DUSP19 mediates spinal cord injury-induced apoptosis and inflammation in mouse primary microglia cells via the NF-kB signaling pathway.

Objective: Spinal cord injury (SCI) is a common injury that seriously threatens human health. NF-κB may be involved in the secondary injury of SCI that is mediated by inflammation and aggravates damage. Our study was aimed to investigate the role of NF-κB signaling in DUSP19-mediated cleaved Caspase-3 expression and the release of inflammatory factors in vivo and in vitro.Materials and Methods: DUSP19 mRNA expression and the content of IL-6 and IL-8 in patients with traumatic SCI (TSCI) were measured by real-time PCR and ELISA, respectively. The levels of p-NF-κBp65, NF-κBp65 and cleaved Caspase-3 expression and the concentrations of IL-6 and IL-8 were measured by western blotting and ELISA, respectively.Results: Patients with TSCI showed lower DUSP19 expression and higher concentration of IL-6 and IL-8 compared with healthy controls. DUSP19 overexpression inhibited p-NF-κBp65 level, cleaved Caspase-3 expression, and production of IL-8 and IL-6 in the mice induced by TSCI. DUSP19 silencing increased p-NF-κBp65 level, cleaved Caspase-3 expression, and concentration of IL-6 and IL-8 in mouse primary microglia cells. DUSP19 overexpression had an inverse effect. Importantly, DUSP19 silencing and overexpression mediated p-NF-κBp65 level, cleaved Caspase-3 expression, and concentration of IL-6 and IL-8 in mouse primary microglia cells were reversed by NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and NF-κB activator 12-myristate 13-acetate (PMA), respectively.Conclusion: These results suggested that DUSP19-mediated SCI-induced apoptosis and inflammation via NF-κB signaling and might therefore serve as a potential therapeutic target for SCI.

Enhancement effect of adenovirus-mediated antisense c-myc and caffeine on the cytotoxicity of cisplatin in osteosarcoma cell lines.

AIMS: Studies on cancer biology have shown that overexpression of oncogenes (with or without functional loss of tumor suppressor genes), which is responsible for the progression of human malignancies via a multistep process, may be reduced by antisense technology. Caffeine enhances the effect of cisplatin (CDDP) chemotherapy on osteosarcoma cells. We constructed the recombinant adenovirus (Myc-AS) encoding the antisense c-myc fragment and investigated the synergic effect of caffeine and Myc-AS on the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin. METHODS: The recombinant adenovirus (Myc-AS) encoding the antisense c-myc fragment was constructed by cloning c-myc cDNA of about 750 bp in a reverse direction into adenovirus vector, then undergoing recombination, amplification and complementation in vivo. Myc-AS and caffeine were used either alone or in combination with CDDP to treat osteosarcoma MG-63 cells in vitro. Western blot, MTT, flow cytometry (FCM) and electron microscopy were used to evaluate the expression of c-myc protein, tumor cell proliferation in vitro and apoptosis and to perform cell cycle analysis. RESULTS: Myc-AS encoding antisense c-myc fragment was obtained with a titer of 2 x 10(9) pfu/ml. Myc-AS downregulated the expression of c-myc protein after transfecting MG-63 cells for 48 h, induced tumor cell apoptosis and inhibited tumor cell proliferation in vitro. Myc-AS or caffeine can enhance the cytotoxic effects of 2.0 and 5.0 microg/ml CDDP on MG-63 cells. Moreover, the significantly enhancing effect of the Myc-AS-caffeine combination on CDDP chemotherapy of MG-63 cells was not restricted to apoptosis but also decreased tumor cell proliferation in vitro. Expression of the apoptosis-associated bcl-2 gene was downregulated and bax was upregulated, with no changes in E2F-1 expression. FCM analysis showed that CDDP treatment induced a block in S phase, and caffeine reversed this block and accelerated cell progression through the S phase. CONCLUSIONS: Myc-AS can induce obvious G2/M phase arrest in transfected cells. Myc-AS combined with caffeine can enhance apoptosis induction and chemotherapeutic effects of CDDP on osteosarcoma MG-63 cells.

[The treatment of knee joint peripheral fractures and/or dislocations with vascular injury].

OBJECTIVE: To investigate the effect and influence factors on knee joint peripheral fractures and/or dislocations with an associated vascular injury through retrospectively study. METHODS: From March 2002 to November 2007 31 patients with knee joint peripheral fractures and/or dislocations with an associated vascular injury were treated, including 24 males and 7 females with a mean age of 41 years (range from 21 to 62 years). Definite diagnosis of vascular injury by combining colored ultrasonic, CTA, operative exploration with clinical signs, fixing fractures and/or dislocations with fixators, plates and screws, reconstructing blood circulation based on the condition of the vascular injury by vascular repair, homograft vein or artificial vascular grafting separately and analysing the effects of PSI, diagnosis and treatment methods on salvage lower extremities. RESULTS: Successful reconstruction was carried out in 31 cases, however there were 1 death because of mult-fractures and brain injury and 6 amputation, 24 cases successful salvage followed up mean 24.2 months, 6 cases bone nonunion and infected bone defect were cured by delayed bone planting or bone transportation. Ligaments repair reconstruction of 7 cases knee joint dislocation were done in delayed 3 or 4 weeks after first operation, the good functional rate was 71.4%. CONCLUSIONS: The patients of PSI under 10 grades in knee joint peripheral fractures and/or dislocations with an associated vascular injury should been carried out treatment, early definite diagnosis and blood circulation reconstruction are the key factors of successful salvage treatment.

Recombinant antisense C-myc adenovirus increase in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin.

C-myc is an oncogene with the important role of cell proliferation controller. It has been found to be amplified and overexpressed in osteosarcoma. Moreover, it can promote cell transformation and induce metastatic features. Some studies showed that overexpression of c-myc could induce resistance in response to antineoplastic agents. Currently, we constructed the recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment and investigated its effect on the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin(CDDP). The osteosarcoma MG-63 cells were transfected by the Ad-Asc-myc in vitro, and Western Blot, MTT assay, RT-PCR, flow cytometry (FCM), and transmission electron microscopy (TEM) were used to study expression of c-myc and caspase-3 protein, tumor cell proliferation in vitro, cell apoptotic morphology and cell cycle change. Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2.0 x 10(9) pfu/ml. Ad-Asc-myc downregulated the expression of c-myc protein after transfected MG-63 cells for 48 hours, combined with the treatment of 2.0, 5.0 microg/ml cisplatin for 2 hours can inhibited tumor cells proliferation in vitro by 33.4 and 54.2 percent, respectively, which had significant difference compared with control recombinant adenovirus (Ad-LacZ) groups (P < 0.05). RT-PCR revealed that Ad-Asc-myc downregulated expression of bcl-2 and upregulated expression of Bax, and no appreciable changes were observed in the expression of E2F-1. Detection of caspase-3 protein TEM, and FCM analysis showed that Ad-Asc-myc could induce apoptosis of transfected cells, which was enhanced by the treatment of cisplatin. Cell cycle analysis showed that obvious G(2)/M phase arrested in transfected cells. In conclusion, Ad-Asc-myc increased the in vitro sensitivity of osteosarcoma MG-63 cells to cisplatin as well as induced apoptosis.

[Adenovirus mediated antisense c-myc gene on the chemotherapy sensitivity of osteosarcoma cells to cisplatin].

OBJECTIVE: To construct the recombinant adenovirus encoding antisense c-myc fragment and to investigate its effect on the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin. METHODS: The recombinant adenovirus (Ad-Asc-myc) encoding antisense c-myc fragment was constructed by cloning c-myc cDNA of about 720 base pairs in a reverse direction into adenovirus vector, then undergoing recombination, amplifying and being complemented in vivo. The osteosarcoma MG-63 cells were transfected by the Ad-Asc-myc in vitro, and Wright staining, Acridine Orange staining, Western Blot, MTT, Flow Cytometry (FCM) were used to study cell morphology, expression of c-myc protein, tumor cell proliferation in vitro, apoptosis and cell cycle change. RESULTS: Ad-Asc-myc encoding antisense c-myc fragment was obtained with the titer of 2 x 10(9) pfu/ml. Ad-Asc-myc down-regulated the expression of c-myc protein after transfected MG-63 cells for 48 h, combined with the treatment of 2.0, 5.0 microg/ml cisplatin for 2 h could inhibit tumor cells proliferation in vitro by 33.4% and 54.2% respectively, which were significantly difference compared with control recombinant adenovirus (Ad-LacZ) groups (P < 0.05). Acridine Orange staining and FCM analysis showed that Ad-Asc-myc could induce apoptosis of transfected cells, which was enhanced by the treatment of cisplatin cell. Cycle analysis showed that obvious G2/M phase arrested in transfected cells. CONCLUSION: Ad-Asc-myc increases the chemotherapy sensitivity of osteosarcoma MG-63 cells to cisplatin as well as induced apoptosis.

[Construction of antisense c-myc recombinant adenovirus and its anti-tumor effects on osteosarcoma cell lines MG-63 and U2OS].

BACKGROUND & OBJECTIVE: c-myc, an oncogene, plays an important role in regulation of cell proliferation, and has been found to be amplified and overexpressed in osteosarcoma. Moreover, it can promote cell transformation, and induce metastasis. This study was to construct recombinant adenovirus encoding antisense c-myc, and to investigate its effects on osteosarcoma cell lines MG-63 (p53-deficient) and U2OS (with wild type p53). METHODS: Recombinant adenovirus Ad-As-c-myc encoding antisense c-myc was constructed by gene reconstruction technique, defined by polymerase chain reaction (PCR), and transfected into human osteosarcoma cell lines MG-63 and U2OS. Western blot, acridine orange staining, reverse transcription-PCR (RT-PCR), and flow cytometry (FCM) were used to detect expression of c-myc, proliferation, apoptosis, and cell cycle of MG-63 and U2OS cells. RESULTS: Ad-As-c-myc encoding antisense c-myc was obtained with the titer of 2x10(9) pfu/ml. Ad-As-c-myc significantly inhibited proliferation of both cell lines, while U2OS with wild type p53 was more susceptible to Ad-As-c-myc. Expression of c-myc mRNA was down-regulated in 2 cell lines 48 h after transfection of Ad-As-c-myc. Acridine orange staining and FCM analysis showed that Ad-As-c-myc induced apoptosis of both cell lines, cell cycle analysis showed obvious G(2)/M phase arrest in MG-63 cells,and G1 phase arrest in U2OS cells after transfection of Ad-As-c-myc. CONCLUSION: Ad-As-c-myc could induce apoptosis through both P53-dependent and P53-independent pathways, and inhibit proliferation of osteosarcoma cells.

[Inhibitory effects of survivin antisense oligonucleotide on drug-resistant human ovarian cancer cell COC1/DDP].

BACKGROUND & OBJECTIVE: Recent studies have shown that survivin is an anti-apoptosis gene, which plays an important role in the carcinogenesis and drug resistance of ovarian cancer. This study was designed to explore the effects of liposome-survivin antisense oligonucleotide (Lip-ASODN) on the growth,apoptosis,and cell cycle distribution of drug-resistant human ovarian cancer cell line COC1/DDP. METHODS: Survivin-ASODN were transfected into COC1/DDP cells mediated by lipofectin. The proliferation of COC1/DDP cells was assessed by cyto-dynamics and MTT assay. The mRNA expression of survivin was determined by reverse transcription- polymerase chain reaction (RT-PCR). The caspase-3 protein activity was measured by Western blot analysis. The apoptotic rate and cell cycle distribution were estimated by flow cytometry (FCM). RESULTS: Compared with Lip-SODN and Lip alone groups, the proliferation of COC1/DDP cells after cultured with Lip-ASODN has been significantly inhibited, its inhibitory rate was (68.3+/-6.2)% after cultured for 72 hours (P< 0.05). The mRNA expression of survivin in Lip-ASODN group was significantly decreased, while the caspase-3 activity increased in a time-dependent manner as compared with Lip-SODN and Lip alone groups (P< 0.05). Cell cycle distribution significantly changed in Lip-ASODN group, many cells have been blocked in G0/G1 phase (79.21%), while G2/M phase (4.92%) and S phase (15.87%) decreased. The apoptotic rate of Lip-ASODN group reached 33.18%, which was much higher than those of Lip-SODN and Lip alone groups (P< 0.05). CONCLUSION: Survivin ASODN can inhibit COC(1)/DDP cell proliferation, reduce the mRNA expression of survivin, and induce cell apoptosis.