While nuclear factor-kappa B (NF-B) and hypoxia-inducible factor 1-alpha (HIF-1) play important roles in the growth and survival of hematopoietic malignancies13C15, VC inhibits the survival and growth of K562 leukemic cells via the downregulation of HIF-1 transcription by inhibiting NF-B activation and suppressing the expression of HIF-1-regulated antiapoptotic proteins of the Bcl-2 family, including myeloid leukemia cell differentiation protein (Mcl-1), B-cell lymphoma (Bcl)-xL, and Bcl-212. increasing ROS accumulation and activating the caspase-3 and p38 pathways11. We also previously found that VC inhibits the growth and induces the apoptosis of various human leukemic cells12. While nuclear factor-kappa B (NF-B) and hypoxia-inducible factor 1-alpha (HIF-1) play important roles in the growth and survival of hematopoietic malignancies13C15, VC inhibits the survival and growth of K562 leukemic cells via the downregulation TTT-28 of HIF-1 transcription by inhibiting NF-B activation and suppressing the expression of HIF-1-regulated antiapoptotic proteins of the Bcl-2 family, including myeloid leukemia cell differentiation protein (Mcl-1), B-cell lymphoma (Bcl)-xL, and Bcl-212. TTT-28 However, TTT-28 these inhibitory effects of VC were not observed in human umbilical cord blood-derived CD34+ normal hematopoietic cells12. Therefore, VC is considered a promising alternative therapy against cancers, including hematopoietic malignancies. Blunting this potential, very few clinical trials have Dll4 addressed the anticancer therapeutic efficacy of VC9,16. A recent study demonstrated using prostate cancer cell lines that the anti-cancer effects of VC were completely abolished by the addition of iron to the culture medium, because increased iron ions in the medium also promoted the decomposition of H2O2, which is mediated by the Fenton reaction. Subsequently, OH produced in the Fenton reaction in the medium is immediately buffered by extracellular proteins owing to its high reactivity, and therefore cannot damage intracellular targets17. TTT-28 The authors also demonstrated that when iron was present at the physiological levels, the decomposition of H2O2 compensates for H2O2 generation and prevents its accumulation. These findings suggested that the anti-cancer effect of VC was overestimated in previous studies. In the present study, using immunodeficient mice transplanted with the human chronic myeloid leukemia-derived leukemic K562 cell line, we demonstrated that the growth inhibitory effect of VC on K562 cells can be completely abolished by the simultaneous administration of iron, and that in the presence of excess iron, K562 cell growth is enhanced by VC and mutations, which cause VC-induced selective cell TTT-28 death in colorectal cancer10, were not detected in K562 cells (data not shown), and those inhibitory effects were attenuated by the addition of ferric ammonium citrate (FAC) (Fig.?1A,B, Supplementary Fig.?1). Open in a separate window Figure 1 Excess iron diminishes the inhibitory effect of VC on K562 cell survival using an experimental transplantation model. On day 0, we transplanted a mixture consisting of Luc-K562 cells and basement membrane matrix subcutaneously into the right flank of NOD/SCID mice. From day 7 after transplantation, we injected the vehicle, VC (0.5?mg/g body weight, once per day), saccharated ferric oxide (SFO; 50?g/g body weight, once per day), or both VC and SFO into the mice for a total of 12 days, and measured tumor sizes on day 23 after transplantation. Bioluminescence imaging of Luc-K562 cells in the mice was also performed. We also measured general toxicity during the experiment, and we did not detect obvious behavioral change, morbid consumption such as significant weight loss, or death of mice. On day 23, tumor growth was significantly suppressed in the mice injected with VC, compared to mice injected with vehicle or SFO (Fig.?3A,B). However, tumor growth was significantly enhanced in the mice injected with both VC and SFO (Fig.?3A,B). We did not detect newly developed tumors other than the tumors initially transplanted, or invasion of the leukemic cells to other organs, including the bone marrow and peripheral blood, of all mice. Open in a separate window Figure 3 High concentrations of iron impair the inhibitory effect of VC on K562 cell growth Imaging System [IVIS]; Xenogen Corporation, Alameda, California, USA). For imaging, mice were transplanted with Luc-K562 cells. After transplantation, the transplanted mice were injected intravenously with D-luciferin (150?mg/kg), placed onto the warmed stage inside the camera box, and were continuously exposed to 2.5% isoflurane to maintain sedation during imaging. Every group of mice was.