Eddie Chan

Interaction of DNA with three dimethyl derivatives of benz(c)acridines

Abstract Dimethylbenz(c)acridines interact with DNA in aqueous solution with the formation of complexes which have lower sedimentation rates and greater thermal stabilities than solutions of free DNA. Low pH and low ionic strength favor the formation of these complexes. The bathochromic shifts in the absorption spectra of the dimethylbenz(c)acridines and the changes in the physical properties of the DNA resulting from such interactions, indicate that these dimethylbenz(c)acridines bind by intercalating between the bases of the DNA. The finding that they bind to solutions of native and denatured DNA to approximately the same extent suggests that they intercalate between adjacent bases on the same strand.

Polycyclic aromatic hydrocarbons: Covalent binding to DNA and effects on template function

Abstract Exposure of solutions of DNA containing intercalated molecules of benz(a)pyrene or pyrene to γ-irradiation resulted in very extensive covalent binding of these hydrocarbons to DNA. The extent of binding for benz(a) pyrene was 1 molecule for every 100 DNA nucleotides and for pyrene was 1 : 270 DNA nucleotides. It was also found that in vitro transcription was inhibited when DNA containing covalently bound benz(a)pyrene was used as template for Escherichia coli DNA dependent RNA polymerase.

The Acetylenic Tricyclic Bis(cyano enone), TBE-31 Inhibits Non–Small Cell Lung Cancer Cell Migration through Direct Binding with Actin

The migratory and invasive potential of the epithelial-derived tumor cells depends on epithelial-to-mesenchymal transition (EMT) as well as the reorganization of the cell cytoskeleton. Here, we show that the tricyclic compound acetylenic tricyclic bis(cyano enone), TBE-31, directly binds to actin and inhibits linear and branched actin polymerization in vitro. Furthermore, we observed that TBE-31 inhibits stress fiber formation in fibroblasts as well as in non–small cell lung cancer cells during TGFβ-dependent EMT. Interestingly, TBE-31 does not interfere with TGFβ-dependent signaling or changes in E-cadherin and N-cadherin protein levels during EMT. Finally, we observed that TBE-31 inhibits fibroblast and non–small cell lung tumor cell migration with an IC50 of 1.0 and 2.5 μmol/L, respectively. Taken together, our results suggest that TBE-31 targets linear actin polymerization to alter cell morphology and inhibit cell migration. Cancer Prev Res; 7(7); 727–37. ©2014 AACR.

aPKC alters the TGFβ response in NSCLC cells through both Smad-dependent and Smad-independent pathways.

ABSTRACT Transforming growth factor &bgr; (TGF&bgr;) signaling controls many cellular responses including proliferation, epithelial to mesenchymal transition and apoptosis, through the activation of canonical (Smad) as well as non-canonical (e.g. Par6) pathways. Previous studies from our lab have demonstrated that aPKC inhibition regulates TGF&bgr; receptor trafficking and signaling. Here, we report that downstream TGF&bgr;-dependent transcriptional responses in aPKC-silenced NSCLC cells were reduced compared with those of control cells, despite a temporal extension of Smad2 phosphorylation. We assessed SARA–Smad2–Smad4 association and observed that knockdown of aPKC increased SARA (also known as ZFYVE9) levels and SARA–Smad2 complex formation, increased cytoplasmic retention of Smad2 and reduced Smad2–Smad4 complex formation, which correlated with reduced Smad2 nuclear translocation. Interestingly, we also detected an increase in p38 MAPK phosphorylation and apoptosis in aPKC-silenced cells, which were found to be TRAF6-dependent. Taken together, our results suggest that aPKC isoforms regulate Smad and non-Smad TGF&bgr; pathways and that aPKC inhibition sensitizes NSCLC cells to undergo TGF&bgr;-dependent apoptosis.

The acetylenic tricyclic bis(cyano enone), TBE-31, targets microtubule dynamics and cell polarity in migrating cells.

Cell migration is dependent on the microtubule network for structural support as well as for the proper delivery and positioning of polarity proteins at the leading edge of migrating cells. Identification of drugs that target cytoskeletal-dependent cell migration and protein transport in polarized migrating cells is important in understanding the cell biology of normal and tumor cells and can lead to new therapeutic targets in disease processes. Here, we show that the tricyclic compound TBE-31 directly binds to tubulin and interferes with microtubule dynamics, as assessed by end binding 1 (EB1) live cell imaging. Interestingly, this interference is independent of in vitro tubulin polymerization. Using immunofluorescence microscopy, we also observed that TBE-31 interferes with the polarity of migratory cells. The polarity proteins Rac1, IQGAP and Tiam1 were localized at the leading edge of DMSO-treated migrating cell, but were observed to be in multiple protrusions around the cell periphery of TBE-31-treated cells. Finally, we observed that TBE-31 inhibits the migration of Rat2 fibroblasts with an IC50 of 0.75 μM. Taken together, our results suggest that the inhibition of cell migration by TBE-31 may result from the improper maintenance of cell polarity of migrating cells.

Synthetic triterpenoids inhibit GSK3β activity and localization and affect focal adhesions and cell migration

Synthetic triterpenoids are a class of anti-cancer compounds that target many cellular functions, including apoptosis and cell growth in both cell culture and animal models. We have shown that triterpenoids inhibit cell migration in part by interfering with Arp2/3-dependent branched actin polymerization in lamellipodia (To et al., 2010). Our current studies reveal that Glycogen Synthase Kinase 3 beta (GSK3β), a kinase that regulates many cellular processes, including cell adhesion dynamics, is a triterpenoid-binding protein. Furthermore, triterpenoids were observed to inhibit GSK3β activity and increase cellular focal adhesion size. To further examine whether these effects on focal adhesions in triterpenoid-treated cells were GSK3β-dependent, GSK3β inhibitors (lithium chloride and SB216763) were used to examine cell adhesion and morphology as well as cell migration. Our results showed that GSK3β inhibitors also altered cell adhesion sizes. Moreover, these inhibitors blocked cell migration and displaced proteins at the leading edge of migrating cells, consistent with what was observed in triterpenoid-treated cells. Therefore, the triterpenoids may affect cell migration via a mechanism that targets and alters the activity and localization of GSK3β.

Abstract LB-345: Acetylenic tricyclic bis-(cyano enone) interacts with cysteine residues of actin and inhibits non-small cell lung cancer cell migration

During epithelial-to-mesenchymal transition (EMT), epithelial cells lose their cell-cell junctions, apical-basal polarity, and reorganize their actin cytoskeleton. These changes, among others, increase the motility of individual cells and promote a highly invasive phenotype. The actin cytoskeleton is also key in cell migration and invasion, as the polymerization of actin at the leading edge provides the necessary driving force for translocation and protrusions. Therefore, the actin cytoskeleton is a potential target for inhibiting tumour progression. In this study we characterized a novel tri-cyclic synthetic triterpenoid derivative, TBE-31, in its ability to associate with actin and inhibit cell migration. Using a pull-down approach, we demonstrate that TBE-31 binds directly to actin. Furthermore, TBE-31 inhibited branched and linear actin polymerization in vitro, and stress fibre formation in cultured cells. Actin cytoskeleton reorganization and stress fiber formation was also inhibited in TGFβ-dependent EMT of non-small cell lung cancer cells. We next used SwissDock software to investigate the potential molecular interaction between TBE-31 and actin. Swissdock predicted that TBE-31 would have a high affinity for binding to the same cleft as Cytochalasin D, a bona-fide actin polymerization inhibitor. As triterpenoids associate with cysteine residues in target proteins, we generated cysteine to alanine mutants of actin and verified TBE-31 binding using pull-down assays. We identified that cysteine 374 is important for TBE-31 interaction with actin. Finally, we observed that TBE-31 inhibited fibroblast and non-small cell lung tumour cell migration with an IC50 of 1.0 and 2.5 μM, respectively. Taken together, our results suggest that TBE-31 targets actin polymerization to alter cell morphology and inhibit cell migration. Citation Format: Eddie Chan, Akira Saito, Tadashi Honda, John Di Guglielmo. Acetylenic tricyclic bis-(cyano enone) interacts with cysteine residues of actin and inhibits non-small cell lung cancer cell migration. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-345.

Abstract 524: Assessing the interaction between acetylenic tricyclic bis-(cyano enone) and cysteine residues of actin to inhibit non-small cell lung cancer cell migration

During epithelial-to-mesenchymal transition (EMT), epithelial cells lose their cell-cell junctions, apical-basal polarity, and reorganize their actin cytoskeleton. These changes, among others, increase the motility of individual cells and promote a highly invasive phenotype. The actin cytoskeleton is also key in cell migration and invasion, as the polymerization of actin at the leading edge provides the necessary driving force for translocation and protrusions. Therefore, the actin cytoskeleton is a potential target for inhibiting tumour progression. In this study we characterized a novel tri-cyclic synthetic triterpenoid derivative, TBE-31, in its ability to associate with actin and inhibit cell migration. Using a pull-down approach, we demonstrate that TBE-31 binds directly to actin. Furthermore, TBE-31 inhibited branched and linear actin polymerization in vitro, and stress fibre formation in cultured cells. Actin cytoskeleton reorganization and stress fiber formation was also inhibited in TGFβ-dependent EMT of non-small cell lung cancer cells. We next used SwissDock software to investigate the potential molecular interaction between TBE-31 and actin. Swissdock predicted that TBE-31 would have a high affinity for binding to the same cleft as Cytochalasin D, a bona-fide actin polymerization inhibitor. As triterpenoids associate with cysteine residues in target proteins, we generated cysteine to alanine mutants of actin and verified TBE-31 binding using pull-down assays. Finally, we observed that TBE-31 inhibited fibroblast and non-small cell lung tumor cell migration with an IC 50 of 1.0 and 2.5 μM, respectively. Taken together, our results suggest that TBE-31 targets actin polymerization to alter cell morphology and inhibit cell migration. Citation Format: Eddie Chan, Akira Saito, Tadashi Honda, John Di Guglielmo. Assessing the interaction between acetylenic tricyclic bis-(cyano enone) and cysteine residues of actin to inhibit non-small cell lung cancer cell migration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 524. doi:10.1158/1538-7445.AM2015-524

Abstract 5008: The acetylenic tricyclic bis(cyanoeneone), TBE-31 inhibits non-small cell lung cancer cell migration through direct binding with actin

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA During metastasis tumor cells undergo epithelial to mesenchymal transition (EMT), where cell-cell junctions dissolve and actin stress fibers are formed. This transition unmasks the migratory and invasive potential of the tumor cells. Here we show that the tricyclic compound TBE-31 binds to purified actin as well as actin from cell lysates. Furthermore, TBE-31 inhibits linear and branched actin polymerization in vitro as well as stress fiber formation in fibroblasts. We also observed that TBE-31 inhibits stress fiber formation in non-small lung cancer cells during TGFβ-dependent EMT. Interestingly, TBE-31 does not interfere with TGFβ-dependent signaling or changes in E- and N-cadherin protein levels during EMT. Finally, we observed that TBE-31 inhibits non-small cell lung tumor cell migration. Our results suggest that TBE-31 targets linear actin polymerization to alter cell morphology and inhibit cell migration. Citation Format: Eddie Chan, Akira Saito, Tadashi Honda, John Di Guglielmo. The acetylenic tricyclic bis(cyanoeneone), TBE-31 inhibits non-small cell lung cancer cell migration through direct binding with actin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5008. doi:10.1158/1538-7445.AM2014-5008

Abstract 476: The effects of the synthetic triterpenoid acetylenic tricyclic bis-(cyano enone) on cell migration and epithelial-to-mesenchymal transition

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL During metastasis, epithelial tumor cells undergo epithelial to mesenchymal transition (EMT) allowing them to migrate to distant organs to establish secondary tumors. Cell migration is initiated by the asymmetrical localization of polarity proteins towards the leading edge. This stimulates the reorganization and polymerization of the microtubule and actin cytoskeletons, which form protrusions in the plasma membrane to drive the cell forwards. Metastasis accounts for over 90% of cancer related deaths, thus a reduction in cancer mortality will require therapies aimed at preventing or delaying the events of metastasis. Our lab has identified the synthetic triterpenoid, CDDO-Im, as a potent inhibitor of cell migration by inhibiting the polymerization of branched actin and disrupting the organization of the microtubule network (To et al., J Biol Chem. 2008, 2010). Recently, TBE-31 has been developed, which is a smaller 3-ring compound that contains the same active functional groups as CDDO-Im. Here we assessed the effects of TBE-31 on cell migration and EMT. TBE-31 was found to displace the polarity proteins from the leading edge of migrating cells and induce multiple cell protrusions. The microtubule network was also observed to be disorganized and the rate of polymerization for both tubulin and actin were lowered in the presence of TBE-31. These effects are believed to allow submicromolar concentrations TBE-31 to inhibit cell migration by as much as 60%. Finally, the reorganization of actin and formation of stress fibers normally associated with EMT was also inhibited by TBE-31 in A549 tumor cells. We are now confirming our results in a panel of lung tumor cell lines. In conclusion, TBE-31 disrupts the cytoskeleton and inhibits cell migration and preliminary data suggest TBE-31 may also hinder EMT. Further research will be needed to determine the overall impact these effects have on the progression of metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 476. doi:1538-7445.AM2012-476