| ObjectiveGlioma is one of the common tumors in central nervous system,which accounts for approximately 35.12%～61.10%of all intracranial tumor.It has such characters as high morbidity,high relapse rate,high mortality and low cure rate.Although the main medical technology such as operation,radiotherapy and chemotherapy has been used for cancer treatments,the prognosis of patients with glioma has remained dismal.As nearly half of gliomas are histologically malignant and most of gliomas are infiltrative growth,patients still cannot avoid relapse even when the fully excision of tumor under the microscope and imaging is performed.Therefore,new diagnostic and therapeutic strategies are required in order to control this devastating disease. Chemotherapy is still the main protocols for glioma other than surgery and radiation.But there are still some barriers for this therapy,mainly because of the adverse side effects of the anticancer agents and the development of chemoresistance. So it requires sequential use of a number of therapeutic agents in an attempt to increase efficacy while maintaining anticancer drugs tolerable toxicity.Studies of molecular biology for tumor are key issue to get insight into the mechanism underlying the pathogenesis of glioma and thus,lead to more effective methods.It is might be of important value to seek for target genes which could enhance chemosensitivity of glioma.Programmed cell death 5(PDCD5),also designated as TF-1 cell apoptosis-related gene-19(TFAR19),is a new identified gene which facilitates apoptosis triggered by certain stimuli.Recent evidences indicate that the expression of PDCD5 protein is down-regulated in some human tumors such as ovarian cancer, gastric cancer,and renal clear cell carcinoma.How about the status of PDCD5 in gliomas and what an effect of PDCD5 is on the progression of glioma? Whether PDCD5 could be one of valuable target genes for glioma chemosensitivity? All these problems remain to be investigated.In this study,we analyzed the relationship between PDCD5 expression and glioma progression according to four main points:1.The status of PDCD5 expression in human primary gliomas and its clinical significance2.The effect of PDCD5 expression on the growth and chemosensitivity of glioma derived cell lines3.The mechanism for PDCD5 influencing chemosensitivity4.The relationship between PDCD5 and other apoptosis related genes(such as PDCD4)Method1.The expression status and clinical significance of PDCD5 in human primary gliomas(1) Case collection:a total of 88 glioma specimens including 30 frozen and 58 paraffin-embedded tissues obtained from patients aged between 30 and 60 years who underwent surgery at the department of Neurosurgery in Qilu Hospital of Shandong University during year 2003-2006.None of the patient had received adjuvant treatments such as radiotherapy or chemotherapy treatment prior to surgery in order to eliminate their effects on gene expression.Also 3 normal nervous tissues were excised from tumor-adjacent sites.(2) The expression of PDCD5 mRNA and protein in 88 glioma tissues and glioma derived cell lines U251 and U87 were detected by RT-PCR and western blot.(3) The correlation between PDCD5 expression and the pathological characteristics was analyzed byχ2 test.Cumulative survival time was calculated by the Kaplan-Meier method and analyzed by the log-rank test.P<0.05 was considered statistically significant.The calculation was performed using the SPSS statistical software.2.The effect of exogenous PDCD5 overexpression or silenced expression on the growth and chemosensitivity of glioma cell lines1.To determine the effect of PDCD5 on the growth and survival of glioma cells, a recombinant plasmid carrying the full-length PDCD5 cDNA transfected into U87 glioma cells which with low expression of PDCD5.Then transfected cells were selected by G418.The analysis of PDCD5 expression after transfection was by RT-PCR,Western blot and immunocytochemistry.2.The morphology detection of the glioma cell line after PDCD5 transfection by microscope.The growth curve was detected by viable cell counts.3.Analysis of the chemosensitivity of PDCD5 transfected cells and controlled cells at different concentration of chemotherapeutic agents(such as cisplatin) by MTT assay.4.The morphology detection for the growth of transfected cells treated with long-term low concentration cisplatin by microscope.5.Synthesis of siRNA targeted PDCD5(HPLC purity >95%) and non-silencing control siRNA.Transfected U251 cells and U87 cells with siRNA,then examined the expression of PDCD5 by RT-PCR and Western blot after 48 hours.6.The morphology detection for the growth of cells transfected with siRNA,and viable cell counts for the proliferation of glioma cells.7.Analysis of the chemosensitivity of U87 cells and U251 cells with silenced expression of PDCD5 by MTT assay.3.The mechanism for PDCD5 enhancing CDDP chemosensitivity on glioma derived cell line(1) The morphology apoptosis was analyzed by Hoechst and late apoptosis was detected by TUNEL assay.(2) The apoptosis pathway was tested by Western blot of cleaved caspase-3, cleaved caspase-8,cleaved caspase-9,Bcl-2 and Bax.4.The facilitating effect of PDCD5 on apoptosis related gene PDCD4(1) Transfected glioma cell line(with high expression of PDCD5 or low expression of PDCD5) with PDCD4 plasmid or empty vector,and then examined PDCD4 expression by RT-PCR or Western blot.(2) The morphology detection for the growth of glioma cells transfected with PDCD4 or empty vector by fluorescence microscope.(3) Detection of changes of apoptosis related protein Bcl-2,Bax.Result1.The expression of PDCD5 in human primary gliomas and its clinical significance(1) Expression of PDCD5 at mRNA and protein levels in glioma cell linesWe first examined the expression of PDCD5 in glioma derived cell lines U87 and U251 by semi-quantitative RT-PCR and Western blot.The result showed that U87 cells with high expression of PDCD5 while U251 cells with low expression of PDCD5,indicating that PDCD5 may involve in the development of glioma.(2) The expression of PDCD5 in human primary gliomasIn order to explore the role of PDCD5,we then collected samples of human primary gliomas.PDCD5 expression at mRNA and protein levels in primary gliomas was also detected by semi-quantitative RT-PCR,Western blot and immunohistochemi -stry.It showed that adjacent tissues of gliomas expressed high levels of PDCD5 both at mRNA and protein levels.Decreased expression of PDCD5 at mRNA level was detected in 16 fresh samples,the decreased rated was 53.33%(16/30);The expression of PDCD5 at protein level also decreased,about 83.33%(25/30),which consistent with the result of mRNA.In order to validate the decreased expression of PDCD5 in gliomas,we explored the status and location of PDCD5 expression by imunohistochemistry in all 88 samples.The result also showed the consistency with mRNA and protein result: adjacent tissues expressed high levels of PDCD5,while tumor tissues expressed low levels of PDCD5.(3) Relationship between PDCD5 expression and pathological characteristics for patients with gliomasTo examine the prognostic value of PDCD5 expression in primary gliomas,we studied a large cohort of patients with this disease.No significant correlation between PDCD5 expression and age,gender and histological type.However,the expression of PDCD5 correlated significantly with the pathological grade(p<0.001).Within the same histological glioma(astrocytoma) type,the rate of reduced expression of PDCD5 was higher in high-grade gliomas(â…¢-â…£) than that in low-grade gliomas(â… -â…¡) (86.00%,43/50 vs.45.16%,14/31).Furthermore,we analyzed the association of the overall survival of the astrocytoma patients with low or high PDCD5 expression. However,as judged by the Kaplan-Meier analysis,PDCD5 expression had no statistically significant impact on the prognosis of astrocytoma patients(p>0.1). Similarly,no prognostic value of PDCD5 expression can be detected for patients with same grades of gliomas.2.The effect of exogenous PDCD5 expression on growth and chemosensitivity of glioma derived cell lineTo further determine effect of PDCD5 expression on glioma progress,we overexpressed PDCD5 in glioma cell line U87 by transfection with exogenous PDCD5 and analyzed its effect on tumor growth.(1) The expression of PDCD5 in glioma cells stably transfected with PDCD5 recombinant plasmidHigh level expression of PDCD5 was detected in U87 cells transfected with PDCD5 compared with untreated control or mock-transfected groups by RT-PCR, Western blot and immunocytochemistry.(2) The exogenous expression of PDCD5 has no influence on morphology and growth of glioma cellsThe morphology of U87 cells transfected with PDCD5 showed little variation compared with untreated control or empty vector transfected groups,also we found no significance for the growth curve among the U87 cells transfected with PDCD5, untreated control or empty vector transfected groups(p>0.05).(3) The exogenous overexpression of PDCD5 enhances the chemosensitivity of glioma derived cell lineWe tested the cell viability of U87 cells transfected with PDCD5 or MOCK control treated with different concentration of CDDP by MTT assay.The cell viability of PDCD5 overexpressed U87 cells was lower than that of mock control.The deference had statistics significance when the concentration of CDDP was 12.5μg/mlor 25μg/ml.We also examined effect of PDCD5 on chemosensitivity of other chemotherapeutic drugs.Consistent with CDDP,the chemosensitivity of U87 cells transfected with PDCD5 for CBDCA and VCR was higher than mock control,having statistics significance,while no significant changes was detected in U87 cells transfected with PDCD5 when treated by VP-16.We then treated the U87 cells with low concentration of CDDP,lower viable cell counts was detected in PDCD5 overexpressed compared with mock control.Also the morphology had the differentiation tendency toward normal glial cells. Remarkably,PDCD5 transfection significantly diminished the capacity of these cells to form colonies treated with low concentration CDDP.3.The effect of PDCD5 silenced expression on growth and chemosensitivity of glioma derived cell line(1) Silenced expression of PDCD5 in glioma cell lines both at mRNA and protein levelsNon-silencing siRNA or the two candidate siRNAs targeted PDCD5 (siPDCD5-1,-2) were transfected into PDCD5 transfected U87 cells or U251 cells.48 hours after transfection,both PDCD5 mRNA and protein levels were significantly decreased in those cells that were transfected with siPDCD5-1 or siPDCD5-2,as assessed by RT-PCR and Western blot.Effect of siPDCD5-2 was more obvious,so we selected it for future experiment. (2) Silenced expression of PDCD5 has no influence on the growth of glioma derived cell lineThe morphology of glioma cells transfected with siPDCD5 showed little variation compared with non-silencing or untreated groups,also we found no significance for the proliferation among the glioma cells transfected with siPDCD5 (p>0.05).(3) Silenced expression of PDCD5 decreases the chemosensitivity of glioma cell lines to CDDPTransfected U87 cells with exogenous PDCD5 expression with siPDCD5 or non-silencing siRNA and then subjected to CDDP and analyzed chemosensitivity by MTT assay.The viable cell rates of U87 cells transfected with PDCD5 were greatly restored at the concentration of CDDP at 10μg/ml or 12.5μg/ml.The viable cell rates of U251 cells also increased when transfected with siRNA targeted PDCD5,which means the chemosensitivity to CDDP decreased.4.The mechanism for PDCD5 enhancing chemosensitivity of glioma cells on CDDP(1) The effect of exogenous PDCD5 overexpression on apoptosisTo furthermore determine the mechanism of PDCD5 enhancing chemosensitivity of CDDP,we tested the apoptosis of U87 cells transfected with PDCD5 or mock control.Hoechst assay revealed that PDCD5-transfected glioma cells appeared conspicuous nuclear condense and underwent more apoptosis than the mock control.Furthermore,TUNEL analysis also clearly showed that PDCD5-transfected glioma cells appeared underwent more apoptosis(44.22%) than the mock control (0.16%) at 25μg/ml of CDDP.Similar results were achieved when at 50μg/ml of CDDP.(2) The influence of exogenous PDCD5 overexpression on the activity of apoptosis related proteinIn order to further expatiate the mechanism for enhanced apoptosis by combination with PDCD5 and CDDP,we further detected the cleavage of caspase-3 caspase-8 and caspase-9,also protein of Bcl-2 and Bax was determined by Western blot.Compared to mock control,Western blot analysis showed that the cleavage of caspase-3,caspase-9 increased greatly in PDCD5 transfected cells compared with controlled groups,whereas caspase-8 showed little difference among the three groups. Expression of Bcl-2 protein also showed obvious decreased in PDCD5 transfected cells,while Bax had no remarkable difference,the ratio of Bcl-2/Bax decreased obviously in PDCD5 stably transfected group.5.The facilitating effect of PDCD5 on apoptosis related gene PDCD4(1) The effect of PDCD5 and PDCD4 expression on the prognosis of glioma patientsWe further studied the role of PDCD5,PDCD4 in the development of glioma in order to explore their relationship.We found that in PDCD4positive group,glioma patients with high PDCD5 expression had better prognosis than that with low expression of PDCD5(p<0.05),while in the PDCD4negative groups,glioma patients with high PDCD5 had no better prognosis than that with low expression of PDCD5. Further in the PDCD5high groups,glioma patients with PDCD4 expression had better prognosis than that with no expression of PDCD4(p<0.05),meanwhile in the PDCD5low Wgroups,glioma patients with PDCD4 expression had no better prognosis than that with no expression of PDCD4,these result suggested that PDCD5 may facilitate the role of PDCD4.(2) The effect of PDCD5 and PDCD4 expression on the growth of glioma cells in vitroPDCD5 is a pro-apoptosis factor while PDCD4 could induce the apoptosis,but whether they had potential interaction remained unknown.Preliminary study was done in vitro.PDCD4 was transfected into U87 cells with high or low expression of PDCD5,and then the morphology was observed by fluorescence microscope.In PDCD5low group,the cells with co-expression of PDCD4 had poor status than the cells with no expression of PDCD4;In PDCD5highgroup,the cells with co-expression of PDCD4 had poor status than the cells with no expression of PDCD4,with cells turning round,shrinkage.Viable cell counts showed that the group with co-expression of PDCD4 and high PDCD5 had slower growth than another three groups,which suggested that PDCD5 and PDCD4 coeffect could enhance the inhibition of glioma cells.(3) The impact of expression of PDCD5 and PDCD4 on the apoptosis related proteinThe balance of proapoptotic and antiapoptotic Bcl-2 family members regulates the susceptibility of cells to apoptosis.The change of the ratio of Bcl-2/Bax may mean the mitochondrial dysfunction with alterations in the permeability transition pore,the release of cytochrome c and apoptosis taken place.So we further detected the expression of Bcl-2 and Bax.By semi-quantitative analysis,the value of Bcl-2/Bax decreased more obviously in PDCD5highPDCD4 psitivegroup of cells compared with another three groups.Conclusion1.Expression of PDCD5 was decreased in primary human glioma tissues and the decreased expression of PDCD5 correlated significantly with the high grade (â…¢-â…£) of astrocytoma.2.We demonstrated that the expression of PDCD5 could enhance the chemosensitivity of CDDP on glioma cell lines from two aspects.3.Exogenous PDCD5 expression enhanced the chemosensitivity of glioma cell line on CDDP mainly by promoting apoptosis of mitochondrial pathway.4.PDCD5 could enhance the inhibition of PDCD4 to glioma cells in vitro.Originality1.We demonstrate,for the first time,that decreased expression of PDCD5 is a frequent event in human glioma and that the expression of PDCD5 significantly correlated with the grade of astrocytomas,which suggested that PDCD5 may involve in the pathological process of glioma.2.We studied the expression of PDCD5 and chemosensitivity in gliomas for the first time.(1) PDCD5 overexpression enhanced the chemosensitivity of glioma cell line to CDDP,while silenced expression of PDCD5 decreased the chemosensitivity,which indicated the potential value of PDCD5 as a sensitizer in glioma treatment. (2)Exogenous PDCD5 overexpression mainly by promoting apoptosis of mitochondrial pathway to exert its role on chemosensitivity,which provide first hand data for the gene therapy targeted PDCD5 for glioma.3.For the first time we found that PDCD5 and PDCD4 co-expression was valuable to the prognosis of glioma patients and better inhibited the glioma cells growth in vitro,which predict the potential role of PDCD5 and PDCD4 for prognosis and outcome of glioma patients.Limitations of this study1.The promoting effect of PDCD5 on CDDP chemosensitivity in vivo and potential clinical application value needs further to be demonstrated.2.The mechanism of decreased PDCD5 expression in gliomas needs further study.
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