• 대한한방종양학회지
• /
• 제6권1호
• /
• pp.67-79
• /
• 2000

Objective : Hedyotis diffusa has been used as an anticancer agent for several decades in oriental medicine. We test whether the methanol extract of the herb affects transcriptional activation factors including $NF-{\kappa}B$ and AP-1. Methods : 1. HL-60 cells were treated with various concentrations(from 200 to $50{\mu}g/ml$) of methanol extract and $H_2O$ extract($200{\mu}g/ml$)of hedyotis diffusa, After 48h later, the cells were tested for viability by MTT assay. 2. The HL-60 cells were treated with $200{\mu}g/ml$ of methanol extract for the indicated periods. First. Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$ and AP-1. Second. Nuclear extracts were isolated and reacted with p50, p65. c-rel pan-Jun, c-Jun, JunB. JunD antibody on ice for 30min. Finally The cell lysates were prepared and analyzed by western blotting using anti-Fas, anti-FasL and anti-p53 antibody. Results : 1. The methanol extract decreases the viability of human lymphoid origin leukemia HL-60 cells in a dose-dependent manner. 2. $NF-{\kappa}B$ is rapidly activated by the addition of the methanol extract, reaches a peak at 30min and gradually returns to resting level. We confirm that $NF-{\kappa}B$ is a heterodimer mainly composed of p65 subunit with c-Rel. 3. Transcriptional activation of AP-1 is detected at 30min and reaches a maximum at 1hr after stimulation of the cells with the methanol extract. AP-1 is mainly composed with Jur-D and partially Jug-B proteins. 4. the methanol extract of Hedyotis diffusa induces the expression of Fas, Fas ligand and p53 proteins of HL-60 cells in a time dependent fashion. Conclusions : These results suggest that the methanol extract of Hedyotis diffusa exerts anticancer effects to induce the death of human leukomic HL-60 cells via activation of trascriptional factors such as $NF-{\kappa}B$ and AP-1, increase in expression of Fas mediated signalling proteins, and induction of tumor suppressor gene. p53.

• 동의신경정신과학회지
• /
• 제21권2호
• /
• pp.125-140
• /
• 2010

Objectives : The purpose of this study is to examine from various angles the protective effect of Gastrodia elata Blume (GEB) against nerve cell death induced by $\beta$-amyloid by using the cell line SH-SY5Y, which is commonly utilized for toxicity testing in nerve cells, and to find out its mechanism of action. Methods : To begin with, as a result of assessing the rate of cell survival by employing MTT reduction assay, the treatment with $\beta$-amyloid at different concentrations caused cytotoxicity, which was inhibited by preprocessing GEB extract. In addition, after $\beta$-amyloid was processed with the cell SH-SY5Y, apoptosis progressed, which was reduced effectively by processing GEB extract. Results : When cytotoxicity was caused by using hydrogen peroxide, a representative ROS, in order to examine the antioxidant effect of GEB, its protective effect was also observed. Apart from ROS, reactive nitrogen species (RNS) are also known to play a crucial role in nerve cell death. The treatment with the NO donor SNAP increased the production of nitric oxide and the expression of iNOS, which was also inhibited by GEB extract. Meanwhile, as an attempt to find out the mechanism of action explaining the antioxidant effect, the intracellular antioxidant enzyme expressions were measured by RT-PCR, which showed that GEB extract increased the expressions of heme oxygenase-1, GAPDH and $\gamma$-glutamate cysteine ligase. Lastly, GEB extract had a protective effect against impaired memory induced by scopolamine in animal models (in vivo). Conclusions : These findings indicate that GEB has a protective effect against the death of cranial nerve cells, suggesting possibilities for the prevention and treatment of AD.

• Asian Pacific Journal of Cancer Prevention
• /
• 제13권8호
• /
• pp.4037-4043
• /
• 2012

Despite progress in elucidating mechanisms associated with colorectal cancer and improvement of treatment methods, it remains a frequent cause of death worldwide. New and more effective therapies are therefore urgently needed. Recent studies have shown that immunogenicity of whole ovarian tumor cells and subsequent T cell response were potentiated by oxidation modification with hypochlorous acid (HOCl) in vitro and ex vivo. These results prompted us to investigate the protective antitumor response with an HOCl treated CT26 colorectal cancer cell vaccine in an in vivo mouse model. Administration of HOCl modified vaccine triggered robust antitumor immunity to autologous tumor cells in mice and prolonged survival period significantly. In addition, increased necrosis and apoptosis were found in tumor tissue from the oxidation group. Interestingly, ELISPOT assays showed that specific T cell responses were not elicited in response to the immunizing cellular antigen, in contrast to raising sera antibody titer and antibody binding activity shown by ELISA assay and flow cytometry. Further evaluation of the mechanisms underlying HOCl modified vaccine mediated humoral immunity highlighted the role of antibody-dependent cell-mediated cytotoxicity. These results combined with previous studies suggest that HOCl oxidation modified whole cell vaccine has wide applicability as a cancer vaccine because it can target both T cell- and B cell-specific responses. It may thus represent a promising approach for the immunotherapy of colorectal cancer.

• Asian Pacific Journal of Cancer Prevention
• /
• 제16권10호
• /
• pp.4215-4218
• /
• 2015

Gastric cancer (GC) is one of the most common malignancies in the world. It is the first cause of cancer deaths in both sexes In Iranian population. Circulating insulin-like growth factor-one (IGF-1) levels have been associated for gastric cancer. IGF-1 protein has central roles involved in the regulation of epithelial cell growth, proliferation, transformation, apoptosis and metastasis. Single nucleotide polymorphism in IGF-1 regulatory elements may lead to alter in IGF-1expression level and GC susceptibility. The aim of this study was to investigate the influence of IGF-1 gene polymorphism (rs5742612) on risk of GC and clinicopathological features for the first time in Iranian population. In total, 241 subjects including 100 patients with GC and 141 healthy controls were recruited in our study. Genotypes were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay with DNA from peripheral blood. The polymorphism was statistically analyzed to investigate the relationship with the risk of GC and clinicopathological properties. Logistic regression analysis revealed that there was no significant association between rs5742612 and the risk of GC. In addition, no significant association between genotypes and clinicopathological features was observed (p value>0.05). The frequencies of the CC, CT, and TT genotypes were 97%, 3%, and 0%, respectively, among the cases, and 97.9%, 2.1%, and 0%, respectively, among the controls. CC genotype was more frequent in cases and controls. The frequencies of C and T alleles were 98.9% and 1.1% in controls and 98.5% and 1.5% in patient respectively. Our results provide the first evidence that this variant is rare in Iranian population and it may not be a powerful genetic predisposing biomarker for prediction GC clinicopathological features in an Iranian population.

• Asian Pacific Journal of Cancer Prevention
• /
• 제13권10호
• /
• pp.5137-5142
• /
• 2012

UHRF2 is a member of the ubiquitin plant homeo domain RING finger family, which has been proven to be frequently up-regulated in colorectal cancer cells and play a role as an oncogene in breast cancer cells. However, the role of UHRF2 in glioma cells remains unclear. In this study, we performed real-time quantitative PCR on 32 pathologically confirmed glioma samples (grade I, 4 cases; grade II, 11 cases; grade III, 10 cases; and grade IV, 7 cases; according to the 2007 WHO classification system) and four glioma cell lines (A172, U251, U373, and U87). The expression of UHRF2 mRNA was significantly lower in the grade III and grade IV groups compared with the noncancerous brain tissue group, whereas its expression was high in A172, U251, and U373 glioma cell lines. An in vitro assay was performed to investigate the functions of UHRF2. Using a lentivirus-based RNA interference (RNAi) approach, we down-regulated UHRF2 expression in the U251 glioma cell line. This down-regulation led to the inhibition of cell proliferation, an increase in cell apoptosis, and a change of cell cycle distribution, in which S stage cells decreased and G2/M stage cells increased. Our results suggest that UHRF2 may be closely related to tumorigenesis and the development of gliomas.

• 한국식품영양과학회지
• /
• 제41권8호
• /
• pp.1072-1078
• /
• 2012

본 연구는 팽이버섯으로부터 열수 및 에탄올 추출물의 항산화와 항염증 활성을 탐색하였다. 라디칼 소거능을 측정한 결과, DPPH 라디칼 소거능은 팽이버섯 열수 추출물의 경우 천연 항산화제인 비타민 C보다 활성이 우수하지는 않지만 다른 종류의 버섯 추출물보다 우수한 효능을 확인하였고 추후 분리, 정제를 통하여 유효 물질을 발굴하여 새로운 소재를 찾아내는 연구가 필요할 것으로 판단된다. ABTS 라디칼 소거능과 FRAP을 이용한 총항산화 효능에 관한 결과에서도 팽이버섯 추출물이 항산화 효과를 나타내는 것을 확인하였고, FTC법을 이용한 지질과산화 억제 효능을 살펴본 결과도 마찬가지로 열수 추출물이 에탄올 추출물보다 항산화 활성이 우수한 것을 확인하였다. 한편, 세포 독성을 살펴보기 위하여 항산화력이 우수한 팽이버섯 열수 추출물을 이용하여 정상 신경세포에 MTT assay를 수행한 결과, 세포의 생존율은 1.0 mg/mL의 이하 농도까지는 생존에 영향을 미치지 않는 것을 확인하였고 신경세포 보호효능 실험에서는 1.0 mM $H_2O_2$로 유도시킨 세포사에 대해 농도의존적인 신경세포 보호 효과가 있는 것을 확인하였으며, 마크로파지 세포에서도 독성에 대하여 신경세포와 유사한 결과를 확인하였다. NO의 저해효능도 농도 의존적으로 감소하였고 추가적으로 세포의 유전학적 분석인 western blot으로 iNOS와 COX-2를 수행하였을 때 LPS군에 비해 팽이버섯 열수 추출물군에서 iNOS 단백질 발현량이 감소하는 것을 확인하였다. 하지만 좀 더 명확한 결과를 위하여 차후 효소활성 및 동물연구 등이 추가적으로 필요할 것으로 판단된다.

• IMMUNE NETWORK
• /
• 제6권1호
• /
• pp.33-42
• /
• 2006

Background: Calcineurin plays a crucial role in T cell activation, cell growth, apoptosis, and angiogenesis, and its over-expression has been implicated in the pathogenesis of cardiomyopathy and stroke. However, the expression and function of calcineurin in the pathologic lesion of chronic inflammatory diseases, like rheumatoid synovium, remain to be defined. This study was aimed to determine the role of calcineurin in inflammatory arthritis and investigate the expression and function of calcineurin in the rheumatoid synovium and synoviocytes, the actual site of chronic inflammation. Methods: Immuno-histochemical staining using specific antibody to calcineurin was perfomed in the synovium of rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLS) from RA and osteoarthritis (OA) patients were isolated from RA and OA patients, and cultured with IL-1${\beta}$ and TNF-${\alpha}$ in the presence or absence of cyclosporin A, a calcineurin inhibitor. The calcineurin expression was assessed by phosphatase assay and Western blotting analysis. IL-6, -10, -17, matrix metalloproteinase (MMP)-1, -2, -3, and -9 released into the culture supernatants were measured by ELISA. After transfection with GFP-Cabin 1 gene into synoviocytes, the levels of IL-6 and MMPs were measured by ELISA. Results: Calcineurin was highly expressed in the lining layer of synovium and cultured synoviocytes of RA patients. The elevated calcineurin activity in the rheumatoid synoviocytes was triggered by proin flammatory cytokines such as IL-1${\beta}$ and TNF-${\alpha}$. In contrast, IL-10, an anti-inflammatory cytokine, failed to increase the calcineurin activity. The targeted inhibition of calcineurin by the over-expression of Cabin 1, a natural calcineurin antagonist, inhibited the production of IL-6 and MMP-2 by rheumatoid synoviocytes in a similar manner to the calcineurin inhibitor, cyclosporin A. Conclusion: These data suggest that abnormal activation of calcineurin in the synoviocytes may contribute to the pathogenesis of chronic arthritis, and thus provide a potential target for controlling inflammatory arthritis.

• 대한본초학회지
• /
• 제31권4호
• /
• pp.101-109
• /
• 2016

Objectives : HT070 is a mixture of herbal extracts from root of Scutellaria baicalensis and stem bark of Eleutherococcus senticosus , which have long been used for stroke therapy in traditional Korean Medicine. The purpose of this study was to investigate the neuroprotective effects of HT070 on global cerebral ischemia and its potential mechanisms.Methods : Transient global cerebral ischemia was produced by 10 min of four-vessel occlusion (4-VO) in male Wistar rats. HT070 was administered orally at a dosage of 200 mg/kg twice at 0 and 90 min after reperfusion. Hippocampal neuronal damage was measured 7 days after reperfusion. To explore the potential mechanisms, we used hydrogen peroxide (H₂O₂)-induced rat pheochromocytoma (PC12) cells as an in vitro model. PC12 cells were pretreated with HT070 for 1 h and then exposed to 100 μM H₂O₂ for 6 h in the presence of HT070. Cell viability was measured by MTT assay and the mRNA expression of Bax, Bcl-2, iNOS and COX-2 were measured by quantitative RT-PCR.Results : Oral administration of HT070 at a dose of 200 mg/kg significantly reduced neuronal death in the hippocampal CA1 region by 13.4% as compared to the vehicle-treated group. HT070 increased cell viability, reversed the down-regulated Bcl-2 mRNA level, and suppressed the up-regulated mRNA expressions of Bax, iNOS, and COX-2 in H₂O₂-treated PC12 cells.Conclusions : HT070 protects against delayed neuronal death after global cerebral ischemia and its neuroprotection properties might be attributed to the inhibition of mitochondrial apoptosis and ROS-generating enzymes.

• 한국식품과학회지
• /
• 제36권6호
• /
• pp.1001-1007
• /
• 2004

암세포 성장 억제효과가 인정된 산수유 methanol 추출물의 chloroform층으로부터 silica gel column과 thin layer chromato-grapies를 이용하여 항암활성물질을 분리하고, IR, mass spectrometer, $^1H-NMR$ 및 $^{13}C-NMRs$을 통해 순수물질을 동정하였다. 이 물질은 triterpenoid로서 $C_{30}H_{48}O_3$의 구조식을 가진 화합물로 분자량이 456인 ursolic acid($3{\beta}$-hydroxyrus-12-ene-28-oic acid)로 동정되었다. 정제된 화합물을 A549 및 MCF-7 암세포주에 48시간동안 처리한 결과 농도 의존적으로 암세포주의 성장을 억제하였으며, 화합물을 처리하지 않은 대조구에 비하여 $30\;{\mu}g/mL$농도로 처리시 40% 이상 그 성장을 억제하였으며, $100\;{\mu}g/mL$농도로 처리시 90% 이상 그 성장을 억제하였다. 정제 화합물을 처리한 암세포를 광학현미경으로 관찰한 결과 뚜렷한 세포수의 감소와 함께 심한 형태학적 변화가 관찰되었다. 암세포 주에 정제시료를 $10\;{\mu}g/mL$농도로 처리하고, 15시간 배양한 후 세포주기를 분석한 결과 sub-G1 phase의 비율이 A549의 경우는 대조구에서 4.0%이었던 것이 11.7%로, MCF-7의 경우는 대조구에서 2.1%이었던 것이 처리구에서는 11.2%로 각각 증가된 것으로 나타나 정제물질은 apoptosis에 의한 세포사멸 유도를 시사하였다.

• 대한한방내과학회지
• /
• 제28권3호
• /
• pp.409-420
• /
• 2007

Objectives : The purpose of this study was to identify the anti-tumor effects of realgar on various cancer cells through molecular biologic and cellular biologic methods. Materials & Methods : We used 5 kinds of cancer cell lines:stomach cancer cell (AGS), glioma cells (T98G, A172, SNU-489) and prostate cancer cells (LNCaP). We injected the boiled extract of realgar. $50{\mu}$g/ml and $100{\mu}$g/ml to culture media (ml) for 24 hours. We examined the morphological changes under an inverted microscope and a fluorescence microscope. We measured the suppressive effect on viability of 5 kinds of cancer cells via XTT assay. We examined the effect on the revelation of PARP cleavage, Bcl-2 protein and Bax protein by western blot analysis. Results : The extract of realgar caused markedly morphological changes on AGS, T98G, SNU-489, and LNCaP. All of them showed withdrawn and floating appearance. The suppressive effect on viability of AGS, T98G, A172, SNU-489, and LNCaP showed that each test group had more suppressive effect on viability of AGS, T98G, A172, SNU-489, and LNCaP than the control group, which was statistically significantly (p<0.01). The extract of realgar did not induce PARP cleavage in AGS, T98G, A 172, SNU-489, or LNCaP. In the revelation of protein related to apoptosis, the protein levels of Bcl-2 decreased and the protein levels of Bax increased in AGS, T98G, SNU-489, and LNCaP treated with realgar. The protein levels of Bcl-2 decreased and the protein levels of Bax did not change in A172 treated with realgar. Conclusions : This experiment showed that realgar has anti-tumor effect on stomach cancer cells (AGS), glioma cells (T98G, SNU-489L and prostate cancer cells (LNCaP)