CTLs, NK cells and NK-derived EVs against breast cancer.

Patients with advanced stage breast cancer need novel therapies. New potential treatments have been developed, such as adoptive cellular therapies and alternative cell-free immunotherapies. The goal of this study was to assess the cytotoxicity of three of the patient-derived immune components, CTLs, NK cells and NK-derived EVs, and evaluate the potential for the development of novel therapy against breast cancer. CTLs were activated against MUC-1 antigen. The in vitro cytotoxic activity of three components was assessed with flow cytometry and in vivo study revealed the efficacy of adoptive cell therapy. Overall, CTLs exhibited the highest cytotoxicity against spheroids of MCF7 breast adenocarcinoma, reaching in all cases higher than double the percentage of NK cells' cytotoxicity. NK-derived EVs exhibited the lowest effect against MCF7 spheroids comparing to the two cell populations. MUC-1 specific CTLs were evaluated with adoptive cell therapy mice study and appeared to be well tolerable and moderately efficacious. More studies need to be performed with CTLs to evaluate safety and efficacy in order to assess their clinical potential, while NK cells and NK-derived EVs are promising candidates that require more experiments to enhance their cytotoxicity.

Adoptive transfer of activated immune cells against solid tumors: A preliminary study.

BACKGROUND: This study presents preliminary results concerning the effectiveness of a novel immunotherapy in cancer. The proposed adoptive cellular therapy product contains a mixture of effector immune cells, specifically macrophages, NK cells, dendritic cells, cytotoxic T lymphocytes and monoclonal antibody producing plasma cells. METHODS: The results were based on both descriptive and inferential statistical analysis of data concerning 17 cancer patients. Particularly, performance scales such as clinical condition, Karnofsky-Index, ECOG index and symptom's scale were evaluated post therapy administration (4 months). Furthermore, circulating tumor cells (CTCs) and a specific tumor marker (EpCAM) were measured pre- and post-cellular therapy. RESULTS: The results revealed a positive evaluation for clinical condition (70.59 %), Karnofsky-Index (88.23 %), ECOG index (94.12 %), and symptoms' scale (64.70 %). In addition, statistically significant reductions were found for both CTCs (p = 0.0016) and EpCAM positive cells (p = 0.0005), post-therapy, which were related to large size effects, namely 0.77 and 0.85, respectively. No cytokine storm, anaphylaxis or severe adverse events were observed with 4 months follow up evaluation. CONCLUSIONS: These preliminary results indicate that the proposed cellular therapy can be considered for further studies in clinical trials.

Teaching an old dog new tricks: The case of Fenbendazole.

The objective of this study is the assessment of the cytotoxic effect of fenbendazole and its commercially available formulation, which is used for its antihelmintic properties. The formulation was tested for its efficacy as well as the determination of the ingredients with proliferation assays and analytical techniques. HPLC, LC-MS and NMR confirmed the stated amount of active ingredient on the label. Dissolution studies were performed to simulate the ability of fenbendazole to dissolve adequately in the fluids of the Gastrointestinal tract, be absorbed in the circulation and reach certain areas of the human body. However, dissolution studies showed that both brands possess issues in their distribution. The in vitro drug screening exhibited potential cytotoxic effect in different types of human cancer cell lines and MDA-MB-231 human breast adenocarcinoma cells appeared to be the most sensitive with IC50 value lower than 10 µM.

Circulating tumour cell gene expression and chemosensitivity analyses: predictive accuracy for response to multidisciplinary treatment of patients with unresectable refractory recurrent rectal cancer or unresectable refractory colorectal cancer liver metastases.

BACKGROUND: Patients with unresectable recurrent rectal cancer (RRC) or colorectal cancer (CRC) with liver metastases, refractory to at least two lines of traditional systemic therapy, may receive third line intraarterial chemotherapy (IC) and targeted therapy (TT) using drugs selected by chemosensitivity and tumor gene expression analyses of liquid biopsy-derived circulating tumor cells (CTCs). METHODS: In this retrospective study, 36 patients with refractory unresectable RRC or refractory unresectable CRC liver metastases were submitted for IC and TT with agents selected by precision oncotherapy chemosensitivity assays performed on liquid biopsy-derived CTCs, transiently cultured in vitro, and by tumor gene expression in the same CTC population, as a ratio to tumor gene expression in peripheral mononuclear blood cells (PMBCs) from the same individual. The endpoint was to evaluate the predictive accuracy of a specific liquid biopsy precision oncotherapy CTC purification and in vitro culture methodology for a positive RECIST 1.1 response to the therapy selected. RESULTS: Our analyses resulted in evaluations of 94.12% (95% CI 0.71-0.99) for sensitivity, 5.26% (95% CI 0.01-0.26) for specificity, a predictive value of 47.06% (95% CI 0.29-0.65) for a positive response, a predictive value of 50% (95% CI 0.01-0.98) for a negative response, with an overall calculated predictive accuracy of 47.22% (95% CI 0.30-0.64). CONCLUSIONS: This is the first reported estimation of predictive accuracy derived from combining chemosensitivity and tumor gene expression analyses on liquid biopsy-derived CTCs, transiently cultured in vitro which, despite limitations, represents a baseline and benchmark which we envisage will be improve upon by methodological and technological advances and future clinical trials.

Adoptive Cellular Transfer Immunotherapies for Cancer.

Patients with cancer require efficient treatment approaches as the mortality rate due to their disease is high. Conventional therapies, like chemotherapy and radiation, have severe side effects. Drug discovery is focusing on the development of alternative strategies that could have beneficial effects to the patients. Cellular therapies are potential therapeutics, and the generation of new products is growing fast. The concept involves the isolation of immune cells, ex vivo activation and reinfusion into the patient. The goal is to boost the immune cells to fight cancer cells. Different immune cells can be used, including dendritic cells, T cells, NK cells, macrophages and B lymphocytes. Some products have already gained FDA approval, while many more are currently in clinical trials. Research is focusing on the improvement of the function of the cells that may require genetic modification or combination with other therapies. Finally, it is crucial to develop novel technologies that could be used in monitoring of the immune profile of patients that have received a cellular therapy to assess the efficacy of the treatment.

Genistein as a dietary supplement; formulation, analysis and pharmacokinetics study.

The objective of this study is to improve and optimize the formulation of Genistein in capsules in order to result in a better pharmacokinetic profile comparing to existing commercial products. In order to do this, five different formulations of Genistein capsules were developed and examined by reviewing their disintegration and dissolution properties. Furthermore, flowability of the powder along with potent incompatibilities between Genistein and its excipients were monitored through their thermal properties. The final formulation of Genistein was quantified using HPLC analysis and then its stability was evaluated thoroughly in real time and accelerated conditions. Finally, with the target to have a product with actual results, in vitro and in vivo studies were conducted. The final product proved to have better results in disintegration and dissolution. Moreover, R.G.C.C.'s capsules exhibited enhanced action in human cell lines as well as impressive pharmacokinetic results in animal models. The in vitro results showed an advantage of the R.G.C.C. product compared to the commercial one, whereas its maximum concertation in vivo was determined 34% higher than the commercial one.

Evaluation of Tegaran Formula ZhenHua cytotoxicity against human cancer cell lines.

The aim of this study is to evaluate the potential health effects of Tegaran Formula ZhenHua, a nutritional supplement used mainly by cancer patients. Its active ingredients and cytotoxicity was assessed with analytical methods and viability assays, respectively. The analytical methods consisted of dissolution, disintegration, HPLC, LC/MS, GC/MS and NMR. Cytotoxicity was assessed by MTT, SRB, CVE colorimetric viability assays in 0, 24, 48 and 72h time points. The results indicate that Tegaran Formula ZhenHua supplement did not present any cytotoxic effects due to issues related to the capsules' solubility, distribution and identification of the active ingredient.

Study and detection of potential markers for predicting metastasis into lymph nodes in prostate cancer.

Hormone-refractory prostate carcinoma has a different cell surface protein profile than hormone-sensitive prostate carcinoma, which provides migration ability and interactions with organs/tissues. Detection and association of these proteins with lymph node metastasis via lymphadenectomy might be beneficial for patients. Gene expression analysis in hormone-refractory and hormone-sensitive commercial cancer cell lines was performed and, after co-cultivation with osteoblasts or endothelial cells, knockdown experiments followed to validate potential biomarkers. "Myeloid-associated differentiation markers, myosin 1b and phosphatidylinositol-4-phosphate-5-kinase type 1 alpha are implicated in metastasis", their knockdown altered the expression of key regulators of endothelial-mesenchymal transition, invasion, motility and migration. In primary prostate tumors, these genes could be an indicator for future metastasis into lymph nodes.

Design, Synthesis, and In Vitro Activity of Pyrazine Compounds.

Despite the fact that there are several anticancer drugs available, cancer has evolved using different pathways inside the cell. The protein tyrosine phosphatases pathway is responsible for monitoring cell proliferation, diversity, migration, and metabolism. More specifically, the SHP2 protein, which is a member of the PTPs family, is closely related to cancer. In our efforts, with the aid of a structure-based drug design, we optimized the known inhibitor SHP099 by introducing 1-(methylsulfonyl)-4-prolylpiperazine as a linker. We designed and synthesized three pyrazine-based small molecules. We started with prolines as cyclic amines, confirming that our structures had the same interactions with those already existing in the literature, and, here, we report one new hydrogen bond. These studies concluded in the discovery of methyl (6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)prolylprolinate hydrochloride as one of the final compounds which is an active and acceptable cytotoxic agent.

Gene expression profiling as a potential predictor between normal and cancer samples in gastrointestinal carcinoma.

Analysis and comparison of gene expression profile among molecules, correlated with essential and crucial biological processes, is of primary importance in cancer research, since it provides significant info regarding the resistance to chemo/radiotherapy, risk for relapse or prediction of metastasis etc. In this study, gene expression profile is used for discriminating efficiently colon cancer cell lines from normal cells and cancer cells in blood samples of colon cancer patients and categorizing different types of gastrointestinal cancer. In particular, blood samples were collected from normal donors as well as from colon cancer patients. Peripheral blood mononuclear cells were isolated and gene expression analysis was performed for more than fifty genes. The same assays were performed for commercial cancer cell lines representing different types of gastrointestinal cancer. In order to examine whether the comparison of gene expression profile can lead to a thorough discrimination between cancer and normal states as well as between different cancer types, we performed clustering analysis based on hierarchical, and k-means algorithms. The clustering analysis efficiently separated: a) colon cancer cell lines from colon patients' samples, b) normal from the colon cancer samples, c) gastric and pancreatic cancer from liver and colon types based. The exploitation of gene expression profile can be successfully used for the discrimination between normal vs cancer samples and/or for categorizing various types of cancer. This of course has important implications in cancer management since it enables the quick discrimination based on cells, isolated from bloodstream, needless of tissue examination or protocols requiring specialized equipment.

Novel small molecule decreases cell proliferation, migration, clone formation, and gene expression through ERK inhibition in MCF-7 and MDA-MB-231 breast cancer cell lines.

The Ras-Raf-MEK1/2-ERK1/2 pathway is an attractive target for the development of anticancer agents because of the high prevalence of ERK activation in human cancers. However, resistance is often developed despite initial clinical response, most likely because of activation of cross-talk pathways. In Research Genetic Cancer Center (RGCC), we are in the process of synthesizing a novel ERK inhibitor, targeting the final stage of the pathway, thus minimizing cross-talk. We have synthesized an intermediate molecule -RGCC416 - and tested its biological activity. MCF-7 and MDA-MB-231 cells were used. Cell viability was measured by crystal violet and cell proliferation by the methyl tetrazolium assay using various compound concentrations. Cell migration and colony formation were determined to assess the ability of invasion and single cancer cell growth, respectively. Expression of genes linked to MAPK/PI3K pathways was determined by PCR. ERK and phospho-ERK levels were determined in both the cytoplasm and the nucleus by western blot. It was found that although the compound did not affect viability, it significantly decreased cell proliferation, migration, and colony formation in both cell lines. In MDA-MB-231, this is possibly through retaining phospho-ERK to the cytoplasm, where it cannot activate cancer-associated genes. There was no difference in ERK levels in MCF-7 cells. This could be because of the different pathways that these cells utilize for survival. We have synthesized a molecule, which could be a promising ERK inhibitor, leading to possible novel treatment options for breast cancer patients.