptome in Akita heart by microarray analyses. (D) Top-ten downregulated transcriptome in Akita heart by microarray analyses. (E) Top-ten upregulated genes in Akita heart common in both NGS and microarray analyses (fold change ≤ 2 or 2 ≥ and <i>p-</i>value < 0.05). All the data are represented as fold change of WT hearts. Values are mean ± SD. N = 3. (F-H) Validation of three upregulated genes in Akita heart using an independent experiment and qPCR analyses. Values are represented as mean ± SEM. N = 3.</p

Schematic representation of workflow.

<p>Cardiac profiling of Akita mice using next generation sequencing (NGS) and microarray, and evaluating the implications of differentially expressed genes on signaling pathways, using Ingenuity Pathway Analyses (IPA).</p

Ingenuity Pathway Analyses (IPA) for key signaling network associated with cardiomyopathy/diabetic heart failure in Akita heart using differentially expressed genes from microarray analyses.

<p>(A-B) The differentially expressed genes in Akita heart obtained from microarray analyses are involved in key signaling networks associated with cardiomyopathy/heart failure.</p