RESUMO
BACKGROUND: Cervical cancer is a prevalent and deadly malignancy in females, with chemotherapy often proving ineffective due to significant side effects and the development of chemo-resistance. This study investigates the medicinal potential of Clerodendrum infortunatum linn. , a genus with approximately 500 species in the Lamiaceae family. Limited research exists on the species of Clerodendrum infortunatum and its various solvent extracts. OBJECTIVE: The study aims to assess the anti-cancer properties of different solvent extracts from this plant on human cervical cancer cells. METHODS: The study examines the plant's phytochemical components and their potential to inhibit cancer growth. Aerial parts of the plant were extracted using the Soxhlet method, and the presence of Rutin, Quercetin, and Gallic Acid in specific solvent extracts was validated through High-Performance Thin Layer Chromatography (HPTLC). In vitro assays, including MTT, Apoptosis, Cell Cycle analysis, Intracellular Reactive Oxygen Species assessment, and Gene expression PCR, were conducted to investigate the plant's anti-cancer properties further. RESULTS: The outcomes of the phytochemical assessment indicated that Rutin was predominantly present in the water extract, with quercetin being more concentrated in the decoction, and the hydro-alcoholic extract showing elevated levels of gallic acid. Notably, the decoction extract demonstrated the highest cytotoxic activity, primarily through early apoptosis and arrests in the S-phase and G2M phases. Clerodendrum infortunatum exhibited a reduction in Intracellular Reactive Oxygen Species. The gene expression analysis disclosed an impact on the BCL-2 gene. CONCLUSION: Notably, Clerodendrum infortunatum exhibited the ability to initiate early apoptosis, halt the cell cycle at the S and G2M phases, and diminish levels of reactive oxygen species significantly. The gene expression analysis revealed an influence on the BCL-2 gene. To sum up, this research underscores the encouraging cytotoxic and antioxidant attributes of Clerodendrum infortunatum, implying its potential for cervical cancer treatment.
Assuntos
Clerodendrum , Neoplasias do Colo do Útero , Humanos , Feminino , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Clerodendrum/química , Neoplasias do Colo do Útero/tratamento farmacológico , Solventes , Quercetina/farmacologia , Espécies Reativas de Oxigênio , Compostos Fitoquímicos , Ácido Gálico , RutinaAssuntos
Colágeno Tipo XVIII/fisiologia , Pele/metabolismo , Adolescente , Adulto , Idoso , Animais , Anticorpos/química , Células Cultivadas , Criança , Colágeno Tipo XVIII/biossíntese , Colágeno Tipo XVIII/metabolismo , Método Duplo-Cego , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Meliaceae/química , Pessoa de Meia-Idade , Extratos Vegetais/farmacologia , Pele/crescimento & desenvolvimento , Envelhecimento da Pele/efeitos dos fármacos , Adulto JovemRESUMO
BACKGROUND: Dark circles around the eyes are a complex issue with two main possible causes, the accumulation of melanin in the skin around the eyes and the accumulation of heme resulting from blood leakage. The free heme produced in this manner is highly cytotoxic, proinflammatory and pro-oxidative. AIMS: To evaluate the effect of Fucus extract on heme oxygenase-1 (HO-1) stimulation activity, and to study its in vitro anti-inflammatory, antioxidative, and collagen stimulation activity. METHODS: The HO-1 stimulation activity was first evaluated at gene level by reverse transcriptase- polymerase chain reaction targeting specific HO-1 gene, and then followed by Western blot in protein level. The in vitro anti-inflammatory effect was measured by quantification of interleukin-8 (IL-8) level. The in vitro antioxidative activity was measured. Collagen stimulation activity was quantitatively measured by the amount of deposited collagen I in the extracellular matrix. RESULTS: Fucus extract was identified to have HO-1 stimulation activity at both gene and protein level. By stimulating this enzyme, it promotes the degradation of toxic heme to its protective catabolites (CO, Ferritin, and bilirubin) and reduces the source of dark circles. In addition, Fucus extract showed good anti-inflammatory efficacy. The strong antioxidation property of Fucus extract can reduce eye bags and wrinkles while its collagen boosting activity will potentially reduce fine lines and wrinkles. CONCLUSION: Fucus extract is a novel product that brings a quadruple approach to the treatment of under-eye dark circles.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Extratos Celulares/farmacologia , Fucus/química , Hiperpigmentação/tratamento farmacológico , Pele/efeitos dos fármacos , Compostos de Bifenilo/metabolismo , Linhagem Celular , Colágeno Tipo I/metabolismo , Face , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Heme Oxigenase-1/metabolismo , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Picratos/metabolismo , Pele/irrigação sanguínea , Pele/metabolismoRESUMO
Oligosaccharyl transferase (OT) catalyzes the transfer of a lipid-linked oligosaccharide to the nascent polypeptide emerging from the translocon. Currently, there is no structural information on the membrane-embedded OT complex, which consists of eight different polypeptide chains. We report a 12 A resolution cryo-electron microscopy structure of OT from yeast. We mapped the locations of four essential OT subunits through a maltose-binding protein fusion strategy. OT was found to have a large domain in the lumenal side of endoplasmic reticulum where the catalysis occurs. The lumenal domain mainly comprises the catalytic Stt3p, the donor substrate-recognizing Wbp1p, and the acceptor substrate-recognizing Ost1p. A prominent groove was observed between these subunits, and we propose that the nascent polypeptide from the translocon threads through this groove while being scanned by the Ost1p subunit for the presence of the glycosylation sequon.
Assuntos
Hexosiltransferases/química , Processamento de Imagem Assistida por Computador/métodos , Proteínas de Membrana/química , Proteínas de Transporte/genética , Clonagem Molecular , Microscopia Crioeletrônica , Digitonina/farmacologia , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Imageamento Tridimensional , Proteínas Ligantes de Maltose , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Modelos Moleculares , Peso Molecular , Complexos Multiproteicos/química , Subunidades Proteicas/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Solubilidade/efeitos dos fármacos , Leveduras/enzimologiaRESUMO
The enzyme complex oligosaccharyl transferase (OT) catalyzes N-glycosylation in the lumen of the endoplasmic reticulum. The yeast OT complex is composed of nine subunits, all of which are transmembrane proteins. Several lines of evidence, including our previous split-ubiquitin studies, have suggested an oligomeric organization of the OT complex, but the exact oligomeric nature has been unclear. By FLAG epitope tagging the Ost4p subunit of the OT complex, we purified the OT enzyme complex by using the nondenaturing detergent digitonin and a one-step immunoaffinity technique. The digitonin-solubilized OT complex was catalytically active, and all nine subunits were present in the enzyme complex. The purified OT complex had an apparent mass of approximately 500 kDa, suggesting a dimeric configuration, which was confirmed by biochemical studies. EM showed homogenous individual particles and revealed a dimeric structure of the OT complexes that was consistent with our biochemical studies. A 3D structure of the dimeric OT complex at 25-A resolution was reconstructed from EM images. We suggest that the dimeric structure of OT might be required for effective association with the translocon dimer and for its allosteric regulation during cotranslational glycosylation.
Assuntos
Hexosiltransferases/química , Proteínas de Membrana/química , Estrutura Quaternária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Animais , Dimerização , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Processamento de Imagem Assistida por Computador , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Modelos Moleculares , Complexos Multiproteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Protein translocation and N-glycosylation are essential coordinated cellular processes that are mediated by the translocon and the oligosaccharyl transferase (OT), respectively. The recent identification of several specific interactions between the OT subunits and the translocon provides a molecular basis for the coupling of these two processes. Data suggest that multiple OT isoforms with different affinities for the translocon and ribosome and with heterogeneous subunit composition might exist in the endoplasmic reticulum (ER) membrane, thereby providing a means of regulating protein N-glycosylation.
Assuntos
Glicoproteínas/metabolismo , Transporte Proteico , Aciltransferases/química , Aciltransferases/metabolismo , Glicosilação , Cinética , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/metabolismoRESUMO
Within hours of turpentine injection to stimulate the acute phase (AP) response in rats, the N-acetylneuraminic acid content of plasma proteins increases and that of fucose decreases, each by about 60%. The two changes are inversely related (r = -0.97). The NeuAc/Gal ratio increases from the normal 0.75 to 1.0 on day 2 of the AP. Whereas 50% of the isolated oligosaccharides of normal plasma proteins are retarded on immobilized Ricinus communis agglutinin, those from day 2 AP plasma fail to do so. This indicates that NeuAc caps the normally Gal-terminated chains. alpha1-Acid glycoprotein (a positive AP protein), alpha1-macroglobulin (a non-AP protein), and alpha1-inhibitor3 (a negative AP protein) also show similar alterations in NeuAc/Gal ratio and decreases in Fuc. alpha2-Macroglobulin, which arises only during the AP, does not contain significant amounts of Fuc. Sambucus nigra agglutinin (alpha2,6-linked NeuAc-specific) binds a majority of plasma proteins, and binding is increased during the AP response. Maackia amurensis lectin (alpha2,3-linked NeuAc-specific) binds only three proteins in normal plasma and three additional proteins in AP plasma. The Fuc-specific Aleuria aurantia agglutinin and Lens culinaris agglutinin each detect five proteins in normal plasma. Their binding decreases during the AP response. These results show that: (1) sialylation and defucosylation of preexisting plasma proteins occur rapidly in the AP response; (2) sialylation caps the preexisting Gal-terminating oligosaccharides; and (3) the oligosaccharides of even the non-AP and negative AP proteins are modified. These changes are distinct from the elevation in the levels of protein-bound monosaccharides and the altered concanavalin A-binding profile the oligosaccharides of AP proteins acquire in diseases.
Assuntos
Reação de Fase Aguda/sangue , Proteínas Sanguíneas/análise , Fucose/sangue , Glicoproteínas/sangue , Ácido N-Acetilneuramínico/sangue , Processamento de Proteína Pós-Traducional , Reação de Fase Aguda/induzido quimicamente , Animais , Fucose/química , Glicoproteínas/química , Glicosilação , Ácido N-Acetilneuramínico/química , Ratos , Ratos WistarRESUMO
Following initiation of translocation across the membrane of the endoplasmic reticulum via the translocon, polypeptide chains are N-glycosylated by the oligosaccharyl transferase (OT) enzyme complex. Translocation and N-glycosylation are concurrent events and would be expected to require juxtaposition of the translocon and the OT complex. To determine whether any of the subunits of the OT complex and translocon mediate interactions between the two complexes, we initiated a systematic study in budding yeast using the split-ubiquitin approach. Interestingly, the OT subunit Stt3p was found to interact only with Sec61p, whereas another OT subunit, Ost4p, was found to interact with all three components of the translocon, Sec61p, Sbh1p, and Sss1p. The OT subunit Wbp1p was found to interact very strongly with Sec61p and Sbh1p and weakly with Sss1p. Other OT subunits, Ost1p, Ost2p, and Swp1p were found to interact with Sec61p and either Sbh1p or Sss1p. Ost3p exhibited a weak interaction with Sec61p and Sbh1p, whereas Ost5p and Ost6p interacted very weakly with Sec61p and failed to interact with Sbh1p or Sss1p. We were able to confirm these split-ubiquitin findings by a chemical cross-linking technique. Based on our findings using these two techniques, we conclude that the association of these two complexes is stabilized via multiple protein-protein contacts. Based on extrapolation of the structural parameters of the crystal structure of the prokaryotic Sec complex to the eukaryotic complex, we propose a working model to understand the organization of the translocon-OT supercomplex.
Assuntos
Retículo Endoplasmático/metabolismo , Hexosiltransferases/química , Proteínas de Membrana/química , Reagentes de Ligações Cruzadas/farmacologia , Cristalografia por Raios X , Galactosidases/metabolismo , Genótipo , Glicosilação , Hexosiltransferases/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Modelos Biológicos , Peptídeos/química , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Canais de Translocação SEC , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo , Ubiquitina/metabolismo , Proteínas de Transporte VesicularRESUMO
Recent evidence indicates that Stt3p plays a central role in the recognition and/or catalytic step in N-glycosylation (asparagine-linked glycosylation) in the lumen of the endoplasmic reticulum. It is known that stt3 mutants exhibit certain phenotypic features that are suggestive of a cell wall defect. To understand the basis of these phenotypes, we devised a genetic screen to isolate strains bearing mutations that lead to synthetic lethality in combination with the stt3-1 mutation. Using this screen, we were surprised to identify two KRE genes (KRE5 and KRE9) that are involved in the biosynthesis of the cell wall beta1,6-glucan. This finding led us to propose that the N-glycosylation process is essential in the biosynthesis of cell wall beta1,6-glucan. This proposal was supported by the observation that several stt3 mutants exhibited a 60-70% reduction in the content of cell wall beta1,6-glucan as compared with WT cells. Transmission electron microscopy revealed that the stt3 mutant strains exhibit a diffused cell wall with loss of the outer mannoprotein layer as compared with the WT cells. Thus, we provide genetic, morphological, and biochemical evidence for the critical involvement of N-glycosylation in some step in assembly of the cell wall beta1,6-glucan in Saccharomyces cerevisiae.
Assuntos
Parede Celular/metabolismo , Glucanos/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , beta-Glucanas , Sequência de Bases , Clonagem Molecular , Primers do DNA , Escherichia coli/genética , Genes Letais , Glucanos/genética , Glicoproteínas/genética , Glicosilação , Mutação , Fenótipo , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Over a decade ago, the gene STT3 was identified in a staurosporine and temperature sensitivity screen of yeast. Subsequently the product of this gene was shown to be a subunit of the endoplasmic reticulum-localized oligosaccharyl transferase (OT) complex. Although stt3 mutants are known to be staurosporine-sensitive, we found that mutants of other OT subunits (except ost4 Delta) are staurosporine-resistant, which indicates that this phenotype of stt3 mutants is not simply a consequence of their defect in glycosylation, as previously speculated. Staurosporine sensitivity was found to be an allele-specific phenotype restricted to cells harboring mutations in highly conserved residues in the N-terminal domain of the STT3 protein. Cells bearing mutations in one of the cytosolic-oriented loops (amino acids 158-168) in the N terminus of Stt3p were found to be specifically susceptible to staurosporine. Staurosporine is a specific inhibitor of Pkc1p, and a genetic link had previously been suggested between PKC1 and STT3. It is known that overexpression of PKC1 suppresses the staurosporine sensitivity of the stt3 mutants in an allele-specific manner, which is typical of mutants of Pkc1p cascade. It has been shown that the pkc1 null mutant exhibits lowered OT activity. Our results combined with these previous observations indicate that the N-terminal domain of Stt3p may interact with members of the Pkc1p cascade and consequently mutations in this domain result in staurosporine sensitivity. We further speculate that the Pkc1p regulates OT activity through the N-terminal domain of Stt3p, the C-terminal domain of which possesses the recognition and/or catalytic site of the OT complex.