Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylitta cytoplasmic polyhedrosis virus

<p>Abstract</p> <p>Background</p> <p><it>Antheraea mylitta </it>cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of <it>Reoviridae </it>family, infects Indian non-mulberry silkworm, <it>Antheraea mylitta</it>, and contains 11 segmented double stranded RNA (S1-S11) in its genome. Some of its genome segments (S2 and S6-S11) have been previously characterized but genome segments encoding viral capsid have not been characterized.</p> <p>Results</p> <p>In this study genome segments 1 (S1) and 3 (S3) of AmCPV were converted to cDNA, cloned and sequenced. S1 consisted of 3852 nucleotides, with one long ORF of 3735 nucleotides and could encode a protein of 1245 amino acids with molecular mass of ~141 kDa. Similarly, S3 consisted of 3784 nucleotides having a long ORF of 3630 nucleotides and could encode a protein of 1210 amino acids with molecular mass of ~137 kDa. BLAST analysis showed 20-22% homology of S1 and S3 sequence with spike and capsid proteins, respectively, of other closely related <it>cypoviruses </it>like <it>Bombyx mori </it>CPV (BmCPV), <it>Lymantria dispar </it>CPV (LdCPV), and <it>Dendrolimus punctatus </it>CPV (DpCPV). The ORFs of S1 and S3 were expressed as 141 kDa and 137 kDa insoluble His-tagged fusion proteins, respectively, in <it>Escherichia coli </it>M15 cells via pQE-30 vector, purified through Ni-NTA chromatography and polyclonal antibodies were raised. Immunoblot analysis of purified polyhedra, virion particles and virus infected mid-gut cells with the raised anti-p137 and anti-p141 antibodies showed specific immunoreactive bands and suggest that S1 and S3 may code for viral structural proteins. Expression of S1 and S3 ORFs in insect cells via baculovirus recombinants showed to produce viral like particles (VLPs) by transmission electron microscopy. Immunogold staining showed that S3 encoded proteins self assembled to form viral outer capsid and VLPs maintained their stability at different pH in presence of S1 encoded protein.</p> <p>Conclusion</p> <p>Our results of cloning, sequencing and functional analysis of AmCPV S1 and S3 indicate that S3 encoded viral structural proteins can self assemble to form viral outer capsid and S1 encoded protein remains associated with it as inner capsid to maintain the stability. Further studies will help to understand the molecular mechanism of capsid formation during cypovirus replication.</p

Increased TGFβ1 and SMAD3 Contribute to Age-Related Aortic Valve Calcification

AimsCalcific aortic valve disease (CAVD) is a progressive heart disease that is particularly prevalent in elderly patients. The current treatment of CAVD is surgical valve replacement, but this is not a permanent solution, and it is very challenging for elderly patients. Thus, a pharmacological intervention for CAVD may be beneficial. In this study, we intended to rescue aortic valve (AV) calcification through inhibition of TGFβ1 and SMAD3 signaling pathways.Methods and ResultsThe klotho gene, which was discovered as an aging-suppressor gene, has been observed to play a crucial role in AV calcification. The klotho knockout (Kl–/–) mice have shorter life span (8–12 weeks) and develop severe AV calcification. Here, we showed that increased TGFβ1 and TGFβ-dependent SMAD3 signaling were associated with AV calcification in Kl–/– mice. Next, we generated Tgfb1- and Smad3-haploinsufficient Kl–/– mice to determine the contribution of TGFβ1 and SMAD3 to the AV calcification in Kl–/– mice. The histological and morphometric evaluation suggested a significant reduction of AV calcification in Kl–/–; Tgfb1± mice compared to Kl–/– mice. Smad3 heterozygous deletion was observed to be more potent in reducing AV calcification in Kl–/– mice compared to the Kl–/–; Tgfb1± mice. We observed significant inhibition of Tgfb1, Pai1, Bmp2, Alk2, Spp1, and Runx2 mRNA expression in Kl–/–; Tgfb1± and Kl–/–; Smad3± mice compared to Kl–/– mice. Western blot analysis confirmed that the inhibition of TGFβ canonical and non-canonical signaling pathways were associated with the rescue of AV calcification of both Kl–/–; Tgfb1± and Kl–/–; Smad3± mice.ConclusionOverall, inhibition of the TGFβ1-dependent SMAD3 signaling pathway significantly blocks the development of AV calcification in Kl–/– mice. This information is useful in understanding the signaling mechanisms involved in CAVD

Broad targeting of resistance to apoptosis in cancer

Apoptosis or programmed cell death is natural way of removing aged cells from the body. Most of the anti-cancer therapies trigger apoptosis induction and related cell death networks to eliminate malignant cells. However, in cancer, de-regulated apoptotic signaling, particularly the activation of an anti-apoptotic systems, allows cancer cells to escape this program leading to uncontrolled proliferation resulting in tumor survival, therapeutic resistance and recurrence of cancer. This resistance is a complicated phenomenon that emanates from the interactions of various molecules and signaling pathways. In this comprehensive review we discuss the various factors contributing to apoptosis resistance in cancers. The key resistance targets that are discussed include (1) Bcl-2 and Mcl-1 proteins; (2) autophagy processes; (3) necrosis and necroptosis; (4) heat shock protein signaling; (5) the proteasome pathway; (6) epigenetic mechanisms; and (7) aberrant nuclear export signaling. The shortcomings of current therapeutic modalities are highlighted and a broad spectrum strategy using approaches including (a) gossypol; (b) epigallocatechin-3-gallate; (c) UMI-77 (d) triptolide and (e) selinexor that can be used to overcome cell death resistance is presented. This review provides a roadmap for the design of successful anti-cancer strategies that overcome resistance to apoptosis for better therapeutic outcome in patients with cancer

Designing a broad-spectrum integrative approach for cancer prevention and treatment

Targeted therapies and the consequent adoption of “personalized” oncology have achieved notable successes in some cancers; however, significant problems remain with this approach. Many targeted therapies are highly toxic, costs are extremely high, and most patients experience relapse after a few disease-free months. Relapses arise from genetic heterogeneity in tumors, which harbor therapy-resistant immortalized cells that have adopted alternate and compensatory pathways (i.e., pathways that are not reliant upon the same mechanisms as those which have been targeted). To address these limitations, an international task force of 180 scientists was assembled to explore the concept of a low-toxicity “broad-spectrum” therapeutic approach that could simultaneously target many key pathways and mechanisms. Using cancer hallmark phenotypes and the tumor microenvironment to account for the various aspects of relevant cancer biology, interdisciplinary teams reviewed each hallmark area and nominated a wide range of high-priority targets (74 in total) that could be modified to improve patient outcomes. For these targets, corresponding low-toxicity therapeutic approaches were then suggested; many of which were phytochemicals. Proposed actions on each target and all of the approaches were further reviewed for known effects on other hallmark areas and the tumor microenvironment. Potential contrary or procarcinogenic effects were found for 3.9% of the relationships between targets and hallmarks, and mixed evidence of complementary and contrary relationships was found for 7.1%. Approximately 67% of the relationships revealed potentially complementary effects, and the remainder had no known relationship. Among the approaches, 1.1% had contrary, 2.8% had mixed and 62.1% had complementary relationships. These results suggest that a broad-spectrum approach should be feasible from a safety standpoint. This novel approach has potential to help us address disease relapse, which is a substantial and longstanding problem, so a proposed agenda for future research is offered

Sequential hTERT knockdown and apigenin treatment inhibited invasion and proliferation and induced apoptosis in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cells. Journal of molecular neuroscience : MN

Abstract Human telomerase reverse transcriptase (hTERT) plays a key role in conferring immortality to human malignant neuroblastomas. We first determined differential expression of hTERT in four human malignant neuroblastoma SH-SY5Y, SK-N-DZ, SK-N-BE2, and IMR-32 cell lines. We then used SK-N-DZ and SK-N-BE2 cell lines, which showed the highest expression of hTERT, to investigate the therapeutic effects of sequential hTERT knockdown and apigenin (APG) treatment. We performed cell invasion assay and studied alterations in expression of matrix metalloproteinases and cell cycle regulatory molecules after this combination therapy. We also investigated induction of apoptosis by using in situ Wright staining, Annexin V staining, and Western blotting. Sequential hTERT knockdown and APG treatment significantly downregulated expression of hTERT so as to cause over 90 % inhibition of cell invasion and 70 % induction of apoptosis in both SK-N-DZ and SK-N-BE2 cell lines. Western blotting demonstrated downregulation of the molecules involved in cell invasion and proliferation, but upregulation of the cell cycle inhibitor and apoptosis-inducing molecules. In conclusion, our current results clearly showed that sequential hTERT knockdown and APG treatment could be a promising therapeutic strategy for effective inhibition of invasion and proliferation and induction of apoptosis in hTERT overexpressing malignant neuroblastoma cells

Sequential hTERT knockdown and apigenin treatment inhibited invasion and proliferation and induced apoptosis in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cells. Journal of molecular neuroscience : MN

Abstract Human telomerase reverse transcriptase (hTERT) plays a key role in conferring immortality to human malignant neuroblastomas. We first determined differential expression of hTERT in four human malignant neuroblastoma SH-SY5Y, SK-N-DZ, SK-N-BE2, and IMR-32 cell lines. We then used SK-N-DZ and SK-N-BE2 cell lines, which showed the highest expression of hTERT, to investigate the therapeutic effects of sequential hTERT knockdown and apigenin (APG) treatment. We performed cell invasion assay and studied alterations in expression of matrix metalloproteinases and cell cycle regulatory molecules after this combination therapy. We also investigated induction of apoptosis by using in situ Wright staining, Annexin V staining, and Western blotting. Sequential hTERT knockdown and APG treatment significantly downregulated expression of hTERT so as to cause over 90 % inhibition of cell invasion and 70 % induction of apoptosis in both SK-N-DZ and SK-N-BE2 cell lines. Western blotting demonstrated downregulation of the molecules involved in cell invasion and proliferation, but upregulation of the cell cycle inhibitor and apoptosis-inducing molecules. In conclusion, our current results clearly showed that sequential hTERT knockdown and APG treatment could be a promising therapeutic strategy for effective inhibition of invasion and proliferation and induction of apoptosis in hTERT overexpressing malignant neuroblastoma cells

miR-30e Blocks Autophagy and Acts Synergistically with Proanthocyanidin for Inhibition of AVEN and BIRC6 to Increase Apoptosis in Glioblastoma Stem Cells and Glioblastoma SNB19 Cells.

Glioblastoma is the most common and malignant brain tumor in humans. It is a heterogeneous tumor harboring glioblastoma stem cells (GSC) and other glioblastoma cells that survive and sustain tumor growth in a hypoxic environment via induction of autophagy and resistance to apoptosis. So, a therapeutic strategy to inhibit autophagy and promote apoptosis could greatly help control growth of glioblastoma. We created hypoxia using sodium sulfite (SS) for induction of substantiated autophagy in human GSC and glioblastoma SNB19 cells. Induction of autophagy was confirmed by acridine orange (AO) staining and significant increase in Beclin-1 in autophagic cells. microRNA database (miRDB) search suggested that miR-30e could suppress the autophagy marker Beclin-1 and also inhibit the caspase activation inhibitors (AVEN and BIRC6). Pro-apoptotic effect of proanthocyanidin (PAC) has not yet been explored in glioblastoma cells. Combination of 50 nM miR-30e and 150 μM PAC acted synergistically for inhibition of viability in both cells. This combination therapy most effectively altered expression of molecules for inhibition of autophagy and induced extrinsic and intrinsic pathways of apoptosis through suppression of AVEN and BIRC6. Collectively, combination of miR-30e and PAC is a promising therapeutic strategy to inhibit autophagy and increase apoptosis in GSC and SNB19 cells

Combination of LC3 shRNA plasmid transfection and genistein treatment inhibited autophagy and increased apoptosis in malignant neuroblastoma in cell culture and animal models.

Malignant neuroblastoma is an extracranial solid tumor that usually occurs in children. Autophagy, which is a survival mechanism in many solid tumors including malignant neuroblastoma, deters the efficacy of conventional chemotherapeutic agents. To mimic starvation, we used 200 nM rapamycin that induced autophagy in human malignant neuroblastoma SK-N-BE2 and IMR-32 cells in cell culture and animal models. Combination of microtubule associated protein light chain 3 short hairpin RNA (LC3 shRNA) plasmid transfection and genistein (GST) treatment was tested for inhibiting rapamycin-induced autophagy and promoting apoptosis. The best synergistic efficacy caused the highest decrease in cell viability due to combination of 50 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated SK-N-BE2 cells while combination of 100 nM LC3 shRNA plasmid transfection and 25 µM GST treatment in rapamycin-treated IMR-32 cells. Quantitation of acidic vesicular organelles confirmed that combination of LC3 shRNA plasmid transfection and GST treatment prevented rapamycin-induced autophagy due to down regulation of autophagy promoting marker molecules (LC3 II, Beclin 1, TLR-4, and Myd88) and upregulation of autophagy inhibiting marker molecules (p62 and mTOR) in both cell lines. Apoptosis assays showed that combination therapy most effectively activated mitochondrial pathway of apoptosis in human malignant neuroblastoma in cell culture and animal models. Collectively, our current combination of LC3 shRNA plasmid transfection and GST treatment could serve as a promising therapeutic strategy for inhibiting autophagy and increasing apoptosis in human malignant neuroblastoma in cell culture and animal models

Real-time qRT-PCR analyses of miR-99a expression in U87MG and U118MG cells after photofrin based PDT and miR transfection.

<p>Cells were seeded, incubated, treated with photofrin, and irradiated with 670 nm light dose of 1 J/cm². After 4 h incubation, cells were transfected with 50 nM anti-miR-99a mimic or miR-99a mimic and incubated for another 24 h. Total RNA was extracted and cDNA was synthesized using gene specific primers, and real-time qRT-PCR analysis was performed for relative expression of miR-99a after normalizing with expression of U6 RNA (control) in glioblastoma U87MG and U118MG cells. Significant difference between untreated control (CTL) and photofrin based PDT or miR-99a transfection was indicated by *<i>P</i><0.05 or **<i>P</i><0.01. Significant difference between a single therapy and combination therapy was indicated by ^#<i>P</i><0.05.</p

Alkaline comet assay and agarose gel electrophoresis to examine DNA fragmentation patterns in U87MG and U118MG cells after photofrin based PDT.

<p>(a) Photomicrographs showing the DNA fragmentation patterns in U87MG cells. Two U87MG cells, one control (CTL) and another cell with highly damaged DNA due to photofrin based PDT, are shown in alkaline comet assay. Also, cells were treated with 50 µg/ml photofrin and irradiated with 670 nm light (1 J/cm²) and incubated for 3 h before isolation of total genomic DNA for DNA laddering assay. The CTL showed intact DNA whereas DNA ladder appeared due to photofrin based PDT. (b) Photomicrographs showing the DNA fragmentation patterns in U118MG cells. Two U118MG cells, one CTL and another cell with highly damaged DNA due to photofrin based PDT, are shown in alkaline comet assay. Also, cells were treated with 50 µg/ml photofrin and irradiated with 670 nm light (1 J/cm²) and incubated for 3 h before isolation of total genomic DNA for DNA laddering assay. The CTL showed intact DNA whereas DNA ladder appeared due to photofrin based PDT.</p