Discussion
The heme precursor ALA accumulates under pathological conditions, such as AIP, tyrosinosis and lead poisoning, diseases often linked with increased liver cancer incidence [17]. The reactive oxygen species generated upon ALA metal-catalyzed oxidation, promote the formation of several radical-induced base degradation products in isolated and cellular DNA [17]. ALA is the precursor of Protoporphyrin IX (Proto IX). Exogenous ALA bypasses the feedback control and may induce Proto IX accumulation. Since heme-containing enzymes are essential for energy metabolism, every nucleated cell should have at least a minimal capacity to synthesize Proto IX and this is the rational for the use of Photodynamic Therapy [18].
ALA proved to be cytotoxic in neuroblastoma cells. This cytotoxic effect of ALA could be diminished by heme, presumably through modulation of ALA synthesis [19].
Here we have demonstrated the cytotoxic effect of ALA on two human hepatocellular carcinoma cell lines HEP G2 and HEP 3B, which was greater on the former. ALA effect would be attributed to its accumulation in HEP G2 cells, as it was shown by its reduction after simultaneous exposure with hemin or glucose, well-known repressors of ALA-S activity. There is evidence that following the exposure to external ALA, there is induction of endogenous ALA synthesis, and that ALA uptake is higher in more rapidly proliferating cells [20]. The strange shape of the viability curves is difficult to explain within the context of these experiments and could probably be due to different ALA and/or PPIX accumulation, which will be measured in future assays.
In HEP G2 cells, protection by hemin would be due to induction of the HO-1 gene, and increased formation of bilirubine, through heme degradation with its known free radicals scavenger effect [21,22]. Glucose protection could be attributed to overcoming reduced glucose uptake provoked by ALA. ALA was reported to affect cultured chick embryo neuronal and glial cells by decreasing glucose uptake [23].
In HEP 3B cells, treatment with ALA and hemin or glucose produced increased cytotoxicity, indicating that the mechanism of ALA action involved would certainly be different.
Apoptosis is a physiological event occurring in response to multiple stimuli including growth factor withdrawal, radiation therapy and presence of chemotherapeutic agents. Several mechanisms regulate the apoptotic process, such as induction of a p53-dependent-pathway after DNA-damaging agents, modulation by the bcl2 family of proteins, and activation of some effectors including the interleukin-converting enzyme (ICE) family of proteases and endonucleases [24,25].
Interestingly, ALA produced apoptosis visualized by DNA ladders, but its action did not appear to correlate with the p53 status; while HEP 3B p53 null cells are relatively resistant to ALA-induced apoptosis, HEP G2 p53 wild type cells are very sensitive to ALA-induced apoptosis. However, the treatment did not alter mRNA p53 levels as revealed by Northern blot analysis suggesting that apoptosis would be occurring through other mechanism, p53 independent.
CDKs have been implicated as modulators of apoptosis [26]. Some important influences that may have an impact on susceptibility to apoptosis include altered generation of reactive oxygen or nitric oxide intermediates or propensity for mitochondrial damage or protease activation.
The mammalian cell cycle is controlled by sequential activation and inactivation of the CDKs family, which are activated by cyclins and inhibited by CKIs. The regulators that operate in the different phases of the cell cycle are various.
In our experiments, CDK2 and CDK4 protein levels decreased with ALA treatment preventing cells proliferation. Considering that both kinases are involved in cell cycle progression, diminution of their levels would induce G1 arrest and lead to the onset of the apoptotic process. When ALA was added to the medium at supraphysiological concentrations (higher than 3 mM), the rate of death cells were so high that the measured kinases levels would only correspond to a minimal proportion of viable cells. Perhaps, these cells represent a part of the population in S phase and therefore, with high levels of the kinases necessary to duplicate. These results prompted us to speculate that ALA toxic effect would be effective only in those cells which would be able to enter in arrest and would not exert any effect on those which are cycling. Further studies are in progress to further support our hypothesis. Disruption of the regulatory system controlling G1 phase progression is a common event in human hepatocarcinogenesis. Overcoming alterations that have occurred in the G1 phase regulatory machinery may provide a novel weapon to treat hepatocellular carcinoma [[27] and references there in].
In summary, ALA has a markedly cytotoxic effect on hepatocarcinoma cell lines at physiological levels, which could be attributed to an enhancement of apoptosis, independent of p53 but due to inhibition of cell cycle regulators CDKs.