Indeed, it has been reported that NSAID ibuprofen controls RNA stability of p75NTR in prostate cancer cells [50]. measured by micro-impedance. Nesprin-2 was selected from two independent microarrays, based on its novelty in relation to cancer and its role in cell organization. SS diminished Nesprin-2 mRNA expression as assessed by reverse transcriptase and real time PCR. Various other NSAIDs were also tested and demonstrated that inhibition of Nesprin-2 mRNA was not unique to SS. Additionally, immunohistochemistry showed higher levels of Nesprin-2 in many tumors in comparison with normal tissues. Further micro-impedance experiments on cells with reduced Nesprin-2 expression showed a proportional loss of cellular adhesion. Conclusions Nesprin-2 is down-regulated by NSAIDs and highly expressed in many cancers. General Significance Our data suggest that Nesprin-2 may be a potential novel oncogene in human cancer cells and NSAIDs could decrease its expression. mice [7, 8], down-regulates -catenin protein apoptosis [9], and induces apoptosis under a number of experimental conditions [10C12]. SS has been DNM1 shown to change colorectal cancer cell morphology [13], alter cytoskeletal organization, and cause loss of actin stress fibers [14, 15]. This is probably due to a dose-dependent reduction of tyrosine phosphorylation of focal adhesion kinase [15]. It has also been demonstrated that SS reduces cell migration and invasion in mouse models and human colorectal cell lines [16, 17]. We speculated that SS alters Entrectinib gene Entrectinib expression related to cell organization, and subsequently we found the structural gene Nesprin-2 (NUANCE/Syne-2) was down-regulated in two independent microarrays using two different Entrectinib doses of SS-treated human colorectal cancer cells. Nesprin-2 is a giant protein with an -actinin-like actin binding domain [18]. To date, together with the closely related Enaptin/Nesprin-1, Nesprin-2 is the largest of the -actinin superfamily, and it encodes a 796 kDa protein containing an N-terminal actin-binding domain, central coiled-coil rod domain, and a C-terminal transmembrane domain [18, 19]. Nesprin-2 also has many truncated alternate splicing forms [20, 21]. The majority of Nesprin-2 is localized to the nuclear envelope, while a very small fraction can be found in the cytoplasm; the tissue distribution of Nesprin-2 mRNA is fairly ubiquitous with most tissues, showing at least trace amounts [18]. Recently Nesprin-2 has been shown to affect nuclear size and to be involved in regulating genes during wound healing [22, 23]. This colossal protein contains multiple binding sites and serves as a framework for protein complexes on the nuclear envelope [24, 25]. In this study, we found that human colorectal cancer HCT-116 cells dramatically changed their morphology and cell adhesion by SS, as assessed using biological, chemical, optical, and electrical methods. Subsequently, Nesprin-2 was identified and confirmed as being down-regulated by SS. Finally, we showed that Nesprin-2 is more highly expressed in tumor tissues, compared to normal tissues, suggesting that Nesprin-2 may be a novel oncogene. 2. Materials and methods 2.1 Reagents The NSAIDs used in this study were purchased as follows: SS, tolfenamic acid, and SC-560 from Cayman Chemical Company (Ann Arbor, Michigan); diclofenac from Sigma-Aldrich (St. Louis, MO); 5,5-dimethyl-3-(3-fluorophenyl)-4-(4-ethylsulfonyl)phenyl-2(5Hybridization Kit protocol. Hybridizations were performed using the Agilent 60-mer oligo microarray processing protocol. Data was obtained using Agilent Feature Extraction software v7.5. Intensity plots were generated for each ratio experiment, and genes were considered signature genes if the p value was less than 0.001. Functional annotation of genes was performed according to the Gene Ontology Consortium (http://www.geneontology.org/index.shtml) by GeneSpring v7.3. 2.6 Reverse Transcriptase PCR and Real Time PCR RNA was isolated from cell cultures using Qiagens RNeasy Mini Kit following the manufacturers protocol. One microgram of RNA was used to generate cDNA Entrectinib using Bio-Rads iScript? cDNA Synthesis Kit following the manufacturers protocol. PCR was performed with the following primers:: Nesprin-2 Giant forward 5-CAGTCCTTACAACTCCTGGACAC-3, Nesprin-2 Giant reverse 5-GACTGATTCTCCTACCCACAGACTC-3; Nesprin-2 all isoforms forward 5-TCACAGAGCAGCAGTCAGGT-3, Nesprin-2 all isoforms reverse 5-GCTCACGTTGACAGAGACCA-3; Nesprin-2 1 forward 5-GCAGAAGCCTATGAGTTG-3, Nesprin-2 1 & 2 reverse 5-TGTAGTGATGCTCGGGACAG-3; Nesprin-2 2 forward 5-CATCCCACAGCAATCATG-3; Nesprin-1 forward 5-GGCTGAAAATCGAAGAGACG-3, Nesprin-1 reverse 5-CATCTC TGTGAGCCAGACCA-3; GAPDH forward, 5-GGGCTGCTTTTAACTCTGGT-3, GAPDH reverse 5-TGGCAGGTTTTTCTAGACGG-3; IDH2 forward 5-GACGGAGATGTGCAGTCAGA-3, IDH2 reverse 5-GTCCGTGGTGTTCAGGAAGT-3; NAG-1 forward 5-CTCCAGATTCCGAGAGTTGC-3, and NAG-1 reverse 5-AGAGATACGCAGGTGCAGGT-3. Densitometric analysis of reverse transcriptase PCR was performed using Scion Image software (Frederick, MD). Real Time PCR was performed using Thermo Scientifics Absolute qPCR SYBR Green Mix (Waltham, MA) on a Bio-Rad MyiQ iCycler thermal cycler using Bio-Rad iQ5 version 2.1 software following the manufactures protocol (Hercules, CA). Measurements were standardized using GAPDH, and each set of three or more trials was averaged. 2.7 Immunohistochemistry Immunostaining on a Biochain Tissue Array Human Tumor Tissue II (Lot# A711214) slide was performed using standard immunohistochemistry procedures (T8235713-2; Newark, CA). The slide was incubated overnight at 4C with undiluted.