ABSTRACT
Non-platinum-metal complexes show great potential as anticancer agents. Herein, a series of dithiocarbazate non-Pt-metal complexes, including [FeIII(L)2]·Cl·2H2O 1, [CoIII(L)2]·NO3·2.5H2O 2, [NiII(L)2] 3, and [ZnII(L)2] 4, have been designed and evaluated for their efficacy as antineoplastic agents. Among them, complex 2 exhibited higher anticancer efficacy than complexes 1, 3, 4, and cisplatin against several cancer cell lines. Hemolysis assays revealed that complex 2 showed comparable hemolysis with cisplatin. In vivo anticancer evaluations showed that complex 2 could retard tumor xenograft growth effectively with low systemic toxicity. Further studies revealed that complex 2 suppressed cancer cells by triggering multiple mechanisms involving the simultaneous inhibition of mitochondria and glycolytic bioenergetics. Overall, our study provides new insights into the anticancer mechanism of Co complexes, which can be used as a good strategy to overcome the flexibility of cancer cells to chemotherapy adaptation.
Subject(s)
Antineoplastic Agents , Coordination Complexes , Neoplasms , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cisplatin/pharmacology , Coordination Complexes/pharmacology , Coordination Complexes/therapeutic use , Ferric Compounds/chemistry , Hemolysis , Humans , Neoplasms/drug therapy , Zinc/chemistryABSTRACT
BACKGROUND: Congenital heart disease (CHD) is the most universal congenital defect disease. This study explores the interrelationship between miR-23b and GTAT6 in the development of CHD. METHODS: We collected clinical samples and constructed in vitro cell models to evaluate the expression of miR-23b, GATA6, and IGF-1. CHD cell models were constructed by hypoxia in H9C2 cells. The expression levels of GATA6 and IGF-1 in H9C2 cells were determined by western blot and qPCR. MiR-23b was knocked down by transfection miR-23b inhibitor. GATA6 knockdown or overexpression vectors were established by the lentiviral approach and cell transfection, respectively. According to the CCK-8 assay and flow cytometry analysis, the proliferation and apoptosis of H9C2 cells were detected. The binding relationship between GATA6 and miR-23b was detected by luciferase reporter assay. RESULTS: The expression level of miR-23b was escalated abnormally, while the expression levels of GATA6 and IGF-1 were decreased in the serum of CHD clinical patients and cell models. miR-23b knockdown in H9C2 cells could up-regulate the expression of GATA6, thus improved the proliferation and decreased apoptosis of H9C2 cells. Overexpression of GATA6 could up-regulate IGF-1 to promote proliferation and inhibit apoptosis in H9C2 cells. MiR-23b could target GATA6 and regulated IGF-1, thus affecting cell proliferation and apoptosis. CONCLUSION: The expression level of miR-23b was remarkably up-regulated in serum of CHD patients and H9C2 cells in vitro, while the expression of GATA6 and IGF-1 was significantly decreased. MiR-23b could influence the proliferation and apoptosis of cardiomyocytes by targeting the down-regulation of the GATA6/IGF-1 axis.
Subject(s)
GATA6 Transcription Factor/metabolism , Heart Defects, Congenital , Insulin-Like Growth Factor I/metabolism , MicroRNAs/metabolism , Apoptosis , GATA6 Transcription Factor/genetics , Heart Defects, Congenital/genetics , Heart Defects, Congenital/metabolism , Humans , MicroRNAs/genetics , Myocytes, Cardiac/metabolismABSTRACT
BACKGROUND: This study was conducted to determine DEPDC1 expression in hepatocelluar carcinomas (HCCs) and to reveal its potential role in diagnosis and prognosis of affected patients. MATERIALS AND METHODS: DEPDC1 expression at the mRNA level was detected by quantitative real-time PCR (qRT-PCR) in 205 cases of HCC and paired adjacent normal liver tissues, and by semi-quantitative RT-PCR in 20 cases. Survival curves were obtained by using Kaplan-Meier method and Log-rank test. Independent predictors associated with regard to disease free survival (DFS) and overall survival (OS) were identified using the Cox proportional hazard model. RESULTS: High DEPDC1 mRNA levels were detected in 144 out of 205 cases (70.24%) of HCC, significantly associated with clinicopathological parameters, including tumor size (≥4cm), alpha-fetoprotein (≥100ng/ml), B-C of BCLC stage and recurrence. Kaplan-Meier survival analysis revealed that HCC patients with high DEPDC1 expression had poor OS and DFS. Multivariate analysis demonstrated that high DEPDC1 expression was an independent predictor of OS (HR=1.651; 95% 95%CI, 1.041- 2.617; p=0.033) and DFS (HR=1.583; 95%CI, 1.01- 2.483; p=0.045). CONCLUSIONS: Our results indicate DEPDC1 might be a novel diagnostic marker and an independent prognostic predictor for HCC patients.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Hepatocellular/genetics , GTPase-Activating Proteins/genetics , Liver Neoplasms/genetics , Neoplasm Proteins/genetics , Neoplasm Recurrence, Local/genetics , alpha-Fetoproteins/genetics , Carcinoma, Hepatocellular/mortality , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/therapy , Female , Follow-Up Studies , Humans , Liver/metabolism , Liver Neoplasms/mortality , Liver Neoplasms/pathology , Liver Neoplasms/therapy , Male , Middle Aged , Neoplasm Recurrence, Local/mortality , Neoplasm Recurrence, Local/pathology , Neoplasm Recurrence, Local/therapy , Neoplasm Staging , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Survival RateABSTRACT
OBJECTIVE: To study the effect of gene expression of mouse uroplakin II (UPII) promoter on human bladder cell cancer cell line. METHODS: The mRNA expression of different cell lines was quantified by RT-PCR. Green fluorescent protein (GFP) and luciferase (Luc) were used as reporter genes. The plasmids carrying UPII or GFP were constructed and transfected into human cell lines of bladder transitional cell cancer (BIU-87), kindey cancer (GRC-1), vascular endothelium (EC), lung cancer cell line (A549) and skin fibroblast cell line (Hs27). GFP activity of cells was detected by confocual microscopy and flow cytometry (FCM). Luciferase value was measured by luminometer (microplate) and luciferase to beta-galactosidase ratios (L/G values) were used for evaluating transfection efficiency. RESULTS: RT-PCR showed high expression level of UPII mRNA in bladder cancer cell line BIU-87, whereas low level or no expression in nonbladder cancer cell lines. The activity of GFP in bladder cancer (BIU-87) cell was higher than that in the other cell lines (5 - 10/HP versus 0 - 2/HP), with 4.34% positive cells in BIU-87 detected by FCM, but no positive cell was found in the other cell lines. L/G values indicated that the luciferase expression in human bladder cancer cells transfected with mouse UPII promoter was 1.8 - 8.2-fold as high as that in the nonbladder cell lines. CONCLUSION: Mouse UPII promoter gene can be expressed in a tissue-specific fashion in human urinary bladder cancer. It is capable of initiating transcription of reporter genes in human bladder cancer cell line.
Subject(s)
Membrane Proteins/genetics , Promoter Regions, Genetic , Urinary Bladder Neoplasms/genetics , Animals , Cell Line, Tumor , Flow Cytometry , Genetic Therapy , Green Fluorescent Proteins , Humans , Luminescent Proteins/genetics , Mice , Organ Specificity , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Urinary Bladder Neoplasms/therapy , Uroplakin IIABSTRACT
Tumor rapid growth depends on neovascularization. Vascular endothelial growth factor, the main mediator during the occurrence and formation of vascularization, has specific receptors whose expression rate shows difference of orders of magnitude between tumor and the normal tissue, so it can be used to transport toxin molecules to the proliferative tumor endothelial and kill cancer cells. In our experiment, we constructed fusion protein DT-VEGF by linking diphtheria toxin's forward 389 amino acids's gene and VEGF165 via a linker. DT-VEGF is expressed in E. coli and purified. Our experiment proves in can kill vascular endothelial cells specifically, and the inhibition of neovascularization of chicken chorionic membrane is also confirmed.
Subject(s)
Diphtheria Toxin/genetics , Immunotoxins/genetics , Recombinant Fusion Proteins/biosynthesis , Vascular Endothelial Growth Factors/biosynthesis , Angiogenesis Inhibitors/biosynthesis , Diphtheria Toxin/biosynthesis , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Vectors , Humans , Recombinant Fusion Proteins/genetics , Vascular Endothelial Growth Factors/geneticsABSTRACT
The NF-kappaB transcription factor plays important regulatory roles in inflammation, apoptosis, immune and stress responses. IkappaB kinase (IKK) composed of two catalytic subunits and a regulator subunit, acts as a key component of NF-kappaB activation pathway through phosphorylation of IkappaB, the inhibitor of NF-kappaB. CIKS (connection to IKK and SAPK), a newly identified cellular protein, is involved in NF-kappaB and JNK activation. Although it has been known that CIKS interacts with IKK complex, and activates both IKK and SAPK when overexpressed; the underling mechanisms are poorly understood. To better understand the physiological roles of CIKS, we have screened human HeLa MATCHMAKER cDNA library for new binding partners of CIKS by using the yeast two-hybrid system with truncated CIKS (151-574) as the bait. The yeast strain AH109 was sequentially transformed with the bait plasmid and the library. The transformants were screened on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal)selective plates. Positive clones were restreaked on SD(-Leu/-Trp / + X-alpha-gal)plates three times to allow loss of some of the AD/library plasmids while maintaining selective pressure on both the DNA-BD and AD vectors. After 3 screenings on SD(-Leu/-Trp / + X-alpha-gal), the positive clones were further verified on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal) plates. The inserts in AD/library plasmids were amplified by PCR and PCR products were characterized by Hae III digestion to eliminate the duplicates. After screening in selective plates, the positive AD/library plasmids were rescued via transformation of E. coli HB101 and the interactions of CIKS (151-574) with positive AD/library plasmids were further assessed by yeast two-hybrid analysis. Finally, the DNA sequences of the positive AD/library plasmids were determined and BLAST analysis against the databases was performed. The BLAST results indicate that the inserts in the positive plasmids encode RIKEN cDNA 473340F03, PLAC8, CD27BP (Siva-1), CDC5L, SnRNP smB, and DVL2. CDC5L is a key component of the multi-protein complex essential for the formation of pre-mRNA splicing product and is not required for spliceosome assembly. A role for CDC5L in the cell division cycle has been precious suggested as its overexpression of this protein in mammalian cells leads to a shortening G2 phase of the cell cycle, and a negative-dominant mutant of CDC5L lacking the N-terminal activation domain delays the G2 phase cell's entry into the mitosis. It has been reported that SnRNP smB participates in pre-mRNA splicing and CD27BP (Siva-1) binds to and inhibits BCL-XL-mediated protection against UV radiation-induced apoptosis and regulates T cell homeostasis. The functions of RIKEN cDNA 473340F03, PLAC8 and DVL2 are unknown. It has been suggested that CIKS functions as a critical component for cross-talk between NF-kappaB and JNK signaling pathways. IKK subunits, which have been demonstrated to interact with CIKS, were not shown up in this experiment. We speculate that the truncated CIKS (151-574) bait may not contain the binding domain that mediates the interaction of IKK subunits with CIKS. Taken together, the above results suggest that CIKS may play a role in cell regulation through interacting with various cellular proteins. Further investigations are required to characterize these interactions.
Subject(s)
Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/metabolism , Two-Hybrid System Techniques , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Dishevelled Proteins , Gene Library , HeLa Cells , Humans , Oncogene Proteins/genetics , Oncogene Proteins/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Plasmids/genetics , Polymerase Chain Reaction , Protein Binding/genetics , Protein Binding/physiology , Proteins , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/geneticsABSTRACT
Human tumor necrosis factor alpha (hTNF-alpha) is one of the most important inflammatory cytokines that acts as a mediator in inflammatory and immune response and plays a key role in host defense against infection. The over expression of hTNF-alpha is associated with serious consequences, such as shock, hypotension, thrombus, septicemia and even death. It has been implicated in many autoimmune and inflammatory diseases, such as rheumatoid arthritis, Crohn's disease, chronic heart failure and septic shock. Inhibiting the bio-activity of hTNF-alpha is one of the strategy for the treatment of these diseases. Compared with traditional recombinant protein drugs, small molecule drugs have many advantages, such as high affinity, low immunogenecity and low cost. Systematic evolution of ligands by exponential enrichment (SELEX) is a powerful method for the selection of oligonucleotides that bind with high affinity and specificity to target proteins. Such oligonucleotides are called aptamers, and are potential therapeutics for blocking the activity of pathologically relevant proteins. To obtain oligonucleotide aptamers specifically binding to TNF, a 40nt random DNA combinatorial library flanked by 31nt fixed sequences was chemically synthesized. The random library was amplified with PCR and subjected to selection by SELEX protocol against hTNFalpha. After incubation of the library with hTNFalpha, the mixture was blotted onto Immobilon-NC transfer membrane. The no-specific binding was washed away and the hTNFa binding aptamers were eluted and detached from the target protein. The eluted oligo nucleotides were amplified with PCR and served as the DNA library for the next round selection. After 12 rounds of such selection, the selected aptamers were cloned to pGEM-T vector. Positive clones were identified by restriction enzyme digestion and DNA sequencing. Oligo DNA were synthesized according to the sequence data and tested for their activities. Binding activity of the aptamers to hTNFalpha were detected by ELISA and dot blot with biotin-streptavidin-horseradish peroxidase system. Mouse L929 cells were used to test the anti-hTNFa activity of the DNA aptamers. The aptamers were incubated with hTNFalpha and added to the L929 cells. The results were read under microscope and with MTT staining. It was shown that these DNA aptamers bound to hTNFalpha with high affinity, and can inhibit the cytotoxicity of hTNFalpha on cell culture. The affinity of these aptamers are different and may related to their structure. These ssDNA aptamers are potential for the treatment and diagnosis of hTNFalpha related diseases.
Subject(s)
Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/pharmacology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism , Animals , Aptamers, Nucleotide/chemical synthesis , Aptamers, Nucleotide/genetics , Biotin/chemistry , Cell Line , Cell Survival/drug effects , Enzyme-Linked Immunosorbent Assay , Gene Library , Humans , Mice , Protein Binding , SELEX Aptamer Technique/methods , Tumor Necrosis Factor-alpha/toxicityABSTRACT
Human T-cell leukemia virus type 1 oncoprotein Tax activates NF-kappaB through direct binding to IKK-gamma, the regulatory component of the IkappaB kinase complex. Mechanisms by which IKK-gamma adapts the Tax signal to the IkappaB kinase are poorly understood. Here we demonstrate that IKK-gamma forms homodimer and homotrimer both in vitro and in yeast or mammalian cells through a C-terminal domain comprising amino acids 251-419. In contrast, Tax protein targets a central region of IKK-gamma, which consists of amino acids 201-250. Interestingly, Tax stimulates the oligomerization of IKK-gamma, likely through direct binding. Taken together, our findings suggest a new model of Tax activation of NF-kappaB, in which Tax interacts with IKK-gamma to stimulate its oligomerization.
Subject(s)
Biopolymers/metabolism , Gene Products, tax/physiology , Protein Serine-Threonine Kinases/metabolism , Biopolymers/chemistry , HeLa Cells , Humans , I-kappa B Kinase , Protein Serine-Threonine Kinases/chemistry , Two-Hybrid System TechniquesABSTRACT
Tumor necrosis factor (TNF) is a key cytokine in immunology system and is related to many human diseases. In order to inhibit the activity of TNF, cDNA coding for soluble TNF receptor II (sTNFRII) and human IgG Fc were linked using a flexible hinge. This gene was expressed in E. coli as a chimeric protein and purified by metal chelate chromatography. The results show that the fusion protein exists in the physiological form as a dimer, has the ability to bind with TNF and inhibits the cytotoxicity of TNF on L929 cells. Contrasting to monomer sTNFRII, the chimeric protein has an improved bioactivity, and displays potential prospects for research and application.