• Journal of Microbiology and Biotechnology
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• 제30권1호
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• pp.38-43
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• 2020

Hand, foot, and mouth disease (HFMD) is caused by enterovirus 71 (EV71) in infants and children under six years of age. HFMD is characterized by fever, mouth ulcers, and vesicular rashes on the palms and feet. EV71 also causes severe neurological manifestations, such as brainstem encephalitis and aseptic meningitis. Recently, frequent outbreaks of EV71 have occurred in the Asia-Pacific region, but currently, no effective antiviral drugs have been developed to treat the disease. In this study, we investigated the antiviral effect of salvianolic acid B (SalB) on EV71. SalB is a major component of the Salvia miltiorrhiza root and has been shown to be an effective treatment for subarachnoid hemorrhages and myocardial infarctions. HeLa cells were cultured in 12-well plates and treated with SalB (100 or 10 ㎍/ml) and 10⁶ PFU/ml of EV71. SalB treatment (100 ㎍/ml) significantly decreased the cleavage of the eukaryotic eIF4G1 protein and reduced the expression of the EV71 capsid protein VP1. In addition, SalB treatment showed a dramatic decrease in viral infection, measured by immunofluorescence staining. The Akt signaling pathway, a key component of cell survival and proliferation, was significantly increased in EV71-infected HeLa cells treated with 100 ㎍/ml SalB. RT-PCR results showed that the mRNA for anti-apoptotic protein Bcl-2 and the cell cycle regulator Cyclin-D1 were significantly increased by SalB treatment. These results indicate that SalB activates Akt/PKB signaling and inhibits apoptosis in infected HeLa cells. Taken together, these results suggest that SalB could be used to develop a new therapeutic drug for EV71-induced HFMD.

• 생명과학회지
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• 제25권4호
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• pp.441-449
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• 2015

본 연구에서는 인체 폐암세포주인 A549 세포와 간암세포주인 HepG2 세포를 사용하여 Endlicheria anomala 메탄올 추출물(Methanol extract of E. anomala, MEEA)의 항암 활성 및 그 분자적 기전에 관하여 분석하였다. 먼저 MEEA가 인체 폐암세포와 간암세포의 증식에 미치는 영향을 분석한 결과, 암세포의 증식을 억제하는 효과가 탁월하였다. 그 후 MEEA에 의한 세포 증식이 억제되는 원인을 분석하기 위하여 Flow cytometry analysis, AnnexinV & 7-AAD 이중 염색을 수행한 결과, 두 세포에서 모두 농도의존적으로 apoptosis 유발군인 SubG1기의 세포 분포가 증가하였고, early apoptosis에서 late apoptosis로 전환되는 공통적인 현상을 확인할 수 있었다. 또한 apoptosis의 유발로 일어날 수 있는 세포의 형태 변화를 관찰하기 위한 DAPI 염색과 DNA fragmentation을 통하여 MEEA 처리에 의한 A549의 염색질 응축, 사멸체 형성 및 DNA의 끌림 현상을 관찰할 수 있었으며, 이와 관련된 분자적 기전 분석을 위한 Western blot을 추가로 수행하여 caspase, PARP, pro-, anti-apoptotic 단백질의 발현을 관찰하였다. 이상의 결과로 MEEA는 인체 폐암세포와 간암세포에서 p53의 발현 증가와 Bcl-2 family의 변화를 유도하며, caspase-3와 관련된 경로를 통하여 농도의존적으로 apoptosis를 유발시킨다는 것을 증명하였다. 이는 MEEA가 항암 활성을 보유하고 있고, 인체 폐암세포와 간암세포 사멸의 기전 연구를 위한 중요한 자료가 될 수 있음을 시사한다.

• 대한한방부인과학회지
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• 제19권3호
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• pp.135-149
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• 2006

Purpose : This study was aimed to investigate the inhibitory effect of Millettia Reticulatas on the proliferation of human uterine leiomyoma cells and the expression of gene related the mechanism of cell apoptosis. Methods : We counted the number of death cells treated with indicated concentration of Millettia Reticulatas and investigated cell death rate by MTS assay. Furthermore, flow cytometry analyis and DNA fragmentation assay were used to dissect between necrosis and apoptosis. and then we observed the differential gene expression by western blot analysis. Results : 1) The inhibitory effect on the growth of uterine leiomyoma cell treated with Millettia Reticulatas was increased in a concentration proportional. 2) The result of flow cytometry analysis. subG1 phase arrest related3 cell apoptosis was investigated 23.49% in uterine leiomyoma cell treated Millettia Reticulatas and showed the fession of proportional concentration. 3) The gene expression of p27, p53, p21, p16 related cell cycle was increased according to increasing concentration but cyclin E was none exchanged. 4) The character of apoptosis, DNA fragmentation was significantly observed the fession of proportional concentration. 5) The expression of pro-caspase3 and PARP were decreased dependent on treatment concentration. Conclusion : This study showed that Millettia Reticulatas have the inhibitory effect on the proliferation of human uterine leiomyoma cell and the effect was related with apoptosis. The apoptotic mechanism was observed that the gene expression of p27, p53, p21, p16 related cell cycle was increased according to increasing treatment concentration, induced G1 phase arrest and finally cell death was occurred. The decreased expression of pro-caspase 3 and PARP were noted that apoptosis was related with caspase pathway.

• Molecules and Cells
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• 제38권5호
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• pp.457-465
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• 2015

The 15-kDa selenoprotein (Sep15) is a selenoprotein residing in the lumen of the endoplasmic reticulum (ER) and implicated in quality control of protein folding. Herein, we established an inducible RNAi cell line that targets Sep15 mRNA in Chang liver cells. RNAi-induced Sep15 deficiency led to inhibition of cell proliferation, whereas cell growth was resumed after removal of the knockdown inducer. Sep15-deficient cells were arrested at the G1 phase by upregulating p21 and p27, and these cells were also characterized by ER stress. In addition, Sep15 deficiency led to the relocation of focal adhesions to the periphery of the cell basement and to the decrease of the migratory and invasive ability. All these changes were reversible depending on Sep15 status. Rescuing the knockdown state by expressing a silent mutant Sep15 mRNA that is resistant to siRNA also reversed the phenotypic changes. Our results suggest that SEP15 plays important roles in the regulation of the G1 phase during the cell cycle as well as in cell motility in Chang liver cells, and that this selenoprotein offers a novel functional link between the cell cycle and cell motility.

• 한국식물생명공학회:학술대회논문집
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• 한국식물생명공학회 2004년도 생명공학 실용화를 위한 비젼
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• pp.81-90
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• 2004

Sialic acid containing glycosphingolipids (gangliosides) have been implicated in the regulation of various biological phenomena such as atherosclerosis. Recent report suggeststhat exogenously supplied disialoganglioside (GD3) serves a dual role in vascular smooth muscle cells (VSMC) proliferation and apoptosis. However, the role of the GD3 synthase gene in VSMC responses has not yet been elucidated. To determine whether a ganglioside is able to modulate VSMC growth. the effect of overexpression of the GD3 synthase gene on DNA synthesis was examined. The results show that the overexpression of this gene has a potent inhibitory effect on DNA synthesis and ERK phosphorylation in cultured VSMC in the presence of PDGF. The suppression of the GD3 synthase gene was correlated with the down-regulation of cyclinE/CDK2. the up-regulation of the CDK inhibitor p21 and blocking of the p27 inhibition,whereas up-regulation of p53 as the result of GD3 synthase gene expression was not observed. Consistently, blockade of GD3 function with anti-GD3 antibody reversed VSMC proliferation and cell cycle proteins. The expression of the CD3 synthase gene also led to the inhibition of TNF--induced matrix metalloproteinase-9 (MMP-9) expression in VSMC as determined by zymography and immunoblot. Furthermore, GD3 synthase gene expression strongly decreased MMP-9 promoteractivlty in response to TNF-. This inhibition was characterized by the down-regulation of MMP-9,which was Iranscriptionally regulated at NF-B and activation protein-1 (AP-1) sites in the MMP-9promoter Finally, the overexpression of MMP-9 in GD3 synthase transfectant cells rescued VSMC proliferation. However MMP-2 overexpression was not affected the cell proliferation. These findings suggest that the fl13 synthase gene represents a physiological modulator of VSMC responses that may contribute to plaque instability in atherosclerosis.

• Environmental Analysis Health and Toxicology
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• 제27권
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• pp.10.1-10.7
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• 2012

Objectives: In recent years, a number of structurally diverse Histone deacetylase (HDAC) inhibitors have been identified and these HDAC inhibitors induce growth arrest, differentiation and/or apoptosis of cancer cells in vitro and in vivo. This study aimed at investigating the antitumor activity of newly synthesized HDAC inhibitor, 3-(4-dimethylamino phenyl)-N-hydroxy-2-propenamide (IN-2001) using human breast cancer cells. Methods: We have synthesized a new HDAC inhibitor, IN-2001, and cell proliferation inhibition assay with this chemical in estrogen receptor-positive human breast cancer MCF-7 cells. Cell cycle analysis on MCF-7 cells treated with IN-2001 was carried out by flow cytometry and gene expression was measured by RT-PCR. Results: In MCF-7 cells IN-2001 showed remarkable anti-proliferative effects in a dose- and time-dependent manner. In MCF-7 cells, IN-2001 showed a more potent growth inhibitory effect than that of suberoylanilide hydroxamic acid. These growth inhibitory effects were related to the cell cycle arrest and induction of apoptosis. IN-2001 showed accumulation of cells at $G_2$/M phase and of the sub-$G_1$ population in a time-dependent manner, representing apoptotic cells. IN-2001-mediated cell cycle arrest was associated with HDAC inhibitor-mediated induction of CDK inhibitor expression. In MCF-7 cells, IN-2001 significantly increased $p21^{WAF1}$ expression. Conclusions: In summary, cyclin-dependent kinase (CDK) induced growth inhibition, possibly through modulation of cell cycle and apoptosis regulatory proteins, such as CDK inhibitors, and cyclins. Taken together, these results provide an insight into the utility of HDAC inhibitors as a novel chemotherapeutic regime for hormone-sensitive and insensitive breast cancer.

• 대한한방부인과학회지
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• 제18권1호
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• pp.181-191
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• 2005

Purpose : ${\beta}$-sitosterol is kind of phytosterols or plant which are structurally similar to cholesterol. This study was aimed to investigate the inhibitory effect of the ${\beta}$-sitosterol on the proliferation of human uterine leiomyoma cells and the expression of gene related the mechanism of cell apoptosis. Methods : We counted the number of death cells treated with indicated time of the ${\beta}$-sitosterol and investigated cell death rate by cell count assay. Furthermore, flow cytometry analysis and DNA fragmentation assay were used to dissect between necrosis and apoptosis. and then we observed the differential gene expression by western blot analysis. Results : 1) The inhibitory effect on the growth of uterine leiomyoma cell treated with the ${\beta}$-sitosterol $16{\mu}M$ was increased in a time dependent. 2) The result of flow cytometry analysis, subG1 phase arrest related cell apoptosis was investigated 16.97% in uterine leiomyoma cell treated with the ${\beta}$-sitosterol $16{\mu}M$ and showed the fashion of proportional time dependent. 3) The gene expression of p27, p21 related cell cycle was increased according to increasing time interval but cyclin E-CDK2 complex was decreased expression. 4) The character of apoptosis, DNA fragmentation was significantly observed on the time dependent. 5) The expression of pro-caspase 3 and PARP were decreased dependent on treatment with time dependent. Conclusion : This study showed that the ${\beta}$-sitosterol have the inhibitory effect on the proliferation of human uterine leiomyoma cell and the effect was related with apoptosis.

• 대한한방부인과학회지
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• 제19권2호
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• pp.199-213
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• 2006

Purpose : This study was aimed to investigate the inhibitory effect of Sparganii Rhizoma on the proliferation of human uterine leiomyoma cells and the expression of gene related the mechanism of cell apoptosis. Methods : This study was evaluated the number of death cells treated with indicated concentration of Sparganii Rhizoma and investigated cell death rate by MTS assay. Furthermore, fluorescence-activated cell sorter analysis and DNA fragmentation assay were used to dissect between necrosis and apoptosis. and then we observed the differential gene expression by western blot analysis. Results :1) The inhibitory effect on the growth of uterine leiomyoma cell treated with Sparganii Rhizoma was increased in a dose dependent manner. 2) As the result of FACS analysis, subG1 phase incrase was observed 23.49% inuterine leiomyoma cell treated with Sparganii Rhizoma at $500\;{\mu}g/ml$ compared to control.. 3) The gene expression of p53, p21 related cell apoptosis was increased according to increasing concentration but p27 was none exchanged. 4) The expression of cyclin A, D and E was decreased in a concentration proportional and then the dephosphorylation of pRb was increased. 5) The character of apoptosis, DNA fragmentation was significantly observed according to increasing concentration. 6) The expression of pro-caspase3 were decreased dependent on treatment concentration and activated PARP took place. Conclusion : The inhibitory effect of Sparganii Rhizoma on the proliferation of human uterine leiomyoma cells was observed with apoptosis and cell cycle arrest. These data suggest that Sparganii Rhizoma might be candidate of medical therapy for uterine leiomyoma.

• Biomolecules & Therapeutics
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• 제30권2호
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• pp.151-161
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• 2022

This study elucidates the anti-cancer potential of gallic acid (GA) as a promising therapeutic agent that exerts its effect by regulating the PI3K/Akt pathway. To prove our research rationale, we used diverse experimental methods such as cell viability assay, colony formation assay, tumor spheroid formation assay, cell cycle analysis, TUNEL assay, Western blot analysis, xenograft mouse model and histological analysis. Treatment with GA inhibited cell proliferation in dose-dependent manner as measured by cell viability assay at 48 h. GA and cisplatin (CDDP) also inhibited colony formation and tumor spheroid formation. In addition, GA and CDDP induced apoptosis, as determined by the distribution of early and late apoptotic cells and DNA fragmentation. Western blot analysis revealed that inhibition of the PI3K/Akt pathway induced upregulation of p53 (tumor suppressor protein), which in turn regulated cell cycle related proteins such as p21, p27, Cyclin D1 and E1, and intrinsic apoptotic proteins such as Bax, Bcl-2 and cleaved caspase-3. The anti-cancer effect of GA was further confirmed in an in vivo mouse model. Intraperitoneal injection with GA for 4 weeks in an A549-derived tumor xenograft model reduced the size of tumor mass. Injection of them downregulated the expression of proliferating cell nuclear antigen and p-Akt, but upregulated the expression of cleaved caspase-3 in tumor tissues. Taken together, these results indicated that GA hindered lung cancer progression by inducing cell cycle arrest and apoptosis, suggesting that GA would be a potential therapeutic agent against non-small cell lung cancer.

• 생명과학회지
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• 제15권3호
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• pp.323-331
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• 2005

남미지역에서 자생하는 Tabebuia avellanedae라는 나무의 수피에서 동정된 quinone계 물질이며, DNA topoisomeras억제제로 알려진 $\beta-lapachone$의 항암작용에 관한 부가적인 자료를 얻기 위하여 인체 간암(HepG2) 및 방광암(T24)세포를 대상으로 조사한 결과 다음과 같은 결과를 얻게 되었다. MTT assay 및 flow cytometry 분석 등의 결과에서, $\beta-lapachone$의 처리에 따라 조사된 두 가지 암세포에서 $\beta-lapachone$처리 농도의존적으로 암세포의 심한 형태적 변형이 동반되면서 암세포의 증식이 억제되었으며, 생존율이 저하되었고 이는 apoptosis유발과 상관성이 있음을 알 수 있었다. $\beta-lapachone$처리에 의한 두 암세포의 증식억제는 종양억제 유전자 p53 및 Cdk inhibitor p21의 발현과는 큰 연관성이 없음을 RT-PCR 및 Western blot analysis를 통하여 확인하였다. 그러나 전사조절인자 Sp-1 및 세포증식 주요조절인자인 PCNA의 단백질 발현은 $\beta-lapachone$처리에 따라 매우 감소되었으며, telomere조절에 중요한 인자들의 선택적 발현 저하 현상도 관찰되었다. 이상의 결과들은 인체 암세포에서 $\beta-lapachone$의 항암작용을 이해하는 중요한 자료가 될 것이며, $\beta-lapachone$과 유사한 화학적 구조 및 성질을 가지는 항암제 후보물질들의 항암기전 비교 및 항암제 개발을 위한 기초 자료로서 응용될 것이다.