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1.
Int J Biol Macromol ; 253(Pt 5): 127742, 2023 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-37923039

RESUMO

Asparaginase has been traditionally applied for only treating acute lymphoblastic leukemia due to its ability to deplete asparagine. However, its ultimate anticancer potential for treating solid tumors has not yet been unleashed. In this study, we bioengineered Erwinia chrysanthemi asparaginase (ErWT), one of the US Food and Drug Administration-approved types of amino acid depleting enzymes, to achieve double amino acid depletions for treating a solid tumor. We constructed a fusion protein by joining an albumin binding domain (ABD) to ErWT via a linker (GGGGS)5 to achieve ABD-ErS5. The ABD could bind to serum albumin to form an albumin-ABD-ErS5 complex, which could avoid renal clearance and escape from anti-drug antibodies, resulting in a remarkably prolonged elimination half-life of ABD-ErS5. Meanwhile, ABD-ErS5 did not only deplete asparagine but also glutamine for ∼2 weeks. A biweekly administration of ABD-ErS5 (1.5 mg/kg) significantly suppressed tumor growth in an MKN-45 gastric cancer xenograft model, demonstrating a novel approach for treating solid tumor depleting asparagine and glutamine. Multiple administrations of ABD-ErS5 did not cause any noticeable histopathological abnormalities of key organs, suggesting the absence of acute toxicity to mice. Our results suggest ABD-ErS5 is a potential therapeutic candidate for treating gastric cancer.


Assuntos
Antineoplásicos , Dickeya chrysanthemi , Neoplasias Gástricas , Humanos , Animais , Camundongos , Asparaginase/genética , Asparaginase/farmacologia , Asparaginase/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Dickeya chrysanthemi/genética , Dickeya chrysanthemi/metabolismo , Asparagina , Glutamina , Neoplasias Gástricas/tratamento farmacológico , Enterobacteriaceae/metabolismo , Albumina Sérica
2.
Invest New Drugs ; 40(5): 895-904, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35857203

RESUMO

Gastric cancer is one of the most common malignant solid tumors in the world, especially in Asia with high mortality due to a lack of effective treatment. The potential usage of the newly constructed arginine-depleting enzyme-mono-PEGylated Bacillus caldovelox arginase mutant (BCA-M-PEG20), an effective drug against multiple cancer cell lines such as cervical and lung cancers, for the treatment of gastric cancer was demonstrated. Our results indicated that BCA-M-PEG20 significantly inhibited argininosuccinate synthetase (ASS)-positive gastric cancer cells, MKN-45 and BGC-823, while another arginine-depleting enzyme, arginine deiminase (ADI, currently under Phase III clinical trial), failed to suppress the growth of gastric cancer cells. In vitro studies demonstrated that BCA-M-PEG20 inhibited MKN-45 cells by inducing autophagy and cell cycle arrest at the S phase under 0.58 U/mL (IC50 values). Significant caspase-dependent apoptosis was induced in MKN-45 after the treatment with 2.32 U/mL of BCA-M-PEG20. In vivo studies showed that administrations of BCA-M-PEG20 at 250 U/mouse twice per week significantly suppressed about 50% of tumor growth in the MKN-45 gastric cancer xenograft model. Taken together, BCA-M-PEG20 demonstrated a superior potential to be an anti-gastric cancer drug.


Assuntos
Neoplasias Pulmonares , Neoplasias Gástricas , Animais , Apoptose , Arginase/farmacologia , Arginina , Autofagia , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Geobacillus , Humanos , Hidrolases/farmacologia , Hidrolases/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Polietilenoglicóis/farmacologia , Polietilenoglicóis/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico
3.
Int J Mol Sci ; 21(20)2020 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-33050217

RESUMO

With our recent success in developing a recombinant human arginase drug against broad-spectrum cancer cell lines, we have explored the potential of a recombinant Bacillus caldovelox arginase mutant (BCA-M) for human cervical cancer treatment. Our studies demonstrated that BCA-M significantly inhibited the growth of human cervical cancer cells in vitro regardless of argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) expression. Drug susceptibilities correlate well with the expressions of major urea cycle genes and completeness of L-arginine regeneration pathways. With the expressions of ASS and ASL genes conferring resistance to L-arginine deiminase (ADI) which is undergoing Phase III clinical trial, BCA-M offers the advantage of a broader spectrum of susceptible cancer cells. Mechanistic studies showed that BCA-M inhibited the growth of human cervical cancer cells by inducing apoptosis and cell cycle arrest at S and/or G2/M phases. Our results also displayed that autophagy served as a protective mechanism, while the growth inhibitory effects of BCA-M could be enhanced synergistically by its combination to the autophagy inhibitor, chloroquine (CQ), on human cervical cancer cells.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Arginase/farmacologia , Autofagia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Geobacillus/enzimologia , Proteínas Recombinantes/farmacologia , Arginase/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Perfilação da Expressão Gênica , Geobacillus/genética , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Proteínas Mutantes , Proteínas Recombinantes/genética , Ureia/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia
4.
Int J Mol Sci ; 21(12)2020 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-32545874

RESUMO

L-arginine (L-Arg) depletion induced by randomly PEGylated arginine deiminase (ADI-PEG20) can treat arginosuccinate synthase (ASS)-negative cancers, and ADI-PEG20 is undergoing phase III clinical trials. Unfortunately, ASS-positive cancers are resistant to ADI-PEG20. Moreover, the yield of ADI production is low because of the formation of inclusion bodies. Here, we report a thermostable arginine-depleting enzyme, Bacillus caldovelox arginase mutant (BCA-M: Ser161->Cys161). An abundant amount of BCA-M was easily obtained via high cell-density fermentation and heat treatment purification. Subsequently, we prepared BCA-M-PEG20, by conjugating a single 20 kDa PEG monomer onto the Cys161 residue via thio-chemistry. Unlike ADI-PEG20, BCA-M-PEG20 significantly inhibited ASS-positive lung cancer cell growth. Pharmacodynamic studies showed that a single intraperitoneal injection (i.p). administration of 250 U/mouse of BCA-M-PEG20 induced low L-Arg level over 168 h. The mono-PEGylation of BCA-M prolonged its elimination half-life from 6.4 to 91.4 h (a 14-fold increase). In an A549 lung cancer xenograft model, a weekly administration of 250 U/mouse of BCA-M-PEG20 suppressed tumor growth significantly. We also observed that BCA-M-PEG20 did not cause any significant safety issue in mouse models. Overall, BCA-M-PEG20 showed excellent results in drug production, potency, and stability. Thereby, it has great potential to become a promising candidate for lung cancer therapy.


Assuntos
Arginase/farmacologia , Geobacillus/enzimologia , Neoplasias Pulmonares/tratamento farmacológico , Mutação , Células A549 , Animais , Arginase/química , Arginase/genética , Arginina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Estabilidade de Medicamentos , Geobacillus/genética , Meia-Vida , Humanos , Hidrolases/administração & dosagem , Hidrolases/farmacologia , Injeções Intraperitoneais , Neoplasias Pulmonares/metabolismo , Camundongos , Modelos Moleculares , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Appl Microbiol Biotechnol ; 104(9): 3921-3934, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32144472

RESUMO

L-Arginine (L-Arg) depletion has attracted great attention in cancer therapy. Although two types of arginine-depleting enzymes, arginine deiminase (ADI) and human arginase I, are undergoing clinical trials, random site of PEGylation, low efficacy of heavy metal as co-factor, and immunogenicity limit the performance of these drugs and cause difficulty in a homogeneous production. Here we screened ten catalytic metal ions and have successfully produced a site-specific mono-PEGylated human arginase I mutant by conjugating the Cys45 residue to PEG-maleimide to minimize the decrease in activity and produce a homogeneous product. The catalytic efficiency trend of metal ion-enriched human arginase I mutant (HAI) was Co2+ > Ni2+ ≫ Mn2+. The overall kcat/KM values of Co-HAI and Ni-HAI were higher than Mn-HAI by ~ 8.7- and ~ 5.2-folds, respectively. Moreover, the results of enzyme kinetics and circular dichroism spectrometry demonstrated that the 20 or 40 kDa linear and branched PEG attached on the HAI surface did not affect the enzyme activity and the protein secondary structures. In vitro studies showed that both Co-HAI-PEG20L and Ni-HAI-PEG20L inhibited the growth of eight types of cancer cell lines. The pharmacodynamic study in mice demonstrated that the i.p. administration of Co-HAI-PEG20L at 13 mg/kg and Ni-HAI-PEG20L at 15 mg/kg was able to maintain a L-Arg level below its detection limit for over 120 h after one injection. The body weights of mice could return to normal levels within 5 days after injection, showing that the doses were well-tolerated. Therefore, both the Ni-HAI-PEG20L and Co-HAI-PEG20L are promising candidates for cancer therapy. KEY POINTS: • Mono-PEGylation applied on human arginase I mutant (HAI) successfully. • The catalytic efficiency of Co- and Ni-enriched HAI was higher than the wild type. • At least eight types of cancer cell lines were inhibited by Co- and Ni-HAI-PEG20L. • Co- and Ni-HAI-PEG20L were able to achieve weekly depletion of L-Arg. Graphical abstract.


Assuntos
Arginase/genética , Arginase/uso terapêutico , Arginina/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Engenharia de Proteínas , Animais , Linhagem Celular Tumoral , Humanos , Íons , Metais , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Estrutura Secundária de Proteína
6.
J Ethnopharmacol ; 122(2): 320-6, 2009 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-19162153

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Agaricus blazei has been used as an adjuvant in cancer chemotherapy and is found to inhibit the growth of various types of tumor cells. AIM OF THE STUDY: Our study has adopted a systematic and bioassay-guided approach to optimize the extraction of Agaricus blazei for anti-leukemic bioactive components. The tumor-selective growth inhibitory activity of the extracts on leukemic cell lines was evaluated in vitro and in vivo using tumor-bearing nude mice. MATERIALS AND METHODS: Agaricus blazei extracts were prepared using different methods. MTT and tritiated thymidine incorporation assays were used to evaluate the in vitro anti-leukemic effects. The most potent extract was further investigated using NB-4 cells-bearing nude mice and mechanistic studies using DNA fragmentation assay and cell death detection ELISA. RESULTS: The JAB80E70 extract showed the most potent tumor-selective growth inhibitory activity against human leukemia NB-4 and K-562 cells. This is the first report of anti-leukemic activity of JAB80E70 in athymic nude mice bearing NB-4 cells. Using DNA fragmentation assays and cell death detection ELISA, JAB80E70 was found to induce apoptosis in NB-4 cells. However, the polysaccharide enriched fractions failed to show significant cytotoxicity on NB-4 cells in vitro. CONCLUSIONS: The JAB80E70 extract exhibited potent anti-leukemic effect in vitro and in vivo. The effect can be attributed, at least in part, to the induction of apoptosis. Besides, polysaccharides in Agaricus blazei may not possess direct anti-leukemic activity in vitro.


Assuntos
Agaricus , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Leucemia Mieloide/tratamento farmacológico , Fitoterapia , Extratos Vegetais/uso terapêutico , Animais , Antineoplásicos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Fígado/citologia , Fígado/efeitos dos fármacos , Camundongos , Camundongos Nus , Extratos Vegetais/farmacologia , Polissacarídeos/farmacologia , Polissacarídeos/uso terapêutico
7.
Oncol Rep ; 16(3): 609-16, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16865263

RESUMO

Coriolus versicolor (CV), also called Yunzhi, has been demonstrated to exert anti-tumor effects on various types of cancer cells. Our previous studies have demonstrated that a standardized aqueous ethanol extract prepared from CV inhibited the proliferation of human leukemia cells via induction of apoptosis. The present study aimed to evaluate the underlying mechanisms of apoptosis through modulation of Bax, Bcl-2 and cytochrome c protein expressions in a human pro-myelocytic leukemia (HL-60) cell line, as well as the potential of the CV extract as anti-leukemia agent using the athymic mouse xenograft model. Our results demonstrated that the CV extract dose-dependently suppressed the proliferation of HL-60 cells (IC50 = 150.6 microg/ml), with increased nucleosome production from apoptotic cells. Expression of pro-apoptotic protein Bax was significantly up-regulated in HL-60 cells treated with the CV extract, especially after 16 and 24 h. Meanwhile, expression of anti-apoptotic protein Bcl-2 was concomitantly down-regulated, as reflected by the increased Bax/Bcl-2 ratio. The CV extract markedly, but transiently, promoted the release of cytochrome c from mitochondria to cytosol after 24-h incubation. In vivo studies in the athymic nude mouse xenograft model also confirmed the growth-inhibitory activity of the CV extract on human leukemia cells. In conclusion, the CV extract attenuated the human leukemia cell proliferation in vivo, and in vitro possibly by inducing apoptosis through the mitochondrial pathway. The CV extract is likely to be valuable for the treatment of some forms of human leukemia.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Leucemia/tratamento farmacológico , Mitocôndrias , Polyporales/química , Animais , Citocromos c/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Células HL-60/efeitos dos fármacos , Células HL-60/metabolismo , Humanos , Leucemia/metabolismo , Leucemia/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transplante Heterólogo , Proteína X Associada a bcl-2/metabolismo
8.
Cancer Biol Ther ; 4(6): 638-44, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15908782

RESUMO

Coriolus versicolor (CV), also called Yunzhi, has been demonstrated to exert anti-tumor effects on various types of cancer cells, but the underlying mechanism has not been fully elucidated. The present study aimed to evaluate the in vitro anti-tumor activity of a standardized aqueous ethanol extract prepared from CV on four breast cancer cell lines using MTT assay, and test whether the mechanism involves apoptosis induction and modulation of p53 and Bcl-2 protein expressions using cell death detection ELISA, p53 and Bcl-2 ELISAs respectively. Our results demonstrated that the CV extract dose-dependently suppressed the proliferation of three breast tumor cell lines, with ascending order of IC50 values: T-47D, MCF-7, MDA-MB-231, while BT-20 cells were not significantly affected. Tumoricidal activity of the CV extract was found to be comparable to a chemotherapeutic anti-cancer drug, mitomycin C. Nucleosome productions in apoptotic MDA-MB-231, MCF-7 and T-47D cells were significantly augmented in a time-dependent manner and paralleled the anti-proliferative activity of CV extract. Expression of p53 protein was significantly upregulated only in T-47D cells treated with the CV extract in a dose- and time-dependent fashion, but not in MCF-7 (except at 400 mug/ml after 16 h) and MDA-MB-231 cells. The CV extract significantly induced a dose-dependent downregulation of Bcl-2 protein expression in MCF-7 and T-47D cells, but not in MDA-MB-231 cells. These results suggested that apoptosis induction, differentially dependent of p53 and Bcl-2 expressions, might be the possible mechanism of CV extract-mediated cytotoxicity in human breast cancer cells in vitro.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Polyporales/química , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Neoplasias da Mama/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Extratos Vegetais/farmacologia , Células Tumorais Cultivadas
9.
Appl Environ Microbiol ; 69(2): 1308-14, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12571064

RESUMO

Here we report the identification of the beta-lactam biosynthesis genes pcbAB and pcbC from a cosmid genomic DNA library of the marine fungus Kallichroma tethys. A BLAST homology search showed that they share high sequence identity with the delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetases and isopenicillin N synthases, respectively, of various fungal and bacterial beta-lactam producers, while phylogenetic analysis indicated a close relationship with homologous genes of the cephalosporin-producing pyrenomycete Acremonium chrysogenum. Expression analysis by reverse transcription-PCR suggested that both genes are highly regulated and are expressed in the late growth phase of K. tethys cultures. Complementation of an Aspergillus nidulans strain deficient in ACV synthetase suggested that at least pcbAB is functional, although attempts to isolate active antibiotic from K. tethys were unsuccessful.


Assuntos
Clonagem Molecular , Hypocreales/enzimologia , Oxirredutases/metabolismo , Peptídeo Sintases/metabolismo , Água do Mar/microbiologia , beta-Lactamas/metabolismo , Sequência de Bases , Meios de Cultura , Regulação Fúngica da Expressão Gênica , Hypocreales/genética , Hypocreales/crescimento & desenvolvimento , Dados de Sequência Molecular , Oxirredutases/genética , Peptídeo Sintases/genética , Filogenia , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Transcrição Gênica
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