Although human telomerase catalytic subunit (TERT) has several cellular functions including telomere homeostasis, genomic stability, cell proliferation, and tumorigenesis, the molecular mechanism underlying anti-apoptosis regulated by TERT remains to be elucidated. Here, we show that ectopic expression of TERT in spontaneously immortalized human fetal fibroblast (HFFS) cells, which are a telomerase- and p53-positive, leads to increases of cell proliferation and transformation, as well as a resistance to DNA damage response and inactivation of p53 function. We found that TERT and a mutant TERT (no telomerase activity) induce expression of basic fibroblast growth factor (bFGF), and ectopic expression of bFGF also allows cells to be resistant to DNA-damaging response and to suppress activation of p53 function under DNA-damaging induction. Furthermore, loss of TERT or bFGF markedly increases a p53 activity and DNA-damage sensitivity in HFFS, HeLa and U87MG cells. Therefore, our findings indicate that a novel TERT-bFGF axis accelerates the inactivation of p53 and consequent increase of resistance to DNA-damage response.
*Apoptosis ; *Catalytic Domain ; Cell Line, Transformed ; Cell Proliferation ; DNA Damage ; Fetus/cytology ; Fibroblast Growth Factor 2/*genetics/metabolism ; Fibroblasts/cytology/metabolism ; Gene Expression Regulation, Neoplastic ; Hela Cells ; Humans ; RNA, Messenger/genetics/metabolism ; Telomerase/deficiency/*metabolism ; Tumor Suppressor Protein p53/*metabolism

PURPOSE: Diallyl disulfide (DADS), an organosulfur compound in garlic, has been reported to be effective in inhibiting the growth of several human tumor cell lines. The aim of this study was to determine whether DADS induced growth inhibition in MCF-7 breast cancer cell lines and to understand the molecular mechanism by which DADS acts. METHODS: MCF-7 cell lines were incubated with various concentrations of DADS for various time intervals and the cytotoxicity was determined by MTT assay. We examined the changes of intracellular proteins related to apoptosis, such as bcl-2, bax and PARP in cells treated with DADS. To study the expression level of bcl-2 and bax, which serve as modulators of apoptosis, we performed RT-PCR and western blot analysis. RESULTS: MCF-7 cells treated with DADS led to the suppression of viability and proliferation in both a time and concentration dependent manner. Microscopic observation revealed typical features of apoptosis in the DADS-treated cells, further verified in nuclear DAPI staining. Flow cyto-metry analysis with FITC-annexinV and propidium iodide (PI) demonstrated that the apoptotic cell population with AnnexinV+/PI- increased dramatically from ~0.8% to ~75% after 24h exposure to 500 microM DADS in MCF-7 cells. Cell cycle analysis demonstrated that the number of apoptotic cells increased with the increasing time of the DADS treatment. Additionally, thermore, we investigated the effects of DADS on apoptosis related gene expression in MCF-7 cells. PARP cleavage was markedly increased in the DADS treated cells with time. This result indicated that DADS induced the caspase-dependent apoptotic pathway. We also found down-regulation of bcl-2, however no significant change of Bax expression was observed after DADS treatment. Conclusion: Taken together, these results indicate that DADS induces apoptosis by activating a caspase pathway involving the activation of Bcl-2 but not of Bax. Our findings suggest chemotherapeutic potentials of DADS in human breast cancer.
Apoptosis ; Blotting, Western ; Breast Neoplasms* ; Breast* ; Cell Cycle ; Cell Line* ; Cell Line, Tumor ; Down-Regulation ; Garlic* ; Gene Expression ; Humans* ; MCF-7 Cells ; Propidium

Hypoxia-inducible factor 1alpha (HIF-1alpha), which transactivates a variety of hypoxia-induced genes, is rapidly degraded under nomoxia through the hydroxylation-ubiquitination-proteasome pathway. In this study, we addressed how HIF-1alpha is stabilized by proteasome inhibitors. The ubiquitin pool was rapidly reduced after proteasome inhibition, followed by the accumulation of non-ubiquitinated HIF-1alpha. The poly-ubiquitination of HIF-1alpha was resumed by restoration of free ubiquitin, which suggests that the HIF-1alpha stabilization under proteasome inhibition is attributed to depletion of the free ubiquitin pool. Ni2+ and Zn2+ also stabilized HIF-1alpha with depletion of the free ubiquitin pool and these effects of metal ions were attenuated by restoration of free ubiquitin. Ni2+ and Zn2+ may disturb the recycling of free ubiquitin, as MG132 does. Based on these results, the state of the ubiquitin pool seems to be another critical factor determining the cellular level of HIF-1alpha.
Cell Hypoxia/physiology ; Cell Line, Tumor ; HCT116 Cells ; HEK293 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis/*metabolism ; Leupeptins/pharmacology ; Nickel/chemistry ; Proteasome Endopeptidase Complex/*metabolism ; Proteasome Inhibitors/*pharmacology ; Ubiquitin/*metabolism ; Ubiquitination/*physiology ; Up-Regulation ; Zinc/chemistry

Inflammation plays an important role in host defense against external stimuli such as infection by pathogen, endotoxin or chemical exposure by the production of the inflammatory mediators that produced by macrophage. Anti-inflammatory factor is important to treat the dangers of chronic inflammation associated with chronic disease. This research aims to analyze the anti-inflammatory effects of Garcinia mangostana L. peel extract (GMPE), α-mangostin, and γ-mangostin in LPS-induced murine macrophage cell line (RAW 264.7) by inhibiting the production of inflammatory mediators. The cytotoxic assay of G. mangostana L. extract, α-mangostin, and γ-mangostin were performed by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) to determine the safe and non-toxic concentration in RAW 264.7 for the further assay. The concentration of inflammatory mediators (COX-2, IL-6, and IL-1β) were measured by the ELISA-based assay and NO by the nitrate/nitrite colorimetric assay in treated LPS-induced RAW 264.7 cells. The inhibitory activity was determined by the reducing concentration of inflammatory mediators in treated LPS-induced RAW 264.7 over the untreated cells. This research revealed that GMPE, α-mangostin, and γ-mangostin possess the anti-inflammatory effect by reducing COX-2, IL-6, IL-1β, and NO production in LPS-induces RAW 264.7 cells.
Cell Line ; Chronic Disease ; Fibrinogen ; Garcinia mangostana ; Inflammation ; Interleukin-6 ; Macrophages ; RAW 264.7 Cells

PURPOSE: Diallyl disulfide (DADS), an organosulfur compound in garlic, has been reported to be effective in inhibiting the growth of several human tumor cell lines. The aim of this study was to determine whether DADS induced growth inhibition in MCF-7 breast cancer cell lines and to understand the molecular mechanism by which DADS acts. METHODS: MCF-7 cell lines were incubated with various concentrations of DADS for various time intervals and the cytotoxicity was determined by MTT assay. We examined the changes of intracellular proteins related to apoptosis, such as bcl-2, bax and PARP in cells treated with DADS. To study the expression level of bcl-2 and bax, which serve as modulators of apoptosis, we performed RT-PCR and western blot analysis. RESULTS: MCF-7 cells treated with DADS led to the suppression of viability and proliferation in both a time and concentration dependent manner. Microscopic observation revealed typical features of apoptosis in the DADS-treated cells, further verified in nuclear DAPI staining. Flow cytometry analysis with FITC-annexinV and propidium iodide (PI) demonstrated that the apoptotic cell population with AnnexinV /PI increased dramatically from ~0.8% to ~75% after 24h exposure to 500nM DADS in MCF-7 cells. Cellcycle analysis demonstrated that the number of apoptotic cells increased with the increasing time of the DADS treatment. Additionally, thermore, we investigated the effects of DADS on apoptosis related gene expression in MCF-7 cells. PARP cleavage was markedly increased in the DADS treated cells with time. This result indicated that DADS induced the caspase-dependent apoptotic pathway. We also found down-regulation of bcl-2, however no significant change of Bax expression was observed after DADS treatment. CONCLUSION: Taken together, these results indicate that DADS induces apoptosis by activating a caspase pathway involving the activation of Bcl-2 but not of Bax. Our findings suggest chemotherapeutic potentials of DADS in human breast cancer.
Apoptosis ; Blotting, Western ; Breast Neoplasms ; Breast ; Cell Line ; Cell Line, Tumor ; Down-Regulation ; Flow Cytometry ; Garlic ; Gene Expression ; Humans ; MCF-7 Cells ; Propidium

In this study, we investigated the anticancer activity of the fin of Thunnus Thynnus (TT ). TT was extracted with methanol (TTM ), and then further fractionated into four subfractions by using solvent partition method, affording hexane (TTMH ), methanol (TTMM ), butanol (TTMB )and aquous (TTMA )soluble fractions. We determined the cyto-toxicity of these four fractions in four kind of cancer cell lines, such as HepG2, MCF-7, B16-F10 and HT29 by MTT assay. The TTMM showed the strongest cytotoxic effect at the concentration of 150 microgram/mL, displaying 95% on the HepG2 cell lines and 82% on MCF-7 cell line. The morphological changes such as membrane shirinking and blebbing of cells were also observed by TTMM treatment in HT29 cell. In addition, we observed that quinone reductase (QR ) activity was elevated by only TTMM and TTMH treatments in HepG2 cell. QR activity was increased to around 2.0 and 1.8 times in TTMM and TTMH treated HepG2 cell at 100 microgram/mL, respectively, compared to that in control. Although further studies are needed, the present work could suggest that the fin of TT has a potential to be usable as a chemo-preventive agent against cancer.
Blister ; Cell Line ; Hep G2 Cells ; HT29 Cells ; Humans ; MCF-7 Cells ; Membranes ; Methanol ; NAD(P)H Dehydrogenase (Quinone)*

An antisense approach was attempted to investigate the role of antisense GLUT1 RNA in suppressing tumor cell phenotypes using N-ras-transformed NIH 3T3 cells. The established cell line transformed by ras showed typical biological characteristics of cancer cells, such as increased glucose transport, GLUT1 mRNA contents, and the ability to form colonies on the soft agar. In this system, the plasmids (pMAM-GLUT1(rev)) which can transcribe the antisense GLUT1 RNA were transfected and the accompanying changes in the phenotypes of the ras-transformed cells were observed. The expression of antisense GLUT1 RNA by induction with dexamethasone reduced the glucose transport by 30% (1.97 +/- 0.13 nmoles) after 4 min incubation when compared to the non-induction group of transformed cell (2.85 +/- 0.19 nmoles). Also, the number of colonies sized over 50 microns on the soft agar was reduced significantly in the antisense RNA expressing group compared to non-induction group. These results suggest that the expression of antisense GLUT1 RNA reduced the glucose transport and transforming potential in soft agar possibly by hybridization with GLUT1 mRNA in N-ras-transformed NIH 3T3 cells.
3T3 Cells/metabolism ; Animal ; Base Sequence ; Cell Line, Transformed ; Cell Transformation, Neoplastic/metabolism/*pathology ; *Genes, ras ; Human ; Mice ; Molecular Sequence Data ; Monosaccharide Transport Proteins/*genetics ; Phenotype ; RNA, Antisense/*metabolism ; Support, Non-U.S. Gov't ; Tumor Cells, Cultured/metabolism/pathology

The Chaga mushroom (Inonotus obliquus) has been used in folk medicine to treat cancers. However, limited information exists on the underlying anticancer effects of the major component of I. obliquus in vivo. We hypothesize that the pure compounds (3beta-hydroxy-lanosta-8,24-dien-21-al, inotodiol and lanosterol, respectively) separated from I. obliquus would inhibit tumor growth in Balbc/c mice bearing Sarcoma-180 cells (S-180) in vivo and growth of human carcinoma cells in vitro. To test this hypothesis, the growth inhibition of each subfraction isolated from I. obliquus on human carcinoma cell lines (lung carcinoma A-549 cells, stomach adenocarcinoma AGS cells, breast adenocarcinoma MCF-7 cells, and cervical adenocarcinoma HeLa cells) was tested in vitro. Then, after S-180 implantation, the mice were fed a normal chow supplemented with 0, 0.1 or 0.2 mg of subfraction 1, 2 or 3 per mouse per day. All of the subfractions isolated from I. obliquus showed significant cytotoxic activity against the selected cancer cell lines in vitro. Subfraction 1 was more active than subfraction 2 and subfraction 3 against the A549, AGS and MCF-7 cancer cell lines in vitro. In in vivo results, subfraction 1 isolated from I. obliquus at concentrations of 0.1 and 0.2 mg/mouse per day significantly decreased tumor volume by 23.96% and 33.71%, respectively, as compared with the control. Subfractions 2 and 3 also significantly inhibited tumor growth in mice bearing S-180 as compared with the control mouse tumor. Subfraction 1 isolated from I. obliquus showed greater inhibition of tumor growth than subfractions 2 and 3, which agrees well with the in vitro results. The results suggest that I. obliquus and its compounds in these subfractions isolated from I. obliquus could be used as natural anticancer ingredients in the food and/or pharmaceutical industry.
Adenocarcinoma ; Agaricales ; Animals ; Breast ; Cell Line ; Drug Industry ; Humans ; Lanosterol ; MCF-7 Cells ; Medicine, Traditional ; Mice ; Stomach ; Tumor Burden ; Ursidae

The Chaga mushroom (Inonotus obliquus) has been used in folk medicine to treat cancers. However, limited information exists on the underlying anticancer effects of the major component of I. obliquus in vivo. We hypothesize that the pure compounds (3beta-hydroxy-lanosta-8,24-dien-21-al, inotodiol and lanosterol, respectively) separated from I. obliquus would inhibit tumor growth in Balbc/c mice bearing Sarcoma-180 cells (S-180) in vivo and growth of human carcinoma cells in vitro. To test this hypothesis, the growth inhibition of each subfraction isolated from I. obliquus on human carcinoma cell lines (lung carcinoma A-549 cells, stomach adenocarcinoma AGS cells, breast adenocarcinoma MCF-7 cells, and cervical adenocarcinoma HeLa cells) was tested in vitro. Then, after S-180 implantation, the mice were fed a normal chow supplemented with 0, 0.1 or 0.2 mg of subfraction 1, 2 or 3 per mouse per day. All of the subfractions isolated from I. obliquus showed significant cytotoxic activity against the selected cancer cell lines in vitro. Subfraction 1 was more active than subfraction 2 and subfraction 3 against the A549, AGS and MCF-7 cancer cell lines in vitro. In in vivo results, subfraction 1 isolated from I. obliquus at concentrations of 0.1 and 0.2 mg/mouse per day significantly decreased tumor volume by 23.96% and 33.71%, respectively, as compared with the control. Subfractions 2 and 3 also significantly inhibited tumor growth in mice bearing S-180 as compared with the control mouse tumor. Subfraction 1 isolated from I. obliquus showed greater inhibition of tumor growth than subfractions 2 and 3, which agrees well with the in vitro results. The results suggest that I. obliquus and its compounds in these subfractions isolated from I. obliquus could be used as natural anticancer ingredients in the food and/or pharmaceutical industry.
Adenocarcinoma ; Agaricales ; Animals ; Breast ; Cell Line ; Drug Industry ; Humans ; Lanosterol ; MCF-7 Cells ; Medicine, Traditional ; Mice ; Stomach ; Tumor Burden ; Ursidae

OBJECTIVE: The efficacy of two-drug combination treatment may be schedule-dependent. We investigated a simulated in-vitro interaction between taxol and doxorubicin in a Cervical cancer cell line HeLa and the role of peroxiredoxin in cytotoxicity. METHODS: Two contradicting schedules of two drugs (taxol followed by doxorubicin or vice versa) were compared each other in terms of cytotoxicity in parental HeLa cell line and the peroxiredoxin (prx)-overexpressing variant. Cytotoxic activity was determined by MTT assay. Cell cycle pertubation was evaluated by flow cytometric analysis. Protein levels were determined by western blot. RESULTS: The sequential treatment of taxol followed by doxorubicin (T--HeLa cells than the reverse sequenced treatement did. In contrast, in parental HeLa cells, no cytotoxic difference was noted in accordance with sequences. In prx-overexpressing HeLa cells, apoptotic change was more prominent in the the sequence of doxorubucin followed by taxol (D--cells, prominent PARP activations were evenly observed in both sequences. Protein level of cyclin D1, a proto-oncogen, was less decreased in the sequence of T--cells compared with the reverese sequence of D--cells. These findings suggest that, in prx-overexpressing situation, it is worthy to concern about the treatment sequence when taxol and doxorubicin are supposed to employ to treat cancer of the uterine cervix.
Appointments and Schedules ; Blotting, Western ; Cell Cycle ; Cell Line ; Cell Line, Tumor* ; Cyclin D1 ; Doxorubicin ; HeLa Cells ; Humans ; Paclitaxel ; Parents ; Peroxiredoxins* ; Uterine Cervical Neoplasms