Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has been shown that the type IV collagenase with its two subtypes, 72 kDa/ MMP-2 and 92 kDa/MMP-9, plays an important role in tumor invasion and metastasis formation that occur through a mechanism of proteolytic degradation of collagen IV in the basement membrane. One possible method to specifically inhibit the function of the targeted protein of a cell is to express intracellular antibody combining site that can block the function or prevent the expression of the targeted molecule. Accordingly, intracellular antibodies against type IV collagenase may have a therapeutic use against tumor invasion and metastasis. As described in our previous reports, an anti-type IV collagenase monoclonal antibody (3D6) was obtained using the hybridoma approach, and its functional single-chain Fv fragment (scFv) named M97 was constructed based on recombinant phage display techniques. In this study, the endoplasmic reticulum (ER)-retained scFv antibody fragment was used to inhibit the function of type IV collagenase. For expression in mammalian cells, the assembled scFv M97 gene with ER retention signal encoding 6 additional amino acid (SEKDEL) was reamplified by PCR. The amplified fragments were cloned into the pcDNA3.1 vector containing the CMV early-intermediate promoter/enhancer. The resulting plasmid was sequenced and then introduced by the lipofectamine method into PG cells, a highly metastatic human lung cancer cell line and G418-resistant cells were obtained by G418 selection. After transfection, the M97 mRNA expression was observed and the type IV collagenase expression was downregulated significantly as measured by ELISA. The biological behavior of PG cells, such as the ability of in vitro invasion of colony formation on soft agar through Matrigel, were also inhibited by scFv M97 transfection. The results indicate that intracellular antibody technology represents a novel and efficient way to selectively abrogate the activity of type IV collagenase, at least in vitro. We are presently exploring the efficacy of this approach in a xenograft model of human lung cancer.
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PMID:Antineoplastic effect of intracellular expression of a single-chain antibody directed against type IV collagenase. 1090 9

Increased expression of matrix metalloproteinases (MMPs) has been associated with human glioblastoma tumor progression. In this study, we sought to down-regulate MMP-9 expression by stably transfecting a high-grade glioblastoma cell line with a plasmid vector capable of expressing an antisense transcript complementary to a 528-bp segment at the 5' end of human MMP-9 cDNA. Stable transfectants were obtained through selection with G418. Of the clones transfected with vector, sense, and antisense constructs, Northern blotting, Western blotting, and gelatin zymography showed that MMP-9 expression was significantly reduced only in the antisense-transfected cells. A Matrigel invasion assay revealed marked reductions in invasiveness for the antisense clones relative to the parental, vector, and sense clones. Cocultures of tumor spheroids and fetal rat brain aggregates showed that the antisense-transfected stable clones showed no invasion of the rat brain aggregates; in contrast, 90% of the parental, vector, and sense clones invaded the rat brain aggregates. Intracerebral injection of antisense stable transfectants in nude mice produced no tumors or very small tumors, but intracerebral injection of parental or vector clones did produce tumors. These results suggest that MMP-9 expression is essential for the invasiveness of glioblastoma cells.
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PMID:Selective suppression of matrix metalloproteinase-9 in human glioblastoma cells by antisense gene transfer impairs glioblastoma cell invasion. 1115 78

Overexpression of urokinase-type plasminogen activator receptor (uPAR) on tumor cell surface is essential for invasion and metastasis in a variety of tumor cells. To establish a retroviral-mediated antisense RNA transfer system of uPAR gene for exploring its function on down-regulation of uPAR expression in leukemia cells, the retroviral vector LaCD87SN was constructed by inserting uPAR gene into LXSN vector in an antisense orientation. An uPAR gene antisense RNA transfer system was established by liposome-mediated transfection in combination with cross infection with retrovirus. Human leukemia cells U937 were transduced with aCD87 amphotropic retrovirus, expressing uPAR antisense RNA, and the U937/aCD87 cells was obtained by G418 selection. The integration and expression of antisense uPAR gene were analyzed by polymerase chain reaction (PCR) assay. The cell surface expression of CD87 and the activities of matrix metalloproteinase (MMP) were assayed by flow cytometry (FCM) and gelatin zymography, respectively. The results showed that the amphotropic retroviral producers Am12/aCD87, which expressed antisense RNA of uPAR gene with a titer of 6.3 x 10(5) cfu/ml in supernatants, were obtained by means of transfection and superinfection. U937/aCD87 cells were established by continuative G418 selection after retrovirus infection. In U937/aCD87 cells, the integrated provirus and the overexpression of antisense uPAR gene was confirmed. Compared with U937/NeoR cells, FCM analysis revealed that CD87 expression on U937/aCD87 cell surface was not downregulated significantly. However, MMP-9 secretion was significantly suppressed in U937/aCD87 cells. In conclusion, although the retroviral-mediated antisense RNA transfer could not efficiently suppress uPAR expression on leukemic cell surface, it may interfere the uPAR-MMP interactions.
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PMID:[Establishment of urokinase receptor gene antisense RNA transfer system and its application in leukemia research]. 1470 41

This study was purposed to investigate the inhibitory effect of short hairpin RNA (shRNA) on expression of vascular endothelial growth factor (VEGF) receptor flt-1 gene in leukemia cells line K562, and to explore the influence of shRNA invasive ability on leukemia cells and its mechanism. The recombinant eukaryotic expression plasmid containing flt-1 shRNA gene was transfected into K562 cells by lipofectamine mediation and positive clones were screened by G418. shRNA gene in K562 cells was confirmed by PCR. RT-PCR and Western blot were employed to detect the expression of flt-1 mRNA and protein in leukemia cells. The invasive ability of K562 cells was studied by Boyden chamber invasion assay before and after flt-1 shRNA transfection. MMP-2 and MMP-9 mRNA expressions were detected by RT-PCR after transfection of the recombinant plasmid C1/U6/FltS2 into K562 cells through liposome. The results showed that the recombinant eukaryotic expression plasmid had been transfected into the human leukemia cell line K562 and positive clones had been screened by G418 for 2 weeks. PCR detection revealed the stable expression of the shRNA gene in K562 cells. Flt-1 gene and protein expressions were inhibited by plasmid-expressed shRNA after transfection of recombinant vetors C1/U6/FltS into K562 cells. The inhibitory efficiency of two different shRNA sequences targeting Flt-1 gene were 46.1% and 65.4% respectively. The expression of MMP-2 and MMP-9 mRNA decreased, and the mean invasion rate in C1/U6/fltS2-transfected K562 cells was lower than that in nontransfected cells. It is concluded that shRNA eukaryotic expression vector specific to VEGF receptor flt-1 gene can high efficiently be transfected into leukemia cell line K562, effectively inhibits the expression of flt-1 gene, weakens the in vitro invasive ability of leukemia cells and the expression levels of MMP-2 and MMP-9 mRNA, which suggests that the VEGF involves in the migration of leukemia cells by regulating the MMP-2 and MMP-9 through joints with the receptor.
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PMID:[Inhibition effect of short hairpin RNA on VEGF receptor flt-1 gene expression in leukemia cell line K562]. 2003 Sep 17

The influence of short hairpin RNA (shRNA)-mediated osteopontin (OPN) gene silencing on the proliferation and invasion of human renal cancer ACHN cells was investigated. Four types of OPN shRNA recombinant plasmids were constructed and RT-PCR assays were used to screen the most highly functional shRNA recombinant plasmids, which were transferred into the cultured ACHN cells by Lipofectamine 2000. The cells transfected by shRNA expression vectors (ACHN/OPN) were visualized under an inverted microscope and screened by G418. Untreated cells (ACHN) and cells transfected by mock vectors (ACHN/Vect) were used as control groups. The expression levels of OPN mRNA and protein were detected by real-time PCR and Western blot respectively. The cell cycle and ratios of apoptotic cells were assessed by flow cytometry. MTT method was used for drawing the growth curve and observing cell proliferation in vitro. The abilities of migration and invasion in three groups were measured by Transwell chamber test. The expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 in three groups were examined by Western blot. Our results showed that the recombinant plasmid could be successfully transferred into ACHN cells by Lipofectamine 2000. Compared with untreated cells, the expression levels of OPN mRNA and protein in ACHN/OPN cells were decreased by 59.68% and 76.42%, respectively (P<0.05), ACHN/OPN cells were blocked in S phase and apoptotic ratio increased significantly (P<0.05), however, no significant differences were found between ACHN/Vect and ACHN. Recombinant plasmid significantly attenuated expression levels of MMP-2 and MMP-9 proteins and suppressed the proliferation, migration, and invasion of ACHN cells. This study suggested that OPN may play an important role in the growth and invasion of human renal cancer ACHN cells, and these processes are correlated with the activations of MMP-2 and MMP-9. Our data provided preliminary experimental evidence for the feasibility of RNA interference technology in gene therapy of human renal cancer.
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PMID:Influence of osteopontin short hairpin RNA on the proliferation and invasion of human renal cancer cells. 2015 57

To explore the potential of the anti-sense nucleic acid of CyclinD1 in lung cancer therapy, the expression vector containing the anti-sense nucleic acid of CyclinD1 was constructed and named pcDNA3.1-CyclinD1. The A549 cells were transfected with pcDNA3.1-CyclinD1 vectors. After being screened by G418, the stable expression positive clones were obtained. MTT method and flow cytometry technique were used to detect cell proliferation and apoptosis, respectively. The results showed the transfected cells exhibited significantly increased apoptosis and inhibited cell growth, compared with negative control and empty vector groups. To investigate the mechanism for anti-sense nucleic acid of CyclinD1 inducing A549 cells apoptosis, the expression levels of retinoblastoma protein (pRb), adenovirus E2 factor-1 (E2F-1), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and MMP-9 were detected by Western blot, and the results showed the expressions of these proteins were all decreased significantly in anti-sense nucleic acid of CyclinD transfected group, compared with those in negative control and empty vector groups. In a word, anti-sense nucleic acid of CyclinD1 induces the apoptosis of lung adenocarcinoma cancer cells, and the depressions of pRb, E2F-1, VEGF, MMP-2 and MMP-9 expressions may be the possible mechanism.
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PMID:[Anti-sense nucleic acid of CyclinD1 induces apoptosis of lung adenocarcinoma cancer cell A549]. 2168 45

Isocitrate dehydrogenase 1 (IDH1) mutation has been reported to be associated with an increased overall survival in patients with glioma in a number of studies. Previous studies have focused on the mutation rate and possible metabolic pathways of the mutated IDH1 gene. However, the effects of IDH1 mutation on the biological behavior of glioma cells and the associated mechanisms, as well as the possible effects they may have on clinical therapy, have not been studied. In the present study, three eukaryotic expression vectors were constructed and transfected into the U87 cell line, specifically, a wild-type form of the IDH1 gene with the enhanced green fluorescent protein (EGFP) gene, a mutated IDH1 gene with the EGFP gene and the EGFP gene only. The three stable cell lines were selected using the G418 antibiotic. The biological behaviors of the cell lines were studied and the mechanisms underlying the biological differences between the cell lines were further investigated. The present study confirmed that IDH1 mutation induced cell cycle arrest in the G1 phase and reduced the proportion of the G2/M phase, by downregulating cell division control protein 2 homolog levels, increasing bromodomain-containing protein 2 levels and markedly limiting cell proliferation. IDH1 mutation had no effect on the apoptosis rate under routine culture conditions. Serum chemotaxis assays showed that IDH1 mutation was markedly associated with a significantly reduced invasion ability, by reducing the levels of matrix metalloproteinase (MMP)-2 and MMP-9. From this study, it may be concluded that IDH1 mutation improves prognosis in glioma patients by altering the cell cycle, inhibiting cell proliferation and downregulating cell invasion ability. The results may provide a partial explanation for the improved prognosis of patients with mutated forms of the IDH1 gene.
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PMID:Mechanisms underlying the biological changes induced by isocitrate dehydrogenase-1 mutation in glioma cells. 2452 Feb 88