Antileukemic Efficacy Of FLT3 Targeting RNA Interference And Roles Of NF-κB Pathway, SMRT In The Signaling Transduction Of FLT3

Acute myelocytic leukemia (AML) is an aggressive hematological malignancy. Presently induction therapies are capable of achieving clinical remission in up to 70% of AML patients; however a large fraction relapse and long-term survival rates still remain 40%, reflecting the need for more effective therapeutic strategies.FLT3 is a member of the class iii receptor tyrosine kinase family, is preferentially expressed in normal human bone marrow selectively in CD34+ hematopoietic stem/progenitor cells and in dendritic cell progenitors. Ligandmediated activation of the FLT3 receptor is important for normal proliferation of primitive hematopoietic cells. However more than 70% of AML blasts express wild-type FLT3, whereas 25% to 35% carry FLT3 mutations including an internal tandem duplication (FLT3-ITD) within the juxtamembrane domain in nearly 30% of patients and possess and point mutations within the activating loop (FLT3-TDK) in 7% of patients. Aberrant activation of FLT3 is closely associated with leukocytosis and poor prognosis of AML patients.FLT3 receptor has received considerable attention as an attractive molecular target in AML. To date, several different FLT3 kinase inhibitors have been developed, however, results of phaseâ… /â…¡of clinical trials seem unsatisfying. None of these inhibitors are truly FLT3 specific because they often recognize other molecular targets such as VEGF-R₂, c-KIT, FMS and PDGF-R, therefore normal cells may experience significant cytotoxicity, and clinical side effects may be unavoidable. The sensitivity of FLT3 inhibitors to the FLT3 structure confers susceptibility to minor structural changes induced by point mutations within the activating loop, and acquired resistance appears. More important, studies of Flt3 signaling suggest involvement of a variety of intermediates, but for most their place in signaling pathways and identification of their physiologic role is unclear. Thus, an improves understanding of how FLT3 interacts with other molecules, as well as how aberrant activation of FLT3 triggers downstream intracellular signaling pathways, will provide more insights for the development of FLT3 targeting therapy.Another methodology for molecularly-targeted inhibition to FLT3 is RNA interference (RNAi), which not only provides a powerful system for analysis of FLT3 mechanism but also illustrate the potential benefit of therapeutic approached. Thus, in this study, we designed and synthesized 3 FLT3 targeting small hairpin RNAs (FLT3-shRNA) and screened the most effective one through evaluating their effects on FLT3 suppression, then in vitro studies we explored the efficacy of downregulation of FLT3 induced by RNAi to cell proliferation, apoptosis in THP-1, HL-60 cell lines.Analyses of signaling passway in the wild-type FLT3 have focused largely on the PI3K-Akt and Ras-MAPK pathways; whereas in the ITD-type FLT3 also involved STAT5 pathways. The aberrant expression of NF-κB, corepressor SMRT(Silencing Medtiator for Retinoic Acid and Thyroid Hormone Receptor) plays an important role in the pathogenesis of AML, However, their roles in FLT3 signaling cascades has not been determined. NF-κB family, taken as transcription factors, participates in cellular processes such as proliferation, apoptosis through regulating transcription of cyclinD1, c-myc, Bcl-2, and so on. SMRT has been identified as fundamental components in the regulation of eukaryotic gene transcription, so we studies whether downregulation of FLT3 influence NF-κB passway, and SMRT in AML cell lines. Under these conditions, we established the subcutaneous xenograft model of THP-1 leukemic tumors in Nu/Nu mice, detected the antitumor activity of FLT3-shRNAi used alone, or combined with NF-κB inhibitor, chemotherapeutic drug in vivo. Partâ… Expression of FLT3 in Five AML Cell Lines and Detection of FLT3-ITD MutationMethods1. Semi-quantitative RT-PCR was used to examine the FLT3 mRNA in five human AML cell lines that is THP-1, HL-60, K562, Dami and Meg-01.2. Western blotting was used to detect the FLT3 whole protein, and Flow cytometer (FCM) was used to detect the part of FLT3 transmembrance-protein.3. In order to determine whether FLT3-ITD Mutation exists in those AML cells, genomic DNA was extracted and was employed for amplifying the sequences of JM domain; their PCR fragments were ligated into T-vector and sequenced.Results1. Just THP-1, HL-60 were detected with basal expression of FLT3 mRNA, which related contents to GAPDH were 0.83±0.07%, 0.48±0.05%, respectively.2. Western blotting showed that just THP-1, HL-60 cells expressed FLT3 protein, FCM showed the percentages of FLT3 positive cells were 53.55±4.44% in THP-1 cells, 27.57±3.42% in HL-60 cell, namely, THP-1 expressed "strong" level as well as HL-60 expressed "intermediate" level.3. Results from sequencing showed the expected nucleotide size in JM region from THP-1, HL-60 cells were 329bp, the same size as that in wild-type FLT3, the concordance rate with the published data in GenBank (NM-004119) were over 98%, indicated no FLT3-ITD Mutation exists in both THP-1 and HL-60 cells..Partâ…¡in vitro Transcription Synthesis, Screening and Efficacy of FLT3 Targeting Short Hairpin RNAMethods1. Designed and synthesized three FLT3 targeted shRNAs (FLT3-shRNA), as well as one non-related sequences shRNA (NC-shRNA) by in vitro transcription system, their concentrations were determined. 2. Firstly THP-1 cells were transfected with 25nM of 3 types of FLT3-shRNA as well as NC-shRNA. For 24h, 48h and 72h cells were collected, FLT3 mRNA was detected by RT-PCR, and the related content of FLT3 mRNA to GAPDH was calculated.3. THP-1 cells were transfected with different contentions FLT3-shRNA1 which was identified with the most effective rate, which is 5nM, 10nM, 15nM, 20nM, 25nM. For 48h cells were collected and FLT3 mRNA was detected.4. According to the results of RT-PCR, FLT3-shRNA1, shRNA3 were used to transfect THP-1 cells. For 48h, 72h FCM was used to tested expression of FLT3 transmembrance protein, at 72h Western blotting was used to detect the expression of FLT3 whole protein.5. HL-60 cells were transfected with 15nM FLT3-shRNA1 depending on the results above, For 48h FLT3 mRNA was detected using RT-PCT, 72h FLT3 protein was detected using FCM and Western blotting.Results1. Three FLT3-shRNA and NC-shRNA were synthesized successfully.2. The expression of FLT3 mRNA was not affected by NC-shRNA, whereas it was suppressed by FLT3-shRNA1, shRNA3 over 50%. the inhibiting rates of shRNA1 were higher than those of shRNA3 (P＜0.001); The most effective time for shRNA1 was 48h with inhibiting rate 72.95±2.07%, as well as for shRNA3 was 48h with inhibiting rate 54.95±2.07%.3. FLT3-shRNA1 suppressed the expression of FLT3 mRNA in a Concentrationdependent manner within 5nM～15nM, the inhibiting rate of 15nM was 71.60±1.46%.4. Transfected with NC-shRNA didn't affect the expression of FLT3 protein, but FLT3-shRNA1, shRNA3 caused special degradation of the FLT3 transmembrance protein for both 48h and 72h detected by FCM, the more effective one was shRNA1 (P＜0.001), the more effective time was 72h (P＜0.001) with inhibiting rate 79.67±0.66%. And the same result was seen in Western blotting assay.5. HL-60 cells were transfected with 15nM FLT3-shRNA1, for 48h its inhibiting rate to FLT3 mRNA was 81.66±10.25%, for 72 its inhibiting rate to FLT3 transmembrance protein was 76.76±11.23%, and result form Western blotting confirmed that the expression of FLT3 protein was significantly suppressed too.Partâ…¢shRNAi-induced FLT3 Suppression Inhibits Myeloproliferation and Induces Apoptosis of AML CellsMethodsAll tests below were settled for 3 groups, treated with 15nM of FLT3-shRNA, 15nM of NC-shRNA, or the same vol of PBS, respectively.1. THP-1 and HL-60 cells (4×10⁴/ml) in 96-well plates were transfected with FLT3-shRNA and cultured for 8 days, 3 wells were added into CCK-8 and cell viabilities were tested for every day, then growth curves were drawn. Furthermore, cells (4×10⁵/ml) were treated as above described, and cell viabilities were tested for 24h, 48h, 72h, and inhibiting rates were calculated.2. Cells were treated as above; cells were stained with PI and analyzed cell cycle by FCM for 48h3. For 48h after transfection, cell apoptosis was analyzed by Annexin V-FITC combined with PI staining, DNA ladder in both THP-1 and HL-60 cells, as well as by TUNEL assay in THP-1 cells.4. For 48h, 72h after transfection, the mRNA level of cyclin D1, cyclin A was determined using RT-PCR, and the protein level was determined in whole cell extracts using Western blotting.Results1. Compared with that treated with PBS, little changes in growth curves were found in cells transtected with NC-shRNA in both THP-1 and HL-60 cells, but growth curves transfected with FLT3-shRNAi were low, fiat which lacked the typical character of exponential growth. Furthermore, an obvious loss of cell viability was detected in cells transfected with FLT3-shRNA, the inhibiting rate was 36.66±3.67% in THP-1 cells, 33.10±3.43% in HL-60 cells.2. FLT3-shRNA transfected cells showed an increase in the percentage of cells in the phase G₀/G₁ (P＜0.01) and a decrease in the percentage of cells in the phase S (P＜ 0.05) in both THP-1 and HL-60 cells.3. Transfection with FLT3-shRNA for 48h resulted in obvious increase of early apoptosis cell detected by Annexin V-FITC staining (P＜0.05), DNA Ladder which is characteristic of apoptotic cell were seen in both THP-1 and HL-60. And the result of TUNEL in THP-1 cells also showed an increase ofapoptotic cells (P＜0.001).4. shRNAi-induced suppression of FLT3 resulted in the downregulation of cyclin D1 expression (P＜0.01). The more effective time for cyclin D1 mRNA was 48h after transfection with the inhibiting rate was 37.09±3.76%, 63.69±21.26% in THP-1, HL-60 respectively. Western blotting showed an obvious decrease in cyclin D1 protein level, and 72h was more significant. Moreover the expression level of cyclin A wasn't affected by FLT3-shRNAi.Partâ…£The Roles of NF-κB Pathway, Corepressor SMRT in the Signaling Transduction of FLT3Methods1. RT-PCR was used to examine the P65, IκB mRNA in THP-1 cell lines, immunohistochemistry and Western blotting was used to detect their protein expression.2. THP-1 (4×10⁵/ml) in 96-well plates treated with different concentrations of Pathernolide (PN) within 0μM～20nM, cell viabilities were tested by CCK-8 and inhibitory concentration 50% (IC₅₀) were calculated for 12h, 24h. According the results above, cells (4×10⁴/ml)were treated with 6μM of PN and cultured for 6 days, 3 wells were tested for cell viabilities for every day, and growth curves were drawn.3. All tests below were settled for 5 groups, treated with PBS (group 1), 15nM of NC-shRNA(group 2), 15nM of FLT3-shRNA(group 3), 6μM of PN(group 4), 15nM of FLT3-shRNA + 6μM of PN(group 5), respectively. All the final time of shRNA was 48h, as well as the final time of PN was 24h. For P65,IκB, cyclinD1 and SMRT, RT-PCR was used to detected their mRNA expression, Western blotting was used to detected their protein expression in whole cell extracts or nuclear extracts. 4. After transfected with 15nM of FLT3-shRNA1 for 48h, THP-1 cell were treated by designed concentrations of PN as described above, the IC₅₀ were determined for another 12h, 24h,5. THP-1 cells were treated with PBS, FLT3-shRNAi, PN, FLT-shRNAi+PN, respectively. After cell collection FCM was used to determine the distribution of cell cycle, Annexin V-FITC staining was employed to analyze cell apoptosis.Results1. RT-PCR showed that P65, IκB mRNA expressed in THP-1 cells, Immunocytochemistry also showed P65, IκB protein, both mainly in cytoplasms, whereas Western blotting indicated P65 expressed not only in cytoplasm but also in nuclear, as well as IκB just in cytoplasm.2. Growth inhibition was seen in cells treated with PN in a concentration-dependant manner, the growth curve treated with 6μM of PN was lower. After 6μM of PN treatment, the IκB mRNA, protein increased obviously, P65 protein in nuclear extracts decreased, cyclin D1 decreased, as well as SMRT in nuclear extracts increased.3. The P65, IκB mRNA, protein in the whole cell extracts weren't affected by transfected with FLT3-shRNA for 48h, but P65 protein in the nuclear extracts decreased, cyclin D1 also downregulated as well. The expression of SMRT mRNA, protein in the whole cell extracts weren't affected but SMRT protein in the nuclear extracts decreased.4. The sensitivity of PN of THP-1 cells was increased due to the downregulation of FLT3, the IC50 for 12h was 6μM～8μM, for 24h was 4μM～6μM, both lower than those in controls. Cells treated with FLT3-shRNAi or PN respectively showed an increase in the percentage of cells in phase G₀/G₁ (P＜0.05), and a decrease in the percentage of cells in phase S (P＜0.05), the results were more prominence while cells treated with FLT3-shRNAi+PN (P＜0.05), Likely, the increased levels of early apoptosis were seen when cells treated with FLT3-shRNAi or PN, the results were more prominence when treated the both (P＜0.05), Results of Western blotting indicated that P65 nuclear protein, cyclin D1 protein decreased in cells treated with FLT3-shRNAi, PN alone, and more decreased when treated FLT3-shRNAi+PN. Partâ…¤Establishment of THP-1 Cell Xenograft Tumor Model in Nu/Nu Mice and Antileukemic Efficacy of FLT3 Targeting RNA Interference in vivoMethods1. THP-1 cells at exponential phase (1×10⁷ cells per mouse) were implanted S.C. into the right flank of Nu/Nu mice, established xenograft tumor model of THP-1 cell line.2. Treatments were initiated when tumors were 100～300mm³, mice were randomly assigned into six groups (5 mice per group). FLT3-shRNAi, PN, daunorubicin (DNR) were administered i.p. alone or combinations as protocol, all for 15days.3. Tumor volumes and body. weights were assessed every 2 day following the treatment. The mice were sacrificed 2 days after the last treatment, tumor massed was weighted, percentage of tumor growth inhibition was calculated...4. For tumor tissues in each group, TUNEL methods were adopted to assay cell apoptosis, immunohistochemistry, RT-PCR, and Western blotting were employed to determine the mRNA, protein expression of FLT3, P65, IκB, cyclin D1, SMRT, respectively.Results1. Treatment with FLT3-shRNAi led to tumor growth inhibition in Nu/Nu mice bearing THP-1 xenograft tumor, the percentage of growth inhibition was 28.95%, the percentage of apoptotic cell increased (P＜0.05). Western blotting showed FLT3 expression decreased in tumor tissues, P65 protein in nuclear as well as cyclin D1 decreased, SMRT protein in nuclear increased.2. Treatment with PN led to tumor growth inhibition in Nu/Nu mice, the percentage of growth inhibition was 32.46%, IκB expression in tumor tissues upregulated as well as p65 protein in nuclear, cyclin D1 protein downregulated, and SMRT protein in nuclear upregulated at the same time. More important, Treatment with FLT3-shRNAi+PN led to more efficiency on tumor inhibition which the percentage of growth inhibition was up to 49.12% (P＜0.05), more apoptotic cells in tumor tissues,(P＜0.01). and the decrease of p65 protein in nuclear, cyclin D1 was more prominence, however the increase of SMRT in nuclear wasn't more than that treated with PN alone.3. DNR had strong effects on tumor inhibition (72.05%), and when it combined with FLT3-shRNAi, the percentage of inhibition increased to 82.52%.Conclusions1. Just THP-1, HL-60 cells constitutively express FLT3 in the five AML cell lines, and no FLT3-ITD mutation exists in them.2. FLT3-shRNA1 can effectively down-regulate FLT3 expression.3. shRNAi-mediated FLT3 suppression inhibits myeloproliferation, induces cell apoptosis in both THP-1, HL-60 cells, thus tentatively confirms the anti-leukemic efficacy of the FLT3 targeting RNA interference in vitro studies. FLT3 suppression also down-regulates the expression of cyclin D1 in mRNA and protein level, and obviouly inhibits cell cycle from phase G₀/G₁ to phase S, which illustrates one of the important reasons for the myeloproliferation inhibition.4. Constitutively activated NF-κB signaling pathway exists in the THP-1 cells, Parthernolide(PN), a NF-κB inhibitor, effectively inhibits NF-κB signaling by upregulate the IκB expression and reverse P65 nuclear transportation, thus, suppress the cell growth.5. shRNAi-mediated FLT3 suppression can inhibit NF-κB activation through downregulates P65 protein in nuclear, thus, cell growth inhibition appears. Besides, SMRT protein in nuclear upregulates, and enhancement of transcriptional repression may be another reason of growth inhibition.6. shRNAi-mediated FLT3 suppression increases the sensitivity of THP-1 to treatment with PN, the combination of FLT3-shRNAi with PN has a cooperation-suppression of NF-κB passway, and illustrates the cooperation effect of anti-leukemic efficacy in vitro studies.