RESUMO
It was aimed at exploring the potential pharmacological effects of alkaloids in Sophora alopecuroides by means of network pharmacology in this study. The main alkaloids in S. alopecuroides were collected for analysis of drug properties, prediction of potential targets and screening of signaling pathways. DAVID analysis tool combined with KEGG database was used to annotate and analyze the signaling pathway. The alkaloids-targets-signaling pathways network was built through Cytoscape software. Results showed that 17 alkaloids in S. alopecuroides involved 49 targets (170 times in all) and 22 important signaling pathways. Three nodes in model of network pharmacology were cross-linked, and the metabolic pathways were coordinated and regulated by each other. It indicated that alkaloids in S. alopecuroides may have therapeutic effect on diseases of cancer, metabolic disorder, endocrine system, digestive system, nervous system and so on.
Assuntos
Alcaloides/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sophora/química , Compostos Fitoquímicos/farmacologiaRESUMO
Previous studies have shown that intensive macropinocytosis occurs in cancer cells and neutral red (NR) is noted for its capability to enter into the cell massively through a process mimetic to macropinocytosis. In addition, trans-cinnamic acid (tCA) has been found to be an inhibitor of histone deacetylase (HDAC). In the present study, cinnamoylphenazine (CA-PZ) that consists of NR and tCA moieties was synthesized and evaluated. As shown, CA-PZ massively entered into colon carcinoma HT-29 cells and pancreatic carcinoma MIA PaCa-2 cells and this entry was blocked by 5-(N-ethyl-N-isopropyl) amiloride (EIPA, an inhibitor of macropinocytosis), indicating a macropinocytosis-mediated uptake. Furthermore, CA-PZ markedly increased the protein expression levels of acetyl-H3, acetyl-H4 and p21 in HT-29 cells and MIA PaCa-2 cells. CA-PZ significantly inhibited the growth of colon carcinoma HT-29 and pancreatic carcinoma MIA PaCa-2 xenografts. By in vivo imaging, CA-PZ displayed prominent accumulation in the tumor xenografts. The study indicates that the newly synthesized CA-PZ acts as an HDAC inhibitor in association with intensive macropinocytosis-mediated intracellular delivery in cancer cells. The use of neutral red for preparation of chimeric molecules with the attribute of macropinocytosis-mediated intracellular delivery might open an alternative way for development of HDAC inhibitors.
Assuntos
Cinamatos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Inibidores de Histona Desacetilases/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Fenazinas/farmacologia , Pinocitose/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Células A549 , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Western Blotting , Linhagem Celular Tumoral , Cinamatos/síntese química , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Bloqueadores do Canal de Sódio Epitelial/farmacologia , Feminino , Células HCT116 , Células HT29 , Inibidores de Histona Desacetilases/síntese química , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia Confocal , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fenazinas/síntese químicaRESUMO
K-Ras mutant pancreatic cancer cells display intensive macropinocytosis, indicating that this process may be exploited in the design of anticancer targeted therapies. In this study, we constructed a macropinocytosis-oriented recombinantly tailored defensin (DF-HSA) which consists of human ß-defensin-2 (DF) and human serum albumin (HSA). The macropinocytosis intensity and cytotoxicity of DF-HSA were investigated in K-Ras mutant MIA PaCa-2 cells and wild-type BxPC-3 cells. As found, the DF-HSA uptake in MIA PaCa-2 cells was much higher than that in wild-type BxPC-3 cells. Correspondingly, the cytotoxicity of DF-HSA to MIA PaCa-2 cells was more potent than that to BxPC-3 cells. In addition, the cytotoxicity of DF-HSA was much stronger than that of ß-defensin HBD2. DF-HSA suppressed cancer cell proliferation and induced mitochondrial pathway apoptosis. Notably, DF-HSA significantly inhibited the growth of human pancreatic carcinoma MIA PaCa-2 xenograft in athymic mice at well tolerated dose. By in vivo imaging, DF-HSA displayed a prominent accumulation in the tumor. The study indicates that the recombinantly tailored ß-defensin can intensively enter into the K-Ras mutant pancreatic cancer cells through macropinocytosis-mediated process and exert potent therapeutic efficacy against the pancreatic carcinoma xenograft. The novel format of ß-defensin may play an active role in macropinocytosis-mediated targeting therapy.
Assuntos
Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Pinocitose , Proteínas Proto-Oncogênicas p21(ras)/genética , Albumina Sérica Humana/metabolismo , beta-Defensinas/metabolismo , Células A549 , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Genes ras , Vetores Genéticos , Humanos , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mutação , Transplante de Neoplasias , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
Multidrug resistance is a major limitation for microtubule-binding agents in cancer treatment. Here we report a novel microtubule inhibitor (2-morpholin-4-yl-5-nitro-benzoic acid 4-methylsulfanyl-benzyl ester, IMB5046), its cytotoxicity against multidrug-resistant cell lines and its antitumor efficacy in animal models. IMB5046 disrupted microtubule structures in cells and inhibited purified tubulin polymerization in vitro. It bound to the colchicine pocket of tubulin. IMB5046 displayed potent cytotoxicity against multiple tumor cell lines with an IC50 range of 0.037-0.426 µM. Notably, several multidrug-resistant cell lines which were resistant to colchicine, vincristine and paclitaxel remained sensitive to IMB5046. IMB5046 was not a P-glycoprotein substrate. IMB5046 blocked cell cycle at G2/M phase and induced cell apoptosis. Microarray assay indicated that the differentially expressed genes after IMB5046 treatment were highly related to immune system, cell death and cancer. In a mouse xenograft model IMB5046 inhibited the growth of human lung tumor xenograft by 83% at a well-tolerated dose. It is concluded that IMB5046 is a tubulin polymerization inhibitor with novel chemical structure and can overcome multidrug resistance. It is a promising lead compound for cancer chemotherapy, especially for treatment of multidrug-resistant tumors.
Assuntos
Benzoatos/administração & dosagem , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Morfolinas/administração & dosagem , Neoplasias/tratamento farmacológico , Nitrobenzoatos/administração & dosagem , Moduladores de Tubulina/administração & dosagem , Células A549 , Animais , Benzoatos/química , Benzoatos/farmacologia , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Células HT29 , Humanos , Camundongos , Morfolinas/química , Morfolinas/farmacologia , Células NIH 3T3 , Neoplasias/genética , Nitrobenzoatos/química , Nitrobenzoatos/farmacologia , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Inhibition of heat shock protein 90 (Hsp90) leads to inappropriate processing of proteins involved in DNA damage repair pathways after DNA damage and may enhance tumor cell radio- and chemo-therapy sensitivity. To investigate the potentiation of antitumor efficacy of lidamycin (LDM), an enediyne agent by the Hsp90 inhibitor geldanamycin (GDM), and possible mechanisms, we have determined effects on ovarian cancer SKOV- 3, hepatoma Bel-7402 and HepG2 cells by MTT assay, apoptosis assay, and cell cycle analysis. DNA damage was investigated with H2AX C-terminal phosphorylation (γH2AX) assays. We found that GDM synergistically sensitized SKOV-3 and Bel-7402 cells to the enediyne LDM, and this was accompanied by increased apoptosis. GDM pretreatment resulted in a greater LDM-induced DNA damage and reduced DNA repair as compared with LDM alone. However, in HepG2 cells GDM did not show significant sensitizing effects both in MTT assay and in DNA damage repair. Abrogation of LDM-induced G2/M arrest by GDM was found in SKOV-3 but not in HepG2 cells. Furthermore, the expression of ATM, related to DNA damage repair responses, was also decreased by GDM in SKOV-3 and Bel-7402 cells but not in HepG2 cells. These results demonstrate that Hsp90 inhibitors may potentiate the antitumor efficacy of LDM, possibly by reducing the repair of LDM-induced DNA damage.
Assuntos
Aminoglicosídeos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Benzoquinonas/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Enedi-Inos/farmacologia , Lactamas Macrocíclicas/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Células Hep G2 , Histonas/efeitos dos fármacos , Histonas/metabolismo , Humanos , FosforilaçãoRESUMO
BACKGROUND: Endostatin (ES) inhibits endothelial cell proliferation, migration, invasion, and tube formation. It also shows antiangiogenesis and antitumor activities in several animal models. Endostatin specifically targets tumor vasculature to block tumor growth. Lidamycin (LDM), which consists of an active enediyne chromophore (AE) and a non-covalently bound apo-protein (LDP), is a member of chromoprotein family of antitumor antibiotics with extremely potent cytotoxicity to cancer cells. Therefore, we reasoned that endostatin-lidamycin (ES-LDM) fusion proteins upon energizing with enediyne chromophore may obtain the combined capability targeting tumor vasculature and tumor cell by respective ES and LDM moiety. METHODS: In this study, we designed and obtained two new endostatin-based fusion proteins, endostatin-LDP (ES-LDP) and LDP-endostatin (LDP-ES). In vitro, the antiangiogenic effect of fusion proteins was determined by the wound healing assay and tube formation assay and the cytotoxicity of their enediyne-energized analogs was evaluated by CCK-8 assay. Tissue microarray was used to analyze the binding affinity of LDP, ES or ES-LDP with specimens of human lung tissue and lung tumor. The in vivo efficacy of the fusion proteins was evaluated with human lung carcinoma PG-BE1 xenograft and the experimental metastasis model of 4T1-luc breast cancer. RESULTS: ES-LDP and LDP-ES disrupted the formation of endothelial tube structures and inhibited endothelial cell migration. Evidently, ES-LDP accumulated in the tumor and suppressed tumor growth and metastasis. ES-LDP and ES show higher binding capability than LDP to lung carcinoma; in addition, ES-LDP and ES share similar binding capability. Furthermore, the enediyne-energized fusion protein ES-LDP-AE demonstrated significant efficacy against lung carcinoma xenograft in athymic mice. CONCLUSIONS: The ES-based fusion protein therapy provides some fundamental information for further drug development. Targeting both tumor vasculature and tumor cells by endostatin-based fusion proteins and their enediyne-energized analogs probably provides a promising modality in cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Endostatinas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapêutico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Endostatinas/farmacocinética , Endostatinas/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Feminino , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/uso terapêutico , Análise Serial de Tecidos , Distribuição Tecidual , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The proteasome inhibitor bortezomib has been applied successfully to treat multiple myeloma (MM). Its synergistic effects with other anticancer drugs have been studied widely. In the present study, it was found that lidamycin (LDM), a member of the enediyne antibiotic family, showed much more potent cytotoxicity than bortezomib to MM cell lines: U266 and SKO-007. Here, we investigated the potential synergy of bortezomib and LDM on MM cells. The results showed that cotreatment of bortezomib and LDM synergistically induced cytotoxicity and apoptosis in MM cell lines, followed by enhanced caspase-3 cleavage and degradation of poly-ADP-ribose polymerase together with the decreased nuclear factor-κB protein. These two drugs synergistically induced apoptosis, which was associated with enhanced activation of two mitogen-activated protein kinases: p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase. Moreover, bortezomib plus LDM synergistically induced apoptosis was also associated with downregulation of extracellular signal-regulated kinase, and induction of endoplasmic reticulum stress response. Overall, our results indicate that the combined regimen of bortezomib and LDM might be a potential therapeutic remedy for the treatment of MM.
Assuntos
Aminoglicosídeos/farmacologia , Antineoplásicos/farmacologia , Ácidos Borônicos/farmacologia , Enedi-Inos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Pirazinas/farmacologia , Aminoglicosídeos/administração & dosagem , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Ácidos Borônicos/administração & dosagem , Bortezomib , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Enedi-Inos/administração & dosagem , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Pirazinas/administração & dosagem , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Targeting and inhibiting angiogenesis is a promising strategy for treatment of cancer. NGR peptide motif is a tumor-homing peptide, which could bind with CD13 expressed on tumor blood vessels. Lidamycin is a highly potent antitumor antibiotic, which is composed of an apoprotein (LDP) and an active enediyne chromophore (AE). Here, an NGR-integrated and enediyne-energized apoprotein composed of cyclic NGR peptide and lidamycin was developed by a two-step procedure. Firstly, we prepared the fusion protein composed of NGR peptide and LDP by recombinant DNA technology. Then, AE was reloaded to the fusion protein to get NGR-LDP-AE. Our experiments showed that NGR-LDP could bind to CD13-expressing HT-1080 cells, whereas the recombinant LDP (rLDP) showed weak binding. NGR-LDP-AE exerted highly potent cytotoxicity to cultured tumor cells in vitro. In vivo antitumor activity was evaluated in murine hepatoma 22 (H22) model and human fibrosarcoma HT-1080 model. At the tolerable dose, NGR-LDP-AE and lidamycin inhibited H22 tumor growth by 94.8 and 66.9%, and the median survival time of the mice was 62 and 37 days, respectively. In the HT-1080 model, NGR-LDP-AE inhibited tumor growth by 88.6%, which was statistically different from that of lidamycin (74.5%). Immunohistochemical study showed that NGR-LDP could bind to tumor blood vessels. Conclusively, these results demonstrate that fusion of LDP with CNGRC peptide delivers AE to tumor blood vessels and improves its antitumor activity.
Assuntos
Apoproteínas/farmacologia , Antígenos CD13/metabolismo , Enedi-Inos/farmacologia , Oligopeptídeos/farmacologia , Aminoglicosídeos/farmacologia , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Apoproteínas/genética , Apoproteínas/metabolismo , Linhagem Celular Tumoral , Feminino , Fibrossarcoma/tratamento farmacológico , Células Hep G2 , Humanos , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Arginine-rich peptides belong to a subclass of cell penetrating peptides that are taken up by living cells and can be detected freely diffusing inside the cytoplasm and nucleoplasm. This phenomenon has been attributed to either an endocytotic mode of uptake and a subsequent release from vesicles or a direct membrane penetration. Lidamycin is an antitumor antibiotic, which consists of an active enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). In the present study, a fusion protein (Arg)(9)-LDP composed of cell penetrating peptide (Arg)(9) and LDP was prepared by DNA recombination, and the enediyne-energized fusion protein (Arg)(9)-LDP-AE was prepared by molecular reconstitution. The data in fixed cells demonstrated that (Arg)(9)-LDP could rapidly enter cells, and the results based on fluorescence activated cell sorting indicated that the major route for (Arg)(9)-mediated cellular uptake of protein molecules was endocytosis. (Arg)(9)-LDP-AE demonstrated more potent cytotoxicity against different carcinoma cell lines than lidamycin in vitro. In the mouse hepatoma 22 model, (Arg)(9)-LDP-AE (0.3mg/kg) suppressed the tumor growth by 89.2%, whereas lidamycin (0.05 mg/kg) by 74.6%. Furthermore, in the glioma U87 xenograft model in nude mice, (Arg)(9)-LDP-AE at 0.2mg/kg suppressed tumor growth by 88.8%, compared with that of lidamycin by 62.9% at 0.05 mg/kg. No obvious toxic effects were observed in all groups during treatments. The results showed that energized fusion protein (Arg)(9)-LDP-AE was more effective than lidamycin and would be a promising candidate for glioma therapy. In addition, this approach to manufacturing fusion proteins might serve as a technology platform for the development of new cell penetrating peptides-based drugs.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Apoproteínas/farmacologia , Peptídeos Penetradores de Células/farmacologia , Enedi-Inos/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Aminoglicosídeos/farmacologia , Animais , Antibióticos Antineoplásicos/metabolismo , Apoproteínas/genética , Apoproteínas/metabolismo , Arginina/genética , Arginina/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Enedi-Inos/metabolismo , Feminino , Células Hep G2 , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
We evaluated the efficacy of a combination strategy, Endostar, a modified recombinant human endostatin, plus dexamethasone, against angiogenesis and hepatoma growth. By colony formation assay, synergistic effects of the combination of Endostar and dexamethasone were observed on the proliferations of human umbilical endothelial cells and hepatoma Bel-7402 cells. Endostar plus dexamethasone inhibited angiogenesis events in vitro. Examined with transwell assay, HUVECs invasion was more efficiently suppressed by combination of Endostar and dexamethasone than by the respective single drug. But the combination treatment of Endostar and dexamethasone to Bel-7402 cells did not alter the migration of HUVECs. The migration of HUVEC tended to coincident with VEGF secretion as determined by ELISA. Tube formation assay, rat aortic ring assay and chick embryo chorioallantoic membrane (CAM) assay indicated that the anti-angiogenesis effects of Endostar were significantly enhanced by dexamethasone. In mouse hepatoma H22 and human hepatoma Bel-7402 subcutaneous xenograft, Endostar plus dexamethasone presented more potent efficacy in tumor growth suppression than the single drug treatments. The above confirms the synergism of Endostar and dexamethasone on anti-angiogenesis and suppression of hepatoma growth. The potentiation effects of the combination indicate that Endostar plus dexamethasone might be a positive strategy for cancer therapy.
Assuntos
Dexametasona/farmacologia , Endostatinas/farmacologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores da Angiogênese/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Aorta/efeitos dos fármacos , Aorta/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/irrigação sanguínea , Sinergismo Farmacológico , Endostatinas/genética , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Humanos , Técnicas In Vitro , Neoplasias Hepáticas Experimentais/patologia , Camundongos , Camundongos Nus , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This study is to investigate inhibitory effects of lidamycin (LDM) on the proliferation of HERG K+ channel highly expressing cancer cells and its synergy with anticancer drugs. MTT assay was used to examine the inhibitory effects of lidamycin combined with various anticancer drugs on the proliferation of human lung cancer A549 cells, human colon cancer HT-29 cells and herg-stably-transfected A549 cells. Using the xenograft model of subcutaneously transplanted HT-29 in nude mice, inhibitory effect was appraised in vivo. The coefficient of drug interaction (CDI) was used to evaluate the synergistic effect of drug combination. LDM significantly inhibited the proliferation ofA549 cells and HT-29 cells with IC50 values of 2.14 and 4.64 ng mL(-1), respectively. The efficacy in HT-29 cells with high HERG potassium expression level is less potent than that in A549 cells with low expression level. In terms of IC50 values, LDM suppressed the growth of herg-stably-transfected A549 cells less potently than pCDNA3.1-stably-transfected A549 cells. There existed synergistic effects in the combinations of fluorouracil (5-FU) and LDM, doxorubicin (DOX) and LDM, or hydroxycamptothecine (HCPT) and LDM. CDI values of the combinations of 5-FU and LDM were more than 0.75. CDI values of LDM and DOX were more than 0.70, but some CDI values of LDM and HCPT were less than 0.70. As for the CDI values, synergistic effects of the combination of LDM and HCPT were the most potent of the three groups. There is no relationship between the inhibitory effect of the growth of cancer cells by 5-FU and HERG potassium expression level. HERG expression level negatively correlated with inhibitory effect on the proliferation of cancer cells by DOX. HERG expression levels and chemosensitivity were positively correlated for HCPT. In the model of subcutaneously xenograft transplanted HT-29 in vivo, LDM and/or HCPT effectively inhibited the growth of HT-29 in nude mice, and the optimum CDI of the combination of LDM and HCPT was less than 1. HERG expression level negatively correlates the chemosensitivity of cancer cells to LDM. There exist synergistic effects in vitro and in vivo in the combination of LDM and HCPT, which inhibitory effects of the proliferation of cancer cells positively modulated by HERG potassium expression level. HERG K+ channel may become a target of combined therapy for choosing anticancer drugs.
Assuntos
Aminoglicosídeos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proliferação de Células/efeitos dos fármacos , Enedi-Inos/farmacologia , Canais de Potássio Éter-A-Go-Go/metabolismo , Neoplasias Pulmonares/patologia , Aminoglicosídeos/administração & dosagem , Animais , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacologia , Antineoplásicos Fitogênicos/administração & dosagem , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Sinergismo Farmacológico , Canal de Potássio ERG1 , Enedi-Inos/administração & dosagem , Fluoruracila/administração & dosagem , Células HT29 , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Potassium channels are essential for the regulation of cell proliferation. As reported, HERG protein is overexpressed in a wide range of human tumors, including colon carcinoma. The aim of this study was to investigate the effects of antibacterial agents sparfloxacin (SPFX), a blocker of HERG channel, on HERG K+ channel highly expressing colon cancer cells. Expression of HERG and apoptosis correlative proteins was examined by Western blotting. The MTT assay was used to detect the cytotoxicity of drugs and drug combination in vitro. Gene transfection was used to examine the changes in herg-related chemosensitivity. Cell apoptosis was analyzed by flow cytometry. The migration and invasion capacity of tumor cells by SPFX was determined by gelatin zymography assay and Boyden chamber. The in vivo efficacy of SPFX was assessed in murine colon carcinoma C26 in BALB/c mice and human colon carcinoma HCT116 xenografts in nude mice. High expression of HERG protein was detected in colon cancer C26, HCT116 and HT-29 cells. The cell viability of the colon cancer cells was inhibited by SPFX in a dose-dependent manner. SPFX induced apoptosis and inhibited migration and invasion of colon cancer HCT116 cells. The increase in apoptosis was associated with a decrease in procaspase-3 and Bcl-2 protein expression. Study with herg-transfected HEK293 cells and siRNA-knock down HCT116 cells confirmed that the cell viability inhibition by SPFX was correlated with HERG expression. When combined with 5-fluorouracil, SPFX showed synergistic anti-proliferation activity in HCT116 and HT-29 cells. Furthermore, SPFX inhibited the growth of human colon carcinoma HCT116 xenografts and showed synergistic effect with 5-fluorouracil in vivo. Our finding suggested that SPFX could be a biochemical modulator in treatment of colon cancer with chemotherapeutic drugs.
Assuntos
Antituberculosos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Canais de Potássio Éter-A-Go-Go/metabolismo , Fluoroquinolonas/farmacologia , Animais , Western Blotting , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Canal de Potássio ERG1 , Feminino , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Cicatrização/efeitos dos fármacosRESUMO
In previous studies, rhein, one of the major bioactive constituents in the rhizome of rhubarb, inhibited the proliferation of various human cancer cells. However, because of its water insolubility, the anti-tumor efficacy of rhein was limited in vivo. In this study, we observed the anti-tumor activity of rhein lysinate (the salt of rhein and lysine easily dissolves in water) in vivo and investigated its mechanism. Inhibition of ovarian cancer SKOV-3 cell proliferation was determined by MTT assay and the mechanism of action of rhein lysinate was investigated by Western blot analysis. The therapeutic efficacy of rhein lysinate was evaluated by intragastric and intraperitoneal administrations in H22 hepatocellular carcinoma mice. Rhein lysinate inhibited the proliferation of SKOV-3 cells and the IC50 value was 80 microM. Rhein lysinate inhibited the phosphorylation of MEK and ERK and increased the anti-tumor activity of Taxol in vitro. It inhibited tumor growth by both intragastric and intraperitoneal administrations and improved the therapeutic effect of Taxol in H22 hepatocellular carcinoma mice. In conclusion, rhein lysinate offers an anti-tumor activity in vivo and is hopeful to be a chemotherapeutic drug.
Assuntos
Antraquinonas/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Paclitaxel/farmacologia , Animais , Antraquinonas/administração & dosagem , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/farmacologia , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Humanos , Concentração Inibidora 50 , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Paclitaxel/administração & dosagem , Fitoterapia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Rheum/química , Resultado do TratamentoRESUMO
In the present study, the effects of lidamycin (LDM), a member of the enediyne antibiotic family, on two human multiple myeloma (MM) cell lines, U266 and SKO-007, were evaluated. In MTS assay, LDM showed much more potent cytotoxicity than conventional anti-MM agents to both cell lines. The IC(50) values of LDM for the U266 and SKO-007 cells were 0.0575 +/- 0.0015 and 0.1585 +/- 0.0166 nM, respectively, much lower than those of adriamycin, dexamethasone, and vincristine. Mechanistically, LDM triggered MM cells apoptosis by increasing the levels of cleaved poly ADP-ribose polymerase (PARP) and caspase-3/7. In addition, activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK) was a critical mediator in LDM-induced cell death. Inhibition of the expression of p38 MAPK and JNK by pharmacological inhibitors reversed the LDM-induced apoptosis through decreasing the level of cleaved PARP and caspase-3/7. Interestingly, phosphorylation of extracellular signal-related kinase was increased by LDM; conversely, MEK inhibitor synergistically enhanced LDM-induced cytotoxicity and apoptosis in MM cells. The results demonstrated that LDM suppresses MM cell growth through the activation of p38 MAPK and JNK, with the potential to be developed as a chemotherapeutic agent for MM.
Assuntos
Aminoglicosídeos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Enedi-Inos/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/enzimologia , Proteínas de Neoplasias/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Aminoglicosídeos/uso terapêutico , Antibióticos Antineoplásicos/uso terapêutico , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Enedi-Inos/uso terapêutico , Ativação Enzimática/efeitos dos fármacos , Humanos , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
This study is to investigate the antitumor activities of the immunoconjugates composed of anti-type IV collagenase monoclonal antibody 3G11 and lidamycin (LDM) prepared by different methods. The immunoconjugates were prepared by linking 2-iminothiolane modified 3G11 to lysine-69 of LDM apoprotein by SPDP and SMBS as the intermediate drug linker. Immunoreactivity of the conjugates was determined by ELISA. The cytotoxicity of the conjugates was examined by clonogenic assay. Antitumor effects of the conjugates in vivo were evaluated in nude mice bearing subcutaneously implanted HT-1080 tumor. ELISA assay showed that the immunoconjugates retained the immunoreactivity of 3G11 against type IV collagenase. The cytotoxicity of the 3G11-SMBS-LDM to HT-1080 cells was significantly more potent than that of free LDM and 3G11-SPDP-LDM. In animal model at the same condition, free LDM inhibited the growth of HT-1080 tumor by 71.2%, while 3G11-SPDP-LDM and 3Gl1-SMBS-LDM reached 77.1% and 86.1%, respectively. The median survival time of the mice treated with free LDM was prolonged by 71.9% compared with that of untreated group. Whereas, the median survival time of 3G11-SPDP-LDM and 3G11-SMBS-LDM was prolonged by 125.3% and 163.7%, respectively, indicating that 3G11-SMBS-LDM was more effective than 3G11-SPDP-LDM in tumor suppression and life span prolongation. 3Gll-SMBS-LDM has more selective antitumor efficacy and lower toxicity, and might be a novel candidate for cancer therapy. LDM was more effective than 3G11-SPDP-LDM in tumor suppression and life span prolongation. 3Gll-SMBS-LDM has more selective antitumor efficacy and lower toxicity, and might be a novel candidate for cancer therapy.
Assuntos
Aminoglicosídeos/uso terapêutico , Antibióticos Antineoplásicos/uso terapêutico , Colagenases/imunologia , Enedi-Inos/uso terapêutico , Fibrossarcoma/terapia , Imunoconjugados/uso terapêutico , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fibrossarcoma/patologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Carga Tumoral/efeitos dos fármacosRESUMO
Single-domain antibodies are attractive as tumor-targeting vehicles because of their much smaller size than intact antibody molecules. Lidamycin is a macromolecular antitumor antibiotic, which consists of a labile enediyne chromophore (AE) and a noncovalently bound apoprotein (LDP). An enediyne-energized fusion protein VH-LDP-AE composed of single-domain antibody directed against type IV collagenase and lidamycin was prepared by a novel two-step method including DNA recombination and molecular reconstitution. VH-LDP-AE demonstrated extremely potent cytotoxicity to cancer cells and marked antiangiogenic activity in vitro. In the mouse hepatoma 22 model, drugs were administered intravenously as a single dose on day 1 with maximal tolerated doses. VH-LDP-AE (0.25 mg/kg) suppressed the tumor growth by 95.9%, whereas lidamycin (0.05 mg/kg) and mitomycin (1 mg/kg) by 79.6 and 51.1%, respectively. In the HT-1080 xenograft model in nude mice, drugs were given intravenously as a single dose on day 4 after tumor implantation. VH-LDP-AE at 0.25 mg/kg suppressed tumor growth by 76% (P<0.05) compared with that of lidamycin at 0.05 mg/kg (53%) on day 18. No obvious toxic effects were observed in all groups during treatments. The results showed that energized fusion protein VH-LDP-AE was more effective than lidamycin and mitomycin. These properties, together with its much smaller size than conventional antibody-based agents, suggested that VH-LDP-AE would be a promising candidate for cancer-targeting therapy. In addition, the two-step approach could serve as a new technology platform for making a series of highly potent engineered antibody-based drugs for a variety of cancers.
Assuntos
Anticorpos/química , Anticorpos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Alantoide/química , Alantoide/efeitos dos fármacos , Sequência de Aminoácidos , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/farmacologia , Animais , Antineoplásicos/síntese química , Western Blotting , Linhagem Celular Tumoral , Embrião de Galinha , Colagenases/química , Colagenases/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Vetores Genéticos , Indicadores e Reagentes , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microscopia de Fluorescência , Microtúbulos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
AIM: Type IV collagenase including MMP-2 and -9 plays an important role in cancer cell invasion and metastasis and is an attractive target for mAb-directed therapy. The immunoreactivity of mAb 3G11, a mAb directed against type IV collagenase in human colorectal carcinomas, was studied by immuno-histochemical (IHC) staining. mAb 3G11 was conjugated to an antitumor antibiotic lidamycin (LDM). The antitumor activity of 3G11-LDM conjugate against colon carcinoma was investigated in mice. METHODS: ELISA, gelatin zymography, and Western blot assay were used for the biological characterization of mAb 3G11. The immunoreactivity of mAb 3G11 with human colorectal carcinomas was detected by IHC staining. The cytotoxicity of LDM and 3G11-LDM conjugate to human colon carcinoma HT-29 cells was examined by clonogenic assay and MTT assay. The therapeutic effect of conjugate 3G11-LDM was evaluated with colon carcinoma 26 in mice. RESULTS: As shown in ELISA, mAb 3G11 reacted specifically with type IV collagenase, while 3G11-LDM conjugate also recognized specifically its respective antigen. In IHC assay, mAb 3G11 showed positive immunoreactivity in most cases of colorectal carcinoma, and negative immunoreactivity in the adjacent non-malignant tissues. By gelatin zymography, the inhibition effect of mAb 3G11 on the secretion activity of type IV collagenase was proved. In terms of IC50 values in MTT assay, the cytotoxicity of LDM to human colon carcinoma HT-29 cells was 10,000-fold more potent than that of mitomycin C (MMC) and adriamycin (ADM). 3G11-LDM conjugate also displayed extremely potent cytotoxicity to human colon carcinoma HT-29 cells with an IC50 value of 5.6 x 10(-19) mol/L. 3G11-LDM conjugate at the doses of 0.05 and 0.1 mg/kg inhibited the growth of colon carcinoma 26 in mice by 70.3 and 81.2%, respectively. CONCLUSION: mAb 3G11 is immunoreactive with human colorectal carcinoma and its conjugate with LDM is highly effective against colon carcinoma in mice.
Assuntos
Aminoglicosídeos/farmacologia , Antibióticos Antineoplásicos/farmacologia , Anticorpos Monoclonais/farmacologia , Colágeno Tipo IV/imunologia , Neoplasias do Colo/tratamento farmacológico , Animais , Neoplasias da Mama , Carcinoma Hepatocelular , Neoplasias do Colo/imunologia , Enedi-Inos , Fibrossarcoma , Células HT29 , Humanos , Imunoconjugados/farmacologia , Técnicas In Vitro , Neoplasias Hepáticas , Camundongos , Camundongos Endogâmicos BALB CRESUMO
AIM: To study the in vitro and in vivo antitumor effect of lidamycin (LDM) on hepatoma and the active moiety of its molecule. METHODS: MTT assay was used to determine the growth inhibition of human hepatoma BEL-7402 cells, SMMC-7721 cells and mouse hepatoma H22 cells. The in vivo therapeutic effects of lidamycin and mitomycin C were determined by transplantable hepatoma 22 (H22) in mice and human hepatoma BEL-7402 xenografts in athymic mice. RESULTS: In terms of IC(50) values, the cytotoxicity of LDM was 10 000-fold more potent than that of mitomycin C (MMC) and adriamycin (ADM) in human hepatoma BEL-7402 cells and SMMC-7721 cells. LDM molecule consists of two moieties, an aproprotein (LDP) and an enediyne chromophore (LDC). In terms of IC(50) values, the potency of LDC was similar to LDM. However, LDP was 10(5)-fold less potent than LDM and LDC to hepatoma cells. For mouse hepatoma H22 cells, the IC(50) value of LDM was 0.025 nmol/L. Given by single intravenous injection at doses of 0.1, 0.05 and 0.025 mg/kg, LDM markedly suppressed the growth of hepatoma 22 in mice by 84.7%, 71.6% and 61.8%, respectively. The therapeutic indexes (TI) of LDM and MMC were 15 and 2.5, respectively. By 2 iv. injections in two experiments, the growth inhibition rates by LDM at doses of 0.1, 0.05, 0.025, 0.00625 and 0.0125 mg/kg were 88.8-89.5%, 81.1-82.5%, 71.2-74.9%, 52.3-59.575%, and 33.3-48.3%, respectively. In comparison, MMC at doses of 5, 2.5, and 1.25 mg/kg inhibited tumor growth by 69.7-73.6%, 54.0-56.5%, and 31.5-52.2%, respectively. Moreover, in human hepatoma BEL-7402 xenografts, the growth inhibition rates by LDM at doses of 0.05 mg/kg X2 and 0.025 mg/kg X2 were 68.7% and 27.2%, respectively. However, MMC at the dose of 1.25 mg/kg X2 showed an inhibition rate of 34.5%. The inhibition rate of tumor growth by LDM was higher than that by MMC at the tolerated dose. CONCLUSION: Both LDM and its chromophore LDC display extremely potent cytotoxicity to hepatoma cells. LDM shows a remarkable therapeutic efficacy against murine and human hepatomas in vivo.
Assuntos
Aminoglicosídeos/uso terapêutico , Antibióticos Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Animais , Sobrevivência Celular/efeitos dos fármacos , Enedi-Inos , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante HeterólogoRESUMO
RNA interference technology is a powerful tool for silencing endogenous or exogenous genes in mammalian cells. Here our results showed that hdm2-siRNA silenced its target mRNA specifically and effectively in human breast cancer cells, reduced tumor cell proliferation and induced apoptotic cell death. Other molecular features modified by hdm2-siRNA included decreased Bcl-2, NF-kappaB, survivin, Ras and Raf levels, elevated p53, p21, BRCA1, Bax, and caspase levels as well as altered expression of other genes. hdm2-siRNA also caused cell cycle arrest at G1 phases with reduction in cyclin and Cdk proteins. In addition, hdm2-siRNA displayed in vivo antitumor activity and increased therapeutic effectiveness of mitomycin in MCF-7 xenografts. Thus, hdm2-siRNA may be a promising gene-specific drug for the treatment of human breast cancer and other tumors.
Assuntos
Neoplasias da Mama/genética , Oncogenes , RNA Interferente Pequeno/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Apoptose/fisiologia , Sequência de Bases , Neoplasias da Mama/fisiopatologia , Ciclo Celular , Linhagem Celular Tumoral , Primers do DNA , Genes Supressores de Tumor , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/fisiologiaRESUMO
BACKGROUND & OBJECTIVE: Matrix metalloproteinases (MMPs)play the important role in many steps of tumor growth and metastasis. Type IV collagenase, which is a key member of MMPs family, has been viewed as a promising target in tumor study. The aim of this study was to evaluate the tumor-specific distribution of the anti-type IV collagenase monoclonal antibody (mAb) 3G11 by radioimaging in tumor-bearing nude mice. METHODS: MAb 3G11 purified by affinity chromatography was labeled with either 131- or 125- iodide by the Iodogen method. Immunoreactivity of mAb 3G11 was determined by ELISA. (131)I-labeled 3G11 was incubated in three different media at 37 Celsius degree and its in vitro stability was tested. Normal BALB/c mice were injected intravenously with 388.5 kBq per mouse of (125)I-labeled 3G11 to explore the pharmacokinetic patterns. The scintigraphic images of human lung carcinoma PG xenografts grown subcutaneously in BALB/c nude mice were made after intravenously administrating of 6.44 MBq per mouse of (131)I-labeled 3G11. RESULTS: MAb 3G11 was more than 98% in purity via affinity purification. The immunoreactivity of mAb 3G11 decreased by approximately 10%-20% after cold iodination. Patterns of blood clearance of mAb 3G11 was defined as two-compartment model, with T(1/2alpha) and T(1/2beta) calculated to be 7.2 h and 345.2 h, respectively. (131)I-labeled 3G11 was almost stable in vitro for 72 h. A clear image of the xenografted tumor was obtained at 72 h, and it further improved at 120 h. CONCLUSION: MAb 3G11 showed high specificity and affinity with tumor tissue through scintiscanning.