Evidence for functional and regulatory cross-talk between Wnt/ß-catenin signalling and Mre11-Rad50-Nbs1 complex in the repair of cisplatin-induced DNA cross-links.

The canonical Wnt/ß-catenin signalling pathway plays a crucial role in a variety of functions including cell proliferation and differentiation, tumorigenic processes and radioresistance in cancer cells. The Mre11-Rad50-Nbs1 (MRN) complex has a pivotal role in sensing and repairing DNA damage. However, it remains unclear whether a connection exists between Wnt/ß-catenin signalling and the MRN complex in the repair of cisplatin-induced DNA interstrand cross-links (ICLs). Here, we report that (1) cisplatin exposure results in a significant increase in the levels of MRN complex subunits in human tumour cells; (2) cisplatin treatment stimulates Wnt/ß-catenin signalling through increased ß-catenin expression; (3) the functional perturbation of Wnt/ß-catenin signalling results in aberrant cell cycle dynamics and the activation of DNA damage response and apoptosis; (4) a treatment with CHIR99021, a potent and selective GSK3ß inhibitor, augments cisplatin-induced cell death in cancer cells. On the other hand, inactivation of the Wnt/ß-catenin signalling with FH535 promotes cell survival. Consistently, the staining pattern of γH2AX-foci is significantly reduced in the cells exposed simultaneously to cisplatin and FH535; and (5) inhibition of Wnt/ß-catenin signalling impedes cisplatin-induced phosphorylation of Chk1, abrogates the G2/M phase arrest and impairs recombination-based DNA repair. Our data further show that Wnt signalling positively regulates the expression of ß-catenin, Mre11 and FANCD2 at early time points, but declining thereafter due to negative feedback regulation. These results support a model wherein Wnt/ß-catenin signalling and MRN complex crosstalk during DNA ICL repair, thereby playing an important role in the maintenance of genome stability.

Genome-wide analysis reveals a regulatory role for G-quadruplexes during Adenovirus multiplication.

The G-quadruplex (GQ) motifs have recently been gaining prominence because of their role as gene cis-regulatory elements in a variety of organisms and as potential druggable targets for anti-cancer therapy and ageing. Several studies have demonstrated the existence of GQs in the genomes of emerging and re-emerging human pathogens, such as hepatitis virus, herpesviruses, Ebola virus, Zika virus and Nipah virus. Human Adenovirus (HAdV) exhibits a large number of clinical manifestations especially infecting the children and the immunocompromised patients. Moreover, the HAdV-based vectors have been widely used to deliver foreign DNAs to cells in gene therapy. However, the DNA secondary structural elements in AdV-based vectors could significantly determine the gene delivery efficacy of the vectors. In this study, using a combination of whole genome sequence analysis, biochemical, biophysical and interaction assays, we revealed fifteen putative GQs that are conserved across the different species of HAdV. We further showed that the GQs are embedded in the sequences of essential viral genes, namely E1B, E2B, and L3 genes (among others), which are involved in the early and late stages of the viral life cycle. Notably, Braco-19 (a well-known GQ binding ligand) interacted specifically with the HAdV GQs and increased their stability and further blocked the HAdV multiplication in human cells. Taken together, our data strongly supported the existence of G-quadruplex structures in the HAdV genome that affect the virus multiplication and posit that such structures may influence the efficacy of the gene-delivery vectors or even the HAdV virus life-cycle.

Identification and characterization of two conserved G-quadruplex forming motifs in the Nipah virus genome and their interaction with G-quadruplex specific ligands.

The G-quadruplex (GQ) motifs are considered as potential drug-target sites for several human pathogenic viruses such as Zika, Hepatitis, Ebola, and Human Herpesviruses. The recent outbreaks of Nipah virus (NiV) in India, the highly fatal emerging zoonotic virus is a potential threat to global health security as no anti-viral drug or vaccine in currently available. Therefore, here in the present study, we sought to assess the ability of the putative G-quadruplex forming sequences in the NiV genome to form G-quadruplex structures and act as targets for anti-viral compounds. Bioinformatics analysis underpinned by various biophysical and biochemical techniques (such as NMR, CD, EMSA, DMS footprinting assay) confirmed the presence of two highly conserved G-quadruplex forming sequences (HGQs) in the G and L genes of NiV. These genes encode the cell attachment glycoprotein and RNA-dependent RNA polymerase, respectively and are essential for the virus entry and replication within the host cell. It remains possible that stabilization of these HGQs by the known G-quadruplex binding ligands like TMPyP4 and Braco-19 represents a promising strategy to inhibit the expression of the HGQ harboring genes and thereby stop the viral entry and replication inside the host cell. Accordingly, we report for the first time, that HGQs in Nipah virus genome are targets for G-quadruplex specific ligands; therefore, could serve as potential targets for anti-viral therapy.

Targeting G-quadruplex DNA with synthetic dendritic peptide: modulation of the proliferation of human cancer cells.

Telomerase, a reverse transcriptase enzyme, is found to over express in most cancer cells. It elongates the telomere region by repeated adding of TTAGGG in the 3'-end and leads to excess cell proliferation which causes cancer. G-quadruplex (G4) formation can inhibit such telomere lengthening. So, stabilization of G4 structure as well as inhibition of telomerase activity is very promising approach in targeted cancer therapy. Herein, the aptitude of a synthetic dendritic peptide, C δ2-(YEE)-E (peptide 1), to target specifically the human telomeric G4 DNA, dAGGG(TTAGGG)3, has been evaluated. Both biochemical and biophysical techniques including gel mobility shift assay, isothermal titration calorimetry and fluorescence spectroscopy have been employed for the purpose. Circular dichroism study reveals that the targeting results an increase in thermal stability of G4 DNA. Interestingly, replacement of N-terminal tyrosine residue of peptide 1 by valine, C δ2-(VEE)-E, (peptide 2) consequences in loss of its G4 DNA targeting ability, although both the peptides exhibit comparable affinity toward double-stranded DNA. Of note, peptide 1 causes cessation of growth of human cancer cells (HeLa and U2OS) and induces apoptosis in vitro. But it has no significant inhibitory effect on the growth of normal human embryonic kidney 293 cells. Mechanistically, Telomeric Repeat Amplification Protocol (TRAP) assay indicates that peptide 1 effectively inhibits the telomerase activity in human cell extracts. Overall, this study demonstrates the usefulness of a synthetic dendritic peptide as an inhibitor of tumor cell growth by inducing apoptosis upon targeting the telomeric G4 DNA.

Targeting G-quadruplex DNA structures in the telomere and oncogene promoter regions by benzimidazole‒carbazole ligands.

Recent studies support the idea that G-quadruplex structures in the promoter regions of oncogenes and telomere DNA can serve as potential therapeutic targets in the treatment of cancer. Accordingly, several different types of organic small molecules that stabilize G-quadruplex structures and inhibit telomerase activity have been discerned. Here, we describe the binding of benzimidazole-carbazole ligands to G-quadruplex structures formed in G-rich DNA sequences containing the promoter regions of human c-MYC, c-KIT1, c-KIT2, VEGF and BCL2 proto-oncogenes. The fluorescence spectroscopic data indicate that benzimidazole-carbazole ligands bind and stabilize the G-quadruplexes in the promoter region of oncogenes. The molecular docking studies provide insights into the mode and extent of binding of this class of ligands to the G-quadruplexes formed in oncogene promoters. The high stability of these G-quadruplex structures was validated by thermal denaturation and telomerase-catalyzed extension of the 3' end. Notably, benzimidazole-carbazole ligands suppress the expression of oncogenes in cancer cells in a dose-dependent manner. We anticipate that benzimidazole-carbazole ligands, by virtue of their ability to stabilize G-quadruplex structures in the promoter regions of oncogenes, might reduce the risk of cancer through the loss of function in the proteins encoded by these genes.

Novel ruthenium azo-quinoline complexes with enhanced photonuclease activity in human cancer cells.

Coordinatively saturated ruthenium complexes with a variable net charge are currently under intense investigation for their anticancer potential. These complexes, possessing long wavelength metal-to-ligand charge transfer with DNA photonuclease activity, have shown promising cytotoxic profiles. Although most of the ruthenium complexes exhibit significant photochemotherapeutic activity, their poor entry into cells hinder their development as potential drug molecules. Here, we report the synthesis and characterization of four new ruthenium (II) azo-8-hydroxyquinoline complexes, their mode of in vitro DNA binding and antiproliferative properties against cultured human cancer cell lines. The activity of these compounds prior to photoirradiation is minimal. However, they could induce DNA photonuclease activity through the generation of reactive oxygen species upon exposure to light. The activities exhibited by these complexes were found to be more efficient (>5-fold) than cisplatin, emphasizing their therapeutic potential. Collectively, these results support the idea that ruthenium (II) azo-8-hydroxyquinoline complexes can serve as potential agents in photodynamic anticancer therapy.