Lung cancer (LC) is one of the leading causes of cancer-related death. As one of the key features of tumor microenvironment, hypoxia conditions are associated with poor prognosis in LC patients. Upregulation of hypoxic-induced factor-1α (HIF-1α) leads to the activation of various factors that contribute to the increased drug resistance, proliferation, and migration of tumor cells. Apurinic/apyrimidinic endonuclease-1 (APEX1) is a multi-functional protein that regulates several transcription factors, including HIF-1α, that contribute to tumor growth, oxidative stress responses, and DNA damage. In this study, we explored the mechanisms underlying cell responses to hypoxia and modulation of APEX1, which regulate HIF-1α and downstream pathways. We found that hypoxia-induced APEX1/HIF-1α pathways regulate several key cellular functions, including reactive oxygen species (ROS) production, carbonic anhydrase 9 (CA9)-mediated intracellular pH, migration, and angiogenesis. Cephalomannine (CPM), a natural compound, exerted inhibitory effects in hypoxic LC cells via the inhibition of APEX1/HIF-1α interaction in vitro and in vivo. CPM can significantly inhibit cell viability, ROS production, intracellular pH, and migration in hypoxic LC cells as well as angiogenesis of HUVECs under hypoxia through the inhibition of APEX1/HIF-1α interaction. Taken together, CPM could be considered as a promising compound for LC treatment.
Molecular crosstalk between the cellular epigenome and genome converge as a synergistic driver of oncogenic transformations. Besides other pathways, epigenetic regulatory circuits exert their effect towards cancer progression through the induction of DNA repair deficiencies. We explored this mechanism using a camptothecin encapsulated in β-cyclodextrin-EDTA-Fe3O4 nanoparticles (CPT-CEF)-treated HT29 cells model. We previously demonstrated that CPT-CEF treatment of HT29 cells effectively induces apoptosis and cell cycle arrest, stalling cancer progression. A comparative transcriptome analysis of CPT-CEF-treated versus untreated HT29 cells indicated that genes controlling mismatch repair, base excision repair, and homologues recombination were downregulated in these cancer cells. Our study demonstrated that treatment with CPT-CEF alleviated this repression. We observed that CPT-CEF exerts its effect by possibly affecting the DNA repair mechanism through epigenetic modulation involving genes of HMGB1, APEX1, and POLE3. Hence, we propose that CPT-CEF could be a DNA repair modulator that harnesses the cell's epigenomic plasticity to amend DNA repair deficiencies in cancer cells.
We constructed a novel chicken (Gallus gallus) lung cDNA library fused inside yeast acting domain vector (pGADT7). Using yeast two-hybrid screening with highly pathogenic avian influenza (HPAI) nucleoprotein (NP) from the strain (A/chicken/Malaysia/5858/2004(H5N1)) as bait, and the Gallus gallus lung cDNA library as prey, a novel interaction between the Gallus gallus cellular RNA export adaptor protein Aly/REF and the viral NP was identified. This interaction was confirmed and validated with mammalian two hybrid studies and co-immunoprecipitation assay. Cellular localization studies using confocal microscopy showed that NP and Aly/REF co-localize primarily in the nucleus. Further investigations by mammalian two hybrid studies into the binding of NP of other subtypes of influenza virus such as the swine A/New Jersey/1976/H1N1 and pandemic A/Malaysia/854/2009(H1N1) to human Aly/REF, also showed that the NP of these viruses interacts with human Aly/REF. Our findings are also supported by docking studies which showed tight and favorable binding between H5N1 NP and human Aly/REF, using crystal structures from Protein Data Bank. siRNA knockdown of Aly/REF had little effect on the export of HPAI NP and other viral RNA as it showed no significant reduction in virus titer. However, UAP56, another component of the TREX complex, which recruits Aly/REF to mRNA was found to interact even better with H5N1 NP through molecular docking studies. Both these proteins also co-localizes in the nucleus at early infection similar to Aly/REF. Intriguingly, knockdown of UAP56 in A549 infected cells shows significant reduction in viral titer (close to 10 fold reduction). Conclusively, our study have opened new avenues for research of other cellular RNA export adaptors crucial in aiding viral RNA export such as the SRSF3, 9G8 and ASF/SF2 that may play role in influenza virus RNA nucleocytoplasmic transport.