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DC Maturation: A Brief Comparison between Three Different Processes

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Journal of Cancer Therapy 2015年第10期6卷 871-880页

作者： Maria Toloudi ioannis papasotiriou Research and Development Department Research Genetic Cancer Centre Ltd. Florina Greece

Dendritic cell (DC) maturation approved to be a pivotal process for initiating immunity. Many protocols were established in order the isolated peripheral blood mononuclear cells (PBMCs) from healthy donors to mature into dendritic cells (DCs). The purpose of this study was to present an effective and reliable DC maturation procedure by comparing three different protocols (Interleukin-4/Tumor Necrosis Factor-appha (IL-4/TNFa) DC protocol, Interferon alpha (IFNa) DC protocol and FAST DC protocol). Whole blood was collected from six healthy donors and PBMCs were isolated by Ficoll gradient centrifugation. The counted cells were incubated with the addition of three different cocktails of supplements for appropriate time period. The final mature DC population was examined either by its phenotypic characteristics under light microscope or by measuring the expression of antigen presenting molecules such as CD80 and CD86 by flow cytometry. It was found that the mature DCs, generated from the IL-4/TNFa DC protocol, expressed higher levels of CD80 and CD86. Furthermore, they sharply exhibited their phenotypic hallmarks.

关键词： DC Maturation TNFa IL-4 IFNa

Cytoplasmic L1 Levels in Cancer

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Journal of Cancer Therapy 2022年第6期13卷 353-361页

作者： Eleana Hatzidaki Panagiotis Apostolou ioannis papasotiriou Research Genetic Cancer Centre SA Florina Greece Research Genetic Cancer Centre International GmbH Zug Switzerland

In addition to shaping genome diversification over evolutionary time, L1 retrotransposition alters gene expression as well. The most notable gene altering process involves insertional mutagenesis. The aim of the study was the examination of both nuclear L1 expression levels and cellular localization in cancer cell lines, PBMCs from healthy volunteers and PBMCs from cancer patients. L1 was detected by FISH in chromosome preparations. L1 probe was custom-made using end-point PCR against L1-ORF2 and conjugated with FITC. It was found that cancer cell lines and clinical samples from cancer patients contained significantly elevated levels of L1 per nucleus compared to healthy volunteers. Cytoplasmic L1 was also increased in the above mentioned samples denoting that cancer could be associated with increased L1 activation and mobility. Our results may provide a novel cancer diagnostic marker and highlight the possibility of cytoplasmic L1 inhibition as a therapeutic intervention for cancer.

关键词： L1 Cancer Retrotransposition FISH Cytoplasmic

Efficacy of 5-FU or Oxaliplatin Monotherapy over Combination Therapy in Colorectal Cancer

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Journal of Cancer Therapy 2015年第4期6卷 345-355页

作者： Maria Toloudi Panagiotis Apostolou ioannis papasotiriou Research Genetic Cancer Centre Ltd. (R.G.C.C. Ltd.) Florina Greece

Recent clinical and research development supports the use of 5-fluorouracil in combination with oxaliplatin for the treatment of patients with advanced colorectal cancer (CRC). 5-Fluorouracil (5-FU), which is as an anti-metabolite, is a widely used cytostatic drug. Although the rate of response, quality of life and overall survival differs between CRC patients, the above combination remains a widely used chemotherapeutic regimen. In some cases, a cancer stem cell (CSC) population may resist the majority of chemotherapeutic models. This study investigated if monotherapy is more efficacious than 5-fluorouracil and oxaliplatin combined for the treatment of CRC, using a CRC cell line and a CSC-like line. Cell viability was evaluated by cellular-based assays, and quantitative polymerase chain reaction (q-PCR) assays were performed to assess the expression of specific genes (TYMS,?DNMT1,?NANOG, DHFR, SHMT1, ERCC1, DPYD) correlated with 5-FU and oxaliplatin resistance. We observed that 5-fluorouracil was more effective in both CRC and CSCs. This finding proved the hypothesis that, in some cases, monotherapy may be more successful in CRC treatment than a drug combination that may be cytotoxic and inflict adverse side effects.

关键词： 5-Fluorouracil Oxaliplatin FOLFOX Colon Cancer Stem Cells

Stemness Gene Profiles of Circulating Tumor Cells

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Journal of Cancer Therapy 2017年第2期8卷 155-167页

作者： Panagiotis Apostolou Maria Papadimitriou ioannis papasotiriou Research Genetic Cancer Centre S.A. Industrial Area of Florina Florina Greece

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.

关键词： Stemness Circulating Tumor Cells RT-qPCR

Evaluation of the Anti-Proliferative Effects of a Green Tea and <i>Capsicum</i>Powder Extract in Cancer Cell Lines

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Journal of Cancer Therapy 2020年第2期11卷 44-54页

作者： Eleana Hatzidaki Maria Papadimitriou ioannis papasotiriou Research Genetic Cancer Centre SA Florina Greece Research Genetic Cancer Centre International GmbH Zug Switzerland

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.

关键词： Proliferation Cancer Green Tea Capsicum Powder

Linearity Comparison of Three Colorimetric Cytotoxicity Assays

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Journal of Cancer Therapy 2019年第7期10卷 580-590页

作者： Maria Papadimitriou Eleana Hatzidaki ioannis papasotiriou Research Genetic Cancer Centre Florina Greece

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.

关键词： Linearity Cytotoxicity Proliferation Metabolism Cancer

MUC1 Antigen-Specific CD8 T Lymphocytes Targeting MCF7 and MDA-MB-231 Human Breast Adenocarcinoma Cell Lines

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Journal of Cancer Therapy 2019年第7期10卷 495-509页

作者： Panagiotis Parsonidis Dimitrios-Athanasios Ntanovasilis ioannis papasotiriou Research Genetic Cancer Centre SA (R.G.C.C. SA) Florina Greece

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.

关键词： MUC1 Dendritic Cells Cytotoxic T Lymphocytes Immunotherapy

Flow Cytometric Detection of Circulating Tumor Cells in Breast Cancer Patients: A Blinded Study

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Journal of Cancer Therapy 2019年第8期10卷 708-715页

作者： Dimitrios-Athanasios Ntanovasilis Panagiotis Apostolou ioannis papasotiriou Research Genetic Cancer Centre S.A. Florina Greece Research Genetic Cancer Centre International GmbH Zug Switzerland

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.

关键词： CTCs Breast Cancer Diagnostics Flow Cytometry

Gene Expression Changes in Colorectal Cancer during Metronomic Chemotherapy and High-Concentration Drug Administration

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Journal of Cancer Therapy 2015年第8期6卷 679-689页

作者： Panagiotis Apostolou Maria Toloudi Irene Kalliara Vasiliki Kipourou Ioanna Tourna ioannis papasotiriou Research Genetic Cancer Centre Ltd. (R.G.C.C. Ltd.) Florina Greece Former Employee in R.G.C.C. Ltd. Florina Greece

5-fluorouracil (5-FU) and oxaliplatin, either alone or in combination, are widely used in chemotherapy for advanced colorectal cancer. Among chemotherapeutic strategies, metronomic chemotherapy has recently demonstrated promising efficacy against otherwise chemoresistant neoplasms. However, data on the efficacy of metronomic applications in cancer stem cells are lacking. This cell population is characterized by resistance to most chemotherapeutic models. In this study, we investigated the efficacy of metronomic chemotherapy and compared it with high-concentration administration of 5-FU and oxaliplatin and their combination in colon adenocarcinoma cells and colon cancer stem cells. We assessed changes in expression levels of specific genes involved in 5-FU and oxaliplatin resistance (thymidylate synthase, DNA (cytosine-5)-methyltransferase 1, dihydrofolate reductase, serine hydroxymethyltransferase, DNA excision repair protein, dihydropyrimidine dehydrogenase) in relation to drug administration schedule using quantitative real-time polymerase chain reaction. We also examined changes in cell viability. Metronomic chemotherapy showed greater efficacy in gene expression levels in colorectal cancer cells, while high, single-concentration administration was more effective in colon cancer stem cells. Regarding cell viability, no significant change was observed between metronomic and single-dose treatments. These results suggest that metronomic chemotherapy may be more effective than high-dose chemotherapy in some patients with colorectal cancer, though high, single-concentration administration may be more effective against cancer stem cells.

关键词： Metronomic Chemotherapy 5-Fluorouracil Oxaliplatin Colorectal Cancer Cancer Stem Cell