The Preliminary Investigation Of The Correlation Between The TrkB/BDNF Signal Pathway And FOXM1in Multiple Myeloma

[Objective] This study aimed to investigate the differential expression of TrkB and FOXM1molecules in multiple myeloma patients. And to further find the correlation of TrkB/BDNF signaling with FOXM1.[Methods] Untreated multiple myeloma patients, complete remission patients and healthy controls were collected and bone marrow mononuclear cells were isolated. The mRNA expression of BDNF, its receptor TrkB and FOXMl were detected by ordinary PCR (RT-PCR) and more sensitive Real time PCR (RQ-PCR) analysis as well. The protein expression of TrkB and FOX1were studied by immunohistochemistry in bone marrow smears from18multiple myeloma patients and9healthy controls.[Results] The expression of BDNF, its receptor TrkB and FOXM1transcripts in marrow myeloma cells from50patients and HMCLs was demonstrated by RT-PCR. TrkB expression was positive in48patients (96%) and FOXM1was positive in40patients(80%). More samples in untreated multiple myeloma patients showed up-regulation of TrkB and FOXM1than in complete remission patients and healthy controls. In bone marrow smears from patients with multiple myeloma, a diffuse cytoplasmic expression of TrkB was demonstrated immunohistochemiacally in17of18(94%)patients, and a nuclei positive expression of FOXM1was also detected in14of18(77%) patients, whereas control bone marrow samples showed weaker TrkB and FOXM1protein staining.[Conclusion] Both TrkB and FOXM1are abnormally high expression in patients with multiple myeloma and their expressions have significant relation. [Objective] We tested the expressions and the relationship of FOXM1and its downstream targets in8226cells, after we have incubated them with TrkB inhibitor K252a. The effect of K252a and Thiostrepton on the apoptosis, cell cycle and proliferation of8226cells were tested.[Method]1.8226cells were divided into eight groups. A groups(l) Oh group;(2)12h group;(3)24h group;(4)48h group; B groups(5) K252a Onm group;(6) K252a100nm group;(7) K252a200nm group;(8) K252a400nm group; The mRNA expressions of FOXMland its downstream targets CyclinBl and CDC25B were detected by real-time PCR to find whether their changes have relations with K252a;2.8226cells were divided into eight groups. A groups(1) Oh group;(2)12h group;(3)24h group;(4)48h group; B groups(5) K252a Onm group;(6) K252a100nm group;(7) K252a200nm group;(8) K252a400nm group; The protein expressions of FOXMland its downstream targets CyclinBl and CDC25B were detected by Western Blot to find whether their changes have relations with K252a;3. HMCL RPMI8226cells were plated in RPMI1640supplemented with1%FBS for4hours, after that8226cells were divided four groups (1) BDNF group (50ng/ml);(2) Thiostrepton group(5μM);(3) BDNF (50ng/ml)+Thiostrepton group(5μM);(4) BDNF (50ng/ml)+Thiostrepton (5μM)+LY294002(50μM) group. Cell apoptosis rate was detected by Annexin V/PI via flow cytometry at24hours;4. After the HMCL RPMI8226cells having been incubated with K252a (0nm,100nm,200nm,400nm) and Thiostrepton (0μM,5μM,10μM,20μM) for24h and48h, Flow CytoMeter stained PI was applied to tested the cell cycle.5. HMCL RPMI8226cells were divided into following groups:K252a (0nm,100nm,200nm,400nm)、 Thiostrepton (0μM,2.5μM,5μM,10μM,20μM) and K252a+Thiostrepton (blank, K252a50nm+Thiostrepton2.5μM, K252a100nm+In Thiostrepton5μM, K252a200nm+Thiostrepton10μM, K252a400nm+Thiostrepton20μM).CFSE labled cell proliferation trail was served to check out the proliferation changes after48hours.[Results]1. After having been incubated with TrkB inhibitor K252a, the mRNA expression of FOXM1and its downstream targets CyclinBl and CDC25B were depressed on dose-dependent and time-dependent manners;2. After having been incubated with TrkB inhibitor K252a, the protein expression of FOXM1and its downstream targets CyclinBl and CDC25B were depressed on dose-dependent and time-dependent manners;3. FOXM1inhibitor Thiostrepton can induce8226cells apoptosis after24h. BDNF delays Thiostrepton-induced death of8226cells, and the ability of BDNF to delay Thiostrepton-triggered apoptosis is partially blocked by LY294002, suggesting that PI3K activation contributes to, but is not fully responsible for, BDNF protection.4. We analyzed cell cycle phase distribution by flow-cytometric analysis of propidium iodide-stained cells after treatment with Onm,1OOnm,200nm and400nm K252a for24h and48h. The data shows that in the HMCL RPMI8226cells,100nm K252a for24h and48h induced G0/G1arrest, whereas treatment with200nm and400nm K252a induced G2/M phase arrest. The HMCL RPMI8226cells’cycle were arrested in G2/M phase, after they having been treated with Thiostrepton for24h and48h, which showed similar trends to the cells treated with high concentration of K252a.5. After the8226cells having been incubated with K252a/Thiostrepton for48h, both K252a and Thiostrepton remarkably inhibited8226cells proliferation ability in a dose-dependent way. These two inhibitors used at the same time appeared synergistic effect.[Conclusion] TrkB inhibitor K252a inhibit the expression of FOXM1and its downstream target genes CyclinBl and CDC25B in HMCL RPMI8226cells. These suggest that FOXM1probably is a new downstream target of TRKB/BDNF signal pathway; Thiostrepton-triggered apoptosis is partially blocked by LY294002, suggesting that PI3K activation contributes to, but is not fully responsible for, BDNF protection. Treatment with high concentration of K252a causes G2/M phase arrest and inhibits the proliferation, suggesting that it may be related to the inhibition of the expression of FOXM1and its targets. [Objective]This study aimed to explore the mechanism of TrkB/BDNF signaling pathway in the regulation of FOXM1by analysis of downstream targets of PtdIns3K and MAPK signaling after the cells were treated by different inhibitors and BDNF.[Method]1. multiple myeloma cell lines8226were treaded by TrkB inhibitor K252a(200nm) for different times:(1) normal control group(Oh);(2)12h group;(3)24h group;(4)48h group. The key components of both P13K/Akt signaling and Raf/MEK/MAPK signaling by Western Blot;2. Cells were serum-starved overnight. To study the effect of different stimuli on the expression of FOXM1, cells were treated with100ng/mL BDNF for24h. Alternatively, cells were pre-treated with PI3K inhibitor LY294002(50μM,30min), FRAP/mTOR inhibitor rapamycin (50nM,30min) or MAPKK inhibitor PD98059(50μM,1h) followed by stimulation with100ng/mL BDNF for24h. The protein expression of FOXM1in8226cells treated from different experimental conditions were detected by Western blot;3. After having been incubated with TrkB inhibitor K252a, FRAP/mTOR inhibitor Rapamycin or FOXM1inhibitor Thiostrepton for24h, we tested the expression of FOXM1, mTOR and TrkB to find whether their changes have relations.[Results]1. Protein lysates were prepared from8226cells after treatment with200nmol/L TrkB inhibitor K252a, and protein expression levels were analyzed by Western blotting using specific antibodies. We compared the changes in protein expression of P-FOXO3a as well as P-Akt and P-MAPK in the8226cells following treatment with K252a for0,12,24, and48h. Except inhibiting protein expression of FOXM1and its downstream target genes CyclinBl and CDC25B, TrkB inhibition was associated with a significant reduction in P-Akt and significant decrease in P-FOXO3a and P-MAPK protein levels.There were also no significant changes in total FOXO3a, Akt, and MAPK levels in response to K252a, indicating that K252a modulates the activity of these signaling intermediates predominantly at post-translational levels.2. TrkB/BDNF signaling pathway regulated the expression of FOXM1is mediated by PI3K/Akt signaling and Raf/MEK/MAPK signaling in8226cells, the comparable effect of BDNF was inhibited by PI3K inhibitor LY294002、 FRAP/mTOR inhibitor Rapamycin and MAPKK inhibitor PD98059.3. After having been incubated with K252a or Rapamycin for24h, the expressions of FOXM1and mTOR were depressed on dose-dependent. Their expressions have significant relation; RPMI8226cells were treated with0,5,10and20μM/L Thiostrepton before cells were collected and total RNA was isolated. FOXM1, mTOR or TrkB mRNA levels were analyzed by real-time PCR. We found that the expressions of FOXM1was depressed on dose-dependent, whereas the expressions of TrkB was up-regulated on dose-dependent.[Conclusion]The above experimental results show that the expression of FOXM1affected by the PI3K/Akt and MAPK signaling pathway. PI3K inhibitor LY294002、 FRAP/mTOR inhibitor Rapamycin and MAPKK inhibitor PD98059, blocked BDNF-induced expression of FOXM1. This finding also suggests that TrkB/BDNF signal pathway maybe through mTOR and FOXO3a to regulate the expression of FOXM1.