RESUMO
BACKGROUND: Concerns about breast cancer had become the most dangerous cancer to women over the world, more and more anti-cancer agents are developed to treat this malignancy. Pharmorubicin is a cytotoxic drug, widely used in the treatment of breast cancer, but its role is limited because of chemoresistance produced by cells. This study focused on exploring the influence of autophagy on the resistance of pharmorubicin in breast cancer cells. METHODS: The cell survival of breast cancer cells was detected by MTT. The mRNA expression of heme oxygenase-1 (HO-1) was tested by qRT-PCR. The protein expression of HO-1, autophagic proteins (LC3-I,LC3-II and Beclin-1), PI3K and Akt was detected by Western blot. Cell autophagy was examined by Cyto-ID Autophagy Detection Kit. RESULTS: After being treated with pharmorubicin, the expression of HO-1 and autophagy related proteins was significantly enhanced, but the cell survival ratio in the two cell lines decreased. After autophagy was inhibited, HO-1 expression in two cells was down-regulated. When pharmorubicin-resistant cells were transfected with si-HO-1, the cell survival decreased and the protein expression of HO-1, autophagic proteins (LC3-II/LC3-I and Beclin-1) as well as autophagy were all down-regulated, while in pharmorubicin-resistant cells transfected with pcDNA3.1-HO-1, the results were reverse. When the PI3K or Akt was inhibited, PI3K, p-Akt, HO-1, autophagic proteins and autophagy were decreased remarkably. CONCLUSION: It was proved that HO-1 induction mediated chemoresistance of pharmorubicin in breast cancer cells by promoting autophagy via PI3K/Akt pathway.
Assuntos
Autofagia/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Epirubicina/farmacologia , Heme Oxigenase-1/genética , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Células MCF-7 , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
The high affinity binding of the anthrax protective antigen (PA) to one of its receptors, capillary morphogenesis protein 2 (CMG2), is essential for the intoxication process of anthrax toxin. To acquire novel research tools to study the PA-CMG2 interaction, we generated several anti-CMG2 monoclonal antibodies (MAbs). We demonstrated that one of the MAbs, 4B5, could inhibit PA-CMG2 binding and could also protect the sensitive cells against an anthrax lethal toxin challenge. We identified the epitope recognized by 4B5 and confirmed that the key residues of the epitope were the residues (119)YI-LK(125) of CMG2. Based on our results, we propose that 4B5 binds to the E122 pocket of CMG2 and interrupts the interaction between the pocket and the PA 2beta3-2beta4 loop. To our knowledge, this is the first report to illustrate that an anti-CMG2 antibody could inhibit the PA-CMG2 interaction and therefore interfere with the intoxication of anthrax toxin.
Assuntos
Anticorpos Monoclonais/imunologia , Toxinas Bacterianas/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Toxinas Bacterianas/química , Toxinas Bacterianas/imunologia , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Estrutura Secundária de Proteína , Receptores de PeptídeosRESUMO
The clinical significance of the dominant-negative Ikaros 6 (DN-IK6) in the treatment of patients with Philadelphia-positive acute lymphoblastic leukemia (Ph+-ALL) with tyrosine kinase inhibitors (TKIs) remains elusive. In the present study, it was demonstrated that DN-IK6 was overexpressed in B-cell (B)-ALL cases compared with T cell-ALL cases at the mRNA and protein levels. Furthermore, nucleotide sequencing revealed that DN-IK6 was due to the deletion of IKAROS family zinc finger 1 exons 4-7. The outcome of patients with Ph+-B-ALL with DN-IK6, and treated with TKIs and hyper-cyclophosphamide/vincristine/doxorubicin/dexamethasone regimen were restrospectively evaluated in a 2 year follow-up. The results demonstrated that those with the DN isoform exhibited significantly lower incidences of remission, shorter median cumulative incidence of relapse times (P<0.05) and shorter median overall survival times (P<0.05) compared with those without the DN isoform. In conclusion, the results of the present study demonstrated that DN-IK6 is overexpressed in the majority of patients with Ph+-ALL, and is significantly associated with resistance to TKI therapy.
RESUMO
Anthrax toxin is the major virulence factor produced by Bacillus anthracis. The toxin consists of three protein subunits: protective antigen (PA), lethal factor, and edema factor. Inhibition of PA binding to its receptors, tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2) can effectively block anthrax intoxication, which is particularly valuable when the toxin has already been overproduced at the late stage of anthrax infection, thus rendering antibiotics ineffectual. Receptor-like agonists, such as the mammalian cell-expressed von Willebrand factor type A (vWA) domain of CMG2 (sCMG2), have demonstrated potency against the anthrax toxin. However, the soluble vWA domain of TEM8 (sTEM8) was ruled out as an anthrax toxin inhibitor candidate due to its inferior affinity to PA. In the present study, we report that L56A, a PA-binding-affinity-elevated mutant of sTEM8, could inhibit anthrax intoxication as effectively as sCMG2 in Fisher 344 rats. Additionally, pharmacokinetics showed that L56A and sTEM8 exhibit advantages over sCMG2 with better lung-targeting and longer plasma retention time, which may contribute to their enhanced protective ability in vivo. Our results suggest that receptor decoys based on TEM8 are promising anthrax toxin inhibitors and, together with the pharmacokinetic studies in this report, may contribute to the development of novel anthrax drugs.
Assuntos
Antitoxinas/farmacologia , Toxinas Bacterianas/antagonistas & inibidores , Receptores de Superfície Celular/química , Receptores de Peptídeos/química , Substituição de Aminoácidos , Animais , Antígenos de Bactérias/metabolismo , Antitoxinas/química , Toxinas Bacterianas/metabolismo , Proteínas Sanguíneas/metabolismo , Linhagem Celular , Rim/efeitos dos fármacos , Rim/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Masculino , Camundongos , Mutação , Ratos , Receptores de Superfície Celular/genética , Receptores de Peptídeos/genética , Solubilidade , Distribuição TecidualRESUMO
PA-binding domain of LF (LFn) or PA-binding domain of EF (EFn) is the anthrax protective antigen (PA) binding domain of anthrax lethal factor (LF) or edema factor (EF). Here we show the development of a novel anthrax toxin inhibitor, fusion protein of N-terminal 27 amino acids deletion of LFn (Delta27LFn) and EFn. In a cell model of intoxication, fusion protein of Delta27LFn and EFn (Delta27LFn-EFn) was a 62-fold more potent toxin inhibitor than LFn or EFn, and this increased activity corresponded to a 39-fold higher PA-binding affinity by Biacore analysis. More importantly, Delta27LFn-EFn could protect the highly susceptible Fischer 344 rats from anthrax lethal toxin challenge. This work suggested that Delta27LFn-EFn has the potential as a candidate therapeutic agent against anthrax.
Assuntos
Toxinas Bacterianas/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/química , Toxinas Bacterianas/toxicidade , Sequência de Bases , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Masculino , Ratos , Ratos Endogâmicos F344RESUMO
Vector based shRNA (short hairpin RNA) expression library has been widely used to screen functional genes. For two main methods that have been used to generate short hairpin RNA libraries, chemical synthesis is too expensive to be widely used and the low efficiency of enzymatic approach makes it difficult to construct. We have developed a protocol to construct a new kind of shRNA library called randomized shRNA library. Within three steps chemically synthesized randomized 19-mers DNA were efficiently converted to double-stranded DNA fragments containing shRNA templates. This kind of shRNA library permits simple and economic construction, providing another choice for whole-genome phenotypic screening of genes.
Assuntos
Biblioteca Gênica , Lentivirus/genética , RNA Viral/genética , RNA/genética , Animais , Células CHO , Clonagem Molecular , Cricetinae , Cricetulus , DNA Viral/síntese química , Humanos , Regiões Promotoras Genéticas , RNA/metabolismo , Interferência de RNARESUMO
AIM: To explore the possibility of using the nontoxic form of anthrax toxin in cancer immunotherapy by LFn-MAGE3 fusion protein expression. METHODS: A fusion expression vector named PET21a-LFn was constructed by inserting LFn coding senquence into PET21a. PET21a-LFn-MAGE3 fusion protein expression vector was constructed by cloning the whole MAGE-3 gene into plasmid PET21a-LFn. Q sepharose FF and Phe HP columns were employed to purify the fusion protein. The biological activity of LFn-MAGE3 was determined by cell test of repressing the cytotoxity of LF and the tests of immunofluscence of mouse macrophage. RESULTS: The resulting plasmid expressed fusion protein LFn-MAGE3 in the soluble form in E.coli BL21, the cell tests showed that purified LFn-MAGE fusion protein was delivered into macrophage effectively with the help of PA(anthrax protective antigen). CONCLUSION: The successful delivery of fusion protein into macrophages coordinated by PA may lay the foundation for its further use in cancer immunotherapy in animal experiments.