RESUMO
Among various homing devices, gonadotropin-releasing hormone-III (GnRH-III) peptide represents a suitable targeting moiety for drug delivery systems. The anti-tumor activity of the previously developed GnRH-III-[4Lys(Bu),8Lys(Dau=Aoa)] conjugate and the novel synthesized GnRH-III-[2ΔHis,3d-Tic,4Lys(Bu),8Lys(Dau=Aoa)] conjugate, containing the anti-cancer drug daunorubicin, were evaluated. Here, we demonstrate that both GnRH-III-Dau conjugates possess an efficient growth inhibitory effect on more than 20 cancer cell lines, whereby the biological activity is strongly connected to the expression of gonadotropin-releasing hormone receptors (GnRH-R). The novel conjugate showed a higher in vitro anti-proliferative activity and a higher uptake capacity. Moreover, the treatment with GnRH-III-Dau conjugates cause a significant in vivo tumor growth and metastases inhibitory effect in three different orthotopic models, including 4T1 mice and MDA-MB-231 human breast carcinoma, as well as HT-29 human colorectal cancer bearing BALB/s and SCID mice, while toxic side-effects were substantially reduced in comparison to the treatment with the free drug. These findings illustrate that our novel lead compound is a highly promising candidate for targeted tumor therapy in both colon cancer and metastatic breast cancer.
Assuntos
Antineoplásicos/farmacologia , Daunorrubicina/análogos & derivados , Daunorrubicina/farmacologia , Hormônio Liberador de Gonadotropina , Ácido Pirrolidonocarboxílico/análogos & derivados , Animais , Antineoplásicos/química , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Daunorrubicina/química , Modelos Animais de Doenças , Feminino , Expressão Gênica , Hormônio Liberador de Gonadotropina/química , Humanos , Masculino , Camundongos , Estrutura Molecular , Ácido Pirrolidonocarboxílico/química , RNA Mensageiro/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Testes de Toxicidade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Compared to classical chemotherapy, peptide-based drug targeting is a promising therapeutic approach for cancer, which can provide increased selectivity and decreased side effects to anticancer drugs. Among various homing devices, gonadotropin-releasing hormone-III (GnRH-III) peptide represents a suitable targeting moiety, in particular in the treatment of hormone independent tumors that highly express GnRH receptors (e.g. colon carcinoma). We have previously shown that GnRH-III[(4)Lys(Ac),(8)Lys(Dau = Aoa)] bioconjugate, in which daunorubicin was attached via oxime linkage to the (8)Lys of a GnRH-III derivative, exerted significant in vivo antitumor effect on subcutaneously developed HT-29 colon tumor. In contrast, results of the study reported here indicated that this compound was not active on an orthotopically developed tumor. However, if Lys in position 4 was acylated with butyric acid instead of acetic acid, the resulting bioconjugate GnRH-III[(4)Lys(Bu),(8)Lys(Dau = Aoa)] had significant tumor growth inhibitory effect. Furthermore, it prevented tumor neovascularization, without detectable side effects. Nevertheless, the development of metastases could not be inhibited by the bioconjugate; therefore, its application in combination with a metastasis preventive agent might be necessary in order to achieve complete tumor remission. In spite of this result, the treatment with GnRH-III[(4)Lys(Bu),(8)Lys(Dau = Aoa)] bioconjugate proved to have significant benefits over the administration of free daunorubicin, which was used at the maximum tolerated dose.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Ácido Butírico/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Daunorrubicina/uso terapêutico , Hormônio Liberador de Gonadotropina/uso terapêutico , Ácido Pirrolidonocarboxílico/análogos & derivados , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/toxicidade , Ácido Butírico/química , Ácido Butírico/toxicidade , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Daunorrubicina/química , Daunorrubicina/toxicidade , Feminino , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/toxicidade , Células HT29 , Coração/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/uso terapêutico , Ácido Pirrolidonocarboxílico/toxicidade , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeted tumor therapy is a perspective procedure to specifically destroy the cancer tissues with eliminating or at least decreasing the side effects of anticancer drugs. For this purpose the drug molecule is attached to a targeting moiety (e.g. peptide hormones) that recognizes tumor specific or overexpressed receptors on cancer cells. The in vitro cytostatic or cytotoxic assays do not give proper information whether the tumor growth inhibitory effect of the conjugate is better than the activity of the free drug. Only in vivo studies are adequate to answer this question. However, the selection of the appropriate tumor model is important to eliminate the false positive results. In our studies a gonadotropin-releasing hormone analog (GnRH-III) was applied as targeting moiety in drug conjugates. The in vivo antitumor activity of these conjugates was investigated on mice bearing subcutaneously or orthotopically szigdeveloped tumors. The subcutaneously implanted tumor model which is isolated from its surroundings may provide false results in tumor growth inhibition. In contrast, the orthotopically developed tumor is a better model representing appropriate anatomical and clinical status of cancer. Therefore, the orthotopical colon cancer developed in our laboratory is a suitable model for the study of the antitumor activity of the conjugates prepared for targeted tumor therapy.
RESUMO
Targeted cancer chemotherapy is a novel approach developed for the specific delivery of anticancer drugs. Tumour targeting can be achieved by combining a chemotherapeutic agent with a targeting moiety that recognizes tumour-specific or highly expressed receptors on cancer cells. We used the gonadotropin-releasing hormone-III (GnRH-III) as a targeting moiety to which the chemotherapeutic agent daunorubicin (Dau) was attached through an oxime bond either directly or by inserting a GFLG tetrapeptide spacer. The in-vivo toxicity of Dau-GnRH-III derivative conjugates was evaluated on healthy BDF-1 female mice, and their tumour growth inhibitory effect was determined on C26 murine and HT-29 human colon carcinoma-bearing mice. Both oxime bond-containing conjugates were well tolerated and exerted significant antitumour activity on C26 colon carcinoma-bearing mice at a dose of 30 mg Dau content in conjugate/kg body weight. Furthermore, the conjugates inhibited the tumour growth more than the free drug at a dose that was still not toxic. Similar tumour growth inhibitory effects were obtained on HT-29 human colon carcinoma-bearing mice using three treatments with 15 mg Dau content in conjugate/kg. The tumour growth inhibitions according to the tumour volume and the tumour weight were 44/41% and 58/50%, respectively. Considering the results, both of the investigated Dau-GnRH-III derivative conjugates were well tolerated and had significant antitumour effect on colon carcinoma-bearing mice.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Daunorrubicina/farmacologia , Hormônio Liberador de Gonadotropina/farmacologia , Ácido Pirrolidonocarboxílico/análogos & derivados , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Daunorrubicina/efeitos adversos , Daunorrubicina/química , Relação Dose-Resposta a Droga , Feminino , Hormônio Liberador de Gonadotropina/efeitos adversos , Hormônio Liberador de Gonadotropina/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Camundongos SCID , Ácido Pirrolidonocarboxílico/efeitos adversos , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Here, we report on the synthesis, enzymatic stability, and antitumor activity of novel bioconjugates containing the chemotherapeutic agent daunorubicin attached through an oxime bond to various gonadotropin-releasing hormone-III (GnRH-III) derivatives. In order to increase the enzymatic stability of the bioconjugates (in particular against chymotrypsin), (4)Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which (4)Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon, and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their stability/degradation (1) in human serum, (2) in the presence of rat liver lysosomal homogenate, and (3) in the presence of digestive enzymes (trypsin, chymotrypsin, and pepsin) was analyzed by liquid chromatography in combination with mass spectrometry. The results showed that (1) all synthesized bioconjugates had in vitro cytostatic effect, (2) they were stable in human serum at least for 24 h, and (3) they were hydrolyzed in the presence of lysosomal homogenate. All compounds were stable in the presence of (1) pepsin and (2) trypsin (except for the (4)Lys containing bioconjugate). In the presence of chymotrypsin, all bioconjugates were digested; the degradation rate strongly depending on their structure. The bioconjugates in which (4)Ser was replaced by N-Me-Ser or Lys(Ac) had the highest enzymatic stability, making them potential candidates for oral administration. In vivo tumor growth inhibitory effect of two selected bioconjugates was evaluated on orthotopically developed C26 murine colon carcinoma bearing mice. The results indicated that the compound containing Lys(Ac) in position 4 had significantly higher antitumor activity than the parent bioconjugate.
Assuntos
Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/uso terapêutico , Daunorrubicina/metabolismo , Daunorrubicina/uso terapêutico , Hormônio Liberador de Gonadotropina/metabolismo , Hormônio Liberador de Gonadotropina/uso terapêutico , Neoplasias/tratamento farmacológico , Ácido Pirrolidonocarboxílico/análogos & derivados , Sequência de Aminoácidos , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Daunorrubicina/química , Daunorrubicina/farmacologia , Estabilidade de Medicamentos , Feminino , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Secundária de Proteína , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/metabolismo , Ácido Pirrolidonocarboxílico/farmacologia , Ácido Pirrolidonocarboxílico/uso terapêutico , Ratos , Soro/metabolismoRESUMO
Here, we report on the synthesis and biological properties of a conjugate in which daunorubicin (Dau) as chemotherapeutic agent was attached through an oxime bond to gonadotropin-releasing hormone-III (GnRH-III) as targeting moiety. In vitro toxicity and the cytostatic effect of the conjugate on MCF-7 human breast and C26 murine colon cancer cell lines were determined, and the results were compared with those obtained for the free daunorubicin, as well as with the doxorubicin containing derivative. In vivo antitumor effect of daunorubicin-GnRH-III was studied on Balb/c female mice transplanted with C26 tumor. Our data indicate that the daunorubicin-GnRH-III conjugate had a lower toxic effect than the free daunorubicin and it was essentially nontoxic up to 15 mg (Dau content)/kg body weight. The treatment of the C26 tumor bearing mice with the conjugate led to tumor growth inhibition and longer survival time in comparison with the controls and with the administration of the free drug. When mice were treated twice with the conjugate (on days 4 and 7 after tumor transplantation), 46% tumor growth inhibition was obtained. In this case, the increase of the median survival time was 38% compared to the controls.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Daunorrubicina/química , Daunorrubicina/farmacologia , Hormônio Liberador de Gonadotropina/química , Oximas/química , Sequência de Aminoácidos , Animais , Antineoplásicos/metabolismo , Antineoplásicos/toxicidade , Catepsina B/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Citostáticos/química , Citostáticos/metabolismo , Citostáticos/farmacologia , Citostáticos/toxicidade , Daunorrubicina/metabolismo , Daunorrubicina/toxicidade , Feminino , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: The aim of this study was to determine whether tumor-associated immune cells may predict response to therapy and disease outcome in head and neck squamous cell carcinoma (HNSCC) patients receiving induction chemotherapy and cetuximab. METHODS: Paraffin-embedded pretreatment biopsy samples from 45 patients with stage III-IV resectable HNSCC were investigated retrospectively by immunohistochemistry for density of different immune cell types based on expression of CD8, FOXP3, CD134, CD137, PD-1, CD20, NKp46, dendritic cell lysosomal-associated membrane protein (DC-LAMP), CD16, CD68, and myeloperoxidase. Results were analyzed for possible correlations with clinicopathologic parameters, response to therapy, and survival. RESULTS: Of the immune cell types studied, we found significant association with response to induction chemotherapy only in the case of DC-LAMP+ mature dendritic cells and PD-1+ lymphocytes; density of DC-LAMP+ cells also correlated with progression-free survival. CONCLUSION: DC-LAMP+ mature dendritic cells and PD-1+ cells may be implicated in response to induction chemotherapy and cetuximab in HNSCC patients.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Cetuximab/uso terapêutico , Células Dendríticas/imunologia , Neoplasias de Cabeça e Pescoço/imunologia , Linfócitos , Receptor de Morte Celular Programada 1/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Adulto , Idoso , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Biópsia , Feminino , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , Quimioterapia de Indução , Linfócitos/imunologia , Linfócitos/fisiologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológicoRESUMO
Development of peptide-based conjugates for targeted tumour therapy is a current research topic providing new possibilities in cancer treatment. In this study, VHLGYAT heptapeptide selected by phage display technique for HT-29 human colon cancer was investigated as homing peptide for drug delivery. Daunomycin was conjugated to the N-terminus of the peptide directly or through Cathepsin B cleavable spacers. Conjugates showed moderate in vitro cytostatic effect. Therefore, sequence modifications were performed by Ala-scan and positional scanning resulting in conjugates with much higher bioactivity. Conjugates in which Gly was replaced by amino acids with bulky apolaric side chains provided the best efficacy. The influence of the cellular uptake, stability and drug release on the anti-tumour activity was investigated. It was found that mainly the difference in the cellular uptake of the conjugates generated the distinct effect on cell viability. One of the most efficient conjugate Dauâ¯=â¯Aoa-LRRY-VHLFYAT-NH2 showed tumour growth inhibition on orthotopically developed HT-29 colon cancer in mice with negligible toxic side effect compared to the free drug. We also indicate that this sequence is not specific to HT-29â¯cells, but it has a remarkable effect on many other cancer cells. Nevertheless, the Phe-containing conjugate was more active in all cases compared to the conjugate with the parent sequence. The literature data suggested that this sequence is highly overlapped with peptides that recognize Hsp70 membrane bound protein overexpressed in many types of tumours.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Daunorrubicina/análogos & derivados , Daunorrubicina/uso terapêutico , Oligopeptídeos/uso terapêutico , Pró-Fármacos/uso terapêutico , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Catepsina B/metabolismo , Proliferação de Células/efeitos dos fármacos , Técnicas de Visualização da Superfície Celular/métodos , Daunorrubicina/metabolismo , Sistemas de Liberação de Medicamentos , Liberação Controlada de Fármacos , Feminino , Células HT29 , Humanos , Camundongos SCID , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Pró-Fármacos/síntese química , Pró-Fármacos/metabolismo , Proteólise , Ratos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Direct antitumor activity of sea lamprey (Petromyzon marinus) gonadotropin-releasing hormone III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2); lGnRH-III) was described on several tumor cells. To improve the selectivity of antitumor effects without increasing the hormone releasing activity and to enhance the enzymatic stability, lGnRH-III dimers were prepared via disulfide bond formation. Our results demonstrate that the lGnRH-III dimer derivatives exhibited higher antiproliferative effect and enzymatic stability in comparison with the native lGnRH-III, while lower LH-releasing potency was determined. In order to find a correlation between the biological and structural features of these compounds, the conformation of lGnRH-III and its dimer derivatives was determined by ECD, VCD, FT-IR and (1)H NMR.
Assuntos
Proliferação de Células/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/farmacologia , Lampreias/metabolismo , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Ácido Pirrolidonocarboxílico/análogos & derivados , Sequência de Aminoácidos , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Quimotripsina/metabolismo , Dicroísmo Circular , Simulação por Computador , Dimerização , Estabilidade Enzimática , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Modelos Químicos , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Ligação Proteica , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/metabolismo , Ácido Pirrolidonocarboxílico/farmacologia , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
BACKGROUND: We investigated in postmenopausal women with primary breast cancer prior to surgical intervention whether, serum levels of different steroid hormones and hormonal precursors associated with tumor tissue estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) status. METHODS: We enrolled 1,042 patients suffering invasive ductal carcinoma undergoing surgical resection in the National Institute of Oncology, Hungary between 2003 and 2011. Serum parameters were measured by RIA/IRMA assays; tumor tissue ER, PR and HER2 status was assessed histologically. Patients were classified according to tumor receptor status. Case-case analysis subjects were categorized into four subgroups based on serum hormone concentrations in ER, PR and HER2 receptor-negative cases, respectively. RESULTS: Serum estrone sulfate and dehydroepiandrosterone sulfate levels correlated with each other and also with serum estrone and estradiol levels. According to case-case study the odds ratios in the highest quartile were 1.517 (p = 0.0305, Ptrend = 0.0394) for androstenedione, 1.495 (p = 0.0317, Ptrend < 0.0105) for estrone and 0.654 (p = 0.0273, Ptrend < 0.0151) for estrone sulfate/estrone ratio in PR+ vs. PR- tumors. Regarding HER2 status (HER2+ vs. HER2-), the odds ratios for estrone, estrone sulfate and estrone sulfate/estrone ratio were 0.530 (p = 0.0234, Ptrend = 0.0595), 2.438 (p = 0.0042, Ptrend < 0.0066) and 3.118 (p = 0.0001, Ptrend < 0.0001) in the highest quartile, respectively. Of note significantly increased BMI associates with PR+ and ER +/PR+ status while significantly decreased BMI was observed in HER2+ cases. CONCLUSIONS: Taken together, measurement of serum estrone and estrone sulfate concentrations prior to surgical intervention might support the individualization of regime in postmenopausal primary breast cancer patients.
RESUMO
Applications of cysteine-insertion and thioether linkage approaches to the preparation of a number of bioactive peptide conjugates are reported. Peptides containing epitopes from (i) herpes simplex virus type 1 glycoprotein D, (ii) a specific N-terminal beta-amyloid epitope recognized by therapeutically active antibodies, and (iii) a GnRH-III peptide from sea lamprey with antitumour activity, were elongated with Cys residues and attached to a chloroacetylated tetratuftsin derivative carrier via a thioether linkage either directly, or by insertion of a spacer. The structures and molecular homogeneity of all the peptide conjugates were ascertained by HPLC, MALDI and electrospray mass spectrometry. The use of a spacer such as an oligoglycine or GFLG-tetrapeptide gave an increased yield in the conjugation reaction and enhanced reaction rates. In the formation of cysteinyl-thioether linkages, it was found that the position of flanking Cys residues markedly influenced the conjugation reaction and the formation of intermolecular epitope disulfide-dimers. C-terminal Cys residues gave thioether conjugates with significantly diminished epitope-dimerization, while Cys at the N-terminal caused rapid disulfide-dimerization, thereby preventing efficient conjugation.