Ganesh Mahidhara

Novel alginate-enclosed chitosan-calcium phosphate-loaded iron-saturated bovine lactoferrin nanocarriers for oral delivery in colon cancer therapy

AIM To develop polymeric-ceramic nanocarriers (NCs) in order to achieve oral delivery of the anticancer neutraceutical iron-saturated bovine lactoferrin (Fe-bLf) protein. MATERIALS & METHODS Fe-bLf or paclitaxel (Taxol®) were adsorbed onto calcium phosphate nanocores, enclosed in biodegradable polymers chitosan and alginate. The Fe-bLf or Taxol-loaded NCs indicated as AEC-CP-Fe-bLf or AEC-CP-Taxol NCs, respectively, were made by combination of ionic gelation and nanoprecipitation. Size distribution, morphology, internalization and release profiles of the NCs were studied along with evaluation of in vitro and in vivo anticancer activities and compared with paclitaxel. RESULTS AEC-CP-Fe-bLf NCs obtained spherical morphology and showed enhanced endocytosis, transcytosis and anticancer activity in Caco-2 cells in vitro. AEC-CP-Fe-bLf NCs were supplemented in an AIN 93G diet and fed to mice in both prevention and treatment human xenograft colon cancer models. AEC-CP-Fe-bLf NCs were found to be highly significantly effective when given orally, as a pretreatment, 1 week before Caco-2 cell injections. None of the mice from the AEC-CP-Fe-bLf NC-fed group developed tumors or showed any signs of toxicity, while the mice fed the control AIN 93G diet showed normal tumor growth. Fe-bLf or Taxol, when given orally in a diet as nanoformulations post-tumor development, showed a significant regression in the tumor size with complete inhibition of tumor growth later, while intratumoral injection of Taxol just delayed the growth of tumors. The pharmacokinetic and bioavailability studies indicated that nanoformulated Fe-bLf was predominantly present on tumor cells compared to non-nanoformulated Fe-bLf. Fe-bLf-loaded NCs were found to help in absorption of iron and thus may have utility in enhancing the iron uptake during iron deficiency without interfering with the absorption of calcium. CONCLUSION With the promising results of our study, the future potential of NC-loaded Fe-bLf in chemoprevention and in the treatment of human colon cancer, deserves further investigation for translational research and preclinical studies of other malignancies.

Recent Advances in Nanoneurology for Drug Delivery to the Brain

The drug development for neurodegenerative disorders are the major challenge to the science in 21st century. Many FDA approved drugs currently available in the market have limitations in crossing the blood brain barrier (BBB) owing to its complicated vasculature posed by the presence of specialized cells. Nanotechnology is an emerging interdisciplinary area, which have many applications including drug delivery. Nanocarrier drug delivery involves targeting drugs enclosed in a particular polymer and/or amphiphilic lipids. Controlled release, nanoplatform availability for combinatorial therapy and tissue specific targeting by using advanced technologies such as molecular Trojan horse (MTH) technology are the promises of nanotechnology. Different problems are associated with drug delivery across the BBB. Some are mostly related to the structure of brain microvasculature system while the others are related to the nanomaterial structure. Different strategies, such as using polymeric/solid lipid nanoparticles and surface modification of nanomaterial with surfactants like polysorbates have been conducted to solve these limitations. Also, nanodrug formulations with double coatings have been designed for oral delivery of drugs to overcome reticulo-endothelial system and to improve their BBB permeability. It seems that the best choice of strategy and material could be achieved with regard to the physical and chemical structure of the drugs. The present review discusses the potential applications of nanotechnology for drug delivery across the BBB.

Antiangiogenic therapy using nanotechnological-based delivery system.

Of the many approaches for the treatment of cancer, angiogenesis and the additional promotion of apoptosis in cancer stem cells by using combinatorial therapy is usually the most recommended. There has been increased interest in the use of antiapoptotic and antiangiogenic biomolecules, such as antiangiogenic microRNA, small interfering RNA, inhibitor of apoptosis protein-binding peptides and Von Hippel-Lindau tumor suppressors, as well as targeting ligands, such as aptamers. Therefore, it is tempting to suggest that such molecules could be used for anticancer therapy. As we review here, such exploitation can be achieved by using nanotechnology and RNA-carrying cationic cell-penetrating peptides, for better protection from the enzymatic digestion and enhanced cellular internalization of these biomolecules.

Fe-bLf nanoformulation targets survivin to kill colon cancer stem cells and maintains absorption of iron, calcium and zinc

AIM To validate the anticancer efficacy of alginate-enclosed, chitosan-conjugated, calcium phosphate, iron-saturated bovine lactoferrin (Fe-bLf) nanocarriers/nanocapsules (NCs) with improved sustained release and ability to induce apoptosis by downregulating survivin, as well as cancer stem cells. MATERIALS & METHODS The stability, nanotoxicity of the modified nanoformulation was evaluated and their anticancer efficacy was re-examined. Their mechanism of internalization was studied and we identified the role of various miRNAs in absorption of these NCs/iron in various body parts of mice. We determined the effect of these NCs on survivin, stem cell markers, red blood cell count, iron, calcium and zinc concentration in mice, determined the antiangiogenic properties of these NCs and studied their effect on cancer stem-like cells. RESULTS Spherical NCs (396.1 ± 27.2 nm) exceedingly reduced viability of Caco-2 cells (32 ± 2.83%). The NCs also showed effective internalization and reduction of cancer stem cell markers in triple-positive CD133, survivin and CD44 cancer stem-like cells. Mice treated with the NCs showed no nanotoxicity and did not develop any tumors in xenograft colon cancer models. We found that the serum iron, zinc and calcium absorption were increased. DMT1, LRP, transferrin and lactoferrin receptors were responsible for internalization of the NCs. Different miRNAs were responsible for iron regulation in different organs. Interestingly, NCs inhibited survivin and its different isoforms. CONCLUSION Our results confirmed that NCs internalized and changed the expression of selected miRNAs that further enhanced their uptake. The NCs activated both extrinsic, as well as intrinsic apoptotic pathways to induce apoptosis by targeting survivin in cancer cells and cancer stem cells, without inducing any nonspecific nanotoxicity. Apart from inhibiting angiogenesis and stem cell markers, NCs also maintained iron and calcium levels.

Oral administration of iron-saturated bovine lactoferrin–loaded ceramic nanocapsules for breast cancer therapy and influence on iron and calcium metabolism

We determined the anticancer efficacy and internalization mechanism of our polymeric–ceramic nanoparticle system (calcium phosphate nanocores, enclosed in biodegradable polymers chitosan and alginate nanocapsules/nanocarriers [ACSC NCs]) loaded with iron-saturated bovine lactoferrin (Fe-bLf) in a breast cancer xenograft model. ACSC-Fe-bLf NCs with an overall size of 322±27.2 nm were synthesized. In vitro internalization and anticancer efficacy were evaluated in the MDA-MB-231 cells using multicellular tumor spheroids, CyQUANT and MTT assays. These NCs were orally delivered in a breast cancer xenograft mice model, and their internalization, cytotoxicity, biodistribution, and anticancer efficacy were evaluated. Chitosan-coated calcium phosphate Fe-bLf NCs effectively (59%, P≤0.005) internalized in a 1-hour period using clathrin-mediated endocytosis (P≤0.05) and energy-mediated pathways (P≤0.05) for internalization; 3.3 mg/mL of ACSC-Fe-bLf NCs completely disintegrated (~130-fold reduction, P≤0.0005) the tumor spheroids in 72 hours and 96 hours. The IC50 values determined for ACSC-Fe-bLf NCs were 1.69 mg/mL at 10 hours and 1.62 mg/mL after 20 hours. We found that Fe-bLf-NCs effectively (P≤0.05) decreased the tumor size (4.8-fold) compared to the void NCs diet and prevented tumor recurrence when compared to intraperitoneal injection of Taxol and Doxorubicin. Receptor gene expression and micro-RNA analysis confirmed upregulation of low-density lipoprotein receptor and transferrin receptor (liver, intestine, and brain). Several micro-RNAs responsible for iron metabolism upregulated with NCs were identified. Taken together, orally delivered Fe-bLf NCs offer enhanced antitumor activity in breast cancer by internalizing via low-density lipoprotein receptor and transferrin receptor and regulating the micro-RNA expression. These NCs also restored the body iron and calcium levels and increased the hematologic counts.

Cancer Targeted Nanoparticles Specifically Induce Apoptosis in Cancer Cells and Spare Normal Cells

Curing cancer is the greatest challenge for modern medicine and finding ways to minimize the adverse effects caused by chemotherapeutic agents is of importance in improving patient’s physical conditions. Traditionally, chemotherapy can induce various adverse effects, and these effects are mostly caused by the non-target specific properties of the chemotherapeutic compounds. Recently, the use of nanoparticles has been found to be capable of minimizing these drug-induced adverse effects in animals and in patients during cancer treatment. The use of nanoparticles allows various chemotherapeutic drugs to be targeted to cancer cells with lower dosages. In addition to this, the use of nanoparticles also allows various drugs to be administered to the subjects by an oral route. Here, locked nucleic acid (LNA)-modified epithelial cell adhesion molecules (EpCAM), aptamers (RNA nucleotide), and nucleolin (DNA nucleotide) aptamers have been developed and conjugated on anti-cancer drug-loaded nanocarriers for specific delivery to cancer cells and spare normal cells. Significant amounts of the drug loaded nanocarriers (92 ± 6 %) were found to distribute to the cancer cells at the tumour site and more interestingly, normal cells were unaffected in vitro and in vivo. In this review, the benefits of using nanoparticle-coated drugs in various cancer treatments are discussed. Various nanoparticles that have been tried in improving the target specificity and potency of chemotherapeutic compounds are also described.

Novel nanoplatform for oral delivery of anti–cancer biomacromolecules

Oral administration of bio–macromolecules is an uphill task and the challenges from varying pH and enzymatic activity are difficult to overcome. In this regard, nanotechnology promises the new hope and offers advantages such as controlled release, target specific delivery, combinatorial therapy and many more. In this study, we demonstrate the formulation of a novel alginate enclosed, chitosan coated ceramic, anti cancer nano carrier (ACSC NC). These NC were loaded with multi functional anti cancer bovine lactoferrin (Lf), a natural milk based protein, for improvement of intestinal absorption, in order to develop a novel platform to carry anti cancer protein and/or peptides for oral therapy. Here we demonstrate the size, morphology, internalisation and release profiles of the nanoparticles (NC) under varying pH as perceived in human digestive system. We further determine the uptake of these particles by colon cancer cell lines by measuring the endocytosis and transcytosis of the NC. These NC can be used for future targeted protein/peptide or nucleic acid based drug delivery to treat difficult diseases including cancer.

Effect of Selenium-Saturated Bovine Lactoferrin (Se-bLF) on Antioxidant Enzyme Activities in Human Gut Epithelial Cells Under Oxidative Stress

Cancer and many chronic inflammatory diseases are associated with increased amounts of reactive oxygen species (ROS). The potential cellular and tissue damage created by ROS has significant impact on many disease and cancer states and natural therapeutics are becoming essential in regulating altered redox states. We have shown recently that iron content is a critical determinant in the antitumour activity of bovine milk lactoferrin (bLF). We found that 100% iron-saturated bLF (Fe-bLF) acts as a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy and thus has a broad utility in the treatment of cancer. Furthermore, we also studied the effects of iron saturated bLFs ability as an antioxidant in the human epithelial colon cancer cell line HT29, giving insights into the potential of bLF in its different states. Thus, metal saturated bLF could be implemented as anti-cancer neutraceutical. In this regard, we have recently been able to prepare a selenium (Se) saturated form of bLF, being up to 98% saturated. Therefore, the objectives of this study were to determine how oxidative stress induced by hydrogen peroxide (H2O2) alters antioxidant enzyme activity within HT29 epithelial colon cancer cells, and observe changes in this activity by treatments with different antioxidants ascorbic acid (AA), Apo (iron free)-bLF and selenium (Se)-bLF. The states of all antioxidant enzymes (glutathione peroxidase (GPx), glutathione reductase (GR), glutathione- s-transferase (GsT), catalase and superoxide dismutase (SOD)) demonstrated high levels within untreated HT29 cells compared to the majority of other treatments being used, even prior to H2O2 exposure. All enzymes showed significant alterations in activity when cells were treated with antioxidants AA, Apo-bLF or Se-bLF, with and/or without H2O2 exposure. Obvious indications that the Se content of the bLF potentially interacted with the glutathione (GSH)/GPx/GR/GsT associated redox system could be observed immediately, showing capability of Se-bLF being highly beneficial in helping to maintain a balance between the oxidant/antioxidant systems within cells and tissues, especially in selenium deficient systems. In conclusion, the antioxidative defence activity of Se-bLf, investigated in this study for the first time, shows dynamic adaptations that may allow for essential protection from the imbalanced oxidative conditions. Because of its lack of toxicity and the availability of both selenium and bLF in whole milk, Se-bLF offers a promise for a prospective natural dietary supplement, in addition to being an immune system enhancement, or a potential chemopreventive agent for cancers.

Nanoformulated nanocarriers with modified lactoferrin for cancer and bio-distribution through MRI

T nuclear receptor pregnane X receptor (PXR) is activated by a range of xenochemicals, including chemotherapeutic drugs, and has been suggested to play a role in the development of tumor cell resistance to anticancer drugs. PXR also has been implicated as a regulator of the growth and apoptosis of colon tumors. Here, we have used a xenograft model of colon cancer to define a molecular mechanism that might underlie PXR-driven colon tumor growth and malignancy. Activation of PXR was found to be sufficient to enhance the neoplastic characteristics, including cell growth, invasion, and metastasis, of both human colon tumor cell lines and primary human colon cancer tissue xenografted into immunodeficient mice. Furthermore, we were able to show that this PXR-mediated phenotype required fibroblast growth factor (FGF) 19 signaling. PXR bound to the FGF19 promoter in both human colon tumor cells and “normal” intestinal crypt cells. However, while both cell types proliferated in response to PXR ligands, the FGF19 promoter was activated by PXR only in cancer cells. Taken together, these data indicate that colon cancer growth in the presence of a specific PXR ligand results from tumor-specific induction of FGF19. These observations may lead to improved therapeutic regimens for colon carcinomas.P dietary choices have long-term health consequences, with important roles in disease development and incidence including heart disease, diabetes, and cancer. It is estimated that 30-40% of all cancers can actually be prevented by adopting a well-balanced diet and other healthy lifestyle choices (e.g. reduced stress, increased exercise). The scope of dietary intervention research with regards to cancer prevention and treatment is large; ranging from dietary restriction such as specific amino acid restriction (tyrosine/phenylalanine) to dietary supplementation including curcumin and saw palmetto.MSI is variable length mutations in tumor DNA caused by deficiency of DNA mismatch repair. Dysfunction of this repair system is caused by inactivation of any of the repair enzymes MLH1, MSH2, MSH6, or PMS2. It can be measured either on the DNA level as MSI or on the protein level with loss of expression of the affected protein. Lynch syndrome is caused by hereditary mutations in any of the four mismatch repair genes. About 15 % of all colorectal cancers have MSI, but not more than one in three of these are caused by germline mutations. The rest is caused by a sporadic phenomenon, promoter hypermethylation of MLH1.The survival rate for women with advanced, metastatic breast cancer has not changed significantly for decades. Regardless of effective therapies, many women still experience recurrences of breast cancer after treatment. Chemotherapy with docetaxel (and other anti-cancer reagents) is widely used for the treatment of breast cancer. Despite an initial response to treatment, the sensitivity to anti-cancer reagents decreased. This leads to progressive disease, normally with fatal consequences. We previously demonstrated that ribophorin II (RPN2), which is part of an N-oligosaccharyl transferase complex, regulates the drug resistance in breast cancer cells and silencing of RPN2 by RNAi-based oligonucleotides is a promising approach to overcome the drug-resistant tumor (Honma K. et al., 2008, Nat Med). In the current study, we examined the role of RPN2 in cancer stem cell (CSC) fraction that is responsible for drugresistance and metastasis. Cell sorting coupled with realtime qPCR analysis showed that RPN2 is highly expressed in CSC fraction. Furthermore, RPN2-knockdown in CSC fraction reduced cancer stem cell frequency, as shown by flow cytometric and in vivo tumorigenicity studies. Thus, these results suggest that RPN2 might be a promising novel target toward the cancer stem cell therapy. L3.2B antibodies can transiently link tumor cells with otherwise inactive cytotoxic T cells in patients for induction of potent redirected lysis of tumor cells. One example is blinatumomab (MT103), a CD19/-CD3-bispecific BiTE for the treatment of human B cell-derived malignancies. Blinatumomab and other BiTE antibodies were shown to activate T cells in a highly conditional manner that is strictly dependent on the presence of target cells. Blinatumomab has commenced a pivotal study for the treatment of adult patients with therapy-refractory acute lymphocytic leukemia (ALL). A phase 2 study in ALL patients has shown an 80% complete molecular response rate at a dose level of 15 micrograms/squaremeter per day. Blinatumomab has also shown high response rates in non-Hodgkin’s lymphoma (NHL) patients with follicular and mantle cell lymphoma, and first signs of efficacy in patients with diffuse large B cell lymphoma. Centrally confirmed complete and partial responses according to Cheson criteria were seen in NHL patients treated at a dose of 60 micrograms/squaremeter/day. The presentation will update on the clinical development of blinatumomab in leukemia and lymphoma.Background: Accumulated evidence suggests that multiple genetic alterations are involved in the complex carcinogenetic process of gastric adenocarcinoma (GAC). Although a number of genetic changes have been reported in GAC, including amplification of CMET and FGFR2, mutation of E-cadherin and KRAS and loss of heterozygosity on 5q and 18q, the molecular events leading to GAC and its progression remain largely unknown. To assess global molecular changes in GAC, we use whole genomic assay to evaluate human GAC samples. Methods: Oligonucleotide array comparative genomic hybridization (aCGH) was performed on 46 GAC samples using a high-density (244K) aCGH system (Agilent Technologies). For each aCGH probe, each sample was classified as having normal, gained, or lost DNA copy number based on log 2 ratio thresholds of 0.15. An independent set of tissue arrayed samples (n=68) was further validated by fluorescent in-situ hybridization (FISH) by using probes visualizing 19q13.3 (red signal) and the centromere (green signal). Amplification of 19q13.3 was defined if the ratio of 19q13.3 to centromere is greater than 2.2. The mean patient’s survival follow-up time was 58 months. Results: aCGH identified 1271 genes with DNA copy loss and 1449 genes with DNA copy gain in gastric cancer. Among these identified genes, 11 deleted and 198 amplified genes were observed to have significant association with patient’s survival. Forty-eight of amplified genes were specifically located on chromosome 19q13.3, including CRX, DACT3, DKKL1, EHD2, EMP3, HIF3A, HRC, IGFL2, IGFL3, KPTN, LIG1, PNKP and PTOV1. Compared with all other patients, those (n=14) with gene amplification on 19q13.3 had a significantly poorer prognosis (p<0.01), independent of other conventional prognosis factors including TNM stage. These results were further confirmed by FISH method and amplification of 19q13.3 was identified in 18 cases with unfavorable clinical outcome. Conclusions: This genome-wide study identified a panel of critical genes associated with progression of GAC. Amplification of the genes on chromosome 19q13.3, a possible signature event in gastric carcinogenesis, represents a potentially useful prognostic biomarker for this aggressive malignancy. Further functional studies are needed to confirm the potential value of these genes in the management of gastric cancer. Signature Molecular Biomarkers of Prognosis of Gastric Adenocarcinoma: A Study of 114 Cases Using Genome-Wide Technique and Fish Dongfeng Tan University of Texas, MD Anderson Cancer Center, USA doi:10.4172/2155-9929.1000002T Keap1 (Kelch-like ECH-associated protein 1) protein tightly regulates the functions of Nrf2 (nuclear factor-erythroid 2-related factor 2 ) which plays a pivotal role in the cellular response to oxidative stress. We determined whether KEAP1 gene is epigenetically regulated in malignant gliomas and if promoter aberrant methylation may impact patient’s outcome. We developed a QMSP assay to analyze 86 malignant gliomas and 20 normal brain tissues. The discriminatory power of the assay was assessed by ROC curve analysis. Th e AUC value of the curve was 0.823 (95%CI: 0.764-0.883) with an optimal cut off value of 0.133 yielding a 74% sensitivity (95%CI: 63%-82%) and an 85% specificity (95%CI: 64%-95%). Bisulfite sequencing analysis confirmed QMSP results and demonstrated a direct correlation between percentage of methylated CpGs and methylation levels (Spearman’s Rho 0.929, P=0.003). Remarkably, a strong inverse correlation was observed between methylation levels and KEAP1 mRNA transcript in tumour tissue (Spearman’s Rho -0.656 P=0.0001) and in a cell line before and after treatment with 2-deoxy-5 Azacytidine (P=0.003). RECPAM multivariate statistical analysis studying the interaction between MGMT and KEAP1 methylation in subjects treated with radiotherapy and temozolomide (n=70), identified three prognostic classes of glioma patients at different risk to progress. While simultaneous methylation of MGMT and KEAP1 promoters was associated with the lowest risk to progress, patients showing only MGMT methylation were the subgroup at the higher risk (HR 5.54, 95% CI 1.35-22.74). Our results strongly indicate that aberrant KEAP1 methylation may represent a novel predictor of outcome in glioma patients.G alteration is the driving force for thyroid tumorigenesis and progression, based upon which novel approaches to the management of thyroid cancer can be developed. The T1799A BRAF mutation in papillary thyroid cancer (PTC) has a great clinical promise and is currently being translated from laboratory to the clinic use. Studies from our and other groups have consistently shown that BRAF mutation is the most common genetic alteration in thyroid cancer, occurring in about 45% of papillary thyroid cancers (PTC) and 25% anaplastic thyroid cancer. The BRAF mutation exerts its oncogenic role through aberrant activation of the MAP kinase signaling pathway. Numerous studies around the world have demonstrated the unique role of BRAF mutation in the development of aggressiveness of PTC, in agreement with our initial findings. For example, BRAF mutation is closely associated with extrathyroidal invasion, lymph node metastasis, advanced tumor stage, and, importantly, disease persistence/recurrence and even decreased patient mortality. BRAF mutation is also associated with loss of radioiodine avidity of PTC, making it difficult to treat this cancer using radioiodine. Numerous studies have demonstrated that BRAF mutation is associated with increased expression of tumor-promoting molecules or suppression of tumor-suppressing molecules, providing a molecular basis for the role of this mutation in the progression and aggressiveness of PTC. Recent studies have also demonstrated an important role of BRAF mutation in the silencing of iodide-handling genes in PTC, providing a molecular explanation for the association of loss of radioiodine avidity of PTC with BRAF mutation. Thus, BRAF mutation is a novel and powerful prognostic molecular marker for poorer prognosis of PTC. Use of BRAF mutation, which can be detected on preoperative thyroid fine needle biopsy specimens, is expected to become an effective strategy for risk stratification of PTC. This may help resolve several clinical dilemmas encountered in the management of PTC, such as how to determine the extent of surgical and medical treatments of PTC in various clinical settings. It is thus expected that BRAF mutation, as a novel prognostic marker in PTC, will have an important impact on thyroid cancer medicine.O previous data demonstrates that a small GTPase hRAB37, which is a member of Rab superfamily, plays a role in lung cancer progression. This study aims to investigate the functions of hRAB37 to regulate vesicle trafficking and its cell signals involved in cell migration. We demonstrated that hRAB37 is a tumor metastatic suppressor protein in lung cancer. Clinical data showed that low hRAB37 protein expression and promoter/exon1 hypermethylation of hRAB37 gene correlated markedly with poor progression-free survival and overall survival in lung cancer patients. Overexpression of hRAB37 resulted in loss of migration/invasion ability in CL1-5 lung cancer cells-based assays and remarkably reduced lung tumor metastasis in animal models. Migration/invasion ability of CL1-5 cells was inhibited under the treatment of conditional medium taken from hRAB37 overexpressed CL1-5 cells resulting from an increased protein level of secreted TIMP-1 protein, which is an inhibitor of matrix metalloproteinases (MMPs). In addition, the decreased expression of MMP2 and MMP9, and FAK-mediated metastasis pathway, including p-FAK, p-AKT and RhoA activity, provided a potential mechanism for the metastasis suppression effects of hRAB37. Furthermore, confocal analysis demonstrated a co-localization of hRAB37 with the secretory marker VAMP2 and the RAB3a exocytosis protein. Confocal images demonstrated a colocalization between RAB37 and TIMP-1, an MMP inhibitor. Our data provided first compelling evidence from cell, animal, and clinical studies that hRAB37 small GTPase is a metastasis suppressor through exocyticly trafficking the anti-metastatic proteins such as TIMP-1. Low expression of hRAB37 due to promoter hypermethylation leads to poor survival of lung cancer.T introduction of preoperative radiotherapy (RT) in the treatment of rectal cancer has reduced the frequency of local recurrence and improved patient survival, and RT is now a part of the standard treatment regime in Sweden. However, it is still a major problem that there are unknown factors contributing to variations in recurrence and survival after RT and surgery among rectal cancer patients with the same tumour stage, therefore it is important to search for biological markers that might influence recurrence and survival, and to identify the patients who benefit from RT.C (diferuloylmethane) has been known to suppress tumor progression. To identify curcumin derivatives having more potent anti-tumor activity, we compared the anti-tumor effect of curcumin and fermented curcumin fermented by bacillus subtillus. Fermented curcumin (diferuloylmethane) markedly suppressed proliferation of various cancer cells through regulation of cell cycle progression compared to curcumin. Expression of apoptosis-associated genes was elevated by treatment of cancer cells with fermented curcumin in a dose-dependent manner. In addition, fermented curcumin suppressed metastasis by downregulating MMP expression. More importantly, the susceptibility to NK cell-mediated killing of cancer cells was modestly more increased in cancer cells treated with fermented curcumin than in those treated with only curcumin. These findings collectively suggest that fermented curcumin plays an essential role in the regulation of tumorigenecity.This book facilitates a better understanding of and draws attention to stem cell and cancer biology two fields that enhance, move, and evolve into each other continuously. It provides an informative study in designing approaches to apply stem cell principles to cancer biology while offering an overview of the challenges in developing combination stem and cancer biology targets for therapeutics. This book serves as a primer for new researchers in the field of cancer biology.F imaging provides non-invasive quantitative information about biological and physiological processes of relevance for the response to treatment, and may as such be critical for the development of individualized cancer therapy. PETimaging with appropriate tracers enables visualization of the tumor energy metabolism, cellular proliferation, apoptosis, angiogenesis, hypoxia as well as receptor status, whereas tracer kinetics can be quantified from 4D PET imaging. Dynamic contrast enhanced (dce) MR imaging can be utilized in deriving voxel-wise information about e.g. blood perfusion of the tumor as well as information about the extra-cellular space. Tumor hypoxia has been shown to correlate to quantitative dce-MR images. Based in diffusion weighted MR imaging, the apparent diffusion of water molecules can be measured and information about cellular integrity of tumor tissue can be derived. MR spectroscopy provides quantitative information about various metabolites in the tumor tissue.P receptors (PRRs) detect molecular signatures of microbes and initiate immune responses to infection. Immune responses generated by prototypical PRRs such as Toll-like receptors (TLRs) have been widely investigated. In contrast, the immune responses initiated by other classes of putative PRRs remain ill defined. C-type lectins are a class of PRRs that recognize carbohydrate structures which are often part of microbial pathogens. Dectin-1 is a C-type lectin receptor present on dendritic cells that recognizes fungal β-glucans. Our investigations suggest that Dectin-1 is not just an antigen uptake receptor but also a modulator or initiator of adaptive immune responses. Human dendritic cells stimulated with Curdlan, Dectin-1 agonist prime CD4 Th17 responses via IL-23 production. Furthermore, these CD4 T cells induce differentiation of B cells to secrete IgG and IgA. More importantly; these dectin-1 stimulated dendritic cells promote the expansion and differentiation of granzyme B expressing cytotoxic T lymphocyte that display high cytolytic activity against target tumor cells in vitro. The capacity of Curdlan-stimulated human DCs to induce differentiation of these cells makes them attractive target for manipulations in clinic against cancer.E receptor-negative (ER-) breast cancer is a heterogeneous disease with limited therapeutic options. Molecular apocrine subtype constitutes 50% of ERbreast tumors and is characterized by a steroid-response signature including androgen receptor (AR) and a high rate of ErbB2 amplification. We have identified a positive feedback loop between the AR and ERK signaling pathways in molecular apocrine breast cancer. In this process, AR regulates ERK phosphorylation and kinase activity. In addition, AR inhibition results in the down-regulation of ERK target proteins including phospho-RSK1. Furthermore, AR-mediated induction of ERK requires ErbB2 and AR activity, in turn, regulates ErbB2 expression as an ARtarget gene. These findings suggest that ErbB2 is an upstream connector between the AR and ERK signaling pathways. Another feature of this feedback loop is an ERK-mediated regulation of AR. In this respect, the inhibition of ERK phosphorylation reduces AR expression and CREB1-mediated transcriptional regulation of AR acts as a down-stream connector between the AR and ERK signaling pathways.O gene in the heart synthesizes four peptide hormones, i.e., long-acting natriuretic peptide, vessel dilator, kaliuretic peptide and atrial natriuretic peptide. These four peptides decrease up to 97% of human pancreatic, breast, colon, ovarian, kidney and prostate adenocarcinomas as well as glioblastomas of the brain, small-cell and squamous cell lung carcinoma cells in cell culture. When infused subcutaneously for 28 days with weekly fresh hormones at 3 nM min-1 kg-1 body weight in athymic mice, they eliminate up to 80% of the human pancreatic adenocarcinomas, 2/3rds of human breast adenocarcinomas, and up to 86% of human small-cell lung cancers with treated mice living a normal lifespan. These cancers never reoccur in the primary site in the lifespan of the mice. Their mechanisms(s) of action in cancer cells includes a 95% inhibition of Ras, 96% inhibition of ERK 1/2 kinases, and 98% inhibition of MEK 1/2 kinases. Mitogens such as epidermal growth factor which stimulate Ras and ERK 1/2 kinases have their effects completely blocked by these cardiac hormones. The cardiac hormones do inhibit ERK 1/2 kinases in healthy cells. In addition to inhibiting the Ras-MEK 1/2-ERK 1/2 kinase cascade, they enter the nucleus as shown by Immunocytochemical techniques where they inhibit DNA synthesis.E of an association between survival time and date of birth would suggest an etiologic role for a seasonally variable environmental exposure occurring within a narrow perinatal time period. Risk factors that may exhibit seasonal epidemicity include diet, infectious agents, allergens, and antihistamine use. Typically data has been analyzed by simply categorizing births into months or seasons of the year and performing multiple pairwise comparisons. This paper present a statistically robust alternative, based upon a trigonometric Cox regression model, to analyze the cyclic nature of birth dates related to patient survival. Disease birth-date results are presented using a sinusoidal plot with peak date(s) of relative risk and a single P value that indicates whether an overall statistically significant seasonal association is present. Advantages of this derivative-free method include ease of use, increased power to detect statistically significant associations, and the ability to avoid arbitrary, subjective demarcation of seasons.W have recently determined that the extracellular protease TACE/ADAM17 is involved in physiological (constitutive) germ cell apoptosis. The mechanism underlying apoptosis induction in cancer cells has been studied in different cell types, but it is not known whether the same factors participate in viable germ cells. Since testicular cancer primarily affects young males, we used pubertal rats (21 days old) as a model to determine whether etoposide induces apoptosis through TACE/ ADAM17 upregulation, akin to physiological process. Germ cell apoptosis induced by DNA damage was associated with an increase in protein levels and cell surface localization of TACE/ADAM17 and ADAM10. On the contrary, apoptosis of germ cells induced by heat stress, another cell death stimulus, did not change levels or localization of these proteins. Pharmacological in vivo inhibition of TACE/ADAM17 and ADAM10 prevents etoposide-induced germ cell apoptosis. Gleevec (STI571) a pharmacological inhibitor of p73, a master gene controlling apoptosis induced by etoposide, prevented the increase of TACE/ ADAM17 levels. In vitro, using a germ cell line model, etoposide was also able to induce up regulation of ADAM10 and TACE/ ADAM17. Pharmacological and genetic inhibition of both enzymes (knockdown) prevented apoptosis induced by etoposide. Our results strongly suggest that TACE/ADAM17 participates in apoptosis of male germ cells induced by DNA damage. Therefore, we have showed a new and a previously unanticipated element in the mechanism of apoptosis induced by etoposide.T efficacy of conventional intravenous cancer chemotherapy is severely limited by systemic drug toxicity. Statistics for the past 20 years indicate little progress in reducing cancer mortality except for distinctive genetically defined subtypes. Reported here are studies showing the efficacy of intratumoral chemotherapy (ITC) as a debulking tool in central tumors applied worldwide in more than 370 published patients over the last decade. ITC has already shown in few observational studies that a surplus of median survival can be achieved if this method is as an adjunct to standard options like reduced intravenous chemotherapy, external or internal radiotherapy even in patients with poor performance status: Cancer drugs are injected directly into the tumor for central tumors or transthoracically for peripheral cancer site and by an EBUS-system for involved mediastinal lymph nodes. In more than 60 published patients (inoperable IIIa – IV) a surplus of up to 77% in median survival (MS) in comparison to expected MS according to UICC 7 data was achieved when ITC was used as an adjunct to standard therapy. Superdoses of cytotoxic drugs may thereby accomplish rapid tumor cell killing without systemic toxic complications but also involved lymph nodes in many studies the hallmark of local recurrence – could be treated directly and specifically. This new paradigm promises to significantly reduce lung cancer morbidity and mortality without the toxic complications associated with conventional systemic chemotherapy. It maybe not only considered in palliative situations but also as preoperative therapy according to the results in animal studies.C which has become the most devastating disease of this era is an uncontrolled growth of cells through division beyond normal limits. Till now in research there are a number of therapies which have come up to fight against this disease. But unfortunately most of them are so toxic and injurious that the medicines become cause of the death of the patient. Among the safer therapeutics, Methylglyoxal is a one suggested by Albert Szent-Gyorgyi in 1963 which he termed as Retine.M human genes undergo alternative splicing, and many abnormal splicing processes are associated with human diseases. However, the molecular relationship between alternative splicing and tumorigenesis is not well understood. Here, we found novel Kruppel-like factor 4 (KLF4) splicing variants produced by exon skipping in human cancer cell lines as well as colon tumor tissues. To elucidate mechanism of the KLF4 alternative splicing, we developed KLF4 minigene system and found that RNA binding motif protein 5 (RBM5) plays an important role in KLF4 splicing, as assessed by gain and loss of functional studies. Several anti-tumorigenic compounds were also tested for the KLF4 splicing. Interestingly, sulindac sulfide restored wild type KLF4 (KLF4L) expression and this is mediated by dephosphorylation of RBM5. Another splicing variant, small KLF4 (KLF4S), localizes in the cytoplasm and nucleus, and antagonizes transcriptional activity of wild type KLF4. Our data suggest that RBM5 plays a pivotal role in the alternative splicing of KLF4, and these splicing variant forms may impact tumorigenesis.C vaccines have signifi cant potential as therapeutics to treat cancer, but they typically only provide a clinical benefi t in a subset of patients. To optimize the clinical use of cancer vaccines and to better understand the factors that aff ect clinical responses, there have major eff orts to identify predictive biomarkers (markers that could be used to select patients that are likely to have a positive response) and biomarkers of effi cacy (markers that could be used to determine if a patient being treated with a cancer vaccine is having a positive response to the treatment). Current biomarker research has focused a variety of factors, such as T cell responses, circulating tumor cells, and cytokine production. One area that has been largely understudied is immune responses to glycans. Cancer cells undergo major changes in carbohydrate expression during the onset and progression of the disease, and aberrantly expressed glycans can serve as important targets for natural immune surveillance and/or for immune responses induced by vaccines. Our group has developed a carbohydrate microarray or “glycan array” which enables us to profi le immune responses to a wide range of carbohydrate antigens in a high-throughput fashion. Th is presentation will focus on the development of the glycan array and its application to the identifi cation of new biomarkers for cancer vaccine research.L cancer remains the most deadly and most difficult cancer to treat effectively. The standard of care for conventional treatment; radiation, systemic chemotherapy and surgery is relatively ineffective for long term survival. CDC statistics indicate a 75% increase in lung cancer mortality during the past 20 years. New concepts for enhancing quality of life and for prolonged survival are needed. Reported here is progress for a new therapeutic paradigm, intratumoral (IT) chemotherapy, a novel localized treatment modality. The procedure, endobronchial intratumoral chemotherapy (EITC) involves direct intratumoral drug injection via a needle bronchoscope. A superdose of drug is thereby made to perfuse the tumor mass and achieve rapid tumor necrosis and massive tumor cell killing. Because of the localized drug delivery, there are no systemic toxic complications with cisplatin or mitoxantrone that are normally associated with conventional IV chemotherapy. Palliation and prolonged survival have been observed clinically for EITC, especially for patients presenting with significant airways obstruction. Collaborative clinical studies has been conducted with Dr. Celikuglu, who has pioneered EITC in Istanbul, and with Dr. Hohenforst-Schmidt in Germany. Favorable clinical outcomes for EITC have now been observed for hundreds of NSCLC patients. In parallel preclinical IT chemotherapy research in Florida, using a murine Lewis lung carcinoma, mitoxantroneloaded albumin nanomesospheres afforded prolonged IT tumoricidal activity and evidence for systemic tumor-specific immune response. Additional research indicates that IT neoadjuvant chemotherapy followed by resection of the necrotic tumor mass may afford a curative response. It is reasonable to conclude that IT chemotherapy represents an important new approach to improved lung cancer treatment.For this purpose, 36 male Wistar growing rats divided into 6 groups (n=6 each) and treated with 0.9% NaCl (control), methylprednisolone 7 mg/kg (MP), MP+ alendronate 20μg/kg, MP+80 mg/kg EPA, MP+160 mg/kg EPA and MP+ 320 mg/kg EPA for 6 weeks. At the end of the experiment, serum CTX, Osteocalcin, Calcium and phosphate concentrations, mineral content of L4 vertebrate and tibia and fibula, trabecular and osteoid widths of femur and cortical widths and marrow area/cortical area ratio of tibial diaphysis determined.C of bioactive compouds could be close to 1 g/day in our diet, making them the largest source of anti-oxidants. Dietary sources include fruits, vegetables, cereals, legumes, chocolate, and plant based beverages such as juices, tea, and wine. Extensive biomedical evidence suggests that bioactive compounds no matter their class may contribute to the prevention of cardiovascular disease, cancer, osteoporosis, diabetes, and neurodegenerative diseases. They have been also shown to exhibit beneficial effects on capillary permeability and fragility, to have anti-platelet, hypolipidemic, anti-hypertensive, antimicrobial, anti-viral, anti-allergenic, anti-ulcerogenic, cytotoxic, anti-neoplastic, anti-inflammatory, anti-atherogenic, and anti-hepatotoxic activities.These potential health benefiting properties may call for development of these compounds into future therapeutic agents. The content of bioactive compounds is also potentially influenced by food processing and storage conditions, which can result in transformation of flavonoids, and loss of flavonoid content. This presentation will briefly cover some relevant current statistics about cancer, dietary recommendations and the different family of bioactive compounds that have exhibited chemopreventive properties in selected foods coverings pre-clinical and clinical studies that have been performed to identify their potential chemopreventive effects after dietary consumption.T emergence of highly aggressive, cancer stem cell-like, subtypes of human squamous cell carcinoma (SCC) reflects increased transforming growth factor-β1 (TGF-β1) synthesis and epidermal growth factor receptor (EGFR) amplification. Cooperative TGF-β/EGFR signaling promotes cell migration and induces expression/activation of proteases (e.g., plasminogen, MMPs) and protease inhibitors that regulate stromal remodeling resulting in the acquisition of an invasive phenotype. Paradoxically, plasminogen activator inhibitor type-1 (SERPINE1,PAI-1), the major inhibitor of plasmin generation, is also upregulated under these conditions and is an early event in tumor progression. Increased PAI-1 expression temporally and spatially modulates plasmin-initiated pericellular proteolysis, preserving a stromal scaffold permissive that facilitates invasive potential. Combined TGF-β1+EGF treratment was used to investigate mechanisms underlying induced epithelial-to-mesenchymal transition (EMT) in ras-transformed human keratinocytes. Dual stimulation with TGF-β1+EGF resulted in keratinocyte “plasticity” and pronounced colony dispersal. Transcriptome analyses indicated that cells undergoing EMT expressed high β1 integrin levels and possessed stem cell-like characteristics. The most up-regulated transcript encoded PAI-1, an established marker of aggressive carcinoma cells and a functional promoter of cell migration suggesting that PAI-1 plays a critical role in epithelial stem cell biology. PAI-1 knockdown alone effectively inhibited TGF-β1+EGF-dependent cell scattering, indicating a functional role for this SERPIN in the dual-growth factor model of induced motility. Identification of signaling networks and their effect on specific invasion-promoting target genes, such as PAI-1, may lead to the development of pathway-specific therapeutics that impact late-stage events in human cutaneous epithelial tumor progression. Supported by grants from the NIH (GM57242) and the NYSDOH Empire State Stem Cell Trust Fund (C024312).Computed tomography perfusion (CTp) is a technique that allows quick qualitative and quantitative evaluation of tissue perfusion by generating maps of blood flow (BF), blood volume (BV), and mean transit time (MTT). Perfusion CT has been found to be useful for non-invasive diagnosis of many diseases like cerebral ischemia and infarction, tumoral neo-angiogenesis, differentiation between malignant and benign process and for tumour response to radioand chemotherapeutic treatment. Recent studies point, that CTp parameters may provide reliable information on vascularization of lymph nodes and may reflect angiogenic activity, helping to understand the changes occurring when malignant process invades the lymph node.Methods and Results: In our study, we demonstrate the formulation of a novel alginate enclosed, chitosan coated ceramic anti cancer nano carriers (ACSC NC). These NC were loaded with multi-functional anti cancer bovine lactoferrin (bLf), a natural milk based protein, for improvement of intestinal absorption, in order to develop a novel platform to carry anti cancer protein and/or peptides for oral therapy. Size, morphology, internalization and release profiles of the ACSC NC under varying pH were determined. Furthermore, uptake of these NC in vitro in colon cancer cell lines was analyzed, by measuring the endocytosis and transcytosis. NCs were characterised through various physical and biological assays. Transcytosis studies indicate the transcytosis of the NC, with minimal damage to the Caco-2 cell monolayer. In conclusion, these NC can be used for future targeted protein/peptide or nucleic acid based drug delivery to treat fiddly diseases such as cancer and neurodegenerative disorders. Lf+ loaded ACSC NC significantly reduced tumour vascularity and blood flow, and increased anti-tumour cytotoxicity, tumour apoptosis and the infiltration of tumours by leukocytes. Lf+ increased the average weight of the spleens of tumourbearing mice by ~20%, accompanied by a major increase in the numbers of particular leukocyte subsets in the spleen. CD4+, CD8+, NK, IFN-γ+-expressing and dendritic cell numbers in the spleen were significantly (P<0.001) increased compared to corresponding cell numbers for mice maintained on the control diet. Lf+ bound to the intestinal epithelium and was preferentially taken up within Peyer’s patches. It increased the production of Th1 and Th2 cytokines within the intestine and tumour, including TNF, IFN-γ, as well as nitric oxide that have been reported to sensitize tumours to doxorubicin chemotherapy. Importantly, it restored both red and white peripheral blood cell numbers depleted by doxorubicin chemotherapy, potentially fortifying the mice against cancer. In summary, bLf is a potent natural adjuvant and fortifying agent for augmenting cancer chemotherapy, but needs to be saturated with iron and administered orally in Lf+ loaded ACSC NC to be effective. Bio-distribution of iron saturated lactoferrin was determined my MRI and confirmed by other imaging techniques. We also compared our results with the doxorubicin and taxol loaded ACNC-NPs.Methods: 323 patients with GEJ who underwent surgical resection had been treated as follows: 256 with proximal gastrectomy, 67 with total gastrectomy 185 with a combined-visceral resection, of which 154 underwent a splenectomy plus partial pancreatectomy, 21 with splenectomy alone and 10 with partial hepatectomy and diaphragmatectomy.The extent of lymphadenectomy was divided into five types( D0 to D4) and the curability of operation was graded as A, B and C.B cancer (BC) is the most common cancer among women worldwide. There are few studies of BC susceptibility alleles in South American populations. Here we describe the identification of risk variants in genes BRCA1, BRCA2 (high penetrance), CHEK2, RAD51, XRCC3 (moderate penetrance), rs2981582 and rs1219648 (low penetrance) in Chilean families at high-risk for breast/ovarian cancer (n=326). Germline BRCA1/2 point mutations were found in 7.1% of families. Families with at least three BC and/or OC cancer cases showed the highest frequency of mutation (15.9%). We identified 14 point mutations, of which 3 in BRCA1 and 3 in BRCA2 were recurrent, possibly reflecting region-specific founder effects. In CHEK2, the 1100delC mutation was not detected in the 1320 samples analyzed. The Thr241Met (XRCC3) polymorphism was associated with increased BC risk (OR=2.44, 95%CI=1.34–4.43). The RAD51 135G>C polymorphism increased BC risk among BRCA1/2negative women with a) a family history of BC and b) age at onset <50 years (OR=2.17, 95%CI=1.11-4.29). The combined Thr/Met–E/G (RAD51D) genotype was associated with increased BC risk among the same group of women (OR=10.5 [95%CI 1.16–94.5]). Our results suggest that variability in XRCC3 and RAD51D plays a role in BC risk via a mutual interaction between the genes. The combined SNPs rs2981582 – rs1219648 (A/A–G/G) of FGFR2 genotypes were associated with increased risk for estrogen-receptor positive BC (OR=2.6, 95%CI=1.2–5.6]). Our results are consistent with a polygenic model for familial BC susceptibility.T in situ proximity ligation assay (in situ PLA) is a novel method for detecting protein-protein interactions in native fixed cells and tissue samples. The assay provides localized single molecule data visualized by fluorescence microscopy and quantified by objective counting. Target protein interaction pairs are bound by primary antibodies in a standard immunostaining reaction, and when bound within a few tens of nanometres distance of each other, an amplified single molecule DNA based reporter is generated. The amplification product of the reporter is visible as a bright spot and remains locally attached to the site of the interaction also revealing sub cellular localization.Ciprofloxacin was given orally to 28 healthy male volunteers for single oral dose of 500mg; Plasma samples were collected at different times interval between 0 and 12h and analyzed both by high pressure liquid chromatography and by a microbiological assay. The detection limits (LOD) were 0.02μg/ml and 0.1μg/ml, for both methods respectively. For each method, coefficients of variation (R(2)) were 0.9995 and 0.9918 in plasma and limit of quantitation (LOQ).02 and 0.5μg/ml. The Comparison of means maximum concentration 2.68 μg/ml at 1.5 hr for test and 2.43 μg/ml are attain in HPLC method of Reference at 2hrs respectively. The plasma concentrations measured by microbiological assay of reference tablet are 3.95μg/ml (mean ± SE) at 1 hour and 3.80μg/ml (mean ± SE) at 1 hour. The concentrations in plasma measured by microbiological method were markedly higher than the high-pressure liquid chromatography values which indicates the presence of antimicrobially active metabolites. The mean ± SE values of pharmacokinetic parameters calculated by HPLC method, for total area under the curve (AUC 0-oo) were 13.11, and 11.91 h.mg/l for both test and reference tablets respectively. The mean ± SE values of clearance measured in l/h were 44.91 and 48.42 respectively. The elimination rate constant Kel [l/h] showed 0.17 l/h for test and 0.15 l/h reference tablets and likewise, absorption half-life expressed in hours shown 0.67 h for test and 1.04 h for reference respectively. The Mean Residence Time for test is 5.48 h and 5.49 h for reference. The mean ± SE values of pharmacokinetic parameters (Microbiological assay) for total area under the curve (AUC 0-oo) were 22.11 and 19.33 h.mg/l for both test and reference tablets respectively. The mean ± SE values of clearance measured in l/h were 29.02 and 31.63 respectively. The elimination rate constant Kel [l/h] showed 0.21 l/h for test and 0.20 l/h reference tablets and likewise, absorption half-life expressed in hours shown 0.86h for test and 0.56 h for reference respectively. The Mean Residence Time for test is 5.27 h and 4.67 h for reference. Significant difference observed between two methods.Title: MicroRNAs induce tamoxifen sensitivity by down-regulation of estrogen receptor alpha signaling in breast cancer Dr. Gary Guishan Xiao, Creighton University Medical Centre, USA Title: Characterisation and impact of circulating tumor cell diversity on therapy response and metastasis formation Dr. Katharina Pachmann, Universitätsklinikum Jena, Germany Title: Lovastatin inhibits T cell proliferation while preserving the cytolytic function of EBV-,CMVand MART-1-specific CTLs Dr. Qing Ma, University of Texas M.D. Anderson Cancer Center, USA Title: Anti-tumor effect of sFlt-1 gene therapy system mediated by Bifidobacterium Infantis on Lewis lung cancer in mice Dr. Hong Zhu, West China Hospital, China Title: An investigation of PD-L1 expression and its association with tumor infiltrating T cells in human cervical carcinomas Dr. Xu Man, Chongqing Medical University, China Title: To compare efficacy and cost effectiveness of different 5ht3 blockers in acute and delayed nausea and vomiting:a randomized study Dr. Shukla Piyush, All India Institute of Medical Sciences, India Title: Functional genomics to identify cancer targets Dr. William C. Hahn, Dana-Farber Cancer Institute, USAC cachexia is a paraneoplasic syndrome affecting the large majority of terminally ill cancer patients and is clinically characterized by a number of symptoms which are not overcome by standard nutritional supplementation or by pharmacological therapy. L-Carnitine has been tested in preliminary studies in human cachexia, resulting in improved fatigue and quality of life. Our results show that in experimental cachexia the marked alterations of lipid metabolism are suppressed by L-carnitine supplementation, and associated with increased survival. The anti-inflammatory effects of L-carnitine supplementation seem to be similar to those elicited by chronic physical exercise in cachectic animals and patients. We have shown that the expression of lipid metabolism-related proteins is restored to normal levels in the liver and muscle after exercise training, re-establishing cellular function. These results are associated with decreased local and systemic inflammation, to which the white adipose tissue markedly contributes in cachexia. The molecular basis of the effects of L-carnitine supplementation and of exercise training upon cancer cachexia, with special focus on the relevance of white adipose tissue, will be examined.Methods: Tumor specimens from 100 untreated patients with ovarian cancer were evaluated for HSD17B12 by immunohistochemistry and correlated with clinicopathologic characteristics, patient outcome and 5 year follow-up. Ovarian carcinoma cell lines OvCa, A2780 and AD10 were used in this study. Since A2780 OvCa cell line expressed the highest level of HSD17B12, this cell line was used for further studies. siRNA knockdown of the enzyme was performed and its effects on tumor cell proliferation and Annexin V binding were determined.P chemotherapy, such as cisplatin, is the primary treatment for ovarian cancer. However, drug resistance has become a major impediment to the successful treatment of ovarian cancer. To date, the molecular mechanisms of resistance to platinum-based chemotherapy remain unclear. In our previous study using a proteomic approach, more than 90 proteins showed significant expression changes when two pairs of ovarian cancer cell lines, A2780/A2780-CP (cisplatinsensitive/cisplatin-resistant) and 2008/2008-C13*5.25 (cisplatin-sensitive/cisplatin-resistant), were compared. Bioinformatics analysis suggested several potential pathways that may be involved in platinum resistance. Among these potential pathways, a redox regulated pathway involving superoxide dismutase 1 (SOD1) was targeted in order to further explore its involvement in drug resistance. Inhibition of SOD1 activity in the resistant cells by either small-molecule inhibitors or siRNA enabled partial reversal of platinum resistance. Our data suggest that targeting SOD1 can potentially lead to sensitization of platinum-resistant ovarian cancer cells, and SOD1 may be used as a therapeutic target for chemosensitization of ovarian cancer.E proteins including Tsg101 are well-established for their role in formation of multivesicular bodies and sorting ubiquitinated endosomal cargoes to lysosomes via their ubiquitin-binding domains. However, genetic ablation studies in mouse models and human cell lines show ESCRT proteins and Tsg101 are required for proliferation, cell viability, and malignant phenotypes of cancerous cells. Utilizing a conditional Tsg101 knockout mouse embryonic fibroblast (MEF) cell line, we show that Tsg101 is indispensible for Src function. Dynamic trafficking of Src at endosomes and translocation of active Src to focal adhesions and invadopodia are impaired when expression of Tsg101 is lost. Blocking Vps4 function by expression of a dominant negative form of Vps4 has similar effect. Viral expression of wildtype Tsg101, but not the N45A mutant, which has lower binding affinity to ubiquitin, restores invadopodia formation and invasivenss of Tsg101-deleted v-Src MEFs. Together, our study reveals a surprising positive role of Tsg101 and probably ESCRT pathway in promoting Src signaling that requires its ability to interact with ubiquitin.N sequencing technology is a powerful and cost-efficient tool for ultra-high-throughput transcriptome analysis. We applied paired-end RNA-seq to generate a deep unbiased transcriptome map of a EBV positive nasopharyngeal carcinoma (NPC) cell C666 and normal cell NPEC2. Using effective bioinformatics pipelines, we unambiguously detected many differentially expressed genes, novel transcripts, a variety of transcript isoforms and chimeric transcripts. Most importantly, we have identified a novel fusion gene which might play a oncogenic function in pathogenesis of NPC. Finally, we found that 78% EBV genes are transcribed, which indicate that the expression pattern of EBV in NPC is more complex than previously expected.Results: Comparing IC50 values showed that MDA-231 cells are more sensitive than MCF7 cells to DOX or DOCT. Immonofluorescent results confirmed the expression of Mdr-1 in these two cell lines after DOX or DOCT treatment. In MDA231 cells the expression of ERK1/2 and pERK was decreased after DOX treatment in a dose-dependent manner. In contrast in MCF7 cells the expression of ERK1/2 and pERK was increased after DOX treatment. DOCT treatment resulted the same result with less significant differences than DOX.T signal transducer and activator of transcription (Stat5) is a cytoplasmic signaling molecule and a transcription factor which confers the effects of cytokines, polypeptide hormones and growth factors into transcriptional activity. In the mammary gland, Stat5 controls epithelial cell proliferation, lactogenesis, lactation and cell survival. In transgenic mice, we have shown that deregulation of Stat5 activity during the reproductive cycle results in parity-dependent development of mammary tumors in post-estropausal females. Overexpression of the native and constitutively active form of Stat5 cause mainly highly differentiated tumors, while its C-terminally truncated form induced mainly the poorly differentiated carcinomas. Several mechanisms may account to the oncogenic effect of Stat5. (i) DNA damage. Production of reactive oxygen species during the vulnerable stage of pregnancy induced significant DNA damage and cellular DNA damage response within three days. Chromosomal breakage, fragmentation and translocations occasionally result in tumorigenesis. (ii) Parity-dependent Histone modifications at the Stat5 binding sites in the cyclin D1 and bcl-x gene promoters during the reproductive cycle. These modifications expose individual cells to the deregulated effect of Stat5 which highly activates these genes. (iii) Silencing tumor suppressers and proliferation antagonists. These distinct metabolic consequences occur during tumor development and mediate the earlier effect of deregulated Stat5. Thus, in association with the protective effect of parity against breast cancer that was reported in epidemiological studies, parity-dependent deregulated levels and activity of Stat5 represent a long-term risk factor. Monitoring Stat5 activity in the breast during the vulnerable stage of pregnancy may help in reducing its oncogenic effect.T study was undertaken to evaluate Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) levels in the peritoneal fluid (PF) and plasma of patients with different stage, grade and histological type of ovarian cancer (n=73) or serous cystadenoma (n=32) in relation to PF and peripheral blood (PB) myeloid and lymphoid dendritic cells (DCs). The PF and plasma level of RANTES was detected using ELISA assay. DCs were estimated using flow cytometry. The following directly conjugated mAbs were used: anti-BDCA-1 (CD1c) FITC, anti-BDCA-2 (CD303) FITC and anti-CD19 CyChrome, anti-CD123 PE.I chemokines, including CCL2, CCL5, and CXCL8 are major contributors to breast malignancy, acting at many different levels. We have analyzed the regulation of CCL2 and CCL5 expression by inflammatory cytokines in breast tumors. Our analyses indicated that TNFα and IL-1β are expressed by the tumor cells in 90% of breast cancer patients, and that both cytokines potently up-regulated the release of CCL2 and CCL5 by breast tumor cells and by normal breast epithelial cells that are as yet non-transformed. Also, we found that TNFα and IL-1β act directly on breast tumor cells and on non-transformed breast epithelial cells to promote cell characteristics leading to increased invasiveness. Combined with additional findings, we suggest that TNFα and IL-1β from autocrine sources are important up-regulators of CCL2 and CCL5 release in early and advanced stages of disease, as well as of progression-related processes. In parallel, we have analyzed the roles played by genetic and signaling components in the regulation of CCL2, CCL5 and CXCL8 in model systems of fibroblasts and of breast tumor cells. In this part, we focused on two components that undergo oncogenic deregulation in breast cancer, namely the tumor suppressor p53 and the Ras signaling pathway. Our findings provide evidence to intricate modes of interaction between p53, Ras and the chemokines, suggesting that inappropriate regulation of these genetic and signaling components promotes the release of pro-malignancy chemokines in breast cancer. Overall, our findings indicate that cooperation between inflammatory mediators and genetic/signaling components in breast cancer support breast tumor growth and metastasis.