Ursolic acid에 의한 자궁 경부암 세포주에서 항증식, 항바이러스 작용 기전 분석

이근호,임은경,윤주희,엄수종,남궁성은,박종섭 대한부인종양 콜포스코피학회 2003 Journal of Gynecologic Oncology Vol.14 No.4

목적 : 동양의 전통적인 약제로 사용되고 있는 ursolic acid는 항암, 항염증 효과가 있다고 알려져 있다. 본 연구는 ursolic acid의 자궁경부암 세포주에 대한 증식 억제와 HPV에 관련된 항바이러스 효과를 관찰하고자 하였다. 연구 방법 : Ursolic acid와 Dexamethasone을 자궁경부암 세포주에 처리한 후 세포 증식 억제 효과를 관찰하기 위하여 MTT assay를 시행하였으며, DNA fragmentation, DAPI staining과 FACS 분석을 통해 apoptosis의 특징을 관찰하였으며, apoptosis와 연관된 단백질들로 western blotting을 시행하였다. 또한, 항바이러스 효과를 관찰하기 위하여 HPV E6, E7의 발현 정도를 RT-PCR로 분석하였다. 결과 : Ursolic acid를 HPV가 함유된 자궁경부암 세포주(HeLa, CaSki, SiHa)에 투여하면 농도와 시간에 의존적으로 비례하여 세포증식이 억제되었으나, HPV를 함유하지 않은 자궁경부암 세포주(C33A)에서는 세포 증식억제 효과는 없었다. HeLa 세포주에서 ursolic acid의 농도를 증가시킴에 따라 apoptosis의 특징을 보여주는 DNA fragmentation, DAPI staining, FACS 분석 소견이 뚜렷하게 나타났으며, westem blottiong을 통하여 Fsa 단백질의 발현이 유도되고, caspase-8, caspase-3, PARP 단백질이 절단됨을 관찰할 수 있었다. HeLa 세포에 ursolic acid를 처리함에 따라 HPV-18 E6, E7 유전자 발현이 감소되었으나, p53과 Rb 단백질의 변화는 관찰할 수 없었다. 결론 : 자궁경부암 세포에 ursolic acid를 투여하면 세포증식 억제가 apoptosis에 의하여 일어나며, HPV E6/E7의 발현저하로 인한 항바이러스 효과도 기여할 것으로 사료된다. 이 연구 결과는 ursolic acid에 의한 항증식, 항바이러스 기전을 이해하는데 도움을 주었으며, 효과적인 약제로서의 사용 가능성을 제시하였다. Objective : Ursolic acid which has been used as a herb medication, was reported for anti-cancer and anti-inflammatory effects. We tried to analyze the anti-proliferative and anti-viral effects of ursolic acid in Human papillomavirus (HPV) associated cervical carcinoma cell lines. Methods : We treated ursolic acid or dexamethasone to cervical carcinoma cell lines. We carried out MTT assay to observe the anti-proliferative effects and tried to find out the characteristics of apoptosis by DNA fragmentation, DAPI staining and FACS analysis. By means of westem blotting, we investigated proteins related with apoptosis. After treating of ursolic acid, we used RT-PCR for the expression of HPV E6 and E7 gene to observe the anti-viral effects. Through proteomics analysis including two-dimensional electrophorosis (2DE) and matrix-associated laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), we found the interaction between cell cycle regulatory factor and apoptosis related protein, and also demonstrated anti-cancer and anti-viral mechanism. Results : While the growth of HPV-positive cervical carcinoma cell lines (HeLa, CaSki, SiHa) is suppressed by ursolic acid in a dose-and time-dependent manner, that of HPV-negative cervical carcinoma cell line (C33A) did not have any inhibitory effects on proliferation. As we increased the concentrations of ursolic acid in HeLa cell, the typical characteristics of apoptosis were noted in DNA fragmentation, DAPI staining and FACS analysis, The expression of Fas protein was induced and caspase-8, caspase-3 and PARP proteins changed into cleavaged forms after treating ursolic acid. HPV-18 E6/E7 gene expression decreased after treating ursolic acid in HeLa cells, but the levels of p53 and Rb proteins did not changed. Total 30 spots were detected by proteomics analtsis, along with 20 up-regulated, 6 down-regulated and 4 unknown protein were founded. Conclusion : These results suggest that ursolic acid inhibit the cell proliferation through apoptosis in HPV-associated cervical carcinoma cell lines. The apoptotic mechanism of ursolic acid in HeLa cells might be through the signal pathway "Fas→caspase-8→PARP". In addition, the antiviral effect of ursolic acid through suppression of HPV E6/E7 gene expression would contribute to growth inhibition in HeLa cells. These results suggest that ursolic acid could be useful for an effective anticancer drug in treatment of HPV-associated cervical neoplasia.

Ursolic acid-induced down-regulation of MMP-9 gene is mediated through the nuclear translocation of glucocorticoid receptor in HT1080 human fibrosarcoma cells

Cha, Hee-Jae,Park, Moon-Taek,Chung, Hae-Young,Kim, Nam-Deuk,Sato, Hiroshi,Seiki, Motoharu,Kim, Kyu-Won 부산대학교 유전공학연구소 1998 분자생물학 연구보 Vol.14 No.-

We have previously reported that ursolic acid, a pentacyclic triterpene acid, inhibited the invasion of HTI1080 human fibrosacrcoma cells by reducing the expression of matrix metalloproteinase-9. Since the chemical structure of ursolic acid is very similar to that of dexamethasone, a synthetic glucocorticoid, we investigated whether ursolic acid acts through the glucocorticoid receptor. The expression of matrix metalloproteinase-9 is thought to be regulated similary with matrix metalloproteinase-1 and matrix metalloproteinase-3 as containing common 2-0-teradecanoylphorbol-acetate responsible region, where AP-1 proteins can bind. Dexamethasone has been studied to respress the 2-0-teradecanoylphorbol-acetate-induced expression of matrix metalloproteinase-1 and matrix metalloproteinase-3 through a glucocorticoid receptor-mediated manner. In Northern biot analysis, we found that ursolic acid reduced the expression of matrix metalloproteinase-1 and matrix metalloproteinase-3 induced by 2-0-teradecanoylphorbol-acetate. Similarly, ursolic acid down-regulated 2-0-teradecanoylphorbol-acetate-induction of matrix metalloproteinase-9 gene in the same manner of dexamethasone. RU486, a potent glucocorticoid receptor antagonist, was used for identifying that ursolic acid-induced down-regulation of matrix metalloproteinase-9 expression is mediated by its binding to glucocorticoid receptor. The effect of ursolic acid on the matrix metalloproteinase-9 expression was blocked by RU486, suggesting that ursolic acid acts via a glucocorticoid receptor in the regulation of matrix metalloproteinase-9. Western blot analysis and immunocytochemistry showed that urslic acid increased glucocorticoid receptor fraction in the muscleus, although it decreased the synthesis of glucocorticoid receptor mRNA. In addition, ursolic acid did not decrease the expression of c-jun and DNA-binding activity of AP-1 to its cognate sequences. Taken together, we suggest that ursolic acid may induce the repression of matrix metalloproteinase-9 by stimulating the muclear translocation of glucocorticoid receptor, and the translocated glucocorticoid receptor probably down-modulating the trans-activating function of AP-1 to 2-0-teradecanoylphorbol-acetate responsible element of matrix metalloproteinase-9 promoter region.

인체 치은섬유모세포에서 Lipopolysaccharides, Ursolic acid와 Oleanolic acid에 의한 Phenytoin 유도 세포활성에 미치는 영향

권오달,김윤성,신형식,Kwon, Oh-Dal,Kim, Yoon-Sung,Shin, Hyung-Shik 대한치주과학회 1994 Journal of Periodontal & Implant Science Vol.24 No.1

Gingival hyperplasia is frequently associated with the long-term use of phenytoin for control of convulsive disorder. The purpose of this study was to investigate on the effects of lipopolysaccharides (LPS), ursolic acid and oleanolic acid to phenytoin-induced cell activity in human gingival fibroblast. Human gingival fibroblasts were cultured form the healthy gingiva of orthodontic patients. Gingival fibroblasts were trypsinized and transferred to the weels of microtest plates. Fibroblast were cultured in growth medium added $5{\mu}g/ml$ of phenytoin, $5{\mu}g/ml$ of LPS, $10^{-7}M$ of ursolic acid and oleanolic acid. The passage number of cultured fibroblasts were fifth and eight. Cell morphology was examined by inverted microscope and the cell activity was measured by proliferation assay. Ursolic acid significantly modulated cell morphology into globular shape at the concentrantion of $10^{-7}M$ in the presence of phenytoin and LPS, and the cell activity was significantl decreased by ursolic acid or oleanolic acid regardless of the presence of phenytoin and LPS. These results suggested that the increased phenytoin-induced cell activity might be modulated by ursolic acid regardless of the presence of phenytoin and LPS. These results suggested that the increased phenytoin-induced cell activity might be modulated by ursolic acid or oleanolic acid. Further study is needed to clarify their toxicological effects on cellular modulation and mRNA expression change.

Ursolic acid의 악성 흑색종 세포주 A375SM과 A375P에서의 항암효능

우중석,김나원,이진규,김재혁,임다영,강신우,김성현,유은선,이재한,한소희,박영석,김병수,김상기,박병권,정지윤 한국식품위생안전성학회 2019 한국식품위생안전성학회지 Vol.34 No.2

Ursolic acid is recognized for various effects such as anti-cancer, antioxidant, and anti-inflamma- tory activity. In this study, we confirmed the anti-cancer effect of ursolic acid on human melanoma cancer cells, A375SM and A375P. Survival rate of the melanoma cells was confirmed by MTT assay and the proliferation rate was confirmed by wound healing assay. The rate of apoptotic bodies was confirmed by DAPI staining, and apoptosis rate was confirmed by flow cytometry. The induction of apoptosis protein was examined by western blotting according to the concentration of ursolic acid in melanoma cells. The survival and proliferation rates of melanoma cells were decreased according to the treatment concentrations of ursolic acid. DAPI staining showed that chromosomal conden- sation of melanoma cells was increased with increasing concentrations of ursolic acid, and increased apoptosis rate of melanoma cells by ursolic acid was confirmed by flow cytometry. We also confirmed by western blotting that cleaved- PARP and Bax were increased and Bcl-2 was decreased at 12 µM concentration of uricolic acid in melanoma cells. This study was carried out at low concentrations of ursolic acid, 0 to 20 µM, and analyzed 24 h after treatment. As a result of this study, it is thought that ursolic acid has the anti-cancer effect through the regulation of apoptosis-related proteins in melanoma cells A375SM and A375P.

Ursolic acid isolated from Uncaria rhynchophylla activates human dendritic cells via TLR2 and/or TLR4 and induces the production of IFN-γ by CD4+ naive T cells

Jung, T.Y.,Pham, T.N.N.,Umeyama, A.,Shoji, N.,Hashimoto, T.,Lee, J.J.,Takei, M. North-Holland ; Elsevier Science Ltd 2010 european journal of pharmacology Vol.643 No.2

Ursolic acid is triterpene isolated from Uncaria rhynchophylla and is a pharmacologically active substance. The induction of dendritic cell maturation is critical for the induction of Ag-specific T-lymphocyte response and may be essential for the development of human vaccine relying on T cell immunity. In this study, we investigated that the effect of Ursolic acid on the phenotypic and functional maturation of human monocyte-derived dendritic cells in vitro. Dendritic cells harvested on day 8 were examined using functional assay. The expression levels of CD1a, CD80, CD83, CD86, HLA-DR and CCR7 on Ursolic acid-primed dendritic cells was slightly enhanced. Ursolic acid dose-dependently enhanced the T cell stimulatory capacity in an allogeneic mixed lymphocyte reaction, as measured by T cell proliferation. The production of IL-12p70 induced by Ursolic acid-primed dendritic cells was inhibited by the anti-Toll-like receptor-2 (TLR2) mAb and anti-TLR4 mAb. Moreover, Ursolic acid-primed dendritic cells expressed levels of mRNA coding for both TLR2 and TLR4. The majority of cells produced considerable interferon-gamma (IFN-γ), but also small amounts of interleukin (IL-4)-4. Ursolic acid-primed dendritic cells have an intermediate migratory capacity towards CCL19 and CCL21. These results suggest that Ursolic acid modulates human dendritic cells function in a fashion that favors Th1 polarization via the activation of IL-12p70 dependent on TLR2 and/or TLR4, and may be used on dendritic cells-based vaccines for cancer immunotherapy.

Ursolic acid increases the secretion of atrial natriuretic peptide in isolated perfused beating rabbit atria

Cui, H.Z.,Wen, J.F.,Choi, H.R.,Li, X.,Cho, K.W.,Kang, D.G.,Lee, H.S. North-Holland ; Elsevier Science Ltd 2011 european journal of pharmacology Vol.653 No.1

Ursolic acid is reported to have beneficial effects on the regulation of cardiovascular homeostasis. However, the effects of ursolic acid on cardiac hormone secretion are yet to be defined. The present study was designed to test the effects of ursolic acid on the secretory and contractile functions of the atria. Experiments were conducted in isolated perfused beating rabbit atria. We measured the changes in atrial dynamics, pulse pressure, stroke volume, cAMP efflux, as well as the secretion of atrial natriuretic peptide (ANP). Ursolic acid increased ANP secretion and mechanical dynamics in a concentration-dependent manner. The inhibition of L-type Ca<SUP>2+</SUP> channels with nifedipine attenuated the ursolic acid-induced increase in ANP secretion but not mechanical dynamics. The inhibition of K<SUP>+</SUP><SUB>ATP</SUB> channels with glibenclamide attenuated the ursolic acid-induced increase in ANP secretion-but not atrial dynamics-in a concentration-dependent manner. The selective Na<SUP>+</SUP>-K<SUP>+</SUP>-ATPase inhibitor ouabain blocked the ursolic acid-induced increase in atrial dynamics but not ANP secretion. These findings show that ursolic acid increases ANP secretion via its activation of K<SUP>+</SUP><SUB>ATP</SUB> channels and subsequent inhibition of Ca<SUP>2+</SUP> entry through L-type Ca<SUP>2+</SUP> channels in rabbit atria. These data also suggest that ursolic acid increases atrial dynamics via its inhibition of Na<SUP>+</SUP>-K<SUP>+</SUP>-ATPase activity.

광나무의 주성분, Ursolic acid와 Oleanolic acid의 항노화 효능

홍용덕 ( Yong Deog Hong ),유대성 ( Dae Sung Yoo ),남미희 ( Mi Hee Nam ),김현정 ( Hyeon Chung Kim ),박시준 ( Si Jun Park ),신송석 ( Song Seok Shin ),천종우 ( Jong Woo Cheon ),박영호 ( Young Ho Park ) 대한화장품학회 2012 대한화장품학회지 Vol.38 No.2

광나무(L. lucidum)는 ursolic acid와 oleanolic acid를 다량 포함하고 있다. 본 연구에서는 광나무 열매, 줄기, 잎 세 부위 추출물의 항주름 효능을 평가하였다. 광나무 추출물은 human skin fibroblasts에서 독성이 없을 뿐만 아니라 MMP-1과 MMP-2의 발현을 감소시키고 COL1A1의 발현을 증가시켰다. 이들 추출물은 모두 농도 의존적으로 COL1A1의 발현을 증가시켰으며 MMP-1과 MMP-2의 발현을 감소시켰다. 광나무 세 부위 추출물 가운데, 열매 부위에 가장 많은 양의 ursolic acid 와 oleanolic acid가 함유되어 있었으며 가장 강한 COL1A1 upregulating 효과와 MMP-1과 MMP-2 downregulating 효과를 나타냈다. 이처럼 항주름 효능을 보이는 광나무 열매 추출물은 기능성 화장품 소재로 개발될 수 있는 가능성이 있다. Ligustrum lucidum (L. lucidum) contains large quantities of ursolic acid and oleanolic acid. In this study, we evaluated anti-wrinkle effects of three parts of L. lucidum extracts. We found that L. lucidum extracts were not only innocuous to human skin fibroblasts but also significantly decreased the expression of both MMP-1 and MMP-2 and increased the expression of COL1A1. Among the three parts of L. lucidum extracts (i.e., fruit, cane, and leaf extracts), the fruit extract was found to contain the greatest amounts of ursolic acid and oleanolic acid. These three parts of L. lucidum extracts increased the expression of COL1A1 and decreased the expression of MMP-1 and MMP-2 in a dose-dependent manner. Especially, the fruit extract of L. lucidum had the greatest upregulating effect on COL1A1 and the greatest downregulating effect on MMP-1 and MMP-2 in a non-toxic and dose-dependent manner. These results suggest that L. lucidum, especially the fruit, could be used as active ingredients for functional cosmetics.

白花蛇舌草 헥산分劃과 多糖體가 抗癌 및 抗轉移 活性에 미치는 影響 : Ursolic acid와 Asperuloside 병용투여시 항암 및 항전이 효과에 관한 연구

宋圭鏞,柳時容,金聖勳 대한동의병리학회 1999 동의생리병리학회지 Vol.13 No.1

산업기술의 발달로 인한 삶의 질의 향상과 더불어 현대의학의 발달에도 불구하고 아직도 불치의 병으로 여겨지고 있는 암에 의한 사망률은 계속 증가되고 있는 추세로 암을 치유하기 위한 연구가 활발히 전개되고 있다. 백화사설초는 항암 본초로 분류되어 암치료를 위해 한방처방에 가미되어 응용되고 있다. 그러나 백화사설초의 성분에 대한 연구는 미흡한 실정이다. 이런 점을 감안하여 본 연구실에서는 백화사설초의 용매 분획 중 헥산 분획이 수종의 암세포에 대해서 세포독성이 있음을 발견하고 물질분리를 실시하여 물질을 단리한 후 물리·화학적인 방법을 통하여 구조를 확인 한 결과 ursolic acid(UA)가 효과를 나타내는 물질이라는 것을 밝혔다. 따라서 UA의 구체적인 항암 및 항전이 효과를 검증하기 위하여 수종의 암세포에 대한 세포독성, 부착저지효과, 폐암전이 억제효과, 혈관형성 억제효과, 암전이에 관련된 유전자인 MMP-2의 발현 정도를 살펴보았고 면역세포에 미치는 영향은 물론 배당체인 asperuloside(AS)를 분리하여 UA와 병용투여하므로써 항암 및 항전이 활성의 변화를 관찰하여 항암제로서의 가능성을 타진하고자 하였다. 실험결과 UA는 수종의 암세포에 농도의존적으로 암의 성장을 억제하였으며, AS의 병용투여시 상승적인 세포독성 효과를 발휘하지 않았다. 그러나 세포부착 저지효과, 혈관형성 억제효과 및 폐암전이 효과와 같은 암전이에 관련된 실험에서 AS는 UA의 효과를 상승적으로 증가시켰을 뿐만아니라 면역세포에 대해서도 증가효과를 나타냈다. 이상의 결과로부터 UA는 항암 및 항전이 효과가 있음을 발견하였으며, AS와 같은 배당체의 병용투여는 이러한 항암 및 항전이 효과를 증가시키는 것으로 보아 암에 유효하게 사용될 수 있으리라 사료된다. Ursolic acid(UA), isolated from the Oldenlandia diffusa(Rubiaceae), is a triterpenoid compound that exist widely in food, medicinal herbs, and other plants and has been recognized to have a wide spectrum of pharmacological activity such as antiinflammatory effect, anti-HIV activity, antihyperlipidemic activity, and antitumor activity. Ursolic acid was treated either alone or in combination with asperuloside(AS), irridoidal glycoside, for the evaluation of antitumor and antimetastatic effects by several experimental parameters, for example, cytotoxicity against various cancer cell line, clonogenic assay, antiadhesion assay, CAM assay, pulmonary colonization assay, MMP-2 expression, and immunological analysis by FACS. The results were obtained as follows; Ursolic acid showed a potent cytotoxic activity in a dose dependent manner against SF295, SK-OV-3, HCT15, and UN-2 cancer cell lines, while combined treatmenmt of UA & AS didn't. However, UA & AS were more effectiv than UA only in inhibitory effect on cell adhesion of A549 cells to complex extracellular matrix in vitro and pulomonary colonization assay by B16BL/6 in vivo. In the clonogenic assay UA & AS more effectively inhibited colony formation by A549 synergistically to 97% than UA to 78%. By analysis by FACS UA & AS activated CD4(T helper cell) and CDB(cytotoxic T cell) as well as macrophage while UA activated macrophage only. UA and AS suppressed MMP-2 gene expression more effectively than UA only in a dose dependent manner. These results suggest that combined therapy of ursolic acid and asperuloside showed antitumor and antimetastatic effects than ursolic aicd only so that Oldenlandiae diffusae Herba can be effectively applied for the prevention and treatment of cancer. It also is very necessary that biological activities and their mechanism should be kept studying between different constituents isolated from same plant.

하고초(夏枯草)에서 추출한 Ursolic acid의파골세포 분화 억제 효과

허자경 ( Ja Kyung Heo ),황덕상 ( Deok Sang Hwang ),이진무 ( Jin Moo Lee ),이창훈 ( Chang Hoon Lee ),장준복 ( Jun Bock Jang ),이경섭 ( Kyung Sub Lee ) 대한한방부인과학회 2014 大韓韓方婦人科學會誌 Vol.27 No.2

This study was conducted to evaluate the inhibitory effect of ursolicacid from Prunella vulgaris on osteoclast differentiation. Methods: MTT-assay was performed to estimate cytotoxicity of ursolic acidfrom Prunella vulgaris in BMMs stimulated with M-CSF. TRAP staining, TRAPactivity and Real-time PCR were performed to know the inhibitory effect onosteoclast differentiation. Actin ring formation were analysed to observe the effectof ursolic acid from Prunella vulgaris. Results: Ursolic acid from Prunella vulgaris has no cytotoxicity at the concentrationof 1 μg/ml or lower. Ursolic acid decreased the number of TRAP positive cells and the expression of NFATc1 gene, c-Fos gene, TRAP and OSCAR in BMMs stimulatedwith RANKL. Ursolic acid restrained the formation of actin ring. Ursolic acidinhibited NF-κB activity by inducing degradation of p-IkBa.Conclusions: Ursolic acid from Prunella vulgaris has the inhibitory effect ofosteoclast differentiation and bone resorption. Futher studies are needed to treatosteoporosis by usolic acid from Prunella vulgaris.

Ursolic acid induced apoptosis in human osteosarcoma cell lines

최인수,김영진,강해미,준드이젬츠 오제렐,김인령,박봉수 대한구강해부학회 2022 대한구강해부학회지 Vol.43 No.1

Ursolic acid is a polyphenolic component present in large amounts in apple peels. It possesses antioxidant, anti-fungal, and anti-inflammatory properties and inhibits the growth and metastasis of various cancer cells. However, studies on the effect of ursolic acid in osteosarcoma cells remain limited. Therefore, we investigated the role of ursolic acid-induced apoptosis and autophagy in HOS and U2OS osteosarcoma cells. Ursolic acid decreased cell survival and proliferation, and evidence of apoptosis, such as nuclear condensation and cleavage, was observed in both cell lines. Ursolic acid also significantly activated the expression of Atg5 and LC3B proteins involved in autophagy. The combination of 3-MA and ursolic acid, an autophagy inhibitor, significantly increased the expression of proteins involved in apoptosis. Therefore, ursolic acid effectively induced apoptosis and autopoiesis in osteosarcoma cells and was more effective when treated with an autopoiesis inhibitor.