Juergen Frank

2-methoxyestradiol induces caspase-independent, mitochondria-centered apoptosis in DS-sarcoma cells.

The anti‐cancer potential of the natural estrogen metabolite 2‐methoxyestradiol is associated with microtubuli interaction, anti‐angiogenetic effects and inhibition of superoxide dismutase leading to apoptosis. The effectors of apoptotic signaling through 2‐methoxyestradiol, however, are cell type‐dependent. We investigated the effect of 2‐methoxyestradiol on several events associated with apoptosis in rat DS‐sarcoma cells. Translocation of the pro‐apoptotic protein Bax to mitochondria was identified as an initial apoptotic event that was accompanied by a decrease in mitochondrial transmembrane potential and the formation of reactive oxygen species (ROS) followed by mitochondrial release of apoptosis inducing factor and endonuclease G. In addition, 2‐methoxyestradiol treatment caused upregulation of death receptor ligands FasL and TNFα and induced caspase‐8 activation. The pan caspase inhibitor Z‐VAD‐FMK did not suppress apoptotic cell death, however, indicating that the major pro‐apoptotic effect of 2‐methoxyestradiol is mediated by a caspase‐independent mechanism. Furthermore, ROS do not seem to play a pivotal role in the toxic/apoptotic effect of 2‐methoxyestradiol in DS‐sarcoma cells because supplementation with various antioxidants provided only limit protection. Colony formation was not affected by antioxidants. Therefore, in DS‐sarcoma cells, the breakdown of mitochondrial integrity with the subsequent release of mitochondrial nucleases is the main factor in 2‐methoxyestradiol mediated cell death.

Intensified oxidative and nitrosative stress following combined ALA-based photodynamic therapy and local hyperthermia in rat tumors.

Oxidative stress‐related changes in tumors upon localized hyperthermia (HT), 5‐aminolevulinic acid‐based photodynamic therapy (ALA‐PDT) and their combination (ALA+HT) were examined after the observation that the antitumor effects of ALA‐PDT could be significantly enhanced upon simultaneous application of HT. Rats bearing s.c. DS‐sarcomas (0.6–1.0 ml) on the hind foot dorsum were anesthetized and underwent one of the following treatments: (i) ALA‐PDT (375 mg/kg 5‐ALA i.v.); (ii) localized HT, 43°C for 60 min; (iii) combined ALA‐PDT and HT [=ALA+HT]. Appropriate control experiments were also performed. After treatment, tumors were excised and rapidly frozen for later analysis of nitrosative stress (protein nitration), apoptotic events (TUNEL, caspase activation, DNA and RNA fragmentation), expression of heat shock proteins (hsp70 and HO‐1), glutathione (GSH) levels and glutathione peroxidase (GPx) activity. Protein nitration was found to increase upon treatment, being especially pronounced in the ALA+HT group, and could partially be related to areas surrounding microvessels. The extent of nitrosative stress also correlated well with the appearance of the markers of apoptosis and the inhibition of in vivo tumor growth as seen in a previous study. GSH levels decreased upon treatment, the reduction being most prominent in the ALA‐PDT and ALA+HT groups. GPx activity, however, showed a significant decrease only in the ALA‐PDT group. Whereas hsp70 expression increased upon HT, ALA‐PDT caused a decrease, and these opposing effects were nullified with ALA+HT. The results obtained point to a number of cellular mechanisms—including effects on cellular defense mechanisms and an abrogation of the heat shock defense mechanism—that may interact to achieve the potentiated tumor response rate seen in vivo upon combined treatment.

Impact of Reactive Oxygen Species on the Expression of Adhesion Molecules in Vivo

Many non-surgical tumor treatments induce reactive oxygen species (ROS) which result in cell damage. This study investigated the impact of ROS induction on the expression of adhesion molecules and whether alpha-tocopherol pre-treatment could have a protective effect. Experimental rat DS-sarcomas were treated with a combination of localized 44 degrees C-hyperthermia, inspiratory hyperoxia and xanthine oxidase which together lead to a pronounced ROS induction. Further animals were pre-treated with alpha-tocopherol. The in vivo expression of E- and N-cadherin, alpha-catenin, integrins alpha v, beta 3 and beta 5 as well as of the integrin dimer alpha v beta 3 was assessed by flow cytometry. The expression of alpha v-, beta 3-integrin, of the alpha v beta 3-integrin dimer and of E-cadherin was significantly reduced by the ROS-inducing treatment. This effect was partially reversible by alpha-tocopherol, indicating that ROS play a role in this process. N-cadherin, alpha-catenin and beta 5-integrin expression were unaffected by ROS. These results indicate that the expression of several adhesion molecules is markedly reduced by ROS and may result in a decrease in the structural stability of tumor tissue. Further studies are needed to clarify the impact of ROS induction on the metastatic behavior of tumors.