• 동의생리병리학회지
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• 제19권5호
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• pp.1370-1374
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• 2005

The Tosyl-JM3 (TJM3) is a modified compound from one of 1,2,3,4-Tetra- hydroisoquinoline (THIQ) derivatives. The THIQs include potent cytotoxic agents that display a range of anti-tumor activities, antimicrobial activity, and other biological properties. In this study, we investigated the effect of TJM3 on the cytotoxicity, induction of apoptosis in human promyelocytic leukemia cells (HL-60 cells). TJM3 showed a significant cytotoxic activity in HL-60 cells (IC50 = approximately $60{\mu}g/m{\ell}$) after a 24 hr incubation. Treatment of HL-60 cells with TJM3 exhibited several features of apoptosis, including formation of DNA ladders in agarose gel electrophoresis, morphological changes of HL-60 cells with DAPI stain. Here we observed that TJM3 caused a decrease of procaspase-3 protein. Further molecular analysis demonstrated that TJM3 led to cleavage of poly(ADP-ribose) polymerase (PARP) by western blot and increase of hypodiploid (Sub-G1) population in the flow cytometric analysis. In conclusion, these above results indicate that TJM3 dramatically suppresses HL-60 cell growth and induces apoptosis. These data may support a possibility for the use of TJM3 in the prevention and treatment of leukemia.

• 동의생리병리학회지
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• 제19권6호
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• pp.1563-1567
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• 2005

Mori Cortex Radicis is distributed in Northwestern China, northern Asia, northern Europe, North America, and Korea. This extracts drops sugar in bloods and inhibits cyclic AMP phophodiesterase. In this study, we investigated whether Mori Cortex Radicis would cause apoptotic death of A549 lung cancer cells. To examine the apoptotic effect of Mori Cortex Radicis, cytotoxicity assay, DNA fragmentation analysis, caspase-3 activity assay, and Western blotting for caspase-3, caspase-9 and poly(ADP-ribose) polymerase (PARP) and cytochrome c were performed. Treatment of cells with Mori Cortex Radicis was shown to induce cell death in a dose-dependent manner. DNA fragmentation was made in response to Mori Cortex Radicis. The active fragments of caspase-3, caspase-9 and PARP were almost completely induced and cytochrome c was released following exposure to Mori Cortex Radicis. To elucidate the apoptotic mechanisms, RT-PCR and Western blot analyses for the expression of Bcl-2, Bu and Cox-2 were carried out. Treatment with Mori Cortex Radicis was expressed the reduction of Bcl-2 and Cox-2 and the induction of Bax. Especially p21 and p53 were increased prior to untreated control, while cyclin E and cyclin D1 decreased in the cytosol. These results suggest that the effect Mori Cortex Radicis is associated with the cell cycle arrest and pro-apoptotic cell death in A549 lung cancer cells.

• 한국자원식물학회지
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• 제28권3호
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• pp.297-304
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• 2015

The flower buds of Sophora japonica L (SF), as a well-known traditional Chinese medicinal herb, have been used to treat bleeding-related disorders such as hematochezia, hemorrhoidal bleeding, dysfunctional uterine bleeding, and diarrhea. However, no specific anti-cancer effect and its molecular mechanism of SF have been described. Thus, we performed in vitro study to investigate if treatment of SF affects activating transcription factor 3 (ATF3) expression and ATF3-mediated apoptosis in human colorectal cancer cells. The effects of SF on cell viability and apoptosis were measured by MTT assay and Western blot analysis against cleaved poly (ADP-ribose) polymerase (PARP). ATF3 activation induced by SF was evaluated using Western blot analysis, RT-PCR and ATF3 promoter assay. SF treatment caused decrease of cell viability and increase of apoptosis in a dose-dependent manner in HCT116 and SW480 cells. Exposure of SF activated the levels of ATF3 protein and mRNA via transcriptional regulation in HCT116 and SW480 cells. Inhibition of extracellular signal-regulated kinases (ERK) 1/2 by PD98059 and p38 by SB203580 attenuated SF-induced ATF3 expression and transcriptional activation. Ectopic ATF3 overexpression accelerated SF-induced cleavage of PARP. These findings suggest that SF-mediated apoptosis may be the result of ATF3 expression through ERK1/2 and p38-mediated transcriptional activation.

• Journal of Microbiology and Biotechnology
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• 제16권9호
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• pp.1399-1404
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• 2006

The roots of Aralia continentalis (AC) have been used traditionally in Korean as a folk medicine for anti-inflammation and as an anti-rheumatic. In this study, we report that the ethyl acetate-soluble traction (ACE) of the methanolic extract of AC root inhibited the cell growth of various human cancer cell lines and induced apoptosis of HL-60, human promyelocytic leukemia cells. Its $IC_{50}$ values on growth inhibition were estimated to be $56.3{\mu}g/ml$ on HL-60, $87.2{\mu}g/ml$ on HepG2, $93.2{\mu}g/ml$ on HeLa, $135.5{\mu}g/ml$ on DU-145, and $135.8{\mu}g/ml$ on HT-29 cells. Interestingly, ACE showed no antiproliferative effect on normal lymphocyte cells used as control. Furthermore, nuclear DAPI staining revealed the typical nuclear features of apoptosis in the HL-60 cells exposed to $80{\mu}g/ml$ ACE, and a flow cytometric analysis of the HL-60 cells using propidium iodide showed that the apoptotic cell population increased gradually from 5% at 0 h to 16% at 12 h and 20% at 24 h after treated with $50{\mu}g/ml$ of ACE. TUNEL assay also revealed the apoptotic induction of the HL-60 cells treated with ACE. To obtain further information on the ACE-induced apoptosis, the expression level of certain apoptosis-associated proteins was examined using a Western blot analysis. Treatment of the HL-60 cells with ACE resulted in the activation of caspase-3, and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). The above results confirmed that the apoptosis in the HL-60 cells was induced by ACE, and that caspase-3-mediated PARP cleavage was involved in the process.

• 생명과학회지
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• 제13권2호
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• pp.154-162
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• 2003

전통 한의학에서 많이 사용되는 길경(Platycodi radix)은 도라지(Platycodon grandiflorum A. DC)의 뿌리 껍질을 벗기거나 또는 그대로 말린 것을 말하며, 염증성 호흡기 질환에 많이 사용되어져 오고 있다. 본 연구에서는 길경 수용액 추출물의 항암 활성 기전해석의 일환으로 A549 인체 폐암세포의 성장에 미치는 영향을 조사하였다. 길경 추출물의 처리에 의하여 처리 농도 의존적으로 암세포의 성장이 억제되었으며, 이는 apoptosis의 유발과 관련이 있었다. 즉 염색체 응축에 의한 apoptotic body의 출현과 sub-Gl기에 속하는 세포의 빈도가 길경 추출물 처리에 의하여 농도 의존적으로 증가하였다. Immunoblot 및 RT-PCR분석에서 길경 추출물의 처리에 의하여 Bcl-2의 mRNA 및 단백질의 발현은 감소되었으며, Bax의 발현이 증가됨을 알 수 있었다. 또한 길경 추출물의 처리에 의하여 caspase-3가 활성화되었으며, 이에 따른 PARP 및 $\beta$-catenin의 단백질 발현 감소 현상을 관찰할 수 있었다. 이러한 결과들은 길경 추출물을 이용한 인체 암세포의 성장억제제의 개발 가능성이 있음을 시사하여 준다.

• Archives of Pharmacal Research
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• 제26권9호
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• pp.739-746
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• 2003

Ultraviolet B (UVB) is known to induce apoptosis in human melanocytes. Here we show the cytoprotective effect of sphingosine-1-phosphate (S1P) against UVB-induced apoptosis. We also show that UVB-induced apoptosis of melanocytes is mediated by caspase-3 activation and poly(ADP-ribose) polymerase (PARP) cleavage, and that S1P prevents apoptosis by inhibiting this apoptotic pathway. We further investigated three major mitogen-activated protein (MAP) kinases after UVB irradiation. UVB gradually activated c-Jun N-terminal kinase (JNK) and p38 MAP kinase, while extracellular signal-regulated protein kinase (ERK) was inactivated transiently. Blocking of the p38 MAP kinase pathway using SB203580 promoted cell survival and inhibited the activation of caspase-3 and PARP cleavage. These results suggest that p38 MAP kinase activation may play an important role in the UVB-induced apoptosis of human melanocytes. To explain this cytoprotective effect, we next examined whether S1P could inhibit UVB-induced JNK and p38 MAP kinase activation. However, S1P was not found to have any influence on UVB-induced JNK or p38 MAP kinase activation. In contrast, S1P clearly stimulated the phosphorylation of ERK, and the specific inhibition of the ERK pathway using PD98059 abolished the cytoprotective effect of S1P. Based on these results, we conclude that the activation of p38 MAP kinase plays an important role in UVB-induced apoptosis, and that S1P may show its cytoprotective effect through ERK activation in human melanocytes.

• 대한의생명과학회지
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• 제29권2호
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• pp.66-74
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• 2023

Triglyceride (TG) accumulation can cause monocytic death and suppress innate immunity. However, the signaling pathways involved in this phenomenon are not fully understood. This study aimed to examine whether inducible nitric oxide synthase (iNOS) is involved in the TG-induced death of THP-1 monocytes. Results showed that iNOS was upregulated in TG-treated THP-1 monocytes, and iNOS inhibition blocked TG-induced monocytic death. In addition, TG-induced poly (ADP-ribose) polymerase (PARP) cleavage and caspase-3 and -7 activation were suppressed by iNOS inhibition. Furthermore, the expression of X-linked inhibitor of apoptosis protein (XIAP) and survivin, which inhibit caspase-3 and -7, was reduced in TG-treated THP-1 monocytes, but iNOS inhibition recovered the TG-induced downregulation of XIAP and survivin expression. Considering that TG-induced monocytic death is triggered by caspase2 and -8, we investigated whether caspase-2 and -8 are linked to the TG-induced expression of iNOS in THP-1 monocytes. When the activities of caspase-2 and -8 were inhibited by specific inhibitors, the TG-induced upregulation of iNOS and downregulation of XIAP and survivin were restored in THP-1 monocytes. These results suggest that TG-induced monocytic death is mediated by the caspase-2/caspase-8/iNOS/XIAP and survivin/executioner caspase/PARP pathways.

• Radiation Oncology Journal
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• 제25권4호
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• pp.227-232
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• 2007

목적: 세포 주기 억제제인 flavopirldol이 후두암과 폐암 세포주에서 방사선으로 인한 아포토시스에 미치는 영향을 알아보고 세포 내 아포토시스 조절 물질들의 발현에 어떤 변화를 가져오는지 알아본다. 대상 및 방법: 사람 후두암 세포주인 AMC-HN3와 폐암 세포주인 NCI-H460을 배양하여 1) 아무 처치도 하지 않은 군, 2) 방사선 조사만 한 군, 3) flavopiridol 약물 처치만 한 군, 4) 방사선과 flavopriodol 동시 병합 치료를 한 군으로 나누어 비교하였다. 방사선 조사시 4 MV 선형가속기의 X-ray를 10 Gy 조사하였고 flavopiridol은 세포 배양액에 100 nM 농도로 희석하여 24시간 동안 투여했다. 치료를 시작한 시점으로부터 24시간 후에 네 군의 아포토시스율을 비교하였다. 아포토시스율은 유세포 분석기를 이용하여 sub-G1 세포의 분율로 구했다. 또한 네 군에서 cleaved caspase-3, cleaved PARP (poly(ADP-ribose) polymerase), p53, p21, cyclin D1, phosphorylated Akt (protein kinase B) 발현 양상을 비교하기 위해 면역단백분석을 시행하였다. 결 과: 방사선 단독 처치 또는 flavoplridol 단독 처치한 군에 비해 방사선과 flavopiridol을 동시 병합 치료한 군에서 아포토시스율이 증가하는 것을 두 가지 암세포주 모두에서 확인할 수 있었다. 면역단백분석에서도 cleaved caspase-3, cleaved PARP 발현이 동시 병합 치료 군에서 높게 나타나는 것을 관찰할 수 있었다. 또한 두 세포주 모두에서 flavopiridol에 의해 cyclin D1 발현이 감소되는 것을 확인하였으나 flavopiridol이 p53, p21 발현에 미치는 영향은 세포주에 따라 다르게 나타났으며 Akt 발현은 두 세포주 모두에서 flavopiridol 투여에 의한 변화가 없었다. 결 론: 본 실험을 통해 사람 후두암 및 폐 암 세포주에서 flavoplridol이 방사선에 의한 아포토시스를 증가시킴으로써 방사선 치료 효과를 증진시킬 수 있음을 확인하였다. Flavopiridol이 p53, p21 발현에 미치는 영향은 세포주에 따라 다른 것으로 나타났으며 phosphorylated Akt 발현은 영향을 주지 않는 것으로 나타났다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권10호
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• pp.4369-4376
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• 2015

Background: To investigate in-vitro antagonistic effect of low-dose liquiritigenin on gemcitabine-induced capillary leak syndrome (CLS) in pancreatic adenocarcinoma via inhibiting reactive oxygen species (ROS)-mediated signalling pathways. Materials and Methods: Human pancreatic adenocarcinoma Panc-1 cells and human umbilical vein endothelial cells (HUVECs) were pre-treated using low-dose liquiritigenin for 24 h, then added into gemcitabine and incubated for 48 h. Cell viability, apoptosis rate and ROS levels of Panc-1 cells and HUVECs were respectively detected through methylthiazolyldiphenyl-tetrazoliumbromide (MTT) and flow cytometry. For HUVECs, transendothelial electrical resistance (TEER) and transcellular and paracellular leak were measured using transwell assays, then poly (ADP-ribose) polymerase 1 (PARP-1) and metal matrix proteinase-9 (MMP9) activity were assayed via kits, mRNA expressions of p53 and Rac-1 were determined through quantitative polymerase chain reaction (qPCR); The expressions of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and PARP-1 were measured via western blotting. Results: Low-dose liquiritigenin exerted no effect on gemcitabine-induced changes of cell viability, apoptosis rate and ROS levels in Panc-1 cells, but for HUVECs, liquiritigenin ($3{\mu}M$) could remarkably elevate gemcitabine-induced decrease of cell viability, transepithelial electrical resistance (TEER), pro-MMP9 level and expression of ICAM-1 and VCAM-1 (p<0.01). Meanwhile, it could also significantly decrease gemcitabine-induced increase of transcellular and paracellular leak, ROS level, PARP-1 activity, Act-MMP9 level, mRNA expressions of p53 and Rac-1, expression of PARP-1 and apoptosis rate (p<0.01). Conclusions: Low-dose liquiritigenin exerts an antagonistic effect on gemcitabine-induced leak across HUVECs via inhibiting ROS-mediated signalling pathways, but without affecting gemcitabine-induced Panc-1 cell apoptosis. Therefore, low-dose liquiritigenin might be beneficial to prevent the occurrence of gemcitabine-induced CLS in pancreatic adenocarcinoma.

• 동의신경정신과학회지
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• 제24권4호
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• pp.419-434
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• 2013

Objectives: The object of this study is to observe the cognition and motor function recovery effects of Joojakwhan (JJW), a traditional Korean poly-herbal formula for treating various neuropsychiatric diseases such as dementia, for the mildly stroke rats, with 60 minutes of reperfusion transient middle cerebral artery occlusion (tMCAO). Methods: In the present study, 125, 250 and 500 mg/kg of JJW were orally administered, once per day for 10 continuous days 2 hours after the tMCAO. The body weight changes, infarct sizes under 2% 2, 3, 5-triphenyl tetrazolium chloride (TTC) stain, sensorimotor functions and cognitive motor behavior tests were serially monitored with cerebral caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP)-immunoreactivities and histopathological changes. The effects of tMCAO on sensorimotor functions were evaluated by using of limb placing and body-swing tests, and the cognitive motor behaviors were also observed with water maze tests. Results: From the results of tMCAO, with marked decreases of body weights, disorders of sensorimotor functions increases the limb placing test scores, and decrease the numbers and percentages of body swings to the ipsilateral sides. The cognitive motor behaviors increases the distances and time to reach the escape platform which included the inhibitions of the decreases with repeated trials that were observed with focal cerebral cortex infarct volumes. In addition, the marked increases of the atrophy, numbers of degeneration, caspase-3- and PARP-immunoreactive cells around peri-infarct ipsilateral cerebral cortex were also observed in tMCAO controls when compared with the sham control rats, respectively. Conclusions: The results obtained from this study suggest that oral administrations of JJW indicate obvious cognitions and motor function recoveries of the rats with tMCAO, mild strokes, which are mediated by neuro-protective effects through known antioxidant effects of components.