Elevation of Fetal Hemoglobin (HbF) by Novel Alkylating Agents in Human Erythroid Cells; Synergistic Effects with Established HbF-Inducing Agents.
2006
Pharmacological induction of fetal hemoglobin (HbF) is beneficial for some patients with β-thalassemia and also ameliorates the severity of pain episodes in sickle cell anemia, mainly by hydroxyurea (HU). However, refractoriness or poor response of some patients treated with HU triggered research for other drugs. In the present study, we evaluated the effects of novel steroidal alkylating agents EA80, XK4 and CS on HbF induction in CD34+ cell cultures from normal donors. Furthermore, we examined the effects of these agents combined with HU, hemin (HE) and butyric acid (BU) on HbF modulation in adult erythroid cells. CD34+ cells from normal donors cultured in serum-free StemSpan medium were exposed to EPO (4 u/ml) + SCF (100ng/ml). Different concentrations of EA80 (0.1–1μM), XK4 (0.1–10 μM) and CS (0.1–1μM) were added to the cultures at day 6 (proerythroblast stage), then cells were washed and harvested 1– 3 days later. The effect of the drugs on cell number was measured by the trypan blue exclusion technique. The number of Hb-containing cells were determined using the benzidine-HCl procedure. EA80, over a wide range of concentrations (0.1–0.8μM), did not compromise cell survival. Continuous exposure of CD34+ to EA80 had a dose (up to 0.4μM)- and-time dependent effect on cell number as well as on globin mRNA levels. Treatment of CD34+ cells with EA80 at 0.4μM for 3 days was followed by a two-fold increase in cell number and a 1.5-fold in benzidine-positive cells. Qualitative and quantitative RT-PCR evaluation of globin-mRNA transcripts in CD34+ demonstrated that EA80 (0.4μM) caused a time- and dose-dependent increase in gamma globin mRNA (1– 3 days: 1.5 to 2.0-fold). The addition of HU and HE in combination with EA80 in normal CD34+ cell cultures led to a 20–30% increase in cell number by day 9. Furthermore, the combination of EA80 with either HU or HE was accompanied by an increase in γ-mRNA content (1.5 and 2.5-fold, respectively).No significant difference was detected in the level of both adult globin mRNAs. In contrast, BU addition had no effect either on erythroid cell proliferation or γ-mRNA levels. The addition of XK4 had a dose dependent effect on cell number and γ-globin mRNA transcripts [highest effect (2.0–5.0-fold) at 5 and 10μM]. Addition of EA80 (0.4μM) concurrently with varied concentrations of XK4 (0.1–10μM) caused a x2–4-fold increase in gamma globin content (highest effect at 5/10μM). For both drugs, the levels of β-and α-mRNAs in normal CD34+ cell cultures were not affected by dose. The addition of the third drug, CS at concentrations between 0.1 and 1μM proved toxic (reduced cell number and γ-globin mRNA transcripts).
Our findings suggest that the beneficial effect of EA80 and XK4 might be threefold:
1. increasing cell number
2. affecting preferentially the rate of transcription of γ-globin mRNA,
3. acting synergisticallly with HE and HU, most likely through transcriptional and posttranscriptional mechanisms.
These results indicate that novel alkylating agents EA80 and XK4, either alone or in combination with other HbF-augmenting drugs, might provide a potentially useful treatment for patients with β-hemoglobinopathies with poor or no response to established Hb-F inducing agents.
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