• 생명과학회지
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• 제31권10호
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• pp.873-884
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• 2021

본 연구에서는 다양한 인체 암포주를 대상으로 NSAID의 항암 효과를 증강시키는 artesunate (ART)의 역할과 이에 대한 분자적 기전을 연구하였다. 다양한 타입의 암세포주를 대상으로 암세포 성장 억제 활성을 조사한 결과, ART는 NSAID인 celecoxib (CCB) 또는 dimethyl-CCB (DMC)와의 병용 효과를 나타내었다. ART 처리로 ATF4/CHOP의 발현 증강과 함께 오토파지 유도 표식인 p62 감소의 결과로서, ATF4/CHOP 경로가 ART의 오토파지 유도 활성에 관여할 것으로 예상되었으며, ART의 오토파지 활성과 관련하여 NRF2 및 암 줄기 세포 관련 단백질인 CD44, CD133, ALDH1, Oct4, mutated p53 (mutp53) 및 c-Myc의 발현이 감소되었다. 또한 DMC 단독처리 보다 ART와 DMC의 병용으로 ATF4/CHOP의 발현 증강과 p62의 감소가 더욱 촉진되고, NRF2 및 암 줄기 세포 관련 단백질 발현 감소도 현저히 촉진되면서 궁극적으로 PARP 활성화에 의해 apoptosis가 유도됨을 알 수 있었다. 이러한 결과는 ART/DMC 병용 처리가 각 물질 단독 처리보다 암세포의 성장 억제 및 apoptosis 유도에 더욱 효과적이고, ART 및 DMC 의 오토파지 유도 활성은 암 줄기 세포 관련 단백질의 분해를 촉진함으로써, 암 줄기 세포가 제거될 수 있음을 시사하였다. 이와 같이 ART는 NSAID 뿐만 아니라 imatinib의 항암 효과를 증강시키는 활성으로, chemosensitizer로서 중요한 후보 물질이 될 수 있음을 밝혔다.

• Biomolecules & Therapeutics
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• 제31권4호
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• pp.446-455
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• 2023

The mechanistic functions of 3-deoxysappanchalcone (3-DSC), a chalcone compound known to have many pharmacological effects on lung cancer, have not yet been elucidated. In this study, we identified the comprehensive anti-cancer mechanism of 3-DSC, which targets EGFR and MET kinase in drug-resistant lung cancer cells. 3-DSC directly targets both EGFR and MET, thereby inhibiting the growth of drug-resistant lung cancer cells. Mechanistically, 3-DSC induced cell cycle arrest by modulating cell cycle regulatory proteins, including cyclin B1, cdc2, and p27. In addition, concomitant EGFR downstream signaling proteins such as MET, AKT, and ERK were affected by 3-DSC and contributed to the inhibition of cancer cell growth. Furthermore, our results show that 3-DSC increased redox homeostasis disruption, ER stress, mitochondrial depolarization, and caspase activation in gefitinib-resistant lung cancer cells, thereby abrogating cancer cell growth. 3-DSC induced apoptotic cell death which is regulated by Mcl-1, Bax, Apaf-1, and PARP in gefitinib-resistant lung cancer cells. 3-DSC also initiated the activation of caspases, and the pan-caspase inhibitor, Z-VAD-FMK, abrogated 3-DSC induced-apoptosis in lung cancer cells. These data imply that 3-DSC mainly increased mitochondria-associated intrinsic apoptosis in lung cancer cells to reduce lung cancer cell growth. Overall, 3-DSC inhibited the growth of drug-resistant lung cancer cells by simultaneously targeting EGFR and MET, which exerted anti-cancer effects through cell cycle arrest, mitochondrial homeostasis collapse, and increased ROS generation, eventually triggering anti-cancer mechanisms. 3-DSC could potentially be used as an effective anti-cancer strategy to overcome EGFR and MET target drug-resistant lung cancer.

• 생명과학회지
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• 제25권8호
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• pp.849-860
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• 2015

본 연구에서는 한약재로 널리 사용되는 건칠, 유근피 및 신석 추출물의 항암 활성을 조사하였다. 생쥐 유래 정상세포(RAW 264.7 대식세포 및 C2C12 근아세포)에서는 건칠, 유근피 및 신석 단독 및 복합 처리에 의하여 유의적인 세포생존율의 억제 현상은 관찰 할 수 없었다. 그리고 건칠, 유근피 및 신석의 복합 처리는 단독 처리군에 비하여 AGS 위암세포의 생존력을 유의적으로 억제하였으나, 폐암(A549), 대장암(HCT116), 간암(Hep3B) 및 방광암(T24) 세포에서는 그 효과가 미비하였다. 아울러 이러한 AGS 위암세포 선택적 생존 억제력은 apoptosis 유도와 밀접한 연관성이 있음을 염색질의 응축 현상, DNA 단편화 및 annexin-V 염색에 의한 flow cytometry 분석을 통하여 확인하였다. 건칠, 유근피 및 신석의 복합 처리는 Fas 및 Fas legand의 발현을 증가시켰으며, XIAP, cIAP-1 및 survivin과 같은 IAP family 단백질과 anti-apoptotic Bcl-xL의 발현은 저하시켰다. 복합 처리는 또한 mitochondrial membrane potential의 손실과 caspases (-3, -8 및 -9)의 활성에 PARP 단백질의 분절화를 유도하였다. 그러나 이러한 복합 처리에 의한 AGS 세포에서 관찰된 세포독성 및 apoptosis 유도 효과는 pan-caspases inhibitor인 z-VAD-fmk의 선처리에 의하여 차단되었다. 이상의 결과는 건칠, 유근피 및 신석의 복합 처리에 의한 AGS 위암세포 선택적 apoptosis 유도가 caspase 의존적으로 일어나고 있음을 보여주는 결과이며, in vivo 모델을 이용한 후속 연구가 진행되어야 할 것이다.

• 동의생리병리학회지
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• 제32권4호
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• pp.261-270
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• 2018

The aim of this study was to evaluate the antitumor activities of Typhae pollen (TP) by confirming in vitro cytotoxicity and in vivo anti-tumor and immune-modulatory effect with anti-cachexia effect. The MTT assay is used in HepG2 cell to detect potential cytotoxic activities of aqueous extract of Typhae pollen (TPe). After HepG2 tumor cell implantation, eight mice per groups were assigned to six groups. Three different dosages of TPe (500, 250 and 125 mg/kg) were orally administered in the amount of $10m{\ell}/kg$ and sorafenib also administered 20mg/kg, every day for 35 days from 28 days after the tumor cell implantation. We observed the changes on body weights, tumor volume and weights, lymphatic organ, serum interferon $(IFN)-{\gamma}$ levels, splenocytes and peritoneal NK cell activity, splenic tumor necrosis factor $(TNF)-{\alpha}$, interleukin $(IL)-1{\beta}$, IL-10 contents. Periovarian fat weights, serum IL-6 levels, thicknesses of deposited periovarian adipose tissue and mean diameters were also detected to monitor the tumor-related anticachexic effects. In tumor masses, the immunoreactivities of cleaved caspase-3 and cleaved poly (ADP-ribose) polymerase (cleaved PARP) - apoptotic marks, cyclooxygenase-2 (COX-2), inducible nitric oxide synthases (iNOS) and tumor necrosis factor $(TNF)-{\alpha}$ were additionally observed by immunohistochemistry. The results were compared with sorafenib. Decreases of COX-2 were demonstrated in sorafenib and TPe treated mice and also increases of iNOS in tumor masses were observed in TPe, not in sorafenib. TPe increased periovarian fat pad weights compared with tumor-bearing controls and sorafenib treated mice. TPe showed increases of splenic $TNF-{\alpha}$, IL-10 and $IL-1{\beta}$, serum $IFN-{\gamma}$ and NK cell activities corresponding to increases of spleen weights, lymph node weights and non-atrophic changes of lymph nodes. Our results show oral treatment of TPe 500, 250 and 125 mg/kg has potent in vitro and in vivo antitumor activities through modest cytotoxic effects, immunomodulatory effects and apoptotic activities in HepG2 tumor cells. In addition, TPe can prevent cancer related cachexia.

• 대한본초학회지
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• 제22권3호
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• pp.27-37
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• 2007

Objectives: Hepatocellular carcinoma is the most common primary malignant tumor of the liver worldwide. In-Jin-Ho-Tang(IJHT) has been used as a traditional Chinese herbal medicine since ancient time. and today it is widely applied as a medication for jaundice which is associated with inflammation in liver. In this study, I investigated whether methanol extract of IJHT induced HepG2 cancer cell death. Methods: Cytotoxic activity of IJHT on HepG2 cells was using XTT assay. Apoptosis induction by Ros A in HCT116 cells was verified by the induction of cleavage of poly ADP-ribose polymerase (PARP). and activation of caspase-3, -8 and -9. The release of cytochrome c from mitochondria to cytosol. the level of Bcl-2 and Bax and the expression of p53 and p21 were examined by western blotting analysis. Furthermore, MAPKs activation was analyzed by western blotting analysis. Results: IJHT induced apoptosis in HepG2 cells. And treatment of IJHT resulted in the release of cytochrome c into cytosol, decreased anti-apoptotic Bcl-2, and increased pri-apoptotic Bax expression. IJHT markedly inactivated extracellular signal-regulated kinase (ERK1/2), and activated p38 mitogen-activated protein (MAP) kinase. Sodium orthovanadate (SOV), a phosphatase inhibitor, to reverse IJHT-induced ERK1/2 inactivation and SB203580, a specific p38 MAP Kinase inhibitor efficiently blocked apoptosis of HepG2. Thus, IJHT induces apoptosis in HepG2 cells via MAP kinase modulation. Conclusion: These results indicated that IJHT has some potential for use as an anti-cancer agent.

• Biomolecules & Therapeutics
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• 제28권2호
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• pp.202-210
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• 2020

Fluoxetine is used widely as an antidepressant for the treatment of cancer-related depression, but has been reported to also have anti-cancer activity. In this study, we investigated the cytotoxicity of fluoxetine to human gastric adenocarcinoma cells; as shown by the MTT assay, fluoxetine induced cell death. Subsequently, cells were treated with 10 or 20 µM fluoxetine for 24 h and analyzed. Apoptosis was confirmed by the increased number of early apoptotic cells, shown by Annexin V- propidium iodide staining. Nuclear condensation was visualized by DAPI staining. A significant increase in the expression of cleaved PARP was observed by western blotting. The pan-caspase inhibitor Z-VAD-FMK was used to detect the extent of caspase-dependent cell death. The induction of autophagy was determined by the formation of acidic vesicular organelles (AVOs), which was visualized by acridine orange staining, and the increased expression of autophagy markers, such as LC3B, Beclin 1, and p62/SQSTM 1, observed by western blotting. The expression of upstream proteins, such as p-Akt and p-mTOR, were decreased. Autophagic degradation was evaluated by using bafilomycin, an inhibitor of late-stage autophagy. Bafilomycin did not significantly enhance LC3B expression induced by fluoxetine, which suggested autophagic degradation was impaired. In addition, the co-administration of the autophagy inhibitor 3-methyladenine and fluoxetine significantly increased fluoxetine-induced apoptosis, with decreased p-Akt and markedly increased death receptor 4 and 5 expression. Our results suggested that fluoxetine simultaneously induced both protective autophagy and apoptosis and that the inhibition of autophagy enhanced fluoxetine-induced apoptosis through increased death receptor expression.

• 한국미생물·생명공학회지
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• 제32권1호
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• pp.67-71
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• 2004

남미 아마존 유역의 열대 우림 자생식물인 Pata de Vaca(Bauhinia forficata)의 추출물로부터 활성분획을 분리하여, 세포성장 억제활성 및 세포사멸 유도에 관해 조사하였다. MTT assay를 통해 그 저해 활성을 비교 분석해 가면서 Pata de Vaca의 70% ethanol 추출물을 HP-20 Diaion column chromatograph 및 C-18 column chromatography에 순차적으로 적용시켰다. 그 결과, 저해 활성이 가장 강하며 수율이 높은 Pata-50을 분리하여, 6종의 암세포주에 대한 MTT assay를 실시하였다. Pata-50에 의한 세포성장 억제는, MCF-7을 제외한 나머지 세포주인 HepG2, HT-29, AGS, A549 및 HeLa세포에 대해서 농도 의존적인 저해활성을 보였으며, $IC_{50}$ 는 40.4-77.5$\mu\textrm{g}$/$m\ell$의 농도 범위에서 나타났다. Pata-50의 세포성장 억제를 확인하였으므로, HepG2 세포에.If Pata-50에 의한 세포주기의 변화와 세포사별로 유도되는 세포의 분포를 측정하기 위하여 flow cytometry analysis를 실시하였다. 그 결과, Pata-50은 HepG2에 대하여 농도 의존적으로 세포사멸을 유도하지만, 세포주기 조절에는 아무런 영향을 미치지 많고 있음을 알 수 있었다. 특히, Pata-50을 50$\mu\textrm{g}$/$m\ell$ 이하로 처리할 경우에는 apoptosis가 유도된 세포기 비율이 10%미만이지만, 80$\mu\textrm{g}$/$m\ell$ 및 110$\mu\textrm{g}$/$m\ell$을 처리하면 28.6%및 56.2%로_급격히 증가하였다. 다음으로, 세사멸에 결정적인 단백질의 발현 양상을 관찰하기 위해, HeLa세포주에 Pata-50을 농도별로 처리하여 Western blot으로 분석하였다. Fig. 4에 나타난 바와 같이, HeLa 세포주에 Pata-50을 80$\mu\textrm{g}$/$m\ell$의 농도로 처리하였을 때, caspase-3.가 활성화되었으며, 이로 인해, PARP가 116 kDa에서 85kDa로 cleavag되어, 불활성화 되는 것을 확인할 수 있었다.

• 대한약침학회지
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• 제19권1호
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• pp.37-44
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• 2016

Objectives: This study examined the relative efficacies of a derivative of betulinic acid (dBA) and its poly (lactide-co-glycolide) (PLGA) nano-encapsulated form in A549 lung cancer cells in vivo and in co-mutagen [sodium arsenite (SA) + benzo[a]pyrene (BaP)]-induced lung cancer in mice in vivo. Methods: dBA was loaded with PLGA nanoparticles by using the standard solvent displacement method. The sizes and morphologies of nano-dBA (NdBA) were determined by using transmission electron microscopy (TEM), and their intracellular localization was verified by using confocal microscopy. The binding and interaction of NdBA with calf thymus deoxyribonucleic acid (CT-DNA) as a target were analyzed by using conventional circular dichroism (CD) and melting temperature (Tm) profile data. Apoptotic signalling cascades in vitro and in vivo were studied by using an enzyme-linked immunosorbent assay (ELISA); the ability of NdBA to cross the blood-brain barrier (BBB) was also examined. The stage of cell cycle arrest was confirmed by using a fluorescence-activated cell-sorting (FACS) data analysis. Results: The average size of the nanoparticles was ~ 110 nm. Confocal microscopy images confirmed the presence of NdBA in the cellular cytoplasm. The bio-physical properties of dBA and NdBA ascertained from the CD and the Tm profiles revealed that NdBA had greater interaction with the target DNA than dBA did. Both dBA and NdBA arrested cell proliferation at G0/G1, NdBA showing the greater effect. NdBA also induced a greater degree of cytotoxicity in A549 cells, but it had an insignificant cytotoxic effect in normal L6 cells. The results of flow cytometric, cytogenetial and histopathological studies in mice revealed that NdBA caused less nuclear condensation and DNA damage than dBA did. TEM images showed the presence of NdBA in brain samples of NdBA fed mice, indicating its ability to cross the BBB. Conclusion: Thus, compared to dBA, NdBA appears to have greater chemoprotective potential against lung cancer.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제35권3호
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• pp.143-154
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• 2013

Purpose: This study was to find efficacy of integrin alpha2 (${\alpha}_2$) and epidermal growth factor receptor (EGFR) as tumor marker of oral squamous cell carcinoma (SCC) and clarify the selective cell death effect of anti-integrin ${\alpha}_2$ and anti-EGFR on SCC cells, additionally testify conjugated gold nanoparticles (GNP) with air plasma for selective cell death of oral SCC. Methods: Expression of integrin ${\alpha}_2$, EGFR on human SCC cells (SCC25) were examined by western blot. SCC25 cells were treated with anti-integrin ${\alpha}_2$, anti-EGFR and analysed by Hemacolor staining, immunoflorescence staining, FACS flow cytometry. Conjugated GNP with integrin ${\alpha}_2$, EGFR antibody were treated by air plasma on SCC cells. Results: Integrin ${\alpha}_2$ and EGFR were over-expressed on SCC25 cells than normal lung WI-38 cells. The cell viability rate of SCC25 cells treated with anti-integrin ${\alpha}_2$, anti-EGFR was lower than WI-38 cells. The concentration changes of nucleus, releasing cytochrome c and apoptosis inducing factor (AIF) from mitochondria to cytosol were observed. The changes of proteins related with apoptosis were observed. Increase of bax, bcl-xL, activation of caspase-3, -7, -9, and fragmentation of PARP, DFF45 and decrease of lamin A/C in SCC25 cells were observed. In FACS, increase of sub-$G_1$ and S phase was observed. Cell cycle related proteins, Such as cyclin D1, cyclin dependent kinase (CDK) 4, cyclin A, cyclin E, CDK 2, p27 were decreased. After SCC25 cells treated with conjugatged GNP-Integrin ${\alpha}_2$, GNP-EGFR, additionally air plasma, the cell death rate was significantly increased. Conclusion: Integrin ${\alpha}_2$, EGFR were over-expressed in oral SCC cells. Anti-integrin ${\alpha}_2$, anti-EGFR in SCC25 cells induced apoptosis selectively. When GNP-anti integrin ${\alpha}_2$, GNP-anti EGFR were treated with air plasma on SCC25 cells, cancer cells were died more selectively. GNP-anti integrin ${\alpha}_2$, GNP-anti EGFR with air plasma could be treatment choice of oral SCC.

• 생명과학회지
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• 제26권12호
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• pp.1458-1465
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• 2016

본 연구는 oxidative stress에 의한 세포죽음 분석의 이상적인 모델로 사용되는 HT22세포를 이용하여 천문동 에탄올추출물의 glutamate에 의한 oxidative toxicity에 대한 신경보호 효과를 살펴보았다. 이를 위해 cell viability, lactate dehydrogenase (LDH), 그리고 세포죽음형태, reactive oxygen species (ROS), mitochondria membrane potential (MMP) 등에 대한 flow cytometry 및 Western blot분석을 이용하였다. 천문동 추출물의 처리는 cell viability 및 LDH분석에서 glutamate에 의한 cell toxicity를 저하시키며, 특히 apoptotic cell death를 현저히 감소시켰다. ROS 및 MMP분석 결과, 천문동 추출물은 ROS의 형성을 저하시키며 glutamate에 의해 저하된 MMP를 현저히 회복시켜 주었다. 이와 관련된 단백질 발현을 보면, 천문동 추출물은 PARP 및 HO-1의 발현을 억제하였다. 이상의 결과는 천문동 추출물이 HT22해마세포에서 ROS형성저해 및 MMP회복에 의해 세포죽음을 완화시켜 보호작용을 하는 것으로 사료되며 oxidative toxicity관련 질환에 적용 가능할 것으로 보여 진다.