Characterization of hepcidin response to holotransferrin in novel recombinant TfR1 HepG2 cells

Hepcidin is the key regulator of systemic iron homeostasis. The iron-sensing mechanisms and the role of intracellular iron in modulating hepatic hepcidin secretion are unclear. Therefore, we created a novel cell line, recombinant-TfR1 HepG2,expressing iron-response-element-independent TFRC mRNA to promote cellular iron overload and examined the effect of excess holotransferrin (5 g/L) on cell-surface TfR1, iron content, hepcidin secretion and mRNA expressions of TFRC, HAMP, SLC40A1,HFE and TFR2. Results showed that the recombinant cells exceeded levels of cell surface TfR1 in wild-type cells under basal (2.8-fold; p<0.03) and holotransferrin supplemented conditions for 24 h and 48 h (4.4- and 7.5-fold, respectively; p<0.01). Also, these cells showed higher intracellular iron content than wild-type cells under basal (3-fold; p<0.03) and holotransferrin-supplemented conditions (6.6-fold at 4 h; p<0.01). However, hepcidin secretion was not higher than wild-type cells. Moreover, holotransferrin treatment to recombinant cells did not elevate HAMP responses compared to untreated or wild-type cells. In conclusion, increased intracellular iron content in recombinant cells did not increase hepcidin responses compared to wild-type cells, resembling hemochromatosis. Furthermore, TFR2 expression altered within 4 h of treatment, while HFE expression altered later at 24 h and 48 h, suggesting that TFR2 may function prior to HFE in HAMP regulation.

Synergy between histone deacetylase inhibitors and DNA- damaging agents is mediated by histone deacetylase 2 in colorectal cancer

Previous studies have associated the overexpression of histone deacetylase 2 (HDAC2) and the presence of TP53 mutations with the progression to advanced stage drug resistant colorectal cancer (CRC). However, the mechanistic link between HDAC2 expression and the TP53 mutational status has remained unexplored. Here, we investigated the function of HDAC2 in drug resistance by assessing the synergistic effects of DNA-targeted chemotherapeutic agents and HDAC inhibitors (HDACis) on two TP53-mutated colorectal adenocarcinoma CRC cell lines (SW480 and HT-29) and on the TP53-wild type carcinoma cell line (HCT116 p53+/+) and its TP53 deficient sub-line (HCT116 p53-/-). We showed that in the untreated SW480 and HT-29 cells the steady-state level of HDAC2 was low compared to a TP53-wild type carcinoma cell line (HCT116 p53+/+). Increased expression of HDAC2 correlated with drug resistance, and depletion by shRNA sensitised the multi-drug resistance cell line HT-29 to CRC chemotherapeutic drugs such as 5-fluorouracil (5-FU) and oxaliplatin (Oxa). Combined treatment with the HDACi suberoylanilide hydroxamic acid plus 5-FU or Oxa reduced the level of HDAC2 expression, modified chromatin structure and induced mitotic cell death in HT-29 cells. Non-invasive bioluminescence imaging revealed significant reductions in xenograft tumour growth with HDAC2 expression level reduced to <50% in treated animals. Elevated levels of histone acetylation on residues H3K9, H4K12 and H4K16 were also found to be associated with resistance to VPA/Dox or SAHA/Dox treatment. Our results suggest that HDAC2 expression rather than the p53 mutation status influences the outcome of combined treatment with a HDACi and DNA-damaging agents in CRC.