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At Research Genetic Cancer Centre, we have developed a novel method for the production of human monoclonal antibodies against a specific antigen of our choice (c-met) using isolated human blood cells. By mimicking nature, dendritic, CD4 and CD19 cells from healthy volunteers were driven towards Th2 immunity. Cell activation was succeeded by a cytokine cocktail, and IgG production was promoted by IgG class switching factors. IgG secretion was determined using both enzyme linked immunosorbent assay (ELISA) and Western blot as well as immunoglobulin heavy chain gamma polypeptide gene expression. Secreted antibody was further purified by affinity column chromatography against c-met peptide. Anti-c-met activity was determined using the purified antibody as primary antibody for c-met detection by ELISA, Western blot and flow cytometry. Finally, anti-c-met antibody efficiency was determined by MCF-7 viability assay. Plasma cell formation and IgG secretion took place after 6 days of culture. Plasma cells produced anti-c-met IgG antibody that significantly decreased MCF-7 breast cancer cell proliferation. To our knowledge, this is the first platform of its kind, generating fully human antibodies-on-demand using patient's own cells, bringing personalized, targeted therapy for cancer one step closer.

Network meta-analysis (NMA) as the quantification of pairwise meta-analysis in a network format has been of particular interest to medical researchers in recent years. As a powerful tool with which direct and indirect evidence from multiple interventions can be synthesized simultaneously in the study and design of clinical trials, NMA enables inferences to be drawn about the relative effect of drugs that have never been compared. In this way, NMA provides information on the hierarchy of competing interventions for a given disease concerning clinical effectiveness, thus giving clinicians a comprehensive picture for decision-making and potential avoidance of additional costs. However, estimates of treatment effects derived from the results of network meta-analyses should be interpreted with due consideration of their uncertainty, because simple scores or treatment probabilities may be misleading. This is particularly true where, given the complexity of the evidence, there is a serious risk of misinterpretation of information from aggregated data sets. For these reasons, NMA should be performed and interpreted by both expert clinicians and experienced statisticians, while a more comprehensive search of the literature and a more careful evaluation of the body of evidence can maximize the transparency of the NMA and potentially avoid errors in its interpretation. This review provides the key concepts as well as the challenges we face when studying a network meta-analysis of clinical trials.

Gastric cancer is one of the most common and deadly cancers worldwide. Screening tests as well as tools for prediction of treatment outcomes and prognosis have been developed, but they have many limitations. The integration of liquid biopsy provided new aspects in screening and diagnosis of gastric cancer. In the present study, we used different techniques, studying the genetic and epigenetic profile of circulating tumor cells. We aimed to acquire all the available information, compare it with already existing studies, and evaluate the benefit of this approach. A blood sample was isolated from 2 gastric cancer patients at stages III–IV, followed by the isolation of CTCs. The circulating tumor cells were used for array comparative genomic hybridization, next-generation sequencing, and whole gene expression microarrays. Different variants were detected, while the microsatellite instability status was stable in both cases. The tumor mutational burden was low to medium. Gene expression assays revealed that >100 genes were overexpressed compared to noncancer samples. Amplifications of X chromosome were also observed in both cases, by using array comparative genomic hybridization. Although there are several techniques for cancer screening, prediction of therapy outcomes, and prognosis, the application of a complete comprehensive cancer panel, combining the study of variants, fusions, chromosomal abnormalities, and gene expression, is more appropriate. Information provided by the above techniques might contribute in designing more efficient treatment protocols and screening tools. Despite the limitation of samples, the data are encouraging, and further study is needed so that they can be used at clinical level.

Objectives Circulating tumour cells (CTCs) from liquid biopsies provide an exceptional opportunity to obtain real-time tumour information and are under current investigation in several cancers, including cutaneous melanoma, but face significant drawbacks in terms of non-standardised methodology, low viable cell numbers and accuracy of CTC identification. In this pilot study, we report that chemosensitivity assays using liquid biopsy-derived metastatic melanoma (MM) CTCs, from 7 patients with stage IIIC, BRAF wild-type metastatic melanomas, localized exclusively to the pelvic region, un-eligible for immunotherapy and treated with melphalan hypoxic pelvic perfusion (HPP), is both feasible and useful in predicting response to therapy. Viable MM CTCs (> 5 cells/ml for all 7 blood samples), enriched by transient culture, were characterised in flow cytometry-based Annexin V-PE assays for chemosensitivity to several drugs. Results Using melphalan as a standard, chemosensitivity cut-off values of > 60% cell death, were predictive of patient RECIST 1.1 response to melphalan HPP therapy, associated with calculated 100% sensitivity, 66.67% specificity, 33.33% positive predictive, 100% negative predictive, and 71.43% accuracy values. We propose that the methodology in this study is both feasible and has potential value in predicting response to therapy, setting the stage for a larger study. Trial registration Clinical Trials.gov Identifier NCT01920516; date of trial registration: August 6, 2013

Background Hepatic artery infusion (HAI) and drug selection by liquid biopsy precision oncotherapy are under investigation for the multidisciplinary treatment of unresectable colorectal liver metastases (CRCLM) in progression after systemic therapy. Here, we compare the safety and efficacy of third-line HAI followed by target therapy with drug regimes selected by liquid biopsy precision oncotherapy to third-line systemic therapy with drug regimes selected partly by tissue biopsy precision oncotherapy, in a retrospective real-life study of 106 unresectable CRCLM patients. Methods Drug regimens for HAI/target therapy were selected by assessing the sensitivity of purified circulating tumor cell (CTCs) to 5-fluorouracil, carboplatin, cisplatin, oxaliplatin, irinotecan, doxorubicin, mitomycin, raltitrexed, and melphalan in-vitro and by real-time qRT-PCR gene expression assays, and for the Systemic therapy cohort were selected by age, comorbidity, performance status, and absence of RAS mutations. Therapeutic responses, adverse events, and quality of life were evaluated by RECIST 1.1, CTCAE 4.03, and ECOG criteria, respectively, and chemo-filtration performed following HAI to reduce systemic toxic effects. Results HAI/target therapy with drugs selected by liquid biopsy precision oncotherapy (44 patients), resulted in 2.27% CRs, 38.63% PRs, 56.81% SD,s and 2.27% PDs; ECOG 2 to 1 improvement, but no infusion-related technical or vascular complications, or deaths. Systemic therapy (62 patients) resulted in 1.6% CRs, 17.74% PRs, 37.09% SDs, and 45.16% PDs; more grade 1–2 adverse events and 4.84% ECOG 1 to 2 worsening. The median 5 month PFS in the HAI/target therapy cohort was significantly longer than 3 months in the systemic cohort (P Conclusions HAI plus chemo-filtration followed by target therapy, with drug regimens selected by liquid biopsy precision oncotherapy, is a safe and efficacious alternative therapeutic strategy for unresectable CRCLM in progression after two lines of systemic therapy and should be considered for a multicentre prospective phase III study, to fully confirm this potential.

In recent years the use of natural supplements in order to prevent, treat or delay recurrence of cancer or reduce chemotherapy toxicity has attracted much attention. One such supplement is Capsol-T which consists of de-caffeinated green tea and chili pepper extracts. The aim of the study was the evaluation of Capsol-T effect on the proliferation of various cancer cell lines representing different cancer types. Cell lines that were used in the study were: DU145, LNCap, MCF7, HCT116 and MOR. The effect of various concentrations and incubation times of Capsol-T on cell viability was determined using the MTT method. The results do not show a common anti-proliferative pattern in all cancer cells. In some cell lines and certain concentrations cell growth was significantly decreased at 24 hr which became more evident at 48 hr. The role of Capsicum powder in cancer is unclear since both cancer cell proliferation and growth arrest have been demonstrated. Green tea on the other hand was found to decrease certain drugs’ bioavailability. Our results suggest that an anti-proliferative effect in certain types of cancer should not be generalized to other types as well. Different concentrations also affect the net result often having opposite effects. Overall, caution should be taken when using natural supplements for their anti-cancer effects.

After two years since the outbreak, the COVID-19 pandemic remains a global public health emergency. SARS-CoV-2 variants with substitutions on the spike (S) protein emerge increasing the risk of immune evasion and cross-species transmission. Here, we analyzed the evolution of the S protein as recorded in 276,712 samples collected before the start of vaccination efforts. Our analysis shows that most variants destabilize the S protein trimer, increase its conformational heterogeneity and improve the odds of the recognition by the host cell receptor. Most frequent substitutions promote overall hydrophobicity by replacing charged amino acids, reducing stabilizing local interactions in the unbound S protein trimer. Moreover, our results identify "forbidden" regions that rarely show any sequence variation, and which are related to conformational changes occurring upon fusion. These results are significant for understanding the structure and function of SARS-CoV-2 related proteins which is a critical step in vaccine development and epidemiological surveillance.

Background/Aim: An early evaluation concerning the effectiveness of supportive oligonucleotide therapy (SOT) in cancer as a monotherapy and in combination with other types of treatment. Patients and Methods: This study evaluated the clinical condition and performance status (Karnofsky-Index) of 95 patients, post-SOT administration. Furthermore, circulating tumor cells (CTCs) from 47 patients’ pre- and post-SOT administration were measured and analyzed by repeated-measures ANOVA. Results: Improvement of the clinical condition was observed in all patients who used SOT (77.89%), SOT in combination with other therapy (69.77%) and SOT as a monotherapy or no information was given concerning another therapy (84.31%). Positive results for Karnofsky-Index were also observed in 71.58%, 61.36%, and 80.39%, respectively. Finally, statistically significant reductions in CTCs were observed for both SOT as a monotherapy and SOT as an adjunctive therapy. Conclusion: The preliminary results indicate that SOT therapy can be used both as monotherapy as well as in combination with other therapies for cancer.

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.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.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.These preliminary results indicate that the proposed cellular therapy can be considered for further studies in clinical trials.

Background Third line innovative systemic treatments and loco-regional chemotherapy by hypoxic pelvic perfusion (HPP) have both been proposed for the treatment of unresectable not responsive recurrent rectal cancer (URRC). In the present study, we have compared the safety and efficacy of HPP/target therapy, using drug regimens selected by liquid biopsy precision oncotherapy, to third-line systemic therapy based on tissue specimens precision oncotherapy. Methods HPP/target therapy regimens were selected based on precision oncotherapy, including assays for chemosensitivity and viability, and qRT-PCR for tumor-related gene expression. In the control group, systemic third-line and further lines of therapy were defined according to clinical and biological parameters. Results From 2007 to 2019, 62 URRC patients were enrolled, comprised of 43 patients in the HPP/target-therapy group and 19 patients in the systemic therapy control group. No HPP related complications were reported and the most common adverse events were skin and bone marrow toxicity. In the HPP/target-therapy group, the ORR was 41.8% whereas in the systemic therapy control group was 15.8%. DCR of the HPP/target-therapy group was significantly improved over the systemic therapy group (P = 0.001), associated with a PFS of 8 vs 4 months (P = 0.009), and OS of 20 vs 8 months (P = 0.046). Conclusions The present data indicate that in URCC patients, the integration of HPP/target-therapy and precision oncotherapy based upon liquid biopsy is as effective and efficacious as third-line treatment in local disease control and, therefore, deserves to be further assessed and compared to conventional systemic treatments in future prospective randomized trials.

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.

In Research Genetic Cancer Center (RGCC), we are in the process of synthesizing a novel ERK inhibitor. We have currently synthesized an intermediate molecule, RGCC169, that needed to be tested to confirm we are using the appropriate synthetic pathways. Because of the limited solubility the compound exhibits, a strategy had to be devised for the free entrance of the molecule into the cell. Extracellular vesicles (EVs) were isolated by polyethylene glycol precipitation and identified by western blot and scanning electron microscopy. Loading was determined by high-performance liquid chromatography, differential scanning calorimetry, and scanning electron microscopy. EV uptake was determined by fluorescent microscopy. The effect of EV-encapsulated RGCC169 was determined by MTT viability assay on MCF7 cells. RGCC169 was incorporated into EVs as shown by high-performance liquid chromatography (26.6%) and scanning electron microscopy. Differential scanning calorimetry peaks shifted from 100.84 to 108.79°C upon encapsulation. EVs were taken up by cells as evident from CD63 fluorescent signal inside the cell's cytoplasm. RGCC169 decreased MCF proliferation (93.5±2.2, P=0.02). EV-encapsulated RGCC169 decreased cell proliferation even further (93.5±2.2 vs. 81.6±2.8, P=0.0002). RGCC169 was successfully loaded into serum EVs possibly by incorporation into the lipid membrane. EVs were taken up by MCF7 cells possibly by endocytic pathways. Although RGCC169 significantly reduced MCF7 viability at 3 μmol/l, the same concentration of RGCC169 encapsulated into EVs decreased cells viability even further. Our findings validate the correctness of our methods and are very promising for the achievement of our final goal, that is, the synthesis of a novel cytotoxic agent.

Cell viability assays, including techniques to assess the proliferation of cancer cell lines, constitute a rapid, inexpensive and sensitive screening method to pre-clinically evaluate the activity of a potential drug or substance. This study investigates and compares seeding densities and linearity of three such methods: MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), SRB (sulforhodamine B) and CVE (crystal violet elution) assays. SRB and CVE are staining assays for proteins, while MTT measures the mitochondrial activity of living cells. Assays were performed on five cancer cell lines, A375, PC3, DU145, HCT116 and COR-L105, and the coefficient of determination (R2) was employed to determine fit into a linear regression model. The results show that CVE is the most linear assay at fixed time points. SRB at 515 nm is better for measurements over time. Seeding densities between 9000 and 12,000 were the optimum. However, seeding densities and doubling times should be taken into consideration when designing an experiment.

MUC1 is an antigen that is overexpressed on the cell surface of many human breast adenocarcinomas and other types of cancer. The cancer immunity cycle has seven steps, starting with release of cancer cell antigen and following with cancer antigen presentation. Priming, activation and trafficking of T cells to tumors are the next steps and the infiltration of T cells into tumors, the recognition of cancer cells by T cells and killing of cancer cells are the final steps. We have tested a synthetic peptide for the MUC1 antigen and generated dendritic cells (DCs) that were pulsed with the specific peptide. Mature DCs were used to activate naive T cells to differentiate into antigen-specific CTLs. CTLs were tested for proliferation, cytokine release (IFNγ), activation markers and cytotoxicity against human breast adenocarcinoma cell lines. The cytotoxic effect of those CTLs was higher against MCF7 human cell line than against MDA-MB-231 human cell line.

Circulating tumor cells are cells that detach from the primary tumor site and migrate to the bone marrow or other tissues where they can initiate a metastatic site. Liquid biopsies are an emerging tool in the past decades that enables us to detect Circulating Tumor Cells in patients’ blood. Flow cytometry is a powerful tool used in liquid biopsy diagnostics. This aims to prove the sensitivity and specificity of a flow cytometric panel for the detection of CTCs in breast cancer patients using healthy individuals’ samples as controls. The study was blinded to the data analyzing researcher. Statistical analysis followed and results show 86.9% area under the curve which indicates that the particular method can be very promising for diagnosing breast cancer.

Background Waldenstrom's macroglobulinemia (WM) is a rare malignancy characterized by bone marrow infiltration by lymphoplasmacytic cells and the presence of a monoclonal IgM paraprotein. The interactions of lymphoplasmacytic cells with other cells in their microenvironment, including mast cells and endothelial cells, support their survival and proliferation and can induce resistance to therapy. von Willebrand factor (vWF) plays a key role in primary hemostasis but is also a marker of endothelial “stimulation.” High levels of vWF have been associated with an adverse prognosis in patients with symptomatic WM and might reflect the interactions between lymphoplasmacytic cells and other cells of their microenvironment. Materials and Methods Considering vWF and ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) as markers of endothelial dysfunction and activation, we evaluated the prognostic importance of vWF and ADAMTS-13 antigen levels in the serum of patients with previously untreated symptomatic WM to validate vWF as a possible prognostic marker for progression-free and overall survival. We also validated the measurement of vWF in the serum instead of citrated plasma and investigated the possible correlations of ADAMTS-13 antigen levels with disease characteristics. The analysis included 42 patients with symptomatic WM and 19 matched healthy controls. Results The serum levels of vWF antigen provided significant prognostic information, and patients with levels ≥ 200 IU/dL had a very poor prognosis compared with patients with lower levels. The ADAMTS-13 antigen levels were decreased in WM patients and correlated with the IgM levels, β2-microglobulin, and extent of bone marrow infiltration. Conclusion vWF levels measured in the serum could become an important prognostic marker in patients with WM and requires further investigation.

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.

Circulating tumour cells (CTCs) from liquid biopsies are under current investigation in several cancers, including epithelial ovarian cancer (EOC) but face significant drawbacks in terms of non-standardised methodology, low viable cell numbers and accuracy of CTC identification. In this pilot study, we report that chemosensitivity assays using liquid biopsy-derived metastatic EOC CTCs, from 10 patients, nine with stage IIIC and one with stage IV disease, in progression after systemic chemotherapy, submitted for hypoxic isolated abdominal perfusion (HAP), are both feasible and useful in predicting response to therapy. Viable metastatic EOC CTCs (&gt, 5 cells/mL for all 10 blood samples), enriched by transient culture and identified by reverse transcription polymerase chain reaction (RT-PCR) and indirect immunofluorescence (IF), were subjected to flow cytometry-based Annexin V-PE assays for chemosensitivity to several chemotherapeutic agents and by RT-PCR for tumour gene expression profiling. Using a cut-off value of &gt, 80% cell death, CTC chemosensitivity tests were predictive of patient RECIST 1.1 responses to HAP therapy associated with 100% sensitivity, 50% specificity, 33% positive predictive, 100% negative predictive and 60% accuracy values. We propose that the methodology employed in this study is feasible and has the potential to predict response to therapy, setting the stage for a larger study.

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.

Checkpoint kinase 2 (CHEK2) is a serine/threonine kinase which is activated upon DNA damage and is implicated in pathways that govern DNA repair, cell cycle arrest or apoptosis in response to the initial damage. Loss of kinase function has been correlated with different types of cancer, mainly breast cancer. CHEK2 functionality is affected by different missense or deleterious mutations. CHEK2*1100delC and I157T are most studied in populations all over the world. Although these variants have been identified in patients with breast cancer, their frequency raises doubts about their importance as risk factors. The present article reviews the recent advances in research on CHEK2 mutations, focusing on breast cancer, based on the latest experimental data.

Circulating tumor cells (CTCs) are a population of tumor-derived cells that detach from the primary tumor and initiate metastasis. However, the mechanisms of this process are still unknown. This phenomenon renders CTCs as a valuable resource for prognosis and diagnosis of cancer. The involvement of stemness transcription factors and markers, such as NANOG, OCT3/4, CD34, NESTIN, and SOX2, in metastasis initiation has been studied recently because their abnormally elevated expression in cancer cells may be highly important in understanding tumor initiation. This study analyzed the genetic profiles of the above genes in CTCs derived from patients with different types of cancer. Blood samples were randomly collected from 71 cancer patients with various cancer types. CTCs were isolated using enrichment protocols and RNA was extracted. RT-qPCR was performed in triplicate using ACTB as the reference gene. The statistical analysis was performed among the ΔCts of the samples using parametric and non-parametric methods. The molecular analysis revealed that the expression of each gene was different than the others. When each type of cancer was analyzed separately, the gene expression profile was not always the same. It is noteworthy that, in all cases, the gene expression of NESTIN differed from that of transcription factors. According to the above data, gene expression profiles might be used as a potential biomarker or constitute a gene signature.

Although monoclonal antibodies are promising, a truly fully human antibody is yet to be produced. Current human antibodies have the human sequence, but are produced in either transgenic animals or in phages. The aim of this paper was to produce a truly human antibody directed against an epitope of our choice, secreted by human plasma cells. The target protein was TMX2 one of the least studied disulfide isomerases. IgG and anti-TMX2 antibody were determined by both Elisa and western blot. TMX2 KD was evaluated by Surface Plasmon Resonance. TMX2 localization was determined by flow cytometry in MCF-7 cells. Efficiency was evaluated by MTT. Gene expression was evaluated by PCR. We have managed to produce two fully human antibodies directed against TMX2 protein. TMX2 protein was found both in the cytoplasm and cell membrane of breast cancer cells. RGCC TMX2 antibody recognizing an extracellular epitope increased cell proliferation. RGCC TMX2 antibody recognizing an intracellular epitope decreased cell proliferation and gene expression related to cancer survival, differentiation and metastasis. These findings suggest this platform is very promising for novel personalized therapies. TMX2 could be a novel target for cancer treatment.

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.

Background: Apples (Malus domestica) are a part of most daily diet. Their positive impact on health and overall wellness is recognized and widely accepted. Nowadays, the demanding industry of cosmetology has raised awareness over the potent dangers of the ingredients and consumers tend to reach out for products more nature based and gentle. In this study, the development of different extraction methods for the isolation of antioxidants from apples’ peels was tried and the evaluation of their efficacy. In addition to this, it was aimed to formulate a simple daily face cream using this extract and prove its action. Materials and Methods: This product was tested for its allergens, its stability and its preservative efficacy with the target to conclude to not only an effective daily cream but to a safe product ready to be used. Moreover, the antioxidant activity of the extract was determined as a single extract as well as after its encapsulation in the face cream. Results: The most common extraction methods were followed and showed that at 200μg/mL concentration the scavenging activity of some extracts is more than 80%. The formulation of those in a product, appeared to have successful results, since the antioxidant value of the peels remained in the product which proved to have a satisfying preservation system, no unforeseen allergens and great stability behavior under different conditions of storage. Conclusion: This study suggests the extraction of apples’ peels and the separation of antioxidant compounds. The findings prove that these kind of ingredients can be used in a product successfully and lead to the daily use by consumers.

BACKGROUND/AIM We developed a novel platform to produce fully human antibodies that could be used as tools in cancer research. This study was based on maturation of B-cells into plasma cells and the subsequent secretion of fully human antibodies against a 9-mer peptide. MATERIALS AND METHODS A peptide was introduced to dendritic cells, followed by co-cultivation with cluster of differentiation 4 (CD4+) cells from healthy donors and CD19(+) cells from patients with cancer. In order to detect plasma cells, we used microscopy, flow cytometry, enzyme-linked immunosorbent assays (ELISAs), and western blotting. RESULTS Oval-shaped cells with eccentric nuclei were found which expressed high levels of CD27 (up to 73%) and CD138 (up to 31%). An antibody against the selected peptide was detected in the culture supernatants of all samples according to ELISA and western blot analyses. CONCLUSION This platform successfully generated fully human antibodies against a specific epitope.

Background: Although cisplatin is used for the treatment of more than half of cancer patients, its use is restricted by serious side effects as well as the development of cisplatin-resistant cancer cells, limiting its use. In RGCC we have synthesized an intermediate molecule in an ERK inhibitor synthesis process. Aims and Objectives: The aim of the study was to evaluate the effects of combined cisplatin plus RGCC molecule treatment on MCF-7 and MDAMB231 breast cancer cell viability, proliferation, ability to form clones and migrate, as well as the effects on cell cycle and gene expression. Materials and Methods: Cell viability and proliferation were measured by Crystal violet exclusion dye and MTT respectively. Clone formation and wound healing assays were also used for clone formation and cell migration evaluation. Cell cycle was studied by flow cytometry, expression of genes was evaluated by PCR and protein expression was evaluated by western blot. Results: It was found that combination therapy decreased cell viability and proliferation, caused growth arrest, decreased cancer cell invasiveness and the ability to form clones as well as perturbed the expression of genes involved in ERK, cell cycle and cell death pathways. Conclusion: Although the exact mechanism of action of the combination therapy remains to be investigated, it was found that it is more effective than cisplatin monotherapy. Our findings could potentially lead to a new therapeutic regime for the treatment of cancer.

Short gold nanorods were synthesized (average length 28.08 nm, average aspect ratio 3.54), which were functionalized with folic acid (FA) and 8-mercaptooctanoic acid (MOA) or 11-mercaptoundecanoic acid (MDA) and loaded with paclitaxel (PCT). FA was conjugated to the nanorods in order to render them targetable for cancer cells overexpressing folate receptors whereas MOA or MDA was attached on the nanorods in order to generate extra hydrophobic areas for entrapment of hydrophobic drugs such as PCT in the nanorods and in order to provide free carboxylic groups, which would allow for the conjugation of drug or other biofunctional molecules to the nanorods. The functionalized gold nanorods (GNRs-MOA-FA and GNRs-MDA-FA) did not exhibit any significant degree of aggregation in cell culture medium and blood plasma even after a prolonged incubation period of 7 days, indicating the adequate colloidal stability of the nanorods in these media. The functionalized nanorods exhibited satisfactory entrapment efficiency (around 40%) for PCT and released less than 25% of their PCT content in phosphate buffer pH 7.4 in 48 h. PCT entrapment efficiency was a little higher and PCT release rate a little lower in the GNRs-MOA-FA. Molecular analysis (qPCR) was used to find out that the MDA-MB-231 cancer cell line expresses the folate receptor (FL1R) whereas the MCF-7 cancer cell line does not. The PCT-loaded GNRs-MOA-FA were more cytotoxic than the PCT-loaded GNRs-MOA nanorods against the MDA-MB-231 cells, which probably relates to the higher uptake of the GNRs-MOA-FA nanorods by these cells. The opposite was true in the case of the MCF-7 cells.