Mechanism of the anti-angiogenic effect of Avemar on tumor cells.

Avemar, a derivative of fermented wheat germ extract, is a non-toxic and natural compound that is used as a dietary supplement by cancer patients undergoing chemotherapy and radiotherapy. Avemar has numerous biological activities, and several recent studies have reported that it may also have metastatic and anti-angiogenic effects. In the present study, the mechanism of the anti-angiogenic effect of Avemar on human cancer cells was investigated. The human cell lines NCI-N87 (gastric tubular adenocarcinoma), PC3 (prostate carcinoma), HeLa (endocervical adenocarcinoma) and A549 (lung adenocarcinoma) were treated with various doses (400, 800, 1,600 and 3,200 µg/ml) of Avemar, and the changes in mRNA and protein levels of two important markers of angiogenesis, vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (Cox-2), were assessed by reverse transcription-quantitative polymerase chain reaction and ELISA. VEGF and Cox-2 protein and mRNA levels were significantly lower in Avemar-treated cells than in untreated cells. The data suggest that Avemar may exert an anti-angiogenic effect on cancer cells. Thus, it is suggested to medical doctors as a potential agent for the anti-angiogenic treatment of cancer.

Caspase-mediated Apoptotic Effects of Ebenus boissieri Barbey Extracts on Human Cervical Cancer Cell Line HeLa.

BACKGROUND: Ebenus boissieri Barbey is an Antalya, Turkey-endemic plant belonging to Fabaceae family. The aerial parts and the roots of E. boissieri Barbey were used in this study. OBJECTIVE: In the present study, we have examined the apoptotic effects of hydroalcoholic extracts of E. boissieri Barbey in human cervical cancer cell line HeLa. MATERIALS AND METHODS: To determine the cytotoxic effect, cells were treated with various concentrations of extracts for 24, 48, and 72 h incubation periods. Cytotoxic effects were examined by Cell Titer 96 aqueous nonradioactive cell proliferation assay and the results were corrected by live/dead viability/cytotoxicity assay and trypan blue exclusion assay. Apoptotic effects were studied with multicaspase kit. Tumor necrosis factor-alpha (TNF-α) and interferon gamma (IFN-γ) release were also measured by enzyme-linked immunosorbent assay. RESULTS: According to the results, E. boissieri Barbey extract caused significant increase in caspase levels. Thus, we suggest that the extract induces cells to undergo apoptosis. Especially, there was a sharp induction in caspase-3 activity. Levels of both TNF-α and IFN-γ in extract-treated groups were significantly and dose dependently exalted as compared to their relative controls. CONCLUSION: The effects of the extract on caspase-3, TNF-α, and IFN-γ levels mediate the plausible mechanism of apoptosis induction in HeLa. To the best of our knowledge, this is the first report indicating any pharmacological properties of E. Boissieri on HeLa cells. SUMMARY: HeLa cell viability was reduced in dose-dependent manner for 72 h with an IC50 of approximate 28.03 µg/mL for aerial and 41.02 µg/mL for rootHeLa cells, exposure to the aerial extract led to 1.9, 3.8, 1.2, 2.4, and 3.45 fold induction of all caspases activities (-2, -3, -6, -8, and -9, respectively)Both 30 µg/mL of aerial and 45 µg/mL of root extracts allowed the production of anticancer cytokines (TNFalpha; IFNgamma) in HeLa cell culture supernatants. Abbreviations used: Tumor necrosis factor-alpha (TNF-α); Interferon gamma (IFN-γ); 3-(4, 5 dimethylthiazol-2-yl)-5-(3- carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS); Phosphate-Buffered Saline (PBS); Fetal Bovine Serum (FBS); para-Nitroanilin pNA; Enzyme-Linked ImmunoSorbent Assay (ELISA); Sodium Dodesyl sulphate -Polyacrilamide gel electrophoresis (SDS-PAGE); Tris-Buffered Saline (TBS); Hydocloric acid (HCl); Standart Error of Mean (SEM); National Cancer Institute (NCI); half maximal inhibitory concentration (IC50).

Endostatin and irradiation modifies the activity of ADAM10 and neprilysin in breast cancer cells.

Angiogenesis, the formation of new blood vessels, is regarded as a key cancer cell property. Endostatin (ES) is a potential antiangiogenic agent and it may be useful when implemented in combination with other cancer therapeutic strategies. The present study investigated the in vitro effects of ES, radiotherapy (RT) or combination therapy (ES + RT) on two important proteases, a disintegrin and metalloproteinase domain­containing protein 10 (ADAM10) and neprilysin (NEP) in 4T1 mouse breast cancer cells and the more metastatic phenotype of 4THMpc breast cancer cells. 4T1 and 4THMpc cells were treated with recombinant murine ES (4 µg/ml) alone, RT (45 Gy) alone or with ES + RT. ADAM10 enzyme activity was determined using a tumor necrosis factor­α converting enzyme (α­secretase) activity assay kit, and NEP enzyme activity was measured with a fluorometric assay based on the generation of free dansyl­D­Ala­Gly from N-dansyl-Ala-Gly-D-nitro-Phe-Gly, the substrate of NEP. Western blotting analysis was performed to determine whether the altered enzyme activity levels of the two cell lines occurred due to changes in expression level. These data indicate that ES independently potentiates the activity of ADAM10 and NEP enzymes in 4T1 and 4THMpc breast cancer cells.

Dimethyl sulfoxide-caused changes in pro- and anti-angiogenic factor levels could contribute to an anti-angiogenic response in HeLa cells.

Dimethyl sulfoxide (DMSO) is widely used in biological research as a general solvent. While it has been previously demonstrated that DMSO possesses a wide range of pharmacological effects, there is no published work regarding the effects of DMSO on pro-angiogenic factor levels. This study was designed to investigate the possible effects of DMSO on the levels of three pro-angiogenic factors released from HeLa cells in vitro. Cells were treated with two different and previously determined concentrations of DMSO. The cytotoxic effects of DMSO concentrations on HeLa cells were determined via MTT. Survival rates of DMSO-treated cells were determined by Invitrogen live/dead viability/cytotoxicity kit and trypan blue exclusion assay. Changes in the pro-angiogenic levels in media were evaluated by Cayman's Substance P Enzyme Immunoassay ELISA kit. Vascular endothelial growth factor ELISA kit and interferon gamma ELISA kit for substance P, VEGF and IFNγ respectively. Changes in substance P levels were corrected by standard western blotting. Changes in VEGF and IFNγ levels were corrected both by western blot and real time PCR. Treatment with 1.4 µM DMSO caused a time-dependent inhibition of cell proliferation at 24, 48 and 72 h. 1.4 µM DMSO caused a significant reduction in VEGF levels at 72 h of incubation and sharp increases in IFNγ levels at both 48 and 72 h of incubation. According to real time PCR analyses, DMSO (1.4 µM) exhibited an inhibitory effect on VEGF but acted as an augmenter of IFNγ release on HeLa cells in vitro. This is the first report showing that the general solvent DMSO suppressed HeLa cell proliferation, decreased the levels of two pro-angiogenic factors (substance P and VEGF) and increased the release of an anti-angiogenic factor IFNγ in vitro.

Cytotoxic and apoptotic effects of Ebenus boissieri Barbey on human lung cancer cell line A549.

BACKGROUND: Fabaceae family members are known to possess preventive and therapeutic potentials against various types of cancers. OBJECTIVE: The aim of this study was to investigate the cytotoxic and apoptotic effects of hydroalcoholic extracts from the aerial parts and roots of an endemic Ebenus species; Ebenus boissieri Barbey in human lung cancer cell line. MATERIALS AND METHODS: After treatment with hydroalcoholic extracts cytotoxic activities of both extracts were measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide assay, whereas caspase-3 activity, tumor necrosis factor-a lpha (TNF-α) and interferon gamma (IFN-γ) releasewere measured by enzyme linked immunosorbent assay. RESULTS: According to in vitro assay results, the increase in all caspases activity suggested that extracts induce cells to undergo apoptosis. Especially, induction in caspase-3 activity was the most remarkable result of this study. Both aerial part and root extracts induced apoptosis by increasing caspase-3 activity, TNF-α and IFN-γ release. When compared to their relative controls, the concentrations of both TNF-α and IFN-γ in extract-treated groups were significantly and dose dependently exalted. CONCLUSION: Taken together, our results indicate that the hydroalcoholic extracts of E. boissieri can be considered as a source of new anti-apoptotic and therefore anti-carcinogenic agent.

Thalidomide combined with irradiation alters the activity of two proteases.

The aim of the present study was to investigate the effects of thalidomide, a drug known for its anti­angiogenic and antitumor properties, at its cytotoxic dose previously determined as 40 µg/ml (according to four cytotoxic test results). The effect of the drug alone and in combination with radiotherapy using Cobalt 60 (60Co) at 45 Gy on the enzymatic activity of substance­P degrading A disintegrin and metalloproteinase (ADAM)10 and neprilysin (NEP) was investigated in the mouse breast cancer cell lines 4T1 and 4T1 heart metastases post­capsaicin (4THMpc). Thalidomide (40 µg/ml) exerted differing effects on the activities of ADAM10 and NEP enzymes. In 4T1 cells, 40 µg/ml thalidomide alone did not alter ADAM10 enzyme activity. 60Co irradiation at 45 Gy alone caused a 42% inhibition in ADAM10 activity, however, the inhibition increased to 89% when combined therapy was used. By contrast, in the 4THMpc cell line, 40 µg/ml thalidomide alone induced a 66.6% increase in ADAM10 enzyme activity. Radiotherapy alone and thalidomide with 60Co combined therapy caused a 33.3 and 40% inhibition of ADAM10 activity, respectively. In 4T1 cells, thalidomide alone caused a 40.9% increase in NEP activity. Radiation therapy alone or in combination with the drug caused a 40.7% increase in NEP activity. In more aggressive 4THMpc cells, thalidomide alone caused a 26.6% increase in NEP activity. Radiotherapy alone and combined therapy caused a 33.3 and 37% increase in enzyme activity, respectively. To the best of our knowledge, the present study is the first to demonstrate that thalidomide alone or in combination with radiotherapy exhibits significant cytotoxic effects on 4T1 and 4THMpc mouse breast cancer cell lines indicating that this drug affects the enzymatic activity of ADAM10 and NEP in vitro.