Effects of ligand activation of peroxisome proliferator-activated receptor γ in human prostate cancer

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear hormone receptor that plays a key role in the differentiation of adipocytes. Activation of this receptor in liposarcomas and breast and colon cancer cells also induces cell growth inhibition and differentiation. In the present study, we show that PPARγ is expressed in human prostate adenocarcinomas and cell lines derived from these tumors. Activation of this receptor with specific ligands exerts an inhibitory effect on the growth of prostate cancer cell lines. Further, we show that prostate cancer and cell lines do not have intragenic mutations in the PPARγ gene, although 40% of the informative tumors have hemizygous deletions of this gene. Based on our preclinical data, we conducted a phase II clinical study in patients with advanced prostate cancer using troglitazone, a PPARγ ligand used for the treatment of type 2 diabetes. Forty-one men with histologically confirmed prostate cancer and no symptomatic metastatic disease were treated orally with troglitazone. An unexpectedly high incidence of prolonged stabilization of prostate-specific antigen was seen in patients treated with troglitazone. In addition, one patient had a dramatic decrease in serum prostate-specific antigen to nearly undetectable levels. These data suggest that PPARγ may serve as a biological modifier in human prostate cancer and its therapeutic potential in this disease should be further investigated.

Stepwise in vitro affinity maturation of Vitaxin, an αvβ3-specific humanized mAb

A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based mutagenesis was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the αvβ3 integrin complex, was used as a model system. Specifically, focused mutagenesis was used in a stepwise fashion to rapidly improve the affinity of the antigen binding fragment by greater than 90-fold. In the complete absence of structural information about the Vitaxin-αvβ3 interaction, phage-expressed antibody libraries for all six Ig heavy and light chain complementarity-determining regions were expressed and screened by a quantitative assay to identify variants with improved binding to αvβ3. The Vitaxin variants in these libraries each contained a single mutation, and all 20 amino acids were introduced at each complementarity-determining region residue, resulting in the expression of 2,336 unique clones. Multiple clones displaying 2- to 13-fold improved affinity were identified. Subsequent expression and screening of a library of 256 combinatorial variants of the optimal mutations identified from the primary libraries resulted in the identification of multiple clones displaying greater than 50-fold enhanced affinity. These variants inhibited ligand binding to receptor more potently as demonstrated by inhibition of cell adhesion and ligand competition assays. Because of the limited mutagenesis and combinatorial approach, Vitaxin variants with enhanced affinity were identified rapidly and required the synthesis of only 2,592 unique variants. The use of such small focused libraries obviates the need for phage affinity selection approaches typically used, permitting the use of functional assays and the engineering of proteins expressed in mammalian cell culture.

A visceral pain pathway in the dorsal column of the spinal cord

A limited midline myelotomy at T10 can relieve pelvic cancer pain in patients. This observation is explainable in light of strong evidence in support of the existence of a visceral pain pathway that ascends in the dorsal column (DC) of the spinal cord. In rats and monkeys, responses of neurons in the ventral posterolateral thalamic nucleus to noxious colorectal distention are dramatically reduced after a lesion of the DC at T10, but not by interruption of the spinothalamic tract. Blockade of transmission of visceral nociceptive signals through the rat sacral cord by microdialysis administration of morphine or 6-cyano-7-nitroquinoxaline-2,3-dione shows that postsynaptic DC neurons in the sacral cord transmit visceral nociceptive signals to the gracile nucleus. Retrograde tracing studies in rats demonstrate a concentration of postsynaptic DC neurons in the central gray matter of the L6-S1 spinal segments, and anterograde tracing studies show that labeled axons ascend from this region to the gracile nucleus. A similar projection from the midthoracic spinal cord ends in the gracile and cuneate nuclei. Behavioral experiments demonstrate that DC lesions reduce the nocifensive responses produced by noxious stimulation of the pancreas and duodenum, as well as the electrophysiological responses of ventral posterolateral neurons to these stimuli. Repeated regional blood volume measurements were made in the thalamus and other brain structures in anesthetized monkeys in response to colorectal distention by functional MRI. Sham surgery did not reduce the regional blood volume changes, whereas the changes were eliminated by a DC lesion at T10.

Neuroprotective activity of a new class of steroidal inhibitors of the N-methyl-d-aspartate receptor

Release of the excitatory neurotransmitter glutamate and the excessive stimulation of N-methyl-d-aspartate (NMDA)-type glutamate receptors is thought to be responsible for much of the neuronal death that occurs following focal hypoxia-ischemia in the central nervous system. Our laboratory has identified endogenous sulfated steroids that potentiate or inhibit NMDA-induced currents. Here we report that 3α-ol-5β-pregnan-20-one hemisuccinate (3α5βHS), a synthetic homologue of naturally occurring pregnanolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3α5βHS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3α5βHS to rats (at a nonsedating dose) following focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduces cortical and subcortical infarct size. The in vitro and in vivo neuroprotective effects of 3α5βHS demonstrate that this steroid represents a new class of potentially useful therapeutic agents for the treatment of stroke and certain neurodegenerative diseases that involve over activation of NMDA receptors.