Browsing by Subject "cancer cells"

Now showing 1 - 4 of 4
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    Discovery of Macrocyclic Inhibitors of Apurinic/Apyrimidinic Endonuclease 1
    (ACS, 2019) Trilles, Richard; Beglov, Dmitri; Chen, Qiujia; He, Hongzhen; Wireman, Randall; Reed, April; Chennamadhavuni, Spandan; Panek, James S.; Brown, Lauren E.; Vajda, Sandor; Porco, John A., Jr.; Kelley, Mark R.; Georgiadis, Millie M.; Biochemistry and Molecular Biology, School of Medicine
    Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential base excision repair enzyme that is upregulated in a number of cancers, contributes to resistance of tumors treated with DNA-alkylating or -oxidizing agents, and has recently been identified as an important therapeutic target. In this work, we identified hot spots for binding of small organic molecules experimentally in high resolution crystal structures of APE1 and computationally through the use of FTMAP analysis (http://ftmap.bu.edu/). Guided by these hot spots, a library of drug-like macrocycles was docked and then screened for inhibition of APE1 endonuclease activity. In an iterative process, hot-spot-guided docking, characterization of inhibition of APE1 endonuclease, and cytotoxicity of cancer cells were used to design next generation macrocycles. To assess target selectivity in cells, selected macrocycles were analyzed for modulation of DNA damage. Taken together, our studies suggest that macrocycles represent a promising class of compounds for inhibition of APE1 in cancer cells.
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    STAINING OF OVCA1 ANTIBODY IN HUMAN MALIGNANCIES
    (Office of the Vice Chancellor for Research, 2012-04-13) Grothaus, Kristen; Temm, Connie; Mayo, Lindsey; Sandusky, George
    Immunohistochemistry biomarkers are currently being developed to tar-get specific proteins found in cancer cells. The biomarker and putative tumor suppressor, OvCa1, has a function that is not well characterized. Due to lack of reagents, we developed monoclonal antibodies of OvCa1 to examine mul-tiple human malignancies. Primary cancers with different histologic grades as well as with metastatic lesions were examined with the monoclonal anti-bodies. Ovarian cancer tissue samples from the IU Simon Cancer Center Tis-sue Bank were used for this study. The samples were fixed in neutral buff-ered formalin and processed into a paraffin block. The slides were microtomed, and immunohistochemistry (IHC) with the OvCa1 antibody was performed. Thirty-one low, medium, and high grade tumors as well as meta-static ovarian carcinomas were evaluated. All cases revealed a range of staining intensity with OvCa1. The results indicated that OvCa1 had the highest immunostaining in the high grade, Stage 3 to 4 ovarian carcinomas. Medium grade tumors had less OvCa1 expression, while the metastatic tu-mors had less staining than any of the other three grades. Immunostaining was observed primarily in the cytoplasm and nucleus of the tumor cells. In addition, we evaluated approximately 20 tumors from various different or-gans. These included prostate, breast, spleen, lung, colon, stomach, and kidney tumors, which were positive for immunostaining with the OvCa1 anti-body. In summary, the results indicate that all histologic grades express the biomarker, OvCa1, and the staining intensity was highest in the high grade, Stage 3 and 4 tumors. Our preliminary studies demonstrate a further need to delineate OvCa1 as a potential biomarker, which could be used for early detection and diagnosis of ovarian cancer.
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    Tissue transglutaminase protects epithelial ovarian cancer cells from cisplatin-induced apoptosis by promoting cell survival signaling
    (Oxford University Press, 2008-10) Cao, Liyun; Petrusca, Daniela N.; Satpathy, Minati; Nakshatri, Harikrishna; Petrache, Irina; Matei, Daniela
    Tissue transglutaminase (TG2), an enzyme involved in protein cross-linking and overexpressed in ovarian tumors, has antiapoptotic effects in cancer cells and may play a role in response to chemotherapy. In this study, we investigated the role of TG2 in the sensitivity of ovarian cancer cells to cisplatin. By using stable knockdown and overexpression strategies, we demonstrate that the level of expression of TG2 regulates apoptosis induced by cisplatin in SKOV3 and OV-90 ovarian cancer cells. Interestingly, not only TG2 knockdown but also a TG2 enzymatic inhibitor (KCC009) sensitized SKOV3 cells to cisplatin. To understand the mechanism by which TG2 exerts its antiapoptotic role, we examined the effects of protein kinase B (Akt) and nuclear factor-kappa B (NF-κB), two survival pathways commonly involved in development of drug resistance. Overexpression of the constitutively active p65 subunit of NF-κB, but not constitutively active Akt, rescued cells with diminished TG2 expression from cisplatin-induced apoptosis. This implicates activation of NF-κB as the main cisplatin resistance mechanism downstream of TG2. Indeed, NF-κB activity is decreased and the level of the inhibitory subunit IκBα is increased in ovarian cancer cells engineered to express diminished levels of TG2 or treated with the enzymatic inhibitor, KCC009. Our data show that TG2 prevents apoptosis induced by cisplatin by activating the NF-κB survival pathway in ovarian cancer cells.
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    Understanding the Efficacy of Inhibitor TRDL-551 on Replication Protein A (RPA) Binding Affinity
    (Office of the Vice Chancellor for Research, 2016-04-08) Kaur, Jasmeet; Balakrishnan, Lata
    Replication protein A (RPA), a single stranded DNA (ssDNA) binding protein, prevents ssDNA from being a target of cellular nucleases, recombining with other complementary sequences, or folding-back on itself to form hairpin structures. Each of these scenarios can cause high rates of genome stability. Due to the protective function of RPA it now used as a therapeutic target for cancer cells, wherein DNA replication and repair often occur at high frequencies. For the current study, we utilized a chemical inhibitor of RPA, TDRL-551, which has been previously characterized as a potent inhibitor of RPA binding to substrate. In this study, we chose to further analyze the TDRL-551 inhibitor by testing different binding conditions either by the unmodified or acetylated form of RPA. Results from our laboratory have shown that acetylated RPA binds with higher affinity to ssDNA compared to the unmodified form. We tested RPA (unmodified and acetylated) binding under two different conditions; (i) RPA was pre-bound to substrate and then incubated with inhibitor or (ii) RPA was pre-bound to inhibitor and then exposed to substrate. Our results showed that in the first case, there was no effect of exposure to inhibitor on both unmodified and acetylated RPA binding. However, when RPA was first incubated with the inhibitor, both the unmodified and acetylated RPA showed reduced binding affinity to the ssDNA substrate. In conclusion, our studies show that the length of the DNA substrate, the posttranslational status of RPA and the time of exposure of the inhibitor to the RPA all play a significant role in determining the potency of this inhibitor. Further studies are being done to ascertain the efficacy of this inhibitor before using it in translational research.