NME1 and DCC variants are associated with susceptibility and tumor characteristics in Mexican patients with colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) ranks third in cancer incidence globally and is the second leading cause of cancer-related mortality. The nucleoside diphosphate kinase 1 (NME1) and netrin 1 receptor (DCC) genes have been associated with resistance against tumorigenesis and tumor metastasis. This study investigates the potential association between NME1 (rs34214448 G > T and rs2302254 C > T) and DCC (rs2229080 G > C and rs714 A > G) variants and susceptibility to colorectal cancer development. METHODS: Samples from 232 colorectal cancer patients and 232 healthy blood donors underwent analysis. Variants were identified using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology. Associations were assessed using odds ratios (OR), and the p values were adjusted with Bonferroni test. RESULTS: Individuals carrying the G/T and T/T genotypes for the NME1 rs34214448 variant exhibited a higher susceptibility for develop colorectal cancer (OR = 2.68, 95% CI: 1.76-4.09, P = 0.001 and OR = 2.47, 95% CI: 1.37-4.47, P = 0.001, respectively). These genotypes showed significant associations in patients over 50 years (OR = 2.87, 95% CI: 1.81-4.54, P = 0.001 and OR = 2.99, 95% CI: 1.54-5.79, P = 0.001 respectively) and with early Tumor-Nodule-Metastasis (TNM) stage (P = 0.001), and tumor location in the rectum (P = 0.001). Furthermore, the DCC rs2229080 variant revealed that carriers of the G/C genotype had an increased risk for develop colorectal cancer (OR = 2.00, 95% CI: 1.28-3.11, P = 0.002) and were associated with age over 50 years, sex, and advanced TNM stages (P = 0.001). CONCLUSIONS: These findings suggest that the NME1 rs34214448 and DCC rs2229080 variants play a significant role in colorectal cancer development.

Disrupted IRF6-NME1/2 Complexes as a Cause of Cleft Lip/Palate.

Mutations and common polymorphisms in interferon regulatory factor 6 ( IRF6) are associated with both syndromic and nonsyndromic forms of cleft lip/palate (CLP). To date, much of the focus on this transcription factor has been on identifying its direct targets and the gene regulatory network in which it operates. Notably, however, IRF6 is found predominantly in the cytoplasm, with its import into the nucleus tightly regulated like other members of the IRF family. To provide further insight into the role of IRF6 in the pathogenesis of CLP, we sought to identify direct IRF6 protein interactors using a combination of yeast 2-hybrid screens and co-immunoprecipitation assays. Using this approach, we identified NME1 and NME2, well-known regulators of Rho-type GTPases, E-cadherin endocytosis, and epithelial junctional remodeling, as bona fide IRF6 partner proteins. The NME proteins co-localize with IRF6 in the cytoplasm of primary palatal epithelial cells in vivo, and their interaction with IRF6 is significantly enhanced by phosphorylation of key serine residues in the IRF6 C-terminus. Furthermore, CLP associated IRF6 missense mutations disrupt the ability of IRF6 to bind the NME proteins and result in elevated activation of Rac1 and RhoA, compared to wild-type IRF6, when ectopically expressed in 293T epithelial cells. Significantly, we also report the identification of 2 unique missense mutations in the NME proteins in patients with CLP (NME1 R18Q in an IRF6 and GRHL3 mutation-negative patient with van der Woude syndrome and NME2 G71V in a patient with nonsyndromic CLP). Both variants disrupted the ability of the respective proteins to interact with IRF6. The data presented suggest an important role for cytoplasmic IRF6 in regulating the availability or localization of the NME1/2 complex and thus the dynamic behavior of epithelia during lip/palate development.

Involvement of nucleoside diphosphate kinase b and elongation factor 2 in Leishmania braziliensis antimony resistance phenotype.

BACKGROUND: Nucleoside diphosphate kinase b (NDKb) is responsible for nucleoside triphosphates synthesis and it has key role in the purine metabolism in trypanosomatid protozoans. Elongation factor 2 (EF2) is an important factor for protein synthesis. Recently, our phosphoproteomic analysis demonstrated that NDKb and EF2 proteins were phosphorylated and dephosphorylated in antimony (SbIII)-resistant L. braziliensis line compared to its SbIII-susceptible pair, respectively. METHODS: In this study, the overexpression of NDKb and EF2 genes in L. braziliensis and L. infantum was performed to investigate the contribution of these proteins in the SbIII-resistance phenotype. Furthermore, we examined the role of lamivudine on SbIII susceptibility in clones that overexpress the NDKb gene, and the effect of EF2 kinase (EF2K) inhibitor on the growth of EF2-overexpressing parasites. RESULTS: Western blot analysis demonstrated that NDKb and EF2 proteins are more and less expressed, respectively, in SbIII-resistant line of L. braziliensis than its wild-type (WTS) counterpart, corroborating our previous phosphoproteomic data. NDKb or EF2-overexpressing L. braziliensis lines were 1.6 to 2.1-fold more resistant to SbIII than the untransfected WTS line. In contrast, no difference in SbIII susceptibility was observed in L. infantum parasites overexpressing NDKb or EF2. Susceptibility assays showed that NDKb-overexpressing L. braziliensis lines presented elevated resistance to lamivudine, an antiviral agent, but it did not alter the leishmanicidal activity in association with SbIII. EF2-overexpressing L. braziliensis clone was slightly more resistant to EF2K inhibitor than the WTS line. Surprisingly, this inhibitor increased the antileishmanial effect of SbIII, suggesting that this association might be a valuable strategy for leishmaniasis chemotherapy. CONCLUSION: Our findings represent the first study of NDKb and EF2 genes overexpression that demonstrates an increase of SbIII resistance in L. braziliensis which can contribute to develop new strategies for leishmaniasis treatment.

Correlation between non-metastatic protein 23 expression and clinicopathological features of colorectal cancer in Asians.

The current meta-analysis was performed to investigate the association between non-metastatic protein 23 (NM23) expression, tumor pathology, and disease prognosis in colorectal cancer (CRC) among Asians. English and Chinese language-based electronic databases (e.g., PubMed, EBSCO, Ovid, Springerlink, Wiley, Web of Science, Wanfang databases, China National Knowledge Infrastructure, VIP databases) were searched using search terms to identify published studies relevant to NM23 and CRC with immunohistochemistry. In total, 289 studies were identified through database searches, and 16 cohort studies (4 studies in English, 12 in Chinese) were chosen for meta-analysis, which included 1592 CRC patients. The results revealed that NM23 protein expression in CRC tissue was higher in patients with Dukes stages A and B than in patients with Dukes stages C and D. The NM23 protein was expressed at higher levels in well- and moderately differentiated tumors than in poorly differentiated tumors. The 5-year survival rate was also higher in CRC patients with NM23-positive tumors than in CRC patients with NM23-negative tumors. Significantly, 5-year tumor relapse and metastasis were lower in patients with NM23-positive tumors than in CRC patients with NM23-negative tumors. The findings suggest that NM23 expression status is associated with tumor aggressiveness and survival in CRC among Asians. Importantly, CRC patients with NM23-positive tumors had a better prognosis, and thus NM23 expression maybe used as a key prognostic indicator for CRC.

Shrimp oncoprotein nm23 is a functional nucleoside diphosphate kinase.

Biosynthesis of nucleoside triphosphates is critical for bioenergetics and nucleic acid replication, and this is achieved by nucleoside diphosphate kinase (NDK). As an emerging biological model and the global importance of shrimp culture, we have addressed the study of the Pacific whiteleg shrimp (Litopenaeus vannamei) NDK. We demonstrated its activity and affinity towards deoxynucleoside diphosphates. Also, the quaternary structure obtained by gel filtration chromatography showed that shrimp NDK is a trimer. Affinity was in the micro-molar range for dADP, dGDP, dTDP and except for dCDP, which presented no detectable interaction by isothermal titration calorimetry, as described previously for Plasmodium falciparum NDK. This information is particularly important, as this enzyme could be used to test nucleotide analogs that can block white spot syndrome virus (WSSV) viral replication and to study its bioenergetics role during hypoxia and fasting.

Sodium valproate inhibits MDA-MB-231 breast cancer cell migration by upregulating NM23H1 expression.

Breast cancer is a common cancer in women, with a highly variable course, from inoffensive to lethal. To find a more effective strategy for its treatment, sodium valproate has been tested as an anti-cancer drug; it is the only clinically available histone deacetylase inhibitor. However, data about the effects of sodium valproate on breast cancer are insufficient in both animals and humans; studies have yielded conflicting conclusions. In particular, little is known about the association between expression of the metastasis suppressor Nm23H1 gene and breast cancer. We hypothesized that sodium valproate regulates NM23H1 expression, and affects migration and/or invasion. We found that sodium valproate at concentrations of 0.8-3.2 mM inhibits migration and modulates Nm23H1 gene expression in a concentration-dependent manner. Confluent MDA-MB-231 cells were scratched by a micropipette tip after VPA treatment for 24 h; 24 h later, the scratch was almostly closed in the 0 mM VPA-treated cells, while the 3.2 mM VPA-treated cells migrated the slowest. The cell migration ratio exposed to 0.8, 1.6 and 3.2 mM VPA was about 66.67, 30.67 and 26.67% (P < 0.05). We also found evidence that sodium valproate upregulates NM23H1 expression, which is a clue to its anti-cancer mode of action. The NM23H1 gene expression was relative fold increased determined by Western blotting at 3.2 mM VPA. Collectively, these observations indicate that sodium valproate has potential for use in breast cancer treatment.

Production, purification, crystallization and preliminary X-ray diffraction studies of the nucleoside diphosphate kinase b from Leishmania major.

Nucleoside diphosphate kinases (NDKs; EC 2.7.4.6) play an essential role in the synthesis of nucleotides from intermediates in the salvage pathway in all parasitic trypanosomatids and their structural studies will be instrumental in shedding light on the biochemical machinery involved in the parasite life cycle and host-parasite interactions. In this work, NDKb from Leishmania major was overexpressed in Escherichia coli, purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The NDK crystal diffracted to 2.2 angstrom resolution and belonged to the trigonal crystal system, with unit-cell parameters a = 114.2, c = 93.9 angstrom. Translation-function calculations yielded an unambiguous solution in the enantiomorphic space group P3(2)21.