Functional buffering via cell-specific gene expression promotes tissue homeostasis and cancer robustness.

Functional buffering that ensures biological robustness is critical for maintaining tissue homeostasis, organismal survival, and evolution of novelty. However, the mechanism underlying functional buffering, particularly in multicellular organisms, remains largely elusive. Here, we proposed that functional buffering can be mediated via expression of buffering genes in specific cells and tissues, by which we named Cell-specific Expression-BUffering (CEBU). We developed an inference index (C-score) for CEBU by computing C-scores across 684 human cell lines using genome-wide CRISPR screens and transcriptomic RNA-seq. We report that C-score-identified putative buffering gene pairs are enriched for members of the same duplicated gene family, pathway, and protein complex. Furthermore, CEBU is especially prevalent in tissues of low regenerative capacity (e.g., bone and neuronal tissues) and is weakest in highly regenerative blood cells, linking functional buffering to tissue regeneration. Clinically, the buffering capacity enabled by CEBU can help predict patient survival for multiple cancers. Our results suggest CEBU as a potential buffering mechanism contributing to tissue homeostasis and cancer robustness in humans.

PRKDC: new biomarker and drug target for checkpoint blockade immunotherapy.

BACKGROUND: Immunological checkpoint blockade is effective in treating various malignancies. Identifying predictive biomarkers to assist patient selection for immunotherapy has become a priority in both clinical and research settings. METHODS: Mutations in patients who responded to immunotherapy were identified through next-generation sequencing. Relationships among protein kinase, DNA-activated, catalytic polypeptide (PRKDC) mutations, mutation load and microsatellite instability (MSI) were analyzed using datasets from The Cancer Genome Atlas. These relationships were validated by conducting an in vitro study and by using tissue samples from 34 patients with gastric cancer. The CT26 animal model was used to evaluate the role of PRKDC as a predictive biomarker and the efficacy of the DNA-PK inhibitor. RESULTS: From the published literature, we found that among patients whose tumors harbored PRKDC mutations, 75%, 53.8%, and 50% of those with lung cancer, melanoma, and renal cell carcinoma, respectively, responded to immunotherapy. Most of these mutations were truncating and located in functional domains or in a destabilizing PRKDC protein structure. Additional analysis showed that a PRKDC mutation was significantly associated with a high mutation load in cervical cancer, colon adenocarcinoma, head and neck squamous cell carcinoma, lung adenocarcinoma, gastric adenocarcinoma and endometrial cancer. Patients with gastric cancer or colon cancer harboring PRKDC mutations were also highly associated with MSI-high status. Finally, we found that knockout PRKDC or DNA-PK inhibitor (PRKDC encodes the catalytic subunit of DNA-dependent protein kinase) enhanced the efficacy of the anti-programmed cell death protein one pathway monoclonal antibody in the CT26 animal model. CONCLUSIONS: PRKDC is not only a predictive biomarker but also a drug target for immune checkpoint inhibitors.

Establishing guidelines to harmonize tumor mutational burden (TMB): in silico assessment of variation in TMB quantification across diagnostic platforms: phase I of the Friends of Cancer Research TMB Harmonization Project.

BACKGROUND: Tumor mutational burden (TMB), defined as the number of somatic mutations per megabase of interrogated genomic sequence, demonstrates predictive biomarker potential for the identification of patients with cancer most likely to respond to immune checkpoint inhibitors. TMB is optimally calculated by whole exome sequencing (WES), but next-generation sequencing targeted panels provide TMB estimates in a time-effective and cost-effective manner. However, differences in panel size and gene coverage, in addition to the underlying bioinformatics pipelines, are known drivers of variability in TMB estimates across laboratories. By directly comparing panel-based TMB estimates from participating laboratories, this study aims to characterize the theoretical variability of panel-based TMB estimates, and provides guidelines on TMB reporting, analytic validation requirements and reference standard alignment in order to maintain consistency of TMB estimation across platforms. METHODS: Eleven laboratories used WES data from The Cancer Genome Atlas Multi-Center Mutation calling in Multiple Cancers (MC3) samples and calculated TMB from the subset of the exome restricted to the genes covered by their targeted panel using their own bioinformatics pipeline (panel TMB). A reference TMB value was calculated from the entire exome using a uniform bioinformatics pipeline all members agreed on (WES TMB). Linear regression analyses were performed to investigate the relationship between WES and panel TMB for all 32 cancer types combined and separately. Variability in panel TMB values at various WES TMB values was also quantified using 95% prediction limits. RESULTS: Study results demonstrated that variability within and between panel TMB values increases as the WES TMB values increase. For each panel, prediction limits based on linear regression analyses that modeled panel TMB as a function of WES TMB were calculated and found to approximately capture the intended 95% of observed panel TMB values. Certain cancer types, such as uterine, bladder and colon cancers exhibited greater variability in panel TMB values, compared with lung and head and neck cancers. CONCLUSIONS: Increasing uptake of TMB as a predictive biomarker in the clinic creates an urgent need to bring stakeholders together to agree on the harmonization of key aspects of panel-based TMB estimation, such as the standardization of TMB reporting, standardization of analytical validation studies and the alignment of panel-based TMB values with a reference standard. These harmonization efforts should improve consistency and reliability of panel TMB estimates and aid in clinical decision-making.

Crystal structures of monometallic dihydropyrimidinase and the human dihydroorotase domain K1556A mutant reveal no lysine carbamylation within the active site.

Dihydropyrimidinase (DHPase) is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase (DHOase), hydantoinase, and imidase. Almost all of these zinc metalloenzymes possess a binuclear metal center in which two metal ions are bridged by a post-translational carbamylated Lys. Crystal structure of Tetraodon nigroviridis DHPase reveals that one zinc ion is sufficient to stabilize Lys carbamylation. In this study, we found that one metal coordination was not sufficient to fix CO2 to the Lys in bacterial DHPase. We prepared and characterized mono-Zn DHPase from Pseudomonas aeruginosa (PaDHPase), and the catalytic activity of mono-Zn PaDHPase was not detected. The crystal structure of mono-Zn PaDHPase determined at 2.23 Šresolution (PDB entry 6AJD) revealed that Lys150 was no longer carbamylated. This finding indicated the decarbamylation of the Lys during the metal chelating process. To confirm the state of Lys carbamylation in mono-Zn PaDHPase in solution, mass spectrometric (MS) analysis was carried out. The MS result was in agreement with the theoretical value for uncarbamylated PaDHPase. Crystal structure of the human DHOase domain (huDHOase) K1556A mutant was also determined (PDB entry 5YNZ), and the structure revealed that the active site of huDHOase K1556A mutant contained one metal ion. Like mono-Zn PaDHPase, oxygen ligands of the carbamylated Lys were not required for Znα binding. Considering the collective data from X-ray crystal structure and MS analysis, mono-Zn PaDHPase in both crystalline state and solution was not carbamylated. In addition, structural evidences indicated that post-translational carbamylated Lys was not required for Znα binding in PaDHPase and in huDHOase.

Structural Basis for pH-Dependent Oligomerization of Dihydropyrimidinase from Pseudomonas aeruginosa PAO1.

Dihydropyrimidinase, a dimetalloenzyme containing a carboxylated lysine within the active site, is a member of the cyclic amidohydrolase family, which also includes allantoinase, dihydroorotase, hydantoinase, and imidase. Unlike all known dihydropyrimidinases, which are tetrameric, pseudomonal dihydropyrimidinase forms a dimer at neutral pH. In this paper, we report the crystal structure of P. aeruginosa dihydropyrimidinase at pH 5.9 (PDB entry 5YKD). The crystals of P. aeruginosa dihydropyrimidinase belonged to space group C2221 with cell dimensions of a = 108.9, b = 155.7, and c = 235.6 Å. The structure of P. aeruginosa dihydropyrimidinase was solved at 2.17 Å resolution. An asymmetric unit of the crystal contained four crystallographically independent P. aeruginosa dihydropyrimidinase monomers. Gel filtration chromatographic analysis of purified P. aeruginosa dihydropyrimidinase revealed a mixture of dimers and tetramers at pH 5.9. Thus, P. aeruginosa dihydropyrimidinase can form a stable tetramer both in the crystalline state and in the solution. Based on sequence analysis and structural comparison of the dimer-dimer interface between P. aeruginosa dihydropyrimidinase and Thermus sp. dihydropyrimidinase, different oligomerization mechanisms are proposed.

Characterization of single-stranded DNA-binding protein SsbB from Staphylococcus aureus: SsbB cannot stimulate PriA helicase.

Single-stranded DNA-binding proteins (SSBs) are essential to cells as they participate in DNA metabolic processes, such as DNA replication, repair, and recombination. The functions of SSBs have been studied extensively in Escherichia coli. Unlike E. coli, which contains only one type of SSB (EcSSB), some bacteria have more than one paralogous SSB. In Staphylococcus aureus, three SSBs are found, namely, SsbA, SaSsbB, and SsbC. While EcSSB can significantly stimulate EcPriA helicase, SaSsbA does not affect the SaPriA activity. It remains unclear whether SsbBs can participate in the PriA-directed DNA replication restart process. In this study, we characterized the properties of SaSsbBs through structural and functional analyses. Crystal structure of SaSsbB determined at 2.9 Å resolution (PDB entry 5YYU) revealed four OB folds in the N-terminal DNA-binding domain. DNA binding analysis using EMSA showed that SaSsbB binds to ssDNA with greater affinity than SaSsbA does. Gene map analysis demonstrated that SAAV0835 encoding SaSsbB is flanked by unknown genes encoding hypothetical proteins, namely, putative Sipho_Gp157, ERF, and HNHc_6 gene products. Structure-based mutational analysis indicated that the four aromatic residues (Phe37, Phe48, Phe54, and Tyr82) in SaSsbB are at positions that structurally correspond to the important residues of EcSSB for binding to ssDNA and are also critical for SaSsbB to bind ssDNA. Similar to EcSSB and other SSBs such as SaSsbA and SaSsbC, SaSsbB also exhibited high thermostability. However, unlike EcSSB, which can stimulate EcPriA, SaSsbB did not affect the activity of SaPriA. Based on results in this study and previous works, we therefore established that SsbA and SsbB, as well as SsbC, do not stimulate PriA activity.

Genome-wide analysis of enhancer RNA in gene regulation across 12 mouse tissues.

Enhancers play a crucial role in gene regulation but the participation of enhancer transcripts (i.e. enhancer RNA, eRNAs) in regulatory systems remains unclear. We provide a computational analysis on eRNAs using genome-wide data across 12 mouse tissues. The expression of genes targeted by transcribing enhancer is positively correlated with eRNA expression and significantly higher than expression of genes targeted by non-transcribing enhancers. This result implies eRNA transcription indicates a state of enhancer that further increases gene expression. This state of enhancer is tissue-specific, as the same enhancer differentially transcribes eRNAs across tissues. Therefore, the presence of eRNAs describes a tissue-specific state of enhancer that is generally associated with higher expressed target genes, surmising as to whether eRNAs have gene activation potential. We further found a large number of eRNAs contain regions in which sequences and secondary structures are similar to microRNAs. Interestingly, an increasing number of recent studies hypothesize that microRNAs may switch from their general repressive role to an activating role when targeting promoter sequences. Collectively, our results provide speculation that eRNAs may be associated with the selective activation of enhancer target genes.

Associations between intronic non-B DNA structures and exon skipping.

Non-B DNA structures are abundant in the genome and are often associated with critical biological processes, including gene regulation, chromosome rearrangement and genome stabilization. In particular, G-quadruplex (G4) may affect alternative splicing based on its ability to impede the activity of RNA polymerase II. However, the specific role of non-B DNA structures in splicing regulation still awaits investigation. Here, we provide a genome-wide and cross-species investigation of the associations between five non-B DNA structures and exon skipping. Our results indicate a statistically significant correlation of each examined non-B DNA structures with exon skipping in both human and mouse. We further show that the contributions of non-B DNA structures to exon skipping are influenced by the occurring region. These correlations and contributions are also significantly different in human and mouse. Finally, we detailed the effects of G4 by showing that occurring on the template strand and the length of G-run, which is highly related to the stability of a G4 structure, are significantly correlated with exon skipping activity. We thus show that, in addition to the well-known effects of RNA and protein structure, the relative positional arrangement of intronic non-B DNA structures may also impact exon skipping.

The impact of trans-regulation on the evolutionary rates of metazoan proteins.

Transcription factor (TF) and microRNA (miRNA) are two crucial trans-regulatory factors that coordinately control gene expression. Understanding the impacts of these two factors on the rate of protein sequence evolution is of great importance in evolutionary biology. While many biological factors associated with evolutionary rate variations have been studied, evolutionary analysis of simultaneously accounting for TF and miRNA regulations across metazoans is still uninvestigated. Here, we provide a series of statistical analyses to assess the influences of TF and miRNA regulations on evolutionary rates across metazoans (human, mouse and fruit fly). Our results reveal that the negative correlations between trans-regulation and evolutionary rates hold well across metazoans, but the strength of TF regulation as a rate indicator becomes weak when the other confounding factors that may affect evolutionary rates are controlled. We show that miRNA regulation tends to be a more essential indicator of evolutionary rates than TF regulation, and the combination of TF and miRNA regulations has a significant dependent effect on protein evolutionary rates. We also show that trans-regulation (especially miRNA regulation) is much more important in human/mouse than in fruit fly in determining protein evolutionary rates, suggesting a considerable variation in rate determinants between vertebrates and invertebrates.

Evolution of cis-regulatory elements in yeast de novo and duplicated new genes.

BACKGROUND: New genes that originate from non-coding DNA rather than being duplicated from parent genes are called de novo genes. Their short evolution time and lack of parent genes provide a chance to study the evolution of cis-regulatory elements in the initial stage of gene emergence. Although a few reports have discussed cis-regulatory elements in new genes, knowledge of the characteristics of these elements in de novo genes is lacking. Here, we conducted a comprehensive investigation to depict the emergence and establishment of cis-regulatory elements in de novo yeast genes. RESULTS: In a genome-wide investigation, we found that the number of transcription factor binding sites (TFBSs) in de novo genes of S. cerevisiae increased rapidly and quickly became comparable to the number of TFBSs in established genes. This phenomenon might have resulted from certain characteristics of de novo genes; namely, a relatively frequent gain of TFBSs, an unexpectedly high number of preexisting TFBSs, or lower selection pressure in the promoter regions of the de novo genes. Furthermore, we identified differences in the promoter architecture between de novo genes and duplicated new genes, suggesting that distinct regulatory strategies might be employed by genes of different origin. Finally, our functional analyses of the yeast de novo genes revealed that they might be related to reproduction. CONCLUSIONS: Our observations showed that de novo genes and duplicated new genes possess mutually distinct regulatory characteristics, implying that these two types of genes might have different roles in evolution.

The longitudinal changes of the visual field in an Asian population with primary angle-closure glaucoma with and without an acute attack.

PURPOSE: To investigate the longitudinal changes of visual field (VF) in Asian patients with primary angle-closure glaucoma (PACG) occurring with and without an acute attack. METHODS: In this retrospective case series, 87 consecutive patients diagnosed with bilateral PACG during the period from 2000 to 2010 were included. The eyes of the enrolled patients were categorized into 2 groups: PACG with 1 documented episode of an acute episode in the affected eye (APACG); PACG without a previous documented acute episode in the fellow eye (CPACG). The historical change in the VF in both groups was assessed every 3 months for >2 years. RESULTS: Of the 174 eyes included in the study, 87 eyes belonged to the APACG group, and 87 eyes belonged to the CPACG group. The mean deviation (MD) in the APACG group decreased significantly from -6.22 to -6.73 dB between the 6- and 9-month follow-up periods (P=0.03). There were significant differences between the 2 groups in the MD index from the 9- to 24-month follow-up. The corrected pattern standard deviation (CPSD) in the APACG group increased significantly from 3.61 to 3.71 dB between the 6- and 9-month follow-up periods (P=0.04). The CPSD index was higher in the APACG group than in the CPACG group from the 9- to 24-month follow-up, which was a statistically significant difference. CONCLUSIONS: Glaucomatous VF damage in Asian patients with APACG was detected after a 6-month follow-up period, despite the presence of laser peripheral iridotomy in this population. In the patients with CPACG, there was no significant difference in the MD and CPSD during the follow-up period. Patients with APACG would benefit from regular monitoring of the decline in the VF.

Synthesis and characterization of para-pyridine linked NHC palladium complexes and their studies for the Heck-Mizoroki coupling reaction.

This paper describes the synthesis of 1-(pyridine-4-ylmethyl) NHC and their Pd(II) and Ag(I) complexes, which are fully characterized. Interestingly, we have also synthesized a Pd complex 3a-CO(3) using a more direct treatment of K(2)CO(3) with PdCl(2). 3a-CO(3) represents the first reported solid structure of a Pd η(2)-carbonato complex stabilized by an NHC framework. 3a-CO(3) can be easily converted to a PdCl(2) derivative by treating it with chloroform. We have found these palladium complexes mediate the Heck-Mizoroki coupling with a low catalyst loading. Furthermore, we also expand such catalytic manifold toward constructing fused polyaromatic substrates, a highly useful class of compounds in optoelectronic chemistry.

Early Ahmed glaucoma valve implantation after penetrating keratoplasty leads to better outcomes in an Asian population with preexisting glaucoma.

BACKGROUND: To evaluate the efficacy of Ahmed Glaucoma Valve (AGV) surgery and the optimal interval between penetrating keratoplasty (PKP) and AGV implantation in a population of Asian patients with preexisting glaucoma who underwent PKP. METHODOLOGY/PRINCIPAL FINDINGS: In total, 45 eyes of 45 patients were included in this retrospective chart review. The final intraocular pressures (IOPs), graft survival rate, and changes in visual acuity were assessed to evaluate the outcomes of AGV implantations in eyes in which AGV implantation occurred within 1 month of post-PKP IOP elevation (Group 1) and in eyes in which AGV implantation took place more than 1 month after the post-PKP IOP evaluation (Group 2). Factors that were associated with graft failure were analyzed, and the overall patterns of complications were reviewed. By their final follow-up visits, 58% of the patients had been successfully treated for glaucoma. After the operation, there were no statistically significant differences between the groups with respect to graft survival (p = 0.98), but significant differences for IOP control (p = 0.049) and the maintenance of visual acuity (VA) (p<0.05) were observed. One year after surgery, the success rates of IOP control in Group 1 and Group 2 were 80% and 46.7%, respectively, and these rates fell to 70% and 37.3%, respectively, by 2 years. Factors that were associated with a high risk of AGV failure were a diagnosis of preexisting angle-closure glaucoma, a history of previous PKP, and a preoperative IOP that was >21 mm Hg. The most common surgical complication, aside from graft failure, was hyphema. CONCLUSIONS/SIGNIFICANCE: Early AGV implantation results in a higher probability of AGV survival and a better VA outcome without increasing the risk of corneal graft failure as a result of post-PKP glaucoma drainage tube implantation.

Autonomic activity difference during continuous positive airway pressure titration in patients with obstructive sleep apnea/hypopnea syndrome with or without hypertension.

BACKGROUND: Current evidence suggests that obstructive sleep apnea-hypopnea syndrome (OSAHS) is an independent risk factor for systemic hypertension. The mechanisms linking OSAHS to hypertension remain unclear. However, recent studies have indicated that abnormal autonomic control may be an important factor. Our study aims to evaulate differences in autonomic activity between hypertensive and normotensive OSAHS patients before and during continuous positive airway pressure (CPAP) therapy. METHODS: Fifty-three OSAHS patients were analyzed in this study. Patients were divided into 2 groups, one group comprising patients with hypertension and the other of patients without hypertension. Heart rate variability (HRV) was assessed by polysomnography, before patients received CPAP titration and during CPAP titration. Then, HRV was compared between the hypertensive and normotensive groups. Multivarate analyses were used to evaluate the influence of clinical variables on autonomic activity. RESULTS: Although HRV before CPAP titration was not statistically different between the 2 groups, low frequency variability was significantly lower in hypertensive subjects who received CPAP titration compared with normotensive subjects. Multivariate analysis revealed that hypertension is a determinant factor of autonomic change during CPAP use. CONCLUSIONS: Our findings demonstrate that CPAP therapy results in a greater and immediate change in autonomic activity in hypertensive OSAHS patients compared with normotensive OSAHS patients. This suggests that CPAP lowers blood pressure by decreasing the patient's autonomic activity.

MetaABC--an integrated metagenomics platform for data adjustment, binning and clustering.

SUMMARY: MetaABC is a metagenomic platform that integrates several binning tools coupled with methods for removing artifacts, analyzing unassigned reads and controlling sampling biases. It allows users to arrive at a better interpretation via series of distinct combinations of analysis tools. After execution, MetaABC provides outputs in various visual formats such as tables, pie and bar charts as well as clustering result diagrams. AVAILABILITY: MetaABC source code and documentation are available at http://bits2.iis.sinica.edu.tw/MetaABC/ CONTACT: dywang@gate.sinica.edu.tw; hktsai@iis.sinica.edu.tw SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Antioxidant xanthone derivatives induce cell cycle arrest and apoptosis and enhance cell death induced by cisplatin in NTUB1 cells associated with ROS.

In an effort to develop novel antioxidant as anticancer agents, a series of xanthones were prepared. In vitro screening, the synthetic xanthones revealed significant inhibitory effects on xanthine oxidase and ABTS radical-cation scavenging activity. The selective compounds 2 and 8 induced an accumulation of NTUB1 cells in the G(1) phase arrest and cellular apoptosis by the increase of ROS level. The combination of cisplatin and 2 significantly enhanced the cell death in NTUB1 cells. Compounds 2 and 8 did not show cytotoxic activity in selected concentrations against SV-HUC1 cells. The present results suggested that antioxidants 2 and 8 may be used as anticancer agent for enhancing the therapeutic efficacy of anticancer agents and to reduce their side effect.

Relief of periorbital pain after acute angle closure glaucoma attack by botulinum toxin type A.

PURPOSE: To assess the efficacy and safety of botulinum toxin type A (BoNT-A) injection in patients suffering from intractable periorbital pain after acute angle closure glaucoma (AACG). PATIENTS AND METHODS: In this prospective randomized intervention study, 19 patients suffering from periorbital pain after an AACG attack were injected with BoNT-A or placebo for pain relief. Patients were assessed on days 1, 2, 7, 14, 30, 60, and 90. The main outcomes were mean change of visual analog rating scale (VARS) and index scores measured through a quality-of-life questionnaire (EQ-5D), and changes in the visual analog scale (VAS), all of which were assessed at each visit. A secondary outcome was the frequency and nature of adverse events and the number of patients who withdrew from the study as a result. RESULTS: In the treatment group (n=10), the mean index score of EQ-5D and VAS changed significantly from the placebo group (by 0.299 and 2.61, respectively) from day 2 (P<0.01). The VARS of EQ-5D also disclosed significant changes from day 2 (17, P<0.01). In addition, efficacy was maintained mainly between days 2 and 60 but declined slightly by day 90. The most frequently reported treatment-related adverse events in the treatment and placebo groups were local tenderness (21%), subcutaneous hemorrhage (10.5%), and conjunctivitis (10.5%). No severe adverse events were reported during the study or follow-up period. CONCLUSIONS: BoNT-A is effective and well tolerated for the treatment of periorbital pain after an AACG attack. Its effects may be maintained for 3 months.

Trabeculectomy in patients with primary angle-closure glaucoma.

PURPOSE: To analyze the results of trabeculectomy in patients with primary angle-closure glaucoma. METHODS: A retrospective and noncomparative case series analysis was performed on data from Taiwan, from 2001 to 2004. The outcomes of trabeculectomy in eyes with acute primary angle-closure glaucoma attack (AACG) and those with chronic primary angle-closure glaucoma (CACG) were assessed in terms of final intraocular pressure (IOP), changes to visual acuity, and the incidence of complications. RESULTS: A total of 52 eyes of 52 patients, 15 patients in AACG group and 37 patients in CACG group, were reviewed. The mean follow-up period was 32 months (range, 26-42 mo). In terms of no visual acuity change after surgery, there were significant differences between the CACG and AACG groups (P=0.02, Fisher exact test). In terms of final IOP control, trabeculectomy outcome was significantly worse in patients in the AACG group than those in the CACG group (P<0.01, Fisher exact test). Moreover, the complication of bleb encapsulation appeared more frequently in the AACG group than in the CACG group (P=0.02, Fisher exact test). CONCLUSIONS: Compared with CACG, trabeculectomy may not be as good for patients of AACG, because it may lead to worsened visual acuity; it also seems to present a greater failure rate, more complications, and fewer cases of surgical survival.

Synthesis and cytotoxic, anti-inflammatory, and anti-oxidant activities of 2',5'-dialkoxylchalcones as cancer chemopreventive agents.

In an effort to develop novel anti-tumor, or cancer chemopreventive agents, a series of 2',5'-dialkoxylchalcones were prepared by Claisen-Schmidt condensation of appropriate acetophenones with suitable aromatic aldehyde. In vitro screening revealed low micromolar activity (IC(50)) against several human cancer cell lines. Selective compound 10 induced an accumulation of A549 cells in the G(2)/M phase arrest which was well correlated with inhibitory activity against tubulin polymerization. Cytotoxic compounds 3 and 12 showed significant inhibitory effects on NO production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophage-like cells while cytotoxic compound 10 revealed potent inhibitory effect on TNF-alpha formation in RAW 264.7 cells in response to LPS. Compounds 3 and 10 also showed significant inhibitory effects on xanthine oxidase. The present results suggested that compounds 3 and 10 were potential to be served as cancer chemopreventive agents.