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AIM:To investigate the effects of Jiawei-Naotai formula (JWNTF) on ATF4/CHOP/Puma pathway in hippocampal neurons of ovariectomized female rats with cerebral ischemia.METHODS:The female rats were randomly divided into sham group, model group, JWNTF group and positive control group.The rats, expect in the sham group, were ovariectomized.The rats in each group were intragastric administration 11 days after ovariectomy.The rats in sham group and model group were given a gavage of 0.9％Na Cl, while the rats in other groups were administrated by corresponding therapy intragastrically for 3 d.The regional cerebral ischemia model was established by middle cerebral artery occlusion (MCAO) suture method 14 days after ovariectomy.The behaviors of the rats were evaluated 24 h after cerebral ischemia.The mRNA levels of Bax, Bcl-2 and caspase-3 were detected by RT-qPCR, and the protein expression of Bax, Bcl-2, caspase-3, ATF4, CHOP and Puma was determined by Western blot.RESULTS:Compared with sham group, the neurobehavioral scores significantly increased in other groups (P<0.05).Compared with model group, the neurobehavioral scores were significantly decreased in positive control group and JWNTF group (P<0.05).The protein expression of Bax, caspase-3, ATF4, CHOP and Puma, and the mRNA expression of Bax and caspase-3 in the hippocampus were much higher, and Bcl-2 was lower in model group than those in sham group (P<0.05).JWNTF significantly reduced the protein expression of Bax, caspase-3, ATF4 and CHOP, and the mRNA expression of Puma, Bax and caspase-3, and markedly increased the expression of Bcl-2 at mRNA and protein levels compared with model group.CONCLUSION:The JWNTF protects against brain damage induced by cerebral ischemia, which may be related to inhibitiing the expression of ATF4/CHOP/Puma pathway-related molecules at mRNA and protein levels.
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Objective To study the expression of apoptosis-associated genes and the role of apoptosis in acute radiation-induced liver injury. Methods A mouse radiation model, which was irradiated with 60Co γ ray, was established in this study, and the pathological changes were observed by light and electron microscopy for 48 hours. Western blotting analysis was used to measure the expression of some proteins (Caspase 3,Caspase 8, Bcl-2, Bcl-xL, Bax, Bad, PUMA, Slug) and TUNEL assay was employed to examine cell apoptosis in mouse liver. Results Degeneration and apoptosis were found in the liver at 4 h after irradiation and necrosis occurred at 12 h after irradiation. The peak of apoptosis with activation of Caspase 3 in liver was detected during 24-48 h after irradiation. Bcl-2 protein expression was up-regulated during 4-24 h after irradiation, then was down-regulated at 48 h; PUMA protein was up-regulated during 4-48 h after irradiation; Bcl-xL and Bad protein were up-regulated during 6-48 h after irradiation; and Bax and Slug protein were up-regulated only at 12 h after irradiation. Conclusion Up-regulation of BH3-only members (PUMA and Bad) after irradiation may be associated with the increase of apoptotic cells in the liver.
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Objective To explore differences of drug resistance of temozolomide(TMZ) on different CD133 immune prototype of glioma cells and study on the changes of their sensitivity to TMZ through increased PUMA. Methods CD133+-U87MG cells sorted by CD133 magnetic beads were cultured in serum-free stem cell medium respectively. The cells were infected with recombinant adenovirus, Ad-PUMA, diluted in cell culture medium with or without TMZ intervention.The inhibitory rate of cell proliferation was detected by MTT assay and 50 ％ inhibition concentration of TMZ was calculated. Apoptosis rates of CD133+-U87MG cells were assessed by flow cytometry (FCM) before and after intervention of exogenous PUMA and TMZ.Results The TMZ IC50 values of CD133+glioma cells were higher than that of CD133- glioma cells. There were significant differences in apoptosis rate between CD133+ glioma cell and CD133- glioma cell (all P＜0.05).Conclusion AdPUMA joint TMZ can promote glioma stem cells apoptosis, thus improve the sensitivity to chemotherapy of glioma.
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Objective To investigate the inhibitive effects of Ad-PUMA combined with temozolomide on human glioblastoma cells growth in vivo experiments. Methods The nude mouse model with human glioblastoma cells subcutaneous transplantation was established. The mice were randomly divided into 4 groups to receive subcutaneous injection at the 14th day separately with: Normal saline 100 μl (control, n=8), Ad-PUMA 2×108 pfu/100 μl (PUMA group, n=8), 10 mg/kg TMZ (TMZ group, n=8) and 2×108 pfu/100 μl Ad-PUMA + 10 mg/kg TMZ (combined group, n=8). Mice were killed after 20 days treatment.Tumor volume, inhibition rates and apoptotic index (AI) were measured, meanwhile, apoptotic tumor cells were detected by TUNEL technology respectively. The expression of MGMT mRNA and MGMT protein were revealed by the methods of RT-PCR and Western blot. Results According to the order: control group, AdPUMA 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 respectively (P＜0.05); the inhibitive rates were 0, 28.5 %, 42.1%, 73.6 % respectively and AI were (2.0±1.2) %, (11.4±2.6) %, (7.6±3.2) %, (20.6±8.6) % (P＜0.05). The results of Western blot and RT-PCR showed that MGMT mRNA and MGMT protein levels in TMZ group were higher than other groups (all P＜0.01). Conclusion Ad-PUMA combined with TMZ greatly enhances the sensitivity of human glioblastoma cells to TMZ and could effectively inhibit the proliferation and promote the apeptosis of glioblastoma cells, its mechanism was probably related Ad-PUMA promote apoptosis and inhibit MGMT expression.
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Objective: To observe the expression of PUMA, P53, Bax and Bcl-2 protein in pancreatic ductal adenocarcinoma (PDA), and to investigate the role of PUMA in tumorigenesis of PDA and its relationship with P53, Bax and Bcl-2 protein expression. Methods: The expression of PUMA, P53, Bax and Bcl-2 protein was examined by immunohistochemical EnVision method in 65 PDA tissues and the corresponding adjacent tissues. Results: The positive rate of PUMA protein expression was 30.8% (20/65) in the PDA tissues, which was significantly lower than that in the corresponding adjacent tissues (49.2%[32/65], P<0.05). The expression of PUMA protein was significantly correlated with the tumor size and lymph node metastasis (P<0.05), but not with patient's age, sex, tumor location, differentiation degree, tumor stage or neural invasion. PUMA expression was negatively correlated with P53 and Bcl-2 expression (P = 0.019, P = 0.015), but was not correlated with Bax expression in PDA tissues. Conclusion: Decreased PUMA protein expression is correlated with tumor size and lymph node metastasis in PDA patients, suggesting that PUMA protein might be involved in the tumorigenesis and development of PDA. Detection of PUMA protein expression may be used for predicting the prognoses of PDA patients.
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Objective To study the effect of PUMA gene mediated by recombinant adenovirus vector combined with radiation on the pancreatic carcinoma. Methods The PANC-1 cells were infected with Ad-PUMA (MOI = 10, 50 and 100, respectively) for 48 h. The expression of PUMA mRNA and protein was detected by RT-PCR and Western blot, respectively. PANC-1 cells were divided into 4 groups: control group, transfection group, irradiation group and combined treatment group. The cell growth inhibition rate and apoptotic rate of PANC-1 cells were assessed by MTT assay and flow cytometry. Human pancreatic carcinomas were transplanted subcutaneously in nude mice, which were randomized into 4 groups: control group, transfection group, irradiation group and combined treatment group. Tumor growth rate and apoptotie index at different time points were recorded in 35 days. Results The expression of PUMA mRNA and protein was increased with the increase of MOI of Ad-PUMA, which was does-dependant (MOI = 10, mRNA = 0.46 ± 0.02, protein = 0. 75 ±0.09;MOI=50, mRNA= 1.12±0.09, protein = 1.01 ±0.18;MOI= 100, mRNA= 1.50±0.08, protein=1.80 ± 0.15 ;P < 0.05). The proliferation of PANC-1 cells was suppressed significantly when transfected by Ad-PUMA in a dose-dependent manner(r = -0.986 55), which was more significant combined with radiation (r = -0.971 26, P < 0.05). Meanwhile, the apoptotie rate was increased in the same manner [for pre- and post-irradiation,which was (45.4 ± 5.26) % and (73.2 ± 6.62) %, respectively, P < 0.05]. From 7 to 35 d after PUMA gene transfection and radiotherapy, the tumor growth was significantly slower than those of irradiation group, transfection group and control group [35 d after therapy, the volume of tumor was (19.82 ± 6.45)mm3 ,(39.5 ± 9.23)mm3 , (33.6 ±3 10.3)mm3 and (52.0 ± 11.43)mm3 , respectively, P < 0.05]. And the apoptotic index was increased in the same manner (AI = 0.43 ± 0.05, 0.29 ± 0.10, 0.24 ± 0.05 and 0.00 ± 0.00, respectively, P < 0.05). Conclusions Recombinant adenoviral-mediated PUMA gene combined with irradiation could increase the cell-killing effect on pancreatic carcinoma. It is better than that of either one kind of therapy.
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objective To construct the recombinant adenovirus containing p53 up-regulated modulator of apoptosis(Ad-PUMA)and investigate its growth inhibition effect on pancreatic callCer cells in vitro and in vivo.Methodls Ad-Easy system was used to construct Ad-PUMA by recombination in E.coli.The virus was Dackaged in 293 cells and subsequently identified valid.The AsPC-1 cells were infected with AdPUMA.Before and after Ad-PUMA infection,the expression of PUMA protein wag investigated by western blot,the inhibition rate of AsPC-1 cells was examined by MTY assay.The in vivo tumor suppressive effect was detected in nude mice with human AsPC-1 xenograft.PUMA protein and the apoptosis of AsPC-1 xenograft were detected by western blot and TUNEL(terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling)method.Results In vitro,the expression of PUMA protein was increased with titer of Ad-PUMA,the proliferation of AsPC.1 cells were suppressed,significantly,and the effect was in a viral dose-dependent nlanner.In vivo,the growth in nude mice of AsPC-1 infected with Ad-PUMA was significantly inhibited with an inhibition rate of 44.2%.The expression of PUMA was significantly up-regulated,and the apoptosis index wa8 significantiv increased in tumor after Ad-PUMA infection as determined by western blot and TUNEL.Conclusion The expression of PUMA call inhibit the proliferation of pancreatic cancer in vitro and in vivo,and may be used 88 a potential tool for cancer therapy
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Objective:To investigate whether PUMA gene transfection can increase sensitivity of pancreatic cancer cells (PC) to 5-FU-induced apoptosis. Methods: PUMA-pCEP4 containing full length PUMA cDNA or pCEP4 was transfected into human pancreatic cancer cell line AsPC-1 by lipofectamine transfection, G418 selection was used to select positive cells. AsPC-1, AsPC-1/PUMA and AsPC-1/pCEP4 cells were separately treated with serial concentrations of 5-FU(0.01-100 ?mol/L). MTT assay was used to determine the cell survival rate in each group and IC50 of 5-FU was calculated. TUNEL,FCM and DNA ladder observation were employed to study cell apoptosis. Western blotting was performed to detect the expression of PUMA protein. Results: The 5-FU IC50 values of AsPC-1, AsPC-1/PUMA and AsPC-1/pCEP4 cells were (12?1.9)?mol/L,(1.6?0.4)?mol/L and (10.4?1.6) ?mol/L, respectively, with the sensitivity of AsPC-1/PUMA cells increased by 7.5 folds. 5-FU induced cell apoptosis of AsPC-1 cells in a dose-dependent manner, with the apoptosis of AsPC-1/PUMA cells more prominent than those of AsPC-1 and AsPC-1/pCEP4 cells. Low concentration of 5-FU (0.1 ?mol/L) induced few apoptosis of AsPC-1/pCEP4 cells([1.14?0.28]%) and AsPC-1 cells ([0.9?0.23]%), and induced apoptosis in AsPC-1/PUMA cells([6.47?1.42]%). High concentration of 5-FU (1.0 ?mol/L) induced apoptosis in all groups, with that in AsPC-1/PUMA cells([34.54?9.36]%) significantly higher than those in AsPC-1/pCEP4 cells([15.8?5.15]%) and AsPC-1 cells ([12.8?3.74]%, both P