The current study demonstrated that FAC acts as a tumor promoter in T51B liver cells. We also found tumor-promoting concentrations of FAC decreased, rather than increased, the proliferation of normal T51B cells. To our knowledge, this is the first report to describe tumor promotion by a physiologically and pathologically relevant form of iron. This is an important point missing from earlier studies of iron overload and neoplastic transformation. Previous experiments used various non-physiological chelating ligands to increase the bioavailability of iron. For example, carbonyl iron caused moderate iron overload in rats, but ferrocene was required for severe iron overload, liver neoplasms, and HCC [28
]. Iron given as the nitrilotriacetate (NTA) complex caused DNA damage and transformation of cells in culture, whereas iron citrate did not [31
]. Iron-NTA was a liver tumor promoter in rats [30
]. Co-administration of an iron ionophore significantly increased iron effects [35
]. Until now it was unclear whether ionic iron alone had transformation-related effects in a mammalian system. Our finding, that FAC had tumor promotion activity in the absence of non-physiological chelating ligands, settles this dispute. This has potential clinical implications, as the goal to reduce the incidence of HCC among iron overload patients may be accomplished through long-term reduction of iron levels [9
]. Novel strategies that target NTBI may be particularly effective in achieving this goal.
By definition, tumor promotion involves the selective proliferation of pre-neoplastic (vs. normal) cells. Classical tumor promoters such as phorbol 12-myristate 13-acetate (TPA) increase DNA synthesis and cell proliferation in cell and animal models of carcinogenesis [50
]. This mitogenic effect is thought to be critical for tumor promotion, acting by positive selection to increase proliferation of initiated cells. Cell proliferation is needed to fix and clonally expand carcinogenic mutations resulting from chemically-induced DNA damage. Alternatively, a tumor promoter may cause growth inhibition and/or cell toxicity, accompanied by outgrowth of a resistant phenotype. This idea was first proposed for liver by Farber and co-workers [51
] as the "resistant hepatocyte model" of tumor promotion. Similarly, a role for compensatory proliferation in liver tumor promotion has been proposed [53
]. Essentially, a certain degree of cell toxicity is tumor promoting in liver because it allows for compensatory proliferation of chemically initiated cells, which would otherwise remain quiescent. These previously described negative selection models are consistent with our findings and offer insight into how NTBI may contribute to HCC in iron overload. We propose that anti-proliferative or other toxic effects of iron loading on normal cells, rather than mitogenic effects on pre-neoplastic cells, explain tumor promotion in the T51B cell model. Consequently, agents which prevent NTBI toxicity are predicted to also block tumor promotion.
HCC may originate from hepatocytes or oval cells, a precursor stem cell type in liver [55
]. Differentiated hepatocytes do not readily proliferate in culture, and so are not suitable for the type of study presented here. To model iron-related HCC, therefore, we used T51B cells, a cell type similar to liver oval cells. In addition, we used 50–200 μM FAC for 12–16 weeks to establish iron overload. Although development of HCC in humans with hemochromatosis occurs at lower serum iron citrate concentrations (5–20 μM) over several decades [9
], several considerations suggest our experimental conditions are appropriate. First, studies of serum NTBI in humans are only partially informative. Iron citrate (unlike transferrin iron) is very rapidly cleared from the blood by the liver [18
], and so the serum concentration likely underestimates liver exposure. Second, iron-related HCC occurs primarily in the setting of liver cirrhosis. The effect of cirrhosis on iron citrate concentrations in the liver itself is unknown, but exposure of preneoplastic cells to levels higher than reported in blood seems possible. Finally, studies of high concentrations of carcinogens and tumor promoters given for short times are generally accepted as useful predictors of effects caused by exposure to lower concentrations for longer times. These points argue that findings from the T51B cell model are applicable to the promotion phase of iron-related HCC in humans.
The route of NTBI uptake in T51B cells is unknown, but there are several possibilities. The divalent metal transporter DMT-1 (NRAMP2) is thought to be important in most cell types [2
]. This protein has been localized to the cell surface in hepatocytes [67
], and iron transport at pH 7.4 has been documented [65
]. However, iron transport by DMT-1 is optimal near pH 5.5, consistent with a primary function in recovery of iron released from transferrin in endosomes. In AML12 hepatocytes, the cell surface zinc transporter zip14 is an additional pathway [68
]. This protein is particularly interesting with respect to neoplastic transformation, as zip14 was reported to be under expressed in HCC [69
]. Downregulation of NTBI uptake is one potential mechanism by which initiated cells could minimize iron-related toxicity and gain a proliferative advantage over normal cells in our model. Alternate NTBI uptake pathways identified in other cell types include the TRP family of cell surface non-selective cation channels [70
], and L-type calcium channels [71
At present, we surmise that NTBI toxicity impaired progression of T51B cells into or through mitosis, based on high levels of cyclin B1. ROS generated by a Fenton-type reaction involving vanadate was shown previously to cause increased cyclin B and M-phase arrest [72
]. Decreases in cyclins D1 and A are expected if proliferating cells become delayed at this point in the cycle. Importantly, these changes were evident at tumor promoting concentrations of FAC (200 μM). Relatively minor phenotypic distinctions may allow pre-neoplastic initiated cells to evade the selective pressure exerted by FAC at this concentration. However, these distinctions were insufficient to overcome additional toxic effects of higher concentrations, since tumor promotion decreased at 500 μM FAC. The cause(s) of increased cyclin B and cell cycle delay are unknown; dissecting potential mechanisms is a goal of future experiments. The step taken here, of demonstrating that these changes are caused by a physiologically and pathologically relevant form of NTBI under conditions of tumor promotion, is a critical one towards understanding and preventing iron-related carcinogenesis in humans.