Study On BCL-XL Antisense Oligodeoxynucleotide Combination With ACNU In Human Glioblastoma

The occurrence and development of glioblastoma(GBM) is not only related to the abnormal hyperplasia of tumor cell,but also to the abnormal apoptosis of the tumor cells.An important mediator of both tumorigenesis and resistance to treatment involves suppression of apoptosis,It is speculated that there are multiple pathways leading to development of GBM tumors.Although the molecular pathways leading to the development of GBMs may differ,almost all GBMs are united by certain common neuropathologic and behavioral features.That is,nearly all GBMs tend to possess high invasive potential, high angiogenesis potential,active proliferation,and resistance to conventional cytotoxic treatments. long-term cure of GBM patients presently is elusive,the most difficult human tumors of all.Nimustine (ACNU) is the first chemotherapeutic agent to treat the GBM,it plays an important role in GBM's postoperation treatment.how to improve the best effects of ACNU in treatment GBM is our task.Bcl-xL has been cloned and characterized as an important member of the inhibitor of apoptosis(bcl-2) family of antiapoptotic proteins.Bcl-xL expression has been found to be undetectable in normal tissues.However,it has been found to be abundantly expressed in fetal tissues and a wide variety of human malignancies.By extending the lifespan of abnormal cells,accumulation of transforming mutations can occur.Its expression has been found to be of prognostic significance in several tumor types.from now on,the study about Bcl-xLis only concentrated on the tumor of hematopoietic,neuroblastoma and gastric system.Bcl-xLexpression patterns and ACNU combination with Bcl-xL-ASODN in GBMs have yet to be reported as of this writing.Our objective in this study was to determine whether the Bcl-xL expression and expression levels are of prognostic significance in human GBM,and to investigate the effects of apoptosis induced by ACNU combination with Bcl-xL-ASODN in human GBM,the methods of immunology and molecular biology were used to systematically study in vitro and in vivo.The following is a brief introduction to this research. Part one Study on the expression and prognostic significance of Bcl-xL mRNA and protein in human glioblastomaAIM:Our objective in this study was to determine the Bcl-xL mRNA and protein expression level in human GBM,and discussion whether Bcl-xL expression and expression levels are prognostic significance in human GBM.to offer an important theoretic foundation to clinical therapy of GBM.METHODS:Using the technology of immunohistochemistry SP,RT-PCR to detect the expression of protein and Bcl-xL mRNA in 20 cases of normal brain tissue,88cases of GBM tissue(include 35cases of recurrent GBM,16 cases of GBM treated by radiotherapy before surgery and 11 cases of GBM treated by chemotherapy before surgery) and A-172 cell line.immunohistochemistry SP and RT-PCR were performed in a blinded manner, without prior knowledge of clinical outcome.For the analysis between the relationship of Bcl-xL expression and survival,univariate and multivariate Cox models were fit with Bcl-xL level as a covariate.Survival curves were calculated using the Kaplan-Meier method.RESULTS:The results indicated that Bcl-xL was expressed highly not only in all glioblastoma tissues but also in A-172 cell line.That may play a important role in the resistance of glioblastoma to ACNU,and the down-regulation of Bcl-xL may contribute to ACNU-induced apoptosis in glioblastoma cells.Bcl-xL expression was detected in most patients,with 76(86.36%) of 88 tumors survivin positive,the survival of patients with Bcl-xL -negative tumors was significantly higher than that of patients with Bcl-xL -positive tumors(P＜0.01) The median survival of Bcl-xL -negative patients was 23.5 months, compared with 10.8 months for Bcl-xL -positive patients.Bcl-xL Expression Levels were significantly associated with reduced survival times(P＜0.05),CONCLUSION:First,Bcl-xL expression and expression levels were significantly associated with reduced survival in glioblastoma patients,we sought to then determine whether inhibition of apoptosis was one mechanism by which Bcl-xL expression enhanced aggressive behavior of glioblastoma.Bcl-xL positivity and protein expression levels are of significant prognostic value in human glioblastoma and seem to be associated with reduced apoptotic capacity of glioblastoma,second,In vitro data on corresponding primary glioma cell lines suggest that Bcl-xL expression may directly be mediating resistance to chemotherapy and radiation.This may,indeed,be a distinct possibility in the clinical setting, because all glioblastoma patients in this series were treated on identical regimens.The other possibility is that the reduced apoptotic capability of these cells secondary to Bcl-xL expression permitted the accumulation of additional mutations,which enhanced the invasive and angiogenic phenotype of these cells.Part two Study on therapeutic effects of ACNU combination with Bcl-xLASODN in A-172 cell line in vitroAIM:In order to investigate the inhibitory effects of Bcl-xL ASODNcombination with ACNU on proliferation of glioblastoma A-172 cells,and the role in down-regulation the expression of Bcl-xL mRNA and protein in vitro.METHODS:The methods of targeted therapy with cationic liposome-mediated ASODN,RT-PCR,MTT assay TR-PCR,Western blot,flow cytometry(FCM) and in situ cell death detection(TUNIL) were used for this study systemlly.RESULTS:The SDH activity of A-172 cells after Bcl-xL-ASODN and ACNU treatment were inhibited greatly.The best doses for ACNU and Bcl-xL ASODN to inhibit the growth of A-172 cells were 25ug/ml and 300nmol/L.the apoptosis rates were 40.82% and 37.88%.The inhibitory rates of A-172 cells for ACNU combination with Bcl-xL -ASODN were 88.63%,for Bcl-xL mRNA were 88.67%.but the level caspase 3 increasd for 430.41%and the apoptosis rates were 80.54%.The were significant difference between the treatment groups and the cell control groups,the vacuity control groups,the non-sense oligonucleotide group,the ASODN group and the ACNU group,respectively(P＜0.05).CONCLUSION:The methods of targeted therapy with cationic liposome-mediated ASODN is the best to put the aim gene into the cells.ACNU combination with Bcl-xL-ASODN can effect to inhibit the proliferation of glioblastoma A-172 cells,it must be kept in mind that Bcl-xL expression is probably one of several mechanisms that glioblastoma cells use to evade apoptosis.This suggests that suppression of apoptosis may be the major mechanism by which Bcl-xL enhances the malignant potential of glioblastomas.The high expression of Bcl-xL in glioblastoma enhanced the resistance of glioblastoma cells to ACNU.Bcl-xL-ASODN can degrade the target gene(Bcl-xL) expression and increase the chemotherapy sensitivity(ACNU).That Bcl-xL overexpression must be an another important effection for resistance to conventional chemotherapy in glioblastoma.it would offer an important theoretic foundation to clinical therapy of glioblastoma.Part three Study on therapeutic effects of ACNU combination with Bcl-xL-ASODN in glioblastoma in vivoAIM:In order to discuss the inhibitory effects of Bcl-xL ASODNcombination with ACNU on growth of human glioblastoma A-172 cells in nude mice,the role in down-regulated the expression of Bcl-xL mRNA and protein and inducing the apoptosis of human glioblastoma cells in vivo.METHODS:Animal test model of A-172 cell line in nude mices were formed by subcutaneous injection of A-172 cells with the dose of 1×10~7.we divided the animal models into five groups and obsevered the changes of transplantation tumour in nude mice treated with ACNU and Bcl-xL-ASODN and ACNU combination with Bcl-xL-ASODN by subcutaneous injection beside tumor.Once a day for fifteen days.The expression of Bcl-xL and caspase-3 in transplantation tumour after treatment were detected by Western blot and RT-PCR.The apoptosis rates of tumor cells in nude mice were determined by TUNEL.RESULTS:Animal test models of A-172 cell line in nude mices were successful.The average time of appearance transplantation tumour in nude mice was 6-8 days.The expression of BcI-Xl was down-regulated while the expression of caspase-3 up-regulated as well in vivo.In ACNU combination with Bcl-xL-ASODN group,the epression of Bcl-xL were down-regulated by 86.46%(mRNA) and 88.31%(protein) respectively.The expression of caspase-3 was up-regulated by 376.64%.There were significant difference between the test group and control group(P＜0.01).The apoptosis rates(TUNIL) of the control groups,the non-sense oligonucleotide group,the ASODN group and the ACNU group and ACNU combination with Bcl-xL-ASODN group were 2.25%,2.75%,36.75%,39.25%and 65.25% respectively,there were significant difference between the last three groups and the top two groups(P＜0.01).After 15d treatment,the volume increasing rate of human glioblastoma in nude mice in TRAIL combination with XIAP-ASODN group was -33.64%,there were significant difference between the last group and control group(P＜0.01).CONCLUSIONS:The growth of transplantation tumour can be inhibited by either ACNU or Bcl-xL-ASODN.But therapeutic effect was more significantly with treating by ACNU combination with Bcl-xL-ASODN.Bcl-xL-ASODN can degrade the target gene(Bcl-xL) and up-regulated the expression of caspase-3 and increase the chemotherapy sensitivity.ACNU combination with Bcl-xL-ASODN can inhibit the growth of glioblastoma cells effectively and increase apoptosis rate of glioblastoma cells greatly.The downregulation of Bcl-xL and The upregulation of caspase-3 contribute to the best therapeutic effect.Our research results showed According to this study,Bcl-xL was found to be over-expressed in most glioblastomas.Bcl-xL overexpression may play an important role in enhancing the malignant behavior of this tumor,increasing the barriers to apoptosis may enhance this transformation process.Bcl-xL expression may be one of several pathways leading to this high invasive potential,high angiogenesis potential,active proliferation,and resistance to conventional cytotoxic treatments.This is the first study on the prognostic significance of Bcl-xLexpression in human glioblastomas.It seems that Bcl-xLexpression patterns indeed may lend additional insight into the molecular pathogenesis of these tumors and may be important prognostic markers.The role of ACNU,Bcl-xL-ASODN and ACNU combination with Bcl-xL-ASODN in treating A-172 cells were tested by using the methods of targeted Therapy With Cationic liposome-Mediated ASODN,these results suggest that Bcl-xL antisense oligodeoxynucleotide combination with ACNU could effectively inhibit the proliferation of glioblastoma cells.The high expression of Bcl-xL in glioblastoma enhanced the resistance of glioblastoma cells to ACNU.Bcl-xL is an important factor for ACNU resistance and the suppression of Bcl-xL expression by the specific antisense oligodeoxynucleotides can significantly increase the sensitivity of glioblastoma cells to ACNU.it would offer an important theoretic foundation to clinical therapy of glioblastoma.