• Biomolecules & Therapeutics
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• 제28권5호
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• pp.465-472
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• 2020

Colorectal cancer (CRC) is one of the most malignant type of cancers and its incidence is steadily increasing, due to life style factors that include western diet. Abnormal activation of canonical Wnt/β-catenin signaling pathway plays an important role in colorectal carcinogenesis. Therefore, targeting Wnt/β-catenin signaling has been considered a crucial strategy in the discovery of small molecules for CRC. In the present study, we found that Nodosin, an ent-kaurene diterpenoid isolated from Isodon serra, effectively inhibits the proliferation of human colon cancer HCT116 cells. Mechanistically, Nodosin effectively inhibited the overactivated transcriptional activity of β-catenin/T-cell factor (TCF) determined by Wnt/β-catenin reporter gene assay in HEK293 and HCT116 cells. The expression of Wnt/β-catenin target genes such as Axin2, cyclin D1, and survivin were also suppressed by Nodosin in HCT116 cells. Further study revealed that a longer exposure of Nodosin induced the G₂/M phase cell cycle arrest and subsequently apoptosis in HCT116 cells. These findings suggest that the anti-proliferative activity of Nodosin in colorectal cancer cells might in part be associated with the regulation of Wnt/β-catenin signaling pathway.

• Journal of Microbiology and Biotechnology
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• 제33권9호
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• pp.1206-1212
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• 2023

The Wnt/β-catenin pathway plays essential roles in regulating various cellular behaviors, including proliferation, survival, and differentiation [1-3]. The intracellular β-catenin level, which is regulated by a proteasomal degradation pathway, is critical to Wnt/β-catenin pathway control [4]. Normally, casein kinase 1 (CK1) and glycogen synthase kinase-3β (GSK-3β), which form a complex with the scaffolding protein Axin and the tumor suppressor protein adenomatous polyposis coli (APC), phosphorylate β-catenin at Ser45, Thr41, Ser37, and Ser33 [5, 6]. Phosphorylated β-catenin is ubiquitinated by the β-transducin repeat-containing protein (β-TrCP), an F-box E3 ubiquitin ligase complex, and ubiquitinated β-catenin is degraded via a proteasome pathway [7, 8]. Colorectal cancer is a significant cause of cancer-related deaths worldwide. Abnormal up-regulation of the Wnt/β-catenin pathway is a major pathological event in intestinal epithelial cells during human colorectal cancer oncogenesis [9]. Genetic mutations in the APC gene are observed in familial adenomatous polyposis coli (FAP) and sporadic colorectal cancers [10]. In addition, mutations in the N-terminal phosphorylation motif of the β-catenin gene were found in patients with colorectal cancer [11]. These mutations cause β-catenin to accumulate in the nucleus, where it forms complexes with transcription factors of the T-cell factor/lymphocyte enhancer factor (TCF/LEF) family to stimulate the expression of β-catenin responsive genes, such as c-Myc and cyclin D1, which leads to colorectal tumorigenesis [12-14]. Therefore, downregulating β-catenin response transcription (CRT) is a potential strategy for preventing and treating colorectal cancer. Plant cytokinins are N⁶-substituted purine derivatives; they promote cell division in plants and regulate developmental pathways. Natural cytokinins are classified as isoprenoid (isopentenyladenine, zeatin, and dihydrozeatin), aromatic (benzyladenine, topolin, and methoxytopolin), or furfural (kinetin and kinetin riboside), depending on their structure [15, 16]. Kinetin riboside was identified in coconut water and is a naturally produced cytokinin that induces apoptosis and exhibits antiproliferative activity in several human cancer cell lines [17]. However, little attention has been paid to kinetin riboside's mode of action. In this study, we show that kinetin riboside exerts its cytotoxic activity against colon cancer cells by suppressing the Wnt/β-catenin pathway and promoting intracellular β-catenin degradation.

• 한국식품영양과학회지
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• 제39권1호
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• pp.54-63
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• 2010

Flavonoid 계열의 한 종류인 baicalin은 항염증, 항암, 항바이러스, 항세균 등의 효능을 가진다. 본 연구진은 선행연구를 통한 이전의 보고에서 baiclain이 adipogenesis pathway(지방세포 형성 경로)의 anti-adipogenic(지방세포 형성억제)과 pro-adipogenic(지방세포 형성 유도) factor들을 조절함으로써 비만 및 adipogenesis를 억제함을 밝혔다. 본 연구에서는, microarray 기술을 이용하여 3T3-L1 세포에서 baiclain이 유도하는 지방세포 형성 억제 효과에 대한 분자적 기작을 보다 상세하게 연구하고자 하였다. 지방세포의 분화 시간(0일, 2일, 4일 및 7일)과 분화 시 baicalin의 처리 유무에 따라 유전자 발현 양상을 분석하기 위해 해당 시료들을 microarray에 적용하였다. Microarray 결과로부터 2배이상의 변화가 있는 3972개의 유전자를 확보하였다. 그 유전자들의 발현 양상을 좀 더 자세히 살펴보기 위해 hierarchical clustering 분석을 진행하였고 그 결과로 20개의 cluster를 분류할 수 있었다. 그들 중 4개의 cluster는 분화의 전반적인 기간에서 baicalin의 첨가에 의해 뚜렷하게 상승(cluster 8과 cluster 10)하거나 반대로 뚜렷하게 감소(cluster 12와 cluster 14)하는 양상을 보였다. Cluster 8과 cluster 10에는 CHOP(CCAAT/enhancer-binding protein homologous protein), INSIG1(insulin induced gene 1), WISP2(WNT1 inducible signaling pathway protein 2), ADM(adrenomedullin), CCND2(cyclin D2), GRN(granulin) 및 TGFB3(transforming growth factor, beta 3)과 같은 세포 증식과 지방세포 형성 억제를 상승시키는 유전자들이 다수 포함되었다. 반대로 cluster 12와 cluster 14에는 세포 증식 억제, 세포 주기 억제 및 세포 성장 억제와 연관되거나 지방세포를 유도하는 유전자인 LTA(lympotoxin A), ACADSB(acyl-Coenzyme A dehydrogenase, short/branched chain), HMGCS2(3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2), IGFBP7(insulin-like growth factor binding protein 7), MERTK(c-merproto-oncogene tyrosine kinase), RASSF2(ras association(RalGDS/AF-6) domain family 2), RHOU(ras homolog gene family, member U) 및 SESN1(sestrin1) 등이 포함되었다. 결론적으로 baicalin은 세포 증식 및 지방세포 형성과 연관된 유전자들을 조절함으로써 지방세포의 분화를 억제하는 것으로 사료된다. 이러한 결과는 baicalin이 유도하는 지방세포 형성 억제 및 비만 억제 효과의 분자적 기작에 대한 중요한 정보를 제시한다.

• Asian Pacific Journal of Cancer Prevention
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• 제15권18호
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• pp.7849-7855
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• 2014

Purpose: To investigate the effect of deacetylase inhibitory trichostatin A (TSA) on anti HepG2 liver carcinoma cells and explore the underlying mechanisms. Materials and Methods: HepG2 cells exposed to different concentrations of TSA for 24, 48, or 72h were examined for cell growth inhibition using CCK8, changes in cell cycle distribution with flow cytometry, cell apoptosis with annexin V-FTIC/PI double staining, and cell morphology changes under an inverted microscope. Expression of ${\beta}$-catenin, HDAC1, HDAC3, H3K9, CyclinD1 and Bax proteins was tested by Western blotting. Gene expression for ${\beta}$-catenin, HDAC1and HDAC3 was tested by q-PCR. ${\beta}$-catenin and H3K9 proteins were also tested by immunofluorescence. Activity of Renilla luciferase (pTCF/LEF-luc) was assessed using the Luciferase Reporter Assay system reagent. The activity of total HDACs was detected with a HDACs colorimetric kit. Results: Exposure to TSA caused significant dose-and time-dependent inhibition of HepG2 cell proliferation (p<0.05) and resulted in increased cell percentages in G0/G1 and G2/M phases and decrease in the S phase. The apoptotic index in the control group was $6.22{\pm}0.25%$, which increased to $7.17{\pm}0.20%$ and $18.1{\pm}0.42%$ in the treatment group. Exposure to 250 and 500nmol/L TSA also caused cell morphology changes with numerous floating cells. Expression of ${\beta}$-catenin, H3K9and Bax proteins was significantly increased, expression levels of CyclinD1, HDAC1, HDAC3 were decreased. Expression of ${\beta}$-catenin at the genetic level was significantly increased, with no significant difference in HDAC1and HDAC3 genes. In the cytoplasm, expression of ${\beta}$-catenin fluorescence protein was not obvious changed and in the nucleus, small amounts of green fluorescence were observed. H3K9 fluorescence protein were increased. Expression levels of the transcription factor TCF werealso increased in HepG2 cells following induction by TSA, whikle the activity of total HDACs was decreased. Conclusions: TSA inhibits HDAC activity, promotes histone acetylation, and activates Wnt/${\beta}$-catenin signaling to inhibit proliferation of HepG2 cell, arrest cell cycling and induce apoptosis.

• 구강회복응용과학지
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• 제38권3호
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• pp.150-161
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• 2022

목적:인간 치은섬유아세포의 세포활동에 관련된 다양한 유전자들의 발현에 마이크로그루브-파이브로넥틴 복합 표면이 미치는 영향을 확인하고자 하였다. 연구 재료 및 방법: 평활한 티타늄시편(NE0), 산처리만 시행한 티타늄시편(E0), 마이크로그루브 및 산처리된 티타늄시편(E60/10), 파이브로넥틴을 고정시킨 평활한 티타늄시편(NE0FN), 산처리 및 파이브로넥틴을 고정시킨 티타늄시편(E0FN), 그리고 마이크로그루브 및 산처리 후 파이브로넥틴을 고정시킨 티타늄시편(E60/10FN) 실험군 제작 후 인간 치은섬유아세포의 세포행동 관련 44개 유전자에 대한 실시간 중합효소연쇄반응 실험을 진행하였다. 결과: 인간치은섬유아세포 부착과 증식 등에 관여하는 4종류 신호전달 경로가 활성화되었다. Focal adhesion 경로에 속하는 Integrin α5, Integrin β1, Integrin β3, Talin-2 유전자들, PI3K-AKT 신호 전달 경로에 속하는 AKT1, AKT2, NF-κB 유전자들, MAPK 신호전달 경로에 속하는 MEK2, ERK1, ERK2 유전자들, 세포주기 신호 전달 경로에 속하는 cyclin D1, CDK4, CDK6 유전자들이 마이크로그루브-파이브로넥틴 복합 티타늄표면(E60/10FN)에서 상향조절되었다. 결론: 마이크로그루브-파이브로넥틴 복합 티타늄표면이 세포행동에 관여하는 다양한 유전자들을 상향조절할 수 있다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권8호
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• pp.3195-3201
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• 2015

Objectives: To investigate the effects of betaine on HeLa cell growth and apoptosis and molecular mechanisms. Materials and Methods: Concentrations of 0.1, 1.0, 5.0, 20.0, 100.0 mg/ml of betaine were used to evaluate the anticancer efficacy for HeLa cells respectively, and MCF-10A was also detected as a normal diploid cell control. Results: We found that proliferation of HeLa cells was inhibited significantly upon exposure to increasing betaine levels with the MTT test (p<0.05). The percentage of S phase cells in the low dose groups (<5mg/ml) were distinctly higher than in high dose groups, and the rates of Sub-G1 phase were the opposite (p<0.01); A high concentration of betaine (>5.0mg/ml) significantly promoted the apoptosis of HeLa cells (p<0.01). SOD activities of the low dose groups were slightly higher than the control group (p<0.05) and there were obvious synchronicity and correlation among the expression of promoting apoptosis genes Bax, P53, Caspase 3 and apoptosis suppression gene Bcl-2. In response to an apoptosis-inducing stimulus, p53 and cyclin D1 could be activated with blockage of the cell cycle at G1/S or S/G2 checkpoints. Conclusions: Our data showed that betaine could promote HeLa cells proliferation in vitro at low concentrations. In contrast, high concentrations could significantly inhibit cell growth and migration, and induce apoptosis of HeLa cells through caspase 3 signaling and further promoted necrosis. This might imply that betaine exhibits tumoricidal effects and acts as a biological response modifier in cancer treatment by inducing apoptosis and cell cycle arrest in a dose and time-dependent manner.

• 대한암한의학회지
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• 제15권1호
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• pp.19-27
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• 2010

The roots and leaves of Angelica keiskei (AK) have been used for the treatment of various diseases including coronary heartdisease, hypertension, and cancer in the Korean folk medicine. However, the mechanism by which methylenechloride fraction (MF) from AK exerts anti-tumorigenic activity in human prostate cancer cells has not been fully understood. In the present study, we report the MF exerted the highest cytotoxicity against prostate cancer DU145 cells compared with other fractions. Especially, MF caused the accumulation of sub-G1 DNA contents of cell cycle and increased annexin V-positive apoptotic bodies and DNA fragmentation. MF down-regulated several proliferative (Cyclin D1) and anti-apoptotic (Bcl-xl, Bcl-2, IAP-1/2, and survivin)gene products in these cells. Hence, MF induced apoptosis through the caspase-3 activation in DU145 cells. We further confirmed that caspase-3 plays an importance role in MF-induced apoptosis in DU145 cells by using caspase-3 inhibitor. Additionally, we observed that MF potentiated Dox-induced apoptosis in DU145 cells. Taken together, our data demonstrate the evidence that MF induces apoptosis depend on caspase-3 activation of and overcomes resistance to chemotherapy in human prostate cancer cells.

• Clinical and Experimental Pediatrics
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• 제49권10호
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• pp.1100-1105
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• 2006

목 적 : 신경세포 분화에 있어 NELL2가 세포 주기와 세포자 멸사에 어떤 영향을 미치는지를 규명하고자 하였다. 방 법 : HiB5 세포에 NELL2를 전달감염시켜 배양하여 세포수를 측정하였다. 세포자멸사를 분석하기 위해 DNA 분절검사를 시행하였으며 PI염색으로 DNA양을 측정하였다. 세포주기의 조절에 관여하는 단백질에 대한 NELL2의 효과를 측정하기 위하여 Western blot과 면역염색을 시행하였다. 결 과 : NELL2를 전달감염시킨 군에서 세포수가 의미가 있게 적었고 DNA 분절검사에서 세포자멸사를 확인하였으며 $G_1$ 정지가 증가하였다. Western blot과 면역염색결과 Rb단백, p53, E2F1(KH-95)이 NELL2를 전달감염시킨 군에서 증가하고 cyclin D가 NELL2를 전달감염시킨 군에서 감소하였다. 결 론 : HiB5 세포에서 NELL2에 의하여 일정 수준에서의 세포자멸사가 동반되고 세포 주기에 있어 $G_1$기의 정지가 일어나며 세포주기를 조절하는 단백질의 변화가 발생함을 확인하였다. 따라서 NELL2가 뇌에서 신경세포 분화에 중요한 역할을 할 걸로 생각된다.

• Journal of Acupuncture Research
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• 제17권2호
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• pp.169-186
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• 2000

To study anti-cancer effect and molecular biological mechanism of bee venom for aqua-acupuncture, the effects of bee venom on cell viability, apoptosis, and cell cycle were analyzed using MTT assay, tryphan blue assay, [3H]thymidine release assay, flow cytometric analysis, activity of caspase-3 protease activity assay, and immunocytometric analysis of PCNA. To explore whether anti-cancer effects of bee venom are associated with the transcriptional control of gene expression, quantitative RT-PCR analysis of apoptosis- and cell cycle-related genes was performed. The obtained results are summarized as follows: 1. The MTT assay demonstrated that cell viability was decreased by bee venom in a dose-dependant manner. 2. Significant induction of apoptosis was identified using tryphan blue assay, [$^3H$]thymidine release assay, and flow cytometric analysis of sub $G_1$ fraction. 3. In analysis of caspase-3 protease activity, the activity had increased significantly, in a dose-dependant manner. 4. Quantitative RT-PCR analysis of the apoptosis-related genes showed that Bcl-2 and $Bcl-X_L$ were down-regulated whereas Bax was up-regulated by bee venom treatment. 5. In flow cytometric analysis of cell cycle and immunocytometric analysis of PCNA expression, cell numbers of $G_1$ phase was increased by a dose-dependant manner. 6. In quantitative RT-PCR analysis of the cell cycle-related genes, p21, p27, and p57 were increased, while Cyclin D1, CDK4, c-Myc, c-Fos, and Histone H3 were decreased. In contrast, there were no remarkable changes in expression levels of CDC2 and c-Jun.

• 대한한방내과학회지
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• 제22권4호
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• pp.579-587
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• 2001

The efficacy of Sagunja-tang and Sagunja-tang plus Mylabris phalerata against the human stomach cancer was examined and molecular biological fight of its actions was studied. In the efficacy test of anti-stomach cancer cells growth using the MTT assay, administration of Sagunja-tang resulted in no significant change of stomach cancer cells growth, with the control group. Administration of Sagunja-tang plus Mylabris phalerata resulted in a decrease of stomach cancer cells growth in proportion to the concentration of mylabris phalerata and time, which was significantly different from the control group(significance recognized when p<0.05). In the test using the apoptosis assay, administration of Sagunja-tang showed an increase in apoptosis of human stomach cancer cells, with no significant difference from the control group. Administrating Sagunja-tang plus Mylabris phalerata showed an increase in apoptosis of stomach cancer cells in proportion to the concentration of mylabris phalerata and time, which was significantly different from the control group(significance recognized when p<0.05). In the test using the quantitative RT-PCR to examine stomach cancer cells growth and revelation of apoptosis related genes, administrating Sagunja-tang plus Mylabris phalerata resulted in a decrease of Bcl-2, an anti-apoptosis gene, in proportiong to concentration. No significant change was examined in the revelation of CDK1, Cdc2, Cyclin D1, PCNA, c-myc, which are genes related to the stomach cancer cells growth, and Bax, Bel-XL, the genes related to apoptosis, and p53. Referring to the results above, Sagunja-tang plus Mylabris phalerata may be considered to have an anti-growth efficacy against human stomach cancer cells, and an inducement efficacy. Therefore, it can be clinically implemented in the human stomach cancer.