Due to the molecular heterogeneity of myelodysplastic syndrome, therapies using single-target drugs are ineffectives. An orchestrated interplay between three important processes: apoptosis, autophagy and cell cycle has been implicated to new anti-cancer therapies. In this concern, natural compounds like quercetin are considered new chemicals for the development of drugs against various molecular targets. Quercetin is ubiquitously found in fruits and vegetables and several beneficial health effects have been associated with the dietary uptake of this bioflavonoid. Accordingly, the goal of this work was to identify the quercetin effects using P39 cell line, derived from a patient with MDS-chronic myelomonocytic leukemia (CMML), kindly provided by Eva Hellstrom-Lindberg, Karolinska Institute Stockholm, as model.
P39 cell line was submitted, in our lab, to karyotyping which showed 46XY,+del(6),-9,-16,-17,+2mar, indicating that this cell line is not contaminated with HL-60. P39 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 U/ ml penicillin, 100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. The quercetin was dissolved in DMSO, final concentration of 0.1% (v/v) in RPMI. P39 cells were treated with quercetin at final concentrations of 10, 50 or 100 µM for 24h. Control cells were treated with vehicle alone. The xenografted model was performed in immunodeficient mice (NOD.CB17-Prkdcscid/J lineage), (n= 6). Mice were inoculated, subcutaneously, with P39 cells (1x107cells/mice) in the dorsal region. Every 7 days the tumor volume was evaluated. The quercetin treatment started after tumors reached 100 to 200 mm3; it was given once every four days by intraperitoneal (i.p) injection at 120mg/Kg body. Control group received equal amounts of vehicle solution as previously described (Wang, et al., 2011). After 21 days, the mice were sacrificed; tumors were removed, minced and homogenized in protein extraction buffer or fixed in formalin immediately for immunohistochemistry. Then detection of apoptosis, autophagy and cell cycle process were performed.
In vitro results show that quercetin inhibited proliferation of P39 cells in a dose-and-time dependent manner and that the cell death induced by quercetin is due to modulation of apoptotic process. The quercetin treatment decreased Bcl2 and McL-1 expression (anti-apoptotic proteins) and increased Bax, an important pro-apoptotic protein. We could also observe changes in membrane potencial (Dym) after quercetin treatment with concomitant release of cytochrome c from mitochondria into the cytosol and increased expression of caspase 9, 8 and 3. Quercetin induced a marked increase in the number of P39 cells in the G1- phase with reduction of CDK2, CDK6, cyclin D, cyclin E, cyclin A and phosphorylation of Rb. Our results also showed increased levels of both p21 and p27 after 24 hours of quercetin treatment. Quercetin promoted pronounced phosphorylation of ERK1/2 and JNK. Using the selective inhibitors PD184352 (ERK inhibitor) and SP600125 (JNK inhibitor) no differences in percentage of apoptotic cells were found after 24 h of incubation. On the other hand, the combination of quercetin and PD184352 or SP600125 significantly decreases the accumulation of P39 cells in the G1 phase. Formation of acidic vesicular organelles (AVOs) was observed in quercetin- treated P39 cells. Then, the main proteins related to the autophagy process were analyzed and we found increased expression of beclin-1 and PI3K class III, ATG5-ATG12, ATG7 and conversion of LC3-I to LC3-II. In addition, quercetin-mediated dephosphorylation of Akt and mTOR which are considered key negative regulators of autophagy. Pharmacological inhibition of autophagy enhanced quercetin-induced suppression of P39 cell growth with no modulation of quercetin in G1 phase of cell cycle. Our results in xenograft model show that after 21 days of quercetin treatment, there was reduction of 31.6% in tumor volume compared to control group. We also observed apoptosis, autophagy and cell cycle activation status in the tumor tissue of animals treated with quercetin and by immunohistochemistry we confirmed the upregulation of caspase 3, p21 and LC3-II confirming in vitro results.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.