• 생명과학회지
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• 제15권3호
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• pp.397-405
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• 2005

브로콜리와 같은 십자화과 식물에서 glucoraphanin의 가수분해를 통해 생성되는 isothiocyanate의 일종인 sulforaphane은 강력한 항암효과를 가지며, 역학적 조사를 포함한 다양한 선행 연구에서 androgen 비 의존적으로 성장하는 전립선 암세포의 증식을 억제하는데 효과가 있었다. 최근 연구 결과에 따르면 sulforaphane은 다양한 인체암세포의 증식을 억제하고 apoptosis를 유발할 수 있는 것으로 알려지고 있으나, 정확한 분자생물학적 기전은 밝혀져 있지 않은 상태이다. 본 연구에서는 sulforaphane의 항암작용 기전을 조사하기 위하여 HeLa 인체자궁경부암세포의 증식에 미치는 sulforaphane의 영향을 조사하였다. Sulforaphane의 처리에 의한 HeLa 세포의 증식억제 및 형태적 변형은 세포주기 C2/M arrest 및 apoptosis 유발과 밀접한 관련이 있음을 알 수 있었다. RT-PCR 및 Western blot 분석 결과, sulforaphane 처리에 의하여 cyclin A 및 cyclin-dependent kinase (Cdk)4 단백질의 발현이 선택적으로 저하되었으며, Cdc2, Cdk inhibitor인 p16 및 p21의 발현은 증가되었다 그러나 sulforaphane은 cyclooxygenases의 발현이나 telomere 조절에 중요한 역할을 하는 인자들의 발현에는 큰 영향을 주지 못하였다. Sulforaphane의 항암 기전을 규명하기 위해서는 더 많은 연구가 부가적으로 필요하겠지만, 본 연구의 결과들에 의하면 sulforaphane은 강력한 인체암세포의 증식 억제 및 항암작용이 있을 것을 시사하여 준다고 할 수 있다.

• Archives of Pharmacal Research
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• 제25권5호
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• pp.685-690
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• 2002

The mitogen-stimulated serine/threonine kinase $p70^{S6k}$ plays an important role in the progression of cells from $G_0/G$_1$$ to S phase of the cell cycle by translational up-regulation of a family of mRNA transcripts family of mRNA transcripts which contain polypyrimidine tract at their 5 transcriptional start site. Here, we report that $p70^{S6k}$ was constitutively phosphorylated and activated to various degrees in serum-deprived AGS, A2058, HT-1376, MG63, MCF7, MDA-MB-435S, MDA-MB-231 and MB-157. Rapamycin treatment induced a significant dephosphorylation and inactivation of $p70^{S6k}$ in all cancer cell lines, while wortmannin, a specific inhibitor of PI3-K, caused a mild dephosphorylation of $p70^{S6k}$ in AGS, MDA-MB-435S and MB-157. In addition, SQ20006, methylxanthine phosphodiesterase inhibitor, reduced the phosphorylation of $p70^{S6k}$ in all cancer cells tested. Consistent with inhibitory effect of rapamycin on $p70^{S6k}$ activity, rapamycin inhibited [$^3H$]-thymidine incorporation and increased the number of cells at $G_{0}G_{1}$ phase. Furthermore, these inhibitory effects were accompanied by the decrease in growth of cancer cells. Taken together, the results indicate that the antiproliferative activity of rapamycin might be attributed to cell cycle arrest at $G_{0}G_{1}$ phase in human cancer cells through the inhibition of constitutively activated $p70^{S6k}$ of cancer cells and suggest $p70^{S6k}$ as a potential target for therapeutic strategies aimed at preventing or inhibiting tumor growth.

• 미생물학회지
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• 제42권4호
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• pp.319-325
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• 2006

칡을 첨가하여 배양한 아가리쿠스버섯(Agaricus blaxei Murill)으로 부터 열수추출, gel filtration chromatography, DEAE ion exchange chromatography를 통하여 아가리쿠스 $\beta$-glucan (AG)을 추출하였다. 추출한 아가리쿠스 $\beta$-glucan에 Bacillus megaterium 유래의 endo-$\beta$-(1$\rightarrow$6)-glucanase를 처리하여 올리고당류(AO)를 얻었다. 이렇게 얻은 AG와 AO를 이용하여 HT-29 인체 대장암 세포에 대한 항암 활성을 조사한 결과, 암세포의 성장 억제 효과는 시료의 처리 농도에 의존적으로 증가하였으며, apoptosis assay에서 암세포의 apoptosis 유발이 농도에 의존적으로 증가되었다. 또한, 암세포의 세포 주기를 분석한 결과, apoptosis 발생을 뜻하는 G0 (sub-G1)기와 G1기의 비율이 증가한 반면 S기와 G2/M기는 대조군에 비해 감소되었다. 이러한 결과를 바탕으로 암세포의 apoptosis 증가에 대한 AO의 작용이 어떤 유전자와 연관이 있는지를 알아보기 위하여 cDNA microarray를 통해 유전자의 발현율을 검색한 결과, apoptosis의 내부 외부 경로에 영향을 주는 유전자(TNESE9, TNFRSF9, FADD, CASP8, BAD, CRADD, CASP9 등)의 발현이 증가되었고 세포 분열 주기의 진행과 관련된 유전자(CCND2와 CDK2)의 발현이 감소되었으며, 세포 분열 주기를 지체시키는 유전자 (CDKN2A)의 발현은 증가되었다. 또한, 사이토카인을 암호하는 유전자 (IL6, IL18, IL6R 등)와 tumor suppressor와 관련된 유전자 (CEACAM1, TP53BP2, IRF1및 PHB)의 발현이 2배 이상 증가된 것을 확인할 수 있었다. 따라서 HT-29 인체 대장암세포에 대한 AO의 성장 억제 작용은 G0/Gl기를 지체시켜 암세포 증식을 억제하고 apoptosis에 의해 암세포를 사멸시키는 항암 활성을 나타내는 것으로 확인되었으며, 특허 AO가 AG보다 현저한 활성을 보였다. 더 나아가 아가리쿠스 $\beta$-glucan (AG)과 올리고당류 (AO)는 항암 활성을 가진 대체 의약 소재로 개발될 수 있을 것으로 기대된다.

• BMB Reports
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• 제50권12호
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• pp.640-646
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• 2017

Regulatory B cells, also well-known as IL-10-producing B cells, play a role in the suppression of inflammatory responses. However, the epigenetic modulation of regulatory B cells is largely unknown. Recent studies showed that the bromodomain and extra-terminal domain (BET) protein inhibitor JQ1 controls the expression of various genes involving cell proliferation and cell cycle. However, the role of BET proteins on development of regulatory B cells is not reported. In this study, JQ1 potently suppressed IL-10 expression and secretion in murine splenic and peritoneal B cells. While bromodomain-containing protein 4 (BRD4) was associated with $NF-{\kappa}B$ on IL-10 promoter region by LPS stimulation, JQ1 interfered the interaction of BRD4 with $NF-{\kappa}B$ on IL-10 promoter. In summary, BRD4 is essential for toll like receptor 4 (TLR4)-mediated IL-10 expression, suggesting JQ1 could be a potential candidate in regulating IL-10-producing regulatory B cells in cancer.

• BMB Reports
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• 제36권2호
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• pp.223-229
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• 2003

The product of a tree (Tabebuia avellanedae) from South America, $\beta$-lapachone, is known to exhibit various pharmacological properties, the mechanisms of which are poorly understood. The aim of the present study was to further elucidate the possible mechanisms by which $\beta$-lapachone exerts its anti-proliferative action in cultured human prostate cancer cells. We observed that the proliferation-inhibitory effect of $\beta$-lapachone was due to the induction of apoptosis, which was confirmed by observing the morphological changes and cleavage of the poly(ADP-ribose) polymerase protein. A DNA flow cytometric analysis also revealed that $\beta$-lapachone arrested the cell cycle progression at the G1 phase. The effects were associated with the down-regulation of the phosphorylation of the retinoblastoma protein (pRB) as well as the enhanced binding of pRB and the transcription factor E2F-1. Also, $\beta$-lapachone suppressed the cyclindependent kinases (Cdks) and cyclin E-associated kinase activity without changing their expressions. Furthermore, this compound induced the levels of the Cdk inhibitor $p21^{WAF1/CIP1}$ expression in a p53-independent manner, and the p21 proteins that were induced by $\beta$-lapachone were associated with Cdk2. $\beta$-lapachone also activated the reporter construct of a p21 promoter. Overall, our results demonstrate a combined mechanism that involves the inhibition of pRB phosphorylation and induction of p21 as targets for $\beta$-lapachone. This may explain some of its anticancer effects.

• 동의생리병리학회지
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• 제21권4호
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• pp.973-981
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• 2007

In the present study, we investigated the antiproliferative activity of the water extract of Samgibopae-tang (SGBPT) in NCI-H460 and A549 non-small-cell lung cancer cell lines. We found that exposure of A549 cells to SGBPT resulted in the growth inhibition in a dose-dependent manner as measured by MTT assay, however SGBPT did not affect the growth of NCI-H460 cells. The antiproliferative effect by SGBPT treatment in A549 cells was associated with morphological changes such as membrane shrinking and cell rounding up. SGBPT treatment did not induce the cell cycle arrest in both cell lines, however the frequency of sub-G1 population was concentration-dependently increased by SGBPT treatment in A549 cells. SGBPT treatment partially induced the expression of tumor suppressor p53 in A549 cells and the expression of cyclin-dependent kinase inhibitor p21(WAF1/CIP1) was markedly increased in both transcriptional and translational levels in A549 cells. The up-regulation of p21 by SGBPT occurred in a similar a concentration dependent manner to that observed with the inhibition of cell viability and induction of sub-G1 population of the cell cycle. However SGBPT treatment did not affect other growth regulation-related genes such as early growth response-1 (Egr-1), nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1), inducible nitric oxide synthease (iNOS), cyclooxygenases (COXs), telomere-regulatory factors in A549 as well as NCI-H460 cells. Taken together, these findings suggested that SGBPT-induced inhibition of human lung carcinoma A549 cell growth was aoosciated with the induction of p21 and the results provided important new insights into the possible molecular mechanisms of the anti-cancer activity of SGBPT.

• 한국수정란이식학회지
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• 제10권1호
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• pp.73-82
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• 1995

large scale production of cloned embryos requires the technology of multiple generation nuclear transplantation(NT) using NT embryos as the subsequent donor nuclei. The purposes of this study were producing the second generation cloned rabbit embryos, and also to determine the electrofusion rate and in vitro developmental potential comparatively in the cloned embryos of the first and second NT generation. The embryos of 16-cell stage were collected from the mated does by flushing oviducts with Dulbecco's phosphate buffered saline(D-PBS) containing 10% fetal calf serum(FCS) at 47 hours after hCG injection In the first generation NT, the nuclear donor embryos were synchronized in the phase of Gi /S transition of 32-cell stage. The first generation NT embryos which were developed to 8-cell were synchronized in Gi /S transition phase of the following 16-cell stage and used as donor nuclei for second generation Synchronization of the cell cycle of blastomeres was induced, first, using an inhibitor of microtuble polymerization, colcemid for 10 hours to arrest blastomeres in M phase, and secondly, using a DNA synthesis inhibitor, aphidicolin for 1.5 to 2 hours to arrest them in Gi /S transition boundary. The recipient cytoplasms were obtained by removing the nucleus and the first polar body from the oocytes collected at 14 hours after hCG injection. The separated donor blastomeres were injected into the enucleated recipient oocytes by micromanipulation and were electrofused by electrical stimulation of three pulses for 60 $\mu$sec at 1.25 kV /cm in 0.28 M rnannitol solution The fused oocytes were co-cultured with a monolayer of rabbit oviductal epithelial cells in M-199 solution containing 10% FCS for 120 hours at 39$^{\circ}C$ in a 5% $CO_2$ incubator. Following in vitro culture of the first and second generation cloned embryos to blastocyst stage, they were stained with Hoechst 33342 dye for counting the number of blastomeres by fluorescence microscopy. The results obtained were summarized as follows: 1. The electrofusion rate was found to be similar as 79.4 and 91.5% in the first and second generation NT rabbit embryos, respectively. 2. The in vitro developmental potential to blastocyst stage of the second generation NT embryos (23.3%) was found significantly(p<0.05) lower, compared with that of the first generation NT embryos (56.8%). 3. The mean blastomeres counts of embryos developed to blastosyst stage following in vitro culture for 120 hours and also their daily cell cycles during the culture period were decreased significantly (p<0.05) to 104.3 cells and 1.33 cylces in the second NT generation, compoared with 210.4 cells and 1.54 cycles in the first NT generation, respectively.

• The Korean Journal of Physiology and Pharmacology
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• 제24권3호
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• pp.223-232
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• 2020

Sesamin, a lipid-soluble lignin originally isolated from sesame seeds, which induces cancer cell apoptosis and autophagy. In the present study, has been reported that sesamin induces apoptosis via several pathways in human lung cancer cells. However, whether mitophagy is involved in sesamin induced lung cancer cell apotosis remains unclear. This study, the anticancer activity of sesamin in lung cancer was studied by reactive oxygen species (ROS) and mitophagy. A549 cells were treated with sesamin, and cell viability, migration ability, and cell cycle were assessed using the CCK8 assay, scratch-wound test, and flow cytometry, respectively. ROS levels, mitochondrial membrane potential, and apoptosis were examined by flow cytometric detection of DCFH-DA fluorescence and by using JC-1 and TUNEL assays. The results indicated that sesamin treatment inhibited the cell viability and migration ability of A549 cells and induced G₀/G₁ phase arrest. Furthermore, sesamin induced an increase in ROS levels, a reduction in mitochondrial membrane potential, and apoptosis accompanied by an increase in cleaved caspase-3 and cleaved caspase-9. Additionally, sesamin triggered mitophagy and increased the expression of PINK1 and translocation of Parkin from the cytoplasm to the mitochondria. However, the antioxidant N-acetyl-L-cysteine clearly reduced the oxidative stress and mitophagy induced by sesamin. Furthermore, we found that cyclosporine A (an inhibitor of mitophagy) decreased the inhibitory effect of sesamin on A549 cell viability. Collectively, our data indicate that sesamin exerts lethal effects on lung cancer cells through the induction of ROS-mediated mitophagy and mitochondrial apoptosis.

• BMB Reports
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• 제41권2호
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• pp.164-169
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• 2008

The tumor suppressor gene p53 regulates apoptotic cell death and the cell cycle. In this study, we investigated the role of p53 in nitric oxide (NO)-induced apoptosis in vascular smooth muscle cells (VSMCs). We found that the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) increased apoptotic cell death in p53-deficient VSMCs compared with wild-type cells. The heme oxygen-ase (HO) inhibitor tin protoporphyrin IX reduced the resistance of wild-type VSMCs to SNAP-induced cell death. SNAP promoted HO-1 expression in both cell types. HO-2 protein was increased only in wild-type VSMCs following SNAP treatment; however, similar levels of HO-2 mRNA were detected in both cell types. SNAP significantly increased the levels of non-heme-iron and dinitrosyl iron-sulfur clusters in wild-type VSMCs compared with p53-deficient VSMCs. Moreover, pretreatment with FeSO4 and the carbon monoxide donor CORM-2, but not biliverdin, significantly protected p53-deficient cells from SNAP-induced cell death compared with normal cells. These results suggest that wild-type VSMCs are more resistant to NO-mediated apoptosis than p53-deficient VSMCs through p53-dependent up-regulation of HO-2.

• Journal of Ginseng Research
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• 제48권2호
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• pp.171-180
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• 2024

Background: Epimers of ginsenoside Rg3 (Rg3) have a low bioavailability and are prone to deglycosylation, which produces epimers of ginsenoside Rh2 (S-Rh2 and R-Rh2) and protopanaxadiol (S-PPD and R-PPD). The aim of this study was to compare the efficacy and potency of these molecules as anti-cancer agents. Methods: Crystal violet staining was used to study the anti-proliferatory action of the molecules on a human epithelial breast cancer cell line, MDA-MB-231, and human umbilical vein endothelial cells (HUVEC) and compare their potency. Cell death and cell cycle were studied using flow cytometry and mode of cell death was studied using live cell imaging. Anti-angiogenic effects of the drug were studied using loop formation assay. Molecular docking showed the interaction of these molecules with vascular endothelial growth factor receptor-2 (VEGFR2) and aquaporin (AQP) water channels. VEGF bioassay was used to study the interaction of Rh2 with VEGFR2, in vitro. Results: HUVEC was the more sensitive cell line to the anti-proliferative effects of S-Rh2, S-PPD and R-PPD. The molecules induced necroptosis/necrosis in MDA-MB-231 and apoptosis in HUVEC. S-Rh2 was the most potent inhibitor of loop formation. In silico molecular docking predicted a good binding score between Rh2 or PPD and the ATP-binding pocket of VEGFR2. VEGF bioassay showed that Rh2 was an allosteric modulator of VEGFR2. In addition, SRh2 and PPD had good binding scores with AQP1 and AQP5, both of which play roles in cell migration and proliferation. Conclusion: The combination of these molecules might be responsible for the anti-cancer effects observed by Rg3.