The Influence Of Survivin On The Biology Behavior Of Neuroblastoma Cell And The Chemotherapy Sensitization Of It’s Inhibitor YM155

BackgroundNeuroblastoma (NB) is one of the most common extracranial solid malignancies in children and infants. Because of its insidious onset, it is usually difficult to treat, especially for children with advanced type. The overall survival rate is still maintained at20%-30%and the recurrence rate is high. Therefore, thorough research about the biology, genetics characteristics and the specific pathogenesis of neuroblastoma has important clinical value for finding new tumor gene therapy and improving the cure rate and long-term prognosis of NB.Survivin, a member of the inhibitor of apoptosis (IAP) family, is known to be expressed in various malignancies. Survivin may participate in the occurrence, development and progression of NB. It may not only be an important reference index for the prognosis of NB, but also be an ideal target for gene therapy. YM155is a water-soluble survivin specific inhibitor. It can specifically inhibit the expression of survivin in vivo and in vitro. It also has strong antitumor effect and at the same time can increase the sensitivity of common tumors to chemotherapy or radiation. What does the regulatory role of survivin play in proliferation, apoptosis, migration and transfer process of NB cells? What will happen if NB cells are treated with YM155? Will YM155improve the sensitivity of NB cells to cisplatin chemotherapy? Combining the in vivo and in vitro experiments, this study further analyzes the relations between survivin and NB as well as YM155and NB, in order to find a new molecular target for NB.Part Ⅰ:The influence of survivin ASODN on migration and invasion of neuroblastoma cells ObjectiveTo observe the effects of survivin on proliferation, apoptosis, migration and invasion of human neuroblastoma cell line SK-N-SH, through transfection of SK-N-SH with different concentrations of survivin ASODN. MethodsSurvivin ASODN was designed and synthesized, and then in-vitro-cultured SK-N-SH was transfected by different concentrations of survivin ASODN with liposome as the vector. RT-PCR and Western blot analysis were used to analyze the survivin mRNA and protein expression level of SK-N-SH cells that were interfered with different concentrations of survivin ASODN. MTT assay was used to determine the inhibition of survivin ASODN on cell growth. Flow cytometry was used to test the apoptosis rate. And Transwell assay was used to analyze the effects of survivin ASODN on migration and invasion of SK-N-SH cell in vitro. ResultsSK-N-SH cells were treated with different concentrations (200,400,800nmol/L) of survivin ASODN for48h. Survivin mRNA expressions and protein expressions were all significantly decreased compared with blank control group, lipofection control group and SODN group (P<0.05). The inhibition effects were dose dependent, which were most obvious at the concentration of800nmol/L.SK-N-SH cell transfected with different concentrations of Survivin ASODN can suppress the cell growth and proliferation to different degrees. Cell inhibitory rates in ASODN transfection groups were significantly higher than that of blank control group, lipofection group and SODN group (P<0.05) in the same duration. Under the same concentration (800nmol/L), cell inhibitory rate of Survivin ASODN was increased significantly with the increase of time. There was obvious time-effect relationship, and cell inhibitory rate rose to the highest at48h.Apoptosis rates of ASODN transfection groups were significantly higher than the blank control group, lipofection control group and SODN group (P<0.05). The cell apoptosis rate was increased with the increase of survivin ASODN concentration, and there was a dose-response relationship. The strongest pro-apoptosis function was found at800nmol/L. SK-N-SH cells transfected by the same concentration (800nmol/L) of survivin ASODN had the maximum cell apoptosis rate at48h. After72h, the cell apoptosis rate began to fall. It showed that the promoting role of survivin ASODN on SK-N-SH apoptosis was time dependent to a certain degree, and it was best at48h.In cell migration and invasion experiment, the cell numbers of migration and invasion in Survivin ASODN groups of different concentrations were significantly decreased at24,48and72h after transfection, compared with the blank control group, lipofection control group and SODN control group (P<0.05).And the changes showed a certain dose and time dependence, with the most obvious function at800nmol/L and the best time was48h. ConclusionSurvivin ASODN can induce apoptosis of SK-N-SH cells, inhibit tumor proliferation and reduce the invasion and migration ability of SK-N-SH cells via specific inhibition on survivin mRNA and protein expression in SK-N-SH cells. Part Ⅱ:The influence of YM155on migration and invasion of neuroblastoma cells ObjectiveTo discuss the effect of survivin inhibitor YM155on the proliferation, apoptosis, migration and invasion of SK-N-SH and its mechanism. MethodsThe SK-N-SH cells were treated with different concentrations of YM155. RT-PCR and Western blot analysis were used to detect the Survivin mRNA and protein expression level of SK-N-SH cells. MTT assay was used to detect the cell growth inhibition. Flow cytometry was used to determine the apoptosis rate. And Transwell experiment was used to analyze the migration and invasion of SK-N-SH cells.ResultsSK-N-SH cells were treated with different concentrations of YM155. Survivin mRNA and protein expression s were significantly decreased in LYM155group of10-500nmol/L (P<0.05), and the effect was dose dependent, namely expressions of survivin mRNA and protein were decreased with the increase of concentration of YM155. Survivin protein expressions in SK-N-SH cells were significantly decreased (P<0.05) after treatment with100nmol/L of YM155for12,24,48and72h, and the inhibition was time dependent.Treated by different concentrations of YM155for72h, the SK-N-SH cells were inhibited in proliferation at different levels. The inhibitory rates of10-500nmol/L YM155groups were significantly higher than that of the control group (P<0.05), and the inhibitory rate was dose dependent, namely it was increased with the increase of the concentration. SK-N-SH cells were inhibited in proliferation at different levels after treatment with100nmol/L of YM155for12,24,48and72h, and the cell inhibitory rates in YM155groups were significantly higher than that of the control group (P<0.05). In addition the cell inhibitory rate was time dependent, namely it was increases with the treating time of YM155prolonged.Treated by different concentrations of YM155for72h, the SK-N-SH cells were inhibited in cell apoptosis at different levels. The apoptosis rate of YM155groups were significantly higher than that of the control group (P<0.05), and the apoptosis rate was dose dependent, namely it was increased with the increase of the concentration (10-500nmol/L). The apoptosis rates of SK-N-SH cells were increased after treatment with100nmol/L of YM155for12,24,48and72h, which were significantly higher than that of the control group (P<0.05), showing time dependent. In cell migration and invasion experiment, the cell numbers of migration and invasion were significantly decreased after treatment with10-500nmol/L of YM155for12,24,48and72h compared with control group (P<0.05), and there showed a certain dose and time dependence.ConclusionYM155as a specific survivin inhibitor can inhibit the NB cell proliferation, induce apoptosis of tumor cells and reduce migration and invasion ability of tumor cells via down-regulating the expression of surviving in NB cells. Part Ⅲ:The effect of YM155on sensitization of neuroblastoma cells to cisplatin chemotherapy ObjectiveTo discuss the effects of combination of YM155and cisplatin on the proliferation and apoptosis of SK-N-SH and the influence of YM155on sensitivity to chemotherapy of cisplatin. MethodsSK-N-SH cells were treated with cisplatin, YM155and combination of YM155and cisplatin respectively. MTT assay was used to determine the cell growth inhibition after treatment. Flow cytometery was used to test the apoptosis rate. Western blot analysis was used to analyze the surviving protein expression of SK-N-SH cells after treatment. ResultsSK-N-SH cells were treated by cisplatin (0.05,1,2μmol/L) with YM155for12～72h, and the cell inhibitory rates were significantly increased, compared to the single cisplatin group, single cisplatin group and the blank control group (P<0.05). There was no significant difference (P>0.05) in inhibitory rate between SK-N-SH cells treated with different concentrations of cisplatin (0.05,1,2μmol/L) plus YM155 for72h. YM155can increase the sensitivity of SK-N-SH cells to cisplatin, and increase the inhibitory effect on cell growth, namely reducing the dosage of cisplatin can also achieve obvious anti-tumor effects.SK-N-SH cells were treated by cisplatin (0.05,1,2μmol/L) and YM155for12-72h, and the cell apoptosis rates were significantly increased compared with the single YM155group, single cisplatin group and the blank control group (P<0.05). There was no significant difference (P>0.05) in apoptosis rate between SK-N-SH cells treated by different concentration of cisplatin (0.05,1,2μmol/L) plus YM155groups. It suggests that YM155has obvious chemosensitization to cisplatin. It can increase the sensitivity of SK-N-SH cells to cisplatin induce cell apoptosis and reduce the dose of cisplatin.Survivin protein expressions were completely suppressed in single YM155group and YM155plus cisplatin group (0.05,1,2μmol/L). And there were no obvious changes of surviving protein expression in single cisplatin group and the blank control group. Single cisplatin plays a role on SK-N-SH cells but has no inhibition effects on surviving protein expression. ConclusionThe sensitization mechanism of YM155to cisplatin may be related to that YM155can specifically inhibit the surviving expression. Part Ⅳ:The sensitization effect of YM155to cisplatin in NB nude rat modelObjectiveTo establish the NB nude rat model in order to further discuss the effects of YM155on chemotherapy sensitivity of cisplatin.MethodsThe NB-burdened rat model was established. Cisplatin, YM155and YM155plus cisplatin were injected in the tumor and around tumor. The growth of tumor was observed for3weeks. And the volume of tumor was calculated. The rats were scarified, and then the tumor was weighed to calculate the inhibitory rate (IR). The tumor was tested by regular pathological examination. TUNEL method was used to observe cell apoptosis in tumor tissue. RT-PCR method and immunohistochemistry were used to detect survivin mRNA and protein expressions.ResultsAfter3-week medication, the nude rats of each group were scarified, and then the tumors were isolated and weighted to calculate IR. IRs of cisplatin group, YM155group, and cisplatin+YM155were33.76%,57.43%and92.34%respectively. The tumor weight of cisplatin+YM155group was obviously lighter than the other two groups, and the IR was significantly higher than the other two groups (P<0.05). During the three weeks of medication, tumor volume growth of cisplatin+YM155group was slowed markedly. The curve of growth was the lowest. And there were significant difference of tumor volume growth compared with YM155group, cisplatin group and the control group (P<0.05). Cisplatin and YM155have certain inhibition on tumor growth in NB nude rats. The combination of cisplatin and YM155can more effectively inhibit the tumor growth in NB nude rats, which is superior to cisplatin orYM155alone.The cell apoptosis in four groups was detected at the end of medication, and it was found that AI was highest in cisplatin+YM155group, followed by YM155group and cisplatin group. AI of cisplatin+YM155group was obviously higher than those of YM155group, cisplatin group and the control group (P<0.05), and AI of YM155group was obviously higher than that of cisplatin group. It suggested that cisplatin and YM155could induce apoptosis of NB, but the role of YM155was more prominent. YM155can more effectively induce tumor cell apoptosis in combined application, which was superior to cisplatin or YM155alone.Survivin mRNA expressions of nude rat transplantation tumors in YM155group and cisplatin+YM155group were decreased significantly, compared with the control group and cisplatin group (P<0.05), and there was no significant difference between the control group and cisplatin group (P>0.05). Immunohistochemical detection results showed that there were different levels of positive survivin protein expression tumor tissue of nude rats of each group. Its positive expression was mainly in the cytoplasm or cell membrane. There were large amount of strong positive survivin expression in the control group and cisplatin group. Positive survivin expression rates were significantly decreased in YM155group and cisplatin+YM155group. Intensity and area of positive survivin expression were significantly lower than those of the control group and cisplatin group.ConclusionCombined application of YM155and cisplatin may be an effective new method for the treatment of NB.