• 한국자원식물학회지
• /
• 제30권6호
• /
• pp.640-646
• /
• 2017

In this study, we elucidated anti-cancer activity and potential molecular mechanism of 70% ethanol extracts from Taxilli Ramulus (Taxillus chinensis (DC.) Danser) (TR-E70) against human colorectal cancer cells. Anti-cell proliferative effect of TR-E70 was evaluated by MTT assay. The effect of TR-E70 on the expression of cyclin D1 in the protein and mRNA level was evaluated by Western blot and RT-PCR, respectively. TR-E70 suppressed the proliferation of human colorectal cancer cell lines, HCT116 and SW480. Although TR-E70 decreased cyclin D1 expression in protein and mRNA level, decreased level of cyclin D1 protein by TR-E70 more dramatically occurred than that of cyclin D1 mRNA. Cyclin D1 downregulation by TR-E70 was attenuated in presence of MG132. In addition, TR-E70 phosphorylated threonine-286 (T286) of cyclin D1. TR-E70-mediated cyclin D1 degradation was blocked in presence of LiCl as an inhibitor $GSK3{\beta}$ but not PD98059 as an ERK1/2 inhibitor and SB203580 as a p38 inhibitor. Our results suggest that TR-E70 may downregulate cyclin D1 as one of the potential anti-cancer targets through $GSK3{\beta}$-dependent cyclin D1 degradation. From these findings, TR-E70 has potential to be a candidate for the development of chemoprevention or therapeutic agents for human colorectal cancer.

• Biomolecules & Therapeutics
• /
• 제23권4호
• /
• pp.339-344
• /
• 2015

Naringenin (NAR) as one of the flavonoids observed in grapefruit has been reported to exhibit an anti-cancer activity. However, more detailed mechanism by which NAR exerts anti-cancer properties still remains unanswered. Thus, in this study, we have shown that NAR down-regulates the level of cyclin D1 in human colorectal cancer cell lines, HCT116 and SW480. NAR inhibited the cell proliferation in HCT116 and SW480 cells and decreased the level of cyclin D1 protein. Inhibition of proteasomal degradation by MG132 blocked NAR-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with NAR. In addition, NAR increased the phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine blocked cyclin D1 downregulation by NAR. p38 inactivation attenuated cyclin D1 downregulation by NAR. From these results, we suggest that NAR-mediated cyclin D1 downregulation may result from proteasomal degradation through p38 activation. The current study provides new mechanistic link between NAR, cyclin D1 downregulation and cell growth in human colorectal cancer cells.

• Tuberculosis and Respiratory Diseases
• /
• 제45권4호
• /
• pp.776-784
• /
• 1998

연구목적: 폐암은 생불학적 및 병리학적인 특성에 따라 비소세포폐암과 소세포폐암으로 구분되는데 전체폐암의 약 80%가 비소세포폐암으로 국소적인 폐암인 경우 수술적 치료가 절대적 완치 방법인데 조기에 발견하여 광범위한 절제로 치료된 경우에도 환자의 약 50% 정도만이 5년간 생존한다. 확실히 비소세포폐암에서 비슷한 병기의 환자들도 생존 가망성은 상당한 차이를 보인다. 이러한 상황이 보다 정확히 생존 가망성을 예측하고 각각의 환자에게 보다 효율적인 치료를 제공할 수 있게 도울 수 있는 새로운 예측 인자를 밝혀야 하는 필요성이 제기되었다. 정상적인 세포주기 뿐만 아니라 암종의 세포주기에 중요한 역할을 하는 cyclin D계중 하나인 cyclin D1이 세포주기에서 G1기에서 S기로의 전환을 유도하므로써 세포주기를 진행시키는데 유방암, 식도암 및 방광암에서 이의 과발현이 암발생에 중요한 요인이 되다는 보고가 있다. 이에 본 연구에서는 광범위 절제된 비소세포폐암에서 cyclin D1의 과발현이 예후에 미치는 영향을 알아보기 위해 연구를 시행하였다. 방 법: 1983년 1월부터 1995년 7월까지 한양대학교 부속병원에서 비소세포폐암으로 진단된 술전 병기 IIIa 이하인 환자 총 81예를 대상으로 치료적 목적으로 광범위 폐절제술을 통해 얻어진 술후 조직에서 cyclin D1에 대한 단클론성 향체를 사용하여 면역조직화화적 염색을 시행하고 더불어 병리조직학적 특정과 임상적 특정, 특히 생존률과의 연관성을 알아보았다. 또한 술후 환자의 예후적 인자를 최소한으로 줄이기 위해 술전에 방사선 치료나 항암제 치료를 받은 환자는 제외하였고 혼합형의 조직학적 진단이 이루어진 경우와 술후 l달 이내에 사망한 경우는 본 연구의 대상에서 제외하였다. 조직병리학적 병기 판정은 TNM 병기 판정 기준에 맞추었고 조직학적 특징은 WHO 표준 기준에 맞추었다. 결 과: Cyclin D1의 발현은 총 81예에서 시행하여 26에에서 발현되어 30.9%의 발현율을 보였고 각각의 조직형이나 병기, 암의 크기에는 통계적 차이를 보이지 않았으나 TNM 병기 판정에 따라 NO와 나머지 N1-3의 두군으로 나누어 비교할 때 cyclin D1에 따른 통계적 의의를 보였고(p=0.035) cyclin D1 양성 발현군의 평균생존기간은 $22.76{\pm}3.507$개월, 음성 발현군의 평균생존기간이 $45.3{\pm}5.64$개월 (p=0.0515)로 생존율과외 상당한 통계적인 연관성의 가능성을 나타내었다. 결 론: 본 연구에서는 여러 가지 세포주기를 직, 간접으로 조절하는 인자중 cyclin D1의 과발현은 비소세포폐암에서 상당히 불량한 예후 인자로 작용할 수 있다고 생각되며 앞으로 보다 초기와 진행된 병기에 따른 cyclin D1의 발현이 예후에 미치는 영향 등 보다 세밀하고 대단위적인 연구가 진행되어 비소세포폐암에서 보다 정확한 치료와 예후를 예측활 수 있는 인자로서의 역할을 밝혀야 한다고 생각된다.

• Journal of Microbiology and Biotechnology
• /
• 제6권6호
• /
• pp.474-481
• /
• 1996

Since the commercially available rabbit anti-cyclin D3, generated from c-terminal 16 amino acid residues which are common to human and murine cyclin D3, is highly cross-reactive with many other cellular proteins of mouse, a new rabbit polyclonal anti-cyclin D3 has been raised by using murine cyclin D3 protein expressed at a high level in Escherichia coli as the immunogen. To express murine cyclin D3 protein in E. coli, the cyclin D3 cDNA fragment encoding c-terminal 236 amino acid residues obtained by polymerase chain reaction (PCR) was inserted into the NcoI/BamHI site of protein expression vector, pET 3d. Molecular mass of the cyclin D3 overexpressed in the presence of IPTG (Isopropyl $\beta$-D-thiogalactopyranoside) was approximately 26 kDa as calculated from the reading frame on the DNA sequence, and the protein was insoluble and mainly localized in the inclusion bodies that could be easily purified from the other cellular soluble proteins. When renaturation was performed following denaturation of the insoluble cyclin D3 protein in the inclusion bodies using guanidine hydrochloride, 4.4 mg of soluble form of cyclin D3 protein was produced from the transformant cultured in 100ml of LB media under the optimum conditions. Four-hundred micrograms of the soluble form of cyclin D3 protein was used for each immunization of a rabbit. When the antiserum obtained 2 weeks after tertiary immunization was applied to Western blot analysis, it was able to detect 33 kDa cyclin D3 protein in both murine lymphoma cell line BW5147.G.1.4 and human Jurkat T cells at 3,000-fold dilution with higher specificity to murine cyclin D3, demonstrating that the new rabbit polyclonal anti-murine cyclin D3 generated against c-terminal 236 amino acid residues more specifically recognizes murine cyclin D3 protein than does the commercially available rabbit polyclonal antibody raised against c-terminal 16 amino acids residues.

• 생명과학회지
• /
• 제16권4호
• /
• pp.598-604
• /
• 2006

$Ca^{2+}$-농도 의존적으로 활성화되는 neutral protease calpain에 의한 단백질 분해는 세포의 성장을 조절하는데 중요한 단백질들의 역할에 매우 중요한 역할을 한다. Cyclin의 분해는 세포주기의 진행을 위한 필연적인 과정이다. D-type cyclins는 외부자극이나 신호에 의하여 세포주기의 G1 초기에 합성이 된 후 cyclin-dependent kinases (cdk4 및 cdk6)와의 결합하여 세포주기 S기 진입을 촉진하는 역할을 한다. 본 연구에서는 MDA-MB-231 인체 유방암세포에서 cyclin D3 단백질이 calpain protease에 의하여 전사 후 수준에서 조절 받고 있음을 제시하였다. 본 실험의 조건에서 lovastatin과 actinomycin D가 처리된 MDA-MB-231 세포에서 cyclin D3 단백질의 발현이 완전히 사라졌지만, calpain inhibitor인 LLnL의 처리에 의하여 정상 수준으로 회복되었음을 알 수 있었다. 그러나 26S proteasome의 선택적 억제제인 lactacystin, the lysosome 억제제인 ammonium chloride 및 chloroquine, serine protease 억제제인 PMSF는 동일 조건에서 lovastatin과 actinomycin D 처리에 의한 cyclin D3의 발현저하를 억제하지는 못하였다. In vitro 조건에서 순수 분리된 calpain은 cyclin D3 단백질을 $Ca^{2+}$ 농도 의존적으로 분해하였으며, cyclin D3 단백질의 half-life는 LLnL 처리에 의하여 매우 유의적으로 증가되었다. 이러한 결과는 cyclin D3 단백질이 $Ca^{2+}$에 의해 활성화 되는 protease calpain에 의해 조절됨을 보여준다.

• 한국자원식물학회지
• /
• 제33권4호
• /
• pp.271-278
• /
• 2020

이상의 연구 결과로 먹넌출 열매 추출물은 GSK3β 의존성 Cyclin D1 단백질의 분해를 통해 대장암세포의 생육 억제와 관련이 있는 것으로 확인된다. 본 결과는 대장암의 항암제 개발을 위한 소재로 먹넌출 열매의 활용이 가능할 것으로 판단된다.

• 한국약용작물학회지
• /
• 제25권5호
• /
• pp.328-334
• /
• 2017

Background: In this study, we evaluated the anti-cancer activity and potential molecular mechanism of 70% ethanol extracts of the root of Aralia cordata var. continentalis (Kitagawa) Y. C. Chu (RAc-E70) against human colorectal cancer cells. Methods and Results: RAc-E70 suppressed the proliferation of the human colorectal cancer cell lines, HCT116 and SW480. Although RAc-E70 reduction cyclin D1 expression at the protein and mRNA levels, RAc-E70-induced reduction in cyclin D1 protein level occurred more dramatically than that of cyclin D1 mRNA. The RAc-E70-induced downregulation of cyclin D1 expression was attenuated in the presence of MG132. Additionally, RAc-E70 reduced HA-cyclin D1 levels in HCT116 cells transfected with HA-tagged wild type-cyclin D1 expression vector. RAc-E70-mediated cyclin D1 degradation was blocked in the presence of LiCl, a $GSK3{\beta}$ inhibitorbut, but not PD98059, an ERK1/2 inhibitor and SB203580, a p38 inhibitor. Furthermore, RAc-E70 phosphorylated cyclin D1 at threonine-286 (T286), and LiCl-induced $GSK3{\beta}$ inhibition reduced the RAc-E70-mediated phosphorylation of cyclin D1 at T286. Conclusions: Our results suggested that RAc-E70 may downregulate cyclin D1 expression as a potential anti-cancer target through $GSK3{\beta}$-dependent cyclin D1 degradation. Based on these findings, RAc-E70 maybe a potential candidate for the development of chemopreventive or therapeutic agents for human colorectal cancer.

• 한국자원식물학회지
• /
• 제31권3호
• /
• pp.218-227
• /
• 2018

In this study, we investigated the effect of the extracts from Vaccinium oldhamii on cell proliferation and the regulatory mechanisms of cyclin D1 protein level in human cancer cells. The branch extracts from Vaccinium oldhamii (VOB) showed higher inhibitor effect against the cell growth than leave extracts (VOL) and fruit extracts (VOF) in human colorectal cancer, breast cancer, prostate cancer, non-small lung cancer, pancreatic cancer and liver cancer cells. In addition, VOB decreased cyclin D1 level at both protein and mRNA level. MG132 treatment attenuated VOB-mediated cyclin D1 downregulation. A point mutation of threonine-286 to alanine attenuated cyclin D1 degradation by VOB. In addition, the inhibition of nuclear export by leptomycin B (LMB) attenuated cyclin D1 degradation by VOB. But, the treatment of PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), LiCl ($GSK3{\beta}$ inhibitor), LY294002 (PI3K inhibitor) or BAY 11-7082 ($I{\kappa}K$ inhibitor) did not affect VOB-induced cyclin D1 degradation. In conclusion, VOB induced cyclin D1 degradation through redistribution of cyclin D1 from the nucleus to cytoplasm via T286 phosphorylation of cyclin D1, which resulted in the inhibition of cancer cell proliferation.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
• /
• 제43권5호
• /
• pp.324-330
• /
• 2017

Objectives: There are only a limited number of studies on cyclin D1 and p63 expression in oral squamous cell carcinoma (OSCC) and leukoplakia. This study compared cyclin D1 and p63 expression in leukoplakia and OSCC to investigate the possible correlation of both markers with grade of dysplasia and histological grade of OSCC. Materials and Methods: The study included a total of 60 cases, of which 30 were diagnosed with OSCC and 30 with leukoplakia, that were evaluated immunohistochemically for p63 and cyclin D1 expression. Protein expression was correlated based on grades of dysplasia and OSCC. Results: Out of 30 cases of OSCC, 23 cases (76.7%) were cyclin D1 positive and 30 cases (100%) were p63 positive. Out of 30 cases of leukoplakia, 21 cases (70.0%) were cyclin D1 positive and 30 (100%) were p63 positive (P<0.05). Conclusion: The overall expression of cyclin D1 and p63 correlated with tumor differentiation, and increases were correlated with poor histological grades, from well-differentiated to poorly-differentiated SCC. Increased cyclin D1 and p63 expression was associated with the severity of leukoplakia. Based on these results cyclin D1 and p63 products can be a useful tool for improved leukoplakia prognosis.

• Plant Biotechnology Reports
• /
• 제2권4호
• /
• pp.227-231
• /
• 2008

D-class cyclins play important roles in controlling the cell cycle in development and in response to external signals by forming the regulatory subunit of cyclin-dependent kinase (CDK) complexes. To evaluate the effects of D-class cyclins in transgenic rice plants, Arabidopsis cyclin D2 gene (CycD2) was linked to the maize ubiquitin1 promoter (Ubi1) and introduced into rice by the Agrobacterium-mediated transformation method. Genomic deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and Western blot hybridizations of the Ubi1:-CycD2 plants revealed copy number of transgene and its increased expression in leaf and callus cells at messenger RNA (mRNA) and/or protein levels. The H1 kinase assay using the immunoprecipitates of protein extracts from the Ubi1:CycD2 plants and nontransgenic controls demonstrated that the introduced Arabidopsis CycD2 forms a functional CycD2/CDK complex with an unidentified CDK of rice. Shoot and root growth was enhanced in the Ubi1:CycD2 seedlings compared with nontransgenic controls, together, suggesting that Arabidopsis cyclin D2 interacts with a rice cyclin-dependent kinase, consequently enhancing seedling growth.