Triptolide Modulates The Sensitivity Of Leukemia Cells To Chemotherapeutics Through Regulation Of MiR-21 Expression

Resistance to anticancer agents is the major clinical obstacle to the successful treatment of leukemia. Drug resistance is considered to be a multifactorial phenomenon involving drug inactivation, enhanced drug efflux, activation of survival pathways and reduced apoptosis; yet the key determinants of drug resistance remain largely unknown. Recent extensive studies have indicated the existence and importance of microRNAs (miRNAs) in cancer initiation and progression, and many miRNAs play an important role in resistance to anticancer drugs by targeting several signaling pathways; thus, the complex role of miRNAs in drug resistance has received increasing attention.MiR-21 was one of the first miRNAs detected in the human genome and to date, is the only miRNA known to be upregulated in all types of human malignancy. Moreover, the level of miR-21 expression is significantly associated with clinicopathological factors and the prognosis of tumor patients, suggesting that it might serve as a diagnostic and prognostic marker for human malignancy. In addition, miR-21 dysregulation has been reported to be a predictor of tumor responses to conventional cytotoxic chemotherapeutic agents, such as gemcitabine, docetaxel, temozolomide and 5-fluorouracil. Recently, several studies have explored the role of miR-21 in drug chemoresistance in both myeloid and lymphocytic leukemia. Studies showed that a specific anti-miR-21 oligonucleotide (AMO-miR-21) improved arsenic trioxide (ATO) cytotoxicity in acute myelogeneous leukemia cells and miR-21 blockage sensitized chronic myelogeneous leukemia cells to ATO and arabinosylcytosine (Ara-C) by inducing apoptosis. These effects of AMO-miR-21 are partly due to the downregulation of PDCD4.Triptolide is a diterpenoid triepoxide firstly purified from the roots of TwHf in 1972, and several synthetic routes have been described. Reports document that triptolide has anti-inflammatory and immunosuppressive, anti-fertility and anticancer abilities. It is well documented that triptolide has a broad spectrum ability to inhibit proliferation and induce apoptosis of various cancer cell lines in vitro and prevent tumor growth and metastases in vivo. Studies demonstrated that triptolide inhibited the expression of genes involved in cell cycle progression and cell survival, such as cyclins D1,B1, and A1, Cdc-25; Bcl-X and c-Jun. Triptolide reduced the expression of apoptosis antagonists XIAP, Bcl-2 and Mcl-1. Triptolide induced caspase-dependent apoptosis of leukemia and cervical cancer cells, and triggered caspase-independent autophagic cell death in pancreatic cancer cells.Objective:The aim of this study was to investigate whether triptolide could modulate the sensitivity of the leukemia drug-resistant cell line K562/A02 to the chemotherapeutic agent doxorubicin by inhibiting miR-21 expression and explore its mechanisms.Methods:(1)The cytotoxicity and reversal effects of Triptolide on multidrug resistance were assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide) assay.(2) Expression of mature miR-21 was determined by SYBR green PCR.(3) NF-κB activation was measured by electrophoretic mobility shift assay (EMSA). Binding of NF-κB to miR-21 gene promoter was evaluated by Chromatin Immunoprecipitation (ChIP) Assay.(4) The miR-21 mimics and inhibitors were chemically synthesized and transfected into K562 cells or K562/A02 cells.(5) P65, T-AKT, P-AKT, PTEN, BCL-2 protein level were determined by western blot.(6)Apoptosis percentage of cells was obtained from Annexin V/fluorescein isothiocyanate (FITC) and propridium iodide (PI) double-staining.(7) The effects of triptolide on P-glycoprotein activity were evaluated by measuring rhodamine 123 (Rh123)-associated mean fluorescence intensity on the basis of the flow cytometric technology.(8) In this study, we hypothesized that PTEN is a direct miR-21 target in K562 cells. To confirm this, the K562 cells were co-transfected with PTEN-3'UTR luciferase reporter and miR-21.(9) To explore the relationship between PTEN and ADR-induced cytotoxicity, we transfected PTEN siRNA or a scrambled siRNA into K562 cells, followed by treatment with various doses of ADR.(10) The mRNA levels of P65, PTEN were determined by SYBR green PCR.Results:(1) Since the reversal multidrug agents was required to be kept at appropriate concentrations, at which the drugs have no inhibitory or toxic effects on the tested cells.Consequently a concentration of 5 nmol/L triptolide was used to further study the reversal effect of multidrug resistance.(2) The miR-21 levels were decreased in K562/A02 cells treated with triptolide. Triptolide could inhibit P65 mRNA levels and proteion levels in K562/A02 cells. Reporter assay further showed that P65 transcriptional activity was reduced by triptolide.(3) Chromatin immunoprecipitation and electrophoretic mobility shift assays both showed that NF-kappaB could specifically bind to the binding sites located in miR-21 promoter. CHIP analysis suggested binding of NF-κB to miR-21 promoter was decreased in K562/A02 cells treatment with triptolide. (4) 5 nmol/L TPL increased the sensitivity of K562/A02 to adriamycin. when adriamycin was combined with 5 nmol/L TPL, the mean apoptotic population of K562/A02 cells was increased from 4.3% to 18.5%, respectively, compared with 3μg/mL of adriamycin treatment alone.(5) K562/A02 cells showed a significant reduction in P-gp expressions after TPL treatment. The results of Rh-123 accumulation assay shown that triptolide inhibited the function of P-gp.(6) The miR-21 levels was increased after infection with miR-21 in K562 cells and the miR-21 levels was decreased after infection with miR-21 inhibitor in K562/A02 cells. K562 cells that were infected with miR-21 had a significantly higher survival than the control group (P< 0.05). K562/A02 cells that were infected with the miR-21 inhibitor had a significantly lower survival than the control group.(7) K562/A02 cells that were transfected with the miR-21 inhibitor had a significantly higher PTEN protein level than the control group. The K562 cells that were transfected with miR-21 had a significantly lower PTEN protein level than the control group. MiR-21 markedly decreased the activity of the PTEN-3'UTR reporter in K562 cells.(8)PTEN siRNA effectively reduced the PTEN protein level (Fig.7A,7B). Furthermore, K562 cells that were pre-treated with PTEN siRNA had increased survival rate compared to the control group. The expression of P-gp was increased in K562 cells pre-treated with PTEN siRNA. (9) TPL effectively down-regulated Bcl-2 protein levels in K562/A02 cells. Western blotting using specific antibodies showed increased Bcl-2 in miR-21 mimic-transfected K562 cells compared with those in negative control cells, whereas opposite trends were found in the cells transfected with miR-21 inhibitor in K562/A02 cells.Conclusions:(1) Triptolide enchanced the sensitivity of doxorubicin in K562/A02 cells.(2) Triptolide decreased the levels of miR-21 in K562/A02 cells and increased the PTEN expression, consequently.(3) Triptolide inhibited the expression and the function of P-gp through PTEN/AKT signal pathway.(4) Triptolide inhibited the BCL-2 expression and enchanced doxorubicin induced apoptosis by modulating miR-21 levels.