IPA-3 Inhibits the Growth of Liver Cancer Cells By Suppressing PAK1 and NF-κB Activation

Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide and is associated with poor prognosis due to the high incidences of metastasis and tumor recurrence. Our previous study showed that overexpression of p21-activated protein kinase 1 (PAK1) is frequently observed in HCC and is associated with a more aggressive tumor behavior, suggesting that PAK1 is a potential therapeutic target in HCC. In the current study, an allosteric small molecule PAK1 inhibitor, IPA-3, was evaluated for the potential in suppressing hepatocarcinogenesis. Consistent with other reports, inhibition of PAK1 activity was observed in several human HCC cell lines treated with various dosages of IPA-3. Using cell proliferation, colony formation and BrdU incorporation assays, we demonstrated that IPA-3 treatment significantly inhibited the growth of HCC cells. The mechanisms through which IPA-3 treatment suppresses HCC cell growth are enhancement of apoptosis and blockage of activation of NF-κB. Furthermore, our data suggested that IPA-3 not only inhibits the HCC cell growth, but also suppresses the metastatic potential of HCC cells. Nude mouse xenograft assay demonstrated that IPA-3 treatment significantly reduced the tumor growth rate and decreased tumor volume, indicating that IPA-3 can suppress the in vivo tumor growth of HCC cells. Taken together, our demonstration of the potential preclinical efficacy of IPA-3 in HCC provides the rationale for cancer therapy.     

REVERSION FROM FLOWERING TO THE VEGETATIVE STATE IN XANTHIUM

Lam , S. L. and A. C. Leopold . (Purdue U., Lafayette, Indiana.) Reversion from flowering to the vegetative state in Xanthium. Amer. Jour. Bot. 47(4): 256—259. Illus. 1960.–Plants of Xanthium pensylvanicum Wallr. which had been induced with short photoperiods were caused to revert to the vegetative state either by decapitation or by pinching and subsequent partial de-budding. The ability of induced plants to revert varies with the intensity of the inductive stimulus, and with the extent to which the plant is cut back. Four successive decapitation treatments caused complete reversion of almost all plants which had been given 7 inductive cycles. Reversion can be obtained after 3 inductive cycles even when all of the induced leaves remain on the plant. It is suggested that under the conditions of these experiments the synthesis of the flowering stimulus is stopped or the stimulus is immobilized in the leaves and made ineffective.     

Strategy combining chiral chromatography with β-cyclodextrin and stereospecific enzyme reaction detection with hydroxysteroid dehydrogenases for resolving steroid epimers

Steroids are chiral molecules with multiple stereogenic centers. Studies of their intermediary metabolism often require analytical techniques to separate the isomers and determine their stereochemistry. Methods for resolving steroid stereoisomers by HPLC using β-cyclodextrin in the mobile phase are reported. Even with the improved selectivity of cyclodextrin chromatography, not all isomers within a steroid series can be resolved. Additional specificity is achieved by reaction detection using postcolumn reactors containing hydroxysteroid dehydrogenases stereospecific for the configuration of the hydroxy functions of steroids. The enzymes catalyze the oxidation of hydroxysteroids and reduction of the coenzyme NAD to NADH. NADH, which is highly fluorescent, is detected at the nanogram levels. Isomers not separated by chromatography were effectively resolved by reaction detection with stereospecific enzymes. © 1992 Wiley-Liss, Inc.     

A Complex RNA-Cleaving DNAzyme That Can Efficiently Cleave a Pyrimidine-Pyrimidine Junction

Several RNA-cleaving deoxyribozymes (DNAzymes) have been reported for efficient cleavage of purine-containing junctions, but none is able to efficiently cleave pyrimidine-pyrimidine (Pyr-Pyr) junctions. We hypothesize that a stronger Pyr-Pyr cleavage activity requires larger DNAzymes with complex structures that are difficult to isolate directly from a DNA library; one possible way to obtain such DNAzymes is to optimize DNA sequences with weak activities. To test this, we carried out an in vitro selection study to derive DNAzymes capable of cleaving an rC-T junction in a chimeric DNA/RNA substrate from DNA libraries constructed through chemical mutagenesis of five previous DNAzymes with a k_obs of ∼ 0.001 min^− 1 for the rC-T junction. After several rounds of selective amplification, DNAzyme descendants with a k_obs of ∼ 0.1 min^− 1 were obtained from a DNAzyme pool. The most efficient motif, denoted “CT10-3.29,” was found to have a catalytic core of ∼ 50 nt, larger than other known RNA-cleaving DNAzymes, and its secondary structure contains five short duplexes confined by a four-way junction. Several variants of CT10-3.29 exhibit a k_obs of 0.3-1.4 min^− 1 against the rC-T junction. CT10-3.29 also shows strong activity (k_obs  > 0.1 min^− 1) for rU-A and rU-T junctions, medium activity (> 0.01 min^− 1) for rC-A and rA-T junctions, and weak activity (> 0.001 min^− 1) for rA-A, rG-T, and rG-A junctions. Interestingly, a single-point mutation within the catalytic core of CT10-3.29 altered the pattern of junction specificity with a significantly decreased ability to cleave rC-T and rC-A junctions and a substantially increased ability to cleave rA-A, rA-T, rG-A, rG-T, rU-A, and rU-T junctions. This observation illustrates the intricacy and plasticity of this RNA-cleaving DNAzyme in dinucleotide junction selectivity. The current study shows that it is feasible to derive efficient DNAzymes for a difficult chemical task and reveals that DNAzymes require more complex structural solutions for such a task.     

Functions of Fun30 Chromatin Remodeler in Regulating Cellular Resistance to Genotoxic Stress

The Saccharomyces cerevisiae Fun30 chromatin remodeler has recently been shown to facilitate long-range resection of DNA double strand break (DSB) ends, which proceeds homologous recombination (HR). This is believed to underlie the role of Fun30 in promoting cellular resistance to DSB inducing agent camptothecin. We show here that Fun30 also contributes to cellular resistance to genotoxins methyl methanesulfonate (MMS) and hydroxyurea (HU) that can stall the progression of DNA replication. We present evidence implicating DNA end resection in Fun30-dependent MMS-resistance. On the other hand, we show that Fun30 deletion suppresses the MMS- and HU-sensitivity of cells lacking the Rad5/Mms2/Ubc13-dependent error-free DNA damage tolerance mechanism. This suppression is not the result of a reduction in DNA end resection, and is dependent on the key HR component Rad51. We further show that Fun30 negatively regulates the recovery of rad5Δ mutant from MMS induced G2/M arrest. Therefore, Fun30 has two functions in DNA damage repair: one is the promotion of cellular resistance to genotoxic stress by aiding in DNA end resection, and the other is the negative regulation of a Rad51-dependent, DNA end resection-independent mechanism for countering replicative stress. The latter becomes manifest when Rad5 dependent DNA damage tolerance is impaired. In addition, we find that the putative ubiquitin-binding CUE domain of Fun30 serves to restrict the ability of Fun30 to hinder MMS- and HU-tolerance in the absence of Rad5.