Viburnum nervosum Leaf Extract Mediated Green Synthesis of Silver Nanoparticles: A Viable Approach to Increase the Efficacy of an Anticancer Drug.

BACKGROUND: There is an urgent need to devise improved alternatives for the efficient delivery of drugs to develop improved therapeutic interventions against cancers. Nanotechnology-based drug delivery vehicles are in-use with obvious issues of toxicity and bio-distribution. Therefore, green synthetic routes are being deployed to replace the conventional nanoparticle formulations for effective drug delivery aiming at developing interventional strategies against cancer. OBJECTIVE: A simple, viable, and fast approach was used for the green synthesis of silver nanoparticles (AgNPs) using aqueous leaf-extract of Viburnum nervosum (VN) and to explore the anti-cancer potential of the crude extract of VN. METHODS: Silver NPs were synthesized by reacting silver nitrate (AgNO3) with leaf extract of VN. Various analytical techniques were used to characterize the AgNPs. Finally, the anti-cancer potential of VN was observed when delivered through AgNPs. RESULTS: The surface plasmon spectra for AgNPs exhibited absorbance peak at 445 nm, and Fourier-Transform Infrared Spectroscopy investigation revealed the presence of biomolecules acting as an effective reducing and capping agent for converting silver nitrate to AgNPs. Further, our results suggest the spherical size of synthesized AgNPs ranging from 12-17 nm. Moreover, in vitro studies conducted for VN extract with breast (MCF-7) and epidermal carcinoma (A431) cells showed biocompatibility. CONCLUSION: Doxorubicin loaded AgNPs documented an increased bioavailability of the drug compared to the free drug, suggesting the use of AgNPs as "novel drug delivery vectors".

Molecular Docking Based Analysis to Elucidate the DNA Topoisomerase IIß as the Potential Target for the Ganoderic Acid; A Natural Therapeutic Agent in Cancer Therapy.

INTRODUCTION: Intermediate covalent complex of DNA-Topoisomerase II enzyme is the most promising target of the anticancer drugs to induce apoptosis in cancer cells. Currently, anticancer drug and chemotherapy are facing major challenges i.e., drug resistance, chemical instability and, dose-limiting side effect. Therefore, in this study, natural therapeutic agents (series of Ganoderic acids) were used for the molecular docking simulation against Human DNATopoisomerase II beta complex (PDB ID:3QX3). METHODS: Molecular docking studies were performed on a 50 series of ganoderic acids reported in the NCBI-PubChem database and FDA approved anti-cancer drugs, to find out binding energy, an interacting residue at the active site of Human DNA-Topoisomerase II beta and compare with the molecular arrangements of the interacting residue of etoposide with the Human DNA topoisomerase II beta. The autodock 4.2 was used for the molecular docking and pharmacokinetic and toxicity studies were performed for the analysis of physicochemical properties and to check the toxicity effects. Discovery studio software was used for the visualization and analysis of docked pose. RESULTS AND CONCLUSION: Ganoderic acids (GS-1, A and DM) were found to be a more suitable competitor inhibitor among the ganoderic acid series with appropriate binding energy, pharmacokinetic profile and no toxicity effects. The interacting residue (Met782, DC-8, DC-11 and DA-12) shared a chemical resemblance with the interacting residue of etoposide present at the active site of human topoisomerase II beta receptor.

RNA-loaded dendritic cells: more than a tour de force in cancer therapeutics.

A wide array of therapeutic strategies has been implemented against cancers, yet their clinical benefit is limited. The lack of clinical efficacy of the conventional treatment options might be due to the inept immune competency of the patients. Dendritic cells (DCs) have a vital role in initiating and directing immune responses and have been frequently used as delivery vehicles in clinical research. The recent clinical data suggest the potential use of DCs pulsed with nucleic acid, especially with RNA holds a great potential as an immunotherapeutic measure with compare to other cancer therapeutics. This review mainly deals with the DCs and their role in transfection with RNA in cancer immunotherapy.

Chickpea Lectin Inhibits Human Breast Cancer Cell Proliferation and Induces Apoptosis Through Cell Cycle Arrest.

BACKGROUND: Breast cancer demands safe adjuvant to overcome the side effects of standard drug tamoxifen. Diet derived bioactive compounds are reported to exhibit modulation of cancer growth leading to cell death. Chickpea is a protein rich edible legume with several bioactive compounds that includes lectin as well. Characterization of chickpea lectin for its effect against cancer cells has been investigated in this study. METHOD: Cicer arietinum L. lectin (CAL) agglutinating trypsin-treated rabbit blood cells was purified employing DEAE-cellulose and SP-sephadex ion exchange chromatography. The lectin was characterized for its biological activity vis-à-vis antiproliferative and apoptotic effects through cell cycle arrest in MCF-7 human breast cancer cells. RESULT: There is a significant inhibition of the survival of breast cancer cells due to chickpea lectin in a dose dependent manner for 24 hr. Lectin treated cells revealed distinct features of apoptosis. Flow cytometric analysis at 80 µg/ml of lectin induced S and G2 phase cell cycle arrest. CAL induced apoptosis in MCF-7 cells associated with lactate dehydrogenase leakage, cell cycle arrest and reactive oxygen species generation. CONCLUSION: Our studies show that chickpea lectin exerted anticancer activity and could be exploited as an essential source for medicine leading to the treatment of breast cancer.

Potential Compounds for Oral Cancer Treatment: Resveratrol, Nimbolide, Lovastatin, Bortezomib, Vorinostat, Berberine, Pterostilbene, Deguelin, Andrographolide, and Colchicine.

Oral cancer is one of the main causes of cancer-related deaths in South-Asian countries. There are very limited treatment options available for oral cancer. Research endeavors focused on discovery and development of novel therapies for oral cancer, is necessary to control the ever rising oral cancer related mortalities. We mined the large pool of compounds from the publicly available compound databases, to identify potential therapeutic compounds for oral cancer. Over 84 million compounds were screened for the possible anti-cancer activity by custom build SVM classifier. The molecular targets of the predicted anti-cancer compounds were mined from reliable sources like experimental bioassays studies associated with the compound, and from protein-compound interaction databases. Therapeutic compounds from DrugBank, and a list of natural anti-cancer compounds derived from literature mining of published studies, were used for building partial least squares regression model. The regression model thus built, was used for the estimation of oral cancer specific weights based on the molecular targets. These weights were used to compute scores for screening the predicted anti-cancer compounds for their potential to treat oral cancer. The list of potential compounds was annotated with corresponding physicochemical properties, cancer specific bioactivity evidences, and literature evidences. In all, 288 compounds with the potential to treat oral cancer were identified in the current study. The majority of the compounds in this list are natural products, which are well-tolerated and have minimal side-effects compared to the synthetic counterparts. Some of the potential therapeutic compounds identified in the current study are resveratrol, nimbolide, lovastatin, bortezomib, vorinostat, berberine, pterostilbene, deguelin, andrographolide, and colchicine.

Potential therapeutic targets for oral cancer: ADM, TP53, EGFR, LYN, CTLA4, SKIL, CTGF, CD70.

In India, oral cancer has consistently ranked among top three causes of cancer-related deaths, and it has emerged as a top cause for the cancer-related deaths among men. Lack of effective therapeutic options is one of the main challenges in clinical management of oral cancer patients. We interrogated large pool of samples from oral cancer gene expression studies to identify potential therapeutic targets that are involved in multiple cancer hallmark events. Therapeutic strategies directed towards such targets can be expected to effectively control cancer cells. Datasets from different gene expression studies were integrated by removing batch-effects and was used for downstream analyses, including differential expression analysis. Dependency network analysis was done to identify genes that undergo marked topological changes in oral cancer samples when compared with control samples. Causal reasoning analysis was carried out to identify significant hypotheses, which can explain gene expression profiles observed in oral cancer samples. Text-mining based approach was used to detect cancer hallmarks associated with genes significantly expressed in oral cancer. In all, 2365 genes were detected to be differentially expressed genes, which includes some of the highly differentially expressed genes like matrix metalloproteinases (MMP-1/3/10/13), chemokine (C-X-C motif) ligands (IL8, CXCL-10/-11), PTHLH, SERPINE1, NELL2, S100A7A, MAL, CRNN, TGM3, CLCA4, keratins (KRT-3/4/13/76/78), SERPINB11 and serine peptidase inhibitors (SPINK-5/7). XIST, TCEAL2, NRAS and FGFR2 are some of the important genes detected by dependency and causal network analysis. Literature mining analysis annotated 1014 genes, out of which 841 genes were statistically significantly annotated. The integration of output of various analyses, resulted in the list of potential therapeutic targets for oral cancer, which included targets such as ADM, TP53, EGFR, LYN, CTLA4, SKIL, CTGF and CD70.

Oncoapoptotic markers in oral cancer: prognostics and therapeutic perspective.

Oral cancer is one of the most commonly found cancers in many South-Asian underdeveloped countries, especially among men in comparison to women. When considering the mortality rate among all types of existing cancers, in India oral cancer is the primary reason for death in men. Some of the major reasons contributing to the high mortality rate are late diagnosis, lack of treatment options and high prevalence of tobacco consumption. Oral cancer is often diagnosed at a stage when cancer cells have already become aggressive and become resistant to standard therapeutic options. Progression, apoptosis, angiogenesis, metastasis and invasion behold great capability to treat and detect cancer at the molecular level. Dysregulation of apoptosis is one of the most common molecular events known to be associated with the development of oral cancer. In this review, we discuss key apoptotic markers which can be used as prognostic and/or therapeutic targets in oral cancer.

Oncoapoptotic signaling and deregulated target genes in cancers: special reference to oral cancer.

Cancer is a class of diseases characterized by uncontrolled cell growth. The development of cancer takes place in a multi-step process during which cells acquire a series of mutations that eventually lead to unrestrained cell growth and division, inhibition of cell differentiation, and evasion of cell death. Dysregulation of oncoapoptotic genes, growth factors, receptors and their downstream signaling pathway components represent a central driving force in tumor development. The detailed studies of signal transduction pathways for mechanisms of cell growth and apoptosis have significantly advanced our understanding of human cancers, subsequently leading to more effective treatments. Oral squamous cell carcinoma represents a classic example of multi-stage carcinogenesis. It gradually evolves through transitional precursor lesions from normal epithelium to a full-blown metastatic phenotype. Genetic alterations in many genes encoding crucial proteins, which regulate cell proliferation, differentiation, survival and apoptosis, have been implicated in oral cancer. As like other solid tumors, in oral cancer these genes include the ones coding for cell cycle regulators or oncoproteins (e.g. Ras, Myc, cyclins, CDKs, and CKIs), tumor suppressors (e.g. p53 and pRb), pro-survival proteins (e.g. telomerase, growth factors or their receptors), anti-apoptotic proteins (e.g. Bcl2 family, IAPs, and NF-kB), pro-apoptotic proteins (e.g. Bax and BH-3 family, Fas, TNF-R, and caspases), and the genes encoding key transcription factors or elements for signal transduction leading to cell growth and apoptosis. Here we discuss the current knowledge of oncoapoptotic regulation in human cancers with special reference to oral cancers.

Designing of novel antigenic peptide cocktail for the detection of antibodies to HIV-1/2 by ELISA.

HIV (human immunodeficiency virus) infection has now become endemic worldwide and AIDS ranks fourth among the world's top killers of mankind. A rapid and accurate HIV testing assay is a pre-requisite for practical applicability of diagnostic tests. The aim of this present study was to design peptide cocktail as an antigen and to develop ELISA test for HIV-1/2 antibody detection, with enhanced sensitivity and specificity. A novel peptide stretch V3-I, covering immunodominant epitope corresponding to V3 hypervariable loop of gp120 antigens of selected Indian isolates, has been studied and incorporated in an antigenic cocktail of gp36, gp41, and rp24 of HIV-1/2. Peptides from these antigens were chemically synthesized and an additional cysteine residue was added at both amino- and carboxyl-terminal sequences of each peptide in order to form inter and intramolecular disulfide bond for the folding of peptides. This generated conformational epitopes with increased oligomericity and stability of peptide sequences; and attachment of antigen to the solid support of ELISA plates. The use of antigenic cocktail of folded peptides and recombinant p24 enhanced sensitivity and specificity of the ELISA test. Evaluation of the test using 1123 serum samples in comparison with Boston Biomedical Incorporation (BBI) panels showed 100% sensitivity and 99.3% specificity with no cross reactivity tribulation. In conclusion, "HIV screen test" detects HIV 1/2 antibodies with a high degree of sensitivity and specificity and could be a promising tool for seroscreening of blood during transfusion, counseling and diagnosis of HIV-1/2.

Induction of apoptosis and sensitization of head and neck squamous carcinoma cells to cisplatin by targeting survivin gene expression.

Survivin is known to be highly-expressed in various carcinomas; and is associated with their biologically aggressive characteristics and drug resistance. We have previously reported survivin as an important anti-apototic protein involved in head and neck squamous cell carcinoma (HNSCC) tumorigenesis and providing resistance to conventional cancer therapies. The purpose of present study was to investigate the potential of survivin as a therapeutic target in HNSCC. This study was designed to explore the effect(s) of survivin-siRNA therapy on the apoptosis in HNSCC cells, and its influence on cisplatin-sensitivity. Lentivirus vector was developed to deliver survivin specific siRNA into cancer cells. The data indicates that silencing of survivin-mediated by Lentivirus-siRNA therapy effectively suppressed cancer cell proliferation and induced caspase-dependent apoptosis in HNSCC cells. The study also shows that the response of HNSCC cells to cisplatin drug treatment at clinically relevant level was limited. We observed survivin to be a key factor involved in this cisplatin-resistance mechanism, and down-regulation of survivin significantly increased sensitivity of cancer cells to cisplatin-mediated apoptosis. Thus, this combination therapy acts as a multimodality regimen with significant potential to improve clinical outcomes in head and neck cancers.

Biology of Cox-2: an application in cancer therapeutics.

Cyclooxygenase-2 (Cox-2) is an inducible enzyme involved in the conversion of arachidonic acid to prostaglandin and other eicosanoids. Molecular pathology studies have revealed that Cox-2 is over-expressed in cancer and stroma cells during tumor progression, and anti-cancer chemo-radiotherapies induce expression of Cox-2 in cancer cells. Elevated tumor Cox-2 is associated with increased angiogenesis, tumor invasion and promotion of tumor cell resistance to apoptosis. Several experimental and clinical studies have established potent anti-cancer activity of NSAID (Non-steroidal anti-inflammatory drugs) and other Cox-2 inhibitors such as celecoxib. Much attention is being focused on Cox-2 inhibitors as beneficial target for cancer chemotherapy. The mode of action of Cox-2 and its inhibitors remains unclear. Further clinical application needs to be investigated for comprehending Cox-2 biological functions and establishing it as an effective target in cancer therapy.

Down-regulation of survivin by oxaliplatin diminishes radioresistance of head and neck squamous carcinoma cells.

BACKGROUND: Oxaliplatin is integrated in treatment strategies against a variety of cancers including radiation protocols. Herein, as a new strategy we tested feasibility and rationale of oxaliplatin in combination with radiation to control proliferation of head and neck squamous cell carcinoma (HNSCC) cells and discussed survivin-related signaling and apoptosis induction. METHODS: Cytotoxicity and apoptosis induced by radiation and/or oxaliplatin were examined in relation to survivin status using two HNSCC cell lines viz., Cal27 and NT8e, and one normal 293-cell line. Survivin gene knockdown by siRNA was also tested in relevance to oxaliplatin-mediated radiosensitization effects. RESULTS: Survivin plays a critical role in mediating radiation-resistance in part through suppression of apoptosis via a caspase-dependent mechanism. Oxaliplatin treatment significantly decreased expression of survivin in cancer cells within 24-72 h. Apoptotic cells and caspase-3 activity were increased parallely with decrease in cell viability, if irradiated during this sensitive period. The cytotoxicity of oxaliplatin and radiation combination was greater than additive. Survivin gene knockdown experiments have demonstrated the role of survivin in radiosensitization of cancer cells mediated by oxaliplatin. CONCLUSIONS: Higher expression of survivin is a critical factor for radioresistance in HNSCC cell lines. Pre-treatment of cancer cells with oxaliplatin significantly increased the radiosensitivity through induction of apoptosis by potently inhibiting survivin.

Detection of survivin and p53 in human oral cancer: correlation with clinicopathologic findings.

BACKGROUND: Survivin, an inhibitor of apoptosis, is overexpressed in cancer. It has been implicated in both prevention of apoptosis and cell cycle regulation. We investigated the distribution of antiapoptotic protein survivin in 29 oral squamous cell carcinoma (OSCC) and 16 oral premalignant lesions. It has been suggested that wild-type p53 represses survivin expression. Therefore, we investigated the status of p53 in relation to survivin to determine the potential involvement in oral tumorigenesis. METHODS: Oral cancer tissues were freshly obtained at the time of surgery and classified as per general rules of head and neck cancer (TNM classification). Immunohistochemistry and reverse transcriptase-polymerase chain reaction were conducted to study the expression of survivin and p53. The Fisher's exact test was employed to determine the association of survivin and p53 with clinicopathologic parameters of the subjects being studied. RESULTS: Positive staining for survivin was found in 72% OSCC and 44% oral premalignant lesions with no immunoreactions in the corresponding normal tissues. For p53, 59% OSCC, 38% premalignant lesions, and 14% normal tissues were positive. Importantly, about half of the p53-positive OSCC and premalignant tissues also showed survivin positivity (28% OSCC and 18% premalignant lesions). Further, it is observed that the number of survivin positive cells was significantly higher in the p53-positive group. Survivin is expressed in a varying proportion of cells, and in majority of patients it was localized in cytoplasm, whereas p53 is strictly restricted to the nucleus. The survivin expression levels in both primary OSCC and premalignant lesions were significantly higher than in normal oral tissues (OSCC, p < .0008; premalignant lesions, p < .04). No significant correlations between survivin and p53 expression with clinicopathologic parameters were found. CONCLUSIONS: Frequent overexpression of apoptosis regulators, survivin and p53, in OSCC as well as in oral premalignant lesions were found. Overexpression of these 2 markers in premalignant lesions suggest a role in early stages of oral carcinogenesis.

A synthetic gag p24 epitope chemically coupled to BSA through a decaalanine peptide enhances HIV type 1 serodiagnostic ability by several folds.

p24 is an immunodominant gag core protein of HIV-1. The synthetic immunodominant epitope of p24 and the recombinant p24 show poor immunoreactivity and specificity, respectively. Their application is, therefore, severely limited in the serodiagnosis of HIV-1, although it is an important marker for early diagnosis. These limitations have been overcome by conjugating the synthetic p24 to BSA through a decaalanine peptide spacer. The engineered p24 shows about 5-fold more efficient immunoreactivity than the synthetic p24, and, at the same time, shows a several fold reduction in nonspecific cross-reactivity as compared to recombinant p24. Our strategy to conjugate the p24 peptide epitope to BSA worked well as a consistent and reliable immunodiagnostic marker. This strategy may also prove useful for the diagnosis of other diseases.

Glycolipids of Mycobacterium tuberculosis strain H37Rv are potential serological markers for diagnosis of active tuberculosis.

A simple and cost-effective diagnostic tool (TB Screen Test) for the screening of patients with pulmonary and extrapulmonary tuberculosis and for differentiation of those individuals from individuals without tuberculosis, other common infections, and healthy controls has been developed. The serological responses of purified mycobacterial glycolipid antigens were examined by a liposome agglutination assay. The assay was able to detect very low antiglycolipid antibody concentrations in the infected individuals. The sera from the tuberculosis patient group had significantly higher concentrations of antiglycolipid antibody than the sera from uninfected control subjects, with 94% sensitivity and 98.3% specificity. Glycolipids of Mycobacterium tuberculosis H37Rv antigens were isolated, purified, and characterized. After interchelation with liposome particles, these purified antigens specifically bound to the antiglycolipid antibodies present in the sera of patients with tuberculosis, resulting in the formation of a blue agglutination. This protocol clearly differentiates healthy controls and M. bovis BCG-vaccinated subjects from those with active tuberculosis. The resultant diagnostic tool, the TB Screen Test, is more economical and rapid (4 min) than other currently available products and can be used for the mass screening of a heavily afflicted population.

Analysis of the shotgun expression library of the Mycobacterium tuberculosis genome for immunodominant polypeptides: potential use in serodiagnosis.

A recombinant DNA strategy was applied to analyze and screen the shotgun expression library from a clinically confirmed local virulent isolate of Mycobacterium tuberculosis with sera from tuberculosis patients, which led to expression and purification of highly immunoreactive and specific mycobacterial antigens expressed during the course of active disease which could be of diagnostic significance. An enzyme-linked immunoassay for diagnosis of tuberculosis was devised by using a shotgun immunoexpression library in the lambdagt11 vector. DNA from a virulent M. tuberculosis patient isolate (TBW-33) confirmed with the BACTEC 460 system was sheared and expressed to generate shotgun polypeptides. beta-Galactosidase fusion proteins capable of demarcating active tuberculosis infections from Mycobacterium bovis BCG-vaccinated healthy subjects or people harboring environmental mycobacteria were selected by comparative immunoreactivity studies. Promising mycobacterial DNA cassettes were subcloned and expressed into the glutathione S-transferase (GST) fusion vector pGEX-5X-1 with a strong tac promoter and were expressed in Escherichia coli BL21. These fusion proteins were severed at a built-in factor Xa recognition site to separate the GST tags and were utilized in an indirect enzyme-linked immunoassay for serodiagnosis of patients with active tuberculosis. The system offered a clear demarcation between BCG-vaccinated healthy subjects and patients with active tuberculosis and proved to be effective in detecting pulmonary as well as extrapulmonary tuberculosis, with an overall sensitivity of 84.33% and an overall specificity of 93.62%.

Immobilization results in sustained calcium transport in Nostoc calcicola Bréb.

The uptake pattern of Ca2+ by the cyanobacterium Nostoc calcicola Bréb in its freely suspended and immobilized form is comprised of two distinct phages; (a) rapid uptake for 1st 10 min followed by (b) slower transport at least up to 60 min. Entrapment of cyanobacterial cells in polyvinyl foam always maintained a higher Ca2+ profile over freely suspended cells. Also, the intracellular Ca2+ concentration was three times more in the former under similar experimental conditions. Whereas, illumination supported maximum Ca2+ transport in all the sets, darkness resulted in drastic reduction (90%) of Ca2+ uptake in freely suspended cells and least (15%) in polyvinyl entrapped cyanobacterial cells. Exogenously added ATP (10 microM) on the other hand, enhanced Ca2+ uptake in dark incubated freely suspended cells; ATP at the same concentration failed to bring out any significant enhancement in cation uptake in immobilized cells facing dark exposure. It was observed that these cells were still able to sustain sufficient ATP preserves to drive active transport of Ca2+ even in the dark. Furthermore, the immobilized cells exhibited remarkable Ca2+ transport rate even at the age of 20 and 50 days at which its free living counterpart took up insignificant Ca2+. These findings suggest the improved metabolic efficiency of polyvinyl foam entrapped cells over freely suspended cells in terms of Ca2+ accumulation and its possible use as a bioreactor for metal accumulation/removal in repetitive cycles without any measurable loss in cell biomass.