Inhibition of the mitochondrial pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase by doxorubicin and brequinar sensitizes cancer cells to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent in selectively killing tumor cells. However, TRAIL monotherapy has not been successful as many cancer cells are resistant to TRAIL. Chemotherapeutic agents, such as doxorubicin have been shown to act synergistically with TRAIL, but the exact mechanisms of actions are poorly understood. In this study, we performed high-throughput small interfering RNA screening and genome-wide gene expression profiling on doxorubicin-treated U1690 cells to explore novel mechanisms underlying doxorubicin-TRAIL synergy. The screening and expression profiling results were integrated and dihydroorotate dehydrogenase (DHODH) was identified as a potential candidate. DHODH is the rate-limiting enzyme in the pyrimidine synthesis pathway, and its expression was downregulated by doxorubicin. We demonstrated that silencing of DHODH or inhibition of DHODH activity by brequinar dramatically increased the sensitivity of U1690 cells to TRAIL-induced apoptosis both in 2D and 3D cultures, and was accompanied by downregulation of c-FLIPL as well as by mitochondrial depolarization. In addition, uridine, an end product of the pyrimidine synthesis pathway was able to rescue the sensitization effects initiated by both brequinar and doxorubicin. Furthermore, several other cancer cell lines, LNCaP, MCF-7 and HT-29 were also shown to be sensitized to TRAIL by brequinar. Taken together, our findings have identified a novel protein target and its inhibitor, brequinar, as a potential agent in TRAIL-based combinatorial cancer therapy and highlighted for the first time the importance of mitochondrial DHODH enzyme and pyrimidine pathway in mediating TRAIL sensitization in cancer cells.

HES6 gene is selectively overexpressed in glioma and represents an important transcriptional regulator of glioma proliferation.

Malignant glioma is the most common brain tumor with 16,000 new cases diagnosed annually in the United States. We performed a systematic large-scale transcriptomics data mining study of 9783 tissue samples from the GeneSapiens database to systematically identify genes that are most glioma-specific. We searched for genes that were highly expressed in 322 glioblastoma multiforme tissue samples and 66 anaplastic astrocytomas as compared with 425 samples from histologically normal central nervous system. Transcription cofactor HES6 (hairy and enhancer of split 6) emerged as the most glioma-specific gene. Immunostaining of a tissue microarray showed HES6 expression in 335 (98.8%) out of the 339 glioma samples. HES6 was expressed in endothelial cells of the normal brain and glioma tissue. Recurrent grade 2 astrocytomas and grade 2 or 3 oligodendrogliomas showed higher levels of HES6 immunoreactivity than the corresponding primary tumors. High HES6 mRNA expression correlated with the proneural subtype that generally has a favorable outcome but is prone to recur. Functional studies suggested an important role for HES6 in supporting survival of glioma cells, as evidenced by reduction of cancer cell proliferation and migration after HES6 silencing. The biological role and consequences of HES6 silencing and overexpression was explored with genome-wide analyses, which implicated a role for HES6 in p53, c-myc and nuclear factor-κB transcriptional networks. We conclude that HES6 is important for glioma cell proliferation and migration, and may have a role in angiogenesis.

Protein lysate microarray analysis to identify microRNAs regulating estrogen receptor signaling in breast cancer cell lines.

Predicting the impact of microRNAs (miRNAs) on target proteins is challenging because of their different regulatory effects at the transcriptional and translational levels. In this study, we applied a novel protein lysate microarray (LMA) technology to systematically monitor for target protein levels after high-throughput transfections of 319 pre-miRs into breast cancer cells. We identified 21 miRNAs that downregulated the estrogen receptor-alpha (ERalpha), as validated by western blotting and quantitative real time-PCR, and by demonstrating the inhibition of estrogen-stimulated cell growth. Five potent ERalpha-regulating miRNAs, miR-18a, miR-18b, miR-193b, miR-206 and miR-302c, were confirmed to directly target ERalpha in 3'-untranslated region reporter assays. The gene expression signature that they repressed highly overlapped with that of a small interfering RNA against ERalpha, and across all the signatures tested, was most closely associated with the repression of known estrogen-induced genes. Furthermore, miR-18a and miR-18b showed higher levels of expression in ERalpha-negative as compared with ERalpha-positive clinical tumors. In summary, we present systematic and direct functional evidence of miRNAs inhibiting ERalpha signaling in breast cancer, and demonstrate the high-throughput LMA technology as a novel, powerful technique in determining the relative impact of various miRNAs on key target proteins and associated cellular processes and pathways.

Identification of target genes in laryngeal squamous cell carcinoma by high-resolution copy number and gene expression microarray analyses.

Molecular mechanisms contributing to initiation and progression of head and neck squamous cell carcinoma are still poorly known. Numerous genetic alterations have been described, but molecular consequences of such alterations in most cases remain unclear. Here, we performed an integrated high-resolution microarray analysis of gene copy number and expression in 20 laryngeal cancer cell lines and primary tumors. Our aim was to identify genetic alterations that play a key role in disease pathogenesis and pinpoint genes whose expression is directly impacted by these events. Integration of DNA level data from array-based comparative genomic hybridization with RNA level information from oligonucleotide microarrays was achieved with custom-developed bioinformatic methods. High-level amplifications had a clear impact on gene expression. Across the genome, overexpression of 739 genes could be attributed to gene amplification events in cell lines, with 325 genes showing the same phenomenon in primary tumors including FADD and PPFIA1 at 11q13. The analysis of gene ontology and pathway distributions further pinpointed genes that may identify potential targets of therapeutic intervention. Our data highlight genes that may be critically important to laryngeal cancer progression and offer potential therapeutic targets.

Systemic physostigmine shows antiallodynic effects in neuropathic rats.

UNLABELLED: The aim of this study was to examine the antiallodynic and antinociceptive effects of subcutaneously administered physostigmine (50, 100, 200 micrograms/kg), compared with morphine (2.5, 5, 10 mg/kg) and NaCl after spinal nerve ligation in rats. The following stimuli were used: acetone (cold allodynia), von Frey hairs (mechanical allodynia), and paw flick test (thermal nociception). Motility boxes were used to investigate the effects of the drugs on motor performance. Physostigmine attenuated both mechanical and cold allodynia dose-dependently but had no effect on the paw flick test. The effect was antagonized by atropine (muscarinic receptor antagonist) but not by mecamylamine (nicotinic receptor antagonist) or naloxone (opioid receptor antagonist). Morphine produced dose-dependent antiallodynic and antinociceptive effects. In the antiallodynic doses, morphine caused severe rigidity. Physostigmine 200 micrograms/kg impaired locomotor activity, but no rigidity was observed. IMPLICATIONS: Physostigmine has different effects on allodynia and nociception, which suggests that different cholinergic (muscarinic) mechanisms may be involved in neuropathic and nociceptive pain.

Behavioural measures of depression and anxiety in rats with spinal nerve ligation-induced neuropathy.

The behaviour of rats with spinal nerve ligation-induced neuropathic pain was studied using tests developed to measure depression and anxiety. Adult male Sprague-Dawley rats were tested with the open field test, elevated plus maze, two compartment test and forced swimming test. Spontaneous motility was measured in a photocell observation box. Mechanical sensitivity was tested with von Frey hairs and cold sensitivity with the acetone drop test. The L5-6 spinal nerves were ligated or a sham operation was performed and the rats were followed for 2 weeks before the same set of tests were repeated. Most of the neuropathy operated rats had mechanical and cold allodynia. With post-injury there was a significant decrease in the activity in the open field test and motility box tests, when compared with the pre-injury results. In the elevated plus maze test there was a significant reduction in the motility, but there was no change in the time spent in the closed wings. In the two compartment test there were no significant differences between the pre- and post-injury results. There were no differences between the rats with spinal nerve ligation injury and the sham operated rats in any of the tests. The results were also comparable when rats that developed a high degree of neuropathy were compared with the rats with low degree of neuropathy and the sham operated group. In conclusion, spinal nerve ligation injury of the spinal nerves L5-6 induces mechanical and cold allodynia, but it does not seem to produce general suffering or measurable anxiety to the animals. Furthermore, tests for anxiety and depression were not able to predict which animals were vulnerable to express symptoms of neuropathic pain after nerve injury.

The effects of the alpha2-adrenergic agonist, dexmedetomidine, in the spinal nerve ligation model of neuropathic pain in rats.

UNLABELLED: Peripheral nerve injury may lead to neuropathic pain. Alpha2-adrenergic agonists acting in the descending inhibitory tracts of the spinal cord are effective in acute nociceptive, inflammatory, and, possibly, neuropathic pain. We studied the prevention and treatment of neuropathy with the selective alpha2-adrenergic agonist dexmedetomidine in male Sprague-Dawley rats with unilateral peripheral mononeuropathy resulting from tight ligation of the L5 and L6 spinal nerves. Rats with ligation injury developed mechanical and cold allodynia, but not heat hyperalgesia. Dexmedetomidine (120 microg/kg subcutaneously [S.C.] 30 min before the injury) did not attenuate mechanical or cold allodynia. Dexmedetomidine infusions (60 microg/d for 7 days after the injury, or 30 microg/d for 7 days started 14 days after the injury) did not attenuate mechanical or cold allodynia in the ipsilateral paw, but they increased mechanical allodynia during the latter treatment in the paw contralateral to the injury. Atipamezole (1 mg/kg S.C.) induced mechanical and cold allodynia in rats that had not developed allodynia in 14 days after the injury. In conclusion, although alpha2-adrenergic mechanisms are recognized as important in the development of neuropathic pain-like symptoms in this animal model, we found no favorable effect from systemic treatment with dexmedetomidine at tolerable doses. IMPLICATIONS: We studied the prevention and treatment of nerve injury-induced pain with the alpha2-adrenergic agonist dexmedetomidine in an animal model. At tolerable doses, systemic dexmedetomidine neither prevented nor attenuated neuropathic pain behavior.

Systemic morphine in the prevention of allodynia in the rat spinal nerve ligation model of neuropathic pain.

Peripheral nerve injury may lead to neuropathic pain that has been considered unresponsive to opioids. In animal models of neuropathic pain, there are previous data of both increased and decreased effect of opioids, but only limited information of the long-term effects of opioid treatment on the development of the symptoms of neuropathy. The possibility of preventing the development of signs of neuropathy with either a single pre-injury injection or chronic postinjury administration of morphine was studied in rats with unilateral peripheral neuropathy due to tight ligation of the L5 and L6 spinal nerves. These rats developed both mechanical and cold allodynia, but no thermal hyperalgesia. Neither subcutaneous morphine given as single injection (10 mg/kg) before the nerve injury nor chronic administration starting immediately after surgery using slow-release pellets (one, two or five pellets containing 75 mg of morphine, resulting in a total dose of 75, 150 or 375 mg) prevented the development of either mechanical or cold allodynia. No autotomy, signs of distress, altered social behaviour or morphine withdrawal was seen in any of the rats. The fact that neuropathic pain-like symptoms were not attenuated by any of the treatments studied could indicate that neither premedication nor postoperative pain management with systemic morphine is effective in preventing postoperative neuropathic pain. Copyright 1998 European Federation of Chapters of the International Association for the Study of Pain.