Detecting functional changes with [(18)F]FAZA in a renal cell carcinoma mouse model following sunitinib therapy.

BACKGROUND: The multitargeting tyrosine kinase inhibitor (TKI) sunitinib is currently the first-line drug therapy for metastasizing renal cell carcinoma (RCC). TKIs have profound effects on tumor angiogenesis, leading to modifications of the tumor microenvironment. The goal of this study was to determine whether these treatment-induced changes can be detected with [(18)F]FAZA. METHODS: The present study utilized positron emission tomography (PET) to analyze tumor oxygenation status during and after sunitinib therapy in the murine Caki-1 RCC tumor model. Dynamic and static scans were performed, as well as ex vivo biodistributions at 3 h post injection (p.i.). Immunohistochemical analysis of tumor tissue was carried out for the quantification of pimonidazole binding and the hypoxia-associated factors CD-31, Ki-67, and Von Willebrand factor (VWF). In addition, in vitro cellular uptake studies were done to analyze the direct effects of sunitinib on the Caki-1 cells. RESULTS: During therapy with sunitinib (40 mg/kg/day), uptake of [(18)F]FAZA into Caki-1 mice decreased by 46 ± 5% (n = 4; 5 days) at 3 h post injection (p.i.) during the first study and 22 ± 5% (n = 8; 9 days) during the long-term study, indicating a decrease in the tumor's hypoxia level. However, when drug therapy was stopped, this effect was reversed completely, and the tumor [(18)F]FAZA uptake increased to 126 ± 6% (n = 6) of the control tumor uptake, indicative of an even higher level of tumor hypoxia compared to the therapy starting point. Sunitinib had no direct effect on [(18)F]FAZA uptake into Caki-1 cells in vitro. CONCLUSION: [(18)F]FAZA PET could be used to monitor drug response during sunitinib therapy in RCC and may guide combination therapies based on the tumor's hypoxia status.

Knockdown of cytoglobin expression sensitizes human glioma cells to radiation and oxidative stress.

Cytoglobin is a recently identified vertebrate globin whose functions include scavenging reactive oxygen and nitrosative species. In tumor cells, CYGB may function as a tumor suppressor gene. Here we show that knockdown of cytoglobin expression can sensitize human glioma cells to oxidative stress induced by chemical inhibitors of the electron transport chain and as well can increase cellular radiosensitivity. When treated with antimycin A, an inhibitor of the mitochondrial electron transport chain, cytoglobin-deficient cells showed significantly higher H2O2 levels, whereas H2O2 levels were significantly reduced in cytoglobin-overexpressing cells. In addition, cytoglobin knockdown significantly decreased the doubling time of glioma cell lines, consistent with a putative tumor suppressor function. These finding suggest that modulating cytoglobin levels may be a promising treatment strategy for sensitizing human glioma cells to oxidative stress that is induced by ionizing radiation, certain chemotherapies and ischemia-reperfusion.

The cytotoxicity of gamma-secretase inhibitor I to breast cancer cells is mediated by proteasome inhibition, not by gamma-secretase inhibition.

INTRODUCTION: Notch is a family of transmembrane protein receptors whose activation requires proteolytic cleavage by gamma-secretase. Since aberrant Notch signaling can induce mammary carcinomas in transgenic mice and high expression levels of Notch receptors and ligands correlates with overall poor clinical outcomes, inhibiting gamma-secretase with small molecules may be a promising approach for breast cancer treatment. Consistent with this hypothesis, two recent papers reported that gamma-secretase inhibitor I (GSI I), Z-LLNle-CHO, is toxic to breast cancer cells both in vitro and in vivo. In this study, we compared the activity and cytotoxicity of Z-LLNle-CHO to that of two highly specific GSIs, DAPT and L-685,458 and three structurally unrelated proteasome inhibitors, MG132, lactacystin, and bortezomib in order to study the mechanism underlying the cytotoxicity of Z-LLNle-CHO in breast cancer cells. METHODS: Three estrogen receptor (ER) positive cell lines, MCF-7, BT474, and T47D, and three ER negative cell lines, SKBR3, MDA-MB-231, and MDA-MB-468, were used in this study. Both SKBR3 and BT474 cells also overexpress HER2/neu. Cytotoxicity was measured by using an MTS cell viability/proliferation assay. Inhibition of gamma-secretase activity was measured by both immunoblotting and immunofluorescent microscopy in order to detect active Notch1 intracellular domain. Proteasome inhibition was determined by using a cell-based proteasome activity assay kit, by immunoblotting to detect accumulation of polyubiquitylated protein, and by immunofluorescent microscopy to detect redistribution of cellular ubiquitin. RESULTS: We found that blocking gamma-secretase activity by DAPT and L-685,458 had no effect on the survival and proliferation of a panel of six breast cancer cell lines while Z-LLNle-CHO could cause cell death even at concentrations that inhibited gamma-secretase activity less efficiently. Furthermore, we observed that Z-LLNle-CHO could inhibit proteasome activity and the relative cellular sensitivity of these six breast cancer cell lines to Z-LLNle-CHO was the same as observed for three proteasome inhibitors. Finally, we found that the cell killing effect of Z-LLNle-CHO could be reversed by a chemical that restored the proteasome activity. CONCLUSIONS: We conclude that the cytotoxicity of Z-LLNle-CHO in breast cancer cells is mediated by proteasome inhibition, not by gamma-secretase inhibition.

mtDNA mutations in invasive cervix tumors: a retrospective analysis.

We tested whether mtDNA mutations are associated with poor outcome in patients with invasive cervix cancer. Tumor samples were banked more than 10 years ago from women with diagnoses of invasive cervix cancer. Automated techniques were used to determine the sequence of the mtDNA-encoded Complex I subunits. Approximately one-third of all tumors had multiple mtDNA sequence alterations. Both univariate and multivariate analysis of the 10 years survival probability showed that the 10 years survival of patients whose tumors had eight or more nucleotide substitutions was significantly worse (P<0.0063 and P<0.012, respectively). The log-rank test also found a significant difference in overall survival (P<0.003). These results suggest that multiple mtDNA mutations are an independent marker of poor prognosis, and that prospective clinical trials that incorporate analysis of mitochondrial genetic alterations in cervix cancer are warranted.