Browsing by Subject "Cancer"
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(2022)Background– The BCL-2 protein family members are major regulators of apoptosis, and the anti-apoptotic (pro-survival) members of the family is commonly targeted with BH3 mimetic drugs in haematological cancers. However, these treatments have not been very impactful when administered as single agents and they have long been investigated for combination therapy with other agents. Acute myeloid leukaemia (AML) is one of the difficult-to-cure haematological malignancies. A recently approved therapy for AML consists of the combinatorial administration of venetoclax (a selective BCL-2 inhibitor) and a DNA methyltransferase (DNMT) inhibitor such as azacitidine or decitabine. Although this novel therapy has shown promising clinical results, the majority of the patients still relapse under this treatment. These relapsed patients typically become highly resistant to treatment and have poor prognosis, emphasising the need for new effective drug combinations. Apart from BCL-2, other family members like BCL-xL and MCL1 are also common targets of BH3-mimetic drugs. This project thus aims to understand and characterise the resistance against BH3-mimetics and investigate new therapeutic approaches to overcome the challenges of resistance. Aims– This study aims (i) to characterise BH3-resistant AML cell lines for uncovering the mechanisms of drug resistance, and (ii) to identify possible combination treatment options for overcoming drug-resistance. Methods– Viability assays with Cell Titer Glo® (CTG) and Drug Sensitivity and Resistance Testing (DSRT). The long-term effectiveness of venetoclax, azacitidine and talazoparib (a PARP inhibitor) as single agents, double combinations and triple combination were investigated with Time-to-Progression (TTP) assay. For the resistant cell line models, underlying resistance mechanisms were assessed by checking protein expression of pro- and/or anti-apoptotic members of the BCL-2 family members with western blot (WB). Real-time quantitative PCR (RT-qPCR) and WB were carried out for transcriptional and translational expression analyses of certain DNA damage-associated genes in PARP inhibitor-resistant cell lines. Results– Drug screening with DSRT has revealed promising results for two combination treatments of a BCL-xL inhibitor (A-1331852) (i) with an Aurora kinase A inhibitor (alisertib) and (ii) with an MCL1 inhibitor (S63845) for BCL-xL inhibitor-resistant cells. WB analyses of BCL-2 family members showed translational upregulation of un-inhibited members of the anti-apoptotic proteins in BH3-mimetic-resistant cell lines. A venetoclax-resistant AML cell line showed increased levels of the DNA damage marker P-γ-H2Ax upon treatments containing venetoclax, as well as increased levels of cleaved-PARP1, indicating induction of apoptosis. RT-qPCR analyses revealed increased mRNA expression of PARP1 in two resistant cell lines, whereas no significant expression changes in other DNA repair mechanism genes on the transcriptional level. Conclusions– In BH3-mimetic-resistant AML cell lines, apoptosis is avoided through translational upregulation of un-inhibited anti-apoptotic members of the BCL-2 family, and this resistance can be countered by combination treatment for additional inhibition of the compensatory anti-apoptotic proteins. Venetoclax is still effective on cells resistant to it, by inducing DNA damage and sensitising these cells against inhibitors of the members of DNA repair pathway. The transcriptional upregulation of PARP1 and the increase in its auto-catalytic activity suggests the DNA damage-inducing effects of the triple combination treatment [Ven + Aza + Tal].
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(2020)Tiivistelmä – Referat – Abstract Ewing sarcoma is a rare bone and soft tissues cancer that occurs mainly among children and young adults. It is an aggressive cancer. Treatment of Ewing Sarcoma Family of Tumours (ESFT) primarily includes surgery, radiation and chemotherapy. The treatment protocol depends on the presence of tumour metastases at the time of diagnosis. In the treatment of local tumours, the 5-year patient survival rate has increased from 50% to 70%. However, patients that have tumour metastases at the time of the diagnosis or have a recurrent disease, the five-year survival rate is only 25%. As the current treatment options have reached their limits, it is important to develop more advanced therapies. DNA methylation is an epigenetic event that affects gene expression. By comparing the methylation level of the DNA in gene promoter regions in ESFT cancer cells to the methylation level of DNA in gene promoter regions in normal cells it could be possible to discover genes and signalling pathways that are important in the development of ESFT and that could be potential drug target molecules. The aim of this study is to find out the genome-wide gene promoter DNA methylation status in Ewing sarcoma cell line samples and Ewing sarcoma patient tumour samples compared to a normal reference sample. Another aim is to find gene promoter regions that are differentially methylated in the Ewing sarcoma cell line samples and the Ewing sarcoma patient tumour samples compared to the normal reference sample. Materials and Methods The Ewing Sarcoma cell line samples (12) were obtained from the Laboratory of Oncologi Research, Instituti Ortopedici Rizzoli Laboratory, Bologna, Italy. The Ewing sarcoma patient tumour samples were pre-isolated DNA samples already in Finland. The normal reference sample was a commercial mesenchymal cell line sample. From the Ewing Sarcoma cell line samples and the normal reference sample, DNA isolation was done by using phenol-chloroform method. DNA methylation profiling of the samples was performed by combining MeDIP (methylated DNA immunoprecipitation) protocol with 2-set promoter microarray hybridization protocol provided by Agilent Tecnologies company. DNA methylation data that was received from the microarrays was normalized and pre-processed with the Feature Extraction software provided also by the Agilent Technologies company. Visualization of the DNA methylation data was performed by using Chipster analysis software provided by CSC. To measure the level of DNA methylation at the gene promoter regions, a log2ratio value was calculated for every gene promoter region in all the sample types. To find gene promoter regions that were differently methylated, a log2 fold change value was calculated from the log2ratio values between the Ewing Sarcoma cell line cancer samples and the normal reference sample and between the Ewing sarcoma patient tumor samples and the normal reference sample for each gene promoter region. The log2 fold change value was also calculated between the Ewing Sarcoma cell line cancer samples and the Ewing Sarcoma patient tumor samples. After this a t-test was performed to determine the statistical significance of the log2 fold change values. Detection of genome-wide DNA methylation levels at the gene promoter regions in the Ewing sarcoma cell line samples and the Ewing sarcoma patient tumour samples compared to the normal reference sample was performed by averaging log2 fold change values. The same calculation method was used to detect the differences in the genome-wide DNA methylation levels at the gene promoter regions between the Ewing sarcoma cell lines and the Ewing Sarcoma patient tumour samples. Results Differences in the DNA methylation levels at the gene promoter regions were detected between the Ewing Sarcoma cell line samples, patient tumour samples, and the normal reference sample. Genome-wide measurement of the DNA methylation levels at the gene promoter areas showed that the Ewing sarcoma cell lines had more DNA methylation at the gene promoter regions than the patient tumour samples and the normal reference sample. The patient tumour samples showed less DNA methylation at the gene promoter regions compared to the Ewing sarcoma cell lines and the normal reference sample. Differentially methylated gene promoter regions between the Ewing sarcoma cell lines and the reference sample were found 16. In the patient tumour samples, also 16 differently methylated gene promoter regions were found compared to the normal reference sample. Differentially methylated gene promoter regions between the Ewing sarcoma cell lines and the patient tumour samples were 56.
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(2019)Tiivistelmä–Referat–Abstract Background: Cancer is one of the leading causes of death around the world and in Finland. Ambitious research projects have been carried out for decades investigating cancer and how it spreads. Over 35 years ago, the systems that regulate vascular formation were discovered; the vascular endothelial growth factor (VEGF)-VEGF receptor VEGFR system and the more recent Angiopoietin-TIE system. These are the main endothelial growth factor receptor pathways involved in regulation of vessel quiescence and angiogenesis. The VEGF-VEGFR system is the first discovered endothelial cell (EC) specific receptor tyrosine kinase (RTK) signaling system. VEGF is a major growth factor involved in proangiogenic activity and vascular permeability when bound to its receptor, VEGFR2. Tumor cells take advantage of the VEGF-VEGFR2 system by secreting VEGF to stimulate angiogenesis in surrounding tissue to create new blood vessels allowing for greater access to nutrients and oxygen for tumor growth. The Angiopoietin-TIE system is the second EC specific RTK signaling system that was discovered. Angiopoietin-1 (ANG1) is the ligand for the TIE2 RTK. ANG1 is an obligatory TIE2 agonist and its effects on intracellular signaling, cell cytoskeleton, and junction-related molecules allows ANG1 to restrict the amount and size of gaps that are formed at EC junctions in inflamed vessels, increasing barrier function and decreasing vascular permeability. Angiopoietin-2 (ANG2) is an autocrine context-dependent TIE2 agonist/antagonist which is implicated in stimulating pathological angiogenesis, inflammation and vascular permeability. Integrins are important cell surface receptors that all cells use to communicate with their environment. Recently, it has been discovered that ANG2 is capable of inducing pathological angiogenesis, and can destabilize ECs when bound to integrin, specifically β1-integrin, via ANG2 N-terminal region. Objectives: The general aim of this study was to discover which part of angiopoiten-2’s N-terminus region was responsible for integrin activation. Materials and Methods: Fibronectin fragment containing type III 7-10 domains was produced and fluorescently labeled with Alexa 647. Integrin activation was measured using the fluorescently labeled Fibronectin III 7-10 and angiopoietins. Fluorescence activated cell sorting (FACS) was used to collect the results, which were analyzed using excel. Chimeric angiopoietins were produced using retroviral vectors and used for FACS experiments. A cell internalization assay was performed in Hela cells using CellTracker™ Orange CMRA and angiopoietin proteins, stained with secondary antibody anti-human Alexa 488 and Texas Red Phallodin. Results: Optimization of the FACS assay defined the minimum number of cells required to reliably measure integrin activation and showed that BD Accuri FACS machine was better suited than Guarva FACS machine for the assay and that the amount of integrin varied between cell passages used for the assay. In addition, it was essential to ensure a homogenous mix of cells and recombinant proteins during the assay and the quality of the produced FN III 7-10 was critical for the success of the assay. Results from the FACS assay confirmed that ANG2 is capable of activating integrin. In addition, chimeric angiopoietins that were expressed and secreted from CHO cells, were capable of activating integrins to a variable degree. The results confirmed the importance of ANG2 N-terminus in integrin activation. Cell internalization assay visually demonstrated angiopoietin binding to Hela cells. ANG2 was internalized by the cell and resistant to the acid wash, while the majority of ANG1 bound to the cell surface was washed away by acid wash. Conclusions: In this thesis work, integrin activation assays were optimized and carried out, along with cell internalization assays, to determine which specific part of ANG2 is responsible for inducing integrin activation. The findings from this work confirmed that ANG2 is capable of activating integrin. Several chimeric constructs were successfully expressed in CHO cells, and the cell supernatants were used to activate integrins. However, more studies are needed to determine which specific region of ANG2 is responsible for integrin activation. Investigating angiopoietin induced integrin activation would allow for a better understanding of the angiopoietin signaling pathway with potential translational significance.
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(2022)Breast cancer is the most common cancer in the world and among women the most cancer deaths causing cancer. MYC is a proto-oncogene, which becomes oncogenic when its expression is deregulated in cancer. MYC is commonly overexpressed in human tumours and this alteration is associated with aggressive cancer phenotype. Furthermore, alterations in the MYC network have been found in the great majority of breast cancers. MYC promotes mitochondrial apoptosis causing a cancer vulnerability, however, in cancer cells the apoptosis is often prevented by antiapoptotic BCL-2 family members. In this study, cell viability and cell death analysis of treated triple-negative breast cancer cell lines together with dendritic cell activation experiments were conducted. This study aimed to find the most potent BCL-2 family antagonist (BH3 mimetic) to combine with metformin to overcome the antiapoptotic BCL-2 family proteins inhibition of MYC-induced apoptosis. In addition, this study determined whether the combinations could induce immunogenic cell death to further intensify cancer cell killing through anti-tumour immunity. In this study, BH3 mimetics combined with metformin were found to induce cell death and reduce cell viability in TNBC cell lines. In addition, metformin and BH3 mimetics were found to activate dendritic cells directly and through immunogenic cell death of cancer cells. However, no MYC-dependent cell death or immunogenic cell death were observed, and this study was unable to indicate the most potent BH3 mimetic to combine with metformin.
Now showing items 1-4 of 4