• 한국식품과학회지
• /
• 제39권6호
• /
• pp.625-629
• /
• 2007

불가사리 골편의 콜라겐으로부터 활성 펩타이드를 분리하기 위하여 초음파를 처리하여 조직을 단편화 시키고 이후 collagnease를 처리하였다. 초음파를 처리할 경우 40kHz의 경우 38.89%의 수율을 나타내었다. 이후 펩타이드의 분자량을 측정하여 12, 20.6, 24, 43, 58a, 100, 116 kDa에서 특정 밴드를 보였다. 이후 Sephadex G-75 컬럼을 이용하여 fraction 별로 모아 사용하였다. 세포 독성을 측정하고 시료 처리 후 형태학적 관찰을 동반한 결과 24 kDa의 경우 최고 농도인 1.0 mg/mL에서 26.7%를 나타내었으며 4번의 계대 이후에도 형태학적 변화가 나타나지 않아 독성이 없다고 해석할 수 있다. 이후 UVA처리 후 MMP-1의 발현을 탐색한 결과 116 kDa부터 24 kDa까지 최고농도인 1.0 mg/mL에서 40, 46.3, 56.8, 57.9, 62.4%의 control 대비 저해율을 보였다. 외부적 스트레스인 UVA에 의한 AP-1의 활성도를 증가시키는 과정을 억제한 것으로 볼 수 있으며 결과적으로 MMP-1의 발현을 효과적으로 조절한 것이라 사료되어 향후 불가사리 콜라겐 유래 펩타이드의 향장소재 활용 가능성이 높다고 할 수 있겠다.

• 대한임상검사과학회지
• /
• 제50권3호
• /
• pp.337-344
• /
• 2018

발생하는 간암 중 가장 주요한 형태인 간세포암은 강한 전이특성으로 인해 높은 재발율과 사망률을 보인다. Silymarin은 엉겅퀴에서 추출한 플라보노이드 성분으로 여러 암세포주에서 상피간엽전환(epithelial mesenchymal transition, EMT) 조절을 통해 항암효과를 보이는 것으로 보고되었다. 본 연구에서는 silymarin이 EMT의 조절을 통해 간세포암 세포주인 Huh7 cell의 침윤과 전이를 억제하는지를 분석하고자 하였다. Huh7 cell의 침윤과 전이 활성을 분석하기 위하여 wound healing assay와 in vitro invasion assay를 시행하였고 EMT 관련 유전자와 상위 신호전달물질의 발현 분석을 위해 Western blot assay를 실시하였다. 그 결과 silymarin은 농도 의존적으로 Huh7 cell의 침윤과 전이를 억제하였다. EMT 관련 유전자 중 세포 부착 단백질인 E-cadherin은 증가하였으나, 중간엽세포의 지표인 vimentin, 종양미세환경 조절에 관여하는 MMP-9의 발현은 억제되었고 이들의 활성에 관여하는 전사인자인 Snail과 nuclear factor $(NF)-{\kappa}B$ 또한 농도 의존적으로 활성이 감소하는 것을 확인할 수 있었다. 특히, 상위신호전달물질 중 silymarin은 phosphoinositide-3-kinase (PI3K)/Akt의 인산화 억제를 통해 EMT 관련 유전자들을 조절하는 것으로 나타났고 이것은 selective inhibitor인 LY294002의 처리 결과로 확인할 수 있었다. 결과적으로, silymarin은 PI3K/Akt 경로를 통해 EMT 관련 유전자의 발현을 조절함으로써 Huh7 cell의 침윤과 전이를 억제하는 것으로 생각된다. 이를 통해 silymarin이 간세포암의 전이 억제에 효과적인 항암물질의 후보가 될 수 있는 잠재력을 가진 후보물질이 될 수 있음을 보여주었다.

• 대한본초학회지
• /
• 제24권3호
• /
• pp.161-167
• /
• 2009

Objectives : Maekmoondong-tang (MMDT), a Korean herbal medicine, has been used to treat severe dry cough in patients with bronchitis and pharyngitis. MMDT has been reported to have anti-inflammatory, anti-allergic, immunomodulatory, secretory-modulating, and metabolic regulatory actions. However, there are no evidence in regard to the effects of MMDT on carcinogenesis and metastasis. Here, we investigated the effects of 70% ethanol extract of MMDT on cell viability, apoptosis, and motility in human hepatocarcinoma HepG2 cells. Methods : Cell viability was measured using the CCK-8 assay, and the apoptosis induction was evaluated by caspase-3 activity. To detect apoptotic features, the cells treated with MMDT were stained with 4'-6-diamidino-2-phenylindole (DAPI). Cell motility was examined by Boyden chamber assay and Real-time Cell Index of Migration assay. Gelatin zymography also performed to measure matrix metalloproteinase (MMP)-2/9 activity. Results : We found that MMDT significantly inhibited cell proliferation and increased caspase-3 activity in a dose-dependent manner in HepG2 cells. Apoptotic features such as chromatin condensation and apoptotic bodies were observed in MMDT-treated cells by DAPI staining. MMDT also suppressed PMA-induced cell motility and activities of MMP-2/9. Conclusions : Our results exhibited that MMDT possess the anti-carcinogenetic and anti-metastatic activities via caspase-3 activation and down-regulation of cell motility and invasion in HepG2 cells. Therefore, these findings suggest that MMDT could be potentially applied to the prevention and treatment of cancer.

• Preventive Nutrition and Food Science
• /
• 제17권4호
• /
• pp.245-253
• /
• 2012

Collagen tripeptide (CTP) is a functional food material with several biological effects such as improving dry skin and wound and bone fracture healing. This study focused on the anti-photoaging effects of CTP on a hairless mouse model. To evaluate the effects of CTP on UVB-induced skin wrinkle formation in vivo, the hairless mice were exposed to UVB radiation with oral administration of CTP for 14 weeks. Compared with the untreated UVB control group, mice treated with CTP showed significantly reduced wrinkle formation, skin thickening, and transepidermal water loss (TEWL). Skin hydration and hydroxyproline were increased in the CTP-treated group. Moreover, oral administration of CTP prevented UVB-induced MMP-3 and -13 activities as well as MMP-2 and -9 expressions. Oral administration of CTP increased skin elasticity and decreased abnormal elastic fiber formation. Erythema was also decreased in the CTP-treated group. Taken together, these results strongly suggest that CTP has potential as an anti-photoaging agent.

• International Journal of Oral Biology
• /
• 제40권1호
• /
• pp.51-61
• /
• 2015

Shikonin, a major ingredient in the traditional Chinese herb Lithospermumerythrorhizon, exhibits multiple biological functions including antimicrobial, anti-inflammatory, and antitumor effects. It has recently been reported that shikonin displays antitumor properties in many cancers. This study was aimed to investigate whether shikonin could inhibit oral squamous carcinoma cell (OSCC) growth via mechanisms of apoptosis and cell cycle arrest. The effects of shikonin on the viability and growth of OSCC cell line, SCC25 cells were assessed by MTT assay and clonogenic assays, respectively. Hoechst staining and DNA electrophoresis indicated that the shikonin-treated SCC25 cells were undergoing apoptosis. Western blotting, immunocytochemistry, confocal microscopy, flow cytometry, MMP activity, and proteasome activity also supported the finding that shikonin induces apoptosis. Shikonin treatment of SCC25 cells resulted in a time- and dose-dependent decrease in cell viability, inhibition of cell growth, and increase in apoptotic cell death. The treated SCC25 cells showed several lines of apoptotic manifestation as follows: nuclear condensation; DNA fragmentation; reduced MMP and proteasome activity; decrease in DNA contents; release of cytochrome c into cytosol; translocation of AIF and DFF40 (CAD) onto the nuclei; a significant shift in Bax/Bcl-2 ratio; and activation of caspase-9, -7, -6, and -3, as well as PARP, lamin A/C, and DFF45 (ICAD). Shikonin treatment also resulted in down-regulation of the G1 cell cycle-related proteins and up-regulation of $p27^{KIP1}$. Taken together, our present findings demonstrate that shikonin strongly inhibits cell proliferation by modulating the expression of the G1 cell cycle-related proteins, and that it induces apoptosis via the proteasome, mitochondria, and caspase cascades in SCC25 cells.

• 대한한의학방제학회지
• /
• 제27권1호
• /
• pp.45-52
• /
• 2019

Objectives : Hyperthermia is a widely used therapeutic tool for cancer therapy and a well-known inducer of apoptosis. Although the Cinnamomi cortex (CC) is a potent anticancer agent for several human carcinomas, it is less potent in the human U937 cell line. To explore any enhancing effects of CC with hyperthermia induced apoptosis, this study investigated the combined effects and apoptotic mechanisms of hyperthermia and CC in U937 cells. Methods : U937 cells were heat treated at $43^{\circ}C$ for 30 min with or without pre-treatment for 1h with CC and then incubated at $37^{\circ}C$ with 5% $CO_2$. Cell viability was analyzed by MTT assay and Trypan blue assay. Morphological changes reflecting apoptosis were visualized under microscope. Synergy effect of CC combined with hyperthermia were calculated by Compusyn software. The expression of proteins related to apoptosis and signaling pathways was determined by western blotting. Results : Hyperthermia with CC reduced cell viability and induced apoptosis. Combined hyperthermia and CC treatment markedly augmented apoptosis by upregulating proapoptotic proteins and suppressing antiapoptotic proteins, culminating in caspase-3 activation. Furthermore, the combined treatment, decreased the expression of in Bcl-2 family, cyclin D1, VEGF, MMP2 and MMP9 expression. Conclusion : This study provides compelling evidence that hyperthermia, in combination with CC, is a promising therapeutic strategy for enhancement of apoptosis and suggests a promising therapeutic approach for cancer.

• BMB Reports
• /
• 제57권5호
• /
• pp.244-249
• /
• 2024

Many types of cancer are associated with excessive angiogenesis. Anti-angiogenic treatment is an effective strategy for treating solid cancers. This study aimed to demonstrate the inhibitory effects of (E)-2-methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) phenol (MMPP) in VEGFA-induced angiogenesis. The results indicated that MMPP effectively suppressed various angiogenic processes, such as cell migration, invasion, tube formation, and sprouting of new vessels in human umbilical vein endothelial cells (HUVECs) and mouse aortic ring. The inhibitory mechanism of MMPP on angiogenesis involves targeting VEGFR2. MMPP showed high binding affinity for the VEGFR2 ATP-binding domain. Additionally, MMPP improved VEGFR2 thermal stability and inhibited VEGFR2 kinase activity, suppressing the downstream VEGFR2/AKT/ERK pathway. MMPP attenuated the activation and nuclear translocation of NF-κB, and it downregulated NF-κB target genes such as VEGFA, VEGFR2, MMP2, and MMP9. Furthermore, conditioned medium from MMPP-treated breast cancer cells effectively inhibited angiogenesis in endothelial cells. These results suggested that MMPP had great promise as a novel VEGFR2 inhibitor with potent anti-angiogenic properties for cancer treatment via VEGFR2/AKT/ERK/NF-κB signaling pathway.

• BMB Reports
• /
• 제43권2호
• /
• pp.91-96
• /
• 2010

The function of macrophage inhibitory cytokine-1 (MIC-1) in cancer remains controversial, and its signaling pathways remain poorly understood. In this study, we demonstrate that MIC-1 induces the transactivation of EGFR, ErbB2, and ErbB3 through the activation of c-Src in SK-BR-3 breast cells. MIC-1 induced significant phosphorylation of EGFR at Tyr845, ErbB2 at Tyr877, and ErbB3 at Tyr1289 as well as Akt and p38, Erk1/2, and JNK mitogen-activated protein kinases (MAPKs). Treatment of SK-BR-3 cells with MIC-1 increased the phosphorylation level of Src at Tyr416, and induced invasiveness of those cells. Inhibition of c-Src activity resulted in the complete abolition of MIC-1-induced phosphorylation of the EGFR, ErbB2, and ErbB3, as well as invasiveness and matrix metalloproteinase (MMP)-9 expression in SK-BR-3 cells. Collectively, these results show that MIC-1 may participate in the malignant progression of certain cancer cells through the activation of c-Src, which in turn may transactivate ErbB-family receptors.

• 한국자원식물학회지
• /
• 제33권4호
• /
• pp.279-286
• /
• 2020

본 연구에서는 부처꽃 에탄올 추출물(ELM)에 대한 항암효능을 알아보기 위하여 인체백혈병 U937 세포의 증식에 미치는 영향과 이와 연관된 apoptosis 유발 여부와 함께 그에 따른 분자생물학적 기전에 대해서 조사하였다. 먼저 ELM 처리에 따른 증식 억제 정도를 조사한 결과, ELM 처리 농도 의존적으로 생존율 및 증식억제 현상이 나타났으며, 핵의 형태 변화, DNA 단편화 및 apoptosis 유발에 관하여 조사한 결과 역시 ELM 처리 농도 의존적으로 증가됨을 확인할 수 있었다. ELM 처리에 따른 U937 세포에서의 apoptosis 유발에 있어서 미토콘드리아 막의 기능 손상이 관여하는 지를 확인하기 위하여 MMP의 변화 정도를 확인한 결과, ELM 처리 농도 증가에 따라 MMP의 소실이 증가하는 것을 관찰할 수 있었다. 이러한 MMP의 소실에 가 관여하는 지를 확인하기 위하여 사멸수용체(DR4, 5, Fas) 및 사멸수용체에 결합하는 리간드(FasL, TRAIL)의 발현 변화를 확인한 결과, DR4 및 DR5의 발현이 증가하는 것으로 관찰되었다. 또한 내인적 경로에 관여하는 Bcl-2 family 유전자들의 발현변화를 확인한 결과, Bcl-2 발현 감소 및 Bax의 발현 증가의 변화를 보였으며, Bid 단백질의 발현감소가 나타났으므로 상대적으로 tBid의 생성이 증가되었음을 추측할 수 있었다. 한편apoptosis 유발에 직접적으로 관여하는 것으로 알려진 caspase-3, -8 및 -9의 발현에 미치는 ELM의 영향에 대해서 조사하였다. 결과에서 알 수 있듯이 ELM은 death receptor에 의하여 활성화 되는 것으로 알려진 caspase-8 및 세포질로 방출된 cytochrome c에 의하여 활성화 되는 것으로 알려진 caspase-9의 활성화를 유발하였으며, caspase cascade에 의하여 apoptosis에 직접적으로 관여하는 caspase-3의 발현도 증가시키는 것으로 나타났다. 또한 활성화된 caspase-3에 의하여 분해가 일어나는 기질 단백질인 PARP의 경우 ELM 처리에 의하여 모두 단편화가 유발되는 것으로 나타났다. 이상의 결과를 종합해 보면 인체 백혈병 U937 세포에 ELM을 처리하였을 경우에 유발되는 apoptosis는 외인적 경로인 DR4 및 DR5의 발현 증가를 통한 caspase-8의 활성화와 이로 인한 Bid 단백질의 단편화와 함께 내인적 경로의 미토콘드리아 기능 손실에 의하여 caspase-9 및 -3의 활성화 유발과 기질단백질들의 분해가 중요한 역할을 하는 것으로 생각되며, IAP family의 발현 감소로 인하여 caspase의 활성이 억제되지 못하는 것도 apoptosis 유도에 어느 정도 관여했을 것으로 생각된다. 따라서 ELM 처리에 의하여 유발되는 apoptosis는 외인적 경로 및 내인적 경로를 모두 경유하는 multiple apoptotic pathway에 의하여 조절되며, 이때 caspases가 중요한 역할을 한다는 것을 알 수 있었다.

• 한방재활의학과학회지
• /
• 제24권2호
• /
• pp.65-81
• /
• 2014

Objectives This study was carried out to find out the anti-osteoarthritic effects of Mahwangbujaseshin- tang (Mahuangfuzixixintang ) on the monosodium iodoacetate (MIA)-induced osteoarthritis rats. Methods Osteoarthritis was induced by injecting MIA ($50{\mu}l$) into the knee joint of rats. Rats were divided into a 3 groups (n=7). The injection did not fit the normal group. A week later, after the injection of MIA, while control group took normal saline 2 ml, the extract of Mahwangbujaseshin-tang (Mahuangfuzixixintang ) (MBST) (200 mg/kg) was injected to treated group. After that, we examined hind paw weight bearing ability, functions of liver and kidney, serum TNF-$\alpha$, IL-$1{\beta}$, IL-6, $PGE_2$, $LTB_4$, TIMP-1, MMP-9 and hematology. Volume of cartilage was measured by micro CT arthrography. Injury of synovial tissue was measured by H & E, Safranin-O immunofluorescence. Results 1) DPPH and ABTS free radical scavenging activity of MBST was increased according to concentration of MBST and total phenolic contents were in high level. 2) In RAW 264.7 cells, ROS production was significantly decreased in MBST (at 10, $100{\mu}g/ml$) and NO was also decreased but meaningless in MBST (at $100{\mu}g/ml$). 3) In RAW 264.7 cells, IL-6 production was significantly decreased in MBST (at $100{\mu}g/ml$) and TNF-$\alpha$ and IL-$1{\beta}$ production were also decreased but meaningless in MBST (at $100{\mu}g/ml$). 4) In hind legs weight-bearing measurement, level of weight-bearing was increased. 5) Functions of liver and kidney were not affected. 6) TNF-$\alpha$, IL-$1{\beta}$, IL-6, $PGE_2$, $LTB_4$, MMP-9 and TIMP-1 production were significantly decreased. 7) In hematology, the levels of neutrophils, monocytes were significantly decreased and the levels of white blood cells, lymphocytes were also decreased but meaningless. 8) In micro CT-arthrography, cartilage volume was significantly increased. 9) Histopathologically, injury on cartilage and synovial membrane of MBST group was decreased. Conclusions Based on all results mentioned above, Mahwangbujaseshin-tang (Mahuangfuzixixintang) is believed to be meaningful for suppressing the progress of osteoarthritis. And it is related to inhibiting the activity of inflammatory cytokine and injury of volume in cartilage.