Current and emerging biomarkers in tumors of the central nervous system: Possible diagnostic, prognostic and therapeutic applications.

Recent investments in research associated with the discovery of specific tumor biomarkers important for efficient diagnosis and prognosis are beginning to bear fruit. Key biomarkers could potentially outweigh traditional radiological or pathological methods by enabling specificity of early detection, when coupled with tumor molecular profiling and clinical associations. Only few biomarkers are approved by regulatory authorities for Central Nervous System Tumors (CNSTs), despite the evaluation of a large number of CNST related markers during clinical trials. Traditional CNSTs biomarkers include 1p/19q co-deletion, O6-Methylguanine-DNA Methyltransferase Methylation, and mutations in IDH1/IDH2. Recently tested CNSTs biomarkers include VEGFR-2, EGFRvIII, IL2, PDGFR, MMPs, BRAF, STAT3, PTEN, TERT, AKT, NF2, and BCL2. Additional studies have highlighted new and novel MicroRNAs, circular RNAs and long non-coding RNAs as promising biomarkers. Studies on microvesicles pinpoint exosomes as promising, less invasive biomarkers that could be isolated from the serum of cancer patients. Furthermore, Cancer Stem Cells (CSCs) related molecules, such as CD133, SOX2 and Nestin, utilized as CNST biomarkers, might enable efficient monitoring of cancer progression, and/or surveillance of emerging drug resistant cells. Approved protocols that implement novel molecular markers in diagnostics, prognostics and drug development will herald a new era of precision and personalized neuro-oncology. This review summarizes and discusses putative CNST biomarkers that are under clinical development, and are ready to move into diagnostic, prognostic and therapeutic applications. Data presented here is predicted to aid in streamlining the process of biomarker's research and development.

Rhazyaminine from Rhazya stricta Inhibits Metastasis and Induces Apoptosis by Downregulating Bcl-2 Gene in MCF7 Cell Line.

BACKGROUND: The role of alkaloids isolated from Rhazya stricta Decne (Apocynaceae family) (RS) in targeting genes involved in cancer and metastasis remains to be elucidated. OBJECTIVE: Identify and characterize new compounds from RS, which inhibit gene(s) involved in the survival, invasion, self-renewal, and metastatic processes of cancer cells. METHODS: Bioinformatics study was performed using HISAT2, stringtie, and ballgown pipeline to understand expressional differences between a normal epithelial cell line-MCF10A and MCF7. NMR and ATR-FTIR were performed to elucidate the structure of rhazyaminine (R.A), isolated from R stricta. Cell viability assay was performed using 0, 25, and 50 µg/mL of total extract of R stricta (TERS) and R.A, respectively, for 0, 24, and 48 hours, followed by scratch assay. In addition, total RNA was isolated for RNA- seq analysis of MCF7 cell line treated with R.A followed by qRT-PCR analysis of Bcl-2 gene. RESULTS: Deptor, which is upregulated in MCF7 compared with MCF10A as found in our bioinformatics study was downregulated by R.A. Furthermore, R.A effectively reduced cell viability to around 50% ( P < .05) and restricted cell migration in scratch assay. Thirteen genes, related to metastasis and cancer stem cells, were downregulated by R.A according to RNA- seq analysis. Additionally, qRT-PCR validated the downregulation of Bcl-2 gene in R.A-treated cells by less than 0.5 folds ( P < .05). CONCLUSION: R.A successfully downregulated key genes involved in apoptosis, cell survival, epithelial-mesenchymal transition, cancer stem cell proliferation, and Wnt signal transduction pathway making it an excellent "lead candidate" molecule for in vivo proof-of-concept studies.

Combination of dipeptidylpeptidase IV inhibitor and low dose thiazolidinedione: preclinical efficacy and safety in db/db mice.

Thiazolidinediones (TZDs) are currently the most efficacious class of oral antidiabetics. However, they carry the burden of weight gain and haemodilution, which may lead to cardiovascular complications. The present study was designed to ascertain whether a combination of dipeptidyl peptidase IV (DPP IV) inhibitor with low dose of a thiazolidinedione absolves TZD associated weight gain and oedema without compromising its efficacy. In this study, we examined the efficacy and safety of lower dose (1 mg/kg/day) of rosiglitazone, a thiazolidinedione, in combination with 5 mg/kg/day dose of LAF-237 (vildagliptin), a known DPP IV inhibitor, in aged db/db mice after 14 days of treatment and compared the combination with therapeutic dose (10 mg/kg) of rosiglitazone. The combination therapy showed similar efficacy as that of 10 mg/kg/day rosiglitazone in lowering random blood glucose (53.8%, p<0.001 and 54.3%, p<0.001 respectively), AUC ((0-120) min) during oral glucose tolerance test (OGTT) (38.6 %, p<0.01; 38.3%, p<0.01 respectively) and triglyceride levels (63.9% and 61% respectively; p<0.01). Plasma active glucagon like peptide-1 (GLP-1) and insulin levels were found to be elevated significantly (p<0.01 and p<0.05 respectively) in both LAF-237 and combination treated groups following oral glucose load. LAF-237 alone had no effect on random glucose and glucose excursion during OGTT in severely diabetic db/db mice. Interestingly, the combination treatment showed no significant increase in body weight as compared to the robust weight gain by therapeutic dose of rosiglitazone. Rosiglitazone at 10 mg/kg/day showed significant reduction (p<0.05) in haematocrit, RBC count, haemoglobin pointing towards haemodilution associated with increased mRNA expression of Na(+), K(+)-ATPase-alpha and epithelial sodium channel gamma (ENaCgamma) in kidney. The combination therapy escaped these adverse effects. The results suggest that combination of DPP IV inhibitor with low dose of thiazolidinedione can interact synergistically to represent a therapeutic advantage for the clinical treatment of type 2 diabetes without the adverse effects of haemodilution and weight gain associated with thiazolidinediones.

Enhanced soluble production of biologically active recombinant human p38 mitogen-activated-protein kinase (MAPK) in Escherichia coli.

The conditions were optimized for maximum soluble yield of biologically active recombinant p38alpha mitogen activated protein kinase (MAPK) vis-à-vis insoluble fraction (inclusion body formation). This study reports a rapid, economical and single step purification process for the overproduction of GST tagged p38alpha MAPK. A yield of 18 mg of highly purified and soluble protein per liter of bacterial culture within 6 h timeframe was achieved. The purified protein was found to be biologically suitable for phosphorylation by upstream kinases and was catalytically active. We further demonstrated that our in-house p38alpha MAPK is more potent (>30%) than a commercially available enzyme.

Targeting signal transduction pathways of cancer stem cells for therapeutic opportunities of metastasis.

Tumor comprises of heterogeneous population of cells where not all the disseminated cancer cells have the prerogative and "in-build genetic cues" to form secondary tumors. Cells with stem like properties complemented by key signaling molecules clearly have shown to exhibit selective growth advantage to form tumors at distant metastatic sites. Thus, defining the role of cancer stem cells (CSC) in tumorigenesis and metastasis is emerging as a major thrust area for therapeutic intervention. Precise relationship and regulatory mechanisms operating in various signal transduction pathways during cancer dissemination, extravasation and angiogenesis still remain largely enigmatic. How the crosstalk amongst circulating tumor cells (CTC), epithelial mesenchymal transition (EMT) process and CSC is coordinated for initiating the metastasis at secondary tissues, and during cancer relapse could be of great therapeutic interest. The signal transduction mechanisms facilitating the dissemination, infiltration of CSC into blood stream, extravasations, progression of metastasis phenotype and angiogenesis, at distant organs, are the key pathologically important vulnerabilities being elucidated. Therefore, current new drug discovery focus has shifted towards finding "key driver genes" operating in parallel signaling pathways, during quiescence, survival and maintenance of stemness in CSC. Understanding these mechanisms could open new horizons for tackling the issue of cancer recurrence and metastasis-the cause of ~90% cancer associated mortality. To design futuristic & targeted therapies, we propose a multi-pronged strategy involving small molecules, RNA interference, vaccines, antibodies and other biotechnological modalities against CSC and the metastatic signal transduction cascade.

Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System.

Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan 5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system.

Optimization of culture parameters and novel strategies to improve protein solubility.

The production of recombinant proteins, in soluble form in a prokaryotic expression system, still remains a challenge for the biotechnologist. Innovative strategies have been developed to improve protein solubility in various protein overexpressing hosts. In this chapter, we would focus on methods currently available and amenable to "desired modifications," such as (a) the use of molecular chaperones; (b) the optimization of culture conditions; (c) the reengineering of a variety of host strains and vectors with affinity tags; and (d) optimal promoter strengths. All these parameters are evaluated with the primary objective of increasing the solubilization of recombinant protein(s) during overexpression in Escherichia coli.

Cancer stem cells: a challenging paradigm for designing targeted drug therapies.

Despite earlier controversies about their role and existence within tumors, cancer stem cells (CSCs) are now emerging as a plausible target for new drug discovery. Research and development (R&D) efforts are being directed against key gene(s) driving initiation, growth, and metastatic pathways in CSCs and the tumor microenvironment (TME). However, the niche signals that enable these pluripotent CSCs to evade radio- and chemotherapy, and to travel to secondary tissues remain enigmatic. Small-molecule drugs, biologics, miRNA, RNA interference (RNAi), and vaccines, among others, are under active investigation. Here, we examine the feasibility of leveraging current knowhow of the molecular biology of CSCs and their cellular milieu to design futuristic, targeted drugs with potentially lower toxicity that can override the multiple drug-resistance issues currently observed with existing therapeutics.

Production of Biopharmaceuticals in E. coli: Current Scenario and Future Perspectives.

Escherichia coli is the most preferred microorganism to express heterologous proteins for therapeutic use, as around 30% of the approved therapeutic proteins are currently being produced using it as a host. Owing to its rapid growth, high yield of the product, cost-effectiveness, and easy scale-up process, E. coli is an expression host of choice in the biotechnology industry for large-scale production of proteins, particularly non-glycosylated proteins, for therapeutic use. The availability of various E. coli expression vectors and strains, relatively easy protein folding mechanisms, and bioprocess technologies, makes it very attractive for industrial applications. However, the codon usage in E. coli and the absence of post-translational modifications, such as glycosylation, phosphorylation, and proteolytic processing, limit its use for the production of slightly complex recombinant biopharmaceuticals. Several new technological advancements in the E. coli expression system to meet the biotechnology industry requirements have been made, such as novel engineered strains, genetically modifying E. coli to possess capability to glycosylate heterologous proteins and express complex proteins, including full-length glycosylated antibodies. This review summarizes the recent advancements that may further expand the use of the E. coli expression system to produce more complex and also glycosylated proteins for therapeutic use in the future.

5-lipoxygenase: a promising drug target against inflammatory diseases-biochemical and pharmacological regulation.

5-Lipoxygenase (5-LO) is the key enzyme involved in the synthesis of pro-inflammatory leukotrienes (LTs) and has become a prime target for new drug discovery research and development efforts by the pharmaceutical and biotech industry. The pathophysiological effects of LTs can be modulated by the selective inhibition of 5-LO. In this review, we summarize the established dogma and recent progress on the biochemical and pharmacological regulation of 5-LO and its diverse cellular partners. In the last decade, significant research efforts have led to the exploitation of 5-LO pathway for developing new drugs against inflammatory diseases. Despite few setbacks, a number of promising molecules have moved into clinical development. These fundamental discoveries and proof-of-concept studies will ultimately be helpful in delineating how 5-LO pathway participates in the development of disease phenotype and what are possible key biomarkers of disease progression and regression. Elucidation of molecular mechanism-of-action of 5-LO in individual cell types will pave the way for improving efficacy parameters. Taken together, this combined knowledge about the 5-LO pathway would be helpful in planning collaborative and targeted R&D efforts, by the academic laboratories and pharmaceutical/ biotech industry, for the discovery and development of novel, efficacious and safer drugs against multiple diseases.

Role of toxicogenomics in the development of safe, efficacious and novel anti-microbial therapies.

Over the last two decades, occurrence of bacterial resistance to commonly used antibiotics has necessitated the development of safer and more potent anti-microbial drugs. However, the development of novel antibiotics is severely hampered by adverse side effects, such as drug-induced liver toxicity. Several antibacterial drugs are known to have the potential to cause severe liver damage. The major challenge in developing novel anti-microbial drugs is to predict, with certain amount of probability, the drug-induced toxicity during the pre-clinical stages, thus optimizing and reducing the time and cost of drug development. Toxicogenomics approach is generally used to harness the potential of genomic tools and to understand the physiological basis of drug-induced toxicity based on the in-depth analysis of Metagenomic data sets, i.e., transcriptional, translational or metabolomic profiles. Toxicogenomics, therefore, represents a new paradigm in the drug development process, and is anticipated to play an invaluable role in future to develop safe and efficacious medicines, by predicting the toxic potential of a new chemical entity (NCE) in early stages of drug discovery. This review examines the toxicogenomic approach in predicting the safety/toxicity of novel anti-microbial drugs, and analyses the promises, pitfalls and challenges of applying this powerful technology to the drug development process.