Effects Of NM ATRA On Proliferation, Apoptosis, Differentiation, The Expression Of TrkB Of NB Cell, And The Relations Between TrkB Signal Transduction Pathway And The Chemoresistance

Introduction and objectiveNeuroblastoma (NB) is a pediatric solid tumor derived from neural crest precursor cells. It is very resistant to current treatment protocols, including high dose chemotherapy. To reverse resistant still remains a major concern. The mechanisms of chemoresistance are very complex, including activation of onco-gene, inactivation of anti-oncogene, and inhibited apoptosis of tumor cells. MDRl ang MRP are studied more and more about the chemoresistant mechanism of NB. Many studys have shown that the chemoresistance of NB is related with the overexpression of p-gp and MRP. However, conflicting reports have appeared.Neurotrophins play an essential role in the growth, survival, and differentiation of neurons in both the peripheral and central nervous systems. The recent studys have shown that the expressions of the receptor of the neurotrophins such as NGF, BDNF, NT-3 have close relationship with the prognosis of NB.Azar first found that the level of TrkA is high in the good-prognosis NB tumors, and low in poor-prognosis NB tumors in 1991. The high level of TrkA is a good prognostic indicator for NB. Inversely, the level of TrkB and BDNF is high in poor prognosis NB tumors. Some of studys have shown that BDNF can promote survival , neurite outgrowth, and cell invasion in NB. So we guessedthat the activation of TrkB signal pathway may be correlated with NB chemoresis-tance.The aim of this research was to approach the effect of nM concentration of ATRA on the growth, apoptosis, differentiation and the expression of TrkB. To approach whether the activation of TrkB-BDNF signal pathway can cause che-moresistance, whether obstraction of TrkB-BDNF pathway can reverse chemore-sistance. We want to find an effective drug to reverse chemoresistance. This study will provide new clews and theoretical basis.Methods1. To detect the level of TrkA, TrkB, BDNF-mRNA before and after the treatment of ATRA by RT-PCR.2. To detect the expression of TrkB protein before and after the treatment of ATRA by cell immunochemistry and Western-blot, to detect the p-TrkB protein before and after the treatment of ATRA by cell immunochemistry and Western-blot.3. MTT analysis; (1) to detect the changes of cell survival rate of the treatment with different nM concentration . (2) the changes of the cell survival rate of different groups :alone CDDP, ATRA + CDDP.BDNF + CDDP,ATRA + BDNF + CDDP. (3) the cell survival rates of different groups: different nM concentration of ATRA + BDNF + CDDP. (4) the cell survival rates of different groups:ATRA + different concentration BDNF + CDDP. (5) the cell survival rates of different groups: ATRA + different concentration K252a + BDNF + CD-DP.4. To detect apoptosis rate by FCM.5. To detect the morphous of the apoptosis cell by transmission electron mocroscope (TEM) and Acridine orange stain .Results1. The expression of TrkA %TrkBN BDNF-mRNA induced by ATRA in differ-ent concentration and time.We can't detect the expression of BDNF%TrkA%TrkB-mNA in SY5Y. The expression of TrkB is positive after the treatment of lOOnM/LATRA in the second day, and the level of TrkB increased in the third, fifth day. The level of TrkB has no difference between the fifth day and seventh day.The expression of TrkA% BDNF were still negtive after the treatment of lOOnM/LATRA in the second^ third% fifth day. The level of TrkB-mRNA increased after the treatment of lOOnM/LATRA, and had dose and time dependence.2. To detect the expression of TrkB protein and p-TrkB protein by cell im-munochemistry.The expression of TrkB protein was undetectable in SY5Y. It was positive after treatment of lOnM/LATRA for five days. The expression of p-TrkB protein was undetectable before the treatment of BDNF (only ATRA) , it was positive after the treatment of adding BDNF for 15 minutes, it was undetectable after the treatment of ATRA + K252a + BDNF.3. To detect the expression of TrkB protein and p-TrkB protein by western-blot. The expression of TrkB protein was undetectable in SY5Y. it was positive after the treatment of 1,10, lOOnM/LATRA for five days. The level of TrkB protein was increased with the adding of the concentration of ATRA.4. To detect the survival rate by MTT;The survival rates of different groups treated by 1,10,100nM/L ATRA were 99.59 ± 9. 36% % 100. 85 ± 15. 94% ,101.24 ±11. 62% , the control group was 100%. the survival rates among the four groups were not significant (p = 0. 993 > 0.05). The difference of the survival rates among the lOnM/LATRA + CDDP group, lOOng/mlBDNF + CDDP group, the CDDP alone group were not significant. The survival rate of the ATRA + BDNF + CDDP group was greatly higher than the CDDP alone group, and the difference of survival rates between the ATRA + BDNF + CDDP group and the control group was not significant. Although the survival rate of lOng/mlBDNF + lOnM/LATRA +5|xg/mlCDDP group was higher than the CDDP alone,but the difference between them was no significant {p =0.363 >0.05). The survival rates of 50ng/ml, lOOng/ml BDNF + ATRA+ CDDP group were greatly higher than CDDP alone group t and the difference of the survival rates between 50ng/mT and lOOng/ml BDNF + ATRA + CDDP group was significant. The difference of the survival rates between the lOOng/ mlBDNF + ATRA + CDDP group and the control group was not significant. 'Hie survival rate of the lnM/LATRA + CDDP + BDNF group was higher than the CDDP alone group. But there was no significance between them. The survival rates of the group 10, lOOnM/LATRA + BDNF + CDDP were greatly higher than the CDDP alone group, and there was obvious difference among them. There was no obvious difference between the lOOnM/LATRA + BDNF + CDDP group and the control group ( p = 0. 558 > 0. 05 ). The survival rate of the ATRA + 0. 25jiM/LK252a + BDNF + CDDP group was 62. 86% , the survival rate of the ATRA + 0. 5^M/LK252a + BDNF + CDDP group was 49. 63% , the survival rate of the ATRA +1. 0yM/LK252a + BDNF + CDDP group was 35.23%. there were obvious difference among them. The difference of the survival rates between the ATRA + 1. 0|xM/LK252a + BDNF + CDDP group and the CDDP a-lone group was no significance.5. To detect the apoptosis rate by FCM:The difference of the early apoptosis rate between the lOOnM/LATRA for 5days group and the control group was no significance. There were no obvious difference among the three groups: ATRA + CDDP, BDNF + CDDP, CDDP a-lone in the early apoptosis rates. The early stage apoptosis rate of the grouplOnM/LATRA + lOOng/mlBDNF + 5jig/mlCDDP was significantly lower than that of the CDDP alone group, but there was no significance between the group lOnM/LATRA + lOOng/mlBDNF +5jjLg/mlCDDP and the control group in the early stage apoptosis rate. The early stage apoptosis rate of the group ATRA + K252a + BDNF + CDDP was greatly higher than that of the group ATRA + BDNF + CDDP. There was no significance between the group ATRA + K252a + BDNF and the control group in the early stage apoptosis rate.6. To survey the shap of the cell apoptosis by TEM:The shape of the cells in the group ATRA + BDNF + CDDP were as follow lower tracing : the intranuclear chromoplasma were well-distributed, the nuclear membrane were clear, the mitochondria ribosome, solvent can be seen, theshape of the cell of the group CDDP alone and the group ATRA + K252a + BD-NF + CDDP wer e as follow lower tracing: The nuclus were pycnosis, cell membrane were damaged, the mitochondria were vacuolar degeneration, nucleolem-ma were shrinkage, Some of the cells were as followed: chromatic agglutination in the intranuclear, the chromatin were side, looking like crescent, the inter-membrancee were not identical. Some of the cells were as followed: nuclus chromatins were side, nuclear type had several notches, nuclear membranes were clear. Much of them were in Middle and advanced stage.7. The cells that are stained by acridine orange can be observed by fluorescence microscope:Control group, ATRA + BDNF + cisplatin group and ARTA + K252a + BD-NF group: the conformation of nucleous is satiation and present a yellow green color of fluorescence. Cisplatin group, ATRA + K252a + BDNF + cisplatin group; the nucleous of most cells are presenting karyopyknosis, becoming punctate or massiveness and concentrating at one side of the cell to form crescent shape or granular shape. The nucleous/plasma is decreasing.8. To observe the differentiation of NB cell:There was no obvious difference among the lnM/LATRA for five days, lOOnM/LATRA for five days and the control group in the differentiation rate, but there were obvious difference between the 5u,M/LATRA and the control group, The differentiation rate of 5uJM/LATRA for five days was significantly higher than the control group.ConclusionsThese data suggest that nM concentration ATRA can induce the expression of TrkB-mRNA and protein, and the induced protein can be phosphorylationed by it' s ligand. nM/LATRA can't induce the expression of TrkA and BDNF-mRNA; nM/LATRA have no effects on the growth, apoptosis and differentiation of the SY5Y cell line.2. The activation of TrkB-BDNF signal pathway can result in chemoresis-tance by inhibiting the SY5Y apoplosis and inhibiting the role of anti-generation...