Resveratrol plus low-dose hydroxyurea compared to high-dose hydroxyurea alone is more effective in γ-globin gene expression and ROS reduction in K562 cells

Abstract Hydroxyurea (HU) is an anti-cancer drug that is used for the treatment of hemoglobinopathies as a γ-globin inducer. However, its dose-dependent effects have hampered its clinical reliability. Resveratrol (RSV) is an antioxidant and γ-globin inducer. The present study aimed to assess their combined effects on the γ-globin gene expression and reactive oxygen species (ROS) level of K562 cells. The results indicated that the γ-globin gene expression was approximately two folds higher in the group treated with RSV 50 µM + HU 25 µM in comparison to HU 100 μM alone (***p < 0.001). However, there was an inverse relationship between the expression of γ-globin gene and HU concentration in the combined groups. Furthermore, the combinations of RSV and HU significantly reduced ROS levels compared to single drugs. Overall, the combination of these compounds was an appropriate strategy for increasing γ-globin expression, reducing oxidant levels, and alleviating the adverse effects of HU. Graphical Abstract


Introduction
The use of fetal hemoglobin (HbF; a2c2) inducers has been considered an appropriate treatment for hemoglobinopathies by reducing the a/b-chain imbalance. Hydroxyurea (HU; CH 4 N 2 O 2 ) is a well-known anti-proliferative agent which the c-globin gene induction of this drug has been proven in several investigations (Arruda et al. 1997;El-Beshlawy et al. 2016). In 1998, the US Food and Drug Administration (FDA) approved HU for the treatment of sickle cell disease. Despite its benefits, HU has dose-dependent side effects due to its potential teratogenic effects (Kumar et al. 2014). The lack of chemical stability and production of reactive oxygen species (ROS) have been reported as other drawbacks of HU (Iyamu et al. 1998).
In recent years, identifying the natural compounds that reactivate the c-globin gene and reduce oxidative stress has been challenged. Resveratrol (RSV; 3,5,4 0 -trihydroxytrans-stilbene) is a member of the stilbene family, which has been identified as an antioxidant, anti-inflammatory, and anti-aging supplement (Kong et al. 2019). Furthermore, the role of RSV as a c-globin inducer has been investigated in several studies (Fibach et al. 2012).
The myelogenous leukemia cell line K562 has been considered a useful in vitro model for studying the erythroid lineage differentiation and gene expression during hematopoiesis (Guo et al. 2012;Wicke et al. 2013).
Previous studies demonstrated the additive effects of HU to other agents on the c-globin gene expression and fetal hemoglobin level (Zhang et al. 2018). Considering these favorable results and the dose-dependent side effects of HU encouraged the in vitro evaluation of the combination therapy on K562 cells with low doses of HU and RSV as an antioxidant and nutritional supplement with the potential of c-globin elevation.

Analysis of cell cytotoxicity by MTT assay
The cytotoxic effect of RSV and HU in K562 cells (25, 50, 75,100 lM) presented dosedependent cytotoxicity of both drugs at 48 h (Figure S1a, supplementary material). For further experiments, the concentration of 25, 50 mM of RSV and HU are used because of the lowest cytotoxicity in MTT assay ( Figure S1b, supplementary material).

Evaluation of the c-globin gene expression by quantitative real-time PCR (qRT-PCR)
As demonstrated in Figure S2, K562 cells treated with HU 25, 50 lM alone did not significantly change the c-globin gene expression. Conversely, the treatment of K562 cells with RSV 25 and 50 lM and HU 100 lM significantly induced the c-globin gene expression ( Ã p 0.05, ÃÃ p < 0.01, and ÃÃÃÃ p < 0.0001, respectively). Figure S3 represents c-globin gene expression of the treatment with the combination of RSV and HU. All of the combination groups were more efficient in the c-globin induction in K562 cells compared to treatment with either agent alone, which indicates the synergistic interaction of these drugs. Besides, a combination group of RSV 50 mM þ HU 25 mM resulted in a significantly higher c-globin induction compared to HU 100 mM (7.99 ± 1.45 folds vs. 4.04 ± 0.95 folds). According to the previous reports, the in vitro 100 lM concentration of HU is the dose that corresponds to the concentration of this drug during in vivo treatment at 20 mg/kg/day (Pecoraro et al. 2014). It is also necessary to mention that the mean dose of HU therapy for patients with betathalassemia is 10-20 mg/kg/day (Algiraigri et al. 2017). Thus in this study, we demonstrate that combined treatment of RSV and HU at lower doses is more effective than the mean dose of HU therapy in the augmentation of the therapeutic potency without undesirable side effects.
As shown in Figure S4, the c-globin gene expression reduced in the combination groups with the higher dose of HU compared to those with the lower dose of HU. This result indicates the inhibitory effect of the higher dose of HU on the c-globin induction potential of RSV.
Thus, considering the efficacy and safety of the low-dose combinations, higher doses of HU may not be effective in combination with RSV.

Measurement of intracellular ROS by flow cytometry
The effect of RSV (25 and 50 lM) and/or HU (25 and 50 lM) on the intracellular ROS level of K562 cells was studied for 48 h by flow cytometry assay. The observations revealed that the ROS level decreased in the K562 cells treated with RSV which was in line with the findings of the previous studies (Fibach et al. 2012). On the other hand, HU alone led to a minor but not significant reduction in the ROS level. Moreover, incubation of K562 cells with the combinations of RSV and HU resulted in a marked reduction in the ROS level compared to the cells treated with a single drug (Figures S5 and  S6). It is clear that excessive ROS plays a significant role in the complications of thalassemia. Thus, the elimination of ROS may provide therapeutic benefits. While excess ROS contributes to pathological processes and cell damage in thalassemia, basal levels of ROS are needed for several critical reactions (Shields et al. 2021). Several studies have mentioned the double sword behavior of ROS by demonstrating that too low levels of ROS may interrupt the physiological processes and lead to immunosuppression as well as several pathological conditions (Perillo et al. 2020). The results indicate that a combined group of RSV 50 mM þ HU 50 mM reduced the ROS level to approximately 70% of the basal level. It was hypothesized that although using the combination of RSV and HU had benefits to prevent oxidative damage through the reduction of ROS level, excessive drops of ROS might exert adverse effects on the physiological processes.

Conclusion
The present study strongly points to new potentials of RSV in enhancing the effects of HU on c-globin expression and ROS level reduction in K562 cells. Our findings suggested that the combined treatment of RSV and lower doses of HU is more effective than HU alone. However, since the level of ROS was extremely low in combined treatment groups, which might be harmful to the physiological processes, further in vivo investigations are recommended to clarify the effect of this combined treatment. Altogether, we recommend combination of low doses of HU and RSV as a promising approach for treating thalassemia and sickle cell anemia.