Changes and significance of Fas and cFLIP in apoptosis of human rhabdomyosarcoma cells induced by combination of TRAIL and cisplatin

AIM: To investigate the synergistic induction of apoptosis in rhabdomyosarcoma cells by the combination of TRAIL or TRAIL gene with cisplatin. METHODS: Rhabdomyosarcoma cells were treated with TRAIL, Ad／GT-TRAIL, cisplatin, respectively or the combination for 3 days. The cytotoxicity was observed by MTT assay. The apoptotic rates and the expression rates of Fas protein were measured by flow cytometry (FCM). The expression of cFLIP mRNA was determined by RT-PCR. RESULTS: Rhabdomyosarcoma cells were treated with Ad／ GT-TRAIL and TRAIL (100.0 μg/L), the cytotoxicity index were 52.5% and 43.5%, the percentage of apoptotic cells were 12.95% and 10.26%, respectively. Combined with cisplatin, the cytotoxicity index and the percentage of apoptotic cells were increased significantly (P<0.05). The expression of Fas protein in rhabdomyosarcoma cells was up-regulated and the expression of cFLIP was down-regulated with cisplatin, which were paralleled by the apoptotic rates. CONCLUSION: Combinatiion of Ad／GT-TRAIL or TRAIL and cisplatin has synergistic apoptosis-inducing effects on rhabdomyosacoma cells.     

Plumbagin enhances TRAIL-induced apoptosis in Kasumi-1 cells

AIM: To investigate the effect of plumbagin and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the apoptosis of leukemic Kasumi-1 cells. METHODS: Kasumi-1 cells were treated with plumbagin alone, recombinant soluble TRAIL(rsTRAIL) alone or the combination of plumbagin with rsTRAIL to induce apoptosis. The cell proliferation was analyzed by CCK-8 assay. Apoptosis was determined by flow cytometry with AnnexinⅤ/PI double staining and TUNEL staining. The expression of DR4 and DR5 at mRNA level was measured by real-time PCR. The expression of signal transduction proteins, such as DR5, caspase-3, caspase-8, caspasep-9, Bid, Bax and c-FLIP was detected by Western blotting. RESULTS: Both rsTRAIL and plumbagin induced the apoptosis in Kasumi-1 cells, and combination of plumbagin with rsTRAIL enhanced the apoptosis. The ratios of Annexin V-positive Kasumi-1 cells were (27.7±2.9)%, (25.6±3.1)% and (52.1±3.3)% in 100 μg/L rsTRAIL group, 2 μmol/L plumbagin group and the combination group, respectively, and the positive rate in combination group was significantly higher than those in other 2 groups. TUNEL assay demonstrated that the number of apoptotic cells in combination group was higher than that in the cells treated with rsTRAIL or plumbagin alone. Plumbagin up-regulated the expression of DR5 at mRNA level in Kasumi-1 cells, and up-regulation of DR5, activation of caspase-8 and down-regulation of c-FLIP at protein level were detected in the cells treated with plumbagin alone and the combination of plumbagin with rsTRAIL. CONCLUSION: Plumbagin enhances TRAIL-induced apoptosis in Kasumi-1 cells by up-regulating DR5, activating caspase-8 and down-regulating c-FLIP.     

Preparation of recombinant human soluble TRAIL and its inducing effect on apoptosis of tumor cells by TRAIL combined with bortezomib

AIM: To construct a prokaryotic expression plasmid to produce recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and to verify the biological activity of TRAIL. METHODS: The prokaryotic expression plasmid pET-28a (+)-TRAIL_114-281 was constructed. Human soluble TRAIL was obtained through optimized inducing protein expression and purification conditions. The biological activity of TRAIL was verified by CCK-8 assay. The apoptosis-inducing effect of TRAIL alone and/or in combination with proteasome inhibitor bortezomib (Velcade, PS-341) on the tumor cell lines H460(TRAIL-sensitive) and K562(TRAIL-resistance) for 24 h was determined. The apoptotic rates of the cells were analyzed by flow cytometry with Annexin V-FITC/PI staining. The activities of caspase-8, -9 and -3 in the cells were detected by colorimetric method. The protein expression of Bax, Bcl-2 and cFLIP was measured by Western blot. The expression of DR4 and DR5 in the H460 cells and K562 cells after treated with bortezomib for 24 h was detected by flow cytometry. RESULTS: The recombinant human soluble TRAIL protein with stable bioactivity was successfully acquired, which induced apoptosis in H460 cells and K562 cells. After treatment with different concentrations of TRAIL, the apoptotic rate of H460 cells was significantly increased with the increase in the concentration of TRAIL (P<0.05), but the apoptotic rate of K562 cells was not affected by the increasing TRAIL concentration. Apoptotic rate in combination group was obviously higher than that in single group (P<0.05). In the process of apoptosis, the activities of caspase-8, -9 and -3 in H460 cells and K562 cells were both increased. The expression of Bcl-2 and cFLIP in treatment groups (especially the combination group) was decreased compared with control group. No significant change of the Bax expression level was observed. The expression of DR4 and DR5 in the H460 cells and K562 cells was significantly up-regulated after treated with bortezomib (P<0.05). CONCLUSION: Bortezomib combined with recombinant human soluble TRAIL synergistically induces apoptosis in tumor cell lines H460 and K562 through initiating intrinsic apoptotic pathways by up-regulating death receptors DR4 and DR5, and reducing the expression of antiapoptotic proteins Bcl-2 and cFLIP.     

控释肥料对杭白菊生长发育及产量品质的影响

以控释复合肥CRF(N∶P2O5∶K2O=20∶8∶10)和普通复合肥CCF(N∶P2O5∶K2O=15∶15∶15)为材料,在大田条件下,研究等氮素用量控释复合肥和普通复合肥对杭白菊产量、品质和养分利用率的影响。结果表明:与普通复合肥相比,控释复合肥能够改善植株农艺性状,提高经济产量,增加水溶性浸出物、可溶性糖、蛋白质、总黄酮类化合物在花中的含量。由于控释复合肥与普通复合肥的养分配比不同,等氮素条件下,控释复合肥的肥料施用量比普通复合肥减少25%。从综合性状看,以施肥量为900 kg/hm2的CRF2处理最佳,节省肥料,提高经济效益,减少环境污染。     

Poly (I∶C) promotes iodine excess-induced chronic lymphocytic thyroiditis in NOD mouse

AIM: To investigate the effects of poly (I∶C) as virus mimics on iodine excess-induced chronic lymphocytic thyroiditis in NOD mouse. METHODS: Female, 32 NOD mice were randomly divided into 4 groups: (1) control; (2) high iodine; (3) poly (I∶C); (4) high iodine+poly (I∶C). Nine weeks after administration, mice were sacrificed. The following parameters were determined: body weight, thyroid weight and anatomic form. Thyroid hormone (T4) in serum was measured by radioimmunoassay, the thyroid morphology was observed through HE staining, apoptosis was detected by TUNEL, the mRNA expression levels of TRAIL, TRAIL-sR1, ICAM-1 and CXCL10 were determined by the method of real time RT-PCR. RESULTS: Compared to control group and poly (I∶C) group, the thyroid absolute weight and relative weight in high iodine group were increased (P<0.01), the level of total T4 in serum was decreased (P<0.05), inflammation and apoptosis were obviously observed, the mRNA expressions of TRAIL, TRAIL-sR1, CXCL10 and ICAM-1 were upregulated (P<0.05). Compared to high iodine group, thyroid absolute weight and relative weight in high iodine+poly (I∶C) group were further increased, the level of total T4 in serum was further decreased (P<0.05), the ratio of inflammatory degree Ⅳ increased to 50.0%, the numbers of apoptosis cells were further enhanced, the mRNA expressions of TRAIL, TRAIL-sR1, ICAM-1 and CXCL10 were further upregulated (P<0.05). Otherwise, the tendency of all parameters in poly (I∶C) group was similar to that in control group (P>0.05). CONCLUSION: Poly (I∶C) aggravates chronic lymphocytic thyroiditis induced by excess of iodine associated with increase in infiltration of lymphocytes and induction of apoptosis.     

杭白菊多糖超声提取工艺的研究

以杭白菊干品为原料,选超声时间、超声温度、超声功率、液料比为考察因素,进行L9(34)正交实验,并用药典方法对多糖含量进行测定,研究菊花多糖的超声波最佳提取工艺。结果表明:菊花多糖的最佳提取工艺为提取温度60℃,提取时间30min,料液比1∶40,提取功率40W;提取温度是影响菊花多糖提取率的最主要因素。     

Evodiamine inhibits growth of Huh7 cells and enhances their sensitivity to TRAIL

AIM: To investigate the effects of evodiamine on the growth and apoptosis of human hepatocellular carcinoma Huh7 cells, and to illustrate the molecular mechanism that evodiamine enhances antitumor activity of tumors necrosis factor-related apoptosis-inducing ligand (TRAIL) in Huh7 cells.METHODS: The cell viability was measured by MTT assay. The cell cycle distribution was analyzed by flow cytometry. The apoptosis rate was determined by TUNEL staining. The protein levels of cell cycle-and apoptosis-related proteins were detected by Western blot analysis.RESULTS: Treatment of Huh7 cells with evodiamine reduced the cell viability (P<0.05). Evodiamine induced cell cycle arrest in G₂/M phase by upregulation of p27, cyclin B1, cell division cycle protein 2 (Cdc2) and p-Cdc2. Evodiamine triggered apoptosis accompanied by cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). Combination of evodiamine with TRAIL significantly reduced the cell viability and increased cleavage of caspase-3 and PARP as compared with the use of each agent alone. Moreover, evodiamine increased the expression of death receptor 5 (DR5) in the Huh7 cells.CONCLUSION: Evodiamine inhibits the cell growth by reducing the cell viability and inducing cell cycle arrest. Evodiamine also triggers cell apoptosis and enhances the sensitivity of Huh7 cells to TRAIL by upregulating the expression of DR5.     

Caudatin sensitizes TRAIL-induced HepG2 cell apoptosis

AIM: To determine whether caudatin, a C₂₁ steroidal aglycone, enhances tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)-associated HepG2 cell apoptosis. METHODS: Cell growth inhibition was determined by MTT assay and cell colony formation assay. The TUNEL apoptosis detection kit was used to analyze cell apoptosis, and the protein expression was examined by Western blotting. RESULTS: Combination of caudatin with TRAIL signi-ficantly reduced cell proliferation and increased the apoptotic rate of HepG2 cells compared with the use of each agent alone. This was evidenced by marked increases in caspase-3, caspase-7, caspase-9 and PARP cleavages in the cells treated with caudatin and TRAIL-compared with control group. Combination of caudatin with TRAIL also led to the strong suppression of survivin. CONCLUSION: Caudatin synergizes HepG2 cells to TRAIL-induced apoptosis by promoting the cleavages of caspase-3, caspase-7, caspase-9 and PARP and inhibiting the expression of survivin.     

Effect of Wip1 gene silencing on chemotherapy sensitivity of human colon cancer cells

AIM: To observe the inhibitory effect of siRNA targeting to Wip1 gene on the Wip1 gene expression in the colon cancer cells and to investigate the influence of Wip1 gene silencing on the chemotherapy sensitivity of colon cancer cells. METHODS: Wip1-811 siRNA targeting to Wip1 gene was transfected into RKO colon cancer cells with high expression of Wip1 gene. The mRNA expression of Wip1 was measured by real-time PCR. The protein level of Wip1 was detected by Western blotting. The viability of RKO colon cancer cells was measured by MTS assay. The cell apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: Wip1-811 siRNA efficiently inhibited the expression of Wip1 at mRNA and protein levels. The enhanced chemotherapy sensitivity of RKO colon cancer cells was observed after inhibition of Wip1 gene expression. The viability of RKO colon cancer cells was decreased from (89.4±6.6)% to (74.7±3.9)% after treated with 5-fluorouracil (P<0.05) and decreased from (77.9±2.4)% to (66.7±2.9)% after treated with oxaliplatin (P<0.05). The cell apoptotic rate was increased from (7.7±0.5)% to (12.3±3.2)% and from (14.7±2.1)% to (34.0±2.1)% when RKO colon cancer cells were treated with 5-fluorouracil and oxaliplatin, respectively (P<0.05). CONCLUSION: Wip1 gene silencing enhances chemotherapy sensitivity of colon cancer cells.     

IFN-γ reinforces the inhibitory effect of tumor necrosis factor-related apoptosis-inducing ligand on apoptosis of human colon cancer cell line RKO cells

AIM: To explore the influence of IFN-γ on the role of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in inducing apoptosis of RKO cell line. METHODS: Survival fraction and apoptosis were measured by MTT method and flow cytometry (FACS). RESULTS: Survival fraction of IFN-γ group, TRAIL and IFN-γ 72 h+TRAIL group were 99.28%, 85.45%, 52.60%, respectively. The percentage of apoptotic cells of IFN-γ group, TRAIL group, IFN-γ 24 h+TRAIL group, IFN-γ 48 h+TRAIL and IFN-γ 72 h+TRAIL group were 1.51%, 2.38%, 4.97%, 13.30%, 21.00%, respectively. The percentage of apoptotic cells of IFN-γ 24 h+TRAIL group was higher than the sum of IFN-γ group and TRAIL group (P<0.01). The longer the IFN-γ pretreated, the higher the percentage of apoptotic cells was observed (P<0.01). CONCLUSION: IFN-γ can reinforce the effect of TRAIL inducing apoptosis in RKO.     

iASPP on apoptosis in breast cancer cells which expressed wild type p53

AIM: To investigate the RNAi effect of the inhibitory member of the ASPP family (iASPP) on the apoptosis of human breast cancer cell MCF-7 which expressed the wild type p53 gene. METHODS: The recombinant plasmid pAd-iASPP-RNAi was transfected into MCF-7 cells. The expression of iASPP mRNA and protein was analyzed by RT-PCR and Western blotting, respectively. The cell apoptosis was detected by FCM, and then the MCF-7 cells were transplanted into nude mice to set up transplantation model. The expression of iASPP RNA and protein in transplanted neoplasm were determined by RT-PCR and Western blotting, the apoptosis index was detected by FCM at the same time. RESULTS: The results showed that the expression of iASPP descended in MCF-7 cells (mRNA 95.4% and protein 96.8%, respectively, P<0.01) and the apoptosis rate and necrosis rate of MCF-7 cells increased (P<0.01) after transfection. As treated with pAd-iASPP-RNAi, the expression of iASPP in transplantation tumor cells descended 87.4% (mRNA) and 89.2% (protein), respectively (P<0.01), and the apoptosis rate and necrosis rate increased accordingly (P<0.01, P<0.05). CONCLUSION: The inhibition of iASPP may resume the ability of p53 to induce apoptosis in breast cancer cells which is able to express wild type p53.     

5-FU upregulates stem cell marker CD133 expression in colon cancer cells

AIM: To investigate the effect of 5-fluorouracil(5-FU)on the expression of the stem cell marker CD133 on colon cancer stem cells. METHODS: CD133 expression on several colon cancer cell lines was detected by flow cytometry. The CD133 positive cells from DLD1 cells were separated by the method of magnetic activated cell separation. Colony assay was used to measure self-renew ability and MTS assay was used to detect the sensitivity to 5-FU after separation. After 5-FU treatment, the change of CD133 mRNA level was measured by qPCR. RESULTS: CD133 expression on the surface of colon cacner cell lines DLD1, HT29, SW480, HCT116, Lovo, RKO was 30.20%, 82.00%, 0.34%, 91.80%, 85.30%, 0.28% respectively. DLD1 cells had two obvious populations according to CD133 expression. CD133 positive cells were separated from DLD1 cells, the positive purity was 87.21%±5.33% and the negative purity was 84.30%±4.65%. CD133 positive cells formed more colonies with limited dilution colony assay(46.33%±4.44% vs 31.00%±2.00%, P<0.05). CD133 positive cells were less sensitive to 5-FU compared to CD133 negative cells(20% less, P<0.01). 5-FU at concentration of 1 mg/L upregulated CD133 mRNA expression in both DLD1 and HT29 cells, the relative quantity was increased from 1 to 1.684±0.012(P<0.01)and 30.702±0.280 to 49.379±0.460(P<0.01)in HT29 and DLD1, respectively. CONCLUSION: Compared to CD133 negative cells, CD133 positive cells show more ability to form colonies in vitro, and are less sensitive to 5-FU. 5-FU upregulats the mRNA expression of CD133, resulting in the CD133 colon cancer stem cells enrichment during 5-FU treatment.     

Aspirin enhances TRAIL-induced apoptosis in HepG-2 cells

AIM: To investigate the enhancing effect of aspirin on the HepG-2 cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its underlying mechanism. METHODS: HepG-2 cells were cultured in DMEM medium with 15% fatal bovine serum. Modified carbol fuchsin staining was used to observe the cellular morphology. Cell proliferation was measured by MTT. The method of terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) was used to examine the apoptotic index of the cells. RT-PCR was applied to detect survivin gene expression. The apoptotic rate and cell cycle were determined by flow cytometry. RESULTS: Aspirin at different concentrations and TRAIL inhibited the proliferation of HepG-2 cells and the cell apoptotic index was obviously higher than that in control group. Aspirin increased the apoptotic rate in a dose-dependent manner. Aspirin at different concentrations and TRAIL significantly down-regulated the mRNA level of survivin. CONCLUSION: Aspirin enhances the apoptosis of HepG-2 cells induced by TRAIL and the mechanism may be related to down-regulating the mRNA expression of survivin.     

焦作市四大怀药资源及评价

怀地黄、怀山药、怀牛膝、怀菊花是焦作市珍贵的地域特产资源,具有独特的药用价值和良好的保健作用.该文对四大怀药的药用、食用、观赏、保健价值进行综述,分析其开发利用现状.     

Sodium valproate induces cycle arrest,apoptosis and elevates p21WAF1 mRNA expression in chronic myeloid leukemia cell line K562

AIM:To investigate the effects of sodium valproate (VPA) on the cell cycle and apoptosis of chronic myeloid leukemia cell line K562, and to explore the possible mechanisms. METHODS:K562 cells were treated with VPA. Cell cycle and apoptosis were analyzed by flow cytometry. The expression of p21WAF1 mRNA was detected by RT-PCR. RESULTS:After treatment with VPA, cell cycle was arrested obviously at G0/G1 phase ［(82.30±9.41)% vs (40.13±2.12)%, P<0.05］. The apoptotic rate was significantly higher in the cells treated with VPA than that in untreated cells ［(11.47%±0.25%) vs (4.77%±0.40%), P<0.05］. The level of p21WAF1 mRNA was increased ［(1.65±0.91) vs (0.25±0.04), P<0.05］. CONCLUSION:VPA induces elevated expression of p21WAF1 mRNA in K562 cells, resulting in G0/G1 phase arrest and apoptosis in vitro.     

Effects of different chemotherapeutic agents on reversing the acquired resistance to TRAIL gene in DLD1 colon cancer cells

AIM:To evaluate effects of different chemotherapeutic agents on reversing the acquired resistance to TRAIL gene and clarify the involved mechanisms in DLD1-TRAIL/R colon cancer cells. METHODS:Human colon cancer cell line DLD1-TRAIL/R cells that were resistant to TRAIL-expressing adenovector (Ad/gTRAIL) were treated with Ad/gTRAIL combined with different chemotherapeutic agents. Then, the cell viability was measured by MTT method, and apoptotic signaling conditions, including activation of caspase-3 and caspase-8, expression of Bax and Bcl-XL, were measured by Western blotting analysis. RESULTS:In vitro data showed that several chemotherapeutic agents, including 5-fluorouracil (5-FU) and mitomycin c (MMC), overcome the acquired resistance to TRAIL gene in DLD1-TRAIL/R colon cancer cells. The combination of Ad/gTRAIL and 5-FU effectively suppressed tumor growth in vivo in subcutaneous tumors established from DLD1-TRAIL/R cells. Further data showed that treatment with the combination of Ad/gTRAIL and 5-FU or MMC led to enhance the activation of caspase-3. Moreover, MMC but not 5-FU induced overexpression of Bax gene that was sufficient to overcome the resistance to TRAIL gene in DLD1-TRAIL/R cells. CONCLUSION:Chemotherapeutic agents, such as 5-FU and MMC, overcome the acquired resistance to TRAIL gene in DLD1-TRAIL/R cells. The candidate mechanisms for MMC but not 5-FU to overcome this resistance might involve the induction of over-expressed Bax protein in DLD1-TRAIL/R cells.     

菊花脂肪酸脱饱和酶基因CmFAD7的克隆与表达分析

以秋菊（Chrysanthemum morifolium Ramat.）抗寒品种‘星光灿烂’为材料,利用RT-PCR和RACE方法从叶片中克隆了ω-3脂肪酸去饱和酶基因,命名为CmFAD7,GenBank登录号为KC567246。该基因cDNA 全长1 284 bp,编码428个氨基酸,相对分子量为48.98 kD,等电点为9.07,编码的氨基酸序列与高山还阳参同源性最高（84%）。该蛋白含有?12-FADs保守结构域和3个跨膜螺旋,N端含有一段叶绿体转运肽,属于膜脂肪酸去饱和酶超级家族。采用实时荧光定量PCR和气相色谱法研究低温胁迫下叶片和根系中CmFAD7的表达量及脂肪酸含量变化,CmFAD7在根系和叶片中均有表达,叶片中的表达量高于根系。根系中5 ℃时表达量最高,叶片中–4 ℃时最高,在–8 ℃时叶片和根系表达量均较低;随着处理温度的降低,叶片中C18:3含量呈逐渐上升趋势,根系中变化不明显。结果表明,菊花CmFAD7与抗寒性有关,低温条件下不同器官的表达量有较大差异。     

Effects of lentivirus-mediated caspase-3 silencing on proliferation and apoptosis of rat bone marrow mesenchymal stem cells

AIM：To investigate the effects of caspase-3 gene silencing on proliferation, cell cycle and apoptosis of rat bone marrow mesenchymal stem cells (MSCs). METHODS：A lentiviral vector expressing caspase-3 shRNA was constructed and transfected into rat bone marrow MSCs．The expression of caspase-3 at mRNA and protein levels was detected by real-time PCR and Western blotting, respectively. Cell proliferation and cell cycle were evaluated by MTS assay and flow cytometry, respectively. The expression of bcl-2 and bax mRNA was detected by real-time PCR. The apoptosis of the cells was evaluated by Hoechst 33258 staining. RESULTS：Recombinant lentivirus was successfully transfected into MSCs. The proliferation of the MSCs transfected with caspase-3 shRNA was significantly promoted (P<0.05) and the proportion of the cells in S phase was increased to (52.66±0.30) %. Compared with control groups, caspase-3 silencing up-regulated the mRNA level of bcl-2 and down-regulated the mRNA level of bax, and the ratio of bcl-2 to bax increased (P<0.05). The apoptotic rate in MSCs-shRNA group was (15.01±1.73) %, which was significantly lower than those in MSCs and MSCs-vector group ［(23.67±1.16) % and (25.67±3.05) %, respectively; P<0.05］. CONCLUSION: Caspase-3 silencing regulates cell cycle, promotes the proliferation and attenuates the apoptosis of rat bone marrow MSCs.