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The genome of Aspergillus niger codes for a fusion protein (EHA25900), which can be aligned with ~50% sequence identity to 9S-dioxygenase (DOX)-allene oxide synthase (AOS) of Fusarium oxysporum, homologues of the Fusarium and Colletotrichum complexes and with over 62% sequence identity to homologues of Aspergilli, including (DOX)-9R-AOS of Aspergillus terreus. The aims were to characterize the enzymatic activities of EHA25900 and to identify crucial amino acids for the stereospecificity. Recombinant EHA25900 oxidized 18:2n-6 sequentially to 9R-hydroperoxy-10(E),12(Z)-octadecadienoic acid (9R-HPODE) and to a 9R(10)-allene oxide. 9S- and 9R-DOX-AOS catalyze abstraction of the pro-R hydrogen at C-11, but the direction of oxygen insertion differs. A comparison between twelve 9-DOX domains of 9S- and 9R-DOX-AOS revealed conserved amino acid differences, which could contribute to the chirality of products. The Gly616Ile replacement of 9R-DOX-AOS (A. niger) increased the biosynthesis of 9S-HPODE and the 9S(10)-allene oxide, whereas the Phe627Leu replacement led to biosynthesis of 9S-HPODE and the 9S(10)-allene oxide as main products. The double mutant (Gly616Ile, Phe627Leu) formed over 90% of the 9S stereoisomer of HPODE. 9S-HPODE was formed by antarafacial hydrogen abstraction and oxygen insertion, i.e., the original H-abstraction was retained but the product chirality was altered. We conclude that 9R-DOX-AOS can be altered to 9S-DOX-AOS by replacement of two amino acids (Gly616Ile, Phe627Leu) in the DOX domain.

Background. The survival of high-grade glioma patients is poor and the treatment of these patients can cause severe side effects. This fosters the necessity to identify prognostic biomarkers, in order to optimize treatment and diminish unnecessary suffering of patients. The aim of this study was to identify prognostic biomarkers for high-grade glioma patients. Methods. Eleven proteins were selected for analysis due to their suggested importance for survival of patients with other types of cancers and due to a high variation in protein levels between glioma patients (according to the Human Protein Atlas, www.proteinatlas.org). Protein expression patterns of these 11 proteins were analyzed by immunohistochemistry in tumor samples from 97 high-grade glioma patients. The prognostic values of the proteins were analyzed with univariate and multivariate Cox regression analyses for the high-grade glioma patients, including subgroup analyses of histological subtypes and immunohistochemically defined molecular subtypes. Results. The proteins with the most significant (univariate and multivariate p < 0.05) correlations were analyzed further with cross-validated Kaplan-Meier analyses for the possibility of predicting survival based on the protein expression pattern of the corresponding candidate. Random Forest classification with variable subset selection was used to analyze if a protein signature consisting of any combination of the 11 proteins could predict survival for the high-grade glioma patients and the subgroup with glioblastoma patients. The proteins which correlated most significantly (univariate and multivariate p < 0.05) to survival in the Cox regression analyses were Myc for all high-grade gliomas and FGF2, CA9 and CD44 for the subgroup of proneural gliomas, with FGF2 having a strong negative predictive value for survival. No prognostic signature of the proteins could be found. Conclusion. FGF2 is a potential prognostic biomarker for proneural glioma patients

Drugs that interfere with microtubule function can prevent cells from mitosis and may cause cell cycle arrest or apoptosis. Various microtubule targeting agents, both stabilizers and inhibitors, are used in a clinical setting to treat cancer. In the current study, we investigated the sensitivity of oesophageal cancer cells to different microtubule targeting agents. The current study demonstrated that different microtubule targeting agents disrupted the microtubule network and inhibited survival of oesophageal cancer cells in a dose-dependent manner. Interestingly, an additional cellular effect with inhibition of tyrosine phosphorylation of the EGFR and subsequent downregulation of EGFR-induced signalling was also observed, suggesting an additional mechanism of action for microtubule destabilising agents. A tyrosine phosphatase inhibitor, sodium orthovanadate, could reverse the EGFR dephosphorylation effects induced by microtubule targeting agents. The EGFR dephosphorylation could be reversed by a tyrosine phosphatase inhibitor, indicating that disruption of the microtubule network may lead to activation of a protein tyrosine phosphatasc (PTP) that can regulate EGFR phosphorylation and activation, an effect of potential clinical relevance for combination therapies in patients.

The insulin-like growth factor-1 (IGF-I) and its receptors play an important role in transformation and progression of several malignancies. Inhibitors of this pathway have been developed and evaluated but generally performed poorly in clinical trials, and several drug candidates have been abandoned. The cyclolignan picropodophyllin (PPP) has been described as a potent and selective IGF-IR inhibitor and is currently undergoing clinical trials. We investigated PPP's activity in panels of human cancer cell lines (e.g., esophageal squamous carcinoma cell lines) but found no effects on the phosphorylation or expression of IGF-IR. Nor was the cytotoxic activity of PPP related to the presence or spontaneous phosphorylation of IGF-IR. However, its activity correlated with that of known tubulin inhibitors, and it destabilized microtubule assembly at cytotoxic concentrations also achievable in patients. PPP is a stereoisomer of podophyllotoxin (PPT), a potent tubulin inhibitor, and an equilibrium between the two has previously been described. PPP could thus potentially act as a reservoir for the continuous generation of low doses of PPT. Interestingly, PPP also inhibited downstream signaling from tyrosine kinase receptors, including the serine/threonine kinase Akt. This effect is associated with microtubule-related downregulation of the EGF receptor, rather than the IGF-IR. These results suggest that the cytotoxicity and pAkt inhibition observed following treatment with the cyclolignan PPP in vitro result from microtubule inhibition (directly or indirectly by spontaneous PPT formation), rather than any effect on IGF-IR. It is also suggested that PPT should be used as a reference compound in all future studies on PPP., De två (2) sista författarna delar sistaförfattarskapet.

INTRODUCTION: Glioblastoma is characterized by chemoresistance. One factor than can contribute to chemoresistance is aberrant DNA methylation of specific genes relevant for drug response, e.g. tumor suppressor genes. AIM: The aim of this study was to investigate whether the tumor suppressor gene SHP1 is epigenetically regulated and if its overexpression affects the sensitivity to chemotherapeutic drugs with different mechanisms of action in glioblastoma cell lines. METHODS: Differences in methylation levels in the SHP1 promoter and SHP1 protein expressions between untreated cells and cells treated with the demethylating agent decitabine were analyzed with bisulfite Pyrosequencing and Western blotting. Differences in drug sensitivity to a panel of chemotherapeutic drugs with different mechanisms of action between SHP1 overexpressing clones and control clones were analyzed with the fluorometric microculture cytotoxicity assay. RESULTS: We demonstrated that SHP1 promoter methylation was correlated to SHP1 expression and that the expression was increased upon demethylation. Overexpression of SHP1 resulted in lower (p < 0.05) sensitivity to the proteasome inhibitor bortezomib and the alkylating agents cisplatin and melphalan. CONCLUSION: SHP1 expression may be epigenetically regulated and its overexpression influences the sensitivity to chemotherapeutic drugs in glioblastoma derived cells.

SOX10 is a transcription factor expressed in nerve cells and melanocytes. The aim of this study was to investigate the protein expression pattern of SOX10 in malignant melanoma tumors and to analyze whether the results correlated with clinical parameters and the proliferation marker Ki-67. Furthermore, proliferation and migration were analyzed in three different cell lines employing SOX10 small interfering RNA-mediated silencing. Expression patterns were determined in 106 primary tumors and 39 metastases in addition to 16 normal skin samples and six benign nevi employing immunohistochemistry and tissue microarrays. The immunohistochemical staining was evaluated manually and with an automated algorithm. SOX10 was strongly expressed in the benign tissues, but for the malignant tumors superficial spreading melanomas stained stronger than nodular malignant melanomas (P=0.008). The staining intensity was also inversely correlated with T-stage (Spearman's ρ=-0.261, P=0.008). Overall survival and time to recurrence were significantly correlated with SOX10 intensity, but not in multivariate analysis including T-stage. With the automated algorithm there was an inverse correlation between the SOX10 staining intensity and the proliferation marker, Ki-67 (ρ=-0.173, P=0.02) and a significant difference in the intensity signal between the benign tissues, the primary tumors and the metastases where the metastases stained the weakest (P≤0.001). SOX10 downregulation resulted in variable effects on proliferation and migration rates in the melanoma cell lines. In conclusion, the SOX10 intensity level differed depending on the tissue studied and SOX10 might have a role in survival. No conclusion regarding the role of SOX10 for in-vitro proliferation and migration could be drawn.

Heat shock protein 90 (Hsp90) has been demonstrated to protect oncogenic variants of signalling molecules from degradation and may consequently serve as a therapeutic target for the treatment of oesophageal cancer for which adequate therapy is often lacking. We studied the expression of Hsp90 in tumour tissues of human oesophageal cancer and the impact of Hsp90 inhibition on oesophageal cancer cell lines using the drug 17-allylamino-17-demethoxygeldanamycin (17-AAG). Quantitative immunohistochemistry was performed on formalin-fixed paraffin-embedded tissues from patients with oesophageal cancer. In squamous cell carcinoma, a marked upregulation of Hsp90 could be noted in dysplastic epithelium and invasive cancer compared with normal epithelium. In adenocarcinoma, Hsp90 was expressed in neoplastic epithelium and also in normal non-neoplastic glands weakly. The inhibition of Hsp90 using 17-AAG led to a significant decrease in cell proliferation and viability in human oesophageal cancer cell lines. Using a clonogenic cell survival assay, Hsp90 inhibition significantly sensitised the cells for gamma-photon irradiation. Heat shock protein 90 was found to be critical for proper signalling induced by both epidermal growth factor and insulin-like growthfactor -1, in which the inhibition of signalling by 17-AAG correlated with the observed reduction in cell proliferation and viability. These results showed that Hsp90 was selectively expressed in oesophageal cancer tissue compared with the corresponding normal tissue, and the inhibition of Hsp90 resulted in decreased proliferation and viability as well as radiosensitisation of oesophageal cancer cells. Heat shock protein 90 represents a potential therapeutic target in the treatment of patients with oesophageal cancer, alone or in combination with radiotherapy.