The Expression Of PUMA In Human Glioma Cell And Its Molecular Mechanism Of PUMA Through Enhanced Chemosensitivity Against Glioma Cells

BACKGROUNDThe reversion of glioma drug-resistance become the important strategy for treatment glioma.But the understanding of mechanisms of glioma drug-resistance occurs remains a matter ofdebate. There is a consensus between glioma drug resistance and high expression drug-resistancegenes. In certain physiological or pathological conditions, apoptosis is programmed cell deathprocess. Apoptosis regulating biological tissue in maintaining steady and individual growth playsan important role. Since most chemotherapy drugs by inducing tumor cell apoptosis is ratherplay a role, so the cell apoptosis pathways of reversion of tumor drug-resistance is anotherimportant mechanism. The tumor drug-resistance research requires that we attach importance tothe apoptosis control mechanism of tumor cell. Looking for key genes is to enhance research inan effective way between apoptosis pathways and tumor drug-resistance. The project is to raisethe p53 up-regulated modulator of apoptosis (PUMA) as the breakthrough point, use of genetransfection, RT - PCR, the Western blot methods, from in vivo and in vitro studies, to explorethe mechanism of PUMA enhance glioma cell chemotherapy drug sensitivity. PUMA highexpression or the PUMA activated promote cell apoptosis and reverse gliomas drug-resistance.PUMA probable control mechanisms: (1) The PUMA lower expression avoid chemotherapydrugs through affectting cell growth cycle; (2) PUMA maintaining cell antiapoptotic andpromote apoptosis system balance, its function lack leads to apoptosis system imbalance to causedrug-resistant; (3) Drug-resistant molecular regulation abnormalities would be closely relatedwith drug resistance. This study try to look for the root of glioma drug resistance from cellapoptosis level, prove the regulation pathways mode of PUMA enhancing glioma drugsensitivity and provide a new approach to prevent and control glioma chemotherapy drugresistance, (4) Activation in G0 / G1 phase of gliomas stem cells, make its into chemotherapydrugs, leading to the effective period within cells apoptosis triggeredOBJECTIVE1. To study the expression of PUMA, Bcl-2 and Bax in human glioma cell and the relation among PUMA, Bcl-2 and Bax.2. To investigate the effect of PUMA on the sensitivity of human glioma U87MG cells totemozolomide and its related mechanisms3. To explore the inhibitive effects of Ad-PUMA combined with temozolomide on humanglioma cells growth in vivo experiments4. To investigate the differences of stem cells based on the glioma CD133 immune prototyperesistant to the effects of drug resistance PUMA..METHODS1. The expression of PUMA, Bcl-2 and Bax protein were examined by Western-blot analysis in52 cases of human glioma cell and 10 cases of normal brain tissues.2. U87MG cells under culture were divided into the normal control group, mock group andexperiment group. After 24 hours 0f culture, the mock and experiment group weretransfected with mock vector (Ad-△BH3) and the recombinant ademnrus carrying PUMA(Ad-PUMA)respectively at a multiplication of infection (MOI) of 50. The proliferationactivity of cells and IC50 were detected by MTT assay, the apoptosis effect and the changeof cell cycle determined by Hoechst stain and flow cytometry (FCM) technologyrespectively．The expression of related proteins was revealed by the method of Western blot.3. The nude mouse model with human glioma cells subcutaneous transplantation wasestablished. The mice were randomly divided into 4 groups to receive subcutaneousinjection at the 14th day separately with: Normal saline (control, n=8), Ad-PUMA2×108pfu/100μl (PUMA group, n=8), 10mg/kg TMZ (TMZ group, n=8) and 2×108pfu/100μlAd-PUMA + 10mg/kg TMZ (combined group, n=8). Mice were killed after 20daystreatment. Tumor volume, inhibition rates and apoptotic index (AI) were measured,meanwhile, apoptotic tumor cells were detected by TUNEL technology respectively．Theexpression of MGMT mRNA and MGMT protein were revealed by the methods of RT-PCRand Western blot.4. Sorting out CD133 + U87 cells, application CD133-WB, RT - PCR, gene carrying flowcytometric analysis method observation exogenous PUMA, TMZ before and after theintervention of cells to grow two changesRESULTS1. There were significant differences in the expression of PUMA, Bcl-2 and Bax protein between the control and gradeⅡ, gradeⅢor gradeⅣ(all P<0.01). There was no significantdifference in the expression of those protein between the control and gradeⅠ(P>0.05).There were correlations between the expression of PUMA, Bax or Bcl-2 and tumordifferentiation grade in human glioma (rPUMA=-0.925, rBax =-0.931, rBcl-2=0.945, allP<0.001). There were correlations between the expression of PUMA and Bax, Bcl-2 inhuman glioma (r1=0.963,r2=-0.937,P＜0.001).2. Exotic PUMA gene was expressed in U87MG cells transfected with Ad-PUMA, whichcould inhibit the proliferation of U87MG cells．The inhibition rate of proliferation 24, 48,72h after transfection was 17.3%, 35.6%, 43.3% and apoptosis rate was 20.3%, 31.4%,45.4% respectively. Results: The TMZ IC50 values of PBS group, Ad-PUMA and Ad-△BH3group cells were (15±1.9), (2.3±0.14) and (13.4±1.6)μmol/L respectively, with thesensitivity to the TMZ of Ad-PUMA group cells increased by 7.0 folds. PUMAoverexpressing U87MG cells showed the DNA synthesis was inhibited and arrested in G2phrase. The results of Western blot showed that after 72 hotms of transfection the PUMAand Bax protein showed increased expression (P<0.01). There was no significant differencein the expression of P53 among these groups (P>0.05).3. According to the order: control group, Ad-PUMA group, TMZ group, combined group,tumor volumes were (3.68±0.09),(2.63±0.13),(2.13±0.07),(0.97±0.02) cm3 in 4 groupsrespectively; the inhibitive rates were 0, 28.5%, 42.1%, 73.6% respectively and the apoptoticindexes (AI) were (2.0±1.2) %, (11.4±2.6) %, (7.6±3.2) %, (20.6±8.6) %. The results ofWestern blot and RT-PCR showed that MGMT mRNA and MGMT protein levels in TMZgroup were higher than other groups (all P<0.01).4. The TMZ IC50 values of CD133 +glioma stem cell are higher than that of CD133- gliomastem cells. There was significant difference in apoptosis rate between CD133 +glioma stemcell and CD133- glioma stem cell. (all P < 0.05)CONCLUSION1. The expression of PUMA may be an important prognostic marker for human glioma. Theexpression of PUMA protein was correlated with tumor differentiation grade, could be usedas a valuable marker for the prognosis of human glioma.2. PUMA gene transfection greatly enhances the sensitivity of U87MG cells to TMZ-inducedapoptosis and can effectively inhibit the proliferation and promote the apoptosis of U87MG cells. The mechanism of apoptosis mainly relates to induce cell cycle G2 arrest and apoptosisin vitro.3. Ad-PUMA combined with TMZ greatly enhances the sensitivity of human glioma cells toTMZ and could effectively inhibit the proliferation and promote the apoptosis of gliomacells, its mechanism is probably related Ad-PUMA promote apoptosis and inhibit MGMTexpression.4. Ad - PUMA joint TMZ can promote glioma stem cell apoptosis, thus improving thesensitivity to chemotherapy gliomas.