ABSTRACT
This study aimed to investigate the synergistic effect of lidamycin (LDM) and rituximab on human B cell lymphoma Ramos cells. Cell proliferation was measured using MTS assay, cell apoptosis was analyzed by Annexin V-FITC/PI assay, the expression of apoptosis related proteins was analyzed by Western blotting, and the in vivo lymphoma inhibition was verified using BALB/c mice inoculated via tail vein using Ramos cells which stably expressed pEGFP-N1 plasmid. The results showed that, after the pretreatment with rituximab for 48 h, rituximab and LDM showed significantly synergistic effects on cell proliferation. Cells in combined treatment group had a higher apoptosis rate than that in LDM treatment group. Compared with the LDM treatment group, the expression of apoptosis-related proteins such as Cleaved caspase-3, Cleaved caspase-7, Cleaved caspase-9 and Cleaved PARP in combined treatment groups increased, and expression of cIAP-2 and Bcl-2 decreased. The result of in vivo experiment showed that, in the combined treatment group, the survival time of BALB/c mice was significantly longer than the mice in control group and LDM treatment group, and the degree of tumor accumulation and metastasis to lymph nodes and spleen was lower.
Subject(s)
Animals , Humans , Mice , Aminoglycosides , Pharmacology , Antibiotics, Antineoplastic , Pharmacology , Antineoplastic Agents , Pharmacology , Apoptosis , Caspase 3 , Metabolism , Caspase 7 , Metabolism , Caspase 9 , Metabolism , Cell Line, Tumor , Cell Proliferation , Drug Synergism , Enediynes , Pharmacology , Inhibitor of Apoptosis Proteins , Metabolism , Lymphoma, B-Cell , Metabolism , Pathology , Mice, Inbred BALB C , Mice, Nude , Neoplasm Metastasis , Neoplasm Transplantation , Poly(ADP-ribose) Polymerases , Metabolism , Proto-Oncogene Proteins c-bcl-2 , Metabolism , Random Allocation , Rituximab , PharmacologyABSTRACT
In the present study, a new compound named 17-(6-cinnamamido-hexylamino-)-17-demethoxygeldanamycin (CDG) was obtained by introducing the cinnamic acid (CA) group into the 17-site of geldanamycin (GDM). The anti-cancer effects of CDG in vitro and in vivo were evaluated. MTT assay was used to examine the inhibitory effect of CDG on the proliferation of MCF-7, HepG2, H460 and SW1990 cells. Immunofluorescent staining flow cytometry combined with Annexin V-FITC/PI staining were used to detect apoptotic cells. Transwell assay was used to analyze the effect of CDG on cell invasion and migration ability. Western blotting was used to detect the expression levels of RAF-1, EGFR, AKT, CDK4 and HER-2 of MCF-7, HepG2 and H460 cells. The toxicities of CDG and GDM were evaluated in mice. Using the subcutaneously transplanted MCF-7 xenograft in nude mice, inhibitory effect was evaluated in vivo. The results showed that CDG inhibited the proliferation of cancer cells (IC50: 13.6-67.4 microg.mL-1). After exposure to CDG for 48 h, most cells presented typical morphologic changes of apoptosis such as chromatin condensation or shrunken nucleus. The rates of apoptosis of MCF-7, HepG2, H460 and SW1990 cells incubated with 10 microg.mL-1 CDG were 23.16%, 27.55%, 22.21%, 20.47%, respectively. A dose-dependent reduction of migration of four cell lines was found after exposure to CDG. The decreased levels of RAF-1, EGFR, AKT, CDK4 and HER-2 showed that CDG possessed HSP90 inhibitory effect. The result of animal toxicity test on the mice suggested that CDG had lower toxicity than GDM. Meanwhile, CDG inhibited the growth of MCF-7 xenografts of athymic mice.
Subject(s)
Animals , Female , Humans , Male , Mice , Antineoplastic Agents , Chemistry , Pharmacology , Apoptosis , Benzoquinones , Chemistry , Pharmacology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cyclin-Dependent Kinase 4 , Metabolism , HSP90 Heat-Shock Proteins , Lactams, Macrocyclic , Chemistry , Pharmacology , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Neoplasm Transplantation , Proto-Oncogene Proteins A-raf , Metabolism , Proto-Oncogene Proteins c-akt , Metabolism , Random Allocation , ErbB Receptors , Metabolism , Receptor, ErbB-2 , Metabolism , Tumor Burden , Xenograft Model Antitumor AssaysABSTRACT
The aim of the present study was to investigate the in vivo anti-metastatic activity of the red pigments of red yolk eggs laid by the ducks dieting on Potamogeton cripus L on the mammary carcinoma (4T1). The pigments were extracted with petroleum ether and acetone (2:1, v/v). BALB/c mice were divided into three groups (n=6), fed with the extracts at 150 mg/kg body weight (BW)/day (DEYE-H) or at 50 mg/kg BW/day (DEYE-L) and identical buffer without the extract (control group). The extracts were administered for 34 days. The treatment significantly inhibited the growth of orthotopical 4T1 tumour (DEYE-H vs control, 1:2; DEYE-L vs control, 2:3) and reduced the metastasis of tumour in the lungs (DEYE-H vs control, 4:7; DEYE-L vs control, 5:7), without statistical difference of body weight among the three groups.
ABSTRACT
This study is to investigate the inhibitory effect of lidamycin (LDM) and its combination with methotrexate (MTX) on lung metastasis of fibrosarcoma by bioluminescence imaging in athymic mice. A stable luciferase transfected HT-1080 cell line was constructed and the capability to establish experimental lung metastasis in athymic mice was confirmed. The optical imaging system was applied to evaluate the formation of lung metastasis in vivo. In addition, metastatic nodules were counted for the evaluation of inhibition rates. As shown, the fluorescent intensity of luciferase-transfected HT-1080 cells was colinear with the cell population and the minimal detected cell population was 100 cells/well. Optical imaging showed that the fluorescent intensity of treated group was apparently lower than that of the control. The inhibition rates of lung metastasis by LDM alone at 0.025 mg x kg(-1) and 0.05 mg x kg(-1) were 53.9% and 75.9%, respectively, while that of MTX alone at 0.5 mg x kg(-1) was 70.2%. The combination of LDM at 0.025 mg x kg(-1) and MTX at 0.5 mg x kg(-1) showed an inhibition rate of 88.7%. The coefficient of drug interaction (CDI) was 0.82. The results herein demonstrated that LDM alone had strong anti-metastasis effect on human fibrosarcoma HT-1080 and the inhibition efficacy is strengthened when combined with MTX.
Subject(s)
Animals , Female , Humans , Mice , Aminoglycosides , Antibiotics, Antineoplastic , Antimetabolites, Antineoplastic , Antineoplastic Combined Chemotherapy Protocols , Therapeutic Uses , Cell Line, Tumor , Drug Synergism , Enediynes , Fibrosarcoma , Pathology , Luminescent Measurements , Lung , Pathology , Lung Neoplasms , Drug Therapy , Pathology , Methotrexate , Mice, Inbred BALB C , Mice, Nude , Random Allocation , Transfection , Xenograft Model Antitumor AssaysABSTRACT
<p><b>OBJECTIVE</b>Lidamycin (LDM) can be dissociated to an apoprotein (LDP) and an active enediyne chromophore (AE). The detached AE can reassemble with its LDP-containing fusion protein to endow the latter with potent antitumor activity. However, the reassembly of AE with LDP is affected by several factors. Our aim was to optimize the assembly efficiency of the AE with a LDP-containing fusion protein and investigate the influence of several factors on the assembly efficacy.</p><p><b>METHODS</b>A method based on RP-HPLC was developed to analyze the assembly rate, and an orthogonal experimental design L(9) (3(4)) was used to investigate the effects of temperature, assembly time, pH and molecular ratio of LDP-containing fusion protein to AE on the assembly rate. Furthermore, the determined optimum conditions for the assembly rate of the LDP-containing fusion protein with AE were applied and evaluated.</p><p><b>RESULTS</b>A calibration curve based on the LDM micromolar concentration against the peak-area of AE by HPLC was obtained. The order in which individual factors in the orthogonal experiment affected the assembly rate were temperature>time>pH>molar ratio of AE to protein and all were statistically significant (P<0.01). The optimal assembly conditions were temperature at 10°C, time of 12 h, pH 7.0, and the molar ratio of AE: protein of 5:1. The assembly rate of AE with a LDP-containing fusion protein was improved by 23% after condition optimization.</p><p><b>CONCLUSION</b>The assembly rate of chromophore of lidamycin with its LDP-containing fusion protein was improved after condition optimization by orthogonal design, and the optimal conditions described herein should prove useful for the development of this type of LDP-containing fusion protein.</p>
Subject(s)
Humans , Aminoglycosides , Chemistry , Pharmacology , Antibiotics, Antineoplastic , Chemistry , Pharmacology , Apoproteins , Chemistry , Cell Line, Tumor , Cell Survival , Chromatography, High Pressure Liquid , Drug Design , Enediynes , Chemistry , Pharmacology , Recombinant Fusion Proteins , Chemistry , Single-Chain Antibodies , ChemistryABSTRACT
Lidamycin (LDM) is a potent antitumor antibiotic. Previous studies have shown that LDM could inhibit proliferation and migration in endothelial cells. In the present report, the effect of LDM on angiogenesis of zebrafish embryo was studied. The results showed that treatment of zebrafish embryos with LDM resulted in significant inhibition of angiogenesis. Morphological observation, quantitative endogenous alkaline phosphatase (EAP) assay, alkaline phosphatase staining, and transgenic zebrafish assay were performed to evaluate vascular development defects in zebrafish. The results indicated that after the zebrafish embryos were exposed to LDM, angiogenesis defects of zebrafish embryos were observed, including pericardial edema, reduced numbers of circulating red blood cells, suppression of zebrafish vessel growth, and absences of SIV (subintestinal vein). The expression of VEGF was detected by RT-PCR assay, quantitative reverse transcriptase real-time PCR (qRT-PCR) assay and Western blotting analysis. The results revealed that LDM could inhibit the expression of VEGF protein, while the expression of mRNA was not significantly affected. The study suggests that LDM could inhibit the zebrafish embryo angiogenesis by down-regulation ofVEGF expression.
Subject(s)
Animals , Aminoglycosides , Pharmacology , Animals, Genetically Modified , Embryology , Genetics , Physiology , Antibiotics, Antineoplastic , Pharmacology , Down-Regulation , Embryo, Nonmammalian , Enediynes , Pharmacology , Neovascularization, Physiologic , Genetics , RNA, Messenger , Metabolism , Vascular Endothelial Growth Factor A , Genetics , Metabolism , Zebrafish , Embryology , Genetics , PhysiologyABSTRACT
Ten pharmacophore models of beta-tubulin inhibitors were established from the training set of seventeen beta-tubulin inhibitors (two categories) with comformer analysis by using the Catalyst software. The optimal pharmacophore model with two hydrophobic units and two hydrogen bond acceptor units were confirmed (RMS = 0.43, Correl = 0.98, Weight = 2.06, Config = 15.97). This pharmacophore model is able to predict the activity of known beta-tubulin inhibitors and can be further used to identify structurally diverse compounds with higher activity.
Subject(s)
Benzamides , Chemistry , Computer-Aided Design , Drug Design , Models, Chemical , Models, Molecular , Molecular Conformation , Molecular Structure , Software , Structure-Activity Relationship , Tubulin Modulators , Chemistry , Urea , ChemistryABSTRACT
In this study, the antitumor activities of VEGF shRNA and tubulin inhibitors on human prostate cancer DU145 cells was investigated, and shRNA transient expression plasmid pCSH1-VEGF targeting VEGF mRNA was constructed. The silence efficiency of pCSH1-VEGF was detected by RT-PCR assay, Western blotting, and Matrigel invasion assay. The sensitivity change of DU145 cells to Taxol and vincristine (VCR) was measured by MTT assay. To detect the effects of pCSH1-VEGF and Taxol in vivo, nude mice model of DU145 xenograft tumor was established by subcutaneous inoculation. The results showed that transcription and expression of VEGF were knocked by pCSH1-VEGF in DU145 cells. Matrigel invasion assay results showed that pCSH1-VEGF significantly reduced the migration of DU145 cells with inhibitory rate of 56.1%. Furthermore, pCSH1-VEGF enhanced the sensitivity of DU145 cells to Taxol and vincristine, and the values of IC50 decreased by 77.3% and 92.6%, respectively. In vivo experiment showed that Taxol, pCSH1-VEGF, combination of pCSH1-VEGF and Taxol inhibited tumor growth by the rates of 48.8%, 56.2% and 81.8%, respectively. The coefficient of drug interaction (CDI) of pCSH1-VEGF and Taxol was 0.82. The data suggested that VEGF shRNA could significantly enhance the sensitivity of human prostate cancer to tubulin inhibitors.
Subject(s)
Animals , Humans , Male , Mice , Antineoplastic Agents, Phytogenic , Pharmacology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Genetic Vectors , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Paclitaxel , Pharmacology , Plasmids , Prostatic Neoplasms , Metabolism , Pathology , RNA Interference , RNA, Small Interfering , Genetics , Transfection , Tubulin Modulators , Pharmacology , Tumor Burden , Vascular Endothelial Growth Factor A , Genetics , Metabolism , Vincristine , PharmacologyABSTRACT
To investigate the effect of lidamycin (LDM) on human gastric carcinoma BGC823 cells and xenograft growth in nude mice, MTT (methyl thiazolyl tetrazolium) assay was used to determine the inhibition of BGC823 cell proliferation by LDM. Induction of apoptosis was studied by flow cytometry and TUNEL assay. The expression of VEGF was detected by Western blotting analysis. Athymic nude mice were used to determine in vivo antitumor activity. Proliferation inhibition and apoptosis induction were studied in lidamycin-treated cells. The expression of VEGF in BGC823 cells decreased in a dose-dependent manner. LDM at 0.02 mg x kg(-1) and 0.04 mg x kg(-1) suppressed the growth of BGC823 xenografts in nude mice by 57% and 72%, respectively. LDM potently induces apoptosis in human gastric carcinoma BGC823 cells and inhibits xenograft growth.
Subject(s)
Animals , Female , Humans , Mice , Aminoglycosides , Pharmacology , Antibiotics, Antineoplastic , Pharmacology , Apoptosis , Cell Line, Tumor , Cell Proliferation , Dose-Response Relationship, Drug , Enediynes , Pharmacology , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Random Allocation , Stomach Neoplasms , Metabolism , Pathology , Vascular Endothelial Growth Factor A , Metabolism , Xenograft Model Antitumor AssaysABSTRACT
<p><b>AIM</b>To investigate the induction of endothelial cell apoptosis and the suppression of VEGF expression in cancer cells by sodium caffeate (SCA).</p><p><b>METHODS</b>Apoptosis of transformed human umbilical vein endothelial cells (ECV304 cell line) was detected by flow cytometry, DNA electrophoresis assay and morphological assessment. Western blotting analysis was applied for determination of VEGF expression in cancer cells. Substrate degradation by type IV collagenase was measured by zymography. ELISA was used to detect the binding of type IV collagenase with relevant monoclonal antibody.</p><p><b>RESULTS</b>SCA induced ECV304 cell apoptosis in a time- and dose-dependent manner. After treatment with 100 and 250 microg X mL(-1) of SCA for 48 h, DNA laddering appeared. SCA treated cells showed strong blue fluorescence and distinct changes of nuclear morphology, such as pyknosis and the occurrence of apoptotic bodies. VEGF expression in hepatoma HepG-2 cells and prostate carcinoma DU145 cells was reduced after SCA treatment. The degradation activity of type IV collagenase including MMP-2 and MMP-9 secreted by giant cell pulmonary carcinoma PG cells was inhibited by SCA in a dose-dependent manner. SCA also reduced the binding of mAb 3D6, a relevant monoclonal antibody, to type IV collagenase.</p><p><b>CONCLUSION</b>SCA can induce endothelial cell apoptosis and inhibit VEGF expression as well as type IV collagenase activity in cancer cells. SCA might be active in modulating tumor angiogenesis and the microenvironment.</p>
Subject(s)
Humans , Male , Apoptosis , Caffeic Acids , Pharmacology , Cell Line, Tumor , Cell Proliferation , Cells, Cultured , Endothelial Cells , Cell Biology , Liver Neoplasms , Metabolism , Pathology , Lung Neoplasms , Metabolism , Pathology , Matrix Metalloproteinase 2 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , Prostatic Neoplasms , Metabolism , Pathology , Umbilical Veins , Cell Biology , Vascular Endothelial Growth Factor A , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To explore the inhibitory effects of endoplasmic reticulum-retained intrabody on the secretion of type IV collagenase and the invasion of human pulmonary giant cell carcinoma PG cells in vitro.</p><p><b>METHODS</b>Two expression plasmids were constructed, pcDNA3.1-CP.scFv and pcDNA3.1-ER.scFv encoding cytoplasm-retained and endoplasmic reticulum-retained single chain antibodies against the type IV collagenase, respectively. The intracellular antibody genes were transfected into the human pulmonary giant cell carcinoma PG cells. Western blot was performed to detect the expression of pcDNA3.1-CP.scFv and pcDNA3.1-ER.scFv. Gelatin zymography was performed to detect seretion of type IV collagenase in PG cells and Matrigel assay was employed for determination of the cell invasiveness.</p><p><b>RESULTS</b>Both of cytoplasm-retained and endoplasmic reticulum-retained introbodies, CP.scFv and ER.scFv, were expressed in PG cells. ER.scFv, significantly inhibited the secretion of type IV collegenase. As shown, matrix metalloproteinase 9 and matrix metalloproteinase 2 were inhibited by 85.7% and by 51.2%, respectively. However, CP.scFv did not show such inhibitory effect. The ER.scFv encoding gene-transfected PG cells were much less invasive than parental or vector control cells, the inhibition rate was 76.3% (P < 0.05), whereas CP.scFv encoding gene-transfected PG cells showed no reduction in invasiveness.</p><p><b>CONCLUSION</b>Those findings demonstrate that endoplasmic reticulum (ER)-retained intracellular antibody technology may selectively abrogate the activity of type IV collagenase in the protein trafficking and secretory pathway and effectively inhibit tumor cell invasion in vitro. Anti-type IV collagenase intrabody may be further used in cancer gene therapy.</p>
Subject(s)
Humans , Carcinoma, Giant Cell , Metabolism , Pathology , Cell Line, Tumor , Cytoplasm , Allergy and Immunology , Endoplasmic Reticulum , Allergy and Immunology , Genetic Vectors , Immunoglobulin Variable Region , Metabolism , Physiology , Lung Neoplasms , Metabolism , Pathology , Matrix Metalloproteinase 2 , Allergy and Immunology , Metabolism , Matrix Metalloproteinase 9 , Allergy and Immunology , Metabolism , Neoplasm Invasiveness , Plasmids , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To assess the effects of early intestinal application of sijunzi decoction (SJZD) on the immune function in post-operational patients of gastrointestinal tumor.</p><p><b>METHODS</b>Ninety-two patients with malignant gastrointestinal tumor were randomly divided into two groups. Patients in both groups were given the isocaloric and isonitrogenous enteral nutritional support starting from the first day after operation for 1 week, but to the tested group, SJZD was given additionally. The concentration of serum cytokines such as interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), the peripheral blood cell counts of total lymphocyte, T-lymphocyte, B lymphocyte, and T lymphocyte subsets (CD3, CD4, CD8, CD4/CD8) as well as the levels of IgA, IgG, IGM and C-reactive protein (CRP) were measured on the day before operation, the first morning after operation and at the end of study.</p><p><b>RESULTS</b>At the end of the study, the concentration of IgA, IgG, 1gM, number of total lymphocyte, CD3, CD4 and CD4 lCD8, and serum IL-2 were obviously higher (P < 0.05), and levels of IL-6, TNF-alpha and CRP were obviously lower in the tested group than those in the control group (P < 0.05).</p><p><b>CONCLUSION</b>Early application of SJZD on the base of enteral nutritional therapy can lessen the degree of post-operational stress and inflammatory response, and enhance the immune function of patients.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , CD4-CD8 Ratio , Drugs, Chinese Herbal , Therapeutic Uses , Gastrointestinal Neoplasms , Drug Therapy , Allergy and Immunology , General Surgery , Immunoglobulin A , Blood , Immunoglobulin G , Blood , Interleukin-1 , Blood , Interleukin-2 , Blood , Phytotherapy , Postoperative PeriodABSTRACT
<p><b>AIM</b>To investigate the inhibitory activity of salvianolic acid A (SAA) on nucleoside transport in cancer cells and its antitumor effect.</p><p><b>METHODS</b>[3H] thymidine and [3H] uridine transport assays were used to determine the inhibitory activity on nucleoside transport in Ehrlich carcinoma cells. The cytotoxicity to cultured cancer cells was examined with clonogenic assay. The antitumor effect in vivo was evaluated with transplantable tumor model in mice.</p><p><b>RESULTS</b>SAA was shown to inhibit thymidine and uridine transport in Ehrlich carcinoma cells with IC50 values of 18.1 and 17.1 micromol x L(-1), respectively. By clonogenic assay, the IC50 of SAA for KB cells was 44.7 micromol x L(-1). SAA markedly potentiated the cytotoxicity of 5-FU and mitomycin C in KB cells as well as the cytotoxicity of MTX in human hepatoma BEL-7402 cells. For in vivo experiment, sarcoma 180 cells were transplanted sc in mice and tested drugs were administered ip. When administered separately, SAA at 200 mg x kg(-1) and 5-FU at 10 mg x kg(-1) inhibited tumor growth by 41% and 27%, respectively. Combination of the two drugs inhibited tumor growth by 63% (CDI = 0.86).</p><p><b>CONCLUSION</b>SAA is active in blocking nucleoside transport in cancer cells and potentiates the cytotoxicity of chemotherapeutic drugs. As an agent showing moderate antitumor effect in vivo, SAA might be useful in combination cancer therapy.</p>