Chemotherapeutic Studies Research Articles

The possibilities of immunohistochemistry for assessing the pathogenetic mechanisms of action of compounds with a suspected antitumor effect. Part I. General indicators of the process activity

Despite the wide arsenal of chemotherapeutic agents, the search and study of new compounds with an alleged antitumor effect is relevant. Morphological diagnostics of pathological processes occurring under the action of pharmacologically active substances is the most important component of preclinical research of compounds with an alleged antitumor effect. It is advisable to use information about the possible cytotoxic effect of candidates for antitumor drugs using an immunohistochemical method for studying organs and systems of experimental animals at different stages of the development of the tumor process by indirect markers of tumor progression activity. Morphological examination of parenchymal organs and tumor tissue in the dynamics of the development of malignant neoplasm is more informative and evidence-based than biochemical research. The aim of the study is to conduct a comparative analysis of markers of tumor process activity for more effective use of morphological and immunohistochemical research methods in the preclinical study of compounds with suspected antitumor activity to assess the prospects for their use with the detection of tumor process activity. The literature search was carried out using the Scopus, Web of Science, PubMed and eLIBRARY databases. The paper presents an overview of current molecular biological markers for assessing the activity of the malignant process in the experiment: Transforming Growth Factor beta 1 (TGF-β1), Ki-67, Tumor necrosis factor alpha (TNF-α), p53, Poly-ADP-ribose polymerase 1 (PARP-1) and Anti-8-Hydroxy-2'-deoxyguanosine (8-OHdG), beta III Tubulin, p120 Catenin, Beta Actin. The listed markers are indirect and can be used in a single mode only for screening studies of antitumor and antimetastatic activity in which a large number of compounds are sorted according to the principle of effectiveness. When conducting an in-depth study of the pharmacological activity of the leader compounds it is necessary to perform a comprehensive immunohistochemical study. Our analysis of the literature data confirms the importance of selecting optimal, sensitive, economically feasible and affordable markers, which in turn leads to the improvement of diagnostic panels and their standardization to simplify their transition into clinical practice.

Wild Daucus Carota Oil Extract: An Innocuous Preventative and Chemotherapeutic Agent Against Chemically Induced Breast Cancer-siftdesk

Daucus carota L. ssp. carota oil extract (DCOE) was shown to possess potent antitumor activity. This study investigates the chemopreventive and chemotherapeutic effects of DCOE against breast cancer induced by 7,12-Dimethylbenz(a)anthracene (DMBA) in rats. In the chemopreventive study, animals were pre-treated with DCOE (25 mg/kg; 1 week) followed by 14 weeks of treatment post-cancer induction with DMBA. In the chemotherapeutic study, animals were allocated into control, DCOE (25 mg/kg) and Cisplatin (2.5mg/kg) groups. When tumor size reached a diameter of 14-15 mm, the animals were divided into three groups each injected by either a vehicle (thrice a week), DCOE (thrice a week) or Cisplatin (twice a week), for 8 weeks to compare and assess the therapeutic value of DCOE with respect to Cisplatin and the vehicle. At 24 weeks, the experiment was terminated. DCOE pre-treatment protected against DMBA-induced toxicity and reduced tumor incidence. While 18% of control died few days post DMBA treatment, 100% survival in DCOE pre-treated group was observed just before week 8. In the chemotherapeutic experiment, treatment with either DCOE or Cisplatin caused significant inhibition of tumor growth. Unlike DCOE, Cisplatin caused drastic decrease in body weight with 75% and 100% death rates at week 6 and 8 respectively. DCOE produced a significant increase in Bax/Bcl2 ratio, Cytochrome c and cleaved caspase 3 proteins. DCOE may be considered a safe chemopreventive and/or chemotherapeutic agent, an effect that is partly due to the induction of the intrinsic apoptotic pathway.

Experimental Hookworm Infection in Laboratory animals: Parasite behavior, Immune response and Chemotherapeutic Studies

Hookworm disease is known to be caused allergic manifestation and severe anemic pathogenicity in man and canine hosts. Attempts have been made to establish laboratory models of Necator americaus, Ancylostoma duodenale, and Ancylostoma ceylanicum, together with canine parasite, Ancylostoma caninum. The studies include pathophysiological aspects of the host-parasite relationship, and develop to establish patent infection. Immunological approach to selecting antigen for diagnosis and protective immunity purpose using larval and adult worm antigens and their secretions became the focus with the subsequent discovery of cloning in vaccine development as main research interest. Chemotherapy of newer drug screening in laboratory models ultimately selected to use for preventive chemotherapy in hookworm endemic areas using recommended drugs.

Gemcitabine in the Era of Cancer Immunotherapy

Gemcitabine has been used as a chemotherapeutic to treat solid cancers for more than 20 years. Whilst its use as chemotherapy has been declining due to the development of more potent chemotherapeutic combinations further studies of gemcitabine have identified varied immune modulatory effects on both tumour cells and leukocyte subsets

Recent Advances and Challenges in Controlling the Spatiotemporal Release of Combinatorial Anticancer Drugs from Nanoparticles.

To overcome cancer, various chemotherapeutic studies are in progress; among these, studies on nano-formulated combinatorial drugs (NFCDs) are being actively pursued. NFCDs function via a fusion technology that includes a drug delivery system using nanoparticles as a carrier and a combinatorial drug therapy using two or more drugs. It not only includes the advantages of these two technologies, such as ensuring stability of drugs, selectively transporting drugs to cancer cells, and synergistic effects of two or more drugs, but also has the additional benefit of enabling the spatiotemporal and controlled release of drugs. This spatial and temporal drug release from NFCDs depends on the application of nanotechnology and the composition of the combination drug. In this review, recent advances and challenges in the control of spatiotemporal drug release from NFCDs are provided. To this end, the types of combinatorial drug release for various NFCDs are classified in terms of time and space, and the detailed programming techniques used for this are described. In addition, the advantages of the time and space differences in drug release in terms of anticancer efficacy are introduced in depth.

Abstract 6338: High-throughput screening program to target ovarian cancer leader cells

Abstract Despite its relatively low incidence ovarian cancer is one of the most lethal gynecological malignancies, characterized by high recurrence rate following primary therapy and almost universal acquisition of chemoresistance. Recently, we identified a subpopulation of “Leader Cells” (LCs) that control invasion, progression and acquired chemoresistance. Therapies targeting leader cells are likely to be pivotal to improving prognosis, particularly in patients who are refractory to standard-of-care. To investigate the role of LCs in chemoresistance, we used CRISPR to integrate an in-frame T2A-GFP expression cassette downstream of the native LC marker gene, Keratin 14 (KRT14). Genomic integration drives the LC specific expression of GFP, facilitating visualization and quantitative tracking of the LC subset. The status of GFP as a surrogate marker of LCs in vitro was confirmed by immunostaining and expression analyses. Chemotherapeutic intervention studies and co-staining with the apoptosis marker cleaved caspase 3, demonstrated a significant enrichment of LCs that represent a non-apoptotic subset of cells resistant to standard chemotherapy. High-throughput drug screening, combining large functional drug libraries with high-throughput viability and invasion assays, was used to identify drugs that could inhibit LC function in vitro. Ovarian cancer cell lines (SKOV3 and COV362.4 expressing KRT14-T2A-GFP) were screened against 1450 FDA-approved drugs, and 218 hits identified that inhibited LC viability, invasive capacity or both. Amongst the hits that we identified, the neural signaling pathway emerged as a potential novel mediator for LC function. Further validation experiments confirmed that inhibition of the neural signaling pathway did not affect viability but synergized with chemotherapy to reduce cisplatin IC50 by 10-20-fold. Our data demonstrate that the high-throughput screen developed is a viable approach to identify opportunities for drug repurposing. Moreover, the neural signaling pathway appears to be important in LC function and inhibiting this pathway results in increased sensitivity to chemotherapy in vitro. Ongoing work will evaluate the applicability of this new tool for clinical screening programs, and further, validate existing drugs for potential repurposing. Citation Format: Nazanin Karimnia, Maree Bilandzic, Magdalena Plebanski, Andrew N. Stephens. High-throughput screening program to target ovarian cancer leader cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6338.

Characterization of an Alginate Encapsulated LS180 Spheroid Model for Anti-colorectal Cancer Compound Screening.

Colorectal cancer is one of the leading causes of cancer-related deaths. A main problem for its treatment is resistance to chemotherapy, requiring the development of new drugs. The success rate of new candidate cancer drugs in clinical trials remains dismal. Three-dimensional (3D) cell culture models have been proposed to bridge the current gap between in vitro chemotherapeutic studies and the human in vivo, due to shortcomings in the physiological relevance of the commonly used two-dimensional cell culture models. In this study, LS180 colorectal cancer cells were cultured as 3D sodium alginate encapsulated spheroids in clinostat bioreactors. Growth and viability were evaluated for 20 days to determine the ideal experimental window. The 3- (4,5- dimethylthiazol- 2- yl)-2,5-diphenyltetrazolium bromide assay was then used to establish half maximal inhibitory concentrations for the standard chemotherapeutic drug, paclitaxel. This concentration was used to further evaluate the established 3D model. During model characterization and evaluation soluble protein content, intracellular adenosine triphosphate levels, extracellular adenylate kinase, glucose consumption, and P-glycoprotein (P-gp) gene expression were measured. Use of the model for chemotherapeutic treatment screening was evaluated using two concentrations of paclitaxel, and treatment continued for 96 h. Paclitaxel caused a decrease in cell growth, viability, and glucose consumption in the model. Furthermore, relative expression of P-gp increased compared to the untreated control group. This is a typical resistance-producing change, seen in vivo and known to be a result of paclitaxel treatment. It was concluded that the LS180 sodium alginate encapsulated spheroid model could be used for testing new chemotherapeutic compounds for colorectal cancer.

842P - CTC-based biomarkers & PSMA-targeted imaging in patients with metastatic castrate-resistant prostate cancer (mCRPC)

Background: Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancers, making it an imaging & therapeutic target of interest. The utility of the PSMA-targeted imaging agent 99mTc-EC0652 is being evaluated, along with biomarker analysis of circulating tumor cells (CTCs), in pts with mCRPC in a completed PSMA-targeted chemotherapeutic study. We now report the PSMA heterogeneity via CTC vs. imaging in the pt population treated to date. Methods: A total of 51 pts evaluated at the time of the data cut had baseline CT & bone scans performed in addition to a 99mTc-EC0652 SPECT/CT as a measure of imaging-based PSMA expression. 41 of 51 pts provided pre-treatment blood samples evaluable for CTC biomarker analysis, which included PSMA expression & predicted genomic instability status (pGI). Images collected from pts were analyzed for both their PSMA+ status as well as sensitivity relative to conventional imaging. Results: 41 of 51 pts (80.4%) had detectable CTCs in their samples prior to therapy while 32 of 38 (84.2%) of the analyzed paired on-therapy samples had CTCs after treatment. At baseline PSMA+ CTCs were found in 19 of 51 (37.2%) pts and overall was expressed on 33% (IQR 11.3% - 40.1%) of CTCs within each pt sample. 15 of 17 (88%) PSMA+ pt samples were also pGI+. 31 of the 51 pts (60.7%) of the pts had bone scan (BS) data available to analyze. 27 of the 51 pts had CT scan data available to analyze for nodal lesions. Concordance between Conventional Imaging & 99mTc-EC0652.Table: 842P99mTc- EC0652+99mTc- EC0652-BS+BS-CT+CT -Bone Lesion (770)770 (100%)0587 (76.2%)183 (23.8%)N/AN/ANodes >1.5cm (54)48 (88.9%)6 (11.1%)N/AN/A54N/ANodes <1.5cm (69)69N/AN/AN/AN/AN/A Open table in a new tab Conclusions: In this cohort, most pts had CTCs for assessment. The majority of pts with CTCs had PSMA-CTCs. The pts CTCs expressing PSMA accounted for a small percentage of the total CTCs observed. These PSMA+ CTCs had high pGI. Pts with CTCs had PSMA imagable disease, especially in bone. The discordance between CTCs & imaging illustrate the disease heterogeneity in regards to PSMA expression. These data should be considered in designing future PSMA-directed regimens. Clinical trial identification: NCT02202447. Legal entity responsible for the study: Endocyte Inc. Funding: Endocyte, Inc. Disclosure: M.J. Morris, O. Sartor: Consultant: Endocyte. A. Armour, R. Messmann, M. Groaning: Employee: Endocyte. All other authors have declared no conflicts of interest.

PSMA heterogeneity analysis in patients with metastatic castrate-resistant prostate cancer (mCRPC): Imaging versus CTCs.

272 Background: Prostate-specific membrane antigen (PSMA) is highly expressed in prostate cancers, making it an ideal imaging and therapeutic target of interest. The utility of the PSMA-targeted imaging agent 99mTc-EC0652 is being evaluated, along with biomarker analysis of circulating tumor cells (CTCs), in pts with mCRPC in a PSMA-targeted chemotherapeutic study. We now report the PSMA heterogeneity via CTC vs. imaging in the pt population treated to date. Methods: Patients were enrolled in 1 of 2 cohorts: mCRPC taxane naïve or taxane exposed. A total of 48 pts evaluated at the time of the data cut had baseline CT &amp; bone scans performed in addition to a 99mTc-EC0652 SPECT/CT as a measure of imaging-based PSMA expression. 40 of 48 pts provided pre-treatment blood samples evaluable for CTC biomarker analysis, which included PSMA expression &amp; were given percent homologous recombination deficiency (%HRD) scores. Four of the best and four of the worst responders were evaluated in more detail for imaging and CTC-based biomarker analysis as it related to clinical outcome. Results: 35 of 40 pts (88%) had detectable CTCs in their samples. 15 of the 35 (43%) pts had PSMA-positive CTCs. The 4 “non-responders” were on study for an average of 41 days and the 4 “responders” for an average of 256 days. Each group contained 2 taxane exposed &amp; 2 taxane naïve patients. Of the 335 bone lesions analyzed by MDP, CT, 99mTc-EC0652, 333 (99.4%) were characterized as PSMApos based on their 99mTc-EC0652 SPECT/CT scan. Of the 26 soft tissue lesions analyzed by CT, all were characterized as PSMApos based on their 99mTc-EC0652 SPECT/CT scan. Seven (26.9%) of those lesions were CT positive (observed on CT scan). Each group had 2 pts that were CTC-based PSMApos &amp; 2 that were PSMAneg. Their percent homologous recombination deficiency (%HRD) scores were of 0.60 [0.333, 1.0] for “non-responders” &amp; 0.01 [0.0, 0.039] for “responders”. Conclusions: PSMA-based imaging showed a high percentage of positive lesions whereas CTC-based PMSA positivity is lower by comparison (43%). The discordance between the imaging results &amp; CTC-based biomarkers, &amp; the relative therapeutic predictive value, requires additional exploration. Clinical trial information: NCT02202447.

An overview on Leishmania (Mundinia) enriettii: biology, immunopathology, LRV and extracellular vesicles during the host-parasite interaction.

One of the Leishmania species known to be non-infective to humans is Leishmania (Mundinia) enriettii whose vertebrate host is the guinea pig Cavia porcellus. It is a good model for cutaneous leishmaniasis, chemotherapeutic and molecular studies. In the last years, an increased interest has emerged concerning the L. (Mundinia) subgenus after the finding of Leishmania (M.) macropodum in Australia and with the description of other new/putative species such as L. (M.) martiniquensis and 'L. (M.) siamensis'. This review focused on histopathology, glycoconjugates and innate immunity. The presence of Leishmania RNA virus and shedding of extracellular vesicles by the parasite were also evaluated.

Gastrointestinal parasitic infections in Indian Gaddi (goat) breed bucks: clinical, hemato-biochemical, parasitological and chemotherapeutic studies.

The present study was envisaged with an aim to evaluate gastrointestinal parasitic infections in a herd of conserved Gaddi (goat) breed bucks (6-9months of age) kept on semi-intensive feeding management. The individuals (n=20) representing the herd were selected and divided into two groups; group I (n=10), clinically ill and group II (n=10), sub clinically infected individuals. The clinical examination revealed anemia, emaciation and rise in body temperature of the individuals of group I as compared to group II. The detailed copro-parasitological examination and copro-culture revealed the presence of eggs of Moniezia expansa and larvae of Haemonchus species, respectively in the fecal samples of both clinically and sub clinically infected individuals. The hemato-biochemical parameters proved vital indicators of the health of group I individuals and exhibited decline in the values of hemoglobin, packed cell volume and total erythrocyte count as compared to group II. Significant (P<0.05) hypoproteinemia, hypoalbuminemia, hypoglycemia and increased levels of alanine aminotransferase and aspartate aminotransferase were observed in infected individuals as compared to treated ones. The detailed parasitological, hemato-biochemical observations and clinical findings elucidated and supported the presence of concurrent gastrointestinal parasitism in the herd. The significant improvement was observed in the health status of the herd after 1month of the therapeutic management, which was carried out using a combination of fenbendazole and praziquantel in both clinically and sub clinically infected individuals.

Immunopathological response of leukocytes against microfilariae and adult worms in white rats infected with Setaria cervi.

Aim:Aim of this study was to see the immunopathological changes against the microfilariae (Mf) and adult worms of a bovine filarid, Setaria cervi in the tissues of vital organs of experimentally infected white rats. The effect of diethylcarbamazine (DEC) was also observed on the Mf, as leukocytes especially lymphocytes produce immunoglobulins which opsonize and increase the efficacy of DEC against circulating Mf. Effect of this drug was also assessed on liver enzymes in the microfilaremic rats.Materials and Methods:Microfilaremia was established by implanting adult worms intraperitoneally and by the infusion of Mf recovered from the uterus of gravid female worms. DEC was administered orally for six consecutive days in the rats having patent infection. Differential leukocyte count was recorded every 3rd day, and liver enzymes were estimated every 10th day in both treated and untreated rats. Pathological changes were observed in HE stained sections of vital organs where Mf or adult worms were trapped.Results:Destruction and reduction in microfilarial density were noticed in microfilaremic rats treated with DEC. Trapped Mf and embedded worms revealed heavy cellular infiltrations by defensive cells which surrounded and attached with the body surface of the Mf as well as adult worms for their destruction and piece meal clearance. Immune-mediated pathology was observed in the tissue sections of lungs, spleen, and liver. Liver enzymes were elevated during the period of higher parasitemia.Conclusions:There was a moderate level of immunopathology against the Mf and adult worms by the leukocytes in experimentally infected microfilaremic rats. Mf were in the process of degeneration where they got trapped. Moderate increase in liver enzyme was noticed which was slightly more in untreated group. Although a fraction of Mf gets killed in the peritoneum, majority of them successfully enter the systemic circulation and survive for about 54 days, which is sufficient enough for conducting immunological and chemotherapeutic studies.

A minimum core outcome dataset for the reporting of preclinical chemotherapeutic drug studies: Lessons learned from multiple discordant methodologies in the setting of colorectal cancer.

In vivo studies in animal models are critical tools necessary to study the fundamental complexity of carcinogenesis. A constant strive to improve animal models in cancer exists, especially those investigating the use of chemotherapeutic effectiveness. In the present systematic review, colorectal cancer (CRC) is used as an example to highlight and critically evaluate the range of reporting strategies used when investigating chemotherapeutic agents in the preclinical setting. A systematic review examining the methodology and reporting of preclinical chemotherapeutic drug studies using CRC murine models was conducted. A total of 45 studies were included in this systematic review. The literature was found to be highly heterogeneous with various cell lines, animal strains, animal ages and chemotherapeutic compounds/regimens tested, proving difficult to compare outcomes between similar studies or indeed gain any significant insight into which chemotherapeutic regimen caused adverse events. From this analysis we propose a minimum core outcome dataset that could be regarded as a standardised way of reporting results from in vivo experimentation.

Structure-based optimization of GRP78-binding peptides that enhances efficacy in cancer imaging and therapy

It is more challenging to design peptide drugs than small molecules through molecular docking and in silico analysis. Here, we developed a structure-based approach with various computational and analytical techniques to optimize cancer-targeting peptides for molecular imaging and therapy. We first utilized a peptide-binding protein database to identify GRP78, a specific cancer cell-surface marker, as a target protein for the lead, L-peptide. Subsequently, we used homologous modeling and molecular docking to identify a peptide-binding domain within GRP78 and optimized a series of peptides with a new protein-ligand scoring program, HotLig. Binding of these peptides to GRP78 was confirmed using an oriented immobilization technique for the Biacore system. We further examined the ability of the peptides to target cancer cells through in vitro binding studies with cell lines and clinical cancer specimens, and in vivo tumor imaging and targeted chemotherapeutic studies. MicroSPECT/CT imaging revealed significantly greater uptake of 188Re-liposomes linked to these peptides as compared with non-targeting 188Re-liposomes. Conjugation with these peptides also significantly increased the therapeutic efficacy of Lipo-Dox. Notably, peptide-conjugated Lipo-Dox significantly reduced stem-cell subpopulation in xenografts of breast cancer. The structure-based optimization strategy for peptides described here may be useful for developing peptide drugs for cancer imaging and therapy.

1,2,3-Triazole-Dithiocarbamate Hybrids, a Group of Novel Cell Active SIRT1 Inhibitors

Background/Aims: Human SIRT1 is reported to be involved in tumorgenesis, mainly due to its modulating effect on p53 by deacetylation on lysine382. A large quantity of SIRT1 inhibitors was applied in chemotherapeutic study, but few of them were applied into clinical trials. Methods and Results: In the current study, a novel series of compounds with 1,4-bispiperazinecarbodithioic acid methyl esters scaffold were characterized to have inhibitory potency to SIRT1 by molecular docking and biochemical evaluation. Further cell level study revealed that one of the most potent SIRT1 inhibitors, compound 3a, is cell active. It can upregulate the amount of p53 by accumulating the K382 acetylation of p53, which lead to the stabilization of p53 in human gastric cancer cell line MGC-803 cells. Meanwhile, we also found compound 3a can inactivate SIRT2 in cells, which suggests the compound as a non-selective SIRT inhibitor. Conclusion: All these findings indicate that compound 3a is a potent, reversible and cell active SIRT1 inhibitor and deserves further investigation as an anticancer agent or a biological tool.

Efficacy of Antiviral Drugs against Feline Immunodeficiency Virus.

Feline immunodeficiency virus (FIV) is one of the most common infectious agents affecting cats worldwide .FIV and human immunodeficiency virus (HIV) share many properties: both are lifelong persistent lentiviruses that are similar genetically and morphologically and both viruses propagate in T-lymphocytes, macrophages, and neural cells. Experimentally infected cats have measurable immune suppression, which sometimes progresses to an acquired immunodeficiency syndrome. A transient initial state of infection is followed by a long latent stage with low virus replication and absence of clinical signs. In the terminal stage, both viruses can cause severe immunosuppression. Thus, FIV infection in cats has become an important natural model for studying HIV infection in humans, especially for evaluation of antiviral compounds. Of particular importance for chemotherapeutic studies is the close similarity between the reverse transcriptase (RT) of FIV and HIV, which results in high in vitro susceptibility of FIV to many RT-targeted antiviral compounds used in the treatment of HIV-infected patients. Thus, the aim of this article is to provide an up-to-date review of studies on antiviral treatment of FIV, focusing on commercially available compounds for human or animal use.

Identification of novel therapeutic target genes in acquired lapatinib-resistant breast cancer by integrative meta-analysis.

Acquired resistance to lapatinib is a highly problematic clinical barrier that has to be overcome for a successful cancer treatment. Despite efforts to determine the mechanisms underlying acquired lapatinib resistance (ALR), no definitive genetic factors have been reported to be solely responsible for the acquired resistance in breast cancer. Therefore, we performed a cross-platform meta-analysis of three publically available microarray datasets related to breast cancer with ALR, using the R-based RankProd package. From the meta-analysis, we were able to identify a total of 990 differentially expressed genes (DEGs, 406 upregulated, 584 downregulated) that are potentially associated with ALR. Gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs showed that "response to organic substance" and "p53 signaling pathway" may be largely involved in ALR process. Of these, many of the top 50 upregulated and downregulated DEGs were found in oncogenesis of various tumors and cancers. For the top 50 DEGs, we constructed the gene coexpression and protein-protein interaction networks from a huge database of well-known molecular interactions. By integrative analysis of two systemic networks, we condensed the total number of DEGs to six common genes (LGALS1, PRSS23, PTRF, FHL2, TOB1, and SOCS2). Furthermore, these genes were confirmed in functional module eigens obtained from the weighted gene correlation network analysis of total DEGs in the microarray datasets ("GSE16179" and "GSE52707"). Our integrative meta-analysis could provide a comprehensive perspective into complex mechanisms underlying ALR in breast cancer and a theoretical support for further chemotherapeutic studies.

Abstract B15: Combination chemotherapy in melanoma using EZH2 inhibitor

Abstract Melanoma is the deadliest variety of skin cancer, which according to the American Cancer Society (ACS) will account for around 9710 deaths and diagnosis of around 76,100 new melanoma cases in the current year. According to the ACS, melanoma is also the fastest growing cancer, and hence it is vital to investigate the pathogenesis involved in the advanced stage of melanoma, and design effective therapeutics for its treatment. Aberrant activity of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) has been reported to be associated with different types of cancer. The EZH2 catalyzes the deposition of the repressive mark histone H3 lysine 27 trimethylation (H3K27me3) on the promoter of the target genes using the catalytic SET domain, thereby epigenetically silencing the expression of the target genes. This is because the H3K27me3 mark results in chromatin compaction which reduces the accessibility of various transcription factors to the gene, and hence adversely affects transcription. Several of such EZH2 target genes have been found to be tumor suppressor genes like RUNX3, DAB2IP, and E-cadherin, and silencing of such tumor suppressor genes is a chief mechanism by which EZH2 leads to cancer progression. Hence derepression of the tumor suppressor genes by inhibition of EZH2 could be an effective treatment strategy in different types of cancer. There has been no study till date investigating the role of EZH2 in melanoma. Our laboratory is investigating the role of EZH2 in the pathogenesis involved in the advanced stage of melanoma, to enable the design of effective therapeutic strategies for the treatment of melanoma. Drugs targeting epigenetic modifiers are becoming increasingly popular because of the fact that most epigenetic modifications and their effects can be reversed using these drugs. Our lab has strong evidence that metastatic (or advanced) melanoma is associated with upregulation of EZH2 and H3K27me3. We have also observed that EZH2 inhibition inhibits proliferation and invasion, and also induces apoptosis in metastatic melanoma cells. Recently our lab is performing combination chemotherapeutic studies in vitro, where EZH2 inhibitor GSK126 (S-adenosyl-methionine competitive inhibitor) is being used in combination with conventional chemotherapeutics like cisplatin and Bcl-2 inhibitors. Our results strongly suggest that when EZH2 inhibitor is used in combination with cisplatin or Bcl-2 inhibitor in metastatic melanoma cell lines, the inhibition is synergistic. This synergistic effect of GSK126 and cisplatin or Bcl-2 inhibitor has been found to be caspase-mediated. Citation Format: Deepanwita Sengupta, Nathan L. Avaritt, Alan J. Tackett. Combination chemotherapy in melanoma using EZH2 inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B15.

Epidemiology of mastitis in Kashmir Valley

A retrospective study was made on some of the epidemiological aspects of mastitis in Kashmir valley. The study was conducted at the clinical services complex of SKUAST Kashmir from 1998–2002.Highest incidence was observed in the year 2000 (10.3%) and lowest incidence in the year 2002 (5.98%). The incidence was higher in cows followed by goat and sheep. Most of the cases were observed in the months of July, August and September with less no of cases in October, November and December. The chemotherapeutic studies of the mastitis milk samples showed 100 per cent sensitivity to ciprofloxacin and gentamicin while there was complete resistance to Ampicillin and Neomycin.

Unveiling the effects of berenil, a DNA-binding drug, on Trypanosoma cruzi: implications for kDNA ultrastructure and replication

Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits a single mitochondrion with an enlarged portion termed kinetoplast. This unique structure harbors the mitochondrial DNA (kDNA), composed of interlocked molecules: minicircles and maxicircles. kDNA is a hallmark of kinetoplastids and for this reason constitutes a valuable target in chemotherapeutic and cell biology studies. In the present work, we analyzed the effects of berenil, a minor-groove-binding agent that acts preferentially at the kDNA, thereby affecting cell proliferation, ultrastructure, and mitochondrial activity of T. cruzi epimastigote form. Our results showed that berenil promoted a reduction on parasite growth when high concentrations were used; however, cell viability was not affected. This compound caused significant changes in kDNA arrangement, including the appearance of membrane profiles in the network and electron-lucent areas in the kinetoplast matrix, but nuclear ultrastructure was not modified. The use of the TdT technique, which specifically labels DNA, conjugated to atomic force microscopy analysis indicates that berenil prevents the minicircle decatenation of the network, thus impairing DNA replication and culminating in the appearance of dyskinetoplastic cells. Alterations in the kinetoplast network may be associated with kDNA lesions, as suggested by the quantitative PCR (qPCR) technique. Furthermore, parasites treated with berenil presented higher levels of reactive oxygen species and a slight decrease in the mitochondrial membrane potential and oxygen consumption. Taken together, our results reveal that this DNA-binding drug mainly affects kDNA topology and replication, reinforcing the idea that the kinetoplast represents a potential target for chemotherapy against trypanosomatids.