Inhibitory effect of Bowman-Birk protease inhibitor on autophagy in MDAMB231 breast cancer cell line.

BACKGROUND: Autophagy has an essential role in cellular energetic balance, cell cycle, and cell death, so the change in autophagy level is crucial in many human diseases such as cancer. Herbal medicine has been widely used to treat cancer. Bowman-Birk protease inhibitor (BBI), a protease inhibitor extracted from soybean, has antitumorigenic, anti-inflammatory, and anti-angiogenic activities. In this study, we evaluated the effect of BBI on the growth of breast cancer cell line and transcript level of autophagy and apoptosis-related genes. MATERIALS AND METHODS: BBI was purified from soybean by ion-exchange chromatography method. The viability of MDA-MB-231 cells that were treated with BBI was measured by MTT assay, and the transcript level of genes involved in autophagy and apoptosis was measured by real-time-polymerase chain reaction (PCR) technique. RESULTS: The results of BBI purification showed that 100 g of the ethanolic fraction yielded 300-mg BBI with more than 95% purity. MTT results revealed that BBI inhibited the cell growth of MDA-MB-231 cell line in a dose-dependent manner, with an IC50 of 200 µg/mL. The results of real-time reverse transcription-PCR exhibited that BBI altered the expression of Atg5, Beclin1, light chain 3-II, and sequestosome1 and increased the Bax/Bcl2 ratio in MDA-MB-231 cell line. CONCLUSION: According to our results, BBI could inhibit autophagy and induce apoptosis in MDA-MB-231 cell line. Thus, BBI may be used as a therapeutic drug in the treatment of breast cancer whether alone or with chemotherapeutic drugs.

Thymoquinone influences the expression of genes involved in self-renewal and immunomodulatory potential of mouse bone marrow-derived mesenchymal stem cells in vitro.

Thymoquinone (TQ) is an active ingredient of some medicinal herbs. Despite extensive studies on the biological and pharmacological properties of TQ, its effect on the characteristics of stem cells remains to be clarified. Therefore, this study was aimed to investigate the effect of TQ on viability, proliferation and immunomodulatory potential of mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) in vitro. The BM-MSCs were isolated from young NMRI mice. The cytotoxic effect of TQ on the BM-MSCs was evaluated using MTT assay. Then, the effect of TQ on the proliferation of BM-MSCs and the mRNA expression of genes involved in self-renewal and immunomodulatory potential of MSCs was assessed by the cell counting and real-time PCR assays. Results showed that TQ reduces the number of BM-MSCs in a dose- and time-dependent manner. In addition, the half-maximal inhibitory concentration values of TQ on the BM-MSCs were 8 µg/ml at 24h and 4 µg/ml at 48 and 72h after treatment. Furthermore, about 90% of the BM-MSCs were alive after treatment with concentrations ≤2 µg/ml of TQ for 24h. The results of cell counting assay indicated that TQ at concentrations of 1-2 µg/ml significantly enhanced the proliferation of BM-MSCs (P < 0.05). The gene expression analysis also showed that Tlr3, Tlr4, Ccl2, Ccl3, Sox2, and Rex1 are overexpressed (Fold change ≥1.5) in the TQ-treated BM-MSCs compared with the untreated samples. In conclusion, these findings propose that TQ may regulate self-renewal and immunomodulatory potential of MSCs. However, the exact mechanisms and the roles of this regulation are required to be elucidated in further study.

Anti-carcinogenic and anti-angiogenic properties of the extracts of Acorus calamus on gastric cancer cells.

OBJECTIVE: Acorus calamus (A. calamus) has been used as a medicinal plant in Asia for its effects on digestive system for the last 2000 years. To investigate the anti-cancer activity of rhizome of A. calamus, the ethanolic and methanolic extracts and essential oil of the rhizome were prepared and their effects were assessed on human gastric cancer cell line (AGS). MATERIALS AND METHODS: The viability of cells which were treated with the extracts and the essential oil was assessed by MTT assay. To evaluate the anti-angiogenic property of the extracts, in vitro tube formation assay was done. Cell cycle distribution and the expression of Oct4 and Nucleostemin, after treatments, were checked by flowcytometry and quantitative RT-PCR, respectively. Furthermore, analysis of essential oil from A.calamus was done by GC-MS. RESULTS: Our results showed that the growth of AGS cells was inhibited by the extracts and essential oil and the extracts inhibited the angiogenesis in HUVEC cells. Our data revealed that the extracts and essential oil of A. calamus caused G1 arrest in AGS cells and downregulation of Oct4 and NS after treatment. By GC-MS analysis, we found new compounds such as epiprezizaene, valencene and isocyclocitral in essential oil of A. CONCLUSION: All together, our results showed that the extracts of A. calamus have anti-proliferative and anti-angiogenic effects on cancer cells.

Ferulago angulata flower and leaf extracts inhibit angiogenesis in vitro through reducing VEGF-A and VEGFR-2 genes expression.

BACKGROUND: Ferulago angulata (Apiacea) has been used in Iranian traditional medicine since ancient times and its various health care and pharmacological benefits have been demonstrated recently. In this study, for the first time, we have investigated the effects of F. angulata flower and leaf ethanol extracts on angiogenesis, as the key process in tumor growth, invasion and metastasis. METHODS: Cytotoxic effects of different concentrations (20-140 µg/mL) of each extract were assessed on human umbilical vein endothelial cells (HUVECs) using neutral red uptake assay. After evaluating the less toxic concentrations (up to 80 µg/mL), we performed three-dimensional angiogenesis, tube formation and migration assays to assess the key properties of HUVECs, including the angiogenesis process, in response to the extracts. Finally, quantitative gene expression analysis of VEGF-A and VEGFR-2, two critical mediators of angiogenesis, was performed using real-time RT-PCR. RESULTS: Both flower and leaf extracts exhibited concentration-dependent inhibition of sprouting, tube formation and migration capacities of HUVECs. For the flower extract, the respective IC50 values were 25.79, 26.52 and 38.92 µg/mL while for the leaf extract, the corresponding IC50 values were 34.18, 41.24 and 28.69 µg/mL. Both flower and leaf extracts downregulated VEGF-A and VEGFR-2 genes relative to the GAPDH gene as the internal control at concentrations of 60 and 80 µg/mL, respectively. CONCLUSION: These findings suggest that both flower and leaf extracts may contain anti-angiogenic compounds and may have the capacity to be utilized in tumor anti-angiogenic therapy strategies.

Ethanolic extract of Ficus carica leave Suppresses Angiogenesis by Regulating VEGF-A and Integrin ß3 mRNA Expression in Human umbilical vein endothelial cells.

BACKGROUND: In the present study, we investigated the anti-angiogenic effects of the ethanol extract of Ficus carica leave on human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were used in this study. The cells were cultured in DMEM medium and then incubated with different concentrations of ethanolic extract of Ficus carica leave (0-25 µg\ml) in the presence or absence of the extract for 24 hours. Cell viability was analyzed using neutral red assay. Endothelial cell tube formation was measured with the Matrigel basement membrane matrix. The level of VEGF and Integrin ß3 mRNA expression in the HUVECs was measured with reverse-transcription quantitative real-time polymerase chain reaction (RT-q real time PCR). RESULTS: We observed that the extract dose dependently inhibited the tube formation of HUVECs. Furthermore, the extract significantly decreased mRNA expression levels of VEGF-A and Integrin ß3 in HUVECs at 20 µg\ml concentration of the extract compared to untreated control cells (P < 0.05). CONCLUSION: Our findings suggest that ethanolic extract of Ficus carica leave contains anti-angiogenic activities and could be a candidate as a potential agent for the prevention of angiogenesis related disorders.

Anti-tumor Activity of Ferulago angulata Boiss. Extract in Gastric Cancer Cell Line via Induction of Apoptosis.

Ferulago angulata Boiss. known in Iran as Chavir, has some bioactive compounds having antioxidant activity. Because of its antioxidant activities, it sounded Chavir extract can be a good candidate for finding chemopreventive agents having inductive apoptosis properties on cancer cells. In this study, the cytotoxic effects and proapoptotic activities of Chavir's leaf and flower extracts were investigated on human adenocarcinoma gastric cell line (AGS). The ferric reducing antioxidant power (FRAP) assay was used to determine antioxidant activity of the extract. Cytotoxic effects of the extract were performed by trypan blue and neutral red assays. For apoptosis detection, we used Annexin V staining, flow cytometry and DNA fragmentation assays. The FRAP assay results showed that antioxidant activity of leaf extract was higher than flower extract. Cytotoxicity and apoptosis-inducing activity of flower and leaf extracts changed coordinately, indicating the cytotoxicity of chavir extracts is due probably to induce apoptosis. Our results revealed that the cytotoxic effects of F. angulate Boiss. extracts on AGS cell line is close to some other plant extracts such as Rhus verniciflua Stokes (RVS) and Scutellaria litwinowii. This is the first study on cytotoxic and apoptosis-inducing effects of chavir leaf and flower extracts against AGS cell line. The Further investigation can be identification of the agent(s) by which these effects is observed.

PlGF gene knockdown in human retinal pigment epithelial cells.

BACKGROUND: To evaluate the knockdown of placental growth factor (PlGF) gene expression in human retinal pigment epithelium (RPE) cells and its effect on cell proliferation, apoptosis and angiogenic potential of RPE cells. METHODS: Human RPE cells were isolated by dispase I solution and cultured in DMEM/F12 supplemented with 10% fetal calf serum (FCS). A small interfering RNA (siRNA) corresponding to PlGF mRNA and a scrambled siRNA (scRNA) were introduced into the cells. Cell proliferation and cell death were examined by ELISA. PlGF mRNA and protein were quantified by real-time polymerase chain reaction (PCR) and western blot. The levels of gene expression for human retinal pigment epithelium-specific protein 65 kDa (RPE65), cellular retinaldehyde-binding protein (CRALBP) and tyrosinase were examined by real-time PCR. The angiogenic activity of RPE cell-derived conditioned media was assayed by a tube formation assay using human umbilical vein endothelial cells (HUVECs). RESULTS: At a final siRNA concentration of 20 pmol/ml, the transfection efficiency was about 80%. The amount of PlGF transcripts was reduced to 10% after 36 h of incubation, and the amount of PlGF protein in culture supernatant was significantly decreased. Suppression of PlGF gene had no effect on RPE cell proliferation and survival, and there were no notable changes in the transcript levels of RPE65, CRALBP or tyrosinase for the cultures treated by siRNA cognate to PlGF. Vascular tube formation was efficiently reduced in HUVECs. CONCLUSIONS: Our findings present PlGF as a key modulator of angiogenic potential in RPE cells of the human retina.