In good agreement with the collapsed mitochondrial potentials, treated cells showed an increase in the oxidized CL (Fig. 6, lower panel). Moreover, we decided to evaluate a correlation between erythroid differentiation and mitochondrial impairment. In particular, the involvement of the mitochondrial pathway
via activation of caspase-3 and caspase-9 was evaluated; to this aim, K562 cells were irradiated in the presence of the pancaspase inhibitor z-VAD.fmk and then benzidine test was performed. As shown in Fig. 7, z-VAD.fmk suppressed erythroid differentiation induced by all furocumarins. We also Duvelisib solubility dmso studied the possible erythroid differentiation activity of irradiated mixtures of some tested furocoumarins. These compounds were 5′-MP, 4′,5′-DMP and 5,5′-DMP and were chosen on the basis of their higher
sensitivity to UV-A photodegradation LY2157299 purchase (followed by UV–vis spectroscopy- data not shown). After their irradiation in methanol solution with different UV-A doses (0, 8, 16 and 32 J/cm2), psoralens were concentrated by solvent evaporation and then resuspended in methanol. The erythroid differentiation of photoproducts was investigated by benzidine test incubating K562 with psoralen irradiated mixtures at two different concentrations (50 and 200 μM) for 5–7 days. Cell growth was also evaluated using the MTT assay after 6 days of treatment (Table 3). After 6 days of incubation, cells treated with 50 μM pre-irradiated mixtures
did not show a clear increase of benzidine positive cells (Fig. 8, upper panel) nor a decrease in cellular viability in comparison to control (Table 3); on the contrary, using the higher concentration, an induction of erythroid differentiation (26–36% benzidine positive cells) (Fig. 8, lower panel) together with a reduction of cellular viability was over observed only with 5,5′-DMP (Table 3); the other POP mixtures exhibited low activity or were inactive. The irreversibility of the erythroid differentiation induction by 5,5′-DMP photoproduct mixtures was also assessed. The first 6 days of treatment were sufficient for K562 cells to differentiate irreversibly since during additional 4 days of culturing in the absence of the inducer of washed cells, the population of benzidine-positive cells still increased (from 26.3 ± 3.1 to 44.3 ± 2.2 in the case of 8 J and from 35.1 ± 2.0 to 40.5 ± 1.1 in the case of 16 J). RT-qPCR was also employed to quantify the expression of globin mRNA following treatment of K562 cells with 5,5′-DMP photoproducts. There is a clear positive relationship between UV-A doses used to obtain the photoproducts and the extent of increased globin mRNAs in respect to control K562 cells (Fig. 9). As far as a possible differential activity of furocoumarin photoproducts on globin gene expression is concerned, the data clearly indicate that accumulation of both the α-like α-globin mRNA and ζ-globin mRNA are strongly induced.