ABSTRACT
The anti-angiogenic activity of tumstatin45-132 is mediated by binding to alphaVbeta3 on endothelial cells and tumor vascular endothelium showing increased expression of alphaVbeta3. Tumor necrosis factor alpha (TNF-alpha) is known to not only possess direct cytotoxicity against tumor cells, but also induces tumor vessel disruption, however, clinical use of TNF-alpha as an anticancer drug is hampered by severe systemic toxicity. In this study, we explore the possibility of fusing tumstatin45-132 with human TNF-alpha in the hope of generating a targeting, bi-functional protein in tumor treatment. Tumstatin45-132-TNF was constructed and expressed in E. coli. The recombinant fusion protein was shown to be insoluble and in an inclusion body form. An effective strategy for refolding and purification of tumstatin45-132-TNF resulted in final purified yields of 3 mg purified fusion protein recovered from 1 liter of E. coli culture. The refolded tumstatin45-132-TNF with a purity of 98% assessed by denaturing SDS - PAGE showed a single band on gels. Endothelial cell proliferation assay and standard cytolytic assays against L929 indicated that the fusion protein maintains tumstatin45-132 and TNF-alpha activity. More importantly, tumstatin45-132-TNF inhibits endothelial cell proliferation more than tumstatin45-132 alone. Cell adhesion assays and competitive binding experiments with anti-integrin antibodies showed that the tumstatin45-132 moiety specifically interacts with alphaVbeta3 integrin. These results lay the solid foundation for further investigation of antitumor activity of tumstatin45-132-TNF in vivo.
Subject(s)
Autoantigens/chemistry , Collagen Type IV/chemistry , Recombinant Fusion Proteins/biosynthesis , Animals , Cell Proliferation/drug effects , Humans , Integrin alphaVbeta3/metabolism , Mice , Protein Folding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/pharmacologyABSTRACT
OBJECTIVE: To investigate the efficiency of a peptide nucleic acid (PNA) delivery system by using liposome via PNA-DNA hybrids and to test the inhibitive action of antisense PNA on expression of multidrug resistance (MDR) related P-glycoprotein (P-gp) in human neuroblastoma cell line SK-N-SH. METHODS: Two antisense PNAs were designed targeting at MDR-1 mRNA and then combined with partially complement DNAs respectively. The hybrids were delivered into cells using cationic liposome. The transfection efficiency, expression of P-gp and MDR-1 mRNA, intracellular adarimycin (ADM) were measured by flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR), and high performance liquid chromatography (HPLC). RESULTS: Transfection of PNA increased the cell average fluorescence intensity significantly and the extent of increase was dependent on the concentration of PNA. After being transfected by both PNAs, P-gp expression of SK-N-SH cells decreased significantly and the intracellular ADM level was increased by about 3 times. The level of MDR-1 mRNA expression slightly decreased after transfection, but no statistical significance was observed. CONCLUSIONS: PNA can be delivered into tumor cells in form of PNA-DNA hybrids by cationic liposome. Properly designed antisense PNA can inhibit MDR related P-gp expression of SK-N-SH cells efficiently and specifically.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , Drug Resistance, Multiple/genetics , Nervous System Neoplasms/metabolism , Neuroblastoma/metabolism , Peptide Nucleic Acids/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Animals , Humans , Oligonucleotides, Antisense/pharmacology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Transfection , Tumor Cells, CulturedABSTRACT
OBJECTIVE: To investigate the effectiveness of phosphorothioate multidrug resistance gene 1 (MDR1) antisense oligodeoxynucleotides (MDR1-AS) suppressing MDR1 expression in multidrug-resistant glioma cell line C6/adr. METHODS: The glioma cell line C6/adr served as the tested model in vitro, MDR1-AS (5'-CTCCATCACCACCTC-3'), complementary to the -9- +6 sequence of first exon, was synthesized and phosphorothioate-modified. As control of sequence specificity, MDR1-S (5'-GAGGTGGTGA TGGAG-3') was used. Both antisense and sense oligodeoxynucleotides were transduced to C6/adr cells by lipofectin. The cytotoxity of MDR1-AS was tested using morphological observation and 3- (4,5-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide assay. Semi-quantitative reverse transcription polymerase chain reaction was used to monitor the expression levels of the MDR1 mRNA in the different groups. The positive rate of the MDR1 gene product P glycoprotein (P-gp) was determined by flow cytometry assessment. RESULTS: No cytotoxicity of MDR1-AS was observed. The MDR1 mRNA expression level was decreased from 106% to about 30.44% 48 h after MDR1-AS treatment. The P-gp positive rate of MDR1-AS treated C6/adr cells decreased from 100% to 32.77%, with that of C6/adr cells considered as 100%. CONCLUSIONS: MDR1-AS can effectively inhibit MDR1 expression in the C6/adr cell line at both the mRNA and protein level, and may be an alternative treatment of drug-resistant gliomas.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , Drug Resistance, Neoplasm , Glioma/genetics , Oligodeoxyribonucleotides, Antisense/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Cell Line, Tumor , Drug Resistance, Multiple , Glioma/metabolism , Humans , RNA, Messenger/biosynthesis , RNA, Messenger/geneticsABSTRACT
OBJECTIVE: To evaluate the animal model of the multidrug resistant glioma cell line C6/adr for further in vivo studies. METHODS: The rat glioma cells C6 and multidrug resistance cells C6/adr were cultured in vitro and implanted into the brain of S-D rats. After implantation, all these animals were examined continually with magnetic resonance imaging (MRI) and histological examination. The growth procedure of intracranial implanted glioma and the survival span of the animal model were evaluated. The statistical analysis was made between the survival data of the two cell lines. RESULTS: The symptoms of intracranial hypertension did not occur until 4 weeks after inoculation. The MRI findings of the implanted glioma in the rat brain were much earlier than the abnormal behavior observed. Pathological results after inoculation demonstrated the MRI findings. The two cell lines had similar growth characteristics and no significant differences in survival times. CONCLUSION: These results suggest that by means of MRI and histology the growth procedure of the implanted glioma in vivo be successfully observed. All these data will proved to be a useful basis for study of glioma in vivo.
Subject(s)
Disease Models, Animal , Drug Resistance, Multiple/genetics , Drug Resistance, Neoplasm/genetics , Glioma/pathology , Animals , Antibiotics, Antineoplastic/pharmacology , Brain Neoplasms/pathology , Doxorubicin/pharmacology , Magnetic Resonance Imaging , Male , Random Allocation , Rats , Rats, Sprague-Dawley , Tumor Cells, CulturedABSTRACT
OBJECTIVE: To establish a drug-resistance cell line of human glioma mediated by MGMT. METHODS: Simulated the clinical usage of BCNU to establish a BCNU-resistant human glioma subline by cyclic exposing the U251 parent cells to a constant concentration of BCNU. The resistance index and the expression of MGMT mRNA of U251/BCNU were detected and compared the difference of in vitro proliferation between U251 and U251/BCNU. RESULTS: A subline--U251/BCNU was successfully established in about 4-month culture, which had a stable resistance to BCNU. U251/BCNU cells showed 17-fold higher resistance to BCNU than did U251 cells by MTT assay, while U251/BCNU cells expressed MGMT mRNA. The doubling time of U251 and U251/BCNU had no statistical difference. CONCLUSION: A drug-resistance cell line of human glioma mediated by MGMT is established, which could provide experimental basis for further studies on the resistance mechanism and reversal methods of glioma chemotherapy.
