The androgen receptor (AR) is the central component in regulation of the androgen signalling within the prostate gland. Deregulation of the AR activity is frequently involved in the development of prostate cancer. Treatment of advanced prostate cancer often involves chemotherapy and most of these drugs exert their function by generating genotoxic stress such as DNA damage. Although many cellular responses to DNA damage have been clarified over the past years, the effects of genotoxic stress on AR function remain to be elucidated. Here, the effects of genotoxic agents used in chemotherapeutic regimes were investigated in relation to endogenously expressed AR function in the hormone responsive prostate cancer cell line LNCaP. This led to the novel finding that the topoisomerase 11 inhibitors, etoposide and doxorubicin, and the DNA crosslinking agent, cisplatin, inhibited the AR activity. It was further discovered that this loss of AR activity could not be explained by changes in cell cycle distribution, altered nuclear translocation of the AR, reduced expression of the receptor or by induction of apoptosis. Activation of the tumour suppressor p53 is a central component in various cellular responses to genotoxic stress, however, the inhibition of AR activity in response to genotoxic stress was found to be mediated by a mechanism independent of p53 function. Etoposide reduced AR ligand binding within the first hour of androgen exposure, a response only observed to a minor degree after cisplatin treatment. In contrast, cisplatin caused a loss of serine 81 phosphorylation on the AR after 8 hours of drug exposure, which was a response not seen in etoposide treated cells. Interestingly, further studies revealed that at early timepoints both agents inhibited the hormone stimulated recruitment of AR to androgen response elements (AREs) in the promoter and enhancer regions of an AR regulated gene. A possible involvement of MAPK, P13K or cell cycle checkpoint signalling pathways was investigated, but none were found to be directly involved, however, preliminary studies suggest that fully functional HSP90 may be involved in this aspect of AR regulation.

Lung cancer is the leading cause of cancer and cancer deaths worldwide, with non-small-cell lung carcinomas (NSCLC) representing 85% of all diagnosed lung cancers. Platinum-combination chemotherapy is the current standard treatment for NSCLC, however, associated toxicities and resistance limit its efficacy. Our laboratory previously identified activating transcription factor 3 (ATF3), a stress-inducible gene whose elevated and sustained expression can trigger apoptosis to a wide variety of stressors, as a key regulator of cisplatin cytotoxicity as well. Thus, enhanced and sustained induction of ATF3 by combining platins with other ATF3 inducers potentially represents an effective therapeutic strategy. A chemical library screen identified vorinostat and topotecan as ATF3 inducers that also enhance cisplatin cytotoxicity. ATF3 plays a significant role in cisplatin, vorinostat and topotecan and their combinations cytotoxicity. Importantly, vorinostat and topotecan induced synergistic cytotoxicity with cisplatin in NSCLC cell lines and their cisplatin resistance sub-lines with enhanced ATF3 expression observed. Our study suggests a potential novel therapeutic approach where ATF3 inducing agents in combination with platins represents a rational combination based therapeutic strategy.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2010. / Härtill 4 uppsatser.

Doctor of Philosophy / Department of Agronomy / Masaaki Tamura / Over the years proteins/peptides have evolved as promising therapeutic agents in the treatment of cancer. Considering the advantages of peptides such as their small size, ease of synthesis, tumor-penetrating ability and bio-compatibility, present report discusses proof of concept for 1. C1B5 peptide of protein kinase Cγ and a low dose of gemcitabine combination treatment for peritoneally disseminated pancreatic cancer and 2. dTAT peptide nanoparticles mediated gene (angiotensin II type 2 receptor gene) therapy for lung cancer. 1. A significant reduction in intraperitoneally (IP) transplanted pancreatic carcinoma growth was demonstrated with C1B5 peptide and gemcitabine co-treatment in an immunocompetent mouse model. Increased number of Granzyme B positive cells was observed in treated mice ascites, suggesting the involvement of immune response in tumor attenuation. The strong effect observed in combination treatment might be because of increase in lymphocyte recruitment by gemcitabine followed by C1B5 peptide mediated CD8+ T-cells or NK cells activation apart from direct cancer cell apoptosis. 2. To test dTAT peptide nanoparticles (dTAT NPs) mediated therapeutic gene delivery, luciferase reporter gene containing dTAT nanoparticles were synthesized (dTAT/pLUC/Ca2+). Synthesis conditions for nanoparticles were optimized based on dTAT/pLUC/Ca2+ nanoparticles transfection efficiency. With the optimized conditions, dTAT NPs containing AT2R, TRAIL or miR-34a pDNA (dTAT/pAT2R, dTAT/TRAIL or dTAT/miR- 34a) were synthesized. Therapeutic potential of these NPs was analyzed in lung adenocarcinoma containing mice by administering them intravenously (IV) or/and intratracheally (IV). Combination treatment with the IV injection of the new dTAT/pAT2R/Ca2+ formulation and the IT injection of the original dTAT/pAT2R/Ca2+ formulation is effective in attenuation of developed human bronchioloalveolar carcinoma in the SCID mouse lungs. Findings from the above mentioned studies have vital clinical relevance as it implies that peptides alone or when used as gene delivery systems may prove to be beneficial in the treatment of various stages of cancer.

No / The azinomycins are potent antitumour antibiotics that are able to crosslink DNA, but are relatively unstable and unlikely to progress as therapeutic candidates. A prototype analogue 4 with more clinical potential has been designed and synthesised and incorporates the epoxide function of the azinomycins and a nitrogen mustard. Two further analogues 5 and 6 that can alkylate DNA but cannot crosslink the duplex have also been synthesised. Compound 4 crosslinks DNA efficiently at nM concentrations. Compounds 4¿6 were submitted to the NCI 60 cell line screen and have similar antitumour activity, although 4 is slightly less active than the non-crosslinking compounds. These observations will be important in the design of further azinomycin analogues with antitumour activity.

Cancer is responsible for one fourth of the total deaths and is the second leading cause of death, behind heart disease, in the United States. However, there are as many approaches to curing cancer as there are types of cancer. One important issue in solid tumors is hypoxia, a lack of oxygen, which promotes angiogenesis and anaerobic metabolism, which can increase cancer progression and metastasis. The HIF transcription factor is responsible for the mediation of many processes involved during hypoxia and is linked to poor patient prognosis, increased cancer progression, and invasiveness of tumors. With this in mind, the HIF pathway has become an attractive target for small molecule inhibition. Herein, we describe the design and synthesis of small molecules that inhibit the HIF pathway. These compounds are based off an initial “hit” compound, KCN-1, from screening of a 10,000 compound library. KCN1 is both highly effective and has a low toxicity profile. Over 200 compounds have been synthesized by the Wang lab, with the best compound IVSR64b having an IC50 of 0.28 μM. Of special interest is that these compounds do not appear to have any inherent toxicity toward healthy tissues, but only affect cancer cells. Moreover, x-ray crystal structures for both KCN-1 and IVSR64b were obtained and used as the basis for computational modeling, which is still in progress.

No description available.

Malignant melanoma is an invasive type of skin cancer with high mortality rates, if not detected promptly. The mortality trends are generally linked to multiple dysplastic nevi, positive family history, genetic susceptibility and phenotypic features including fair skin, freckles, numerous atypical nevi, light coloured hair and eyes, inability to tan and prolonged exposure to ultraviolet radiation B (UVB). To date, the major anti-cancer therapeutics for melanoma include surgery, chemotherapy, radiotherapy, and immunotherapy. Recently, extracellular vesicles, especially exosomes, have been highlighted for their therapeutic benefits in numerous chronic diseases such as cancer. Exosomes display multifunctional properties, including inhibition of cancer cell proliferation and initiation of apoptosis. Hence, this study aimed to evaluate the genotoxicity and cytotoxicity of cord blood stem cell-derived (CBSC) exosomes on 6 samples of peripheral blood lymphocytes taken from healthy individuals and melanoma patients and on 3 samples of melanoma (CHL-1) cells. The limited number of samples was due to the time limitations and restrictions that were in place due to the COVID-19 pandemic. In this in vitro study, the optimal concentration of CBSC-derived exosomes (0, 100, 200, 300, 400 μg/ml protein at 24, 48 and 72h treatments) was confirmed by the CCK-8 assay. CBSC exosomes (300 μg/ml) were used to treat lymphocytes and CHL-1 cells in the Comet assay and evaluated using the real-time polymerase chain reaction (qPCR) and Western blotting (WB). The data of the CCK-8 and Comet assays illustrated that exosomes exerted genotoxic effects on CHL-1 cells (CCK-8 assay, ****p < 0.0001), (Comet assay, *p <0.05, **p < 0.01). However, the data portraying a reduction in the viability of lymphocytes needs further investigation as the number of samples was limited, therefore, further clarification is required. Importantly, no significant adverse effect was observed in healthy lymphocytes when treated with the same exosomes (p = ns). When further challenged with UVA+B radiation, the exosomes did not induce any genoprotective effect on ROS-induced CHL-1 cells, compared to the positive control (p = ns). Our data insinuates that the damage might be caused by inducing apoptosis. The anti-tumourigenic potential of exosomes was observed by activating the p53-mediated apoptotic pathway in CHL-1 cells, up-regulating p53, p21 and caspase 3 and down-regulating BCL-2 at mRNA (**p < 0.01, ***p <0.001, ****p <0.0001) and protein levels (*p < 0.05, **p <0.01). The potency of CBSC exosomes in inhibiting cancer progression in CHL-1 cells whilst causing no harm to the healthy lymphocytes makes it an ideal potential candidate for anti-cancer therapy. More samples are required to evaluate the therapeutic effect of exosomes on lymphocytes from cancer patients to fully understand their mechanism of action.

No description available.

Anticancer nucleosides are effective against solid tumors and hematological malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4’-ethynyl-2’-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (dCK) phosphorylation for its activity and induces replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å co-crystal structure of dCK bound to EdC and UDP reveals how the rigid 4’-alkyne of EdC fits within the active site of dCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared to FDA approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a pre-clinical nucleoside prodrug candidate for DLBCL and ALL. / Biomedical Sciences